UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS
AGRÓNOMOS
INFLUENCE OF NEW INTEGRATED PEST MANAGEMENT
STRATEGIES ON APHIDOPHAGOUS PREDATORS IN
HORTICULTURAL CROPS
TESIS DOCTORAL
AGUSTÍN GARZÓN HIDALGO
Ingeniero Agrónomo
2015
DEPARTAMENTO DE PRODUCCIÓN AGRARIA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
INFLUENCE OF NEW INTEGRATED PEST MANAGEMENT
STRATEGIES ON APHIDOPHAGOUS PREDATORS IN
HORTICULTURAL CROPS
Autor: AGUSTÍN GARZÓN HIDALGO
Ingeniero Agrónomo
Directora: FLOR BUDIA MARIGIL
Dra. en Ciencias
2015
UNIVERSIDAD POLITÉCNICA DE MADRID
Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad Politécnica de
Madrid, el día
de
de 2015.
Presidente:
Secretario:
Vocal:
Vocal:
Vocal:
Suplente:
Suplente:
Realizado el acto de defensa y lectura de Tesis el día
de
de 2015
en la Escuela Técnica Superior de Ingenieros Agrónomos de Madrid.
EL PRESIDENTE
EL SECRETARIO
Fdo.:
Fdo.:
LOS VOCALES
Fdo.:
Fdo.:
Fdo.:
A mis padres y a mi hermano
AGRADECIMIENTOS
En primer lugar, quiero agradecer a la Universidad Politécnica de Madrid la concesión de
la beca que me ha permitido realizar esta Tesis.
Quiero dar las gracias a Elisa Viñuela por aceptarme en el laboratorio de la Unidad de
Protección Cultivos de la ETSIA. A Flor Budia, mi directora de Tesis, por transmitirme su
confianza y apoyo a lo largo de estos cuatro años. A Pilar Medina, por su constante ayuda en
el desarrollo de las investigaciones, por su ejemplo y por su cariño. A Ángeles Adán y a
Pedro del Estal por todas sus enseñanzas y su buen talante. He aprendido mucho con todos
vosotros.
Gracias a Luis Quirós y a Yara Quirós por su colaboración en los ensayos de laboratorio. A
Ignacio Morales por su inestimable ayuda tanto en los ensayos de laboratorio como en los de
invernadero, en los que me acompañó y me ayudó a desenvolver el trabajo. Vuestra ayuda fue
fundamental.
Una parte importante de esta Tesis se llevó a cabo en colaboración con el Instituto de
Ciencias Agrarias del CSIC. Por ello, agradezco a Alberto Fereres y a Arantxa Moreno la
posibilidad de realizar los ensayos de diagnóstico de virosis en su laboratorio. A Elisa Garzo,
María Plaza, Beatriz Dáder, Sara Hernando, Saioa Legarrea y Michele Sousa por tratarme
siempre con amabilidad y ayudarme con el desarrollo de los ensayos. Fue un placer trabajar
con vosotros.
Ao professor Geraldo Andrade, por ter me recebido tão calorosamente no Laboratório de
Ecotoxicologia durante minha estadia de pesquisa realizada na UFLA, por sua imensa
hospitalidade, seus ensinamentos e sua amizade. A Leia, por seu carinho, preocupação,
dedicação, alegria, bom trabalho e empenho para que os ensaios fossem bem sucedidos. A
Jader, por ser como um irmão no Brasil. A Valeria e a Pablo por serem excelentes
companheiros e amigos. A Brenda e a Rodrigo por todo o trabalho que realizaram para a
conclusão dos ensaios, muito obrigado. A Fernanda, Rafaella, Thaís, Andrea, Ronelza,
Dejane, Frontino e Lucas, pela colaboração e bondade. A Irene e Julio por serem pessoas tão
boas. Levo todos no meu coração.
Gracias a aquellos investigadores con los que he coincidido en el laboratorio durante algún
periodo de la Tesis, Antonio Gobbi, Sotero Aguilar, Francisco Avendaño y Clifford Sadof,
por sus valiosas enseñanazas y el intercambio cultural.
Gracias a Beatriz Díaz por esclarecer todas las dudas que surgieron a la hora de hacer el
análisis espacial de vectores y virus, y por su gran amabilidad.
Gracias a Agustín Fernández y a María Luisa García del Centro Nacional de Microscopía
Electrónica por su profesionalidad y diligencia.
Gracias a Román Zurita por la asistencia técnica en los ensayos de invernadero.
Gracias de corazón a todos mis compañeros y amigos, con los que compartí vivencias
durante estos cuatro años y que contribuyeron a crear un estupendo ambiente donde hubo
tiempo tanto para el trabajo como para las fiestas. Empiezo por los veteranos Fermín y
Paloma, que ejercieron de cicerones en mis primeros años en el laboratorio y con quienes
compartí muchos momentos de risas. A Mar, con quien he compartido largos ratos de
escritura en los últimos meses. A Rosa, por ser una excelente compañera y amiga, además de
buenísima persona. A mi querida Andrea por ser tan alegre y tener tan buen humor siempre,
por su amistad y todos los buenos momentos que hemos pasado. A mis colegas lusoparlantes
Valentim, Alexandre y Lara, con quienes pasé muy buenos ratos y que me dieron la
oportunidad de practicar portugués. A Rubén, con quien tuve una conexión especial desde el
principio, por su buena filosofía de vida y su amistad. A Aline, por haber compartido con ella
muchos momentos de felicidad. Y por supuesto a Guillermo, Mario, Celeste, Gonzalo, Sergio,
Ana y Míriam, con quienes he pasado muy buenos momentos también.
A Jelena, con quien coincidí al inicio de esta etapa, durante la cual hemos compartido
sinsabores, alegrías e inquietudes (además de épicas noches en los karaokes de Madrid),
fraguando en una gran amistad. A Luis Hiernaux, por proponerme una colaboración en la
redacción de dos libros, fruto de la cual surgió una buena amistad.
A mi familia en general, y en especial a mi tía Flor, por su constante cariño y
preocupación.
Y por supuesto, a mis padres por darme la vida, la educación y el amor que me han dado,
gracias infinitas. Y a mi querido hermano Javier, por ser tan bueno y quererme tanto.
INDEX
ABBREVIATIONS .................................................................................................................. v
FIGURE INDEX .................................................................................................................... vii
TABLE INDEX ....................................................................................................................... xi
RESUMEN .............................................................................................................................. xv
SUMMARY............................................................................................................................ xix
CHAPTER 1. GENERAL INTRODUCTION....................................................................... 1
1.1. HORTICULTURAL CROPS PRODUCTION ............................................................... 1
1.2. APHIDS AND ASSOCIATED VIRAL DISEASES ...................................................... 2
1.2.1. NON-PERSISTENTLY TRANSMITTED VIRUSES ............................................. 4
1.2.2. PERSISTENTLY TRANSMITTED VIRUSES ....................................................... 5
1.3. INTEGRATED PEST MANAGEMENT (IPM) IN HORTICULTURAL CROPS ....... 7
1.4. BIOLOGICAL CONTROL STRATEGIES .................................................................... 8
1.4.1. APHIDOPHAGOUS PREDATORS ........................................................................ 8
1.4.2. TROPHIC INTERACTIONS IN THE AGROECOSYSTEM ............................... 11
1.5. PHYSICAL CONTROL STRATEGIES ....................................................................... 12
1.5.1. UV-ABSORBING PLASTIC COVERS ................................................................ 12
1.6. CHEMICAL CONTROL STRATEGIES ..................................................................... 13
1.6.1. SIDE EFFECTS OF PESTICIDES ON NATURAL ENEMIES ........................... 13
CHAPTER 2. OBJECTIVES ................................................................................................ 15
CHAPTER 3. INFLUENCE OF APHIDOPHAGOUS PREDATORS ON THE SPREAD
OF NON-PERSISTENTLY AND PERSISTENTLY TRANSMITTED VIRUSES BY
APHID VECTORS ................................................................................................................. 17
SUB-CHAPTER 3.1. THE EFFECT OF Chrysoperla carnea AND Adalia bipunctata ON
THE SPREAD OF Cucumber mosaic virus BY Aphis gossypii........................................... 17
ABSTRACT ......................................................................................................................... 17
3.1.1. INTRODUCTION ...................................................................................................... 18
3.1.2. MATERIALS AND METHODS ............................................................................... 20
3.1.2.1. INSECTS ............................................................................................................. 20
3.1.2.2. PLANT MATERIAL ........................................................................................... 21
i
3.1.2.3. VIRUS ACQUISITION ....................................................................................... 21
3.1.2.4. EXPERIMENTS .................................................................................................. 21
3.1.2.5. STATISTICAL ANALYSIS ............................................................................... 24
3.1.3. RESULTS ................................................................................................................... 26
3.1.3.1. A. gossypii DENSITY ON THE CENTRAL SOURCE PLANT ........................ 26
3.1.3.2. A. gossypii DISPERSAL FROM THE CENTRAL SOURCE PLANT .............. 26
3.1.3.3. CMV SPREAD .................................................................................................... 29
3.1.3.4. ASSOCIATION PATTERNS BETWEEN VECTOR AND VIRUS .................. 33
3.1.4. DISCUSSION ............................................................................................................. 36
SUB-CHAPTER 3.2. THE EFFECT OF Chrysoperla carnea AND Adalia bipunctata ON
THE SPREAD OF Cucurbit aphid-borne yellows virus BY Aphis gossypii........................ 39
ABSTRACT ......................................................................................................................... 39
3.2.1. INTRODUCTION ...................................................................................................... 40
3.2.2. MATERIALS AND METHODS ............................................................................... 41
3.2.2.1. INSECTS ............................................................................................................. 41
3.2.2.2. PLANT MATERIAL ........................................................................................... 42
3.2.2.3. VIRUS ACQUISITION ....................................................................................... 42
3.2.2.4. EXPERIMENTS .................................................................................................. 43
3.2.2.5. STATISTICAL ANALYSIS ............................................................................... 45
3.2.3. RESULTS ................................................................................................................... 46
3.2.3.1. A. gossypii DENSITY ON THE CENTRAL SOURCE PLANT ........................ 46
3.2.3.2. A. gossypii DISPERSAL FROM THE CENTRAL SOURCE PLANT .............. 47
3.2.3.3. CABYV SPREAD ............................................................................................... 50
3.2.3.4. ASSOCIATION PATTERNS BETWEEN VECTOR AND VIRUS .................. 54
3.2.4. DISCUSSION ............................................................................................................. 57
CHAPTER 4. DEVELOPMENT AND REPRODUCTION OF Chrysoperla externa FED
ON Myzus persicae VECTORING Potato leafroll virus ...................................................... 61
ABSTRACT ......................................................................................................................... 61
4.1. INTRODUCTION ......................................................................................................... 62
4.2. MATERIALS AND METHODS .................................................................................. 64
4.2.1. INSECTS REARING METHODS ......................................................................... 64
4.2.2. BIOASSAY............................................................................................................. 66
4.2.3. STATISTICAL ANALYSIS .................................................................................. 67
ii
4.3. RESULTS ...................................................................................................................... 68
4.3.1. DEVELOPMENTAL TIME AND SURVIVAL OF LARVAL AND PUPAL
STAGES ........................................................................................................................... 68
4.3.2. REPRODUCTIVE PARAMETERS AND SURVIVAL OF ADULTS ................. 70
4.4. DISCUSSION ................................................................................................................ 72
CHAPTER 5. DIRECT AND PLANT-MEDIATED INFLUENCE OF LOW UV
RADIATION ON Aphis gossypii AND THE PREDATOR Chrysoperla carnea IN
ABSENCE AND PRESENCE OF Cucumber aphid-borne yellows virus ........................... 77
ABSTRACT ......................................................................................................................... 77
5.1. INTRODUCTION ......................................................................................................... 78
5.2. MATERIALS AND METHODS .................................................................................. 81
5.2.1. INSECTS ................................................................................................................ 82
5.2.2. PLANT MATERIAL .............................................................................................. 82
5.2.3. UV RADIATION SOURCE AND UV-ABSORBING PLASTIC COVERS ........ 83
5.2.4.
VIRUS
ACQUISITION
IN
THE
CABYV-INFECTED
CUCUMBER
EXPERIMENT ................................................................................................................. 85
5.2.5. EXPERIMENTS ..................................................................................................... 86
5.2.5.1. EXPERIMENTAL DESIGN ........................................................................... 86
5.2.5.2. A. gossypii INFESTATION AND C. carnea RELEASING ........................... 87
5.2.5.3. PHYSIOLOGICAL MEASURES ON CUCUMBER PLANTS .................... 89
5.2.5.3.1. FRESH WEIGHT, DRY WEIGHT AND STEM LENGTH................... 89
5.2.5.3.2. CELL WALL AND CUTICLE ANALYSIS OF LEAF SAMPLES ...... 90
5.2.5.3.3. CARBON, HYDROGEN, NITROGEN AND SULFUR ANALYSIS OF
LEAF SAMPLES .................................................................................................... 93
5.2.6. STATISTICAL ANALYSIS .................................................................................. 93
5.3. RESULTS ...................................................................................................................... 94
5.3.1. HEALTHY CUCUMBER PLANTS EXPERIMENT ............................................ 94
5.3.1.1. PLANT-MEDIATED EFFECTS OF LOW UV RADIATION ON THE
FITNESS OF A. gossypii.............................................................................................. 94
5.3.1.2. PLANT-PREY-MEDIATED EFFECTS OF LOW UV RADIATION ON C.
carnea ........................................................................................................................... 97
5.3.1.3. DIRECT EFFECTS OF LOW UV RADIATION ON CUCUMBER PLANTS
...................................................................................................................................... 98
iii
5.3.2. CABYV-INFECTED CUCUMBER PLANTS EXPERIMENT .......................... 100
5.3.2.1. PLANT-MEDIATED EFFECTS OF LOW UV RADIATION ON THE
FITNESS OF A. gossypii............................................................................................ 100
5.3.2.2. PLANT-PREY-MEDIATED EFFECTS OF LOW UV RADIATION ON C.
carnea ......................................................................................................................... 103
5.3.2.3. DIRECT EFFECTS OF LOW UV RADIATION ON CUCUMBER PLANTS
.................................................................................................................................... 104
5.4. DISCUSSION .............................................................................................................. 106
CHAPTER 6. TOXICITY AND SUBLETHAL EFFECTS OF SIX INSECTICIDES TO
LAST INSTAR LARVAE AND ADULTS OF THE BIOCONTROL AGENTS
Chrysoperla carnea AND Adalia bipunctata ....................................................................... 113
ABSTRACT ....................................................................................................................... 113
6.1. INTRODUCTION ....................................................................................................... 114
6.2. MATERIALS AND METHODS ................................................................................ 116
6.2.1. INSECTS .............................................................................................................. 116
6.2.2. INSECTICIDES .................................................................................................... 117
6.2.3. BIOASSAYS ........................................................................................................ 118
6.2.3.1. LARVAE BIOASSAYS................................................................................ 118
6.2.3.2. ADULTS BIOASSAYS ................................................................................ 120
6.2.4. STATISTICAL ANALYSIS ................................................................................ 121
6.3. RESULTS .................................................................................................................... 122
6.3.1. EFFECTS OF INSECTICIDES ON C. carnea .................................................... 122
6.3.2. EFFECTS OF INSECTICIDES ON A. bipunctata............................................... 126
6.4. DISCUSSION .............................................................................................................. 129
CONCLUSIONS................................................................................................................... 133
REFERENCES ..................................................................................................................... 135
iv
ABBREVIATIONS
BBCH: Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie
BCA: biological control agent
C: centroid
CE: Comunidad Europea
CABYV: Cucurbit aphid-borne yellows virus
cm: centimeter
CME: Centro de Microanálisis Elemental
CMV: Cucumber mosaic virus
CNME: Centro Nacional de Microscopía Electrónica
CSIC: Consejo Superior de Investigaciones Científicas
cv.: cultivar
DAS-ELISA: Double Antibody Sandwich - Enzyme Linked Immunosorbent Assay
Dr.: Doctor
EC: European Community
EELM: Estación Experimental “La Mayora”
ETSIA: Escuela Técnica Superior de Ingenieros Agrónomos
EU: European Union
g: gram
GIP: Gestión Integrada de Plagas
h: hour
ha: hectare
Ia: SADIE index of aggregation
IOBC: International Organization for Biological Control
IPM: Integrated Pest Management
IRAC: Insecticide Resistance Action Committee
kJ: kilojoule
kPa: kilopascal
l: liter
L:D: light:dark
LS: solution for seed treatment
v
LSD: least significant difference
m: meter
MFRC: maximum field recommended concentration
min: minute
ml: milliliter
MoA: mode of action
n: number of replicates
nm: nanometer
PAR: photosinthetically active radiation
PLRV: Potato leafroll virus
PVC: polyvinyl chloride
RH: relative humidity
RNA: ribonucleic acid
SADIE: Spatial Analysis by Distance IndicEs
S.E.: standard error
t: ton
UCM: Universidad Complutense de Madrid
UFLA: Universidade Federal de Lavras
UPM: Universidad Politécnica de Madrid
UV: ultraviolet
μm: micrometer
vi: SADIE index of clustering in patches
vj: SADIE index of clustering in gaps
W: watt
X: SADIE index of spatial association
vi
FIGURE INDEX
CHAPTER 1
Figure 1.1. Commercial greenhouse of cucumber in Almería (Spain) (left) and potato crop
in Segovia (Spain) (right). ..................................................................................................... 2
Figure 1.2. Alate and nymphs of A. gossypii on a cucumber cotyledon (left) and colony of
apterae M. persicae on a potato leaf (right). .......................................................................... 4
Figure 1.3. Symptoms of Cucumber mosaic virus (CMV) in a cucumber leaf. .................... 5
Figure 1.4. Symptoms of Cucurbit aphid-borne yellows virus (CABYV) in a cucumber leaf
(left) and symptoms of Potato leafroll virus (PLRV) in a potato leaf (right). ...................... 7
Figure 1.5. Larva of C. carnea on a cucumber leaf (left) and larva of C. externa predating
an aphid (right). ................................................................................................................... 10
Figure 1.6. In the aphidophagous coccinellids as A. bipunctata, both larvae (left) and adults
(right) have predaceous habits. ............................................................................................ 11
CHAPTER 3
SUB-CHAPTER 3.1
Figure 3.1.1. Insect-proof net covered cages where replicates were placed, at greenhouse
facilities of the ETSIA-UPM. .............................................................................................. 22
Figure 3.1.2. Spatial distribution of the experimental unit showing in the central point the
CMV-source plant where alate aphids and natural enemies were released, surrounded by
the 48 test plants at one true leaf stage. ............................................................................... 23
Figure 3.1.3. Recording of the number of A. gossypii alatae and nymphs in the surrounding
test plants. ............................................................................................................................ 24
Figure 3.1.4. A. gossypii dispersal and CMV spread (centroids) from the central source
plant in the short and long term (1 day and 5 days) in the absence (z) or presence (S) of
natural enemies. Short-term evaluation (1 day): C. carnea (a, e); A. bipunctata (b, f). Longterm evaluation (5 days): C. carnea (c, g); A. bipunctata (d, h). The axes show the
coordinates for each cucumber plant in the plot; the real distance between each plant was
12 cm. .................................................................................................................................. 35
vii
SUB-CHAPTER 3.2
Figure 3.2.1. Inoculation of CABYV by transferring viruliferous A. gossypii individuals
from a previously infected source plant (left) to receptor healthy plants (right)................. 43
Figure 3.2.2. Natural enemies releasing in the central CABYV-source cucumber plant. ... 44
Figure 3.2.3. Microplates used for CABYV diagnosis in test plants by means of ELISA
serological technique. .......................................................................................................... 45
Figure 3.2.4. A. gossypii dispersal and CABYV spread (centroids) from the central source
plant in the short and long term (7 days and 14 days) in the absence (z) or presence (S)
of natural enemies. Short-term evaluation (7 days): C. carnea (a, e); A. bipunctata (b, f).
Long-term evaluation (14 days): C. carnea (c, g); A. bipunctata (d, h). The axes show the
coordinates for each cucumber plant in the plot; the real distance between each plant was
12 cm. .................................................................................................................................. 56
CHAPTER 4
Figure 4.1. C. externa adults rearing cage draped with filter paper to collect the eggs to be
used in the experiments. ...................................................................................................... 64
Figure 4.2. Uninfected potato leaves (left) and PLRV-infected potato leaves (right) used to
rear the M. persicae individuals used in the experiments. .................................................. 65
Figure 4.3. Experimental units used to follow the C. externa larval development. ............ 66
Figure 4.4. Experimental unit used for a couple (female and male) of C. externa adults to
asses the fecundity (left) and microplate to evaluate the egg hatching (right). ................... 67
Figure 4.5. Survival curve of C. externa adults (30 days after emergence) developed from
larvae fed with E. kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M.
persicae, in laboratory conditions (25 ± 2°C, 70 ± 10% RH, photoperiod 12:12 L:D).
Curves significantly differed (Kaplan-Meier Log-Rank, Holm-Sidak, p < 0.05). .............. 72
CHAPTER 5
Figure 5.1. Experimental cages covered by two different plastic films: control treatment
(Térmico Blanco®) (left) and abs-UV treatment (Térmico Antivirus®) (right). .................. 85
Figure 5.2. Cucumber plant used as CABYV source (left) for the inoculation of the
experimental plants by transferring viruliferous A. gossypii individuals (right). ................ 86
viii
Figure 5.3. A. gossypii infestation of cucumber plants (BBCH scale = 14) (left) and L1 C.
carnea larvae ready to be released two weeks after (right). ................................................ 88
Figure 5.4. Chronogram of the experiments, with the four different UV radiation
treatments (control, plants grown under Térmico Blanco® plastic film along all the trial;
control > abs-UV, plants grown under Térmico Blanco® plastic film until aphid infestation
and transferred under Térmico Antivirus® plastic film until the end of the trial; abs-UV >
control, plants grown under Térmico Antivirus® plastic film until aphid infestation and
transferred under Térmico Blanco® plastic film until the end of the trial; abs-UV, plants
grown under Térmico Antivirus® plastic film along all the trial), dates of A. gossypii
infestation and C. carnea releasing and collecting, and cucumber plants harvesting. ........ 89
Figure 5.5. Fixation process of the vegetal samples (left) and ethanol graded series for their
dehydration (right). .............................................................................................................. 91
Figure 5.6. Silicon mold with the vegetal samples embedded in the hardened resin. ......... 92
Figure 5.7. Transmission electron microscope images of the cell wall (left) and the cuticle
(right) of adaxial epidermal cells of cucumber leaves. ........................................................ 92
Figure 5.8. Evolution of A. gossypii apterae population during the experiment on healthy
cucumber plants. The releasing of C. carnea larvae was done in S7 (7th sampling)........... 96
Figure 5.9. Evolution of A. gossypii alatae population during the experiment on healthy
cucumber plants. The releasing of C. carnea larvae was done in S7 (7th sampling)........... 96
Figure 5.10. Evolution of A. gossypii apterae population during the experiment on
CABYV-infected cucumber plants. The releasing of C. carnea larvae was done in S7 (7th
sampling). .......................................................................................................................... 102
Figure 5.11. Evolution of A. gossypii alatae population during the experiment on CABYVinfected cucumber plants. The releasing of C. carnea larvae was done in S7 (7th sampling).
........................................................................................................................................... 102
CHAPTER 6
Figure 6.1. Experimental units used for testing pesticides side-effects in C. carnea (left)
and A. bipunctata (right) larvae. ........................................................................................ 119
Figure 6.2. Experimental cages where C. carnea (left) and A. bipunctata (right) emerged
adults were transferred to for the evaluation of the reproductive parameters. .................. 120
ix
Figure 6.3. Experimental units used for testing pesticides side-effects in C. carnea (left)
and A. bipunctata (right) adults. ........................................................................................ 121
x
TABLE INDEX
CHAPTER 3
SUB-CHAPTER 3.1
Table 3.1.1. Influence of the presence of C. carnea L3 or A. bipunctata adults on the
density of A. gossypii (adult alate morphs and nymphs) in the central CMV-source plant 1
day and 5 days after infestation with 100 adult alatae. The data are the means ± SE of the
number of aphids/plant (n = 3 replicates)1. ......................................................................... 28
Table 3.1.2. Influence of the presence of C. carnea L3 or A. bipunctata adults on the
percentage of peripheral plants occupied by at least one A. gossypii (adult alate or nymph)
and percentage of CMV-infected plants 1 and 5 days after infestation with 100 adult alatae
in the central CMV-source plant. The data are the means ± SE (n = 3 replicates)1. ........... 28
Table 3.1.3. Spatial pattern indexes of A. gossypii populations and CMV spread after 1 day
in the presence and absence (control) of C. carnea L3 or A. bipunctata adults. The data are
the means ± SE of three replicates/natural enemy1. ............................................................ 31
Table 3.1.4. Spatial pattern indexes of A. gossypii population and CMV spread after 5 days
in the presence and absence (control) of C. carnea L3 or A. bipunctata adults. The data are
the means ± SE of three replicates/natural enemy1. ............................................................ 32
Table 3.1.5. Association index (X) in every replicate (1, 2, 3) between A. gossypii and
CMV infection, after 1 day or 5 days, in the presence of C. carnea L3 or A. bipunctata
adults.................................................................................................................................... 34
SUB-CHAPTER 3.2 Table 3.2.1. Influence of the presence of C. carnea larvae or A. bipunctata adults on the
density of A. gossypii (adult alate morphs and nymphs) in the central CABYV-source plant
7 days and 14 days after infestation with 100 adult alatae. The data are the means ± SE of
the number of aphids/plant (n = 3 replicates)1..................................................................... 49
Table 3.2.2. Influence of the presence of C. carnea larvae or A. bipunctata adults on the
percentage of peripheral plants occupied by at least one A. gossypii (adult alate or nymph)
and percentage of CABYV-infected plants 7 and 14 days after infestation with 100 adult
alatae in the central CABYV-source plant. The data are the means ± SE (n = 3 replicates)1.
............................................................................................................................................. 49
xi
Table 3.2.3. Spatial pattern indexes of A. gossypii populations and CABYV spread after 7
days in the presence and absence (control) of C. carnea larvae or A. bipunctata adults. The
data are the means ± SE of three replicates/natural enemy1. ............................................... 52
Table 3.2.4. Spatial pattern indexes of A. gossypii population and CABYV spread after 14
days in the presence and absence (control) of C. carnea larvae or A. bipunctata adults. The
data are the means ± SE of three replicates/natural enemy1. ............................................... 53
Table 3.2.5. Association index (X) in every replicate (1, 2, 3) between A. gossypii and
CABYV infection, after 7 days or 14 days, in presence of C. carnea larvae or A. bipunctata
adults.................................................................................................................................... 55
CHAPTER 4 Table 4.1. Developmental time of larval instars, total larval period and pupal period of C.
externa when fed with E. kuehniella eggs, non-PLRV-infected M. persicae and PLRVinfected M. persicae, in laboratory conditions (25 ± 2°C, 70 ± 10% RH, photoperiod 12:12
L:D). .................................................................................................................................... 68
Table 4.2. Survival of larval instars, total larval period and pupal period of C. externa
when fed with E. kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M.
persicae, in laboratory conditions (25 ± 2°C, 70 ± 10% RH, photoperiod 12:12 L:D). ..... 69
Table 4.3. Reproductive parameters of C. externa adults when larvae were fed with E.
kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M. persicae, in
laboratory conditions (25 ± 2°C, 70 ± 10% RH, photoperiod 12:12 L:D). ......................... 71
CHAPTER 5 Table 5.1. Radiation conditions under the UV-source lamp (50 cm from the light bulb) and
inside the experimental cages (below the plastic cover). Transmission percentages
represent the radiation transmitted inside both cages with regard to the same level outside
the cages. ............................................................................................................................. 84
Table 5.2. Evolution of A. gossypii apterae population on healthy cucumber plants under
different UV radiation conditions. The releasing of 20 adult apterae individuals/plant was
done on 14/10/2013. The temperature and relative humidity conditions during the
experiment were controlled (15-27 ºC, 50-85% RH). ......................................................... 95
Table 5.3. Evolution of A. gossypii alatae population on healthy cucumber plants under
different UV radiation conditions. The releasing of 20 adult apterae individuals/plant was
xii
done on 14/10/2013. The temperature and relative humidity conditions during the
experiment were controlled (15-27 ºC, 50-85% RH). ......................................................... 95
Table 5.4. Reproductive parameters of C. carnea adults developed from the larvae released
inside the experimental cages under different UV radiation conditions with healthy
cucumber plants. Fecundity (±SE) and fertility (±SE) were measured along the first week
of oviposition in laboratory conditions (25 ± 2 ºC, 75 ± 10% RH, 16:8 L:D photoperiod).97
Table 5.5. Stem lenght (cm), fresh weight (g) and dry weight (g) of healthy cucumber
plants grown under different UV radiation conditions. The temperature and relative
humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH)........ 98
Table 5.6. Percentages (%) of C, H, N and S of healthy cucumber plants grown under
different UV radiation conditions. The temperature and relative humidity conditions during
the experiment were controlled (15-27 ºC, 50-85% RH). ................................................... 99
Table 5.7. Cell wall thickness (μm) and cuticle thickness (nm) of epidermal cells of the
upper face of leaves of healthy cucumber plants grown under different UV radiation
conditions. The temperature and relative humidity conditions during the experiment were
controlled (15-27 ºC, 50-85% RH). ..................................................................................... 99
Table 5.8. Evolution of A. gossypii apterae on CABYV-infected cucumber plants under
different UV radiation conditions. The releasing of 20 adult apterae individuals/plant was
done on 28/03/2014. The temperature and relative humidity conditions during the
experiment were controlled (15-27 ºC, 50-85% RH). ....................................................... 101
Table 5.9. Evolution of A. gossypii alatae on CABYV-infected cucumber plants under
different UV radiation conditions. The releasing of 20 adult apterae individuals/plant was
done on 28/03/2014. The temperature and relative humidity conditions during the
experiment were controlled (15-27 ºC, 50-85% RH). ....................................................... 101
Table 5.10. Reproductive parameters of C. carnea adults developed from the larvae
released inside the experimental cages under different UV radiation conditions with
CABYV-infected cucumber plants. Fecundity (±SE) and fertility (±SE) were measured
along the first week of oviposition in laboratory conditions (25 ± 2 ºC, 75 ± 10% RH, 16:8
L:D photoperiod). .............................................................................................................. 104
Table 5.11. Stem lenght (cm), fresh weight (g) and dry weight (g) of CABYV-infected
cucumber plants grown under different UV radiation conditions. The temperature and
xiii
relative humidity conditions during the experiment were controlled (15-27 ºC, 50-85%
RH). ................................................................................................................................... 104
Table 5.12. Percentages (%) of C, H, N and S of CABYV-infected cucumber plants grown
under different UV radiation conditions. The temperature and relative humidity conditions
during the experiment were controlled (15-27 ºC, 50-85% RH). ...................................... 105
Table 5.13. Cell wall thickness (μm) and cuticle thickness (nm) of epidermal cells of the
upper face of leaves of CABYV-infected cucumber plants grown under different UV
radiation conditions. The temperature and relative humidity conditions during the
experiment were controlled (15-27 ºC, 50-85% RH). ....................................................... 106
CHAPTER 6 Table 6.1. Effects on development, mortality and reproduction in C. carnea when L3 larvae
were exposed to fresh residues of insecticides on a glass surface, using the maximum field
recommended concentration (MFRC) for horticultural crops. .......................................... 124
Table 6.2. Effects on mortality and reproduction in C. carnea when adults were exposed to
fresh residues of insecticides on a glass surface, using the maximum field recommended
concentration (MFRC) for horticultural crops. ................................................................. 125
Table 6.3. Effects on development, mortality and reproduction in A. bipunctata when L4
larvae were exposed to fresh residues of insecticides on a glass surface, using the
maximum field recommended concentration (MFRC) for horticultural crops. ................ 127
Table 6.4. Effects on mortality and reproduction in A. bipunctata when adults were
exposed to fresh residues of insecticides on a glass surface, using the maximum field
recommended concentration (MFRC) for horticultural crops. .......................................... 128
xiv
RESUMEN
El 1 de enero de 2014 entró en vigor la Directiva Europea 2009/128/CE sobre uso
sostenible de plaguicidas y el Real Decreto 1311/2012 por el cual se traspone dicha normativa
comunitaria al ámbito nacional. Estos reglamentos establecen el marco legal por el que las
explotaciones agrícolas deben cumplir los principios generales de la Gestión Integrada de
Plagas (GIP). Los principios de la GIP dan preferencia a aquellos métodos de control que sean
sostenibles y respetuosos con el medio ambiente, dando prioridad al control biológico, al
físico y a otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos con los
enemigos naturales es necesario en ocasiones para el adecuado manejo de las plagas en
cultivos hortícolas. Por ello, el objetivo general de esta Tesis ha sido aportar conocimientos
para la mejora del control de plagas en cultivos hortícolas, mediante la integración de
estrategias de lucha biológica, física y química.
