ABSTRACT
UZSÁK, ADRIENN ILDIKÓ. Social Facilitation of Reproduction in the German Cockroach.
(Under the direction of Dr. Coby Schal, Dr. Yasmin J. Cardoza, Dr. Jules Silverman, and Dr.
Wes Watson).
The German cockroach, Blattella germanica, is an economically and medically
important cosmopolitan pest in urban settings. All life stages produce allergens that trigger
human allergies and asthma, they are potential vectors of microbial pathogens, and large
amounts of insecticides are used to control German cockroach populations. Social
interactions among individuals within cockroach aggregations have been shown to lead to
physiological changes such as accelerated female reproduction. Understanding the proximate
mechanisms through which social interactions accelerate reproduction in the German
cockroach is of great interest because it has potential significance in uncovering new targets
for cockroach control.
In the first study, we investigated the effects of social interactions on juvenile
hormone-III (JH) production and on various reproductive events throughout the ovarian cycle
of B. germanica. We show that both JH production and oocyte development are densitydependent; both were stimulated by social interactions with as few as only one conspecific
female, but high density reversed these effects. We found that all JH-dependent events are
influenced by social interactions, including oocyte development, sexual maturation and the
onset of sexual receptivity. We also show that females are most sensitive to social
interactions when 2-3 days old when the corpra allata (CA) produce JH at higher levels.
In the second study, we extended our studies to the effects of social interactions on
the reproductive physiology and behavior of male German cockroaches. We show that social
interactions facilitate certain aspects of male reproductive physiology, such as JH
biosynthesis and protein production by the accessory reproductive glands; however, a
number of other events, such as sexual readiness and mating success are unaffected by social
conditions.
In the third study, we evaluated the sensory modalities that participate in the social
facilitation of female reproduction. We also investigated whether the stimuli responsible for
group effects are species-specific in adult females, and whether these effects are rhythmically
regulated. Females respond to social cues only during their scotophase and 2 hours of social
interactions with a conspecific female is sufficient to accelerate reproduction. We reveal that
tactile stimulation is the primary sensory input associated with social conditions—with no
evidence for involvement of the visual and olfactory systems—and that the antennae play an
important role in the reception of tactile cues. We also show that the stimuli responsible for
group effects are not species-specific in adult females.
In the fourth study, we examined social facilitation of the reproductive rate in B.
germanica females using motor-driven artificial tactile stimulation and we investigated the
key elements, or stimulus features, of antennal contact that evoke this phenomenon. We
establish and validate a motorized system that can deliver tactile stimuli mimicking the
cockroach antenna. Furthermore, we show that tactile stimulation with a broad array of
artificial materials, some deviating significantly from the native antennal morphology, can
facilitate female reproduction.
© Copyright 2012 by Adrienn Ildikó Uzsák
All Rights Reserved
Social Facilitation of Reproduction in the German Cockroach
by
Adrienn Uzsak
A dissertation submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the Degree of
Doctor of Philosophy
Entomology
Raleigh, North Carolina
2013
APPROVED BY:
_______________________________
Dr. Coby Schal
Committee Chair
______________________________
Dr. Yasmin J. Cardoza
________________________________
Dr. Jules Silverman
________________________________
Dr. Wes Watson
DEDICATION
To my dearest family and closest friends that I have been fortunate enough to have been
blessed with.
ii
BIOGRAPHY
Adrienn Ildikó Uzsák was born in Budapest, Hungary on December 20, 1980. Along
with her sister Edina, she was raised in Gödöllő by her parents, Miklós and Mária, who
taught her the love of plants, animals, and the environment that surrounds us and soon she
began to develop her passion for Nature.
In 2000, she enrolled to the University of Debrecen to study Biology. Beside the
required courses, she took many additional ones in different fields and she also participated
in plant management in the Botanical Garden and in the organization of the insect collection
at the Entomology Department. This was the time when she really started to fall for insects.
She was amazed by their diversity and she was so eager to study their behavior. She also
realized that the English language plays a crucial role in science writing. Ultimately, in 2008,
Adrienn received her Master of Science in Biology with a minor in Ecology, as well as a
Master of Science in Specialized English Translation.
Adrienn joined North Carolina State University in January 2009 to pursue a Ph.D.
degree in Entomology. Under the direction of Dr. Coby Schal, she studied differential
physiological changes due to social facilitation of reproduction in the German cockroach and
she investigated the sensory modalities and pathways that mediate these changes. In
collaboration with engineers from the Electrical and Computer Engineering Department,
NCSU, she also established and validated an artificial motorized system to test sensory
modalities involved in this phenomenon. In 2012, Adrienn received the Outstanding Ph.D.
Student Award from the North Carolina Entomological Society.
iii
ACKNOWLEDGEMENTS
This dissertation would not have been possible without the expertise and support of
several people who contributed in many ways to the completion of my studies. I owe my
deepest gratitude to my advisor, Dr. Coby Schal for his support, patience, and
encouragement. His professional and editorial advice was essential and taught me valuable
insights about academic research.
Sincere thanks go to the members of my advisory committee, Dr. Yasmin J. Cardoza,
Dr. Jules Silverman, and Dr. Wes Watson for their valuable advice and guidance to improve
my work and for providing critical reading of and suggestions on my dissertation.
My thanks also go to past and current members of the Schal lab, especially to my
great postdoctoral fellows, Katalin Böröczky, Alonso Suazo-Calix, and Ayako WadaKatsumata for their precious time and expertise with which they greatly enhanced my
scientific knowledge. I also thank to Rick Santangelo for his endless technical support.
I am thankful to my collaborators from the Bozkurt lab at the Electrical and Computer
Engineering Department, NCSU, Dr. Alper Bozkurt, James Dieffenderfer, and Tahmid Latif
for their input into our project together.
I express gratitude to the Department of Entomology, to Consuelo Arrellano for
statistical support, and to sources of funding for my project, the Blanton J. Whitmire
endowment and the Structural Pest Management Training and Research Facility.
I owe so much to my family, who has been understanding and supportive of my
pursuit for a higher education. Despite the distance of thousands of miles between us, I am
conscious of their love and support. I will eternally be grateful to my parents and my sister.
iv
I also thank to friends who have helped balance my life and provided encouragement
throughout such a demanding period of my life. Finally, my heartfelt thanks goes to my
boyfriend, Zach Miller for his understanding, encouragement, and unconditional love in the
past few years, and I only hope I can be an anchor for him, as he has been for me.
v
TABLE OF CONTENTS
LIST OF FIGURES ................................................................................................................. xi
LITERATURE REVIEW ......................................................................................................... 1
Social interactions and the Allee effect in gregarious species........................................... 2
Endocrine control in social facilitation of reproduction .................................................... 5
Sensory modalities in social facilitation of reproduction .................................................. 6
The impact of the German cockroach on human health and the environment .................. 7
Overview of the dissertation .............................................................................................. 8
Future directions ................................................................................................................ 9
References ........................................................................................................................... 12
CHAPTER 1. Differential physiological responses of the German cockroach to social
interactions during the ovarian cycle ...................................................................................... 22
ABSTRACT ........................................................................................................................ 23
INTRODUCTION ............................................................................................................... 24
MATERIALS AND METHODS ........................................................................................ 27
Insects .............................................................................................................................. 27
Oocyte dissection and measurements .............................................................................. 28
Effects of female density on oocyte maturation and juvenile hormone biosynthesis ..... 28
Time course of oocyte maturation in isolated and paired females in the first and second
ovarian cycles .................................................................................................................. 29
Effects of social isolation and social interactions on sexual receptivity ......................... 30
Differential responses of females to duration of social interactions ............................... 31
vi
Responses to social interactions during gestation ........................................................... 32
Statistical analyses ........................................................................................................... 33
RESULTS............................................................................................................................ 34
Effects of female density on oocyte maturation and juvenile hormone biosynthesis ..... 34
Time-course of oocyte maturation in socially isolated and paired females in the first and
second ovarian cycles ...................................................................................................... 35
Effects of social isolation and social interactions on sexual receptivity ......................... 36
Differential responses to duration of social interactions during the first preoviposition
period ............................................................................................................................... 36
Effects of social isolation and social interactions during the gestation period ................ 37
DISCUSSION ..................................................................................................................... 38
ACKNOWLEDGMENTS................................................................................................... 46
REFERENCES .................................................................................................................... 47
FIGURES ............................................................................................................................ 52
CHAPTER 2. Social interaction facilitates reproduction in male German cockroaches,
Blattella germanica ................................................................................................................. 59
ABSTRACT ........................................................................................................................ 60
MATERIALS AND METHODS ........................................................................................ 66
Insects .............................................................................................................................. 66
JH-III biosynthesis by the CA ......................................................................................... 67
Bla g 4 protein content of the ARG ................................................................................. 68
Quantification of cuticular hydrocarbons ........................................................................ 68
vii
Courtship readiness.......................................................................................................... 69
Copulation and mating success........................................................................................ 70
Data analyses ................................................................................................................... 70
RESULTS............................................................................................................................ 71
Social facilitation of JH-III synthesis in paired B. germanica males .............................. 71
Social facilitation of ARG protein ................................................................................... 72
Accumulation of CHC in males is not socially facilitated .............................................. 72
Effects of social conditions on male courtship behavior ................................................. 73
Social facilitation of copulation success in males ........................................................... 73
DISCUSSION ..................................................................................................................... 75
ACKNOWLEDGMENTS................................................................................................... 81
REFERENCES .................................................................................................................... 83
FIGURES ............................................................................................................................ 94
CHAPTER 3. Sensory cues involved in social facilitation of reproduction in Blattella
germanica females ................................................................................................................ 102
ABSTRACT ...................................................................................................................... 103
INTRODUCTION ............................................................................................................. 104
