Acta Tropica 81 (2002) 47 – 52
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Aggregation behaviour in Panstrongylus megistus and
Triatoma infestans: inter and intraspecific responses
H.H.R. Pires a,*, M.G. Lorenzo a, L. Diotaiuti a, C.R. Lazzari b,
A.N. Lorenzo Figueiras b
a
Laboratório de Triatomı́neos e Epidemiologia da Doença de Chagas, Centro de Pesquisas René Rachou-FIOCRUZ,
A6. Augusto de Lima 1715, CEP 30190 -002 Belo Horizonte, MG, Brazil
b
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Uni6ersidad de Buenos Aires, Pabellón II,
Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina
Received 29 January 2001; received in revised form 10 September 2001; accepted 17 September 2001
Abstract
We tested the aggregation response to inter and intraspecific chemical signals in Panstrongylus megistus and
Triatoma infestans. As previously described for T. infestans, larvae of P. megistus significantly aggregated on papers
impregnated with their own excrement and on papers marked with cuticular substances deposited on surfaces on
which these insects had walked. T. infestans bugs also aggregated on papers impregnated by faeces or by cuticular
substances of P. megistus, and P. megistus aggregated on papers contaminated by faeces or by cuticular substances
of T. infestans. The response of P. megistus to its cuticular substances was significantly stronger than that to its faeces.
The non-specificity of the two signals is discussed in the context of the ecological relationship between both species.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: Triatominae; Aggregation; Behaviour; Pheromones; Communication; Chagas disease
1. Introduction
Panstrongylus megistus is an haematophagous
insect, which feeds on endothermic vertebrates. It
acts as a vector species for the flagellate Trypanosoma cruzi, which is the causative agent of
Chagas disease. P. megistus is found both in
sylvatic habitats and in rural houses of eastern
Protocols for animal use followed the code of ethics of the
COBEA (Colégio Brasileiro de Experimentação Animal).
* Corresponding author. Fax: +55-31-329-53115.
E-mail address: [email protected] (H.H.R. Pires).
Brazil, and is considered the second most important vector of this disease in Brazil (Silveira et al.,
1984).
Triatoma infestans has been the main vector of
Chagas disease during previous decades, but has
now been virtually eliminated from much of its
range, particularly in Brazil, Uruguay and Chile
(Dias and Schofield, 1999). Many reports showed
an apparent lack of simultaneous colonisation of
human dwellings by both T. infestans and P.
megistus. In certain zones of Brazil, e.g. Minas
Gerais (Dias, 1965) and São Paulo (Silva et al.,
0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 1 - 7 0 6 X ( 0 1 ) 0 0 1 8 5 - 1
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H.H.R. Pires et al. / Acta Tropica 81 (2002) 47–52
1971), where T. infestans was eliminated, P.
megistus colonised the vacant ecotopes.
Aggregation behaviour, as a response to chemical signals, has been reported in several species of
Triatominae (Velázquez Antich, 1968; Baldwin et
al., 1971; Schofield and Patterson, 1977; Ondarza
et al., 1986; Cruz-López et al., 1993; Lorenzo
Figueiras et al., 1994; Manrique and Lazzari,
1995). The specificity of these signals has been
studied by Cruz-López et al. (1993), who reported
that interspecific aggregation signals are present in
the faeces of larvae and adults of five species of
the subfamily Triatominae. Lorenzo Figueiras
and Lazzari (1998a) showed that an aggregating
substance present in the dry faeces of T. infestans,
Triatoma sordida and Triatoma guasayana acts
both inter and intraspecifically. In contrast, according to Neves and Paulini (1982), T. infestans,
P. megistus and T. sordida may emit substances
that repel members of the other two species.
Lorenzo Figueiras and Lazzari (1998b) also
described an aggregation factor in T. infestans, in
addition to the one present in faeces, left by bugs
walking on substrates. These authors proposed
that this species presents a chemical substance in
its cuticle that is transferred onto surfaces and
provokes assembling by arresting the bugs. This
signal constitutes an assembling mark acting as a
chemical footprint.
Previous authors that analysed the aggregation
behaviour of triatomines have shown that the
active compounds of faeces and footprints do not
have a common origin. The available evidence
suggests that they are two different substances or
mixtures of them. Footprints can be extracted by
non-polar solvents (Lorenzo Figueiras and Lazzari, 1998b), while the active compounds in the
faeces elute in polar solvents (Cruz-López and
Morgan, 1995; Lorenzo Figueiras and Lazzari,
1995). Whereas faecal odours are able to attract
bugs when added to an air current, i.e. involve
olfactory chemoreception, footprints require the
physical contact of the insects with the marked
surfaces, suggesting contact chemoreception as
mediating the perception of the stimulus. Other
authors have also demonstrated that cuticular
chemical signals promote aggregation behaviour
in Blatella germanica (Rivault et al., 1998). They
described the substances as cuticular hydrocarbons and indicated that they are better extracted
with non-polar solvents as dichloromethane and
pentane, but not with methanol.