La primera de las líneas de investigación de esta Tesis se centró en el estudio del efecto de
la presencia dos depredadores, larvas Chrysoperla carnea y adultos de Adalia bipunctata, en
la dispersión del virus de transmisión no persistente Cucumber mosaic virus (CMV) y del
virus de transmisión persistente Cucurbit aphid-borne yellows virus (CABYV), transmitidos
por el pulgón Aphis gosypii en cultivo de pepino. La tasa de transmisión de CMV fue baja
para los dos tiempos de evaluación ensayados (1 y 5 días), debido al limitado movimiento de
su vector A. gossypii. Las plantas que resultaron infectadas se localizaron próximas a la fuente
de inóculo central y la presencia de ambos enemigos naturales no incrementó
significativamente el porcentaje de plantas ocupadas por pulgones ni la tasa de transmisión de
CMV. Los patrones de distribución de A. gossypii y de CMV tan solo fueron coincidentes en
las proximidades de la planta central infectada en la que se liberaron los insectos. En los
ensayos con CABYV, la presencia de C. carnea y de A. bipunctata respectivamente provocó
xv
un incremento significativo de la dispersión de A. gossypii tras 14 días, pero no tras 7 días
desde la liberación de los insectos. La reducción en el número inicial de pulgones en la planta
central infectada con CABYV fue siempre mayor tras la liberación de C. carnea en
comparación con A. bipunctata. Sin embargo, la tasa de transmisión de CABYV y su
distribución espacial no se vieron significativamente modificadas por la presencia de ninguno
de los depredadores, ni tras 7 días ni tras 14 días desde el inicio de los ensayos.
Al igual que se estudió el efecto de la presencia de enemigos naturales en el
comportamiento de las plagas y en la epidemiología de las virosis que transmiten, en una
segunda línea de investigación se evaluó el posible efecto del consumo de pulgones
portadores de virus por parte de los enemigos naturales. Este trabajo se llevó a cabo en el
Laboratorio de Ecotoxicología del Departamento de Entomología de la Universidade Federal
de Lavras (UFLA) (Brasil). En él se evaluó la influencia en los parámetros biológicos del
enemigo natural Chrysoperla externa al alimentarse de Myzus persicae contaminados con el
virus de transmisión persistente Potato leafroll virus (PLRV). El consumo de M. persicae
contaminados con PLRV incrementó significativamente la duración de la fase larvaria,
reduciendo también la supervivencia en comparación a otras dos dietas a base de M. persicae
no contaminados con el virus y huevos del lepidóptero Ephestia kuehniella. La duración de la
fase de pupa de C. externa no difirió significativamente entre las dietas a base de pulgones
contaminados con PLRV y pulgones no contaminados, pero ambas fueron menores que con la
dieta con huevos de E. kuehniella. Sin embargo, ni la supervivencia en la fase de pupa ni los
parámetros reproductivos de los adultos emergidos mostraron diferencias significativas entre
las dietas evaluadas. Por el contrario, la supervivencia de los adultos durante los 30 primeros
días desde su emergencia sí se vio significativamente afectada por la dieta, siendo al término
de este periodo del 54% para aquellos adultos de C. externa que durante su fase larvaria
consumieron pulgones con PLRV.
xvi
Dentro de la GIP, una de las estrategias de carácter físico que se emplean para el control de
plagas y enfermedades en cultivos hortícolas protegidos es el uso de plásticos con
propiedades fotoselectivas de absorción de la radiación ultravioleta (UV). Por ello, la tercera
línea de investigación de la Tesis se centró en el estudio de los efectos directos e indirectos
(mediados por la planta) de condiciones especiales de baja radiación UV sobre el crecimiento
poblacional del pulgón A. gossypii y los parámetros biológicos del enemigo natural C.
carnea, así como sobre las plantas de pepino en las que se liberaron los insectos. Los ensayos
se realizaron en jaulones dentro de invernadero, utilizándose en el primero de ellos plantas de
pepino sanas, mientras que en el segundo las plantas de pepino fueron previamente infectadas
con CABYV para estudiar de qué manera afectaba la incidencia del virus en las mismas
condiciones. Las condiciones de baja radiación UV (bajo plástico Térmico Antivirus®)
ejercieron un efecto directo en las fases iniciales del cultivo de pepino, promoviendo su
crecimiento, mientras que en fases más avanzadas del cultivo indujeron un aumento en el
contenido en nitrógeno de las plantas. Las plantas de pepino que fueron sometidas a mayor
intensidad de radiación UV (bajo plástico Térmico Blanco®) al inicio del cultivo mostraron un
engrosamiento significativo de las paredes de las células epidérmicas del haz de las hojas, así
como de la cutícula. El uso del plástico Térmico Antivirus®, utilizado como barrera
fotoselectiva para crear condiciones de baja radiación UV, no alteró con respecto al plástico
Térmico Blanco® (utilizado como control) el desarrollo poblacional del pulgón A. gossypii ni
los parámetros biológicos evaluados en el depredador C. carnea. En el segundo experimento,
realizado con plantas infectadas con CABYV, la incidencia de la virosis enmascaró las
diferencias encontradas en experimento con plantas sanas, reduciendo aparentemente la
influencia de las distintas condiciones de radiación UV.
Por último, para el desarrollo de las estrategias de GIP es importante estudiar los posibles
efectos secundarios que los plaguicidas pueden tener en los enemigos naturales de las plagas.
xvii
Es por ello que en la Tesis se evaluaron la toxicidad y los efectos subletales (fecundidad y
fertilidad) de flonicamida, flubendiamida, metaflumizona, spirotetramat, sulfoxaflor y
deltametrina en los enemigos naturales C. carnea y A. bipunctata. Los efectos secundarios
fueron evaluados por contacto residual tanto para larvas como para adultos de ambos
enemigos
naturales
en
condiciones
de
laboratorio.
Flonicamida,
flubendiamida,
metaflumizona y spirotetramat fueron inocuos para larvas de último estadio y adultos de C.
carnea y A. bipunctata. Por este motivo, estos insecticidas se presentan como buenos
candidatos para ser incorporados dentro de programas de GIP en combinación con estos
enemigos naturales para el control de plagas de cultivos hortícolas. Sulfoxaflor fue
ligeramente tóxico para adultos de C. carnea y altamente tóxico para larvas de último estadio
de A. bipunctata. Para A. bipunctata, sulfoxaflor y deltametrina fueron los compuestos más
dañinos. Deltametrina fue también el compuesto más tóxico para larvas y adultos de C.
carnea. Por tanto, el uso de deltametrina y sulfoxaflor en programas de GIP debería tomarse
en consideración cuando se liberasen cualquiera de estos dos enemigos naturales debido al
comportamiento tóxico que mostraron en condiciones de laboratorio.
xviii
SUMMARY
On 1 January 2014 came into effect the Directive 2009/128/EC of the European Parliament
about sustainable use of pesticides and the Royal Decree 1311/2012 that transposes the
regulation to the Spanish level. These regulations establish the legal framework that
agricultural holdings must adhere to in order to accomplish the general principles of
Integrated Pest Management (IPM). The guidelines of IPM give priority to sustainable and
eco-friendly pest control techniques, such as biological and physical measures. Nevertheless,
the use of pesticides that are selective to natural enemies is sometimes a necessary strategy to
implement accurate pest management programs in horticultural protected crops. Therefore,
the general objective of this Thesis was to contribute to the improvement of pest management
strategies in horticultural crops, by means of the integration of biological, physical and
chemical techniques.
The first research line of this Thesis was focused on the evaluation of the effects of two
aphidophagous predators, Chrysoperla carnea larvae and Adalia bipunctata adults, on the
spread of the non-persistently transmitted Cucumber mosaic virus (CMV, Cucumovirus) and
the persistently transmitted Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), by
the aphid vector Aphis gossypii in a cucumber crop under greenhouse conditions. The CMV
transmission rate was generally low, both after 1 and 5 days, due to the limited movement of
its aphid vector A. gossypii. Infected plants were mainly located around the central virusinfected source plant, and the percentage of aphid occupation and CMV-infected plants did
not differ significantly in absence and presence of natural enemies. The distribution patterns
of A. gossypii and CMV were only coincident close to the central plant where insects were
released. In the CABYV experiments, the presence of C. carnea larvae and A. bipunctata
adults induced significant A. gossypii dispersal after 14 days but not after 7 days. The
xix
reduction in the initial aphid population established in the central plant was always higher for
C. carnea than for A. bipunctata. Nevertheless, CABYV spread was not significantly
modified by the presence of each predator either in the short term (7 days) or in the long term
(14 days). Furthermore, the percentage of CABYV-infected plants did not significantly differ
when each natural enemy was present in any evaluation period.
It is important to evaluate the influence that natural enemies have on pest dynamics and on
the spread of viral diseases, but it should be also taken into account the possible effect on the
performance of natural enemies when they feed on preys that act as vectors of viruses. Thus,
in a second research line developed in the Laboratory of Ecotoxicology, Department of
Entomology, of the Universidade Federal de Lavras (UFLA) (Brazil), it was evaluated the
performance of Chrysoperla externa under the condition of consuming Myzus persicae acting
as vector of Potato leafroll virus (PLRV). The diet composed of PLRV-infected M. persicae
significantly increased the length and reduced the survival rate, of the larval period in regard
to the other two diets, composed of non-infected M. persicae and Ephestia kuehniella eggs.
The lengths of the pupal stage were not significantly different between the aphid diets, but
both were significantly shorter than that of E. kuehniella eggs. Neither pupal survival nor
reproductive parameters revealed significant differences among the diets. Nevertheless, the
adult survival curves during the first 30 days after emergence showed significant differences,
reaching at the end of this interval a value of 54% for those C. externa adults fed on PLRVinfected aphids during their larval period.
According to the IPM guidelines, one of the physical strategies for the control of pests and
diseases in horticultural protected crops is the use of plastic films with photoselective
properties that act as ultraviolet (UV) radiation blocking barriers. In this sense, the third
research line of the Thesis dealt with the study of the direct and plant-mediated influence of
low UV radiation conditions on the performance of the aphid A. gossypii and on the biological
xx
parameters of the natural enemy C. carnea, as well as on the cucumber plants where insects
were released. The experiments were conducted inside cages under greenhouse conditions,
using for the first one healthy cucumber plants, while for the second experiment the cucumber
plants were previously infected with CABYV in order to assess the influence of the virus in
the same conditions. The low UV radiation conditions (under Térmico Antivirus® plastic
film) seemed to exert a direct effect in the early stages of cucumber plants, enhancing their
growth, and in an increasing nitrogen content at further developmental stages. The higher UV
radiation exposure (under Térmico Blanco® plastic film) in the early stages of the cucumber
crop induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves. The
use of Térmico Antivirus® plastic film as a photoselective barrier to induce low UV radiation
conditions did not modify, in regard to Térmico Blanco® plastic film (used as control), neither
the population development of A. gossypii nor the studied biological parameters of the
predator C. carnea. In the second experiment, done with CABYV-infected cucumber plants,
the incidence of the virus seemed to mask the direct and plant-mediated influence of the
different UV radiation conditions.
In last term, for the development of IPM strategies it is important to study the potential
side effects that pesticides might have on natural enemies. For this reason, in the Thesis were
tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide,
metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies C. carnea
and A. bipunctata. The side effects of the active ingredients of the insecticides were evaluated
with residual contact tests for the larvae and adults of these predators under laboratory
conditions. Flonicamid, flubendiamide, metaflumizone and spirotetramat were innocuous to
last instar larvae and adults of C. carnea and A. bipunctata. Therefore, these pesticides are
promising candidates for being incorporated into IPM programs in combination with these
natural enemies for the control of particular greenhouse pests. In contrast, sulfoxaflor was
xxi
slightly toxic to adults of C. carnea and was highly toxic to last instar larvae of A. bipunctata.
For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging compounds.
Deltamethrin was also the most toxic compound to larvae and adults of C. carnea. In
accordance with this fact, the use of sulfoxaflor and deltamethrin in IPM strategies should be
taken into consideration when releasing either of these biological control agents, due to the
toxic behavior observed under laboratory conditions.
xxii
General Introduction
CHAPTER 1. GENERAL INTRODUCTION
1.1. HORTICULTURAL CROPS PRODUCTION
The horticultural production in Spain represents an important sector of its agriculture, with
a surface of 348.853 ha and a yield of 13.199.258 t of vegetables (not including potatoes)
(MAGRAMA, 2015), heading the list of European Union (EU) countries in extension
dedicated to these crops (EUROSTAT, 2011). The surface of protected horticultural crops is
approximately the 20% of the total horticultural crops surface (MAGRAMA, 2015), mainly
represented by tomato, pepper and cucurbits. Other horticultural crops are cultivated in open
fields and also play a relevant economical role in Spanish agriculture, such as potatoes, with
an annual yield value in 2014 of 759 million euros (MAGRAMA, 2015). In this Thesis, the
crops chosen to work with were cucumber and potato.
Cucumber (Cucumis sativus L.) is an annual herbaceous plant native to Southern Asia that
belongs to the Cucurbitaceae family (Figure 1.1). It is a coarse and climbing plant that grows
up trellises or any other supporting frames, with thin, spiraling tendrils. The plant has large,
prickly, hairy triangular leaves that form a canopy over the fruit, and yellow male and female
flowers. The female flowers are recognized by the swollen ovary at the base, which will
become the edible fruit (Maroto, 1983). The surface dedicated to this crop in Spain was 8.902
ha in 2014, with a production of 753.941 t. Almost the 90% of the crop surface is located in
the Southeastern region of Spain, where cucumber is grown in protected conditions inside
greenhouses (MAGRAMA, 2015). In the year 2012, Spain occupied the 7th position in the
world’s main cucumbers and gherkins producer countries (FAOSTAT, 2012).
Potato (Solanum tuberosum L.) is an annual herbaceous plant native to South America,
member of the Solanaceae family (Figure 1.1). It propagates by forming tubers which enables
it to grow as perennial, but it is invariably cultivated as an annual crop. Potato plants grow to
1
General Introduction
a height of 30 to 100 cm, present angular stems, alternate and pinnate leaves, white or reddish
flowers and green berries. The edible tubers develop as lateral swellings of storage tissue at
the tips of the rhizomes (Oerke et al., 1999). In 2014, the cultivated surface of potato in Spain
was 72.431 ha, yielding 2.182.082 t (MAGRAMA, 2015). In the year 2012, Spain occupied
the 31st position in the world’s potatoes producer countries (FAOSTAT, 2012).
Figure 1.1. Commercial greenhouse of cucumber in Almería (Spain) (left) and potato crop in Segovia (Spain)
(right).
1.2. APHIDS AND ASSOCIATED VIRAL DISEASES
Aphids (Hemiptera: Aphididae) are one of the most damaging pests groups (van Emden &
Harrington, 2007). They are small insects with a powerful reproductive potential and of
immense importance in horticultural crops. They are considered major pests because they
remove plant assimilates (Miles, 1989), induce galls (Brown et al., 1991), excrete honey dew
that acts as a growing medium for unwanted fungi (Rabbinge et al., 1981; Fokkema et al.,
1983) and transmit plant viruses (Sylvester, 1989).
Aphids are by far the most important group of virus vectors (Katis et al., 2007). In fact,
over 190 species of aphids have been reported to transmit plant viruses (Nault, 1997), with
many species able to transmit more than one virus (Eastop, 1983; Nault, 1997; Hull, 2002).
The majority of reported aphid virus vectors belong to the genera Acyrthosiphon, Aphis,
2
General Introduction
Macrosiphum and Myzus (Kennedy et al., 1962). The cotton aphid Aphis gossypii Glover and
the green peach aphid Myzus persicae (Sulzer) are among the 14 aphid species of most
agricultural importance (Blackman & Eastop, 2007).
The cotton aphid A. gossypii is a polyphagous cosmopolitan pest that has been recorded on
over 320 plant species worldwide, belonging to about 46 families (Ebert & Cartwright, 1997;
Blackman & Eastop, 1984) (Figure 1.2). These include plants of economic importance, such
as cucumber (van Schelt et al., 1990). A. gossypii feeding results in significant yield reduction
and economic loss (Vendramin & Nakano, 1981). Direct damage is caused by phloem
sucking mainly from the leaves and stems, but at high densities individuals may feed from
any plant parts except for the mature reproductive structures and roots (Saha & Raychaudhuri,
1995). Nevertheless, the most important economic impact caused by A. gossypii is due to its
ability for transmitting more than 60 plant viral diseases (Kersting et al., 1999; Blackman &
Eastop, 1984; Henneberry et al., 2000). In fact, A. gossypii is the most important vector of
Cucumber mosaic virus (CMV) in cucurbits (Blackman & Eastop, 1984).
The green peach aphid, M. persicae is found throughout the world and has an exceptionally
wide host range (van Emden et al., 1972) (Figure 1.2). M. persicae feeds on hundreds of host
plants belonging to over 40 plant families (Blackman & Eastop, 1984). It is considered a
serious problem due to its ability to transmit over 100 virus diseases of plants in about 30
different families, including many major crops (Blackman & Eastop, 2007). For example, in
potato crops M. persicae is the most efficient vector of Potato leafroll virus (PLRV) (Álvarez
et al., 2007).
3
General Introduction
Figure 1.2. Alate and nymphs of A. gossypii on a cucumber cotyledon (left) and colony of apterae M. persicae on
a potato leaf (right).
1.2.1. NON-PERSISTENTLY TRANSMITTED VIRUSES
Non-persistent virus transmission is characterized by the following aspects: acquisition and
inoculation during brief probes of the aphid (1–2 min), no latent period after acquisition, short
retention times (up to 12 hours), loss of virus after aphid moulting, low virus-vector
specificity and acquisition of the virus from the epidermis of the infected plant (Pirone &
Harris, 1977; Loebenstein & Raccah, 1980; Hooks & Fereres, 2006; Ng & Falk, 2006).
Cucumber mosaic virus (CMV) is a representative example of this type of transmission.
CMV is the type member of the genus Cucumovirus in the family Bromoviridae (Figure 1.3).
It has three functional pieces of single-stranded RNA, packaged in three icosahedral,
isometric, not enveloped particles about 28-30 nm in diameter (Agrios, 2005). CMV is
transmitted by many aphid species, being A. gossypii the most effective vector (Labonne et
al., 1982). In addition, it may be transmitted by mechanical contact, seed and pollen
(Palukaitis & García-Arenal, 2003). CMV has the widest host range of any virus (Palukaitis et
al., 1992), attacking important horticultural crops, including solanaceous, cucurbits and
legumes, but also ornamentals and woody plants (Scholthof et al., 2011). The main symptoms
4
General Introduction
in infected plants are yellowish leaf areas, green and yellow mottling and reduced plant
growth.
Figure 1.3. Symptoms of Cucumber mosaic virus (CMV) in a cucumber leaf.
1.2.2. PERSISTENTLY TRANSMITTED VIRUSES
In persistent transmission, aphids require longer times (hours to days) to acquire the virus,
and can inoculate it for much longer periods (days to weeks), transmitting the virus after
moulting and often for their entire lifespan (Hogenhout et al., 2008; Hull, 2013). Persistent
plant viruses move through the insect vector, from the gut lumen into the hemolymph or other
tissues and finally into the salivary glands from which these viruses are introduced back into
the plant during insect feeding (Sylvester, 1980). Persistent viruses can be propagative if they
replicate in the vector and nonpropagative if they do not replicate inside it (Hull, 2013). In the
second group it can be found the Cucurbit aphid-borne yellows virus (CABYV) and the
Potato leafroll virus (PLRV) (Figure 1.4).
5
General Introduction
CABYV is a member of genus Polerovirus, family Luteoviridae (Mayo & D’Arcy, 1999).
Its viral particles are icosahedrical, approximately 25 nm in diameter. Like other
poleroviruses, CABYV is limited to phloem tissues in infected plants (Lecoq & Desbiez,
2012). It is transmitted in a persistent, circulative nonpropagative manner by A. gossypii, M.
persicae and Macrosiphum euphorbiae (Thomas), and its host range includes cucumber,
melon, squash, watermelon and other non cucurbit species such as lettuce and fodder beet
(Lecoq et al., 1992). Typical symptoms include yellowing and thickening of the older leaves.
CABYV severely reduces yield in melon and cucumber by reducing the number of fruits per
plant but it does not alter fruit shape or quality (Dogimont et al., 1996). Among the viruses
identified in Spanish cucurbit crops, CABYV has been shown to be the prevalent one in
southeastern Spain (Kassem et al., 2007; Juárez et al., 2013).
PLRV is a single stranded RNA virus and is also a member of the genus Polerovirus,
family Luteoviridae. PLRV is transmitted by aphids in a persistent, circulative
nonpropagative manner (Harrison, 1984). The most efficient vector is M. persicae (Bradley &
Rideout, 1953; Syller, 1994), which remains infective for life once acquired the virus
(Radcliffe & Ragsdale, 2002). The vector requires up to 15 minutes to acquire PLRV and
become infective within 8-72 hours (latent period) (Radcliffe, 1982; Radcliffe & Ragsdale,
2002). PLRV infects potato crops worldwide, but among its host are other solanaceous and
non-solanaceous species included in families such as Cucurbitaceae, Asteraceae and
Brassicaceae (Natti et al., 1953; Harrison, 1984). The symptoms induced in potato include
paleness or reddening of leaf tips, which may roll and become erect, stunting of shoots and
reduction in tuber number and size, which leads to yield losses (Harrison, 1984).
6
General Introduction
Figure 1.4. Symptoms of Cucurbit aphid-borne yellows virus (CABYV) in a cucumber leaf (left) and symptoms
of Potato leafroll virus (PLRV) in a potato leaf (right).
1.3. INTEGRATED PEST MANAGEMENT (IPM) IN HORTICULTURAL CROPS
In order to reduce harm caused by important pests of plants, animals and humans, the
purpose of Integrated Pest Management (IPM) is to use a combination of low cost biological,
cultural and chemical tactics that reduce pests to tolerable levels, with minimal effects on the
environment (van Lenteren & Woets, 1988; van Lenteren, 1995). In the definition of the Food
and Agriculture Organization of the United Nations (FAO, 2015), IPM is an ecosystem
approach to crop production and protection that combines different management strategies
and practices to grow healthy crops and minimize the use of pesticides. Therefore, IPM gives
priority to biological and physical measures versus chemical strategies.
The Directive 2009/128/EC of the European Parliament and the Royal Decree 1311/2012
that transposes the regulation to the Spanish level, establish the legal framework to achieve a
sustainable use of pesticides by reducing the risks and impacts of pesticide use on human
health and the environment and promoting the use of IPM and of alternative approaches or
techniques such as non-chemical alternatives to pesticides (EEC/CEE, 2009; BOE, 2012). In
Spain, the agricultural extension dedicated to IPM reaches 831.702 ha, being 13.726 ha of
them dedicated to horticultural crops (MAGRAMA, 2013).
7
General Introduction
1.4. BIOLOGICAL CONTROL STRATEGIES
Biological control can be defined as the use of an organism to reduce the population
density of another organism and thus includes the control of animals, weeds and diseases
(Bale et al., 2008). Two reasons for the demand to control aphids biologically are the
application of insecticides, which has led to a high level of resistance in several species, and
the increasing use of biological control against other pests, which necessitates compatible
control measures against aphids (Rabasse & van Steenis, 1999). Many natural enemies of
aphids are known, ranging from monophagous to polyphagous species. Aphids are commonly
attacked by predators, parasitoids, and pathogens, often collectively termed Aphidophaga
(Völkl et al., 2007). Amongst predatory insects controlling aphids, species of ladybirds
(Coleoptera: Coccinellidae), hoverflies (Diptera: Syrphidae), lacewings (Neuroptera:
Chrysopidae) and midges (Diptera: Cecidomyiidae) are relevant (van Steenis, 1992; Robledo
Camacho et al., 2009). Amongst aphid parasitoids, hymenopterans from the families
Braconidae and Aphelinidae have been used in biological control and IPM programs much
more often than other natural enemies (Powell & Pell, 2007). Some species of fungi are
entomopathogenic, infecting aphids through the cuticle, eventually killing the host (Pell et al.,
2001; Shah & Pell, 2003).
1.4.1. APHIDOPHAGOUS PREDATORS
Amongst aphid predators, chrysopids and coccinellids are two important families of
aphidophagous insects. In the case of chrysopids, only larvae are predaceous, while in
coccinellids both larvae and adults feed on aphids (Völkl et al., 2007). In this Thesis, the
experiments have been conducted with two lacewing species (Neuroptera: Chrysopidae),
Chrysoperla carnea (Stephens) and Chrysoperla externa Hagen (Figure 1.5), and with the
coccinellid Adalia bipunctata (L.) (Coleoptera: Coccinellidae).
8
General Introduction
Larval lacewings fulfill many of the requirements of an effective biological control agent.
They are voracious, active predators, with an excellent searching capacity (Sundby, 1966;
Bond, 1980). Lacewing larvae are also polyphagous, feeding on a large range of small, softbodied arthropods (Principi & Canard, 1984); their prey include pests of economic
importance such as aphids, whitefly, mites, mealybugs and various lepidopteran pests (Wang
& Nordlund, 1994; McEwen et al., 2001). Adults are not predacious and live on pollen,
nectar, and honeydew (Principi & Canard, 1984). Because of their resistance to several
common insecticides, lacewings are suitable for use in integrated pest management programs
(New, 1975; Senior & McEwen, 2001; Medina et al., 2003a).
Amongst chrysopids, the green lacewing C. carnea is a cosmopolitan predator with great
potential in integrated pest management (Hassan, 1974; Stelzl & Devetak, 1999; Duelli,
2001), found in a wide range of natural, agricultural and forestry habitats and widely used for
biocontrol of aphids in greenhouse crops (Greeve, 1984; Thompson, 1992). Besides, C.
carnea is amongst the selected indicator species to be submitted to higher tier testing for
pesticide registration in the European Union (Candolfi et al., 2001).
In the Neotropical Region, C. externa is a widespread species, distributed from the
southeast of the United States and the Antilles, to South America (Adams, 1983; Albuquerque
et al., 1994). This predator is being considered a potentially biological control agent in South
America because it feeds on some important agricultural pest and it has a strong preference
for open habitats (Albuquerque et al., 1994; Carvalho et al., 1998).
9
General Introduction
Figure 1.5. Larva of C. carnea on a cucumber leaf (left) and larva of C. externa predating an aphid (right).
Aphidophagous coccinellids are primarily found in the subfamily Coccinellinae (Frazer,
1988). Ladybirds are considered the most conspicuous aphid predators in crops, and they have
a long tradition in biological control (Völkl et al., 2007). The characteristics that make
ladybirds successful aphid predators are a high searching capacity, high voracity, appropriate
food range, and the capability to develop on alternative food if aphids are scarce (Hodek &
Honěk, 1996). Nevertheless, a disadvantage of the use of coccinellids is that they are not able
to form a self-perpetuating population in the greenhouse, so repeated introductions are
necessary (Hämäläinen, 1977, 1980).
The two-spotted ladybird A. bipunctata is a polyphagous coccinellid native to the
Palearctic and Nearctic regions (Figure 1.6). Since the use of the non-indigenous Harmonia
axyridis (Pallas) (Coleoptera: Coccinellidae) as a biological control agent has been
compromised (Brown et al., 2008; van Lenteren et al., 2008), there is growing interest in
commercializing A. bipunctata for biological control of aphids in Europe (Bonte et al., 2010).
The two-spotted ladybird predates a high number of aphid species but can also use coccids,
diaspidids and lepidopteran eggs as alternative prey (De Clercq et al., 2005; Omkar & Pervez,
2005). In times of prey scarcity, A. bipunctata has been observed feeding on pollen
(Hemptinne & Desprets, 1987).
10
General Introduction
Figure 1.6. In the aphidophagous coccinellids as A. bipunctata, both larvae (left) and adults (right) have
predaceous habits.
1.4.2. TROPHIC INTERACTIONS IN THE AGROECOSYSTEM
In IPM programs, the inoculative release of aphidophagous predators is a frequently used
strategy for controlling aphid foci in commercial greenhouses (Yano, 2006). The benefits in
the reduction of the herbivore pressure by natural enemies are known, but their inclusion in a
plant-virus-vector system may trigger complex interactions between the agents, as they may
modify the disease incidence within the crop (Dicke & van Loon, 2000). In fact, virus spread
is directly linked to the behavioural responses of its vectors when encountering natural
enemies, performing strategies such as kicking, walking away, dropping off the plant or
displaying a coordinated movement within the colony (Dixon, 1958). Aphid vector responses
and therefore virus epidemiology, might be influenced as well by the foraging habit that
natural enemies exhibit within the system (Smyrnioudis et al., 2001; Belliure et al., 2011).
Depending on the disturbance level caused in the colony by the searching behaviour of the
natural enemy, aphids will secrete alarm pheromones that act as a chemical signals, causing
aphids to stop feeding and disperse, either walking or dropping off their host plant
(Montgomery & Nault, 1977a; Losey & Denno, 1998; Day et al., 2006).
11
General Introduction
In a reciprocal sense, vegetal viruses may exert an indirect influence on the performance of
natural enemies, because modifications in their aphid preys have been reported when feeding
on infected plants (Fiebig et al., 2004; Stout et al., 2006). The physiological (morphology,
development and reproduction) and behavioral (predation or parasitization rate, host
localization, walking and flying) characteristics of natural enemies are influenced indeed by
the nutritive properties of the food consumed (Bigler, 1989; Grenier & De Clercq, 2003).
1.5. PHYSICAL CONTROL STRATEGIES
Physical control methods are intended to create a less favorable environment for pest
reproduction and survival by deliberately manipulating some component of the
agroecosystem (Hilje et al., 2001). Among the physical strategies to control pests are included
the use of organic and inorganic mulches, sticky barriers, traps, slippery surfaces, windbreak
screens, flooding, thermal methods and plastic covers (Vincent et al., 2009).
1.5.1. UV-ABSORBING PLASTIC COVERS
Plastic-covered greenhouses provide a protected environment, and facilitate improved
control of temperature, humidity and light intensity and quality (Antignus, 2000). By the
inclusion of additives in the plastic film, the UV-absorbing properties can be modified,
obtaining materials that block transmission of UV (200-400 nm) but do not interfere with the
transmission of the visible range of the light spectrum (400-700 nm) (Antignus et al., 1996).
Many authors have pointed out that the use of UV-absorbing films as greenhouse cover leads
in decreased insect population (Halevy, 1997; Antignus et al., 1998; Costa & Robb, 1999;
Costa et al., 2002). This is due to the sensitivity of insects ocular photoreceptors in the
bandwidth of UV radiation (200-400 nm), which has a direct incidence on insects behaviour,
such as, orientation, navigation, host finding and feeding (Antignus & Ben-Yakir, 2004).
12
General Introduction
The direct deleterious effects of UV radiation on insect performance are well documented
(e.g. Bothwell et al., 1994; McCloud & Berenbaum, 1994; Bacher & Luder, 2005), but the
indirect or host-mediated effects must also be taken into account. It has been demonstrated
that different conditions of UV radiation can produce changes in plant tissues that affect the
choice of sites for insect feeding (Ballaré et al., 1996; Rousseaux et al., 1998) and oviposition
(Caputo et al., 2006; Foggo et al., 2007), as well as insect growth and survival (Zavala et al.,
2001; Izaguirre et al., 2003; Kuhlmann & Müller, 2009a, 2010).
An important issue of UV-absorbing covers is to ensure the compatibility of this measure
with biological control to guarantee success in IPM, which has been recorded by several
authors until now (Chyzik et al., 2003; Chiel et al., 2006; Doukas & Payne, 2007; Legarrea et
al., 2010).
1.6. CHEMICAL CONTROL STRATEGIES
The use of chemical pesticides that cause undesired side effects on non-target beneficial
organisms may lead to pest outbreaks (Albajes et al., 1999). Thus, in IPM, the process of
developing the selectivity of a pesticide aims to maximize its specific effect against pests and
diseases and minimize its effect on non-target organisms (Hull & Beers, 1985).
1.6.1. SIDE EFFECTS OF PESTICIDES ON NATURAL ENEMIES
One of the challenges of a successful implementation of biological control strategies is
making it compatible with chemical control. Despite a more rational use of pesticides in the
last years, there are still products with unproved selectivity with some species of natural
enemies, so testing the side effects of pesticides on beneficial organisms has become
obligatory in several countries (Hassan, 1994; Medina et al., 2008).