MATERIALS AND METHODS ...................................................................................... 109
Insects ............................................................................................................................ 109
Oocyte dissection and measurements ............................................................................ 109
Diel periodicity of the reproductive response to social interactions ............................. 110
The effects of visual cues on reproductive rate ............................................................. 111
viii
The effects of chemical cues on reproductive rate ........................................................ 111
Species-specificity of the “grouping effect” in B. germanica ....................................... 112
The effects of tactile cues on the reproductive response ............................................... 113
Function of the antennae in receiving cues that stimulate reproduction ....................... 115
Statistical analyses ......................................................................................................... 116
RESULTS.......................................................................................................................... 116
Social facilitation of reproduction is more effective in the scotophase ......................... 116
Chemical and visual cues appear to not play a role in social facilitation of reproduction
....................................................................................................................................... 117
Effects of social interactions with different insect species on reproductive rate ........... 118
Antennal contact mediates the social facilitation of reproduction in B. germanica ...... 118
The antennae receive cues that modulate the reproductive rate in B. germanica ......... 120
DISCUSSION ................................................................................................................... 121
Sensory inputs that facilitate reproduction in B. germanica ......................................... 122
How do females receive the mechanosensory information relevant to the “grouping
effect”?........................................................................................................................... 126
What sensory-CNS-neuroendocrine pathways are involved in social facilitation of
reproduction? ................................................................................................................. 127
ACKNOWLEDGMENTS................................................................................................. 129
REFERENCES .................................................................................................................. 131
FIGURES .......................................................................................................................... 140
CHAPTER 4. Social facilitation of insect reproduction with motor-driven tactile stimuli .. 148
ix
ABSTRACT ...................................................................................................................... 149
INTRODUCTION ............................................................................................................. 150
MATERIALS AND METHODS ...................................................................................... 153
Insects and rearing conditions ....................................................................................... 153
Ovary dissection and oocyte measurements .................................................................. 153
Effects of tactile cues on the reproductive response to social interactions ................... 154
Motorized tactile stimulation system ............................................................................. 154
Validation and use of the motorized system .................................................................. 155
Fabrication of synthetic antennae .................................................................................. 157
Statistical analyses ......................................................................................................... 158
RESULTS.......................................................................................................................... 159
Antennal contact mediates the social facilitation of reproduction ................................ 159
Validation of the motorized tactile stimulation system ................................................. 160
Stimulus features related to artificial “antenna” movement .......................................... 160
Stimulus features related to artificial “antenna” morphology ....................................... 161
DISCUSSION ................................................................................................................... 162
ACKNOWLEDGMENTS ................................................................................................. 168
FIGURES .......................................................................................................................... 174
x
LIST OF FIGURES
CHAPTER 1. .......................................................................................................................... 22
Figure 1. The effect of female density on reproductive rate in B. germanica. ................... 52
Figure 2. Time-course of oocyte maturation in B. germanica females during the
preoviposition period of the first and second ovarian cycles. ............................................. 53
Figure 3. Effects of social isolation and group-housing on the onset of sexual receptivity in
B. germanica females. ......................................................................................................... 54
Figure 4. Experimental design of assays to determine whether certain stages during the
preoviposition period are more or less affected by transient social isolation or transient
social interactions. ............................................................................................................... 55
Figure 5. Differential responsiveness of B. germanica females to social interaction and
social isolation during the preoviposition period. ............................................................... 56
Figure 6. Effects of social isolation and group-housing on oocyte length at the end of the
first gestation period. ........................................................................................................... 58
Figure 1. The effect of B. germanica adult male density on JH-III biosynthesis. .............. 94
Figure 2. In vitro JH-III release rates during the first 19 days of adult B. germanica males.
............................................................................................................................................. 95
Figure 3. Social facilitation of protein accumulation in B. germanica adult male accessory
reproductive glands. ............................................................................................................ 96
xi
Figure 4. Effects of social isolation and social interactions on the amount of hydrocarbons
on the cuticular surface of B. germanica adult males. ........................................................ 97
Figure 5. Effects of isolation and social interactions on courtship behaviour in B.
germanica males. ................................................................................................................ 98
Figure 6. Effects of social isolation and social interactions in B. germanica adult males on
the frequency and latency of copulation, and the frequency of egg case abortion. .......... 100
CHAPTER 3. ........................................................................................................................ 102
Figure 1. Response of B. germanica females to transient social interaction during the
photophase and scotophase. .............................................................................................. 140
Figure 2. Effects of visual and chemical stimuli on the reproductive rate of B. germanica
females. ............................................................................................................................. 141
Figure 3. Effects of social interaction with different insect species on the reproductive rate
of B. germanica females.................................................................................................... 142
Figure 4. Effects of contact stimuli on the reproductive rate of B. germanica females. ... 143
Figure 5. Effects of interaction with the antennae on the reproductive rate of B. germanica
females. ............................................................................................................................. 145
Figure 6. Function of the antennae in receiving social cues that stimulate reproduction. 147
CHAPTER 4. ........................................................................................................................ 148
Figure 1. Motorized tactile stimulation system showing the power supply (right), controller
(center), and 20 Petri dishes each mounted on a stepper motor (top and bottom). ........... 174
xii
Figure 2. Effects of interaction with P. americana antennae only on the reproductive
maturation of B. germanica females. ................................................................................ 175
Figure 3. Validation of the motorized tactile stimulation system. .................................... 176
Figure 4. Dose-response histogram for the effects of motor speed and duration of
stimulation on the reproductive rate of B. germanica females. ........................................ 177
Figure 5. Evaluation of the significance of the structural complexity of an artificial tactile
stimulus on the reproductive rate of B. germanica females. ............................................. 178
Figure 6. Evaluation of the significance of the flexibility and thickness of artificial tactile
stimuli on the reproductive rate of B. germanica females. ............................................... 179
xiii
LITERATURE REVIEW
1
Social interactions and the Allee effect in gregarious species
Living in groups is critical for various adaptive behaviors and may provide various direct
fitness benefits such as increased foraging efficiency, decreased risk of predation, or
increased encounters with potential mates (Wilson, 1971; Krause and Ruxton, 2002; review:
Blumstein et al., 2010). When closely related individuals share these benefits, then groupliving potentially can increase indirect fitness of group members as well (Hamilton, 1964). A
positive correlation between mean overall fitness and population size or density has been
referred to as the Allee effect (Allee and Bowen, 1932). However, group-living is not always
beneficial; costs of grouping can include competition over limited resources, increased risk
of predation, inbreeding depression, sex ratio bias, and greater pathogen and disease
transmission (Krause and Ruxton, 2002).
Social interactions among individuals within a group can also lead to morphological,
physiological, and behavioral changes that can result, for example, in faster development and
reproduction. This phenomenon—known as “social facilitation” or “grouping effect”
(Grassé, 1946; Gervet, 1968)—is well described in single-celled and multi-cellular
organisms. For example, in quorum-sensing and biofilm forming bacteria, cell-cell
communication leads to the formation of protective biofilms in order to tolerate
environmental changes (review: Elias and Banin, 2012). When resources are depleted,
individual amoebae of the cellular slime mold Dictyostellium discoideum aggregate to form a
multicellular migratory slug, find a suitable location, and produce a fruiting body that
facilitates spore dispersal (Marée and Hogeweg, 2001). Group-induced phenotypic plasticity,
2
or “phase-polyphenism”, is well documented in some insect species. In locusts, for example,
exposure to a group shifts individuals from solitarious to gregarious coloration, morphology,
and behavior (Ellis, 1963; Roessingh and Simpson, 1994; Bouaichi et al., 1995; Lester et al.,
2005). In other insect species social interactions can affect larval or adult morphology,
resulting in the production of winged forms in aphids (Johnson, 1965; Lees, 1967;
Sutherland, 1969) and wing dimorphism in plant hoppers (Iwanaga and Tojo, 1986) and
crickets (Zera and Tiebel, 1988). Many examples have emerged of social facilitation of the
rates of growth and development in insects, and prominent among these are species where
nymphs (e.g., cockroaches and crickets) or larvae (e.g., moth caterpillars) reared in groups
develop faster than isolated or solitary individuals (Chauvin, 1946; Long, 1953; Wharton et
al, 1986; Izutsu et al., 1970; Woodhead and Paulson, 1983; McFarlane et al., 1984; Weaver
and McFarlane, 1990; Holbrook and Schal, 1998; Lihoreau and Rivault, 2008; Ronnas et al.,
2010).
Behavioral changes in foraging, mate finding, mate choice, and reproduction in
response to social interactions have been documented in both vertebrate and invertebrate
species (Wilson, 1971; review: Clayton, 1978; Krause and Ruxton, 2002; Pulliam and
Caraco, 1984). For example, in some social vertebrate species only dominant individuals
mate (e.g., Hodge et al., 2008), and such is the case in social hymenoptera where the queen
produces specific pheromones that inhibit reproductive maturation in workers and maintain a
division of labor within the colony (review: Le Conte and Hefetz, 2008). Male facilitation of
female sexual maturation has also been shown in a number of vertebrate and insect species as
well (review: Rekwot at al., 2001). Social facilitation of reproduction in non-social insects is
3
less prevalent (review: Rekwot et al., 2001) but it has been reported in orthopterans (review:
Pener and Simpson, 2009). For example, in the desert locust, Schistocerca gregaria, crowded
adults (gregarious) reach sexual maturation earlier than those reared in isolation (solitarious)
(Norris, 1954). Solitary S. gregaria females show a significantly higher preference for
ovipositing on plants with egg pods laid by conspecific gregarious females than on plants
without egg pods, showing a gregarizing effect on the progeny (Bashir et al., 2000). Socially
facilitated oviposition has also been shown in mosquitoes and flies (e.g., Davis, 1998;
Prokopy and Reynolds, 1998; Reiter, 2007). Moreover, in some insect species, the presence
of individuals of various developmental stages, both sexes, and even different species has a
facilitating effect on reproduction (Lees, 1967; Bradley, 1985; review: Braendle et al., 2006;
Allen, 2010).