Considering that excrement acts as a chemical
mark for guiding triatomines towards protected
sites (Lorenzo and Lazzari 1996), this landmark
(and the refuges) could be potentially exploited by
other species that would benefit from the interaction. Once inside the refuge, both mechanical
stimulation (thigmotaxis) and footprints could arrest the animals during daytime hours (non-active
time).
In the present study, we analysed the aggregation response of P. megistus to substances present
in their faeces and to cuticular compounds left on
papers by walking insects. We also analysed the
responsiveness of both P. megistus and T. infestans to faeces and footprints of the other species,
to test the existence of attraction or repellence
between them.
2. Materials and methods
2.1. Insects
Our experiments were conducted with larvae of
P. megistus and T. infestans, reared on chicken.
All the insects used for measuring behavioural
responses were 1-week-old 4th instar larvae, i.e.
after ecdysis, and unfed at that stage.
2.2. Sources of stimuli
Faeces from larvae of each species were collected separately on pieces of filter paper (3× 2
cm) placed below plastic containers with recently
fed bugs. Donor insects corresponded to several
larval stages. Faeces released by the bugs fell
down through a plastic mesh closing the containers at the bottom, and were absorbed by the
paper pieces. Excrement was collected during 4
days following feeding. Care was taken in avoiding that the insects could make physical contact
with filter papers. The pieces of filter paper impregnated with faeces were used 3 days after the
end of their collection (dry faeces).
H.H.R. Pires et al. / Acta Tropica 81 (2002) 47–52
To analyse the potential response of the bugs to
cuticular footprints, a group of 20 unfed 3rd
instar larvae of P. megistus or T. infestans were
placed separately in plastic containers with pieces
of filter paper (3×2 cm) inside, and allowed to
walk freely. These insects had the anus sealed with
wax to avoid the deposition of faeces. After 7
days, the insects were removed and the papers
used for the assays.
49
paper with wax versus two clean ones was also
performed with T. infestans, to test the effect of
the wax used to seal the anuses of larvae in this
species (N= 105, k= 9).
After each assay, the arena was cleaned and the
bottom paper replaced by a clean one. Sectors
associated with clean and impregnated papers
were rotated between assays, in order to avoid
any external bias. Each bug was used only once
and then discarded.
2.3. Experimental design
The arena was similar to that previously used
by Lorenzo Figueiras et al. (1994). Briefly, it
consisted of a Petri dish, 13 cm in diameter,
divided in three sectors and lined with a filter
paper. The assays were carried out using a 3×2
cm piece of filter paper placed on each sector. One
paper was impregnated with the test stimulus,
while the other two were clean controls. A group
of larvae (between 9 and 15) was placed in an
inverted container in the centre of the arena and
after 10 min carefully released lifting the recipient
by means of a fine thread. After 1 h, the position
of the bugs was recorded by counting the numbers of insects at each sector.
2.4. Experimental series
Five experimental series were performed in order to test the aggregation response of P. megistus
to: (a) a clean paper in each sector (control assay)
(N = 51, k =5); (b) a paper impregnated with wax
versus two clean ones (control assay for testing
the effect of the wax used to seal the anuses of
larvae) (N = 68, k= 9); (c) a paper impregnated
with faeces of P. megistus versus two clean ones
(N =142, k= 13); (d) a paper impregnated with
cuticular substances of P. megistus versus two
clean ones (N =120, k =8); (e) a paper impregnated with faeces of T. infestans versus two clean
ones (N = 131, k =10) and (f) a paper impregnated with footprints of T. infestans versus two
clean ones (N = 120, k =8). In addition, two series were performed in order to test the aggregation behaviour of T. infestans to: (a) faeces
(N=141, k = 11) and (b) footprints (N = 82, k =
7) of P. megistus. A control series presenting a
2.5. Statistical analysis
For each experimental series the distribution of
the insects in the arena was analysed by means of
a G-test for goodness of fit to a random distribution (i.e. 1/3 for experimental sector and 2/3 for
control sectors). An unpaired t-test or Mann–
Whitney U-statistic was used to compare the aggregation response to faeces or to footprints by P.
megistus and by T. infestans.
3. Results
Figure 1 depicts the intensity of the aggregation
response of P. megistus to their own footprints
and faeces, and to footprints and faeces of T.
Fig. 1. Aggregation response of P. megistus to footprints and
faeces of P. megistus (dash square) or T. infestans (empty
square). The horizontal line indicates the expected value from
a random distribution (33%). Letters indicate the statistical
significance of the aggregation response in comparison to the
corresponding control sectors: (a) PB 0.001; (b) PB0.005.
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H.H.R. Pires et al. / Acta Tropica 81 (2002) 47–52
Concerning the origin of faeces according to the
species, i.e. P. megistus or T. infestans, no difference has been found in their attractiveness to bugs
of the same or the other species (t-test, NS).