13
General Introduction
The main ways of exposure to pesticides are by direct contact, residual contact and
ingestion. The most common type of exposure of natural enemies to pesticides is by direct
and residual contact (Croft, 1990), but depending on the pesticide, its effect via ingestion or
other routes of uptake can be more severe (Candolfi et al., 2000; Medina et al., 2008). The
International Organization for Biological and Integrated Control (IOBC), throughout the
Working Group “Pesticides and Beneficial Organisms”, develops standard methods to test the
side effects of pesticides on the most important natural enemies, choosing selective pesticides
suitable for use in integrated control programs (Hassan, 1994). The side effects sequential
testing procedure proposed by the IOBC starts with laboratory studies, which are considered
the worst-case scenario (Barrett et al., 1994). The most susceptible life stage and the less one
must be exposed to the compounds. Additionally, it can be performed extended laboratory
tests, which have the purpose of studying the toxicity of the insecticides sprayed on vegetal
tissue. In the case of finding toxic effects under laboratory conditions, the sequential testing
scheme should be followed to check if the harmful effect remains under more realistic
conditions, developing firstly semi-field assays, and in the last term field experiments (Bigler
& Waldburger, 1994; Hassan, 1994, van de Veire et al., 2002).
The purpose of these studies is to test the short term direct effects, such as mortality, and
the long term possible effects or sublethal effects, such as those related with the reproductive
parameters (fecundity and fertility) (Medina et al., 2008). Depending on the total effect
caused by the pesticides in the natural enemies, the IOBC establishes four toxicity categories:
(1) harmless, (2) slightly harmful, (3) moderately harmful, (4) harmful (Hassan, 1994).
14
Objectives
CHAPTER 2. OBJECTIVES
The general objective of this Thesis was to evaluate the compatibility of new Integrated
Pest Management tactics, such as UV-photoselective barriers and novel pesticides, with
biological strategies to improve the control of aphids and plant viruses in horticultural crops.
In addition, the trophic interactions involved in the use of natural enemies were also studied.
Among these interactions, the influence of aphidophagous predators on the spread of aphid
vectors and viral diseases, and their performance when consuming plant virus aphid vectors
were studied.
To achieve this objective, the following specific targets were defined:
1. To evaluate the effects of two aphidophagous predators, Chrysoperla carnea
(Stephens) larvae and Adalia bipunctata (L.) adults, on the spread of Aphis gossypii
Glover and the plant viruses with different modes of transmission Cucumber mosaic
virus (CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV,
Polerovirus) in a cucumber crop under greenhouse conditions.
2. To determine the influence on the biology of Chrysoperla externa (Hagen) of the
predation of Myzus persicae (Sulzer) acting as a vector of Potato leafroll virus (PLRV,
Polerovirus) in potato under laboratory conditions.
3. To study the direct and plant-mediated impact of low UV radiation conditions on the
performance of the aphid A. gossypii and the predator C. carnea, and the direct impact
on the physiology of cucumber plants under greenhouse conditions. To assess these
interactions in a CABYV-infected cucumber plants scenario.
4. To evaluate the lethal and sublethal effects of the pesticides flonicamid, flubendiamide,
metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on C. carnea and A.
15
Objectives
bipunctata larvae and adults under laboratory conditions to determine their
compatibility with these biological control agents.
16
Influence of aphidophagous predators on CMV spread
CHAPTER 3. INFLUENCE OF APHIDOPHAGOUS PREDATORS ON
THE SPREAD OF NON-PERSISTENTLY AND PERSISTENTLY
TRANSMITTED VIRUSES BY APHID VECTORS
SUB-CHAPTER 3.1. THE EFFECT OF Chrysoperla carnea AND Adalia
bipunctata ON THE SPREAD OF Cucumber mosaic virus BY Aphis
gossypii1
ABSTRACT
The effects of two aphidophagous predators, the larvae of Chrysoperla carnea and adults
of Adalia bipunctata, on the spread of Cucumber mosaic virus (CMV) transmitted in a nonpersistent manner by the cotton aphid Aphis gossypii were studied under semi-field
conditions. Natural enemies and aphids were released inside insect-proof cages (1 x 1 x 1 m)
with a central CMV-infected cucumber plant surrounded by 48 healthy cucumber seedlings,
and the spatiotemporal dynamics of the virus and vector were evaluated in the short and long
term (1 and 5 days) in the presence and absence of the natural enemy. The spatial analysis by
distance indices (SADIE) methodology together with other indices measuring the dispersal
around a single focus was used to assess the spatial pattern and the degree of association
between the virus and its vector. Both natural enemies significantly reduced the number of
aphids in the CMV source plant after 5 days but not after 1 day. The CMV transmission rate
was generally low, especially after 1 day, due to the limited movement of aphids from the
1
Published in: Garzón, A., Budia, F., Medina, P., Morales, I., Fereres, A., Viñuela, E. (2015) The effect of
Chrysoperla carnea (Neuroptera: Chrysopidae) and Adalia bipunctata (Coleoptera: Coccinellidae) on the
spread of cucumber mosaic virus (CMV) by Aphis gossypii (Hemiptera: Aphididae). Bulletin of
Entomological Research 105, 13-22.
17
Influence of aphidophagous predators on CMV spread
central CMV-source plant, which increased slightly after 5 days. Infected plants were mainly
located around the central virus-infected source plant, and the percentage of aphid occupation
and CMV-infected plants did not differ significantly in absence and presence of natural
enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the
central plant. The complexity of multitrophic interactions and the role of aphid predators in
the spread of CMV are discussed.
3.1.1. INTRODUCTION
Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus in the family
Bromoviridae, is one of the most important plant viruses. Its host range is very wide; it infects
more than 1200 plant species in 100 families, including ornamentals, woody plants and
important crops such as pepper, lettuce, beans and cucurbits (Scholthof et al., 2011). Affected
plants exhibit yellowish leaf areas, green and yellow mottling and stunting. CMV is a nonpersistent stylet-borne transmitted pathogen by more than 80 aphid species in 33 genera, with
Aphis gossypii (Glover) (Hemiptera: Aphididae) the most effective vector (Labonne et al.,
1982). CMV is efficiently acquired and inoculated during very brief (5-10 s) intracellular
probes on epidermal plant tissues without a latent period. Its retention time in the vector is
under 12 hours, and it is unable to cross the insect’s cuticle at the tip of the stylets (Ng &
Falk, 2006, Pirone & Perry, 2002, Fereres & Moreno, 2009). In addition, CMV may be
transmitted by mechanical contact, seed and pollen (Palukaitis & García-Arenal, 2003).
The prevention of non-persistent virus diseases is particularly difficult due to the quick
transmission process, and chemical control strategies are often not sufficiently efficient
(Raccah, 1986; Collar et al., 1995), because they do not act fast enough to prevent aphids
from inserting their mouthparts into plants (Loebenstein & Raccah, 1980). This fact forces the
search for better alternatives for their control (Thackray et al., 2000), developing strategies
18
Influence of aphidophagous predators on CMV spread
that maximise the impact of biological control agents, which are currently the focus of
Integrated Pest Management (IPM) programs. The inoculative release of aphidophagous
predators is a commonly used strategy in IPM programs aiming at the control of aphid foci in
commercial greenhouses (Yano, 2006). Aphidophagous parasitoids are also a major
component in the management of some aphids (Abney et al., 2008).
The potential of biological control agents to limit the spread of aphid-borne non-persistent
viruses is still not well known (Hooks & Fereres, 2006), and their effect depends on their
capacity to reduce the number of aphid individuals by predation or parasitisation, on the level
of disturbance of the aphid colonies and on the time aphids require to acquire and inoculate
the virus (Belliure et al., 2011). Additionally, different species of natural enemies even within
the same genus can induce very different responses in vector colonies and virus
epidemiology, resulting in either a reduction or an increase in viral dispersion (Smyrnioudis et
al., 2001; Hodge et al., 2008, 2011; Dáder et al., 2012).
Because the spread of viruses seems to be correlated with the foraging habits of natural
enemies (Smyrnioudis et al., 2001; Belliure et al., 2011), the objective of this study was to
determine the dissemination of CMV by A. gossypii in the absence and presence of the
following two predators: Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) larvae
and adult Adalia bipunctata (L.) (Coleoptera: Coccinellidae). C. carnea larvae are very
mobile and voracious polyphagous predators in many agricultural systems (Viñuela et al.,
1996; Medina et al., 2003b) and one of the indicator species considered for higher testing in
the EU for pesticide registration (Candolfi et al., 2001). A. bipunctata adults are generalist
aphidophagous predators with a wide range of prey (Omkar & Pervez, 2005). Both species,
which are commercially available, are currently widely used in Spain in protected
horticultural crops (Robledo et al. 2009).
19
Influence of aphidophagous predators on CMV spread
3.1.2. MATERIALS AND METHODS
3.1.2.1. INSECTS
A single virginiparous apterae of A. gossypii collected in a melon crop in Almería (Spain)
was used to initiate a clonal population. The aphid colony was kept at low densities on
Cucumis melo L. plants cv. ‘Primal’ in a climatic chamber (20 ºC, 60-80% RH, 16:8 L:D
photoperiod) in the Crop Protection Laboratory of the ETSIA-UPM (Madrid, Spain). For the
trials, alate individuals were collected with an aspirator and placed in groups of 100 in
Falcon® tubes just before being transferred to the experimental cages. This aphid is a suitable
prey species for the two natural enemies used in the tests (van Steenis, 1992).
A laboratory colony of C. carnea was established from L1 larvae obtained from Agrobío
(CHRYSOcontrol®, Almería, Spain) following the rearing procedure of Medina et al. (2001).
Larvae were fed Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs ad libitum, and
adults were provided with a very nutritious artificial diet (Vogt et al., 2000). For the trials,
gauze with < 24-h-old eggs from the adult rearing cages were allowed to develop up to the L3
instar. Larvae were collected and starved for 1 h before starting the test.
A. bipunctata L1 larvae purchased from Agrobío (ADALIAcontrol®, Almería, Spain) were
placed in cages with zig-zag folded filter paper and a mixture of E. kuehniella eggs and bee
pollen (1:1) that was replaced three times per week (Bonte et al., 2010) up to L3-L4
emergence. Mature larvae were placed in individual arenas with a small amount of food to
avoid cannibalism and guarantee successful pupation. After 6-7 days, newly emerged adults
were transferred to cages with a water source (baize soaked with water), and the same diet
was provided for the larvae. Once a week, melon leaves with A. gossypii nymphs were added
as a dietary supplement and habituation to feeding on live prey. The environmental conditions
20
Influence of aphidophagous predators on CMV spread
were similar to those of C. carnea rearing. A. bipunctata adults, 2-3 weeks old, were used in
the experiments.
3.1.2.2. PLANT MATERIAL
Cucumber (Cucumis sativus L. cv. ‘Ashley’) plants were grown from seed in 150 alveoli
trays (56 cm x 36 cm x 6 cm) filled with a 1:1 mixture of soil substrate (Projar, Valencia,
Spain) and vermiculite Termita® (Aminco, Donostia, Spain) in an insect-proof chamber at 2325 ºC, 60-80% relative humidity (RH) and a 16:8 L:D photoperiod.
3.1.2.3. VIRUS ACQUISITION
Cucumber plants were mechanically inoculated 13 days after sowing (BBCH scale = 11)
and used as viral sources 4 weeks post-inoculation (BBCH scale = 65). The CMV isolate used
was M96/9 (IA subgroup), obtained from a melon crop in 1996 in Tarragona (Spain) and
kindly supplied by Dr. E. Moriones (EELM-CSIC, Spain) (Díaz et al., 2003). CMV-infected
cucumber plants were maintained in an insect-proof chamber at 23-25 ºC, 60-80% RH and a
16:8 L:D photoperiod. Plants to be used as CMV-infected source in trials were previously
tested for virus infection by double antibody sandwich enzyme-linked immunosorbent assay
(DAS-ELISA) (Clark & Adams, 1977) using specific commercial antibodies against CMV
(Agdia Inc., Indiana, USA).
3.1.2.4. EXPERIMENTS
The methodology used in this study was similar to that of Dáder et al. (2012) with
adaptations due to the different natural enemy or virus studied. The trials started one week
after transplanting the cucumber test plants (48 seedlings/replicate) and four weeks after the
inoculation of the CMV-infected source plants (1 plant/replicate). Cucumber seedlings at the
one true leaf stage (BBCH scale = 11) were transplanted to 4 plastic trays (54 cm x 38.5 cm x
21
Influence of aphidophagous predators on CMV spread
10 cm) filled with a mixture of soil substrate and vermiculite (2:1) (distance between plants
was 12 cm), moved to the greenhouse and placed inside cubic cages (1 x 1 x 1 m) covered by
an insect-proof net (90 μm pore Ø) (Figure 3.1.1). The temperature and relative humidity
were remotely controlled in the greenhouse through a central computer, guaranteeing a range
of 15-27 ºC and 50-85% RH during the experiment.
Figure 3.1.1. Insect-proof net covered cages where replicates were placed, at greenhouse facilities of the ETSIAUPM.
For each natural enemy, the experiments consisted of 3 replicates of 3 cages with the
natural enemy and three without. In order to allow acclimation to the greenhouse
environment, one day before insect release a potted CMV-infected cucumber source plant was
placed in the centre of the tray surrounded by 48 non-infected cucumber plants placed in a
grid but without physical contact with the virus-infected plant (Figure 3.1.2). Twenty-four
hours later, 100 winged A. gossypii adults were confined in the CMV-infected source plant by
means of a plastic cylinder with a ventilated lid and allowed to establish and acquire the virus
for 15 minutes. Immediately afterward, the cylinder was removed, and 3 L3 larvae of C.
22
Influence of aphidophagous predators on CMV spread
carnea or 3 adults of A. bipunctata were released in the source plant in half of the cages used
for each natural enemy. Natural enemies were released one by one with the help of a brush in
those leaves where aphids had been previously established.
Figure 3.1.2. Spatial distribution of the experimental unit showing in the central point the CMV-source plant
where alate aphids and natural enemies were released, surrounded by the 48 test plants at one true leaf stage.
Because CMV is acquired and transmitted in very short time periods, lasting few minutes,
aphid dispersion and the spread of CMV infection from the source plant were evaluated in the
short term (after 1 day) and long term (after 5 days) in the presence and absence of each of the
two natural enemies. Immediately after the evaluation, the virus source plant was placed in a
bag (number of winged aphids and nymphs was evaluated later with binoculars); the
absence/presence, number and development status of A. gossypii individuals in the peripheral
plants was recorded (Figure 3.1.3), and plants were sprayed with the systemic insecticide
Confidor® 20 LS (Bayer CropScience, 200 g l-1 w/v imidacloprid as a soluble concentrate,
dilution rate 0.75 ml l-1) in order to kill the aphids and prevent further viral transmission. Four
23
Influence of aphidophagous predators on CMV spread
weeks after evaluation, leaf samples of receptor plants were frozen at -80 ºC, and CMV
infection was diagnosed by DAS-ELISA as previously described.
Figure 3.1.3. Recording of the number of A. gossypii alatae and nymphs in the surrounding test plants.
The mean temperature during short-term trials (evaluation after 1 day) was 19.6 ± 0.2 ºC
(C. carnea) and 22.6 ± 0.7 ºC (A. bipunctata); during long-term trials (evaluation after 5
days), it was 20.6 ± 0.6 ºC (C. carnea) and 18.4 ± 0.3 ºC (A. bipunctata).
During assays, watering was performed by placing the trays with plants in a larger tray
partially filled with water in order to not disturb the insects.
3.1.2.5. STATISTICAL ANALYSIS
The density of aphids in the CMV source plant, dispersal of aphids towards peripheral noninfected plants (percentage of plants occupied by at least one aphid morph) and percentage of
CMV-infected plants at the two evaluation periods (1 d and 5 d) were compared by Student’s
t test (p < 0.05) after normality checks (Kolmogorov-Smirnov test) in the presence and
absence of C. carnea or A. bipunctata. The Statgraphics Plus 5.1 statistical package
24
Influence of aphidophagous predators on CMV spread
(Statistical Graphics Corporation, 1994-2000) was used for calculations. The percentage
reduction in the number of aphids in the central virus-infected plant in the presence of each
natural enemy was calculated with Abbott’s formula: Reduction (%) = (nc – nt) / nc x 100;
where nc is the number of aphids in absence of natural enemy, nt is the number of aphids in
presence of natural enemy (Abbott, 1925). Spatially referenced counts in every sampling unit were used to calculate several indexes.
The spatial distribution of aphids and virus spread was studied through indexes used by Korie
et al. (1998) and the spatial analysis by distance indices (SADIE) methodology, a biologically
based method (Perry, 1995) currently considered a powerful tool for the study of viral spread
and pest and natural enemy displacement in a crop (Díaz et al., 2010; Legarrea et al., 2014).
Korie’s indexes (Korie et al., 1998) were calculated to analyse the dispersal around a single
focus: the distribution of the aphid dispersal or viral infection from the central plant (δ)
measured as the distance between the central plant (x = 4, y = 4) and the centroid (C),
considering C as the average position of aphids or CMV infected plants in the experimental
plot; the spread of the infection as the average distance of infected plants from the central
release focus (Δ). The spatial pattern of viral infection and aphid dispersal was described by
the SADIE index of aggregation (Ia) (aggregated if Ia > 1; regular if Ia < 1 or random if Ia = 1)
and the indexes of clustering in patches and gaps (vi, vj) (> 1.5 patchiness, < -1.5 insects in the
plant are members of a gap; close to 1, a random placement of insects in that plant in relation
to others nearby) (Perry, 1998). The last indexes determine the extent to which each unit
contributes to the global clustering; units with numbers greater than the overall mean are
assigned to a positive patch cluster index (vi) and those with counts under the mean to a
negative gap cluster index (vj) (Perry et al., 1999). Moreover, the degree of spatial association
between aphids and CMV spread was measured by the index of spatial association (X), which
correlates the cluster indexes of the two data sets previously calculated (Perry & Nixon,
25
Influence of aphidophagous predators on CMV spread
2002). Similar indexes (vi or vj) are indicative of a local association (P < 0.025) at the sample
site; if they are different, a local dissociation is indicated (P > 0.975) (Díaz et al., 2010). The
number of natural enemies/replicate was too low to meet the requirements of SADIE analysis,
and their spatial distribution could not be calculated.
3.1.3. RESULTS
3.1.3.1. A. gossypii DENSITY ON THE CENTRAL SOURCE PLANT
Both enemies promoted aphid movement from the central CMV-source plant to the
peripheral plants (Table 3.1.1). The presence of C. carnea caused a significant percentage
reduction (Abbott, 1925) in the number of A. gossypii adult alatae in the central CMVinfected plant both after 1 day (54.88%, t = 4.46, p < 0.01) and 5 days (93.63%, t = 12.17, p <
0.01) and in the number of nymphs after 5 days (85.36%, t = 5.74, p < 0.01). Adalia
bipunctata only caused significant reductions in the numbers of adult alatae (84.40%, t = 6.39,
p < 0.01) and nymphs (77.10%, t = 3.37, p = 0.03) after 5 days.
3.1.3.2. A. gossypii DISPERSAL FROM THE CENTRAL SOURCE PLANT
In the peripheral plants, the occupancy rate by at least one aphid (adult alate or nymph)
was never significant (p > 0.05) (Table 3.1.2). The effect of the chrysopid and coccinellid was
different, however. The percentage of occupancy after 1 day was slightly higher in the
presence of L3 C. carnea (alatae 7.0 ± 1.8%; nymphs 5.6 ± 1.4%) than in the control (alatae
3.5 ± 2.5%; nymphs 3.5 ± 3.5%) but after 5 days, the dispersal of aphids towards the receptor
plants increased enormously and similarly for both aphid developmental stages (control 30.6
± 2.5% alatae, 34.8 ± 3.5% nymphs; with natural enemy 30.6 ± 10.8% alatae, 36.8 ± 9.8%
nymphs). In contrast, when A. bipunctata was present, the number of alate aphids in the
peripheral plants after 5 days was lower than after 1 day (10.4 ± 4.8% and 18.8 ± 1.2%), while
the number of nymphs was similar (27.1 ± 8.4% and 20.2 ± 2.5%). Aphids showed limited
26
Influence of aphidophagous predators on CMV spread
movement after 1 day, mostly restricted to the vicinity of the central CMV-source plant, as
indicated by the lower values of Δ index (Table 3.1.3), and a little more movement after 5
days (higher Δ index values for alatae + nymphs, Table 3.1.4), which is graphically shown in
Figure 3.1.4 (a,b and c,d, respectively), where the centroid position (C) of A. gossypii (alatae
+ nymphs) populations are represented in presence and absence of both enemies.
27
Influence of aphidophagous predators on CMV spread
Table 3.1.1. Influence of the presence of C. carnea L3 or A. bipunctata adults on the density of A. gossypii (adult alate morphs
and nymphs) in the central CMV-source plant 1 day and 5 days after infestation with 100 adult alatae. The data are the means ±
SE of the number of aphids/plant (n = 3 replicates)1.
Control
C. carnea
t
p
R(%)a
Control
A. bipunctata
Short term (1 day)
Alatae
Nymphs
68.7±5.0
83.0±42.2
31.0±6.8
23.0±5.1
4.46
1.41
0.01
0.23
54.88
72.29
53.0±8.2
131.3±12.6
32.7±6.8
106.0±32.0
1.91 0.13
0.74 0.50
38.30
19.27
Long term (5 days)
Alatae
Nymphs
26.7±1.2
1.7±1.7 12.17 <0.01 93.63
314.3±37.0 46.0±28.5 5.74 <0.01 85.36
75.0±8.7
253.3±55.4
11.7±4.7
58.0±17.0
6.39 <0.01
3.37 0.03
84.40
77.10
t
p
R(%)a
1
a
Student's t test (p < 0.05). Significant data in bold.
reduction in aphid population (Abbott, 1925).
Table 3.1.2. Influence of the presence of C. carnea L3 or A. bipunctata adults on the percentage of peripheral plants occupied
by at least one A. gossypii (adult alate or nymph) and percentage of CMV-infected plants 1 and 5 days after infestation with
100 adult alatae in the central CMV-source plant. The data are the means ± SE (n = 3 replicates)1.
Control
C. carnea
t
p
Control
A. bipunctata
Short term (1 day)
Alatae
Nymphs
CMV
3.5±2.5
3.5±3.5
-
7.0±1.8
5.6±1.4
4.2±4.2
-1.13
-0.56
-
0.32
0.60
-
16.7±5.5
16.0±6.6
0.7±0.7
18.8±1.2
20.2±2.5
0.7±0.7
-0.37 0.73
-0.59 0.59
0.00 1.00
Long term (5 days)
Alatae
Nymphs
CMV
30.6±2.5
34.8±3.5
2.1±2.1
30.6±10.8
36.8±9.8
10.4±6.4
0.00
-0.20
-1.24
1.00
0.85
0.28
4.9±1.8
20.2±10.9
9.0±7.0
10.4±4.8
27.1±8.4
6.9±1.8
-1.08 0.34
-0.50 0.64
0.29 0.79
1
Student's t test (p < 0.05).
28
t
p
Influence of aphidophagous predators on CMV spread
3.1.3.3. CMV SPREAD
The CMV transmission rate by A. gossypii from the central source plant was very low after
1 day and did not change in presence or absence of both natural enemies (p > 0.05; Table
3.1.2). In presence of L3 C. carnea larvae, only 12.5% of peripheral plants (6 out of 48)
became infected in one replicate and none in the other two. Because of that, the average value
was very low, and the S.E. was very high (4.2 ± 4.2%). In presence of A. bipunctata adults,
the average viral transmission rate was even lower (0.7 ± 0.7%). After 1 day, infected plants
were concentrated in the central area of the experimental unit, near the CMV source plant, as
shown in Figure 3.1.4 (e, f). After 5 days, the viral transmission slightly increased, but values
were always under 11% and not very homogeneous, so no significant differences were
observed between treatments. However, the percentage of infected plants seemed to be higher
in the presence of L3 C. carnea larvae (10.4 ± 6.4%) and in the absence of A. bipunctata
(9.0±7.0% compared to 6.9±1.8% in presence of the natural enemy) (Table 3.1.2). After 5
days, the viral spread from the source to the receptor plants increased somewhat [Figure 3.1.4
(g, h)] as indicated by the higher values of indices δ and Δ (Table 3.1.4).
The low values of the indices measuring the spread of the infection from the CMV-source
plant after 1 day [δ, Δ] are also indicative of the limited movement of the aphid colony in the
short term, both in the absence and presence of predators, which meant that no statistically
significant differences were obtained (Table 3.1.3). Based on the higher values of index δ in
presence of C. carnea and A. bipunctata, both enemies initially (after 1 day) seemed to
promote the dispersal of A. gossypii and CMV infection (2.55 ± 0.87 and 4.55 ± 1.03 cm,
respectively) compared to a more centralised distribution in control cages (0.64 ± 0.60 and
1.70 ± 0.39 cm, respectively). Despite this fact, the aggregation index in the short term was
lower than 1 (Ia < 1), irrespective of the presence of the predators, which indicated in this case
a concentration of A. gossypii individuals in the central infected plant, surrounded by plants
29
Influence of aphidophagous predators on CMV spread
with no aphids, and the lack of significance of the clustering indices vi (< 1.5) and vj (> -1.5)
(Table 3.1.3).
When the evaluation period was extended (5 days), an increase in the value of the dispersal
parameters was observed, especially in presence of natural enemies, but the viral and aphid
distribution patterns were heterogeneous (Table 3.1.4). Irrespective of the natural enemy
present, no statistically significant difference in the spread of CVM could be detected in the
displacement and distribution indices with and without natural enemies. When C. carnea was
present, the spread of the infection (Δ) by alatae (t = -4.93, p < 0.01) or total morphs (t = 4.91, p < 0.01) was significantly higher than in its absence (p < 0.05). A. bipunctata increased
both aphid dispersal (δ; t = -3.47, p = 0.03) and the spread of infection (Δ; t = -8.03, p < 0.01).
Both natural enemies seemed to promote the dispersal of aphids because the population
centroids were more distant from the CMV-source plant in their presence (Figure 3.1.4, c, d).
In contrast to the values obtained after 1 day, the distribution pattern of aphid colonies (alatae
+ nymphs) was only concentrated in the central plant (Ia < 1) in the absence of predators and
aggregated (Ia > 1) when they were present, but neither of the clustering indices were
significant (vi < 1.5; vj > -1.5) (Table 3.1.4). A random CMV distribution (Ia ≈ 1) was observed
in the presence of C. carnea and A. bipunctata based on the Ia aggregation index (1.002 ±
0.072 and 1.020 ± 0.060, respectively) and on the clustering indices (vi < 1.5 and v j > -1.5)
(Table 3.1.4). The randomness of the CMV spread in the presence of either of the two
predators was also reflected by the distribution of infected plants shown in Figure 3.1.4 (g, h).
30
Influence of aphidophagous predators on CMV spread
Table 3.1.3. Spatial pattern indexes of A. gossypii populations and CMV spread after 1 day in the presence and absence (control) of C. carnea L3 or A. bipunctata
adults. The data are the means ± SE of three replicates/natural enemy1.
δa
Δb
Iac
vid
vje
0.73±0.65
0.64±0.60
0.00±0.00
0.80±0.73
0.70±0.66
0.00±0.00
0.737±0.003
0.737±0.002
0.735±0.001
1.004±0.005
1.011±0.007
0.999±0.002
-0.708±0.002
-0.711±0.001
-0.710±0.001
1.57±0.62
2.55±0.87
1.28±1.28
2.23±1.02
3.56±1.43
6.55±6.55
0.780±0.007
0.805±0.018
0.827±0.092
1.101±0.103
1.081±0.106
1.016±0.014
-0.748±0.009
-0.782±0.017
-0.805±0.096
2.11±0.52
1.70±0.39
2.83±2.83
6.12±2.29
5.16±1.99
2.83±2.83
0.748±0.007
0.752±0.008
0.775±0.040
0.815±0.042
0.875±0.041
1.017±0.019
-0.720±0.007
-0.725±0.006
-0.748±0.039
4.48±0.82
4.55±1.03
2.00±2.00
7.66±1.54
7.93±1.82
2.00±2.00
0.748±0.025
0.752±0.025
0.780±0.044
0.832±0.023
0.842±0.071
1.025±0.024
-0.736±0.030
-0.732±0.019
-0.753±0.043
C. carnea
Control
Alatae
Total (alatae+nymphs)
CMV
Natural enemy
Alatae
Total (alatae+nymphs)
CMV
A. bipunctata
Control
Alatae
Total (alatae+nymphs)
CMV
Natural enemy
Alatae
Total (alatae+nymphs)
CMV
1
Student’s t test was used to compare δ and Δ indices between control and natural enemy replicates, and no statistically significant differences were detected (p >
0.05).
a
mean distance between the centroid of the aphid/virus distribution and the central release plant ± SE, in cm; bmean distance of aphid/virus-infected plants
positions from the central release focus ± SE, in cm; cmean aggregation index calculated by the SADIE method ± SE; d,evi, vj are the mean cluster indices
calculated by the SADIE methodology and refer to patches and gaps, respectively ± SE.
31
Influence of aphidophagous predators on CMV spread
Table 3.1.4. Spatial pattern indexes of A. gossypii population and CMV spread after 5 days in the presence and absence (control) of C. carnea L3 or A. bipunctata
adults. The data are the means ± SE of three replicates/natural enemy1.
δa
Δb
Iac
vid
vje
5.53±1.09
5.62±1.32
2.83±2.83
14.22±2.092
12.74±1.93
4.83±4.83
0.754±0.030
0.747±0.025
0.796±0.060
0.885±0.029
0.854±0.019
1.007±0.002
-0.754±0.029
-0.750±0.031
-0.770±0.060
13.7±5.35
12.99±4.17
10.57±4.38
29.26±2.24
26.04±1.89
20.27±6.35
1.221±0.197
1.181±0.075
1.002±0.072
1.235±0.182
1.292±0.118
1.059±0.008
-1.227±0.200
-1.172±0.092
-0.991±0.081
0.47±0.123
4.25±1.14
17.54±8.84
1.06±0.384
11.66±4.87
19.84±10.35
0.734±0.007
0.761±0.023
1.304±0.387
1.121±0.132
0.876±0.089
1.387±0.322
-0.713±0.004
-0.747±0.016
-1.295±0.397
4.13±1.05
13.79±3.64
5.14±4.18
6.55±0.57
25.25±2.36
21.76±6.95
0.849±0.039
1.154±0.242
1.020±0.060
1.076±0.103
1.166±0.227
1.062±0.026
-0.842±0.047
-1.131±0.233
-1.006±0.065
C. carnea
Control
Alatae
Total (alatae+nymphs)
CMV
Natural enemy
Alatae
Total (alatae+nymphs)
CMV
A. bipunctata
Control
Alatae
Total (alatae+nymphs)
CMV
Natural enemy
Alatae
Total (alatae+nymphs)
CMV
1
Student’s t test was used to compare δ and Δ indices between control and natural enemy replicates. Significant data in bold. 2alatae (t = -4.93, p < 0.01), total
morphs (t = -4.91, p < 0.01). 3alatae (t = -3.47, p = 0.03).4alate (t = -8.03, p < 0.01).
a
mean distance between the centroid of the aphid/virus distribution and the central release plant ± SE, in cm; bmean distance of aphid/virus-infected plants
positions from the central release focus ± SE, in cm; cmean aggregation index calculated by the SADIE method ± SE; d,evi, vj are the mean cluster indices
calculated by the SADIE methodology and refer to patches and gaps, respectively ± SE.
32
Influence of aphidophagous predators on CMV spread
3.1.3.4. ASSOCIATION PATTERNS BETWEEN VECTOR AND VIRUS
After 1 day, irrespective of the presence or absence of one of the two predators, the
distribution patterns of aphid colonies and viral spread around the central cucumber CMVsource plant were similar, and significant association indices (X) were obtained (P < 0.025)
(Table 3.1.5). After 5 days, however, a general lack of significance in the association indices
between the virus and the vector was detected (Table 3.1.5).
33
Influence of aphidophagous predators on CMV spread
Table 3.1.5. Association index (X) in every replicate (1, 2, 3) between A. gossypii and CMV infection, after 1 day or 5 days, in the presence of C. carnea L3 or A.
bipunctata adults.