In many insects, copulation stimulates reproduction in females, due to either social
stimuli or male transfer of modulatory peptides into the female’s reproductive tract (reviews:
Wolfner, 1996; Gillott, 2003; Avila et al., 2011). Reproductive effects due to non-copulatory
social interactions are much less common in non-polyphenic species, and have been
investigated in the German cockroach, Blattella germanica and the brownbanded cockroach,
Supella longipalpa (Gadot et al., 1989b; Chon et al., 1990). In both species, females exhibit
faster oocyte development due to social stimuli during copulation; however, only in the
German cockroach do females respond to social stimuli associated with the presence of
conspecifics. When reared in groups B. germanica females exhibit faster oocyte development
than solitary-reared females (Gadot et al., 1989b; Holbrook et al., 2000). However, the
4
proximate mechanisms involved in social facilitation of reproduction in B. germanica have
not been investigated.
Endocrine control in social facilitation of reproduction
The juvenile hormones (JH) play a pivotal role in insect development and reproduction
(Raikhel et al., 2005; Goodman and Cusson, 2012). JH has been well investigated as the
major regulatory hormone of reproductive events in a wide array of insect taxa (Raikhel et
al., 2005). As a major gonadotropic hormone, JH is produced and released by the corpora
allata (CA). In the German cockroach, JH production shows a stage-specific pattern: it
steadily increases after adult eclosion, declines through ovulation and remains low during the
gestation period (Bellés et al., 1987; Gadot et al., 1989a). JH stimulates vitellogenesis and a
single basal oocyte matures in each ovariole; however, during gestation, oocyte development
is suppressed via inhibition of the CA by the central nervous system (CNS), in response to
signals from the externally incubated ootheca (Roth and Stay, 1962; Burns et al., 1991; Liang
and Schal, 1994; Schal and Chiang, 1995). Finally, at the end of gestation, JH biosynthesis
increases again and the new basal oocytes start to develop as well (Gadot et al., 1989a; Gadot
et al., 1989b; Schal and Chiang, 1995). JH has been shown to regulate female reproductive
physiology in the German cockroach, including the synthesis and uptake of vitellogenin and
other female-specific proteins, onset of sexual receptivity, production of sexual signals,
mating, and the time-course of oviposition (Bellés et al., 1987; Gadot et al., 1989a; Burns et
al., 1991; Schal and Chiang, 1995; Schal et al., 1997). The potential effects of social
5
interactions on JH synthesis, and with that on female reproduction, have not been
investigated.
Sensory modalities in social facilitation of reproduction
Social interactions can modulate the reproductive physiology of individuals through various
sensory pathways, including visual, auditory, olfactory, gustatory and tactile sensing. For
example, acoustic stimulation attracts colonial birds to new colony sites, which increases
reproductive behaviors and per capita reproductive success increases as colony size increases
(Clark et al., 2012). Similarly, in flamingos, visual cues stimulate pair formation, nesting and
breeding by group displays of large flocks (Stevens and Pickett, 1994). The sight of
displaying males stimulate oocyte development in female lizards (Crews, 1975), while
females of the green swordtail, Xiphophorus helleri, mature faster when they see high-quality
males (determined by ornament size) (Walling et al., 2007). The importance of pheromones
in the context of male facilitation of female sexual maturation has been widely studied in
mammals and insects (review: Rekwot et al., 2001). The most prominent examples of
chemical cues affecting reproduction can be found in eusocial insects, where pheromones are
crucial for the coordination and integration of the colony, facilitating caste differentiation,
division of labor, and foraging activities (review: Robinson, 1992).
The importance of tactile cues in social facilitation of reproduction is probably best
described in locusts. Tactile stimulation is the main sensory modality responsible for adult
phase change in S. gregaria (Roessingh et al., 1998; Hägele and Simpson, 2000; Simpson et
6
al., 2001; Rogers et al., 2003) and for maternal determination of progeny characteristics
where grouping causes isolated females to produce gregarious offspring (Maeno, et al., 2011;
Maeno et al., 2012). In cockroaches, the sensory stimuli involved in the social facilitation of
nymphal development have been investigated in B. germanica and in the American
cockroach, Periplaneta americana, where tactile cues from conspecifics and even from
different taxa accelerate development in nymphs (Wharton et al., 1968; Izutsu et al., 1970;
Nakai and Tsubaki, 1986; Lihoreau and Rivault, 2008). However, the nature of the sensory
stimuli in the social facilitation of reproduction in B. germanica, the mechanism(s) by which
the perceived stimuli are processed as social signals, and the CNS pathways that couple
social cues and JH production remain unknown.
The impact of the German cockroach on human health and the environment
The German cockroach is both economically and medically important, having a significant
impact on humans and livestock. It is closely associated with human-built structures in
agricultural and urban environments, where cockroaches are a significant source of indoor
allergens. Potent allergens produced by German cockroaches have been linked to the
development and aggravation of asthma and other respiratory diseases (Arbes et al., 2005),
especially in relatively closed environments, resulting in an important risk factor in homes,
schools and hospitals. Although there is no definitive evidence that cockroaches are direct
vectors of pathogens, they can carry and transfer pathogens either mechanically or in their
digestive system while moving between sewers and human food materials (review: Schal and
7
Hamilton, 1990; Rivault et al., 1993; Pai et al., 2003; Ahmad et al., 2011). Since large
infestations may increase their potential role in human disease, effective suppression of
cockroach populations is needed to reduce health-related problems. Extensive use of
insecticides for cockroach control in the U.S. and globally has been associated with
environmental concerns and insecticide resistance. Thus, there is a need to develop safe,
effective, and environmentally compatible insect control techniques targeting groups of
cockroaches. Understanding the effects of social interactions on cockroach reproduction is
important, not only because it will delineate basic circuits in insect neuroethology, but
because it has potential significance in uncovering new targets for cockroach control.
Overview of the dissertation
My dissertation focuses on understanding the proximate mechanisms through which social
interactions accelerate reproduction in the German cockroach. In Chapter 1, I describe the
effects of social interactions on JH production and on the reproductive rate of B. germanica
throughout the ovarian cycle. In Chapter 2, I investigate whether reproduction is facilitated
by social interaction in German cockroach males. In Chapter 3, I identify the sensory
modalities that participate in the social facilitation of female reproduction. I also investigate
whether the stimuli responsible for group effects are species-specific in adult females, and
whether these effects are rhythmically regulated. And finally, in Chapter 4, I dissect the
tactile sensory channel, focusing on antennal contact, which I determined to be the main
sensory modality responsible for accelerated reproduction in B. germanica females.
8
Together, this research comprises the most thorough and integrative study of the mechanisms
of social facilitation in a non-polyphenic insect species.
Future directions
The brain integrates a multitude of extrinsic and intrinsic stimuli, including grouping stimuli,
and paces the activity of the CA, dictating the rate and magnitude of all JH-dependent events
(Schal and Chiang, 1995, Schal e al., 1997). How tactile stimuli get transduced into neuronal
and neuroendocrine signals that regulate CA activity and JH titer in B. germanica is
unknown. Neuroregulation of reproduction has been studied in many insect species. Biogenic
amines have been found to function as regulatory factors, for example, in reproductive
division of labor (e.g. Bloch et al., 2000), in ovarian and egg development (e.g. Neckameyer,
1996; Pendleton et al., 1996; Andersen et al., 2006) and in ootheca formation (Owen and
Foster, 1988). In the German cockroach fluctuations in biogenic amine levels were reported
to be significant in correlation with JH production and JH-regulated oocyte growth (Pastor et
al., 1991a; Pastor et al., 1991b), which are known to be indirectly modulated by social
interactions (Gadot et al., 1989a; Schal et al., 1997, Holbrook et al., 2000; Uzsák and Schal,
2012). A relationship between brain biogenic amines and social interactions has been studied
in females of the gregarious cockroach Blaberus craniifer, where biogenic amine levels are
generally higher in the brain of individuals kept in isolation after eclosion compared to those
of crowded females through the first ovarian cycle (Goudey-Perriere et al., 1992).The
transduction pathway has been best described in S. gregaria, where tactile stimulation of
9
specific mechanoreceptive sensilla on the hind legs causes an increase in serotonin levels in
the metathoracic ganglion which appears to mediate the process of phase transition and
behavioral gregarization (Rogers et al., 2003; Rogers et al., 2004; Anstey et al., 2009).
Scientific knowledge about the neurochemistry of the German cockroach, however, is
extremely limited. Perhaps social tactile stimuli affect neurons expressing different
neurochemicals and the neurochemical changes in the CNS lead to accelerated reproduction
in B. germanica females. Thus, identification of the sensory-CNS-neuroendocrine
transduction pathways that mediate the socially-induced acceleration of reproduction in this
species is of great interest. A comprehensive effort to increase our understanding of the
mechanism could add an important new dimension to cockroach pest management as well.
How mechanosensory information relevant to the “grouping effect” is received by B.
germanica females is also not well understood. The neural circuits that integrate
mechanosensory stimuli associated with grouping might include exteroceptive signals from
the surface of the antenna and proprioceptive signals that naturally result from the inward
displacement of the antenna on contact with a conspecific. The inputs to these neuronal
circuits come from mechanosensory structures on the antenna (i.e., hairs). These structures,
however, have multiple types and are differentially distributed across various antennal
segments, and arranged as single sensilla or as specialized arrays of mechanosensory units
(e.g., hair plates) (Seelinger and Tobin, 1981), making it difficult to determine where and
how socially relevant tactile stimuli are received. It would be important to know whether
there is mechanosensory specialization in the antennae in terms of the types of
mechanosensory structures that respond to social stimuli, as well as their distribution.