The control series testing the effect of wax,
both with P. megistus and T. infestans, showed no
significant aggregation response of the bugs (Gtest, NS).
4. Discussion
Fig. 2. Aggregation response of T. infestans to footprints and
faeces of T. infestans (dash square) or P. megistus (empty
square). The horizontal line indicates the expected value from
a random distribution (33%). Letters indicate the statistical
significance of the aggregation response in comparison to the
corresponding control sectors: (a) PB 0.001.
infestans. The control series presenting three clean
papers showed no significant preference of the
bugs for any sector of the arena (G-test, NS).
Larvae of P. megistus aggregated significantly
around papers impregnated with their own faeces
(G-test, PB 0.005), as well as to papers on which
they had walked (G-test, P B 0.001). P. megistus
also showed significant aggregation on papers impregnated with excrement of T. infestans (G-test,
PB 0.001) and around papers contacted by T.
infestans (G-test, PB 0.001). The response of P.
megistus to their own footprints was significantly
stronger than that to their faeces (Mann– Whitney
P= 0.02; 77.5–45.8%). The same tendency was
observed in the response of the P. megistus to
footprints and faeces of T. infestans (Mann –
Whitney NS; 71.9–53.9%), though that trend fell
short of statistical significance.
Fig. 2 shows the intensity of the aggregation
response of T. infestans to its own faeces and
footprints from data obtained by Lorenzo Figueiras (1997), and to faeces and footprints of P.
megistus, respectively. T. infestans larvae significantly assembled on papers impregnated with excrement of P. megistus (G-test, P B0.001) and
aggregated around papers contacted by the latter
(G-test, PB 0.001) (Fig. 2).
This paper reports the existence of an assembling substance in the dry faeces of P. megistus
and a contact factor that is left on walked substrates, both of which promote aggregation. These
signals from P. megistus and T. infestans show a
similar activity for both species.
Taking into account the results by previous
authors and those obtained in the present report,
we suggest that a common aggregation factor or
blend that promotes aggregation is present in the
faeces of these insects. This implies that
pheromone specificity, at least for aggregation
signals, does not contribute to the isolation of
triatomine species.
The results presented here also suggest that
similar cuticular compounds promoting aggregation are present in both species studied. This
aspect should be analysed for other triatomines to
establish whether the same kind of signal exists
for them.
Several authors reported the isolation between
some triatomine species in the field (Pellegrino
and Brener, 1951; Dias, 1965, 1968; Freitas,
1968). Neves and Paulini (1982) suggested that a
repellent effect exerted by volatiles from T. infestans, T. sordida and P. megistus could be responsible for their isolation. Our results, however,
appear as consistent with those of other authors
that reported reciprocal attractive responses to
aggregation signals in several triatomine species
(e.g. Cruz-López et al., 1993; Lorenzo Figueiras
and Lazzari, 1998a). Thus, the fact that the chemical signals from faeces and footprints attract
both species similarly would indicate that the lack
of symmetry between them can not be explained
H.H.R. Pires et al. / Acta Tropica 81 (2002) 47–52
by mutual repellence. Other possible reasons, for
example, different capabilities for obtaining
blood meals on non-anaesthetised mice have been
reported for these two species (Pereira et al.,
1995), which could be related to the presence of
anaesthetic-like factors in the saliva of T. infestans (Dan et al., 1999). As a consequence, different species could be better or worse adapted to
quickly obtain a blood meal inside housings, a
decisive task in a competition between species for
colonising a given place.
Considering the application of communication
signals in the manipulation of bug’s behaviour in
order to control them, the signals here characterised appear as potential baits for sensors and
traps. In this sense, it deserves to be emphasised
that the response of P. megistus to its footprints
was stronger than that to its faeces, suggesting
that this chemical signal could be a better candidate for its use for triatomine sensors. Therefore, the identification of the active compounds
present in that signal is required in order to
allow its application for that purpose. The detection of triatomine bugs is a fundamental task in
Chagas disease control campaigns. Current methods that include manual detection and cardboard
sensor boxes are applied depending on the vector
species and the region (Garcia Zapata, 1990;
Schofield, 1994). Nevertheless, these are not always effective given that different triatomine species show variations in their behaviour and
environmental preferences. Currently, there is
particular concern about the detection of nondomiciliated triatomines, principally for their capability of re-invading households from wild
locations (Silveira et al., 1984). Vector species as
Triatoma brasiliensis, Triatoma dimidiata and others that re-invade artificial environments, may
require more sensible detection methods (Dias,
1995) to avoid the establishment of new colonies
from few invading individuals. For other insect
pests, chemical baits are used in detection/control
tools of broad application (Foil and Hogsette,
1994; Vale et al., 1986, 1988; Rajendran, 1999),
and which in many cases represent cost-effective
solutions.
51
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