1 day
C. carnea
a
Control 1
Alatae-CMV
Total (alatae+nymphs)-CMV
Control 2
Alatae-CMV
Total (alatae+nymphs)-CMV
Control 3
Alatae-CMV
Total (alatae+nymphs)-CMV
Natural enemy 1
Alatae-CMV
Total (alatae+nymphs)-CMV
Natural enemy 2
Alatae-CMV
Total (alatae+nymphs)-CMV
Natural enemy 3
Alatae-CMV
Total (alatae+nymphs)-CMV
5 days
A. bipunctata
b
C. carnea
A. bipunctata
X
P
X
P
X
P
X
P
0.9994
0.9996
<0.0001
<0.0001
0.7909
0.7385
0.0041
0.0038
0.2192
0.3794
0.1122
0.0273
0.1109
0.2836
0.2297
0.0377
0.9996
0.9994
<0.0001
<0.0001
0.5649
0.5727
0.0162
0.0130
0.3021
0.2139
0.0982
0.1307
0.3280
0.2362
0.0510
0.0824
0.7500
0.7572
0.0002
0.0001
0.4307
0.4756
0.0210
0.0095
0.5100
0.2918
0.0010
0.0274
0.9994
0.2195
<0.0001
0.1073
0.7785
0.7204
0.0044
0.0028
0.4111
0.5385
0.0080
0.0008
-0.0049
-0.0126
0.5151
0.5277
0.3429
-0.1290
0.0342
0.7316
0.5564
0.5660
0.0010
0.0007
0.3768
0.5950
0.0200
0.0010
0.3307
0.4411
0.0181
0.0006
0.3292
-0.0610
0.0569
0.5926
0.8277
0.7298
<0.0001
0.0049
0.3265
0.3736
0.0325
0.0239
0.0188
0.1424
0.4546
0.2156
0.4842
0.3058
0.0082
0.0460
a,b
X is the measure of the degree of association, and P is the probability level. Associations are significant (in bold in the table) when P < 0.025, and dissociations
are significant when P > 0.975.
34
Influence of aphidophagous predators on CMV spread
Figure 3.1.4. A. gossypii dispersal and CMV spread (centroids) from the central source plant in the short and long term (1 day and 5 days) in the absence (z) or presence (S)
of natural enemies. Short-term evaluation (1 day): C. carnea (a, e); A. bipunctata (b, f). Long-term evaluation (5 days): C. carnea (c, g); A. bipunctata (d, h). The axes show
the coordinates for each cucumber plant in the plot; the real distance between each plant was 12 cm.
35
Influence of aphidophagous predators on CMV spread
3.1.4. DISCUSSION
As could be expected based on the sedentary behaviour of A. gossypii compared with other
aphid species (Adams & Hall, 1990), the dispersal of the aphid colony after the release of
adult morphs in the central virus-infected plant was limited, especially after 1 day, and did not
increase consistently after 5 days. The preference that many vectors seem to have for staying
on infected plants where they exhibit an improvement in their fitness could also have
contributed to the observed behaviour (Kennedy, 1951; McKenzie et al., 2002; Maris et al.,
2004; Jiu et al., 2007; Jeger et al., 2012). In the presence of natural enemies and in contrast to
other aphid species, A. gossypii does not exhibit a typical escape response (Nelson &
Rosenheim, 2006); thus, in our trials, we did not expect immediate movement from the viral
source plant after their release. After 5 days, the two natural enemies assessed, C. carnea and
A. bipunctata, played the expected conventional role of benefiting the host plant by reducing
the pest pressure, and a significantly lower number of A. gossypii were detected in the central
source plant. In contrast, after 1 day, a significant decrease in the number of aphids in the
central plants was observed only in the lacewing plots. The differences in consumption rate
and mobility between the two natural enemies might account for this. C. carnea seems to be a
more voracious species than A. bipunctata based on their larval aphid consumption rates
(Gurney & Hussey, 1970; Liu & Chen, 2001), which are even a bit lower than those of A.
bipunctata adult (Ellingsen, 1969). Larvae of C. carnea must be highly mobile in order to
follow their soft-bodied prey, which are mostly homopterous (Viñuela et al., 1996; Duelli,
2001), but they exhibit a much lower movement capacity compared to coccinellid adults,
which tend to fly away from the crop soon after release even in greenhouses (Lommen et al.,
2008). Consequently, Chrysoperla larvae most likely spent more time in the central virusinfected plant than the coccinellid, so its aphid consumption rate was higher.
36
Influence of aphidophagous predators on CMV spread
However, dispersion of aphids in the presence or absence of either of the two natural
enemy studied (C. carnea or A. bipunctata) was scarce and similar. The percentage of noninfected peripheral plants occupied by at least one aphid after 1 or 5 days did not significantly
differ in either case, as shown by the centroids of the aphid populations, which were located
around the central arena point.
The CMV transmission rate observed in our trials was low, and no differences in the viral
and vector spatial distributions were detected. CMV is a non-persistent virus, and as such, its
transmission is highly promoted if the vector acquires the viral particles during short periods
(seconds), as the non-colonising aphids, the main vectors of this type of virus, typically do. In
this case, the general rule is a lack of virus-vector association patterns. However, A. gossypii,
which is a good CMV vector, can settle in plants and form colonies, so the viral acquisition
time can be increased, and therefore the efficiency of transmission decreases due to the loss of
the viral particles linked to the stylet-specific receptors during the ingestion of phloem sap
(Ng & Perry, 2004; Moreno et al., 2012). In agreement with this, in our assays, the
transmission rate was low, and at a short evaluation period (1 day), the SADIE association
indexes (X) between vector and virus were significant in practically every replicate (P <
0.025). In contrast, after 5 days, non-coincident distribution patterns between A. gossypii and
CMV were observed, which is typical in non-persistent transmitted viruses due to the low
transmission success after the first visit of an aphid to a plant (low rate of secondary spread)
(Kennedy et al., 1962).
The epidemiology of aphid-borne viruses is highly linked to vector movement (Jeger et al.,
2004), and the presence of natural enemies can modify that of viruliferous aphids, but this
effect is likely imperceptible when compared to environmental influences (Hooks & Fereres,
2006). In our trials, there was not a significant effect of the two aphidophagous predators, C.
carnea and A. bipunctata, in the incidence and spread of CMV. Infected plants were mainly
37
Influence of aphidophagous predators on CMV spread
located around the central virus-infected source plant, and the percentage of aphid occupation
and CMV-infected plants did not significantly differ in the absence and presence of natural
enemies. However, other studies on the spread of viruses transmitted in a non-persistent
manner show that the foraging habit of natural enemies can have an influence on vector
dispersal and viral spread (Tamaki et al., 1970; Roitberg & Myers, 1978; Smyrnioudis et al.,
2001; Belliure et al., 2011). Nelson & Rosenheim (2006) observed that 96% of the encounters
between C. carnea larvae and A. gossypii individuals in a cotton field ended with aphid
consumption, while only 4% induced an anti-predator response. In the case of coccinellids,
their more energetic searching behaviour has been identified as a key factor in the dispersal
and epidemiology of several virus-vector systems (Roitberg & Myers, 1978; Bailey et al.,
1995; Losey & Denno, 1998; Smyrnioudis et al., 2001; Belliure et al., 2011). However, in our
assays, this characteristic was not reflected by an increase of viral spread because CMV
transmission was equal to or even lower in the presence of A. bipunctata after 1 day or 5 days,
although any differences were not statistically significant. Dáder et al. (2012) with a similar
methodology observed that the parasitoid Aphidius colemani Viereck increased CMV
transmission by A. gossypii in the short term (2 days) but not in the long term (7 days) in the
cucumber.
In conclusion, the results obtained in this work are once again indicative of the complexity
involved in trophic interactions among crops, vectors, viruses and predators. Consequently, it
is necessary to perform large-scale trials in commercial greenhouses in order to obtain further
information about the benefit/damage ratio that aphidophagous predators induce in aphid
populations and about the spread of non-persistent transmitted viruses in protected crops.
38
Influence of aphidophagous predators on CABYV spread
SUB-CHAPTER 3.2. THE EFFECT OF Chrysoperla carnea AND Adalia
bipunctata ON THE SPREAD OF Cucurbit aphid-borne yellows virus BY
Aphis gossypii2
ABSTRACT
The effects of two aphidophagous predators, the larvae of Chrysoperla carnea and the
adults of Adalia bipunctata, on the spread of Cucurbit aphid-borne yellows virus (CABYV)
transmitted by the cotton aphid Aphis gossypii were studied under semi-field conditions.
Aphids and their natural enemies were released inside insect-proof cages with a central
CABYV-infected cucumber plant surrounded by 48 healthy cucumber seedlings. The
spatiotemporal dynamics of the virus and vector were evaluated in the short (7 days) and long
term (14 days) in the presence and absence of each predator. The spatial analysis by distance
indices (SADIE) methodology, together with other indices measuring the dispersal around a
single focus, was used to assess the spatial pattern and the degree of association between the
virus and its aphid vector. The presence of C. carnea larvae and A. bipunctata adults induced
significant A. gossypii dispersal after 14 days but not after 7 days. The reduction in the initial
aphid population established in the central plant was always higher for C. carnea than for A.
bipunctata. Nevertheless, CABYV spread was not significantly modified by the presence of
each predator either in the short term or in the long term. Furthermore, the percentage of
CABYV-infected plants did not significantly differ when each natural enemy was present in
any evaluation period. The present study suggests an influence of both predators on the spread
2
Accepted in Annals of Applied Biology: Garzón, A., Budia, F., Morales, I., Fereres, A., Viñuela, E.,
Medina, P. Do Chrysoperla carnea and Adalia bipunctata influence the spread of Cucurbit aphid-borne
yellows virus and its vector Aphis gossypii?
39
Influence of aphidophagous predators on CABYV spread
of the aphid, mainly in the long term, but no significant effect of the predators in the spread of
the viral disease was demonstrated.
3.2.1. INTRODUCTION
The major cultivated cucurbit species (watermelon, melon, pumpkin, squash and
cucumber) are important vegetable crops worldwide. In Spain, the cultivated surface
dedicated to these crops in 2013 reached 66.732 ha (MAGRAMA, 2014a).
Virus-induced diseases are a cosmopolitan problem for cucurbits and cause severe
economic losses. In fact, more than 35 viruses have been isolated from cucurbit species
(Provvidenti, 1996), and at least ten of them have been identified in Spanish cucurbit crops
(Luis-Arteaga et al., 1998; Kassem et al., 2007; Juárez et al., 2013). Amongst these viruses,
CABYV (Cucurbit aphid-borne yellows virus) has been shown to be the prevalent virus in
cucurbit crops in southeastern Spain, increasing its spread since its first report in 2003 (Juárez
et al., 2004; Juárez et al., 2013).
CABYV is a member of the genus Polerovirus in the family Luteoviridae (Mayo &
D’Arcy, 1999). Its viral particles are icosahedral, approximately 25 nm in diameter, and
encapsidate the CABYV genome, which consists of a single-stranded positive sense RNA
molecule of 5.7 kb. Similar to other members of the Luteoviridae, CABYV is obligately
transmitted by aphids in a persistent, circulative and vector-specific manner (Guilley et al.,
1994; Herrbach, 1999). CABYV is efficiently transmitted by two aphid species, Myzus
persicae (Sulzer) (Hemiptera: Aphididae) and Aphis gossypii Glover (Hemiptera: Aphididae)
(Lecoq et al., 1992).
Kassem et al. (2013) observed that A. gossypii was the main aphid species involved in the
spread of CABYV in melon crops in the southeastern region of Spain. This aphid requires
40
Influence of aphidophagous predators on CABYV spread
prolonged feeding times and direct contact with vascular tissues for virus transmission
(Fereres & Moreno, 2009).
The behaviour of this type of pathosystem depends on virus-plant, plant-vector and virusvector interactions. These relationships are biologically complex, but once elucidated, they
shed light on the implementation of innovative management strategies to reduce the impact of
the virus disease (Bosque-Pérez & Eigenbrode, 2011). Furthermore, aphids are attacked by a
wide range of predators and parasitoids (Blümel, 2004) that promote escape responses and
rapid dispersion (Dixon, 1958). The foraging habits of their natural enemies seem to modify
the spread of viruses vectored by aphids (Smyrnioudis et al., 2001; Belliure et al., 2011).
Thus, the objective of this study was to determine if the spread of CABYV by A. gossypii
was affected by the presence of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae)
larvae and Adalia bipunctata (L.) (Coleoptera: Coccinellidae) adults. C. carnea larvae are
very mobile and voracious polyphagous predators in many agricultural systems, exerting an
effective control over soft-bodied insects such as aphids (Viñuela et al., 1996; Medina et al.,
2003b). Moreover, it is one of the indicator species considered for higher testing in the EU for
pesticide registration (Candolfi et al., 2001). A. bipunctata adults are predators with a wide
range of aphid prey (Omkar & Pervez, 2005). Both species, which are commercially
available, are currently widely used in Spain in protected horticultural crops (Robledo et al.
2009).
3.2.2. MATERIALS AND METHODS
3.2.2.1. INSECTS
A single virginiparous apterae of A. gossypii collected in a melon crop in Almería (Spain)
was used to initiate a clonal population, according to the procedure explained in Garzón et al.
(2015). For the trials, alate individuals were collected with an aspirator and placed in groups
41
Influence of aphidophagous predators on CABYV spread
of 100 in Falcon® tubes before being transferred to the experimental cages. A. gossypii is a
suitable prey species for the two predators used in the tests (van Steenis, 1992).
A laboratory colony of C. carnea was established from L1 larvae obtained from Agrobío
(CHRYSOcontrol®, Almería, Spain) following the rearing procedure of Medina et al. (2001)
and Vogt et al. (2000). For the trials, gauze with < 24-h-old eggs was taken from the adult
rearing cages, and the eggs were allowed to develop up to the L2 instar used in the
experiments.
A. bipunctata L1 larvae were purchased from Agrobío (ADALIAcontrol®, Almería, Spain)
and reared according to Bonte et al. (2010). L4 larvae were placed in individual arenas for the
emergence of adults as explained in Garzón et al. (2015). Two weeks old A. bipunctata adults
were used in the experiments.
3.2.2.2. PLANT MATERIAL
Cucumber (Cucumis sativus L. cv. ‘Ashley’) plants were grown from seed in 150 alveoli
trays (56 cm x 36 cm x 6 cm) filled with a 1:1 mixture of soil substrate (Projar, Valencia,
Spain) and vermiculite Termita® (Aminco, Donostia, Spain) in an insect-proof chamber at 2325 ºC, 60-80% relative humidity (RH) and a 16:8 L:D photoperiod.
3.2.2.3. VIRUS ACQUISITION
The CABYV isolate was provided by Dr. H. Lecoq, obtained in Montfavet (France) from
zucchini squash in 2003, and maintained in cucumber plants by aphid serial transmission. The
cucumber plants were inoculated by means of CABYV vectoring aphids, following the
protocol explained in Moreno et al. (2011) and Dáder et al. (2012). After the inoculation
process (Figure 3.2.1), plants were sprayed with Biopirot® (Aragonesas Agro, natural
pyrethrin 1.5% + rotenone 3%, dilution rate 2 ml l-1) to kill the aphids.
42
Influence of aphidophagous predators on CABYV spread
Figure 3.2.1. Inoculation of CABYV by transferring viruliferous A. gossypii individuals from a previously
infected source plant (left) to receptor healthy plants (right).
The plants to be used as CABYV-infected cucumber plants were maintained in an insectproof chamber at 23–25 °C, 60–80% RH and a 16:8 L:D photoperiod. These plants were
previously tested for virus infection by a double antibody sandwich enzyme-linked
immunosorbent assay (DAS-ELISA) (Clark & Adams, 1977) using specific commercial
antibodies against CABYV (Sediag, France).
3.2.2.4. EXPERIMENTS
The methodology used in this study was similar to that of Garzón et al. (2015) with
adaptations due to the different virus studied. For each natural enemy, two evaluation times
were considered (7 days and 14 days, respectively), and the experiments (4 in total) consisted
of 3 replicates of 3 cages with the natural enemy and 3 without. In order to allow acclimation
to the greenhouse environment, one day before the insect release a potted CABYV-infected
cucumber source plant was placed in the centre of the tray surrounded by 48 non-infected
cucumber plants at the one true leaf stage (BBCH scale = 11) placed in a grid. Twenty-four
hours later, 100 winged A. gossypii adults were confined in the CABYV-infected source plant
by means of a plastic cylinder with a ventilated lid and allowed to establish. After one hour,
the cylinder was removed, releasing 3 L2 C. carnea larvae in the source plant in half of the
43
Influence of aphidophagous predators on CABYV spread
cages used for the C. carnea experiment and 3 A. bipunctata adults in the source plant in half
of the cages used for A. bipunctata experiment (Figure 3.2.2). C. carnea larvae and A.
bipunctata adults were collected and starved for 2 h before starting the test in order to
increase the voracity. The temperature and relative humidity were remotely controlled in the
greenhouse through a central computer, guaranteeing a range of 15-27 ºC and 50-85% RH
during the experiment.
Figure 3.2.2. Natural enemies releasing in the central CABYV-source cucumber plant.
Because CABYV is acquired and transmitted in prolonged feeding times and requires
direct contact with vascular tissues (Fereres & Moreno, 2009), the aphid dispersion and the
spread of CABYV from the source plant were evaluated after 7 days (short term) and after 14
days (long term) in the presence and absence of each of the two natural enemies. The number
of A. gossypii individuals in the virus source plant and in the peripheral plants was recorded,
as well as their developmental status. The plants were sprayed with the systemic insecticide
Confidor® 20 LS (Bayer CropScience, 200 g l-1 w/v imidacloprid as a soluble concentrate,
dilution rate 0.75 ml l-1) in order to kill the aphids and prevent further viral transmission. Four
44
Influence of aphidophagous predators on CABYV spread
weeks after aphid spread evaluation, leaf samples of every receptor plant were frozen at -80
ºC, and CABYV infection was diagnosed by DAS-ELISA as previously described (Figure
3.2.3).
Figure 3.2.3. Microplates used for CABYV diagnosis in test plants by means of ELISA serological technique.
3.2.2.5. STATISTICAL ANALYSIS
The density of aphids in the CABYV source plant, dispersal of aphids towards peripheral
non-infected plants (percentage of plants occupied by at least one aphid morph) and
percentage of CABYV-infected plants at the two evaluation periods (7 d and 14 d) were
compared by Student’s t test (p < 0.05) after normality checks (Kolmogorov-Smirnov test) in
the presence and absence of C. carnea or A. bipunctata. The Statgraphics Plus 5.1 statistical
package (Statistical Graphics Corporation, 1994-2000) was used for calculations. The
percentage reduction in the number of aphids in the central virus-infected plant in the
presence of each natural enemy was calculated with Abbott’s formula: Reduction (%) = (nc –
nt) / nc x 100; where nc is the number of aphids in the absence of a natural enemy, nt is the
number of aphids in the presence of a natural enemy (Abbott, 1925). 45
Influence of aphidophagous predators on CABYV spread
Spatially referenced counts in every sampling unit (receptor plant) were used to calculate
several indexes. The distribution of aphids and virus spread was studied through indexes used
by Korie et al. (1998) and the spatial analysis by distance indices (SADIE) methodology, a
biologically based method (Perry, 1995). The spatial pattern of viral infection and aphid
dispersal was described by the index of aggregation (Ia) (aggregated if Ia > 1; regular if Ia < 1
or random if Ia = 1) and the indexes of clustering in patches and gaps (vi, vj) (> 1.5 patchiness,
< -1.5 insects in the plant are members of a gap; close to 1, a random placement of insects in
that plant in relation to others nearby) (Perry, 1998; Perry et al., 1999). Moreover, the degree
of spatial association between aphids and CABYV spread was measured by the index of
spatial association (X) (local association if P < 0.025; local dissociation if P > 0.975) (Perry &
Nixon, 2002; Díaz et al., 2010).
To obtain a more global picture, the following indexes used by Korie et al. (1998) to
analyse dispersal around a single focus were also calculated: the distribution of the viral
infection or aphid dispersal from the central plant (δ) measured as the distance between a
given plant (x = 4, y = 4) and the centroid (C, average position of aphids or CABYV infected
plants), and the spread of the infection as the average distance of infected plants from the
central release focus (Δ).
3.2.3. RESULTS
3.2.3.1. A. gossypii DENSITY ON THE CENTRAL SOURCE PLANT
Considering both short-term (7 days) and long-term (14 days) assays, C. carnea promoted
a higher reduction than A. bipunctata in aphid density in the central CABYV-source plant
(Table 3.2.1). The presence of C. carnea caused a significant percentage reduction (Abbott,
1925) in the number of A. gossypii adult alatae in the central CABYV-infected plant both
after 7 days (90.32%, t = 3.88, p = 0.02) and 14 days (82.73%, t = 2.98, p = 0.04) and in the
46
Influence of aphidophagous predators on CABYV spread
number of nymphs after 7 days (92.39%, t = 5.09, p = 0.01). A. bipunctata only caused
significant reductions in the numbers of nymphs (85.10%, t = 8.77, p < 0.01) after 14 days.
3.2.3.2. A. gossypii DISPERSAL FROM THE CENTRAL SOURCE PLANT
As an overview, the occupancy rate in the peripheral plants by at least one aphid (adult
alate or nymph) in the presence of any of the predators was never significant (p > 0.05)
compared with the absence of predators (Table 3.2.2). However, the effect of the chrysopid
and coccinellid was slightly different, at least in the short time (7 days).
The percentage of occupancy after 7 days was slightly higher in the presence of L2 C.
carnea (alatae 20.83 ± 3.18%; nymphs 27.10 ± 1.21%) than in the control (alatae 19.43 ±
2.49%; nymphs 25.00 ± 4.32%). After 14 days, the dispersal of aphids towards the receptor
plants increased for both aphid developmental stages (with natural enemy 32.63 ± 6.83%
alatae, 45.83 ± 12.71% nymphs; control 27.10 ± 5.23% alatae, 34.73 ± 5.95% nymphs) (Table
3.2.2).
When A. bipunctata was present, the number of aphids in the peripheral plants after 7 days
(16.00 ± 0.70% alatae, 19.47 ± 1.82% nymphs) was lower than in its absence (19.47 ± 5.95%
alatae, 22.93 ± 7.31% nymphs), in contrast to the results observed with C. carnea. After 14
days this trend reverses, with an increase of the aphid individuals in the peripheral plants
when A. bipunctata was present (34.73 ± 9.71% alatae, 39.60 ± 11.45% nymphs), compared
with its absence (22.23 ± 2.50% alatae, 31.23 ± 2.07% nymphs) (Table 3.2.2).
In the short term (7 days), aphids showed a more dispersive behaviour in the presence of
L2 C. carnea larvae than that recorded with A. bipunctata adults (Table 3.2.3 and Figure
3.2.4). Nevertheless, this aphid dispersal was never significant when compared with the
control, as reflected by the indexes δ and Δ (Table 3.2.3). On the contrary, the long-term
assays (14 days) revealed a significantly higher aphid (alatae + nymphs) dispersion when
47
Influence of aphidophagous predators on CABYV spread
individuals of either natural enemy were released, according to Δ index (Table 3.2.4); the
value obtained for C. carnea presence was 31.54 ± 0.38 cm vs 23.75 ± 1.60 cm of control (t =
-4.75, p < 0.01), while in the case of A. bipunctata presence the value was 26.33 ± 4.36 cm vs
10.36 ± 2.53 cm of the respective control (t = -3.17, p = 0.03). The Ia of alate aphids when C.
carnea was present took a value of 1.707 ± 0.086, indicating a significant aggregated
distribution (vi = 1.522 ± 0.071; vj = -1.709 ± 0.086), as did the values for total morphs (alatae
+ nymphs) (Ia = 1.643 ± 0.141; vi = 1.575 ± 0.113; vj = -1.630 ± 0.110) (Table 3.2.4). These
numeric values can be graphically observed in Figure 3.2.4 (a,b and c,d, respectively), where
the centroid position (C) of A. gossypii (alatae + nymphs) populations are represented in the
presence and absence of both predators.
48
Influence of aphidophagous predators on CABYV spread
Table 3.2.1. Influence of the presence of C. carnea larvae or A. bipunctata adults on the density of A. gossypii (adult alate morphs and
nymphs) in the central CABYV-source plant 7 days and 14 days after infestation with 100 adult alatae. The data are the means ± SE of the
number of aphids/plant (n = 3 replicates)1.
R(%)a
Control
C. carnea
Short term (7 days)
Alatae
Nymphs
10.33±2.33
188.33±32.84
1.00±0.58
14.33±9.49
Long term (14 days)
Alatae
Nymphs
9.67±2.60
1.67±0.67 2.98 0.04 82.73
296.00±78.02 109.67±17.94 2.33 0.08 62.95
1
a
t
p
Control
3.88 0.02 90.32
5.09 0.01 92.39
17.67±4.18
147.33±36.02
A. bipunctata
t
R(%)a
p
20.33±5.24 -0.40 0.71
189.67±74.09 -0.51 0.63
-15.05
-28.74
17.00±4.51
4.33±1.20
2.71 0.05
1535.00±133.67 228.67±65.81 8.77 <0.01
74.53
85.10
Student's t test (p < 0.05). Significant data in bold.
reduction in aphid population (Abbott, 1925).
Table 3.2.2. Influence of the presence of C. carnea larvae or A. bipunctata adults on the percentage of peripheral plants
occupied by at least one A. gossypii (adult alate or nymph) and percentage of CABYV-infected plants 7 and 14 days after
infestation with 100 adult alatae in the central CABYV-source plant. The data are the means ± SE (n = 3 replicates)1.
Control
C. carnea
t
p
Control
A. bipunctata
t
p
Short term (7 days)
Alatae
Nymphs
CABYV
19.43±2.49
25.00±4.32
5.57±1.83
20.83±3.18
27.10±1.21
8.37±4.17
-0.35
-0.47
-0.62
0.75
0.66
0.57
19.47±5.95
22.93±7.31
13.90±4.84
16.00±0.70
19.47±1.82
4.20±1.21
0.58
0.46
1.94
0.59
0.67
0.12
Long term (14 days)
Alatae
Nymphs
CABYV
27.10±5.23
34.73±5.95
4.17±2.40
32.63±6.83
45.83±12.71
2.10±0.00
-0.64
-0.79
-
0.56
0.47
-
22.23±2.50
31.23±2.07
5.57±2.49
34.73±9.71
39.60±11.45
3.50±1.85
-1.25 0.28
-0.72 0.51
0.67 0.54
1
Student's t test (p < 0.05).
49
Influence of aphidophagous predators on CABYV spread
3.2.3.3. CABYV SPREAD
The CABYV transmission rate by A. gossypii from the central source plant was lower in
the presence of C. carnea just in the long-term assay (14 days) when compared with the
control. Notwithstanding, the releasing of A. bipunctata caused a decrease of viral infection in
the peripheral plants both for short (7 days) and long term (14 days) with regard to its absence
(Table 3.2.2). Despite that fact, these reductions in CABYV transmission in the presence of
natural enemies were never significant (p > 0.05; Table 3.2.2). The percentages of CABYV
infected plants were always under 14%, reaching the highest value for the control replicates of
the A. bipunctata experiment after 7 days (13.90 ± 4.84%) and the lowest in the presence of
C. carnea larvae in the 14 days assay (2.10 ± 0.00%) (Table 3.2.2).
The indexes of aggregation (Ia) for CABYV after 7 and 14 days revealed a random
distribution (Ia ≈ 1) of the virus infection, contrasted with the lack of significance of the
clustering indices vi (< 1.5) and vj (> -1.5) (Table 3.2.3 and Table 3.2.4). This randomness can
be observed in Figure 3.2.4, especially for control replicates in short-term experiments (e, f)
and for replicates with the presence of a natural enemy in long-term experiments (g, h). The
spatial distribution of CABYV is numerically translated by the indexes δ and Δ (Table 3.2.3
and Table 3.2.4). In the 7-days assays, the mean distance indexes showed higher values in the
absence of both C. carnea (δ = 1.24 ± 0.26 cm; Δ = 22.42 ± 3.45 cm) and A. bipunctata (δ =
1.81 ± 0.36 cm; Δ = 28.60 ± 2.39 cm) in comparison with their presence (δ = 0.99 ± 0.39 cm;
Δ = 18.69 ± 4.86 cm for C. carnea; δ = 0.83 ± 0.08 cm; Δ = 16.40 ± 4.40 cm for A.
bipunctata) (Table 3.2.3). On the contrary, in the 14-days assays the indexes δ and Δ proved
to be higher in the presence of C. carnea (δ = 1.34 ± 0.47 cm; Δ = 17.14 ± 5.79 cm) compared
with the control (δ = 0.68 ± 0.37 cm; Δ = 14.17 ± 9.75 cm), and in the presence of A.
bipunctata (δ = 0.95 ± 0.56 cm; Δ = 14.41 ± 7.72 cm) with regard to its respective control (δ
50
Influence of aphidophagous predators on CABYV spread
= 0.81 ± 0.20 cm; Δ = 11.10 ± 3.43 cm) (Table 3.2.4). None of these differences was
statistically significant.
51
Influence of aphidophagous predators on CABYV spread
Table 3.2.3. Spatial pattern indexes of A. gossypii populations and CABYV spread after 7 days in the presence and absence (control) of C. carnea larvae or A.
bipunctata adults. The data are the means ± SE of three replicates/natural enemy1.
δa
Δb
Iac
vid
vje
1.08±0.33
0.78±0.22
1.24±0.26
19.85±2.25
13.32±1.74
22.42±3.45
0.965±0.129
0.820±0.033
0.935±0.075
0.953±0.098
0.897±0.044
0.990±0.082
-0.967±0.121
-0.809±0.025
-0.924±0.073
1.52±0.73
1.34±0.64
0.99±0.39
30.03±7.27
28.43±7.77
18.69±4.86
1.373±0.115
1.206±0.088
1.032±0.069
1.338±0.109
1.129±0.095
1.083±0.033
-1.369±0.118
-1.216±0.084
-1.014±0.075
0.43±0.16
0.42±0.10
1.81±0.36
13.55±4.05
15.25±2.49
28.60±2.39
0.811±0.063
0.789±0.043
1.358±0.142
0.775±0.049
0.871±0.072
1.371±0.118
-0.814±0.067
-0.791±0.044
-1.349±0.144
0.77±0.27
0.75±0.23
0.83±0.08
14.44±3.99
15.42±4.51
16.40±4.40
0.864±0.077
0.886±0.092
0.885±0.020
1.002±0.084
0.892±0.088
0.970±0.042
-0.860±0.077
-0.882±0.103
-0.867±0.025
C. carnea
Control
Alatae
Total (alatae+nymphs)
CABYV
Natural enemy
Alatae
Total (alatae+nymphs)
CABYV
A. bipunctata
Control
Alatae
Total (alatae+nymphs)
CABYV
Natural enemy
Alatae
Total (alatae+nymphs)
CABYV
1
Student’s t test was used to compare δ and Δ indices between control and natural enemy replicates and no statistically significant differences were detected (p >
0.05).
a
mean distance between the centroid of the aphid/virus distribution and the central release plant ± SE, in cm; bmean distance of aphid/virus infected plants
positions from the central release focus ± SE, in cm; cmean aggregation index calculated by SADIE method ± SE; d,e ʋҧi, ʋҧj are mean cluster indices calculated by
SADIE methodology and refers to patch and gaps, respectively ± SE.
52
Influence of aphidophagous predators on CABYV spread
Table 3.2.4. Spatial pattern indexes of A. gossypii population and CABYV spread after 14 days in the presence and absence (control) of C. carnea larvae or A.
bipunctata adults. The data are the means ± SE of three replicates/natural enemy1.
δa
Δb
Iac
vid
vje
0.87±0.31
0.93±0.39
0.68±0.37
21.99±2.722
23.75±1.60
14.17±9.75
0.967±0.015
1.029±0.053
0.898±0.083
1.036±0.029
1.060±0.070
1.012±0.039
-0.975±0.012
-1.015±0.053
-0.885±0.090
1.78±0.29
1.60±0.41
1.34±0.47
31.57±1.70
31.54±0.38
17.14±5.79
1.707±0.0864
1.643±0.1415
0.931±0.053
1.522±0.071
1.575±0.113
1.034±0.024
-1.709±0.086
-1.630±0.110
-0.908±0.055
0.61±0.28
0.43±0.20
0.81±0.20
16.65±3.12
10.36±2.533
11.10±3.43
0.827±0.059
0.740±0.019
0.983±0.073
0.859±0.060
0.756±0.046
1.110±0.027
-0.832±0.063
-0.717±0.025
-0.958±0.077
1.06±0.11
1.19±0.24
0.95±0.56
24.84±3.70
26.33±4.36
14.41±7.72
1.241±0.179
1.305±0.264
0.903±0.086
1.154±0.196
1.240±0.243
0.994±0.002
-1.246±0.175
-1.269±0.249
-0.887±0.091
C. carnea
Control
Alatae
Total (alatae+nymphs)
CABYV
Natural enemy
Alatae
Total (alatae+nymphs)
CABYV
A. bipunctata
Control
Alatae
Total (alatae+nymphs)
CABYV
Natural enemy
Alatae
Total (alatae+nymphs)
CABYV
1
Student’s t test was used to compare δ and Δ indices between control and natural enemy replicates. Significant data in bold. 2alate (t = -2.99, p = 0.04), total
morphs (t = -4.75, p < 0.01). 3total morphs (t = -3.17, p = 0.03). 4,5Significant aggregated distribution (vi > 1.5; vj < -1.5).
a
mean distance between the centroid of the aphid/virus distribution and the central release plant ± SE, in cm; bmean distance of aphid/virus infected plants
positions from the central release focus ± SE, in cm; cmean aggregation index calculated by SADIE method ± SE; d,e ʋҧi, ʋҧj are mean cluster indices calculated by
SADIE methodology and refers to patch and gaps, respectively ± SE.