10
Another difficulty is that the effectiveness of tactile cues in a social context depends on
whether the receiver is able to identify the source of the stimulus by being responsive to
specific stimulus characters (e.g. shape, texture, biomechanics). Studies testing stimulus
characters that guide an individual’s behavior date back to Niko Tinbergen who used
cardboard models to define the key elements of relevant stimuli (Tinbergen 1951). However,
such studies have been heavily biased in the visual, auditory, and chemosensory modalities
since stimuli associated with these modalities are relatively easy to mimic. On the other hand,
to disentangle social tactile stimulus characters is considerably more challenging because the
insect antenna is a multi-sensory organ and tactile cues often involve complex and contextdependent active movements by both the signaler’s and the receiver’s antennae (review:
Staudacher et al., 2005), making it difficult to isolate and quantify the salient tactile features
that elicit behavior. By decoding the tactile stimulus characters we could identify neuronal
and endocrine circuit elements in the transduction pathway through which mechanoreceptors
communicate relevant tactile cues through the antennal nerve to the CNS which ultimately
accelerate female reproduction in B. germanica by disinhibiting the activity of the CA.
This framework predicts the conditions under which we should be able to enhance
our understanding of the mechanisms of socially-mediated facilitation of reproduction in B.
germanica females. However to understand the CNS pathways that couple social cues and JH
production, we urge future studies to move beyond investigating sensory reception, and
instead design studies that examine how stimuli get transduced into neuronal and
neuroendocrine signals that regulate CA activity and JH titer using perhaps real-time
measurements in the CNS.
11
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21
CHAPTER 1.
Differential physiological responses of the German cockroach to social interactions
during the ovarian cycle
(This work was published in the Journal of Experimental Biology: Uzsák, A. and Schal, C.
2012. Differential physiological responses of the German cockroach to social interactions
during the ovarian cycle. Journal of Experimental Biology, 215: 3037-3044. DOI:
10.1242/jeb.069997)
22
ABSTRACT
In many animal species social interactions can influence the morphology, physiology, and
behavior of individuals, including their rate of development and reproduction. Reproduction
in cockroaches is regulated by juvenile hormone III (JH) and social interactions have been
shown to accelerate female reproduction in the German cockroach, Blattella germanica
(Linnaeus), by stimulating JH production. However, it is not clear in this or any other insect
species whether social facilitation of the reproductive rate occurs throughout the ovarian
cycle or only at certain stages. We compared the effects of social interactions during the preoviposition period when JH production is high and during gestation when little JH is
produced, as well as during the first ovarian cycle when females are virgin and the second
ovarian cycle after females had mated. Social interaction with one conspecific female was
sufficient to accelerate JH production and oocyte maturation, but this effect was reversed by
crowding. Social interactions also accelerated the onset of sexual receptivity in virgin
females. However, social interactions failed to shorten gestation, suggesting that social cues
stimulate JH production only when the corpora allata (CA) are active and not when CA
activity is suppressed by the central nervous system. Females were most responsive to
transient social isolation and transient social interactions when 2–3 days-old, suggesting that
they are particularly sensitive to social interactions when their CA become active. Overall,
these results show that all JH-dependent events in the reproductive cycle of B. germanica
females are under the strong influence of social interactions.
23
INTRODUCTION
The onset and pace of reproduction in most animals are dependent upon specific internal and
external stimuli. Internal stimuli include the animal’s nutritional state, blood composition and
osmolarity, and whether it has mated. External stimuli include environmental conditions
(e.g., temperature, humidity, photoperiod, and host plants) and social interactions
(Engelmann, 1970; Gilmore and Cook, 1981). In many animal species, social interactions can
affect fitness by altering the morphology, physiology, and behavior of individuals, thus
influencing growth, development, or reproduction (Krause and Ruxton, 2002; Wilson, 1971).
Behavioral modulation of reproduction is expected in social animals. For example, in some
vertebrate social systems only dominant individuals mate (e.g. Hodge et al., 2008), and in
some eusocial insects the queen uses pheromones to inhibit reproductive maturation in
workers (reviewed in Le Conte and Hefetz, 2008).
Social facilitation or social suppression of reproduction is less common in
facultatively gregarious or solitary animals. Nevertheless, here too, some examples of
reproductive effects due to the presence of conspecifics have been documented. For example,
interaction of adult females with sexually mature males has been shown to accelerate the
onset of reproduction in a number of vertebrate species and some insects (reviewed in
Rekwot et al., 2001). Social interactions dramatically shift the desert locust from solitary to
gregarious phase, and social interactions also influence female reproduction; solitary females
are preferentially attracted to vegetation containing eggs of gregarious females, resulting in
phase-dependent gregarization of their progeny (Bashir et al. 2000). Moreover, in some
24
insect species, reproduction can be accelerated in females by the presence of individuals of
various developmental stages, both genders, and even different species (e.g. Lees, 1967;
Bradley, 1985; reviewed in Braendle et al., 2006; Allen, 2010).
Previous studies on social facilitation in cockroaches have focused on nymphal
development, where social interactions greatly accelerate the pace of development (Roth and
Willis, 1960; Wharton et al., 1968; Izutsu et al., 1970; Woodhead and Paulson, 1983;
Lihoreau and Rivault, 2008). Little is known, however, about socially-facilitated
physiological changes in adult cockroaches. This phenomenon has been investigated only in
the German cockroach, Blattella germanica and the brownbanded cockroach, Supella
longipalpa. Whereas females of both species respond to mating stimuli with faster oocyte
maturation, only B. germanica females, and not S. longipalpa females (Chon et al., 1990),
respond to social interactions with a shorter preoviposition period and hence faster
reproduction (Gadot et al., 1989b; Holbrook et al., 2000).
The German cockroach represents a unique system for studies of insect reproduction.
Females exhibit a reproductive pattern that is functionally intermediate between oviparity
and ovoviviparity (Roth and Stay, 1962). Females undergo a period of sexual maturation
after eclosion, followed by mating. Like oviparous cockroaches, the female ovulates and
extrudes an egg case. However, unlike oviparous species, the egg case is then rotated 90o and
retained externally by the female, attached at the vestibulum (an outer chamber that lies
above the enlarged seventh abdominal sternite), until the nymphs hatch approximately 21–22
days later at 27oC (Roth and Stay, 1962). This incubation period is functionally similar to
gestation in ovoviviparous cockroaches, as the corpora allata (CA) are inhibited in both
25
groups, suppressing juvenile hormone III (JH) production (Tobe and Stay, 1985; Gadot et al.,
1989a; Gadot et al., 1989b; Gadot et al., 1991; reviews: Schal et al., 1997; Treiblmayr et al.,
2006). As the major gonadotropic hormone, JH is produced and released by the CA in a
stage-specific manner and it paces the female’s reproductive rate. The JH biosynthetic rate
increases after eclosion, stimulating vitellogenesis and its uptake by the oocytes, declines
through ovulation, and remains low during gestation (Belles et al., 1987; Gadot et al., 1989a).
Juvenile hormone also stimulates the production and release of the attractant sex pheromone
blattellaquinone (Liang and Schal, 1994; Nojima et al., 2005) and a courtship-inducing
contact sex pheromone blend (Eliyahu et al., 2008), the production of accessory gland
proteins that form the egg case (Burns et al., 1991), and JH controls the expression of sexual
receptivity in the female (Schal and Chiang, 1995).
In B. germanica, oocyte development is cyclic and interrupted by a protracted
gestation. Its panoistic paired ovaries each comprises approximately 20 ovarioles, and only a
single basal oocyte in each ovariole (i.e., next to be oviposited) matures during the
preoviposition period (Roth and Stay, 1962). At oviposition, the penultimate oocyte becomes
the new basal oocyte, but its growth is suppressed during most of the 21-day gestation
period. This suppression is due to CNS inhibition of CA activity, originating from
mechanosensory signals associated with the presence of the externally incubated egg case in
the genital vestibulum (Roth and Stay, 1962; Liang and Schal, 1994; Schal and Chiang,
1995). Thus, during gestation JH production remains low, but at the end of gestation, before
egg hatch, JH titers slightly increase and the basal oocytes grow slightly as well (Gadot et al.,
1989a; Gadot et al., 1989b). The average length of the basal oocytes is a reliable measure of
26
the reproductive stage because all basal oocytes mature synchronously and their length
correlates well with both the activity of the CA (as measured by their JH biosynthesis in
vitro), and with JH titer in the hemolymph (as measured by GC-MS) (Sevala et al., 1999;
Treiblmayr et al., 2006).
In this study we sought to determine whether social interactions could hasten the
reproductive rate throughout the ovarian cycle, or only during certain limited stages of the
cycle. We hypothesized that the ovarian cycle of B. germanica would be affected most by
social interactions when the CA are active and produce JH, and not when the CA are
practically inactive during gestation. First, we showed that both JH production and basal
oocyte growth were density-dependent: social interactions increased both, but crowding at
high female density reversed these effects. We also found that social interactions stimulated
oocyte maturation in both virgin and mated females. In order to determine if only JHdependent events are influenced by social interactions we investigated the effects of social
interactions at different periods of the ovarian cycle.
MATERIALS AND METHODS
Insects
A laboratory colony of insecticide-susceptible German cockroaches (American Cyanamid
strain) was maintained at 27 ± 1ºC, ambient relative humidity (40–70%), a photoperiod of
12L:12D, and provided with shelter, water and food pellets (LabDiet 5001 Rodent Diet, PMI
27
Nutrition International, Brentwood, MO, USA). This large, developmentally synchronous
colony permitted the use of many newly eclosed females from a single cohort in most
experiments. Newly-eclosed (day 0) adult females of similar size and degree of sclerotization
with intact wings were selected for each experiment. Experiments were conducted under the
same conditions as described above, controlled temperature and photoperiod, with water and
food available ad libitum.
Oocyte dissection and measurements
Females were ice-anesthetized and dissected under cockroach saline (Kurtti and Brooks,
1976) and the ovaries were removed. Each ovary contains approximately 20 ovarioles and
only a single basal oocyte in each ovariole takes up yolk proteins. Because all 40 basal
oocytes in the two ovaries grow synchronously, 10 randomly selected basal (vitellogenic)
oocytes were measured with an ocular micrometer in the eyepiece of a dissecting
microscope. Measured oocyte lengths were averaged for each female, but because all basal
oocytes of B. germanica mature synchronously, there was little variation in oocyte length
within each female.
Effects of female density on oocyte maturation and juvenile hormone biosynthesis
The effect of female density on oocyte development was investigated by placing 1, 2, 8, or
20 newly-eclosed females in a plastic Petri dish (90 mm diameter, 15 mm high, Fisher
28
Scientific, Pittsburgh, PA, USA) with food and water for 6 days. Females were carbon
dioxide-anesthetized on day 6, their CA were incubated in vitro to measure JH biosynthetic
rates and their basal oocytes were measured. We set up 40 females per treatment (40 dishes
of 1 female each, 20 dishes of 2, 5 dishes of 8, and 2 dishes of 20 females), but some
replicates were lost because of the difficulty of dissecting and incubating the CA. Only
females from which we could measure both parameters were used, resulting in 26–33
females per treatment.
JH biosynthesis was measured in vitro using a radiochemical assay (Pratt and Tobe,
1974; Gadot et al., 1989b; Holbrook et al., 2000). Briefly, the CA-corpora cardiaca
complexes were dissected and incubated in modified methionine-free TC-199 medium
(Specialty Media, Lavalette, NJ, USA), supplemented with 100 µM L-[3H-methyl]methionine (NEN, Wilmington, DE, USA), 5 mM CaCl2, and 20 mg/ml Ficoll type 400.
Incubation and extraction conditions followed Holbrook et al. (2000).
Time course of oocyte maturation in isolated and paired females in the first and second
ovarian cycles
Newly-eclosed (day-0) females were either socially isolated or paired in plastic Petri dishes
and provisioned with food and water. Cohorts of virgin isolated and paired females were
dissected daily for 6 days and their oocytes were measured. Sample size was 10 females per
treatment on each day.
29
B. germanica females may obtain and store enough sperm when first mated to last
their entire reproductive life of several months (Cochran, 1979). Thus, the first ovarian cycle
is unlike subsequent ovarian cycles as females mature sexually, become sexually receptive,
and mate during the first preoviposition period (Schal et al., 1997). Because the first ovarian
cycle requires social interaction with males, we hypothesized that the female’s endocrine
system would be affected by social interactions during the first, but not subsequent ovarian
cycles. For the second ovarian cycle we reared newly-eclosed females in large groups in
plastic cages (185 X 130 mm, 105 mm high, Althor Products, Windsor Locks, CT, USA)
with egg carton shelters and allowed them to mate on day 6. Only mated females with a fully
rotated egg case were selected on day 9 and monitored in a separate plastic cage during
gestation. Just before the end of the gestation period (day 28), we removed the egg case, thus
synchronizing the start of the second ovarian cycle among all females. Females were housed
in social isolation or in pairs during the second ovarian cycle. Oocyte length was measured in
cohorts of females daily 1–4 days after the removal of the egg case (day 29–32).
Effects of social isolation and social interactions on sexual receptivity
B. germanica females undergo several days of sexual maturation before they become
sexually receptive, and the onset of sexual receptivity is regulated by JH (Schal et al., 1997).
We hypothesized that in the presence of conspecifics, females would become sexually
receptive faster than females raised in social isolation. Newly-eclosed females were either
isolated or paired with other females in plastic Petri dishes. On day 6 all isolated females
30
were transferred into a cage (300 mm diameter, 90 mm high, Althor) with sexually mature
males (9–10 d old), and all paired females were likewise transferred into a second cage and
provided males from the same cohort. We removed copulating pairs every 15–30 min for 3 h
and placed them in a refrigerator for later dissection. Copulation averages ~90 min in this
species, so this design guaranteed that successful copulations would be discovered. We then
measured oocyte length in all 4 treatment groups: (a) isolated mated; (b) isolated unmated;
(c) paired mated; and (d) paired unmated. Because few socially isolated females mated on
day 6, this assay was repeated on day 7 with another cohort of females and males. Sample
size was 36 females per treatment on each day.
Differential responses of females to duration of social interactions
To test whether females are differentially responsive to social interaction during successive
stages of their preoviposition period, we housed individual newly-eclosed females in
isolation, transiently paired some females with another 10–12 day old female (marked by
clipping its wing tips) for only 24 h on either day 0, 2, 4 or 6, and then again socially isolated
the females until the beginning of day 7 (to allow day 6 pairing to be completed), when they
were dissected and their basal oocytes were measured (see design Fig. 4A). Other females
were treated in the same way, but they were transiently paired with other females for 48 h
instead of 24 h on either days 0–1, 2–3, or 4–5; these females were dissected at the end of
day 6 (see Fig. 4B).
31
Opposite experiments were also conducted, with newly-eclosed females socially
paired, then transiently isolated for 24 h or 48 h, and again paired for the remainder of the
experiment (see design Fig. 4A and B). Sample size was 17–21 females per treatment.
Responses to social interactions during gestation
If social interactions during gestation were to accelerate reproduction during the ~21 day
gestation period, as they do in the preoviposition period, then gestation and embryogenesis
would be disrupted. We therefore hypothesized that the female’s reproductive physiology
would be unresponsive to social interactions during gestation. Nevertheless, because JH
biosynthesis is known to increase slightly before egg hatch, and the basal oocytes grow
slightly as well (Gadot et al., 1989b; Schal et al., 1997; Treiblmayr et al., 2006), we
hypothesized that social interactions in the late phase of gestation might influence the second
ovarian cycle.
To examine the effects of social isolation and social interactions on the duration of
gestation, newly-eclosed females were reared in large groups in plastic cages, as before.
Males were introduced to 6-day-old females. We monitored for mating every 15 min during a
2 h period, and because copulation in B. germanica lasts for approximately 90 min (Schal
and Chiang, 1995) we are confident that only mated females were retained for these
experiments. To generate a synchronous cohort of gestating females, only females with a
fully-rotated egg case on day 9 were either socially isolated or housed in pairs during the
32
ensuing gestation period. Egg hatch was monitored daily at the end of gestation. Sample size
was 41 isolated and 42 paired females.
To investigate the influence of social isolation or interactions during the first
gestation period on the development of second-cycle oocytes, the experimental procedure
described above was repeated. The effect of these treatments was assessed by measuring the
length of the basal oocytes on day 24 (day 15 of gestation = 6 days before hatch = 71%
embryogenesis time), and again on day 29 (day 20 of gestation = 1 day before hatch = 95%
embryogenesis time). Sample size was 10–15 females per treatment.
Statistical analyses
Data were analyzed with Student’s t-test to compare two-sample means and with one-way
ANOVA for multiple comparisons using SAS® 9.1.3 software (SAS Institute Inc. 2002-2003,
Cary, North Carolina). The LSD test was used for comparisons of means and PROC GLM
for residuals from adjusted model as well as to test whether residuals met the assumption of
independence and homogeneous variances among the treatments. For unbalanced data, we
used LSMEANS. Significance level for rejecting the null hypothesis was α = 0.05. Variation
around the mean is represented by the standard error of the mean (s.e.m.).
33
RESULTS
Effects of female density on oocyte maturation and juvenile hormone biosynthesis
Socially isolated virgin females matured their oocytes slowly, from ~0.5 mm at eclosion to
only 0.90 ± 0.04 mm (N = 26) by day 6 (Fig. 1). Females housed with a single conspecific
female, on the other hand, exhibited significantly faster oocyte maturation (day 6: 1.30 ± 0.03
mm, N = 26) and this effect was even more pronounced at higher density with 8 females per
Petri dish (1.44 ± 0.02 mm, N = 33). However, crowded females, at 20 per Petri dish,
exhibited significantly stunted oocyte maturation (day 6: 1.14 ± 0.06 mm, N = 29) (ANOVA,
P < 0.001) (Fig. 1).
The patterns of oocyte maturation matched the activity of the CA in these females, as
expected (Fig. 1). Because paired females demonstrated clear social facilitation of
reproduction (i.e., faster oocyte development) by day 6 (ANOVA, P < 0.001), this minimal
experimental design (socially isolated vs. pair-housed) was used in all subsequent
experiments.
Social isolation might be stressful for nocturnal cockroaches in the absence of shelter.
Because long-term presence of shelters contributes to faster nymphal development, greater
adult body mass, and greater female fertility (Gemeno et al., 2011), we examined whether
shelter ameliorated the effects of social isolation. There was no significant difference
between socially isolated females with or without shelters (0.95 ± 0.05 mm vs. 0.88 ± 0.06
mm, respectively) (Student’s t = 2.0262, P = 0.3575); similarly, oocyte maturation in pair-
34
housed females that were provided with either one or two shelters was not significantly
different from that of paired females housed without shelter (1.50 ± 0.07 mm, 1.58 ± 0.04
mm, 1.55 ± 0.06 mm, respectively) (ANOVA, F2, 54 = 0.290, P = 0.7495). These results
indicate that the differential oocyte growth observed in socially isolated and paired B.
germanica females is independent of any stress associated with lack of shelters in the
remainder of our experiments.
Time-course of oocyte maturation in socially isolated and paired females in the first and
second ovarian cycles
For the first 2 days after adult eclosion, oocyte maturation was similar in solitary B.
germanica females that remained socially isolated and in females paired with a conspecific
female (Fig. 2). Subsequently, however, paired females diverged significantly from isolated
females, and by day 6 their oocytes were 1.76-times longer than the oocytes of isolated
females (1.63 ± 0.05 mm vs. 0.92 ± 0.07 mm) (Student’s t-test: t = 2.1009, P < 0.0001) (Fig.
2).