53
Influence of aphidophagous predators on CABYV spread
3.2.3.4. ASSOCIATION PATTERNS BETWEEN VECTOR AND VIRUS
Considering an overall perspective of the four assays, the association between A. gossypii
and CABYV seemed to take place mainly in the presence of each natural enemy and in the
long term (14 days) (Table 3.2.5). The significant (P < 0.025) association values (X)
apparently appeared both for alate individuals-CABYV and total morphs (alatae + nymphs)CABYV, although it is slightly higher in the second case, reflecting the A. gossypii trend to
establish in colonies and the CABYV persistent, circulative manner of transmission in which
it is transferred from adults to nymphs.
54
Influence of aphidophagous predators on CABYV spread
Table 3.2.5. Association index (X) in every replicate (1, 2, 3) between A. gossypii and CABYV infection, after 7 days or 14 days, in presence of C. carnea larvae or A.
bipunctata adults.
7 days
C. carnea
a
Control 1
Alatae-CABYV
Total (alatae + nymphs)-CABYV
Control 2
Alatae-CABYV
Total (alatae + nymphs)-CABYV
Control 3
Alatae-CABYV
Total (alatae + nymphs)-CABYV
Natural enemy 1
Alatae-CABYV
Total (alatae + nymphs)-CABYV
Natural enemy 2
Alatae-CABYV
Total (alatae + nymphs)-CABYV
Natural enemy 3
Alatae-CABYV
Total (alatae + nymphs)-CABYV
14 days
A. bipunctata
b
C. carnea
A. bipunctata
X
P
X
P
X
P
X
P
0.5197
0.6629
0.0003
0.0001
0.2856
0.3966
0.0340
0.0067
0.2330
0.2109
0.1186
0.1186
0.2423
0.1936
0.0755
0.1502
0.3085
0.2538
0.0243
0.0574
-0.3428
-0.2127
0.9898
0.9248
0.4638
0.4122
0.0030
0.0095
0.2069
0.3201
0.1000
0.0487
0.1984
0.1849
0.1031
0.1160
0.0270
-0.0402
0.4214
0.5999
0.2286
0.1911
0.1064
0.1441
0.2746
0.1551
0.0545
0.2397
0.2561
0.2616
0.0676
0.0664
0.2107
0.2841
0.1229
0.0772
0.3347
0.3481
0.0256
0.0149
0.3179
0.3555
0.0190
0.0072
0.3212
0.2450
0.0393
0.0833
0.3626
0.4343
0.0322
0.0074
0.3425
0.3057
0.0138
0.0213
0.3002
0.4703
0.0184
0.0090
-0.4819
0.1385
0.9998
0.1879
0.5646
0.5672
0.0023
0.0029
0.0400
0.2238
0.4011
0.0766
0.3097
0.1239
0.0060
0.2109
a,b
X is the measure of the degree of association and P the probability level. Associations are significant (in bold in the table) when P < 0.025 and dissociations are
significant when P > 0.975.
55
Influence of aphidophagous predators on CABYV spread
Figure 3.2.4. A. gossypii dispersal and CABYV spread (centroids) from the central source plant in the short and long term (7 days and 14 days) in the absence (z) or presence
(S) of natural enemies. Short-term evaluation (7 days): C. carnea (a, e); A. bipunctata (b, f). Long-term evaluation (14 days): C. carnea (c, g); A. bipunctata (d, h). The axes
show the coordinates for each cucumber plant in the plot; the real distance between each plant was 12 cm.
56
Influence of aphidophagous predators on CABYV spread
3.2.4. DISCUSSION
The host plant, vector and virus become interdependent components of a complex
pathosystem (Irwin & Thresh, 1990), and the addition of a fourth element represented by a
natural enemy makes their interactions even more intricate.
In the present study, the release of C. carnea and A. bipunctata induced significant A.
gossypii dispersal in the long term (14 days) but not in the short term (7 days). The number of
peripheral plants occupied by aphids was lower only in the 7-day experiment in the presence
of A. bipunctata compared with its absence; this reduction in the aphid population of the
surrounding plants contrasts with the results for C. carnea at 7 days and can be explained in
terms of mobility. Although C. carnea larvae are highly mobile (Viñuela et al., 1996; Duelli,
2001), they exhibit a much lower movement capacity compared to coccinellid adults
(Lommen et al., 2008). This means that once released in the central infected plant, C. carnea
larvae stayed on it, inducing the alarm in the aphid colony and thus promoting the A. gossypii
spread. Meanwhile, it was observed that some A. bipunctata adults flew away from the initial
releasing point after a few minutes, making their behaviour much more unpredictable. This
difference in the displacement capacity of both predator morphs is reflected also in the aphid
density on the central CABYV-infected plant; the remaining number of A. gossypii
individuals on this plant, especially nymphs, was always lower in the presence of C. carnea
larvae in comparison with A. bipunctata adults. In fact, this aphid reduction in the presence of
the chrysopid larvae was significant in the short term (7 days) both for A. gossypii alatae and
nymphs.
At 14 days, the aphid reduction in the central CABYV-infected plant decreased similarly
for both predators, and the aphid occupancy in the peripheral plants increased in their
presence but in a non-significant manner compared with the control. The significance was
57
Influence of aphidophagous predators on CABYV spread
found in the mean distance index Δ for A. gossypii (alatae + nymphs) spread, which means
that the presence of any of the natural enemies induced a further displacement of aphid
individuals from the colony in the long term. Nonetheless, the overall increase in aphid spread
is directly correlated with the increase in the evaluation time, irrespective of the absence or
presence of natural enemies. Despite the sedentary behaviour of A. gossypii (Adams & Hall,
1990; Nelson & Rosenheim, 2006), the percentage of plants infested by aphid individuals
increased in regard to the results of Garzón et al. (2015). In that study, the evaluation intervals
after the initial release were lower (1 day for short term; 5 days for long term), as the authors
were investigating the spread of A. gossypii as well, although in this case A. gossypii acting as
the vector of the non-persistently transmitted virus CMV (Cucumber mosaic virus).
Therefore, aphid spread was higher in the present study, regardless of the manner of
transmission, which is known to affect the vector’s behaviour. Mauck et al. (2010) observed
that A. gossypii is initially attracted by the volatile organic compounds emitted by CMVinfected squash plants. However, sometime after landing, they prefer non-infected plants. In
contrast, there are examples of a coupling of enhanced vector performance and preference for
virus-infected hosts in the persistently transmitted viruses of the Luteoviridae family
(Eigenbrode et al., 2002; Hodge & Powell, 2008a), as is the case of CABYV.
The CABYV spread at 7 days was lower in the presence of the predators, as reflected by
the δ and Δ mean distance indexes and the spatial distribution, in spite of an increase of virusinfected plants in the presence of C. carnea, correlated with higher aphid occupancy in the
peripheral plants. The presence of natural enemies may induce vector movement from the
infected source plants in small-scale movements that result in the increased incidence of plant
disease (Bailey et al., 1995; Losey & Denno, 1998; Smyrnioudis et al., 2001). The virusaphid correlation also existed in the 7-days experiment with A. bipunctata, but in this case
both A. gossypii and CABYV appeared in lower percentages in the presence of the
58
Influence of aphidophagous predators on CABYV spread
coccinellid. These results contrast with those obtained by Smyrnioudis et al. (2001), who
observed significantly more infected plants in the presence of Coccinella septempunctata L.
(Coleoptera: Coccinellidae); they found twice as many Barley yellow dwarf virus (BYDV)
infected plants after two weeks in the presence than in the absence of predator. Bailey et al.
(1995) also found that the introduction of coccinellid predators increased the infection rate
when compared to controls. Nevertheless, different natural enemy species may have different
effects on small and large-scale migration and therefore on virus transmission (Montgomery
& Nault, 1977b; Kunert & Weisser, 2003).
In contrast, in the long-term assays (14 days), the tendency was reversed in terms of
percentages, and the rise in the aphid occupation in the presence of each predator was not
aligned with CABYV transmission, whose percentage was lower than in the controls.
Nevertheless, the limited CABYV transmission rates irrespective of the absence/presence of
the predators resulted in a lack of significance. Despite a reduction in the number of plants
infected, the CABYV spread increased when each natural enemy was released, according to
the higher δ and Δ indexes and the graphical representation (S further from centre than z in
g and h graphs). This displacement was higher in the presence of C. carnea, most likely due
to the centrifugal spread that larvae cause in aphids, whereas A. bipunctata winged adults
have the capacity of flying and performing a more centripetal effect on the aphid colony.
Differences in the escaping aphid response appear to be primarily due to the searching
behaviour of their natural enemies (Lossey & Denno, 1998). The aphid grouping that A.
bipunctata caused in the centre-located plants was reflected in a higher concentration of
CABYV-infected plants around the central one, and resulted in significant vector-virus
association indexes (X), both for the short and long term. The different degrees of activity of
the aphid predators may be an important aspect of the epidemiology of aphid-transmitted
viruses (Smyrnioudis et al., 2001; Jeger et al., 2004), but sometimes this effect might be
59
Influence of aphidophagous predators on CABYV spread
imperceptible when compared with environmental influences (Hooks & Fereres, 2006), which
becomes a limitation to extrapolating the results.
In any case, the collateral effects of the use of biological control agents should be taken
into account when the crop is affected by viruses. Therefore, the consideration of virus-plantvector-natural enemy interactions is a key factor for understanding virus epidemiology (Jeger
et al., 2012). Because the effects of natural enemies of the vector population on CABYV
spread have not been exhaustively studied, further research is necessary regarding the
mechanisms that aphidophagous predators trigger in aphid populations and the spread of
persistent transmitted viruses in protected crops.
In this study, the presence of C. carnea larvae and A. bipunctata adults induced the spread
of A. gossypii, which was fairly observed after 14 days from the initial release. When the
evaluation period increased, the activity of both predators led to higher aphid displacement, in
spite of its sedentary nature. Nevertheless, the searching behaviour of each natural enemy and
their disparate locomotive skills triggered slightly different responses in the aphid colony. For
example, the reduction in the initial aphid population established in the central plant was
always higher for C. carnea versus A. bipunctata, due to the increased time spent in the
release point. In contrast to aphid results, the CABYV spread was not significantly affected
by the presence of the predators in either the short term or the long term. Moreover, the
number of CABYV-infected plants did not significantly differ in any evaluation time when
either natural enemy was present. Therefore, it is suggested an influence of the
aphidophagous predators on the spread of the aphid, mainly in the long term, but no
significance in the epidemiology of the viral disease.
60
C. externa fed on M. persicae vectoring PLRV CHAPTER 4. DEVELOPMENT AND REPRODUCTION OF Chrysoperla
externa FED ON Myzus persicae VECTORING Potato leafroll virus3
ABSTRACT
The aim of this study was to evaluate the biological parameters of Chrysoperla externa
(Hagen) under the condition of consuming Myzus persicae (Sulzer) acting as vector of Potato
leafroll virus (PLRV). Therefore, three different diets were offered ad libitum in laboratory
experiments to the chrysopid during the larval period. The feeding regimes were as follows:
M. persicae fed on PLRV-infected potato leaves, M. persicae fed on uninfected potato leaves,
and an artificial diet composed of Ephestia kuehniella Zeller eggs was used as the control.
The following parameters were studied: the developmental time and survival rate of the larval
and pupal stages, the reproductive functions of the emerged adults (sex ratio, percentage of
fertile females, fecundity and egg viability) and the survival curve of the first 30 days after
adult emergence. When C. externa was fed with aphids vectoring PLRV showed significant
differences regarding developmental time and survival of larval period in comparison with the
C. externa fed on diets of non-contaminated aphids and E. kuehniella eggs. The length of the
pupal stage did not show significant differences between the aphid diets, but both were
significantly shorter in comparison with that of the C. externa fed the control diet. Any
significant differences were detected in the pupal survival as well as in the reproductive
parameters. Concerning adult survival, the percentages of individuals alive 30 days after
3
Submitted to Neotropical Entomology: Garzón, A., Freire, B.C., Carvalho, G.A., Oliveira, R.L., Medina,
P., Budia, F. Development and Reproduction of Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae)
Fed on Myzus persicae (Sulzer) (Hemiptera: Aphididae) Vectoring Potato leafroll virus (PLRV)
61 C. externa fed on M. persicae vectoring PLRV emergence were 0.75, 0.51 and 0.54 for the diets of E. kuehniella eggs, uninfected aphids and
PLRV-infected aphids, respectively, presenting significant differences among the treatments.
4.1. INTRODUCTION
Potato (Solanum tuberosum L.) is one of the main horticultural crops in Brazil and
worldwide. In 2014, the Brazilian annual yield of potatoes was 3.675.611 t for approximately
130.730 ha (Agrianual, 2015). Among the pests that cause damage to the potato, the aphid
Myzus persicae (Sulzer) (Hemiptera: Aphididae) is one of the most important because of its
sucking activity, which reduces vegetative growth, and its potential as a transmitter of vegetal
viruses (Souza-Dias, 1995). M. persicae is the most efficient vector of Potato leafroll virus
(PLRV) (genus Polerovirus, family Luteoviridae) (Harrison, 1984). PLRV is a persistently
transmitted circulative virus that depends on aphids for its dispersal and transmission to host
plants. In this tritrophic relationship, virus infections can change hosts in such a way that the
interactions between the host and vector are influenced (Álvarez et al., 2007). M. persicae
preferentially settles on potatoes infected by PLRV rather than on uninfected potato plants,
achieving faster development and higher fecundity rates (Castle & Berger, 1993; Castle et al.,
1998). Eigenbrode et al. (2002) found an increased emission of several volatiles by PLRVinfected plants when compared with non-infected plants, which may act as attractants and
arrestants of M. persicae.
To control aphids, larvae of chrysopids are efficient natural enemies because of their prey
searching activity and predatory capacity (Fonseca et al., 2000; Medina et al., 2003b). In the
Neotropical Region, the chrysopid species Chrysoperla externa (Hagen) (Neuroptera:
Chrysopidae) is widely found because of its facility of adaptation to different ecosystems,
being present in many relevant crops in Brazil (Albuquerque et al., 1994; Carvalho & Souza,
2000). The broad distribution of C. externa makes it suitable for use in biological control and
62 C. externa fed on M. persicae vectoring PLRV in a variety of cropping systems (Olazo, 1987), as is the case of potato fields infested with M.
persicae.
The host plant, vector and virus become interdependent components of a complex
pathosystem (Irwin & Thresh., 1990), and the addition of a new trophic level represented by a
natural enemy increases the complexity of their interactions. The virus and aphids can interact
via competition for plant resources and can indirectly induce changes in plant physiology,
such as the activation or suppression of plant defenses, which will have not only direct effects
on the aphids and viruses but also at the highest trophic level, the aphid’s natural enemies
(Christiansen-Weniger et al., 1998; Belliure et al., 2005; Hodge & Powell, 2008b). In this
regard, several studies have demonstrated that chrysopids are attracted by aphid sex
pheromones in olfactometer and field trapping experiments (Boo et al., 1998; Zhu et al.,
2005).
There are also numerous research studies about the effects that natural enemies have on
vectors and associated virus spread (e.g., Smyrnioudis et al., 2001; Belliure et al., 2011;
Garzón et al., 2015), but the reciprocal plant virus-mediated effect on the performance of
natural enemies should be taken into account as well. Some plant viruses may have a negative
impact on beneficial arthropods, as is the case of the parasitoid Aphidius ervi Haliday
(Hymenoptera: Aphidiidae) when parasitizing Sitobion avenae (F.) (Hemiptera: Aphididae)
after having acquired Barley yellow dwarf virus (BYDV) (Christiansen-Weniger et al., 1998).
Nevertheless, there is little information about the possible effect of plant viruses on insect
predators via vector consumption.
Therefore, the objective of this study was to determine the influence of the ingestion of
PLRV-infected M. persicae on C. externa biological parameters.
63 C. externa fed on M. persicae vectoring PLRV 4.2. MATERIALS AND METHODS
The insect rearing and the experiment were conducted in the Laboratory of Ecotoxicology,
Department of Entomology, Federal University of Lavras, Brazil. The environmental
conditions were controlled in a climatic chamber (25 ± 2°C, 70 ± 10% RH, photoperiod 12:12
light:dark).
4.2.1. INSECTS REARING METHODS
The C. externa individuals used in the experiments were obtained from a laboratory
colony, which was provided an artificial diet composed of brewer’s yeast and bee honey (1:1)
and distilled water, and reared in PVC cages (15 cm Ø, 20 cm high) with the upper side
covered by polyethylene film and the lower part supported in a plastic tray. The inside surface
of the cage was draped in filter paper where the eggs were collected (Figure 4.1).
Figure 4.1. C. externa adults rearing cage draped with filter paper to collect the eggs to be used in the
experiments.
64 C. externa fed on M. persicae vectoring PLRV The aphid colony of M. persicae that was used to feed the chrysopid larvae was established
on potato plants (S. tuberosum) and divided into uninfected plants and PLRV-infected plants.
To follow a controlled rearing procedure, potato leaves were placed in Petri dishes (15 cm Ø)
with agar-water solution (1%) to keep the turgidity of the vegetal tissues (Figure 4.2). Leaves
were previously disinfected in sodium hypochlorite solution (1%) for 5 minutes and then
rinsed with water. Approximately 200 apterae aphids (mainly adults) were placed with the
help of a camel brush in each glass dish containing a potato leaf. The Petri dishes were
covered with punctured polyethylene film to allow transpiration and avoid an excess of
moisture. Every 4 days, aphids were transferred to new dishes with leaves from uninfected or
PLRV-infected potato plants. To test the viral infection of M. persicae reared on PLRVinfected leaves, samples of aphids were diagnosed every two weeks following the serological
protocol of the double antibody sandwich enzyme-linked immunosorbent assay (DASELISA) (Clark & Adams, 1977) using specific commercial antibodies against PLRV (Agdia
Inc., Indiana, USA).
Figure 4.2. Uninfected potato leaves (left) and PLRV-infected potato leaves (right) used to rear the M. persicae
individuals used in the experiments.
65 C. externa fed on M. persicae vectoring PLRV 4.2.2. BIOASSAY
The eggs of C. externa (< 24 hours) were isolated from the rearing cage in Petri dishes (5.5
cm Ø, 1 cm high) with filter paper in the base and covered by polyethylene film (Figure 4.3).
C. externa larvae were fed from emergence until pupation with three different diets: Ephestia
kuehniella Zeller (Lepidoptera: Pyralidae) eggs, M. persicae reared on uninfected potato
leaves and M. persicae reared on PLRV-infected potato leaves. Each treatment was formed of
60 replicates in a completely randomized design. The food was replaced daily and both E.
kuehniella eggs and aphids were offered ad libitum to C. externa larvae. The larvae
development and survival were evaluated every day until pupation. The length and survival in
the pupal stage was also determined.
Figure 4.3. Experimental units used to follow the C. externa larval development.
For the assessment of the emerged adults reproductive parameters, 12 couples for each
treatment were isolated in PVC cages (10 cm Ø, 10 cm high) containing an artificial diet
(brewer’s yeast and bee honey 1:1) and distilled water, and draped in filter paper to collect the
eggs laid by each female (fecundity) for 4 weeks (Figure 4.4). The egg viability was
66 C. externa fed on M. persicae vectoring PLRV determined by the isolation in microplates of 8 of the eggs of each couple 3 times per week (a
total of 12 evaluations per couple) and posterior assessment of egg hatching by observation of
the hatched larvae (Figure 4.4).
Figure 4.4. Experimental unit used for a couple (female and male) of C. externa adults to asses the fecundity
(left) and microplate to evaluate the egg hatching (right).
The evaluated parameters were: developmental stage, length and survival of each larval
instar and pupal period, reproduction of emerged adults (sex ratio, proportion of fertile
females, fecundity, egg viability) and survival curve in the first 30 days from adult
emergence.
4.2.3. STATISTICAL ANALYSIS
Data (means ± SE) were analyzed using one-way analysis of variance (ANOVA, p < 0.05)
with the Statgraphics Plus 5.1 statistical software package (Statistical Graphics Corporation,
1994-2000). The nonparametric tests of Kruskal-Wallis and Mann-Whitney (p < 0.05) were
used to establish differences when data violated the premises of the ANOVA (Heath, 1995).
The adults’ survival curves were analyzed with the statistical software package SigmaPlot
11.0 (Systat Software Inc., 2008), using Kaplan-Meier Log-Rank Test and Holm-Sidak
method (p < 0.05) to determine significant differences between curves.
67 C. externa fed on M. persicae vectoring PLRV 4.3. RESULTS
4.3.1. DEVELOPMENTAL TIME AND SURVIVAL OF LARVAL AND PUPAL
STAGES
The C. externa fed with PLRV-infected aphids showed significant differences (KruskalWallis, p < 0.05) in terms of the larval developmental time (H = 48.21; p < 0.001) (Table 4.1)
compared with the C. externa fed the other two diets. The larval developmental time for
PLRV-infected M. persicae diet was the highest (8.59 days).
Table 4.1. Developmental time of larval instars, total larval period and pupal period of C. externa when fed with
E. kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M. persicae, in laboratory conditions
(25 ± 2°C, 70 ± 10% RH, photoperiod 12:12 L:D).
Developmental time (days)
Larval instar
Feeding regime
Larval period
Pupal period
2.75 ± 0.07 c
(52)
7.65 ± 0.08 c
(52)
10.79 ± 0.08 a
(52)
3.15 ± 0.08 a
(54)
3.19 ± 0.08 b
(53)
8.34 ± 0.08 b
(53)
10.40 ± 0.07 b
(52)
3.18 ± 0.10 a
(49)
3.46 ± 0.09 a
(37)
8.59 ± 0.08 a
(37)
10.22 ± 0.07 b
(36)
I
II
III
E. kuehniella eggs
2.26 ± 0.06 a
(58)
2.71 ± 0.08 b
(55)
Non-PLRV-infected
M. persicae
2.02 ± 0.02 b
(57)
PLRV-infected M.
persicae
2.04 ± 0.04 b
(54)
Data (mean ± SE) followed by different letters in the same column indicate significant differences (KruskalWallis, p < 0.05). Mann-Whitney’s Test, (p < 0.05) was used to determine the different letters.
Number of replicates in parentheses
The survival of the C. externa larvae showed significant differences (Kruskal-Wallis, p <
0.05) among the diets (H = 15.76; p < 0.001) (Table 4.2). The lowest value was obtained
when the chrysopid larvae were fed with PLRV-infected M. persicae (67.27%). The critical
instar seemed to be the third one because 20% of the initial number of larvae (12 out of 60)
died in this stage when they had consumed PLRV-infected aphids. In this stage, a significant
(Kruskal-Wallis, p < 0.05) extension in developing time (3.46 days) was also observed when
68 C. externa fed on M. persicae vectoring PLRV compared with the uninfected aphids and artificial diets (3.19 and 2.75 days, respectively) (H
= 13.89; p < 0.001).
Table 4.2. Survival of larval instars, total larval period and pupal period of C. externa when fed with E.
kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M. persicae, in laboratory conditions (25 ±
2°C, 70 ± 10% RH, photoperiod 12:12 L:D).
Survival (%)
Larval instar
Feeding regime
Larval period
Pupal period
98.11 ± 1.89 a
89.66 ± 4.03 a
100.00 ± 0.00 a
94.74 ± 2.98 a
100.00 ± 0.00 a
92.98 ± 3.41 a
98.11 ± 1.89 a
90.74 ± 3.98 a
84.09 ± 5.58 b
67.27 ± 6.39 b
97.30 ± 2.70 a
I
II
III
E. kuehniella eggs
96.67 ± 2.34 a
94.83 ± 2.93 a
Non-PLRVinfected M.
persicae
98.28 ± 1.72 a
PLRV-infected M.
persicae
90.00 ± 3.91 a
Data (mean ± SE) followed by different letters in the same column indicate significant differences (KruskalWallis, p < 0.05). Mann-Whitney’s Test, (p < 0.05) was used to determine the different letters.
The length of the pupal period did not show significant differences between the aphid
diets, but the pupal periods associated with both experimental diets were significantly shorter
(Kruskal-Wallis, p < 0.05) than the pupal period of the control diet (H = 22.93; p < 0.001)
(Table 4.1). The diet composed of PLRV-infected aphids was the one that induced the shorter
time in the pupal stage (10.22 days) for C. externa, which is in accordance with the results of
Oliveira (2014) for C. cubana (13.27 days). Nevertheless, the percentage of survival of the
pupal stage of C. externa did not show significant differences (Kruskal-Wallis, p < 0.05)
among any of the diets, reaching values close to 100% for the E. kuehniella eggs diet, for the
non-infected M. persicae diet (98.11%) and the PLRV-infected M. persicae diet (97.30%) (H
= 1.27; p = 0.531) (Table 4.2).
69 C. externa fed on M. persicae vectoring PLRV Therefore, considering the whole preimaginal period (larva + pupa) of C. externa, the
length of this period tends to be balanced when comparing the PLRV-M. persicae diet with
the control. In the case of the non-infected M. persicae diet, the developmental times for
larval and pupal period were intermediate in regard to the other two diets.
4.3.2. REPRODUCTIVE PARAMETERS AND SURVIVAL OF ADULTS
The diets did not have a significant influence on the reproductive parameters of C. externa
adults (Kruskal-Wallis, p < 0.05 for sex ratio; ANOVA, p < 0.05 for fecundity and egg
viability) (Table 4.3). The female-to-male ratio was lower in the case of the PLRV-infected
aphids diet (0.38) (H = 5.24; p = 0.073). Nonetheless, 100% of the females in this treatment
were fertile, as recorded for the diets of the non-infected aphids and E. kuehniella eggs. The
fecundity was higher for those females that evolved from larvae fed with PLRV-infected aphids
(16.44 eggs/♀ day-1) than for females evolved from larvae fed with non-infected aphids (12.35
eggs/♀ day-1) and E. kuehniella eggs (14.69 eggs/♀ day-1), despite not being significant (F2,33 =
1.93; p = 0.161). Non-significant differences were found as well in the viability of laid eggs
(F2,33 = 2.52; p = 0.096), with an intermediate value in the case of PLRV-infected aphids diet
(70.90%) in regard to the other two diets.
70 C. externa fed on M. persicae vectoring PLRV Table 4.3. Reproductive parameters of C. externa adults when larvae were fed with E. kuehniella eggs, nonPLRV-infected M. persicae and PLRV-infected M. persicae, in laboratory conditions (25 ± 2°C, 70 ± 10% RH,
photoperiod 12:12 L:D).
Sex ratio*a
Proportion of fertile
females**
Fecundity
(eggs/♀ day-1)b
Egg viability
(%)b
E. kuehniella eggs
0.52 a
1.00
14.69 ± 1.49 a
80.04 ± 4.29 a
Non-PLRV-infected M.
persicae
0.63 a
1.00
12.35 ± 4.09 a
60.33 ± 6.68 a
PLRV-infected M.
persicae
0.38 a
1.00
16.44 ± 5.93 a
70.90 ± 7.25 a
Feeding regime
*The sex ratio was obtained with the formula: Σ♀ / Σ(♀ + ♂).
**The proportion of fertile females was obtained by the relationship between the females that oviposited viable
eggs during the assay and the total number of females.
a
Data followed by the same letter did not significantly differ (Kruskal-Wallis, p < 0.05).
b
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p <
0.05).
Concerning the survival of the adults during the 30 first days after emergence, significant
differences (Kaplan-Meier Log-Rank Test, p < 0.05) were found among all curves (p <
0.001), obtaining final values of 0.75, 0.51 and 0.54 for the proportion of live adults for the
diets of E. kuehniella eggs, non-infected M. persicae and PLRV-infected M. persicae,
respectively (Figure 4.5).
71 C. externa fed on M. persicae vectoring PLRV Survival (alive adults proportion)
1,0
E. kuehniella eggs
Non-PLRV-infected M. persicae
PLRV-infected M. persicae
0,9
0,8
0,7
0,6
0,5
0
5
10
15
20
25
30
Time (days)
Figure 4.5. Survival curve of C. externa adults (30 days after emergence) developed from larvae fed with E.
kuehniella eggs, non-PLRV-infected M. persicae and PLRV-infected M. persicae, in laboratory conditions (25 ±
2°C, 70 ± 10% RH, photoperiod 12:12 L:D). Curves significantly differed (Kaplan-Meier Log-Rank, HolmSidak, p < 0.05).
4.4. DISCUSSION
The results obtained in this work can be directly compared with those of Oliveira (2014),
who studied the same biological parameters in the neuropteran predator Ceraeochrysa cubana
(Hagen) when fed with PLRV-infected M. persicae in the same laboratory conditions. The
shorter developmental time for both predators was observed with the E. kuehniella eggs diet
(7.65 days for C. externa and 12.04 days for C. cubana), as could be expected because E.
kuehniella eggs are known to be a food that ensures rapid growth and development, high
fecundity and survival (Tauber et al., 2000). The differences observed in the present work
between E. kuehniella eggs and M. persicae may indicate that eggs are more nutritious and/or
72 C. externa fed on M. persicae vectoring PLRV that chrysopid larvae expended more energy in handling the aphids. This is in accordance
with the results obtained by López-Arroyo et al., (1999) and Pappas et al., (2007), who also
found a faster preimaginal development of different species of the genus Ceraeochrysa and
Dichochrysa (Neuroptera: Chrysopidae) when fed on E. kuehniella eggs compared with M.
persicae aphids.
It is possible that PLRV-induced modifications in prey physiology may reduce the prey’s
suitability as a nutrient source for C. externa larvae. Calvo & Fereres (2011) studied the
multitrophic interactions between lettuce, the Turnip yellows virus (TuYV), its aphid vector
Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae) and the aphid parasitoid A. ervi,
determining that parasitoid performance is highly dependent on the timing when parasitism
occurs with respect to the exposure of aphids to virus-infected leaves. This could explain why
significant differences in larvae survival rates did not appear until the third instar. The longer
time the larvae spent feeding on PLRV-infected aphids, together with the fact that L3 C.
externa larvae food ingestion can reach 65% in weight of the food they consume during the
larval period (Tavares et al., 2011), might have had considerable influence on their survival
rates. Christiansen-Weniger et al., (1998) also observed that the development of A. ervi larvae
in the cereal aphid S. avenae could be inhibited by the presence of BYDV. In contrast, Gupta
et al., (2013) did not find significant variations in the developmental time and survival rate of
the predator Eocanthecona furcellata Wolff (Hemiptera: Pentatomidae) when reared on
different proportions of nucleopolyhedrovirus (NPV) infected Spodoptera litura Fabricius
(Lepidoptera: Noctuidae), despite NPV being an entomopathogenic virus; this may be due to
the gut conditions of many predators, which do not degrade the proteinaceous matrix of the
virus, thus the virions that would trigger the infection are not released (Granados & Lawler,
1981).
73 C. externa fed on M. persicae vectoring PLRV Numerous insects accumulate nutrients during immature stages that are used in the
imaginal stage for reproduction (Johansson, 1964; Wigglesworth, 1972), but in this case, the
differences in the nutritive properties of the three diets did not seem to have a clear effect on
the adult period, at least in terms of reproduction. In this regard, the results obtained are in
accordance with those of Oliveira (2014) for the neuropteran C. cubana in terms of sex ratio,
fecundity and eggs viability; this author used the same diets that were used in the present
study and did not find significant differences among them. Nevertheless, other authors found
significant differences in terms of fecundity for different species of chrysopids, such as
Dichochrysa prasina Burmeister and Chrysoperla sinica (Tjeder), when fed with different
aphid diets, observing that those which presented higher preimaginal development and
survival showed better reproductive performance of adults (Pappas et al., 2007; Khuhro et al.,
2012).
In contrast, the consumption of PLRV-infected aphids by C. externa larvae affected the
survival of the adults that developed from them. This agrees with the findings of other authors
who found an evident influence of the quality of the diet consumed by chrysopid larvae on the
longevity of adults (Wang & Nordlund, 1994; Pappas et al., 2007; Huang & Enkegaard,
2009). On the contrary, Armenta et al., (2003) studied the effects of Spodoptera frugiperda (J.
E. Smith) (Lepidoptera: Noctuidae) multinucleocapsid polyhedrovirus (SfMNPV) on
Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) and did not find any negative
influence on its survival.