In the second ovarian cycle, mated females proceed directly into vitellogenesis and
rapid oocyte maturation, bypassing the processes of sexual maturation and mating that they
experience in the first ovarian cycle. Therefore, their basal oocytes grow faster during the
second ovarian cycle. Still, a significant difference in oocyte length of socially isolated
females and paired females could be seen as early as day 2 in the second ovarian cycle, and
these differences increased during the subsequent 2–3 days (Fig. 2).
35
Effects of social isolation and social interactions on sexual receptivity
Social isolation significantly delayed not only oocyte maturation but sexual receptivity as
well. The basal oocyte length of 1.25 mm is the approximate stage for the onset of sexual
receptivity (Schal and Chiang, 1995). Only 8.1% of the socially isolated females mated on
day 6, whereas 66.6% of the paired females mated on day 6. All the socially isolated females
had oocytes <1.25 mm, whereas 58.3% of the socially paired females developed their oocytes
to >1.25 mm (Fig. 3A). Similar results were obtained with a separate cohort of females on
day 7, but with 58.3% of the socially isolated females mating (85.8% with oocytes >1.25
mm) compared to 91.7% of paired females (100% with oocytes >1.25 mm) (Fig. 3B). These
results show that all JH-regulated events—both physiological and behavioral—appear to be
under the strong influence of social interactions.
Differential responses to duration of social interactions during the first preoviposition
period
When females were socially isolated since adult emergence, but then transiently paired with a
conspecific female for only 24 h on day 0 or on days 2, 4, or 6 (see design Fig. 4A), their
basal oocytes were not significantly different from the negative controls (socially isolated
females during the entire experiment), indicating a lack of response to the transient social
interaction (Fig. 5A). Likewise, when females were paired with conspecific females since
adult emergence, but then transiently isolated for 24 h on any day on day 0 or on days 2, 4, or
36
6 (see design Fig. 4A), the length of their basal oocytes was approximately the same as in
females that were socially paired during the entire experiment (positive controls) (Fig. 5A).
Thus, either social isolation or social interactions for only 24 h during a 6 day oocyte
maturation period appear to be insufficient to change the course of the ovarian cycle.
However, when socially isolated females were paired transiently for 48 h (see design
Fig. 4B), the basal oocytes of only those females that were paired on days 2–3 were
significantly larger than the oocytes of females that were isolated during the entire
experiment (Fig. 5B). When pairs of females were split and isolated transiently for 48 h (see
design Fig. 4B), there was no significant difference between the oocyte length of females
isolated on any two days during the 6-day-long experiment and the positive control (females
paired during the entire experiment). Notably, however, oocyte maturation in paired females
that were isolated only on days 2–3 was slightly delayed compared with the positive control
(Fig. 5B), and by comparing them with the standard curve of oocyte growth (Fig. 2), we
could estimate that these females experienced a 1 day delay in their ovarian cycle. Overall,
the most significant influence on oocyte maturation was transient social interaction or
transient social isolation on days 2–3, indicating that females at this age are particularly
responsive to social cues (Fig. 5A and B).
Effects of social isolation and social interactions during the gestation period
While social isolation significantly delayed oocyte development and sexual maturation in
German cockroach females in both the first and second preoviposition periods, it did not
37
affect the duration of gestation. The mean duration of gestation was the same in socially
isolated females (21.49 ± 0.31 days) and females paired with another conspecific female
(21.78 ± 0.79 days) (Student’s t = 1.7198, P = 0.0901). This suggests that when the CA are
inactive, social interactions fail to stimulate oocyte maturation.
In 24-day old females (day-15 of gestation), the length of the basal oocytes was
similar whether females were socially isolated (0.50 ± 0.02 mm) or paired with other females
(0.52 ± 0.01 mm) (t = 1.7291, P = 0.2004) during the gestation period. However, by day 29,
one day before normal egg hatch, the oocytes of socially paired females grew significantly
larger (0.66 ± 0.02 mm) than in socially isolated females (0.61 ± 0.02 mm) (t = 1.7081, P =
0.0314) (Fig. 6). Thus, growth of the basal oocytes is suppressed during most of the gestation
period. However, just before the eggs hatch, the basal oocytes begin to grow and their growth
rate is affected by social isolation or social interactions.
DISCUSSION
The principal observation emerging from our investigation is that the reproductive rate of
adult B. germanica females can be modulated by social conditions; social isolation slows
both oocyte maturation and the onset of sexual receptivity whereas social interactions as
minimal as between just two females speed up reproduction. Moreover, our results show that
social conditions influence the reproductive rate only when the CA are active or competent to
produce JH, the major gonadotropic hormone in cockroaches. Notably, the endocrinegonadal response to social interactions was independent of mating status, as both virgin
38
females in the first ovarian cycle and mated females in the second ovarian cycle reproduced
faster in response to social cues. Even pregnant females responded to social interactions late
in gestation, as their CA became competent to produce and release JH. On the other hand,
throughout most of gestation, when CA activity was suppressed by the brain, social
interactions and social isolation did not affect the female’s reproductive physiology.
Reproduction in B. germanica females is under the control of JH-III, which paces the
reproductive rate and governs the duration of the preoviposition period, and whose absence
enables a protracted gestation (reviews: Schal et al., 1997; Treiblmayr et al., 2006). The rate
of oocyte maturation in socially isolated, group-housed, and over-crowded females reflected
the rates of JH production by the respective CA—smaller basal oocytes in isolated and
crowded females (20 per dish) corresponded well with lower rates of JH synthesis, whereas
larger oocytes in pair-housed and grouped females (8 per dish) paralleled higher rates of JH
synthesis on day 6. These results indicate that social interactions, through yet unknown
sensory modalities and CNS integration, strongly influence the rates of JH biosynthesis in
German cockroach females. They also validate, yet again, that the size of the basal oocytes is
an excellent indirect measure of the influence of social conditions on the female’s JH
biosynthetic rates.
Socially facilitated nymphal development has been documented in all cockroach
species that have been rigorously examined (Roth and Willis, 1960; Izutsu et al., 1970;
Woodhead and Paulson, 1983; Holbrook and Schal, 2004; Lihoreau and Rivault, 2008).
German cockroach nymphs reared in social isolation develop much more slowly than groups
of 2–100 individuals; however there were no significant differences in growth rates among
39
the grouped treatments, presumably because the sizes of the experimental arenas in these
tests were adjusted to maintain the same nymphal density (Ishii and Kuwahara, 1967; Izutsu
et al., 1970). To test whether social facilitation of reproduction in females is densitydependent, we maintained adult females at different densities (1, 2, 8, and 20) in same-size
experimental arenas. Oocyte maturation was significantly delayed in socially isolated
females compared to pair-housed females and this effect was even more pronounced with 8
females per dish. However, at a density of 20 females per dish delays in oocyte maturation
were observed again. Because crowding induces the production of salivary proteins that also
function as repellents for alarm or defensive purposes (Ross and Tignor, 1988), it would be
interesting to know whether these proteins are responsible in part for impeding JH production
and oocyte maturation.
Faster oocyte maturation in group-housed females may be related to differential food
intake by females under different social conditions. Holbrook et al. (2000) found, however,
that although feeding is socially stimulated in adult female B. germanica—food consumption
was higher in females that were paired with conspecific females since adult eclosion than in
females reared in social isolation—oocyte development in these two treatments differed even
when all females were provided with the same amount of food. Therefore, other nonnutritional factors likely mediate the social facilitation of reproduction in B. germanica.
Physiological responses to stressful conditions are often linked to the circadian clock
(see review: Boerjan et al., 2010). Therefore, it is possible that stressful conditions, such as
absence of a shelter during the photophase of nocturnally-active cockroaches, could result in
slower oocyte maturation in socially isolated females. This is especially relevant because in
40
large cage demographic studies of B. germanica the presence of shelters has been shown to
contribute to faster nymphal development, greater adult body mass, and greater female
fertility (Gemeno et al., 2011). However, in our 6-day assays the presence of shelters did not
enhance oocyte maturation in either socially isolated females or in pair-housed females.
These results further support the idea that social interactions affect reproduction through
specific sensory and neuronal pathways that affect the neuroendocrine system rather than
through stress-induced effects.
Four major life history characteristics of B. germanica feature in the evolution of
social facilitation of reproduction. First, like many pest species, these cockroaches are highly
gregarious. Second, unlike some other cockroach species that can reproduce
parthenogenetically, German cockroach females must mate in order to successfully
reproduce. Third, while females may mate multiple times during their 8–12 month adult
lifespan, they obtain and store enough sperm in their first mating to last their entire
reproductive life, so multiple matings are not essential (Cochran, 1979). Finally, unlike many
insect species, including some cockroaches, B. germanica virgin females do not resorb their
oocytes, and instead oviposit a large, malformed, and inviable egg case that represents a
significant nutritional and energetic investment. One hypothesis to account for the evolution
of social facilitation of reproduction is that a single colonizer female that finds herself
socially isolated from conspecifics ought to slow down her oocyte development until a
potential mate arrives, in order to prevent or at least delay ovipositing an inviable egg case.
Under these conditions, social interactions would convey to the female the presence of a
potential mate, and she should then accelerate her reproductive rate to become sexually
41
receptive faster to maximize her fitness. A corollary of this hypothesis would further predict
that females should respond to the inhibitory effects of social isolation only as virgins in the
first ovarian cycle, but not in subsequent ovarian cycles after being mated, because mates are
no longer essential. This prediction, however, was not borne-out by our results—oocyte
maturation in mated females in the second ovarian cycle was also subject to both the
inhibitory effects of social isolation and the stimulatory effects of social interactions.
Therefore, it is plausible that socially facilitated reproduction evolved because social
interactions with conspecifics offer to the adult female other benefits, such as greater
foraging efficiency, protection from predators, exchange of symbiotic microbes, or even
modification of the microclimate.
Not surprisingly, based on the modulation of JH production during the ovarian cycle,
we found that females were differentially responsive to social cues during the preoviposition
period. Transient social interactions, or transient social isolation, for 24 h had no statistically
discernible effects on oocyte maturation. However, longer transient social interactions or
social isolation for 48 h significantly influenced oocyte maturation, mostly around days 2–3.
These observations suggest that females respond maximally to social conditions when their
CA become competent to produce JH. Around 2–3 days after eclosion the CA attain a high
competence to produce large amounts of JH, as determined by stimulating the CA in vitro
with the JH precursor farnesoic acid (Gadot et al., 1989a). It seems reasonable that females
would be most responsive to social/isolation stimuli at times when their CA are most
responsive to CNS disinhibition, but we were surprised to find that females with maximally
active CA on days 4–6 (Schal et al., 1997) were less responsive to social stimuli than 2–3
42
day-old females. It is possible that at later stages of oocyte maturation the CA are committed
to produce JH at high biosynthetic rates and they are therefore less responsive to CNS
directives associated with social stimuli. However, further experiments will be required to
confirm our speculations.
Schal and Chiang (1995) established that for females to develop and express sexual
receptivity the CA must be active and produce JH, and females mate only when the JH titer
reaches a certain threshold level. Although the ovaries are not needed for B. germanica CA
to become active and for females to mate (Gadot et al., 1991), the inextricable relationship
between JH production and the rate of oocyte maturation in normal B. germanica females
suggests that oocyte size could be used to pinpoint a minimal threshold of JH production
below which most females would refuse to mate. Indeed, in the ovoviviparous Leucophaea
maderea females this threshold was found to be 1.08 ± 0.01 mm (Engelmann, 1960), and
most virgin B. germanica females accept males when their oocytes are >1.25 mm in length
(Schal and Chiang, 1995; Schal et al., 1997). In controlled experiments that examined sexual
receptivity and oocyte length in socially isolated females and in females paired with a
conspecific female, we found that significantly more paired females mated on either day 6 or
day 7 compared with socially isolated females. Moreover, the great majority of paired
females that mated on day 6 or day 7 had oocytes >1.25 mm. These results confirm that
social interactions not only stimulate oocyte maturation in B. germanica females but also
hasten the onset of sexual receptivity by at least a day. These results also verify that both
physiological and behavioral reproductive events are JH-regulated and that these events are
strongly influenced by social conditions.
43
A previous study showed that social interactions accelerate and synchronize the
timing of oviposition in virgin B. germanica females, whereas social isolation delays
oviposition and prompts isolated females to oviposit asynchronously (Gadot et al., 1989b). In
addition, Lihoreau and Rivault (2008) reported that social conditions also influence the
number of ovarian cycles (i.e., number of egg cases produced) during the female’s lifespan,
resulting in significantly fewer cycles per socially isolated virgin female than in socially
grouped virgin females. Our results with mated females show that the disparity between
socially isolated and socially grouped females is due to different lengths of preoviposition
periods, and not to differences in their gestation periods; egg hatch occurs approximately 20–
21 days after oviposition in both sets of females. This is consistent with the hypothesis that
during periods which require the JH titer to remain low for a protracted gestational period,
stimulatory social cues, such as those associated with social interactions, are somehow
uncoupled from the CNS pathways that activate the CA, rendering females reproductively
unresponsive to social cues. Notably, however, slight differences were evident in the
maturation of the basal oocytes of socially isolated and socially paired females in the last few
days of gestation, corresponding with a slight elevation in the rates of JH production and the
JH titer in preparation for the next ovarian cycle (Treiblmayr et al., 2006). This mechanism
of integrating social stimuli with endocrine function is adaptively consistent with the strategy
of inhibiting vitellogenesis during gestation, because oocyte maturation would result in the
abortion of the incubated egg case.
Our study sheds light on the effects of social interactions on JH production, which in
turn regulates the rate of female reproduction in the German cockroach. We demonstrated
44
that female density influences oocyte development, indicating that the quantity of social
stimulation is an important factor in the social facilitation of female reproduction; the
presence of one conspecific female was sufficient to accelerate oocyte maturation, but high
female density delayed oocyte maturation through a crowding effect. In aggregate, our results
on the stage-specific responses of females to social interactions support the conclusion that
social interactions represent a powerful environmental stimulus that modulates the activity of
the CA in concert with other extrinsic and internal cues. Therefore, through their effects on
the synthesis and release of JH, social interactions indirectly modulate all JH-dependent
activities including the synthesis of vitellogenin and other female-specific proteins,
attainment of sexual receptivity, production of sexual signals, mating, and the time-course of
oviposition.
The sensory modalities that participate in the social facilitation of reproduction and
the sensory and CNS pathways that couple social cues and JH production are largely
unknown. B. germanica thus is a fascinating model system in which to delineate which
sensory cues influence JH production and how these cues influence CA activity during
vitellogenesis but are somehow uncoupled from influencing the CA during gestation. We are
also interested in comparative studies to understand why socially facilitated reproduction has
evolved in some cockroach species, like B. germanica, and not in others (e.g., S. longipalpa).
45
ACKNOWLEDGMENTS
We thank to Katalin Böröczky, Jan Buellesbach, Yasmin J. Cardoza, Jules Silverman, Ayako
Wada-Katsumata, Wes Watson, and two anonymous reviewers for critical comments on this
manuscript. This project was supported by the Blanton J. Whitmire endowment at North
Carolina State University and scholarships from the North Carolina Pest Management
Association and the Structural Pest Management Training and Research Facility.
46
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51
FIGURES
Oocyte length
JH biosynthesis
1.50
14
d
D
C
Oocyte length (mm)
1.25
b
10
B
8
1.00
A
a
6
4
0.75
2
JH biosynthesis rate (pmol/hr/CA pair)
12
c
0
0.50
1
2
8
20
Females per Petri dish
Figure 1. The effect of female density on reproductive rate in B. germanica. Females were
housed in Petri dishes for 6 days at densities of 1, 2, 8, or 20 females per dish. Basal oocyte
length was measured and JH biosynthetic rates of the CA were quantified in vitro. Sample
sizes were 26, 26, 33, and 29 respectively, for 1, 2, 8, and 20 females per dish. Variation
around the mean is represented by the standard error of the mean (s.e.m.). ANOVA for
oocyte length: F3, 110 = 32.829, P < 0.001; ANOVA for JH synthesis: F3, 110 = 22.384, P <
0.001. Means were compared by LSD and means not sharing a letter are significantly
different (P < 0.05).
52
1.75
*
Paired females
Isolated females
*
*
1.50
Oocyte length (mm)
*
1.25
*
*
1.00
*
0.75
0.50
0.25
0
1
2
3
4
5
6
First ovarian cycle
29
30
31
32
Second ovarian cycle
Female age (day)
Figure 2. Time-course of oocyte maturation in B. germanica females during the
preoviposition period of the first and second ovarian cycles. For the first ovarian cycle,
newly-eclosed (day–0) females were socially isolated or housed in pairs in Petri dishes. For
the second ovarian cycle, females were reared in groups after adult emergence, mated on
day-6 and reared in groups during gestation. The egg case was removed on day 28, 2 days
before the end of gestation, thus synchronizing their reproductive cycle. Females were then
socially isolated or housed in pairs. Basal oocyte length was measured on each day indicated.
Each point shows the mean ± s.e.m of 10 females in the first ovarian cycle and 10–15
females in the second cycle. Significant differences between treatments are shown with an
asterisk (Student’s t-test: t = 2.1009, P < 0.05).
53
1.75
A. day 6
1.50
c
bc
b
1.25
Oocyte length (mm)
a
1.00
1.75
B. day 7
c
b
1.50
b
1.25
a
1.00
Isolated unmated
Isolated mated
Paired unmated
Paired mated
Figure 3. Effects of social isolation and group-housing on the onset of sexual receptivity in B.
germanica females. Newly-eclosed females were either socially isolated or paired with
another female in a Petri dish and allowed to mate on day 6 (A) or day 7 (B). Basal oocyte
length was measured in all females (N = 36 per treatment). Variation around the mean is
represented by the standard error of the mean (s.e.m.). Means were compared by LSD and
means not sharing a letter are significantly different (P < 0.05).
54
Figure 4. Experimental design of assays to determine whether certain stages during the
preoviposition period are more or less affected by transient social isolation or transient social
interactions. Open rectangles represent social isolation, closed rectangles represent socially
paired conditions. Each row corresponds to a treatment (bar) in Fig.5.
55
Figure 5. Differential responsiveness of B. germanica females to social interaction and social
isolation during the preoviposition period. (A) Newly-eclosed females were either socially
isolated and then transiently paired with another female or paired and then transiently
socially isolated for 24 h either on day 0, 2, 4, or 6, and their basal oocytes were measured on
day 7 (N = 19–21). See design in Fig. 4A. (B) Other groups of females were similarly treated,
but they were transiently paired or transiently socially isolated for 48 h instead of 24 h either
on days 0–1, 2–3, or 4–5, and their basal oocytes were measured on day 6 (N = 17–19). See
design in Fig. 4B. Variation around the mean is represented by the standard error of the mean
(s.e.m.). Means not sharing a letter are significantly different (P < 0.05).
56
2.00
A. 24 hrs, measured day 7
b
b
1.75
b
1.50
b
b
a
a
a
a
a
Oocyte length (mm)
1.25
1.00
Isolated
d-0
d-2
d-4
d-6
Paired 24 hrs
d-0
d-2
d-4
d-6
Paired
Isolated 24 hrs
1.75
B. 48 hrs, measured day 6
d
1.50
d
cd
bcd
bcd
1.25
abc
ab
a
1.00
Isolated
d-0-1 d-2-3
d-4-5
Paired 48 hrs
d-0-1
d-2-3
d-4-5
Paired
Isolated 48 hrs
57
0.70
Isolated
Paired
*
Oocyte length (mm)
0.65
0.60
0.55
0.50
29
24
Female age (day)
Figure 6. Effects of social isolation and group-housing on oocyte length at the end of the first
gestation period. Females were mated on day-6 and females with a fully-rotated egg case on
day-9 were either socially isolated or paired with another female during the ensuing gestation
period. The effects of these treatments on oocyte length were assessed by measuring the
basal oocytes on day 24 (day-15 of gestation), and again on day 29 (day-20 of gestation). N =
10–15 females. Each point shows the mean ± s.e.m. Significant differences between
treatments are shown with an asterisk (Student’s t-test for day 24: t = 1.7291, P = 0.2004,
Student’s t-test for day 29: t = 1.7081, P = 0.0314).