To summarize, the diet composed of PLRV-infected M. persicae significantly increased
the length and reduced the survival rate, of the larval period in regard to the diets of noninfected M. persicae and E. kuehniella eggs. The lengths of the pupal stage were not
significantly different between the aphid diets; for both of these diets, the length of the pupal
stage was significantly lower than that for the control diet. Neither pupal survival nor
74 C. externa fed on M. persicae vectoring PLRV reproductive parameters revealed significant differences among the diets. Nevertheless, the
adult survival curves during the first 30 days after emergence showed significant differences.
In conclusion, the ingestion by C. externa of PLRV-infected M. persicae seemed to have an
effect during the larval period and on the survival of the adults but not on the pupal stage and
adult reproductive parameters. Further laboratory and field research are required to gain a
better understanding of whether C. externa could act as an effective control of M. persicae in
potato crops, even in circumstances of PLRV infection.
75 76 Influence of UV on the performance of A. gossypii, C. carnea and cucumber CHAPTER 5. DIRECT AND PLANT-MEDIATED INFLUENCE OF LOW
UV RADIATION ON Aphis gossypii AND THE PREDATOR Chrysoperla
carnea IN ABSENCE AND PRESENCE OF Cucumber aphid-borne yellows
virus
ABSTRACT
The direct and plant-mediated influence of low UV radiation conditions were studied on
the aphid Aphis gossypii and on the chrysopid Chrysoperla carnea, as well as on the
cucumber plants themselves, both in absence and presence of Cucurbit aphid-borne yellows
virus (CABYV). The experiments were conducted under semi-field conditions, growing the
cucumber plants inside cages covered with two different types of plastic film, the UVabsorbent Térmico Antivirus® and the control Térmico Blanco®. A 300 W Ultra-Vitalux®
lamp was used as UV-radiation supplement. Four treatments were established both in absence
and presence of CABYV: plants grown under Térmico Blanco® (control), plants grown under
Térmico Antivirus® (abs-UV), plants grown under Térmico Blanco® until BBCH stage 14 and
then transferred under Térmico Antivirus® (control>abs-UV), and plants grown under
Térmico Antivirus® until BBCH stage 14 and then transferred under Térmico Blanco® (absUV>control). The aphid infestation was done at BBCH stage 14 and two weeks after the L1
chrysopid larvae were released.
In the experiment with non-infected cucumbers only the control>abs-UV plants showed a
significant increase in stem length, cell wall thickness and cuticle thickness in regard to the
rest of treatments. The plants of this treatment together with those of the abs-UV one,
presented a significantly higher content in N. In the experiment with CABYV-infected
77 Influence of UV on the performance of A. gossypii, C. carnea and cucumber cucumbers only a significant increase in the S content was recorded for control and abs-UV
treatments. No significant effects amongst the different UV conditions were observed in any
of the experiments neither in A. gossypii performance nor in C. carnea adults reproductive
parameters.
5.1. INTRODUCTION
In protected horticulture, Integrated Pest Management (IPM) is one of the most consistent
strategies to control insect pests (Kogan, 1998). Despite that fact, crop losses still occur, so it
continues to be important to look for supplementary pest control measures. In this regard, the
technology of ultraviolet (UV) blocking films shows considerable potential, because they
interfere with insects visual responses and behaviour, affecting their ability to orient in the
crop (Antignus et al., 1996; Doukas, 2002; Diaz & Fereres, 2007). Photoselective plastics are
a quite recent development that can block or modify the transmitted light to obtain specific
benefits (Catalina et al., 2000).
UV radiation spans the wavelength region from 400 to 100 nm. Within the UV portion is
possible to distinguish three types of wavelengths: UV-C (100-280 nm), UV-B (280-315 nm)
and UV-A (315-400 nm) (Diffey, 2002), although the radiation relevant for plants is
composed of UV-B, UV-A, photosinthetically active radiation (PAR, 400-700 nm) and nearinfrared radiation (700 nm - 1mm) (Kuhlmann & Müller, 2011). UV-C is extremely harmful
to organisms, but not relevant under natural conditions of solar radiation; UV-B is
characterized by inducing a variety of damaging effects in plants despite representing the
1.5% of the total spectrum; UV-A is the less hazardous part of UV radiation (Hollósy, 2002).
The effects of a high UV radiation on plants have been studied in order to understand the
consequences that the reduction of the ozone layer would have on nature (Caldwell et al.,
1989; Newsham et al., 1996; Björn et al., 1997; Jayakumar et al., 2003). In contrast to an
78 Influence of UV on the performance of A. gossypii, C. carnea and cucumber artificial increase of UV, another approach is to filter specific wavelengths of the ambient
radiation to diminish selectively UV-B or UV-B and UV-A (Rousseaux et al., 1998; Mazza et
al., 1999; Zavala et al., 2001; Jayakumar et al., 2004; Paul et al., 2005; Kuhlmann & Müller,
2009b). The stress caused in plants by UV-A and UV-B radiation, both for excess or deficit,
results in unusual growth patterns and coloration, leaf ultrastructure and anatomy,
photosynthetic pigments, protective mechanisms (e.g. cuticular waxes, secondary metabolites)
phenology and biomass yield (Smith et al., 2000; Kakani et al., 2003; González et al., 2009;
Dáder et al., 2014).
The majority of studies dealing with the ecological impacts of different UV radiation
conditions have focused on the hazardous effects of the UV-B fraction, while the influence of
UV-A has been less investigated. Recently, Dáder et al., (2014) have shed light on the effects
of UV-A on several crops and their pests, finding a modulation in plants chemistry and
anatomy, and therefore modifying the performance of insects that feed on them. In regard to
UV-B the literature is profuse, demonstrating that changes in its levels can affect host plants
and organisms at other trophic levels, altering the relationships between primary producers
and consumers (Rousseaux et al., 1998). The host-mediated effects of UV-B on phytophagous
arthropods are induced by changes in plant morphology, physiology and biochemistry. Some
of the morphological changes best documented are related to leaf thickness and epicuticular
waxes (Bornman & Vogelmann, 1991; Ballaré et al., 1996; Kakani et al., 2003). In regard to
biochemical changes, UV-B is known to affect the levels of flavonoids, leaf nitrogen,
hemicelluloses and lignin (Caldwell et al., 1983; Hatcher & Paul, 1994; Gehrke et al., 1995).
All these factors potentially affect leaf attractiveness and acceptability to phytophagous
insects (Coley, 1983), and therefore their fitness and biological parameters.
It must be considered that UV radiation influence has a dual component, affecting insects
directly or indirectly, through food quality changes (Kuhlmann & Müller, 2011). Insects
79 Influence of UV on the performance of A. gossypii, C. carnea and cucumber compound eyes are sensitive to wavelengths in the range from UV light to red (Prokopy &
Owens, 1983; Qiu & Arikawa, 2003), so radiation changes in this interval directly affect their
visual cues to orient, navigate, avoid predators and locate host plants, prey and mates
(Prokopy & Owens, 1983; Warrant & Nilsson, 2006). In fact, Antignus et al. (1996)
suggested that the ability of UV-absorbing plastic sheets to protect crops from Bemisia tabaci
(Gennadius) (Homoptera: Aleyrodidae), Aphis gossypii Glover (Hemiptera: Aphididae) and
Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is due to the presence of UV
photoreceptors in their compound eyes.
This UV direct influence might not only affect herbivorous insects, but also their natural
enemies performance (Chyzik et al., 2003; Doukas, 2003; Chiel et al., 2006; Legarrea et al.;
2012), which is susceptible as well to prey-mediated changes induced by host biochemical
modification due to UV radiation conditions. In a trophic system, a variation in the food
consumed by the prey, which results in differences in the chemical composition of the
herbivore, can affect its susceptibility to predator attack and the performance of the predator
itself (Price et al., 1980). Amongst aphid predators, larvae of chrysopids are efficient natural
enemies because of their prey searching activity and predatory capacity (Fonseca et al., 2000;
Medina et al., 2003b). Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) is the most
widely distributed chrysopid in the Iberian Peninsula (Díaz-Aranda & Monserrat, 1990); its
larvae are voracious aphid predators in many agricultural systems (Viñuela et al., 1996;
Medina et al., 2003b). There are numerous studies reporting the prey quality influence on the
pre-imaginal development, survival, fecundity and longevity of chrysopid adults (Muma,
1957; Principi & Canard, 1984; Obrycki et al., 1989; Zheng et al., 1993).Thus, it becomes
relevant to analyse the biological parameters of the predator C. carnea under the plant-prey
mediated influence of special UV radiation conditions.
80 Influence of UV on the performance of A. gossypii, C. carnea and cucumber In addition to the study of the influence of UV radiation on plants, pests and natural
enemies, the scenario appears even more intricate with the possibility of a viral disease
infecting the crop. In this case it has been studied the Cucurbit aphid-borne yellows virus
(CABYV) (genus Polerovirus, family Luteoviridae), which has been shown to be the
prevalent virus in cucurbit crops in southeastern Spain (Juárez et al., 2004; Juárez et al.,
2013), being efficiently transmitted by Aphis gossypii Glover (Hemiptera: Aphididae) (Lecoq
et al., 1992). The changes that CABYV induces in the host add complexity to the plantmediated effects on the performance of A. gossypii.
Taking into consideration all the mentioned above, the objective of these experiments was
to evaluate the direct and plant-mediated influence of low UV radiation conditions on the
fitness of A. gossypii and on the biological parameters of the predator C. carnea, both with
healthy cucumber plants and with CABYV-infected cucumber plants. The direct influence of
low UV radiation on cucumber plants physiology was also analysed.
5.2. MATERIALS AND METHODS
The evaluation of the plant-mediated low UV radiation influence on A. gossypii, C. carnea
and the cucumber itself was composed of two different experiments:
a) With healthy cucumber plants (autumn 2013).
b) With CABYV-infected cucumber plants (spring 2014).
The methodology developed for both experiments was identical in terms of evaluation,
with the difference that the cucumber plants of the second experiment had to be inoculated
according to the process explained in section 5.2.4.
81 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.2.1. INSECTS
A single virginiparous apterae of A. gossypii was collected in a melon crop in Almería
(Spain) and used to initiate a clonal population. The aphid colony was kept at low densities on
Cucumis melo L. plants cv. ‘Primal’ in a climatic chamber (20ºC, 60-80% RH, 16:8 L:D
photoperiod) in the Crop Protection Laboratory of ETSIA-UPM (Madrid, Spain). For the
trials, adult apterae individuals were collected with the help of a camel brush just before being
transferred to the experimental cages. This aphid is a suitable prey species for C. carnea, the
natural enemy used in the tests (van Steenis, 1992).
The natural enemy C. carnea was established in a laboratory colony from L1 larvae
obtained from Agrobío (CHRYSOcontrol®, Almería, Spain), following the rearing procedure
of Medina et al. (2001).
Larvae were fed Ephestia kuehniella (Zeller) (Lepidoptera:
Pyralidae) eggs ad libitum, and adults were provided with a very nutritious artificial diet
according to Vogt et al. (2000). For the trials, gauze with <24-h-old eggs from the adult
rearing cages was used; these eggs were allowed to hatch and the L1 instar larvae with 2 days
of life (to be better handled than newly emerged ones) were collected for the experiments.
5.2.2. PLANT MATERIAL
Cucumber (Cucumis sativus L. cv. ‘Ashley’) seeds were sown and germinated in 150
alveoli trays (56 cm x 36 cm x 6 cm) filled with a 1:1 mixture of soil substrate (Projar,
Valencia, Spain) and vermiculite Termita® (Aminco, Donostia, Spain), inside an insect-proof
chamber at 23-25ºC, 60-80% relative humidity (RH) and a 16:8 L:D photoperiod. After
germination, the seedlings were transferred to the definitive pots (2000 cm3 capacity) and
placed inside the experimental cages (0.3 m x 0.3 m x 0.6 m) covered with the two plastic
films (UV-absorbent and control) tested in the trials in greenhouse conditions (15-27ºC and
82 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 50-85% RH). Cucumber plants were grown approximately for a month until they reached the
four true leaves stage (BBCH scale = 14) and were infested with the aphids.
5.2.3. UV RADIATION SOURCE AND UV-ABSORBING PLASTIC COVERS
The UV radiation source used for the experiments was an Ultra-Vitalux® 300W E27 lamp
(Osram, Munich, Germany). The radiated power of this lamp in the UVA spectrum (315-400
nm) is 13.6 W and in the UVB spectrum (280-315 nm) is 3.0 W. Ten of these lamps were
installed inside the greenhouse, so that two cages (one of each plastic cover) received the
radiation from one lamp. The distance between the light bulb and the upper part of the cages
was 50 cm. Lamps were switched on for a period of 14 hours every day during the whole
length of the experiments.
The experimental cages where cucumber plants were introduced were covered in their
lateral upper 30 cm and top part with two different plastic films, depending on the treatment
(Figure 5.1). The plastic film used as UV-absorbent barrier was Térmico Antivirus® (Solplast
S.A., Murcia, Spain), with a thickness of 200 μm and 85% of PAR transmission. The plastic
film used as control was Térmico Blanco® (Solplast S.A., Murcia, Spain), with a thickness of
200 μm and 85% of PAR transmission. Despite having the same properties in regard to PAR
radiation, the UV-transmission characteristics of both plastic films were different (Table 5.1).
The UV daily doses were 158.16 kJ m-2 day-1 UV-A and 0.71 kJ m-2 day-1 UV-B for control
treatment, and 8.62 kJ m-2 day-1 UV-A and 0.10 kJ m-2 day-1 UV-B for abs-UV treatment. The
measures were done every sampling day with a radiometer ALMEMO 25904S (Ahlborn
GmbH, Holzkirchen, Germany).
83 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.1. Radiation conditions under the UV-source lamp (50 cm from the light bulb) and inside the
experimental cages (below the plastic cover). Transmission percentages represent the radiation transmitted inside
both cages with regard to the same level outside the cages.
control treatment
(Térmico Blanco®)
abs-UV treatment
(Térmico Antivirus®)
UV-Aa
UV-Ba
UV-A
UV-B
Top of the cage
(50 cm from the UV-source)
17.192
0.456
17.192
0.456
Inside the cage
(below the plastic cover)
3.138
0.014
0.171
0.002
Transmission inside the cage (%)
18.25
3.07
0.99
0.44
a
Radiation measures in W m-2
To guarantee an optimal ventilation inside the cages, their lower lateral 30 cm were
covered with nets with similar UV properties. For the UV-absorbing treatment, it was used POptinet 50 (50 mesh) (Polysack Plastic Industries Ltd., Nir Yitzhak, Israel), that filters the
72% of radiation in the UV spectrum of 280-400 nm (Legarrea et al., 2010). For the control
treatment, it was used the standard Criado y López net (50 mesh) (El Ejido, Spain), which has
no special UV-absorbing properties.
84 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Figure 5.1. Experimental cages covered by two different plastic films: control treatment (Térmico Blanco®) (left)
and abs-UV treatment (Térmico Antivirus®) (right).
5.2.4.
VIRUS
ACQUISITION
IN
THE
CABYV-INFECTED
CUCUMBER
EXPERIMENT
In the experiment for assessing the plant-mediated influence of low UV radiation with
virus-infected cucumbers, the plants were inoculated with CABYV (Figure 5.2). The CABYV
isolate was kindly provided by Dr. H. Lecoq, obtained in Montfavet (France) from zucchini
squash in 2003, and maintained in cucumber plants by aphid serial transmission. Cucumber
plants were inoculated by means of CABYV vectoring aphids, following the protocol
explained in Moreno et al. (2011) and Dáder et al. (2012). After the inoculation process,
plants were sprayed with Biopirot® (Aragonesas Agro, natural pyrethrin 1.5% + rotenone 3%,
dilution rate 2 ml l-1) to kill the aphids.
85 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Figure 5.2. Cucumber plant used as CABYV source (left) for the inoculation of the experimental plants by
transferring viruliferous A. gossypii individuals (right).
Plants to be used as CABYV-infected cucumber plants were maintained inside cages (1 m
x 0.6 m x 0.6 m) covered with the two plastic films tested in the experiments (Térmico
Blanco® and Térmico Antivirus®) under UV radiation, in the same conditions as the ones of
the experiment with healthy cucumbers. CABYV-inoculated cucumber plants were grown
approximately for a month until they reached the four true leaves stage (BBCH scale = 14),
when they were tested for virus infection by double antibody sandwich enzyme-linked
immunosorbent assay (DAS-ELISA) (Clark & Adams, 1977) using specific commercial
antibodies against CABYV (Sediag, France). Those plants that became CABYV-positive in
the DAS-ELISA test were used for the experiment and were immediately infested with
aphids.
5.2.5. EXPERIMENTS
5.2.5.1. EXPERIMENTAL DESIGN
Each experiment (virus free and CABYV-infected cucumbers) consisted of four different
treatments to test both the plant-mediated effect and the direct effect of the low UV radiation
on A. gossypii and C. carnea. Therefore, when cucumber plants reached the four true leaves
stage (BBCH scale = 14) and before being infested with aphids, half of the plants that had
86 Influence of UV on the performance of A. gossypii, C. carnea and cucumber been growing in cages covered with Térmico Blanco® plastic film were transferred to cages
covered with Térmico Antivirus® plastic film, and vice versa. Thus, the design of each
experiment was as follows:
a) 5 cucumber plants grown under Térmico Blanco® plastic film along all the trial (named
in Results as control treatment).
b) 5 cucumber plants grown under Térmico Antivirus® plastic film along all the trial
(named in Results as abs-UV treatment).
c) 5 cucumber plants grown under Térmico Blanco® plastic film until aphid infestation and
transferred to Térmico Antivirus® plastic film covered cages until the end of the trial (named
in Results as control > abs-UV treatment).
d) 5 cucumber plants grown under Térmico Antivirus® plastic film until aphid infestation
and transferred to Térmico Blanco® plastic film covered cages until the end of the trial
(named in Results as abs-UV > control treatment).
5.2.5.2. A. gossypii INFESTATION AND C. carnea RELEASING
Once defined the four treatments, cucumber plants (BBCH scale = 14) were infested with
adult apterae A. gossypii. A total of 20 A. gossypii individuals were placed on the leaves of
each plant (5 aphids/leaf) (Figure 5.3). From that moment on, 3 evaluations per week along 2
weeks were done to assess the evolution of the aphid population in every plant, counting the
number of apterae individuals and the number of alate individuals as well. After these two
weeks that the aphids were left to develop on cucumber plants, 5 two-days-old L1 C. carnea
larvae were released in each plant (Figure 5.3). The frequency of the evaluations was
maintained in 3 per week, counting the number of A. gossypii apterae and alate individuals in
every replicate and monitoring the C. carnea larvae released (number of recaptured
87 Influence of UV on the performance of A. gossypii, C. carnea and cucumber individuals and developmental stage). The evaluations were done until C. carnea larvae
pupated. Afterwards, the pupae were collected and joined in treatments to follow their
evolution in the laboratory (25 ± 2 ºC, 75 ± 10% RH, 16:8 L:D photoperiod). The adult
emergence was recorded and as many couples (1 ♀ and 1 ♂) as possible were made for each
treatment to assess the reproductive parameters (fecundity and viability of eggs) according to
Vogt et al. (2000). Every adult couple was maintained in a plastic oviposition cage (12 cm in
diameter × 5 cm in height) and provided food and water; a piece of cotton gauze was placed
in the upper part of the oviposition cage, where the eggs of C. carnea were collected to
measure fecundity. The reproduction test started 5 to 7 days after the first egg was laid. Along
one week, two samples of < 24-h eggs were collected from each replicate and kept in separate
plastic cages (9 cm in diameter × 2 cm in height) for incubation until hatching, and the egg
viability was determined (Giolo et al., 2009).
Figure 5.3. A. gossypii infestation of cucumber plants (BBCH scale = 14) (left) and L1 C. carnea larvae ready to
be released two weeks after (right).
In Figure 5.4 it is represented a chronogram of the experiments, from the germination of
the cucumber plants until their harvesting.
88 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Figure 5.4. Chronogram of the experiments, with the four different UV radiation treatments (control, plants
grown under Térmico Blanco® plastic film along all the trial; control > abs-UV, plants grown under Térmico
Blanco® plastic film until aphid infestation and transferred under Térmico Antivirus® plastic film until the end of
the trial; abs-UV > control, plants grown under Térmico Antivirus® plastic film until aphid infestation and
transferred under Térmico Blanco® plastic film until the end of the trial; abs-UV, plants grown under Térmico
Antivirus® plastic film along all the trial), dates of A. gossypii infestation and C. carnea releasing and collecting,
and cucumber plants harvesting.
5.2.5.3. PHYSIOLOGICAL MEASURES ON CUCUMBER PLANTS
5.2.5.3.1. FRESH WEIGHT, DRY WEIGHT AND STEM LENGTH
Once C. carnea pupae were removed from the greenhouse experimental cages and taken to
laboratory to follow their evolution, cucumber plants were cut from the stem basis to analyse
several physiological parameters. The stem length from the basis to the apex was measured to
determine a possible influence of the low UV radiation on plant growth. Besides, the fresh
89 Influence of UV on the performance of A. gossypii, C. carnea and cucumber weight was recorded, and after leaving the cucumber plants for 72 hours inside a drying oven
at 60 ºC, the dry weight was determined as well.
5.2.5.3.2. CELL WALL AND CUTICLE ANALYSIS OF LEAF SAMPLES
Before drying the cucumber plants, samples of foliar tissue (0.2 x 1 cm) were cut from
every plant to be studied by means of a transmission electron microscope model JEOL JEM
1010® (JEOL Ltd., Tokyo, Japan) in the CNME (Centro Nacional de Microscopía
Electrónica). The technique used to prepare the samples was a conventional resin
ultramicrotomy. In this technique the sample is fixed, stained, dehydrated, embedded in resin,
sectioned and then stained once more. Fixation is a process of cross-linking the biological
molecules together so that in the subsequent steps these molecules and the structure are
maintained. This process also reduces the amount of damage the beam can cause to the
sample. The fixatives used were formaldehyde, glutaraldehyde and osmium tetroxide (Figure
5.5). Formaldehyde has a small molecule that can diffuse rapidly through the tissue and fix
the core, but it is not a strong fixative. A better fixation is achieved by using glutaraldehyde,
which permanently cross-links tissues, but the drawback is that glutaraldehyde does not
penetrate tissue as well as formaldehyde. A third fixative is osmium tetroxide (OsO4), which
is a strong fixative and also a stain. Typically, to get the best ultrastructural preservation,
tissue blocks are fixed by a combination of formaldehyde and glutaraldehyde followed by an
OsO4 treatment (Dykstra, 2003).
90 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Figure 5.5. Fixation process of the vegetal samples (left) and ethanol graded series for their dehydration (right).
After this step the tissue is dehydrated in a graded series of ethanol in increasing
concentrations (40%, 50%, 60%, 70%, 80%, 90% and 100%) (Figure 5.5) and transitioned
into a solvent (ethanol) that can dissolve the resin. The resin is then transitioned into the tissue
and oriented into a silicone mold and cured (70 ºC for 3 days) (Figure 5.6). The hardened
tissue block is then trimmed into a trapezoid and sectioned using an ultramicrotome. This
microtome is used in conjunction with a diamond knife to cut ultrathin sections from the
block. The sections float on top water contained within a trough behind the knife. These
sections are then picked up on a copper grid, which may have a polymeric or carbon supportfilm to prevent sagging and tearing of sections. Once dried, grids are stained with uranyl
acetate and lead citrate to enhance contrast.
91 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Figure 5.6. Silicon mold with the vegetal samples embedded in the hardened resin.
The transmission electron microscope JEOL JEM 1010® was used to determine the cell
wall thickness (20.000 x magnification) and the cuticle thickness (200.000 x magnification)
(Figure 5.7). The images were analysed with the help of Soft Imaging Viewer® software (Soft
Imaging System Corp., Lakewood, US).
Figure 5.7. Transmission electron microscope images of the cell wall (left) and the cuticle (right) of adaxial
epidermal cells of cucumber leaves.
92 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.2.5.3.3. CARBON, HYDROGEN, NITROGEN AND SULFUR ANALYSIS OF LEAF
SAMPLES
From every dried plant, a sample of 0.3 g was taken to analyse the percentages of carbon
(C), hydrogen (H), nitrogen (N) and sulfur (S). Every sample was micronized by submersing
the Eppendorf® tubes (Hamburg, Germany) in liquid nitrogen and grinding them with the help
of a glass stick. After that process, samples were sent to the CME-UCM (Centro de
Microanálisis Elemental, Universidad Complutense de Madrid) and analysed by means of a
LECO CHNS-932 Elemental Analyser® (Michigan, US). This technique involves combustion
of test sample in an oxygen rich environment. The products of combustion in a CHNS
analysis (CO2, H2O, N2 and SO2) are carried through the system by helium (He) carrier gas.
The combustion products are measured quantitatively by means of a non-dispersive infrared
(IR) absorption detection system, except for the N2 which is determined via a thermal
conductivity detector (TC).
5.2.6. STATISTICAL ANALYSIS
Data, presented as the means ± SE, were analyzed using one-way analysis of variance
(ANOVA) with the Statgraphics Plus 5.1 statistical software package (Statistical Graphics
Corporation, 1994-2000). The means were separated using the Fisher least significant
difference (LSD) multiple range test (p < 0.05). The nonparametric test of Kruskal-Wallis was
used when data violated the premises of the ANOVA (Heath, 1995).
93 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.3. RESULTS
5.3.1. HEALTHY CUCUMBER PLANTS EXPERIMENT
5.3.1.1. PLANT-MEDIATED EFFECTS OF LOW UV RADIATION ON THE
FITNESS OF A. gossypii
Regarding the evolution of A. gossypii population, no significant differences (ANOVA, p <
0.05) were found amongst treatments in any of the samplings (Table 5.2). The apterae aphid
population experimented a sustained increase until the 6th sampling, with a pronounced
growth from the 6th to the 7th sampling (Figure 5.8). It was recorded a decrease in aphid
population (except for control treatment) after C. carnea larvae releasing (8th sampling)
(Figure 5.8). In the 11th sampling (the last one), it was observed a reduction of aphid
population in every treatment. This overall A. gossypii density reduction at the end of the trial
(from the 9th sampling) was mainly due to a dropping off behaviour of individuals in search of
another food source, in response to the damaged status of cucumber plants.
A similar evolution was observed for A. gossypii alatae individuals. Once again, no
significant differences (ANOVA, p < 0.05) were observed amongst the different treatments in
any of the samplings (Table 5.3). The highest increase of alatae aphid population in all
treatments was observed from the 8th to the 9th sampling (Figure 5.9), coincident with the
increase of apterae population and the presence of C. carnea larvae inside the cages. From the
9th sampling, the alatae aphid population decreased until the 11th sampling in all treatments
(Figure 5.9).
94 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.2. Evolution of A. gossypii apterae population on healthy cucumber plants under different UV radiation conditions. The releasing of 20 adult apterae individuals/plant
was done on 14/10/2013. The temperature and relative humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
1st sampling
(16/10/2013)
2nd sampling
(18/10/2013)
3rd sampling
(21/10/2013)
4th sampling
(23/10/2013)
5th sampling
(25/10/2013)
6th sampling
(28/10/2013)
7th sampling
(30/10/2013)
8th sampling
(01/11/2013)
9th sampling
(04/11/2013)
10th sampling
(06/11/2013)
11th sampling
(08/11/2013)
control
150.2±16.9a
225.8±32.7a
359.4±45.5a
874.8±123.9a
1434.7±137.3a
1994.6±152.6a
3707.6±231.5a
3946.8±450.8a
4172.0±277.6a
3026.0±342.0a
1862.5±433.4a
abs-UV
155.4±23.8a
246.2±37.0a
372.0±59.5a
893.4±176.2a
1328.2±179.0a
1819.0±160.0a
3526.8±266.7a
3300.4±263.3a
3825.2±301.2a
3628.6±471.4a
2905.4±574.2a
control>abs-UV
138.0±24.1a
252.8±34.1a
356.8±38.3a
823.4±79.5a
1323.4±144.2a
1823.4±226.3a
3926.4±481.9a
3592.6±193.2a
4835.4±544.4a
4570.8±559.4a
4408.0±1216.7a
abs-UV>control
141.6±12.2a
216.6±24.9a
309.6±45.3a
690.2±58.0a
1215.4±128.6a
1740.6±222.5a
3962.8±301.8a
3189.2±172.6a
4198.4±639.2a
4263.2±286.9a
3907.8±451.0a
F3,16 = 0.16
F3,16 = 0.27
F3,16 = 0.33
F3,16 = 0.60
F3,16 = 0.36
F3,16 = 0.31
F3,16 = 0.37
F3,16 = 1.35
F3,16 = 0.81
F3,16 = 2.57
F3,14 = 2.40
p = 0.922
p = 0.844
p = 0.805
p = 0.624
p = 0.781
p = 0.820
p = 0.776
p = 0.293
p = 0.506
p = 0.090
p = 0.111
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p < 0.05).
Table 5.3. Evolution of A. gossypii alatae population on healthy cucumber plants under different UV radiation conditions. The releasing of 20 adult apterae individuals/plant
was done on 14/10/2013. The temperature and relative humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
1st sampling
(16/10/2013)
2nd sampling
(18/10/2013)
3rd sampling
(21/10/2013)
4th sampling
(23/10/2013)
5th sampling
(25/10/2013)
6th sampling
(28/10/2013)
7th sampling
(30/10/2013)
8th sampling
(01/11/2013)
9th sampling
(04/11/2013)
10th sampling
(06/11/2013)
11th sampling
(08/11/2013)
control
0.2±0.2a
0.2±0.2a
1.0±0.3a
6.0±4.1a
8.8±6.3a
11.4±6.5a
21.8±14.3a
36.6±25.1a
71.2±23.2a
47.0±13.0a
18.25±5.6a
abs-UV
0.2±0.2a
0.2±0.2a
0.4±0.2a
4.6±2.4a
6.6±3.2a
10.2±5.2a
18.8±5.4a
28.6±9.0a
80.2±16.8a
66.2±14.7a
38.6±10.4a
control>abs-UV
0.4±0.2a
0.4±0.2a
1.2±0.4a
4.0±1.9a
7.0±3.6a
11.2±5.6a
30.8±12.5a
60.2±34.4a
129.6±31.6a
96.0±22.6a
59.0±11.3a
abs-UV>control
0.0±0.0a
0.0±0.0a
0.2±0.2a
2.2±1.1a
4.0±1.2a
9.0±3.8a
17.8±4.7a
36.8±10.4a
115.6±28.6a
83.8±23.9a
54.4±13.7a
F3,16 = 0.76
F3,16 = 0.76
F3,16 = 2.67
F3,16 = 0.37
F3,16 = 0.24
F3,16 = 0.04
F3,16 = 0.34
F3,16 = 0.30
F3,16 = 1.18
F3,16 = 1.24
F3,14 = 2.51
p = 0.532
p = 0.532
p = 0.083
p = 0.779
p = 0.867
p = 0.988
p = 0.797
p = 0.826
p = 0.348
p = 0.328
p = 0.101
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p < 0.05).
95 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 6000
control
number of individuals
5000
abs-UV
4000
control>abs-UV
3000
abs-UV>control
2000
1000
0
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
Figure 5.8. Evolution of A. gossypii apterae population during the experiment on healthy cucumber
plants. The releasing of C. carnea larvae was done in S7 (7th sampling).
140
number of individuals
120
control
100
abs-UV
80
control>abs-UV
60
abs-UV>control
40
20
0
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
Figure 5.9. Evolution of A. gossypii alatae population during the experiment on healthy cucumber plants.
The releasing of C. carnea larvae was done in S7 (7th sampling).
96 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.3.1.2. PLANT-PREY-MEDIATED EFFECTS OF LOW UV RADIATION ON C.
carnea
The number of recaptured C. carnea pupae from the experimental cages was never the
initial number released (5 L1 larvae/plant; 25 L1 larvae/treatment). From the control treatment
17 pupae were collected, 15 pupae from the abs-UV treatment, 17 pupae from the
control>abs-UV treatment and 15 pupae from the abs-UV>control treatment. Once the pupae
were collected, they were taken to the laboratory and joined in treatments to record the adult
emergence. The emergence values were 100% for control treatment, 93.3% for abs-UV
treatment, 94.1% for control>abs-UV treatment and 80% for abs-UV>control treatment.
With the emerged adults, as many couples (1 ♀ and 1 ♂) as possible were made to assess
the fecundity (control, 8; abs-UV, 7; control>abs-UV, 7; abs-UV>control, 7) and the fertility
(control, 7; abs-UV, 5; control>abs-UV, 4; abs-UV>control, 6). No significant differences
were recorded amongst treatments (ANOVA, p < 0.05) in terms of fecundity and fertility. The
highest fecundity value was observed in control treatment (289.38 ± 44.03 eggs/♀ week-1),
while for fertility all values were similar (between 73% and 77%) (Table 5.4).
Table 5.4. Reproductive parameters of C. carnea adults developed from the larvae released inside the
experimental cages under different UV radiation conditions with healthy cucumber plants. Fecundity (±SE) and
fertility (±SE) were measured along the first week of oviposition in laboratory conditions (25 ± 2 ºC, 75 ± 10%
RH, 16:8 L:D photoperiod).
Fecundity (eggs/♀ week-1)
Fertility (%)
control
289.38 ± 44.03 a
73.04 ± 5.33 a
abs-UV
205.86 ± 47.08 a
72.91 ± 8.73 a
control>abs-UV
107.43 ± 52.43 a
77.23 ± 2.08 a
abs-UV>control
187.57 ± 37.35 a
73.16 ± 7.01 a
F3,25 = 2.78
F3,18 = 0.08
p = 0.062
p = 0.969
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p <
0.05).
97 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.3.1.3. DIRECT EFFECTS OF LOW UV RADIATION ON CUCUMBER PLANTS
In regard to the influence of the different UV radiation conditions on the growth and
biomass yield of cucumber plants, only were recorded significant differences (ANOVA,
Fisher’s LSD Test, p < 0.05) in terms of stem length (Table 5.5). This parameter was
significantly higher for abs-UV>control treatment (46.8 ± 0.8 cm). Concerning the fresh
weight and the dry weight, no significant differences were found amongst treatments.
Table 5.5. Stem lenght (cm), fresh weight (g) and dry weight (g) of healthy cucumber plants grown under
different UV radiation conditions. The temperature and relative humidity conditions during the experiment were
controlled (15-27 ºC, 50-85% RH).
control
abs-UV
control>abs-UV
abs-UV>control
Stem lenght (cm)
Fresh weight (g)
Dry weight (g)
36.0 ± 1.8 a
40.3 ± 2.2 a
38.8 ± 2.9 a
46.8 ± 0.8 b
F3,16 = 4.91
p = 0.013
13.0 ± 1.0 a
17.1 ± 2.4 a
20.7 ± 5.1 a
21.3 ± 1.6 a
F3,16 = 1.94
p = 0.163
2.4 ± 0.1 a
2.3 ± 0.2 a
3.1 ± 0.5 a
2.7 ± 0.2 a
F3,16 = 1.78
p = 0.191
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p <
0.05).
The analysis of the percentages of C, H, N and S showed that nitrogen (N) was the only
element that presented significant differences amongst treatments (ANOVA, Fisher’s LSD
Test, p < 0.05) (Table 5.6). The highest N contents were recorded in abs-UV (1.47 ± 0.16%)
and control>abs-UV (1.27 ± 0.23%) treatments, while the lowest was obtained in control
treatment (0.76 ± 0.05%). The percentages of C, H and S were very similar between
treatments, and this is the reason for not recording significant differences amongst them.
98 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.6. Percentages (%) of C, H, N and S of healthy cucumber plants grown under different UV radiation
conditions. The temperature and relative humidity conditions during the experiment were controlled (15-27 ºC,
50-85% RH).
control
abs-UV
control>abs-UV
abs-UV>control
C (%)
H (%)
N (%)
S (%)
31.92 ± 1.17 a
33.55 ± 0.50 a
33.20 ± 0.94 a
33.27 ± 0.88 a
F3,16 = 0.65
p = 0.596
5.29 ± 0.12 a
5.41 ± 0.07 a
5.36 ± 0.12 a
5.44 ± 0.12 a
F3,16 = 0.52
p = 0.672
0.76 ± 0.05 a
1.47 ± 0.16 b
1.27 ± 0.23 b
1.18 ± 0.15 ab
F3,16 = 3.45
p = 0.042
0.22 ± 0.05 a
0.29 ± 0.03 a
0.26 ± 0.05 a
0.23 ± 0.03 a
F3,16 = 0.56
p = 0.651
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p <
0.05).
The measurements of the cell wall and cuticle thickness of epidermal cells of the upper
face of cucumber leaves under different UV conditions revealed significant differences
(ANOVA, Fisher’s LSD Test, p < 0.05) amongst treatments (Table 5.7). The highest values
were observed in the control>abs-UV treatment, both for cell wall thickness (1.35 ± 0.06 μm)
and cuticle thickness (48.0 ± 3.02 nm). The significantly lowest values of cell wall thickness
were recorded for abs-UV and abs-UV>control treatments (0.66 ± 0.05 μm and 0.69 ± 0.02
μm, respectively). The abs-UV>control treatment also presented the lowest cuticle thickness
value (30.86 ± 2.09 nm) in comparison with the other treatments.
Table 5.7. Cell wall thickness (μm) and cuticle thickness (nm) of epidermal cells of the upper face of leaves of
healthy cucumber plants grown under different UV radiation conditions. The temperature and relative humidity
conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
control
abs-UV
control>abs-UV
abs-UV>control
Cell wall thickness (μm)
Cuticle thickness (nm)
0.92 ± 0.04 a
0.66 ± 0.05 b
1.35 ± 0.06 c
0.69 ± 0.02 b
F3,24 = 51.92
p < 0.001
38.29 ± 1.71 a
36.57 ± 2.68 ab
48.0 ± 3.02 c
30.86 ± 2.09 b
F3,24 = 8.60
p = 0.005
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p <
0.05).
99 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.3.2. CABYV-INFECTED CUCUMBER PLANTS EXPERIMENT
5.3.2.1. PLANT-MEDIATED EFFECTS OF LOW UV RADIATION ON THE
FITNESS OF A. gossypii
The analysis of the evolution of the apterae A. gossypii population, only revealed
significant differences (ANOVA, Fisher’s LSD Test, p < 0.05) amongst treatments in the 1st
sampling (Table 5.8). This initial differences disappeared in the 2nd sampling, maintaining this
constant until the end of the trial (10th sampling). In this experiment, the apterae aphid
population experienced a sustained increase until the 9th sampling, with no abrupt changes,
and then slightly decreased in the 10th one (Figure 5.10). This reduction in the number of
apterae aphids was general, affecting all treatments in the same extent. Despite the positive
population growth until the 9th sampling, the curve slope in every treatment started to
decrease from the 7th sampling, coincident with the moment of C. carnea larvae release. The
abs-UV treatment curve was over the curves of the rest of treatments along all the experiment,
while the control>abs-UV treatment curve was under them in all samplings (Figure 5.10).
The evolution of the A. gossypii alatae population did not experience significant
differences (Kruskal-Wallis, p < 0.05; ANOVA, p < 0.05) amongst treatments in any of the
samplings (Table 5.9). The alatae population growth was always positive until the end of the
experiment for every treatment, although the bigger increase took place from the 7th sampling,
coincident with the moment of C. carnea larvae release (Figure 5.11).
100 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.8. Evolution of A. gossypii apterae on CABYV-infected cucumber plants under different UV radiation conditions. The releasing of 20 adult apterae
individuals/plant was done on 28/03/2014. The temperature and relative humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
1st sampling
(31/03/2014)
2nd sampling
(02/04/2014)
3rd sampling
(04/04/2014)
4th sampling
(07/04/2014)
5th sampling
(09/04/2014)
6th sampling
(11/04/2014)
7th sampling
(14/04/2014)
8th sampling
(16/04/2014)
9th sampling
(18/04/2014)
10th sampling
(21/04/2014)
control
135.0±21.2a
183.2±24.9a
260.8±55.1a
628.0±119.8a
1176.6±318.6a
1979.4±410.7a
2622.0±486.9a
3113.8±565.0a
3348.4±579.5a
3260.0±649.8a
abs-UV
104.8±21.3ab
155.6±27.1a
245.8±49.5a
762.0±150.6a
1511.0±433.0a
2394.0±495.3a
3272.0±623.5a
3868.8±701.9a
4171.0±812.4a
4070.0±850.4a
control>abs-UV
64.6±7.4b
122.0±14.7a
165.2±28.2a
379.6±76.5a
792.0±166.5a
1427.2±296.9a
2227.2±402.9a
2621.0±450.9a
2888.4±529.1a
2764.2±593.4a
abs-UV>control
62.0±10.9b
143.2±22.6a
213.6±36.6a
469.8±93.0a
1008.4±185.0a
1635.4±327.3a
2563.6±479.0a
3191.0±595.2a
3451.4±674.7a
3410.2±795.6a
F3,16 = 4.54
F3,16 = 1.25
F3,16 = 0.94
F3,16 = 2.23
F3,16 = 1.05
F3,16 = 1.18
F3,16 = 0.75
F3,16 = 0.77
F3,16 = 0.65
F3,16 = 0.54
p = 0.018
p = 0.323
p = 0.444
p = 0.125
p = 0.398
p = 0.350
p = 0.538
p = 0.528
p = 0.594
p = 0.659
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p < 0.05).
Table 5.9. Evolution of A. gossypii alatae on CABYV-infected cucumber plants under different UV radiation conditions. The releasing of 20 adult apterae
individuals/plant was done on 28/03/2014. The temperature and relative humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
1st sampling
(31/03/2014)
2nd sampling
(02/04/2014)
3rd sampling
(04/04/2014)
4th sampling
(07/04/2014)
5th sampling
(09/04/2014)
6th sampling
(11/04/2014)
7th sampling
(14/04/2014)
8th sampling
(16/04/2014)
9th sampling
(18/04/2014)
10th sampling
(21/04/2014)
control
0.4±0.2a
0.4±0.2a
0.4±0.2a
0.4±0.2a
1.6±0.7a
2.4±1.2a
9.2±4.8a
14.2±7.0a
22.2±8.4a
43.8±13.1a
abs-UV
0.0±0.0a
0.0±0.0a
0.0±0.0a
1.0±0.5a
2.0±0.9a
2.2±0.9a
10.0±5.3a
26.4±10.4a
41.8±16.1a
49.4±13.6a
control>abs-UV
0.0±0.0a
0.4±0.4a
0.2±0.2a
0.2±0.2a
0.2±0.2a
0.2±0.2a
0.8±0.4a
3.4±1.0a
6.6±1.8a
19.6±9.0a
abs-UV>control
0.2±0.2a
1.0±0.4a
0.4±0.2a
0.6±0.4a
0.8±0.4a
1.6±0.9a
3.0±1.6a
12.2±8.8a
19.4±12.6a
27.6±9.2a
H = 4.10
F3,16 = 1.62
F3,16 = 0.92
F3,16 = 0.83
F3,16 = 1.81
F3,16 = 2.31
F3,16 = 2.23
F3,16 = 1.41
F3,16 = 1.40
F3,16 = 1.47
p = 0.251
p = 0.224
p = 0.455
p = 0.495
p = 0.187
p = 0.116
p = 0.124
p = 0.277
p = 0.278
p = 0.260
Data (mean ± SE) followed by the same letter in the 1st sampling column did not significantly differ (Kruskal-Wallis, p < 0.05).
Data (mean ± SE) followed by the same letter in the same column (2nd – 10th samplings) did not significantly differ (ANOVA, p < 0.05).
101 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 4500
number of individuals
4000
control
3500
3000
abs-UV
2500
control>abs-UV
2000
abs-UV>control
1500
1000
500
0
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
Figure 5.10. Evolution of A. gossypii apterae population during the experiment on CABYV-infected
cucumber plants. The releasing of C. carnea larvae was done in S7 (7th sampling).
number of individuals
60
50
control
40
abs-UV
control>abs-UV
30
abs-UV>control
20
10
0
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
Figure 5.11. Evolution of A. gossypii alatae population during the experiment on CABYV-infected
cucumber plants. The releasing of C. carnea larvae was done in S7 (7th sampling).
102 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 5.3.2.2. PLANT-PREY-MEDIATED EFFECTS OF LOW UV RADIATION ON C.
carnea
As happened in the experiment with healthy cucumber plants, the number of recaptured C.
carnea pupae from the experimental cages was never the initial number released (5 L1
larvae/plant; 25 L1 larvae/treatment). From the control treatment 18 pupae were collected, 18
pupae from the abs-UV treatment, 16 pupae from the control>abs-UV treatment and 13 pupae
from the abs-UV>control treatment. Once the pupae were collected, they were taken to the
laboratory and joined in treatments to record the adult emergence. The emergence values were
94.4% for control treatment, 72.2% for abs-UV treatment, 93.8% for control>abs-UV
treatment and 84.6% for abs-UV>control treatment.
With the emerged adults, as many couples (1 ♀ and 1 ♂) as possible were made to assess
the fecundity and fertility (control, 7; abs-UV, 3; control>abs-UV, 6; abs-UV>control, 5). No
significant differences were recorded amongst treatments (ANOVA, p < 0.05) in terms of
fecundity and fertility. The highest fecundity value was observed in abs-UV treatment (315.67
± 10.73 eggs/♀ week-1), while for fertility the highest value was for abs-UV>control
treatment (88.66 ± 2.13%) (Table 5.10).
103 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.10. Reproductive parameters of C. carnea adults developed from the larvae released inside the
experimental cages under different UV radiation conditions with CABYV-infected cucumber plants. Fecundity
(±SE) and fertility (±SE) were measured along the first week of oviposition in laboratory conditions (25 ± 2 ºC,
75 ± 10% RH, 16:8 L:D photoperiod).
Fecundity (eggs/♀ week-1)
Fertility (%)
control
258.71 ± 30.87 a
86.60 ± 1.92 a
abs-UV
315.67 ± 10.73 a
80.29 ± 3.40 a
control>abs-UV
313.17 ± 15.96 a
81.21 ± 3.17 a
abs-UV>control
284.80 ± 21.12 a
88.66 ± 2.13 a
F3,17 = 1.20
F3,17 = 2.16
p = 0.339
p = 0.130
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p <
0.05).
5.3.2.3. DIRECT EFFECTS OF LOW UV RADIATION ON CUCUMBER PLANTS
Concerning the influence of the different UV radiation conditions on the growth and
biomass yield of CABYV-infected cucumber plants, no significant differences (ANOVA, p <
0.05) were found for stem length, fresh weight and dry weight, with very similar values for
every treatment (Table 5.11).
Table 5.11. Stem lenght (cm), fresh weight (g) and dry weight (g) of CABYV-infected cucumber plants grown
under different UV radiation conditions. The temperature and relative humidity conditions during the experiment
were controlled (15-27 ºC, 50-85% RH).
control
abs-uv
control>abs-uv
abs-uv>control
Stem lenght (cm)
Fresh weight (g)
Dry weight (g)
52.6 ± 0.7 a
53.5 ± 1.1 a
51.3 ± 1.1 a
51.5 ± 1.5 a
F3,16 = 0.80
p = 0.512
40.1 ± 2.1 a
42.8 ± 2.5 a
38.9 ± 3.1 a
41.1 ± 1.6 a
F3,16 = 0.47
p = 0.704
4.4 ± 0.3 a
4.3 ± 0.3 a
4.6 ± 0.4 a
4.6 ± 0.2 a
F3,16 = 0.23
p = 0.875
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p <
0.05).
104 Influence of UV on the performance of A. gossypii, C. carnea and cucumber The analysis of the percentages of C, H, N and S revealed that sulfur (S) was the only
element that presented significant differences amongst treatments (ANOVA, Fisher’s LSD
Test, p < 0.05) (Table 5.12). The highest S contents were recorded in abs-UV (0.32 ± 0.11%)
and control (0.30 ± 0.06%) treatments, while the lowest was obtained in abs-UV>control
treatment (0.06 ± 0.03%). The percentages of C, H and N were very similar between
treatments, and this is the reason for not finding significant differences amongst them.
Table 5.12. Percentages (%) of C, H, N and S of CABYV-infected cucumber plants grown under different UV
radiation conditions. The temperature and relative humidity conditions during the experiment were controlled
(15-27 ºC, 50-85% RH).
control
abs-uv
control>abs-uv
abs-uv>control
C (%)
H (%)
N (%)
S (%)
39.27 ± 0.42 a
39.12 ± 0.66 a
39.31 ± 0.21 a
39.39 ± 0.35 a
F3,16 = 0.06
p = 0.979
6.06 ± 0.06 a
6.01 ± 0.05 a
6.03 ± 0.04 a
6.09 ± 0.07 a
F3,16 = 0.42
p = 0.742
1.46 ± 0.18 a
1.73 ± 0.47 a
1.76 ± 0.27 a
1.32 ± 0.14 a
F3,16 = 0.51
p = 0.678
0.30 ± 0.06 a
0.32 ± 0.11 a
0.11 ± 0.05 ab
0.06 ± 0.03 b
F3,16 = 3.53
p = 0.039
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p <
0.05).
The measurements of the cell wall and cuticle thickness of epidermal cells of the upper
face of CABYV-infected cucumber leaves under different UV conditions did not reveal
significant differences (ANOVA, p < 0.05) amongst treatments (Table 5.13).
105 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Table 5.13. Cell wall thickness (μm) and cuticle thickness (nm) of epidermal cells of the upper face of leaves of
CABYV-infected cucumber plants grown under different UV radiation conditions. The temperature and relative
humidity conditions during the experiment were controlled (15-27 ºC, 50-85% RH).
control
abs-uv
control>abs-uv
abs-uv>control
Cell wall thickness (μm)
Cuticle thickness (nm)
0.51 ± 0.03 a
0.56 ± 0.04 a
0.52 ± 0.04 a
0.55 ± 0.04 a
F3,24 = 0.35
p = 0.787
21.71 ± 2.88 a
21.71 ± 3.25 a
24.57 ± 4.57 a
30.29 ± 3.25 a
F3,24 = 1.30
p = 0.298
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p <
0.05).
5.4. DISCUSSION
The adoption of UV-blocking covers by growers as an alternative technique to chemical
use for pest and diseases control seems to be promising if it might not modify the behaviour
of the crop in a negative way and the economic return of this cropping system is maintained
(Monci et al., 2004; Kittas et al., 2006). In the present study, it was not found a clear
influence of the different UV-radiation conditions evaluated in the development and
physiology of cucumber plants, both for healthy plants and CABYV-infected plants.
Depending on a number of factors, such as species, cultivar and growth conditions, plant
growth may be inhibited or stimulated by different levels of UV radiation (Staxén &
Bornman, 1994).
In terms of growth, only the plants that were initially grown under the UV-absorbent
plastic film Térmico Antivirus® and then transferred under control plastic film Térmico
Blanco® (abs-UV>control treatment) experienced a significant increase in stem length,
followed by the abs-UV treatment. One of the most characteristic acclimatisation responses of
plant morphology to UV is the inhibition of plant growth (Jansen et al., 1998; Zavala &
Ravetta, 2002; Caldwell et al., 2003), so its absence in the early stages of the crop could
106 Influence of UV on the performance of A. gossypii, C. carnea and cucumber explain the increase in stem length. Kuhlmann & Müller (2009b) detected that broccoli plants
(Brassica oleracea L. convar. botrytis) grown under low-UV and subsequently transferred to
different UV-conditions were not affected in growth. Amudha et al. (2005) observed that the
removal of UV-B from natural solar radiation caused a large increase in the growth of
Cyamopsis species and a marginal increase in Vigna radiata. Also, Varalakshmi et al. (2003)
determined that the exclusion of solar UV-B and UV-B/A caused enhanced vegetative growth
and yield parameters in soybean (cv. JS 7105). On the other hand, the lower stem length of
the control treatment plants, with no UV-absorbent properties, can be due to an inhibition in
hypocotyl elongation and a shortening of the internodes caused by UV-B exposition
(Caldwell et al., 2003). Kuhlmann & Müller (2009b) observed that broccoli plants germinated
under ambient UV radiation levels were smaller. In the case of the experiment with CABYVinfected cucumbers, the stem growth tends to the equality in all treatments, not recording any
significant differences. This fact could be linked to the attenuation of the different UVradiation conditions in favour of a predominant influence of CABYV-induced changes and
symptoms, which include yellowing and thickening of basal and older leaves (Lecoq et al.,
1992).
The other developmental parameters, fresh weight and dry weight, did not experience any
effect derived from UV radiation, neither in the experiment with healthy cucumbers nor in the
experiment with CABYV-infected ones. In contrast, Kadur et al. (2007) observed that
soybean plants experienced a dramatic change in dry weight, fresh weight and leaf size in
UV-B/A and UV-B excluded sunlight along with control plants. The results of previous UV
influence studies indicate that plants vary widely in their response to different levels of UV-A
and UV-B radiation (Fiscus et al., 1999; Robson et al., 2003; Comont et al., 2012; Dáder et
al., 2014). Concerning UV-A, there is little evidence about its effects on plant development,
but a recent research showed that exposure to higher levels induced a growth reduction in
107 Influence of UV on the performance of A. gossypii, C. carnea and cucumber pepper and eggplant (Dáder et al., 2014). In some species (e.g. cucumber, mung bean, New
Zealand spinach and ‘‘New Fire’’ lettuce) growth is inhibited by solar UV-B (Adamse et al.,
1997; Krizek et al., 1997; Pal et al., 1997; Krizek et al., 1998). In contrast, some plants (e.g.
tomato) experience an enhanced growth (Krizek et al., 1997; Cybulski & Peterjohn, 1999),
whereas in others (e.g. cotton, oat) it is unaffected (Adamse et al., 1997; Krizek et al., 1997).
Regarding the contents of C, H, N and S, it seemed that the different UV-exposure
treatments did not significantly affect these levels except for N in the non-infected cucumbers
experiment and S in the CABYV-infected cucumbers experiment. In the non-infected plants
experiment, higher N content percentages were obtained for plants that grew under low UV
conditions since the beginning or from the 14 BBCH stage (abs UV and control>abs-UV
treatments). Nevertheless, other authors such as Hatcher & Paul (1994), found out that tissue
nitrogen in pea increased with increasing UV-B; Dáder et al. (2014) also obtained a raise in N
levels derived from free amino acids in pepper plants grown under supplemental UV-A
radiation. In contrast, Ghisi et al. (2002) found that nitrate content was not influenced by the
UV-B exposure, neither in root nor in leaf tissue when they studied the effect of an UV-B
enriched environment on the level of nitrates in barley. In the CABYV-infected plants
experiment only S content percentages varied significantly amongst treatments, but it cannot
be affirmed that it is a consequence of the different UV-exposure conditions, because the
higher contents were found in control and abs-UV treatments. The influence of CABYV
presence might have had a direct effect on the levels of the studied elements, because it is
known that pathogen infection can modify plant chemistry (Moran, 1998; Felton et al., 1999;
Hatcher et al., 2004). The contents of C and H did not significantly differ in any of the
experiments, despite there are reports of some authors that detected changes in carbohydrates
composition in response to different UV exposure conditions (Takeuchi et al., 1989; Santos et
al., 1993; He et al., 1994).
108 Influence of UV on the performance of A. gossypii, C. carnea and cucumber Concerning the measurements of the cell wall and cuticle thickness of epidermal cells of
the upper face of non-infected cucumber leaves, it was recorded a significant increase in those
treatments in which plants were grown along all the experiment or in the beginning of their
development (until 14 BBCH stage) under control UV conditions (control and control>absUV treatments). Verdaguer et al. (2012) observed that plants exposed to UV-A radiation
increased the thickness of the adaxial epidermal cells in regard to those that were grown
without UV-A. Also, enhanced UV-B radiation can cause an increase in leaf wax layers and
cuticle thickness of different crop plant species (Steinmüller & Tevini, 1985;
Grammatikopoulos et al., 1998). In the CABYV-infected cucumbers experiment no
significant differences were found in terms of cell wall and cuticle thickness, which can be
due again to an attenuation of the UV radiation conditions in favour of a major influence of
virus presence.
In a trophic system, the changes that occur in one of its levels have an impact on the other
ones, as part of a complex structure (Irwin & Tresh, 1990). Therefore, the physiological
changes that could be potentially induced in cucumber plants both by the influence of low UV
radiation conditions and CABYV infection were expected to exert an effect on aphid
population performance, and eventually on chrysopid fitness. Several investigations of plantinsect relationships on differently UV-exposed plants recorded that UV-B irradiated plants
were damaged to a lesser extent by insect herbivores than non-irradiated plants (Ballaré et al.,
1996; Zavala et al., 2001; Rousseaux et al., 2004).
Nevertheless, it was not recorded a significant variation in the aphid density of cucumber
plants grown under different conditions of UV radiation, neither in apterae individuals nor in
alatae ones. The higher content in N of the plants from the abs-UV and control>abs-UV
treatments could have been a factor for triggering a faster development of aphids in those
plants, because aphid growth and reproduction mainly depend on the total amount and quality
109 Influence of UV on the performance of A. gossypii, C. carnea and cucumber of phloem-derived amino acids as a nitrogen source (Douglas, 2006; Dáder et al., 2014), but it
did not happen. Another factor that could have had an influence on aphid settlement is the
thickness of cell wall of the epidermal cells, cuticle and the presence of waxes. Aphid
performance is known to be influenced by the quality and quantity of cuticular wax coverage
(Thompson, 1963; Powell et al., 1999). Despite that fact, the significant differences detected
in cell wall and cuticle thickness of plants grown under different UV radiation conditions did
not have an effect in aphid performance. In the experiment with CABYV-infected cucumbers
the results were almost identical, detecting only significant differences in the apterae aphid
population after the first sampling, which disappeared in subsequent samplings until the end
of the assay. Virus infections can change hosts in such a way that interactions between host
and vector are influenced (Álvarez et al., 2007), so it is possible that plant-mediated effects
derived from UV radiation conditions could have been pushed into the background as a
consequence of CABYV infection induced effects. It is known that virus infection can alter
the suitability of host plants for aphid vectors (Bosque-Pérez & Eigenbrode, 2011), and as in
the case of Luteoviridae family enhance their performance (Eigenbrode et al., 2002; Hodge &
Powell, 2008b).
UV receptors are present in all known insect species that have been examined (Briscoe &
Chittka, 2001), and not only insect pests but also their arthropod natural enemies may use
similar mechanisms (including vision and olfaction) to locate food sources (Brown et al.,
1998; Storeck et al., 2000; Blackmer & Cross, 2001). In the present work no significant
differences were found in the reproductive parameters of C. carnea adults developed from
larvae released on plants under the different UV radiation conditions, both in the experiment
with non-infected cucumber plants and the experiment with CABYV-infected cucumber
plants. It is important to establish a clear distinction between direct and plant-mediated effects
of UV on insects, although sometimes it is not possible (Newsham et al., 1996; Zavala et al.,
110 Influence of UV on the performance of A. gossypii, C. carnea and cucumber 2001; Veteli et al., 2003). Regarding the direct influence of low UV radiation on the prey
searching effectiveness, it is expected that it did not have much impact on lacewing larvae,
because they are known to use tactile cues when searching for prey (Hutchins et al., 2003;
Hajek, 2004). Concerning the indirect or plant-prey-mediated influence of low UV radiation
conditions, the quality of the larval food can affect the reproduction of the ensuing adults
(New, 1975), so the practical absence of variations in the other elements of the trophic
system, plants and aphids, was reflected in the lack of differences in the biology of the
chrysopid adults amongst treatments. Although natural enemies are commonly used in
commercial greenhouse crops, there is little published work on the effects of UV-blocking
films on biological control, and the scarce literature deals with the influence of low UV
conditions on parasitoids. Chyzik et al. (2003) observed that the fecundity and host finding
activity of the parasitoid Aphidius matricariae (Haliday) (Hymenoptera: Braconidae) was not
influenced by UV radiation. Nevertheless, Doukas (2003) found that Encarsia formosa Gahan
(Hymenoptera: Aphelinidae) exhibited preference to non-UV-absorbing plastic film in
choice-chamber tests. Three other hymenopteran parasitoid species, Aphidius colemani
Viereck (Hymenoptera: Braconidae), Diglyphus isaea Walker (Hymenoptera: Eulophidae)
and Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae), showed a preference for
ambient light over UV-depleted light, when their orientation behaviour was evaluated in a Ytube with two different filters. However, their parasitism rates did not differ in greenhouses
covered either with regular plastic or UV-absorbing plastic sheet (Chiel et al., 2006).
Regarding non-parasitoid natural enemies, Legarrea et al. (2012) studied the impact of UVabsorbing nets on the visual cues of the predators Orius laevigatus (Fieber) (Hemiptera:
Anthocoridae) and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), observing that
the anthocorid was caught in higher numbers in traps under regular nets, whereas the
phytoseid preferred environments with attenuated UV radiation. In view of the above, the
111 Influence of UV on the performance of A. gossypii, C. carnea and cucumber performance of natural enemies is highly dependent on several factors, such as the species,
the properties of the plastic films or nets tested and the experimental conditions.
The development of non-chemical methods for a successful integrated pest management,
such as biological control or photoselective barriers, requires a proper understanding of the
interactions between insects and their host plants as well as the ecology and behaviour of the
pest species and their natural enemies (van Lenteren & Noldus, 1990). According to the
literature reviewed, UV-mediated changes are highly dependent on the plants and insects
studied. The results obtained in this study do not provide clear effects of the different UV
radiation conditions in the biological system considered. In fact, Kuhlmann & Müller (2011)
after reviewing 38 studies about the comparison of high and low UV exposure levels on plantinsect interactions, found unclear/neutral effects in the 29% of them. Therefore, broader
research is needed to draw applicable conclusions to each biological system studied.
112 Side effects of pesticides on C. carnea and A. bipunctata CHAPTER 6. TOXICITY AND SUBLETHAL EFFECTS OF SIX
INSECTICIDES TO LAST INSTAR LARVAE AND ADULTS OF THE
BIOCONTROL AGENTS Chrysoperla carnea AND Adalia bipunctata4
ABSTRACT
To further develop Integrated Pest Management (IPM) strategies against crop pests, it is
important to evaluate the effects of insecticides on biological control agents. Therefore, we
tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide,
metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies
Chrysoperla carnea and Adalia bipunctata. The side effects of the active ingredients of the
insecticides were evaluated with residual contact tests for the larvae and adults of these
predators in the laboratory. Flonicamid, flubendiamide, metaflumizone and spirotetramat
were innocuous to last instar larvae and adults of C. carnea and A. bipunctata. Sulfoxaflor
was slightly toxic to adults of C. carnea and was highly toxic to the L4 larvae of A.
bipunctata. For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging
compounds with a cumulative larval mortality of 100%. Deltamethrin was also the most toxic
compound to larvae and adults of C. carnea. In accordance with the results obtained, the
compounds flonicamid, flubendiamide, metaflumizone and spirotetramat might be
incorporated into IPM programs in combination with these natural enemies for the control of
4
Published in: Garzón, A., Medina, P., Amor, F., Viñuela, E., Budia, F. (2015) Toxicity and sublethal
effects of six insecticides to last instar larvae and adults of the biocontrol agents Chrysoperla carnea
(Stephens) (Neuroptera: Chrysopidae) and Adalia bipunctata (L.) (Coleoptera: Coccinellidae).
Chemosphere 132, 87-93.
113 Side effects of pesticides on C. carnea and A. bipunctata particular greenhouse pests. Nevertheless, the use of sulfoxaflor and deltamethrin in IPM
strategies should be taken into consideration when releasing either of these biological control
agents, due to the toxic behavior observed under laboratory conditions. The need for
developing sustainable approaches to combine the use of these insecticides and natural
enemies within an IPM framework is discussed.
6.1. INTRODUCTION
The impact of synthetic pesticides on the environment, the beneficial arthropods and the
human health by exposure to these chemicals are issues of growing concern. In response to
this, the European Union Directive 2009/128/EC established a legal framework to achieve the
sustainable use of pesticides and to implement Integrated Pest Management (IPM) strategies
in all member states by 1st January 2014 (EEC/CEE, 2009). The priority of IPM is the use of
biological control, but biocontrol may not always be effective enough to manage insect pest
populations, and corrective insecticide treatments may be needed, particularly in greenhouses,
where the incidence of pests is higher (Medina et al., 2008; Jalali et al., 2009). Chemical
control may be required to suppress pests that have no efficient biological control agents, and
thus the identification of products that are not harmful to beneficial organisms and are
respectful of the environment is a primary concern in IPM programs (Viñuela et al., 2000;
Hassan & van Veire, 2004). The main premise for the use of pesticides in IPM systems is to
use products with proven selectiveness to biological control agents (BCAs), low mammalian
and avian toxicity, minimal environmental persistence, and low risk of developing resistance
in the target populations, yet with a fairly broad spectrum of insecticidal activity against pests
(van Lenteren & Woets, 1988; Harris, 2000; Viñuela et al., 2000). An accurate evaluation of
the potential side effects of insecticides on BCAs is critical for developing effective IPM
strategies (Desneux et al., 2006; Stark et al., 2007).
114 Side effects of pesticides on C. carnea and A. bipunctata The green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) is found in
a wide range of agricultural habitats and ranks as one of the most commonly used and
commercially available natural enemies (Tauber et al., 2000; New, 2001). The lacewing is
considered a key generalist biological control agent, which is used primarily through
augmentative periodic releases of larvae for the control of various aphid species in
greenhouses and outdoor crops (Medina et al., 2002; van der Blom, 2008; Turquet et al.,
2009). However, C. carnea larvae also consume a wide variety of soft-bodied arthropods,
such as scales, leafhoppers, whiteflies, psyllids, thrips, eggs and larvae of lepidopterans, and
mites (Principi & Canard, 1984; Rimoldi et al., 2008).
The ladybird Adalia bipunctata (L.) (Coleoptera: Coccinellidae) is commercially available
and widely used in Spain to protect horticultural crops (Robledo Camacho et al., 2009). Its
diet is composed of aphids, which are consumed both by larvae and adults (Hodek & Honěk,
1996). The contribution of coccinellids to decreased population growth rates of aphids is well
known (Hodek & Honěk, 1996; Völkl et al., 2007).
Amongst phytophagous insects, aphids, thrips and whiteflies are the most serious pests of
greenhouse crops (Rabasse & van Steenis, 1999). These insects cause mechanical damage
with their sucking activity and transmit numerous plant viruses (Ng & Perry, 2004). Despite
outstanding characteristics as aphid predators, C. carnea and A. bipunctata do not always
maintain populations of aphid pests below economic thresholds, and thus insecticides remain
an important management tool in greenhouse IPM programs to control key pests (Desneux et
al., 2007; Obrycki et al., 2009). To generate the IPM guidelines for natural enemy
conservation requires not only the assessment of insecticide lethal effects but also the possible
sublethal effects in individuals who survive an exposure to a particular product (Desneux et
al., 2007; Moscardini et al., 2013) because these effects could have an important effect on
natural enemy population dynamics (Stark & Banks, 2003).
115 Side effects of pesticides on C. carnea and A. bipunctata Therefore, the objective of this study was to evaluate the lethal and sublethal effects of six
pesticides: deltamethrin, flonicamid, flubendiamide, metaflumizone, spirotetramat (included
in the Phytosanitary Products Registry of the Spanish Ministry of Agriculture, Food and
Environment; MAGRAMA, 2014b), and sulfoxaflor. C. carnea and A. bipunctata larvae and
adults were tested in the laboratory to classify the toxicity of the insecticides in accordance
with the International Organization for Biological and Integrated Control of Noxious Animals
and Plants (IOBC) directives.
6.2. MATERIALS AND METHODS
The bioassays were conducted in the Laboratory of Crop Protection, Department of
Agrarian Production, Technical University of Madrid, Spain. The laboratory conditions were
controlled at 25 ± 2 ºC, with relative humidity at 75 ± 10%, and a photoperiod of 16:8
(light:dark).
6.2.1. INSECTS
A laboratory colony of C. carnea was established with L1 larvae obtained from Agrobío
(CHRYSOcontrol®, Almería, Spain) following the rearing procedure of Medina et al. (2001).
The larvae were fed Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs ad libitum,
and adults were provided a nutritious artificial diet (Vogt et al., 2000). For the trials, gauze
with < 24-h-old eggs was used and let to develop the L3 instar for the larvae bioassays and the
adults (< 48-h post emergence) for imaginal bioassays.
A. bipunctata L1 larvae were purchased from Agrobío (ADALIAcontrol®, Almería, Spain)
and were placed in cages with zigzag folded filter paper and a mixture of E. kuehniella eggs
and bee pollen (1:1) replaced three times per week (Bonte et al., 2010) up to the L4 moult; the
fourth larval instar was used for testing the insecticides for side effects. For the adult
experiments, L4 larvae were placed in individual arenas with a small amount of diet to avoid
116 Side effects of pesticides on C. carnea and A. bipunctata cannibalism and to guarantee successful pupation. After 6–7 d, the newly emerged adults
were transferred to cages with a water source (baize soaked with water) and the same diet
provided to larvae. A. bipunctata adults, < 48-h from emergence, were used in the imaginal
experiments.
6.2.2. INSECTICIDES
In the experiments, six commercial formulations of insecticides were tested. The active
ingredients that were evaluated are currently used in IPM programs on protected crops of
southeastern Spain, except for sulfoxaflor, which is a product under evaluation process. All
compounds were tested at the maximum field recommended concentrations for horticultural
crops, according to the product labels and following the instructions provided by the Spanish
Ministry of Agriculture, Food and Environment. The insecticides, concentrations of active
ingredient tested, and modes of action (IRAC, 2012; MAGRAMA, 2014b) were as follows:
flonicamid 60 mg a.i. L-1, a selective homopteran feeding blocker (Teppeki® 50% w/w water
dispersible granules; Ishihara Sangyo Kaisha Ltd., Osaka, Japan); flubendiamide 60 mg a.i. L1
, a ryanodine receptor modulator (Fenos® 24% w/w water dispersible granules; Bayer S.A.S.,
Lyon, France); metaflumizone 240 mg a.i. L-1, a voltage-dependent sodium channel blocker
(Alverde® 24% w/v suspension concentrate; BASF SE, Ludwigshafen, Germany);
spirotetramat 75 mg a.i. L-1, an inhibitor of acetyl CoA carboxylase (Movento® 150 O-TEC
15% w/v oil dispersion; Bayer CropScience AG, Monheim am Rhein, Germany); sulfoxaflor
63.6 mg a.i. L-1, a nicotinic acetylcholine receptor (nAChR) agonist (GF-2626 11.43% w/w
suspension concentrate; Dow AgroSciences, Indianapolis, IN, US); and deltamethrin 12.45
mg a.i. L-1, a sodium channel modulator (Decis® EW 15 1.5% w/v emulsion in water; Bayer
CropScience S.L., Paterna, Valencia, Spain), which is used as a broad spectrum insecticide in
horticultural crops.
117 Side effects of pesticides on C. carnea and A. bipunctata 6.2.3. BIOASSAYS
Residual contact experiments were conducted because this is the primary way the larvae
and adults of both biocontrol agents are exposed to insecticide contamination. Glass plates (12
× 12 × 0.5 cm) were sprayed with a Potter precision spray tower (Burkard Manufacturing Co.,
UK) to test the residual contact activity of the pesticides. The standard application was
between 1.5 and 2 mg cm-2 (1 ml at 55 kPa); this is the concentration interval recommended
by the IOBC validity criteria for ecotoxicological experiments on beneficial arthropods
(Hassan, 1994). As soon as the glass plates were dry, larvae and adults were exposed to the
insecticides with two different methods.
6.2.3.1. LARVAE BIOASSAYS
The larvae bioassays used seven individualized L3 or L4 (for C. carnea and A. bipunctata,
respectively) larvae < 48-h from moulting per replicate and 6 replicates per treatment,
following the procedure of Medina et al. (2004) (Figure 6.1). Larval mortality, percentage of
pupae formed and successful adult emergence from those pupae were recorded. Cumulative
mortality was the percentage of individuals who failed to reach the complete adult form. All
the adults that emerged from the same treatment were collected and placed together until the
beginning of oviposition.
118 Side effects of pesticides on C. carnea and A. bipunctata Figure 6.1. Experimental units used for testing pesticides side-effects in C. carnea (left) and A. bipunctata (right)
larvae.
For the C. carnea assays, as many replicates as possible of three pairs of adults (depending
on the survivorship: control, 7; flonicamid, flubendiamide, metaflumizone and spirotetramat,
6; sulfoxaflor, 5; deltamethrin, 3) were maintained in plastic oviposition cages (12 cm in
diameter × 5 cm in height) and provided food and water to assess the reproductive parameters
according to Vogt et al. (2000) (Figure 6.2). A piece of cotton gauze was placed in the upper
part of the oviposition cages, where the eggs of C. carnea were collected to measure
fecundity. The reproduction test started 5-7 d after the first egg was laid. Along one week,
two samples of < 24-h eggs were collected from each replicate and kept in separate plastic
cages (9 cm in diameter × 2 cm in height) for incubation until hatching, and the egg viability
was determined (Giolo et al., 2009).
For A. bipunctata, because of the scarce morphological evidence of sexual dimorphism
(Hodek & Honěk, 1996), the adults that emerged were divided into as many groups of 10
individuals each as possible (control, flonicamid, flubendiamide, metaflumizone and
spirotetramat, 4; sulfoxaflor and deltamethrin, 0), and placed into plastic oviposition cages
(12 cm in diameter × 5 cm in height), providing food and water (E. kuehniella eggs and bee
pollen, 1:1; Bonte et al., 2010) to assess the reproductive parameters (Figure 6.2). In the upper
119 Side effects of pesticides on C. carnea and A. bipunctata part of the oviposition cages was placed a cotton gauze and inside them wrinkled filter paper,
where the eggs of A. bipunctata were collected. The reproduction tests began 4-5 d after adult
emergence. The eggs were sampled and treated as described for C. carnea, but the evaluation
was conducted for two weeks. The individuals of each group were sexed a posteriori (by
dissection and extraction of the genitalia) to determine the sex ratio and the number of eggs
laid per female.
Figure 6.2. Experimental cages where C. carnea (left) and A. bipunctata (right) emerged adults were transferred
to for the evaluation of the reproductive parameters.
6.2.3.2. ADULTS BIOASSAYS
For bioassay with the adults of C. carnea, individuals (< 48-h from emergence) were
exposed to dried residues on the glass surfaces. Three pairs (3 ♂ and 3 ♀) of adults were a
replicate, and each treatment had five replicates. The test units were described by Giolo et al.
(2009) and were used as soon as the plates were dry (Figure 6.3). The mortality was recorded
after 3 d of residual contact. After the residual contact, the surviving adults were transferred to
the plastic cages described above to evaluate reproduction (Figure 6.2), maintaining the initial
number of replicates (5) for each treatment. The effects on reproductive parameters (fecundity
and fertility) were determined as described for the larvae bioassay.
120 Side effects of pesticides on C. carnea and A. bipunctata In the bioassay with A. bipunctata adults, 14 individuals (<48 h from emergence) per
replicate were exposed to dried residues on the glass surfaces, and each treatment had five
replicates. The test units were the same as in the experiment with C. carnea (Figure 6.3), and
the performance of the assay and the evaluation process were identical. The initial number of
replicates per treatment (5) was maintained to assess the reproductive parameters. The
surviving individuals of each replicate were sexed a posteriori to evaluate the reproductive
parameters.
Figure 6.3. Experimental units used for testing pesticides side-effects in C. carnea (left) and A. bipunctata (right)
adults.
6.2.4. STATISTICAL ANALYSIS
Data, presented as the means ± SE, were analyzed using one-way analysis of variance
(ANOVA) with the Statgraphics Plus 5.1 statistical software package (Statistical Graphics
Corporation, 1994-2000). The means were separated using the Fisher least significant
difference (LSD) multiple range test (p < 0.05). The nonparametric tests of Kruskal-Wallis
and Mann-Whitney (p < 0.05) were used to establish differences only when data violated the
premises of the ANOVA (Heath, 1995).
The total effect (E) of each insecticide was determined taking into account the percentage
of mortality observed in each treatment in relation to the mortality observed in the control
121 Side effects of pesticides on C. carnea and A. bipunctata treatment, and corrected using the Schneider-Orelli’s formula (Püntener, 1981). It was used
the formula proposed by Overmeer & van Zon (1982): E (%) = 100 – (100 – Mc) × ER; where
Mc is the final corrected mortality and ER is the ratio of mean number of eggs laid weekly by
treated females versus control females.
According to the IOBC laboratory scale (Hassan, 1994) and based on their total effects, the
pesticides were classified in four toxicity categories: (1) harmless (<30%); (2) slightly
harmful (30–79%); (3) moderately harmful (80–99%); and (4) harmful (>99%).
6.3. RESULTS
6.3.1. EFFECTS OF INSECTICIDES ON C. carnea
Significant differences were found in the L3 larvae bioassay among treatments in pupation
(F6,35 = 14.49; p < 0.001) and cumulative mortality (F6,35 = 12.39; p < 0.001), in particular for
deltamethrin compared with the control. Regarding adult emergence, the results did not show
significant differences (H = 9.61; p = 0.142). No statistically significant differences were
found for fecundity among treatments (F6,32 = 0.42; p = 0.863). For fertility of eggs laid by
females significant differences were obtained (F6,32 = 2.68; p = 0.032), among the insecticides
flubendiamide and sulfoxaflor compared with the control treatment (Table 6.1).
When testing the insecticides on adults, only sulfoxaflor and deltamethrin had significant
effects on the mortality at 3 d compared with the control (F6,28 = 17.49; p < 0.001).
Statistically significant differences were found between these two insecticides as well, with
the mortality (at 3 d) of sulfoxaflor reaching 56.67% (Table 6.2). For the reproductive
parameters (eggs/♀ week-1 and viable eggs laid), only deltamethrin significantly decreased
fecundity (15.20 eggs/♀ week-1) (F6,28 = 8.72; P < 0.001) and fertility (3.54%) (F6,26 = 16.21;
p < 0.001) compared with the rest of the treatments (Table 6.2).
122 Side effects of pesticides on C. carnea and A. bipunctata Flonicamid, flubendiamide, metaflumizone and spirotetramat were harmless (IOBC class
1) to L3 larvae and adults of C. carnea. Sulfoxaflor proved to be harmless (IOBC class 1) only
to L3 larvae of C. carnea, whereas it was slightly toxic (IOBC class 2) to C. carnea adults.
Deltamethrin was slightly toxic (IOBC class 2) and moderately toxic (IOBC class 3) to L3
larvae and adults of C. carnea respectively.
123 Side effects of pesticides on C. carnea and A. bipunctata Table 6.1. Effects on development, mortality and reproduction in C. carnea when L3 larvae were exposed to fresh residues of insecticides on a glass surface, using the
maximum field recommended concentration (MFRC) for horticultural crops.
Treatment
Control
MFRC
(mg a.i. l-1)
Pupation
(%)a
Adult emergence
(%)b
Cumulative mortality
(%)c
Fecundity
(eggs/♀ week-1)d
Fertility
(%)e
E (IOBC Cat.)f
-
100.00 ± 0.00 a
100.00 ± 0.00 a
0.00 ± 0.00 a
217.88 ± 17.80 a
76.94 ± 1.98 a
-
Flonicamid
60
100.00 ± 0.00 a
95.24 ± 3.01 a
4.76 ± 3.01 a
190.55 ± 16.80 a
75.39 ± 2.91 a
16.69 (1)
Flubendiamide
60
92.85 ± 3.19 a
94.44 ± 5.55 a
11.91 ± 6.82 a
204.06 ± 29.27 a
66.22 ± 3.50 b
17.49 (1)
Metaflumizone
240
97.62 ± 2.38 a
100.00 ± 0.00 a
2.38 ± 2.38 a
207.83 ± 26.91 a
71.80 ± 1.25 ab
6.90 (1)
75
90.47 ± 7.06 a
100.00 ± 0.00 a
9.52 ± 7.06 a
203.72 ± 20.49 a
71.68 ± 1.91 ab
15.42 (1)
63.6
90.47 ± 3.01 a
97.22 ± 2.78 a
11.91 ± 4.39 a
225.00 ± 22.89 a
67.78 ± 1.64 b
9.04 (1)
12.45
54.76 ± 6.82 b
86.11 ± 6.69 a
52.38 ± 7.06 b
241.22 ± 8.47 a
70.67 ± 3.30 ab
47.29 (2)
Spirotetramat
Sulfoxaflor
Deltamethrin
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p < 0.05) for pupation, cumulative mortality, fecundity and fertility.
Data (mean ± SE) followed by the same letter did not significantly differ (Kruskal-Wallis, p < 0.05) for adult emergence.
a.i. = active ingredient. E = total effect, according to Overmeer & van Zon (1982).
a
Percentage of formed pupae compared with the total number of treated larvae.
b
Percentage of emerged adults compared with the number of formed pupae.
c
Percentage of dead larvae, pupae and adults that failed to moult.
d
Number of eggs laid per female measured 5–7 d after the first egg laying.
e
Percentage of hatched eggs versus laid eggs.
f
IOBC Toxicity category for laboratory tests based on the total effect caused by each insecticide: (1) harmless (<30%); (2) slightly harmful (30–79%); (3) moderately harmful (80–99%); and (4)
harmful (>99%).
124 Side effects of pesticides on C. carnea and A. bipunctata Table 6.2. Effects on mortality and reproduction in C. carnea when adults were exposed to fresh residues of insecticides on a glass surface
using the maximum field recommended concentration (MFRC) for horticultural crops.
Treatment
Control
MFRC
(mg a.i. l-1)
Adult mortality at 3 d
(%)
Fecundity
(eggs/♀ week-1)a
Fertility
(%)b
E (IOBC Cat.)c
-
0.00 ± 0.00 a
253.70 ± 12.40 a
71.03 ± 1.40 a
-
Flonicamid
60
6.67 ± 4.08 ab
259.20 ± 20.26 a
70.06 ± 1.20 a
4.65 (1)
Flubendiamide
60
0.00 ± 0.00 a
227.40 ± 17.21 a
73.04 ± 2.99 a
10.37 (1)
Metaflumizone
240
13.34 ± 3.33 ab
245.60 ± 11.61 a
73.44 ± 2.59 a
16.10 (1)
75
0.00 ± 0.00 a
227.00 ± 15.90 a
66.49 ± 5.61 a
10.52 (1)
63.6
56.67 ± 8.50 c
184.80 ± 67.18 a
61.55 ± 16.10 a
68.44 (2)
12.45
20.00 ± 8.16 b
15.20 ± 10.16 b
3.54 ± 2.40 b
95.21 (3)
Spirotetramat
Sulfoxaflor
Deltamethrin
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p < 0.05).
a.i. = active ingredient. E = total effect, according to Overmeer & van Zon (1982).
a
Number of eggs laid per female measured 5–7 d after the first egg laying.
b
Percentage of hatched eggs versus laid eggs.
c
IOBC Toxicity category for laboratory tests based on the total effect caused by each insecticide: (1) harmless (<30%); (2) slightly
harmful (30–79%); (3) moderately harmful (80–99%); and (4) harmful (>99%).
125 Side effects of pesticides on C. carnea and A. bipunctata 6.3.2. EFFECTS OF INSECTICIDES ON A. bipunctata
In the A. bipunctata larvae bioassay, sulfoxaflor and deltamethrin were much more
detrimental than with C. carnea. Statistically significant differences were found for pupation
(H = 38.62; p < 0.001), adult emergence (H = 27.34; p < 0.001) and cumulative mortality (H
= 36.76; p < 0.001) between these two compounds and the rest of treatments, including the
control. Sulfoxaflor reduced significantly the percentage of pupation (7.14%), and
furthermore, of those scarce pupae that formed, no adults emerged. The effects of
deltamethrin were stronger than sulfoxaflor because no pupae were formed and every larva
died (Table 6.3). The reproductive parameters were not significantly different (fecundity: F4,15
= 2.45; p = 0.092; fertility: F4,15 = 1.62; p = 0.221). No significant differences were found for
fertility of eggs, and values of all treatments were below 60% (Table 6.3).
The results with adults showed a similar scenario; mortality at 3 d after treatment was
significantly higher than the control for sulfoxaflor (22.86%) and deltamethrin (100.00%;
Table 6.4) (F6,28 = 55.72; p < 0.001). The treatments did not significantly differ for fecundity
(F5,23 = 0.42; p = 0.828) and viability of the eggs (F5,23 = 1.98; p = 0.120). In the adults
bioassay, the overall fecundity reached higher values than those of the larvae assay, but all
were less than 70% (Table 6.4).
As occurred with C. carnea, flonicamid, flubendiamide, metaflumizone and spirotetramat
were harmless (IOBC class 1) to L4 larvae and adults of A. bipunctata. Sulfoxaflor was
harmless (IOBC class 1) just to adults of A. bipunctata, while it was toxic (IOBC class 4) to
L4 larvae. Deltamethrin was toxic (IOBC class 4) to both L4 larvae and adult stages of the
coccinellid.
126 Side effects of pesticides on C. carnea and A. bipunctata Table 6.3. Effects on development, mortality and reproduction in A. bipunctata when L4 larvae were exposed to fresh residues of insecticides on a glass surface, using the
maximum field recommended concentration (MFRC) for horticultural crops.
Treatment
MFRC
(mg a.i. l-1)
Control
Pupation
(%)a
Adult emergence
(%)b
Cumulative mortality
(%)c
Fecundity
(eggs/♀ week-1)d
Fertility
(%)e
E (IOBC Cat.)f
-
100.00 ± 0.00 a
100.00 ± 0.00 a
0.00 ± 0.00 a
79.15 ± 11.73 a
58.38 ± 1.84 a
-
Flonicamid
60
100.00 ± 0.00 a
100.00 ± 0.00 a
0.00 ± 0.00 a
103.64 ± 9.84 a
48.07 ± 3.56 a
-30.94 (1)
Flubendiamide
60
100.00 ± 0.00 a
100.00 ± 0.00 a
0.00 ± 0.00 a
105.00 ± 15.78 a
52.69 ± 3.91 a
-32.66 (1)
Metaflumizone
240
97.62 ± 2.38 a
92.86 ± 3.20 a
9.53 ± 3.01 a
63.80 ± 3.10 a
59.88 ± 7.74 a
27.08 (1)
75
100.00 ± 0.00 a
97.62 ± 2.38 a
2.38 ± 2.38 a
102.12 ± 14.20 a
47.00 ± 3.73 a
-25.95 (1)
63.6
7.14 ± 4.88 b
0.00 ± 0.00 b
100.00 ± 0.00 b
-
-
100.00 (4)
12.45
0.00 ± 0.00 b
100.00 ± 0.00 b
-
-
100.00 (4)
Spirotetramat
Sulfoxaflor
Deltamethrin
-
Data (mean ± SE) followed by different letters in the same column significantly differed (Kruskal-Wallis, Mann-Whitney, p < 0.05) for pupation, adult emergence and cumulative mortality.
Data (mean ± SE) followed by the same letter in the same column did not significantly differ (ANOVA, p < 0.05) for fecundity and fertility.
a.i. = active ingredient. E = total effect, according to Overmeer & van Zon (1982).
a
Percentage of formed pupae compared with the total number of treated larvae.
b
Percentage of emerged adults compared with the number of formed pupae.
c
Percentage of dead larvae, pupae and adults that failed to moult.
d
Number of eggs laid per female measured 4–6 d after the emergence of adults.
e
Percentage of hatched eggs versus laid eggs.
f
IOBC Toxicity category for laboratory tests based on the total effect caused by each insecticide: (1) harmless (<30%); (2) slightly harmful (30–79%); (3) moderately harmful (80–99%); and (4)
harmful (>99%).
127 Side effects of pesticides on C. carnea and A. bipunctata Table 6.4. Effects on mortality and reproduction in A. bipunctata when adults were exposed to fresh residues of insecticides on a glass
surface, using the maximum field recommended concentration (MFRC) for horticultural crops.
Treatment
Control
Adult mortality at 3 d
(%)
MFRC
(mg a.i. l-1)
Fecundity
(eggs/♀ week-1)a
Fertility
(%)b
E (IOBC Cat.)c
-
1.43 ± 1.43 a
81.08 ± 6.24 a
67.81 ± 6.60 a
-
Flonicamid
60
10.00 ± 3.64 ab
75.58 ± 11.25 a
57.39 ± 2.43 a
14.88 (1)
Flubendiamide
60
14.29 ± 4.52 ab
85.23 ± 13.91 a
50.30 ± 5.64 a
8.60 (1)
Metaflumizone
240
10.00 ± 3.64 ab
97.91 ± 17.98 a
59.95 ± 3.72 a
-10.26 (1)
75
10.00 ± 7.00 ab
94.69 ± 5.92 a
44.27 ± 10.60 a
-6.64 (1)
63.6
22.86 ± 6.93 b
101.83 ± 28.61 a
62.91 ± 3.32 a
1.71 (1)
12.45
100.00 ± 0.00 c
Spirotetramat
Sulfoxaflor
Deltamethrin
-
-
100.00 (4)
Data (mean ± SE) followed by different letters in the same column significantly differed (ANOVA, LSD, p < 0.05).
a.i. = active ingredient. E = total effect, according to Overmeer & van Zon (1982).
a
Number of eggs laid per female measured 4–6 d after the emergence of adults.
b
Percentage of hatched eggs versus laid eggs.
c
IOBC Toxicity category for laboratory tests based on the total effect caused by each insecticide: (1) harmless (<30%); (2) slightly
harmful (30–79%); (3) moderately harmful (80–99%); and (4) harmful (>99%).
128 Side effects of pesticides on C. carnea and A. bipunctata 6.4. DISCUSSION
Among the six compounds evaluated, only deltamethrin significantly affected the pupation
and cumulative larval mortality of C. carnea, in the L3 bioassay. The toxic effect of
deltamethrin by residual contact was also observed in the L1 larvae of C. carnea by Bigler &
Waldburger (1994), although other authors found innocuous effects of this insecticide to first
instar larvae and adults following exposure (Giolo et al., 2009). Larvae of predators can be
more easily damaged by pesticides than adults because they walk on treated surfaces and
cannot fly, but some exceptions have been reported in the case of C. carnea, that seems to be
more resistant in the larval stage (Medina et al., 2004). Despite its slightly toxic effect on L3
larvae, this developmental stage showed less susceptibility than the adult one. Ishaaya &
Casida (1981) reported that esterases present in the larvae of C. carnea have high hydrolyzing
activity against several synthetic pyrethroids and are a major factor in the natural tolerance to
these compounds.
The higher susceptibility of chrysopid adults to deltamethrin was reflected in significantly
higher mortality in regard to the control, and furthermore, in a significant reproductive
dysfunction. Low oviposition and viability of eggs with deltamethrin was because of the
mode of action of pyrethroids, which act functionally on the voltage-sensitive sodium channel
(Sattelle & Yamamoto, 1988). These compounds induce a shock effect in individuals that
causes muscular convulsions and results in the disruption of physiological functions, such as
reproduction, eventually leading to death (Bigler & Waldburger, 1994; Amarasekare &
Shearer, 2013).
Regarding the novel insecticide sulfoxaflor, which acts to disturb the nervous system as a
nicotinic acetylcholine receptor (nAChR) agonist (IRAC, 2012), no previous studies have
evaluated the side effects in C. carnea. Nevertheless, compared with results with other
129 Side effects of pesticides on C. carnea and A. bipunctata insecticides with the same mode of action such as imidacloprid, the adult mortality at 3 d was
similar (66.6%) (Huerta et al., 2003) to that obtained with sulfoxaflor (56.7%) in the present
study. The developmental stage had again a relevant influence on pesticide effects, because in
sulfoxaflor exposure to L3 larvae this compound proved to be harmless.
Concerning the coccinellid A. bipunctata, the susceptibility to insecticides was higher in L4
larvae than in adults, the opposite of that observed for the chrysopid C. carnea. This likely
was due to their closer adhesion to the substrate with anal pads and their higher foraging
activity, both of which increase the likelihood of contact with a contaminated surface (Jalali et
al., 2009). In a qualitative sense, the toxicity of the evaluated compounds was similar to both
natural enemies because for A. bipunctata sulfoxaflor and deltamethrin were again the most
damaging active ingredients. However, in a quantitative sense, because the cumulative larval
mortality reached 100%, and thus, no adults emerged in any treatment, both compounds were
substantially more toxic to A. bipunctata. According to the literature, the same toxic effects
were observed with insecticides that had the same mode of action, such as imidacloprid and
thiacloprid (IRAC MoA: 4) and lambda-cyhalothrin (IRAC MoA: 3), killing both larvae and
adults (Jalali et al., 2009; Jansen, 2010). In a study by Olszak (1999) with different stages of
A. bipunctata, pyrethroids were the most toxic compounds. In the adult bioassay, sulfoxaflor
was harmless, as were the rest of compounds, except for deltamethrin, which caused 100%
mortality after 3 d. This result was similar to that of Jalali et al. (2009), who found that adults
were three-fold more susceptible to lambda-cyhalothrin (pyrethroid, IRAC MoA: 3) than to
imidacloprid (neonicotinoid, IRAC MoA: 4).
The effects of the insecticides flonicamid, flubendiamide, metaflumizone and spirotetramat
on C. carnea and A. bipunctata were not comparable to those of sulfoxaflor and deltamethrin.
A reasonable explanation to their harmless behavior can be found in the selectivity and mode
of action of these compounds, because they are active mainly by ingestion (Tomlin, 2009),
130 Side effects of pesticides on C. carnea and A. bipunctata while the experiments evaluated the residual contact effects. For example, the selective
homopteran feeding blocker flonicamid was found previously to be innocuous to L4 larvae
and adults of A. bipunctata even at a concentration ten-fold higher than recommended (Jalali
et al., 2009). No literature references on the effects of flubendiamide, metaflumizone and
spirotetramat on these BCAs were found, so the results obtained in this study will clarify the
possibilities for combined use of both pest control strategies. Pesticide compatibility with
biological control agents is a major concern in IPM and knowledge about the activity of
insecticides toward the pests, the non-target insects, and the environment is a necessity (Stark
et al., 2004). The present work studies the physiological selectivity of the tested insecticides
in laboratory, but further research is required to determine the ecological selectivity. The use
of selective pesticides at recommended concentrations, and spatio-temporal separation of
pesticide applications and natural enemies when possible, are main factors for integrating
natural enemies with pesticides in pest management programs (Ruberson et al., 1998).
In conclusion, flonicamid, flubendiamide, metaflumizone and spirotetramat were nontoxic
to last instar larvae and adults of C. carnea and A. bipunctata. These insecticides are good
candidates to be incorporated into IPM programs in combination with these BCAs for the
control of specific greenhouse pests, such as aphids, caterpillars, whiteflies and scales. By
contrast, sulfoxaflor was slightly toxic to adults of C. carnea and was highly toxic to fourth
instar larvae of A. bipunctata. Deltamethrin was the most toxic compound to larvae and adults
of both natural enemies. Therefore, the use of deltamethrin and sulfoxaflor should be
considered when releasing C. carnea and A. bipunctata as part of an IPM strategy.
The degree of toxicity of pesticides is influenced by the exposure method and the life
stage, among others. Therefore, additional laboratory studies that assess more sublethal
effects and field studies are needed to fully understand the selectivity of the tested insecticides
to C. carnea and A. bipunctata, especially those that were toxic in laboratory.
131 132 Conclusions CONCLUSIONS
Specific conclusions
1. Neither Chrysoperla carnea larvae nor Adalia bipunctata adults significantly enhanced the
dispersal of Aphis gossypii in any of the evaluation periods considered (1, 5, 7 and 14
days). The reduction in the A. gossypii alatae population was always higher for C. carnea
larvae than for A. bipunctata adults.
2. The Cucumber mosaic virus (CMV) transmission rates were low in general due to the
limited spread of its vector Aphis gossypii. There was not a significant effect of the two
aphidophagous predators, Chrysoperla carnea and Adalia bipunctata, in the incidence and
spread of CMV.
3. The Cucurbit aphid-borne yellows virus (CABYV) spread was not significantly affected
by the presence of Chrysoperla carnea and Adalia bipunctata respectively, in either the
short term or the long term experiments.
4. The ingestion of Myzus persicae vectoring Potato leafroll virus (PLRV) by Chrysoperla
externa significantly increased the larval period length and decreased the survival of the
adults, but not affected the pupal stage and the adults reproductive parameters.
5. The low UV radiation conditions seemed to have a direct effect in the early stages of
cucumber plants, enhancing their growth, and in an increasing nitrogen content at further
developmental stages. The higher UV exposure in the early stages of the cucumber crop
induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves.
6. No direct and plant-mediated UV effects were found neither in the development of the
aphid Aphis gossypii nor in the performance of its predator Chrysoperla carnea with the
use of Térmico Antivirus® photoselective cover in the current experimental conditions.
133 Conclusions 7. In the experiment with cucumber plants infected with Cucurbit aphid-borne yellows virus
(CABYV), the presence of the viral disease appeared to mask the direct and plant-mediated
influence of UV radiation.
8. Flonicamid, flubendiamide, metaflumizone and spirotetramat were nontoxic to last instar
larvae and adults of Chrysoperla carnea and Adalia bipunctata when tested by residual
contact on a glass surface under laboratory conditions. These insecticides are promising
candidates to be incorporated into IPM programs in combination with these natural
enemies.
9. Sulfoxaflor was slightly toxic to adults of Chrysoperla carnea and was highly toxic to
fourth instar larvae of Adalia bipunctata when tested by residual contact on a glass surface
under laboratory conditions. In the same conditions, deltamethrin was the most toxic
compound to larvae and adults of both natural enemies. The use of sulfoxaflor and
deltamethrin should be considered when releasing C. carnea and A. bipunctata as part of
an IPM strategy.
General conclusion
The agroecosystem must be considered from a holistic perspective, taking into account that
any environmental or human disruption has a direct or indirect influence on all its parts. There
is no ideal strategy to defend crops against pests and diseases, but the approaches to improve
the implementation of IPM programs are necessary to protect the environment and guarantee
the food supply in a balanced manner.
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