58
CHAPTER 2.
Social interaction facilitates reproduction in male German cockroaches, Blattella
germanica
(This work was submitted to Animal Behaviour)
59
ABSTRACT
Sociality can be beneficial for individuals because it may increase access to resources,
provide greater protection from predators, and create opportunities for mating. In some
species, social conditions and experience also influence the physiology and behavioral
responses of individuals. Although social facilitation of sexual maturation is poorly
understood in insects, the German cockroach has served as an important model system for
this phenomenon as group-housed females mature faster than solitary females. However,
social modulation of male reproduction has not been investigated in this or any other nonpolyphenic insect species. We investigated the relative effects of social interactions and
isolation on endocrine, reproductive and behavioral events during sexual maturation of B.
germanica males. Results show that social interactions significantly facilitate juvenile
hormone biosynthesis as well as protein production by the accessory reproductive glands,
whereas isolation suppresses these processes. Other processes, such as accumulation of
cuticular hydrocarbons, maturation of the male’s courtship readiness and his readiness to
copulate appear to be relatively independent of social experience. However, the latency of
the courtship response as well as the latency to copulation were shorter in socially paired
males than in isolated males, and more egg cases were aborted (a measure of sperm viability)
by females that mated with isolated males than those that mated with paired males. Our study
is the first to reveal that social interactions have a profound effect on certain aspects of male
reproductive physiology and behavior in an insect species that does not exhibit phase or
developmental polyphenism.
60
INTRODUCTION
A positive correlation between population density and fitness is known as the Allee effect, as
was elegantly demonstrated in goldfish (Allee and Bowen 1932). Accordingly, some species
realise greater fitness in aggregation, through greater survival and reproduction.
Aggregations can provide various ecological benefits, including group defence and
cooperative hunting, environmental conditioning (modification of the microclimate), and
efficient mate-finding. In some species, group-living can also provide significant
physiological benefits, including sharing of symbiotic microbes among group members and
density-dependent social facilitation of growth and reproduction. However, the benefits of
aggregations can be tempered by negative density-dependent phenomena such as inbreeding
depression, sex ratio bias, disease transmission, and greater apparency of the group to
predators and parasites.
The social environment can exert a powerful influence on an individual’s
development, growth, metabolism and reproduction (Pulliam and Caraco 1984; Krause and
Ruxton 2002). Some of the best studied examples are from quorum-sensing and biofilm
forming bacteria, where cell-cell communication can drive dramatic physiological changes,
including the formation of a protected biofilm environment in which resident bacteria can
tolerate extreme changes in pH, temperature, limited nutrient supply and antibiotic exposure
(review: Elias and Banin 2012). Many examples have emerged of social facilitation of
growth and development rates in various insect species (Chauvin 1946; Long 1953; Wharton
et al. 1968; Izutsu et al. 1970; Woodhead and Paulson 1983; McFarlane et al. 1984; Weaver
61
and McFarlane 1990; Holbrook and Schal 1998; Lihoreau and Rivault 2008; Ronnas et al.
2010) and Allee effects on polyphenic development (review: Simpson et al. 2011) in
response to the presence of conspecifics. Moreover, the sensory mechanisms that underlie
these phenomena have also been delineated recently in some species (see Lihoreau and
Rivault 2008; Maeno et al. 2011; Uzsák and Schal 2012 (Chapter 1)).
There are also some prominent examples of social facilitation of reproduction, with
most describing the effects of male facilitation of female sexual maturation; studies on the
social modulation of male reproduction have been limited mainly to vertebrates (review:
Rekwot et al. 2001). Surprisingly, however, apart from eusocial species, examples of social
facilitation of reproduction are considerably scarcer for insects, and most involve behavioral
rather than physiological changes in females. Socially facilitated oviposition is most
common, as for example in Aedes aegypti mosquitoes, where oviposition decisions are
governed in part by the presence and density of conspecific eggs and larvae (e.g., Reiter
2007). In Rhagoletis pomonella flies females oviposit more in groups than singly (Prokopy
and Reynolds 1998), and egg production and oviposition in blowflies exhibit positive density
dependence, suggesting an Allee effect (Davies 1998). Although group-living has been
shown to facilitate growth rates in most cockroach species examined to date (Roth and Willis
1960; Woodhead and Paulson 1983; Holbrook and Schal 2004; Lihoreau and Rivault 2008),
faster reproduction due to social influence has been documented in females of only one
species, the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) (Gadot et
al. 1989a). Females of the German cockroach reproduce faster in groups, whereas isolated
females exhibit slower oocyte maturation and delayed onset of sexual receptivity (Gadot et
62
al. 1989a; Holbrook et al. 2000; Lihoreau and Rivault 2008; Uzsák and Schal 2012 (Chapter
1)).
Density-dependence of male reproductive behavior appears to be common, and most
often expressed as a decline in sexual thresholds in isolated adult males, as shown for
example in Drosophila paulistorum courtship (Kim and Ehrman 1998). Social facilitation of
male reproductive physiology, on the other hand, is a rare and relatively uninvestigated
phenomenon, limited to cases of phase and developmental polyphenism (e.g., aphids,
crickets, locusts). The best studied case involves locusts, in which sexual maturation of males
can be socially facilitated or retarded, as part of a general density-dependent phase
polyphenism that affects a wide-range of phase characteristics including anatomy,
morphology, colouration, behavior, ecology, development and reproduction (review: Pener
and Simpson 2009). The colour of adult Schistocerca gregaria changes with crowding, and
both males and females sexually mature faster in the presence of sexually mature older males
(Norris 1954). On the other hand, Locusta migratoria migratorioides males reared in
isolation mature faster than those reared under crowded conditions (Norris 1964). Socially
facilitated reproduction may be mediated through direct contact between conspecifics, or via
visual, olfactory, gustatory or auditory cues associated with group members; in some cases
the use of indirect cues does not require the concurrent presence of conspecifics in the
aggregation. For example, a “maturation-accelerating pheromone”, produced by sexually
mature S. gregaria males, is partly responsible for social facilitation of maturation, and other
chemotactile stimuli also appear to be involved (review: Hassanali et al. 2005). Interestingly,
63
a maturation-retarding effect, exerted by young males on other young males, has also been
described in the same species (Norris 1954).
The mechanisms that underlie social facilitation of insect sexual maturation are
poorly understood. The juvenile hormones (JH) are pivotal gonadotropic hormones in insect
reproduction (Raikhel et al. 2005), and in German cockroach females JH-III is produced and
released by the corpora allata (CA) in a stage-specific manner: JH biosynthesis increases and
stimulates oocyte maturation, declines through ovulation and remains low during gestation
(Bellés et al. 1987; Gadot et al. 1989b; Sevala et al. 1999; Treiblmayr et al. 2006). JH
controls and paces many processes associated with reproduction in B. germanica females,
including the synthesis of yolk and oothecal proteins, sexual receptivity, production of sex
pheromones, mating, and the time-course of oviposition (reviews: Schal et al. 1997;
Treiblmayr et al. 2006). Sensory cues associated with social interactions in females
significantly affect CA activity, demonstrating that social facilitation of reproduction is
coupled to the endocrine system (Uzsák and Schal 2012 (Chapter 1)). It is not known,
however, whether B. germanica males have a similar response to social interactions as do
females. JH is responsible for social facilitation of sexual maturation in male locusts, as well
as changes in colouration and the production of the maturation-accelerating pheromones in
crowded S. gregaria (review: Pener and Simpson 2009).
Although JH release rates are considerably lower in B. germanica males than in
females, JH production in males increases steadily after adult emergence, as in females
(Piulachs et al. 1992). This pattern suggests that JH might play an important role in male
reproduction as well. Indeed, JH has been found to regulate male accessory reproductive
64
gland (ARG) development during sexual maturation in B. germanica: CA removal resulted in
lower total protein content of the ARG, and administration of JH restored normal ARG
development (Piulachs et al. 1992). Furthermore, as the spermatophore forms during
copulation, conglobate gland (a part of the ARG) proteins are depleted and JH production by
the CA declines; but within several hours after copulation JH synthesis rates are elevated and
proteins accumulate in the conglobate gland (Vilaplana et al. 1996b). The amount of a
specific ARG protein, Blattella germanica allergen 4 (Bla g 4), increases gradually after
adult emergence and reaches a plateau by day 14 (Fan et al. 2005). This age-modulated
production of Bla g 4 is regulated by JH and topical administration of JH results in elevated
Bla g 4 levels (Fan et al. 2005). Thus, ARG size and protein content appear to be reliable
measures of reproductive maturation in males—as oocyte size and yolk content are in
females—to investigate whether males experience social facilitation of reproduction.
Other B. germanica male physiological and behavioral events that might be socially
facilitated during reproductive maturation include the accumulation of cuticular
hydrocarbons (CHC), courtship and copulation readiness, and the ability to successfully
inseminate females. After adult emergence, CHC accumulate on the cuticular surface and in
the haemolymph of B. germanica females (Schal et al. 1994), so it is plausible that
production of CHC might follow a similar pattern in males. Moreover, accumulation of CHC
might be affected by social experience, as shown in Drosophila melanogaster, where
isolation during development leads to greater quantities of CHC (Kim et al. 2004) and the
genotypic composition of adult social groups affects the pattern of male pheromone (CHC)
accumulation on the cuticle (Krupp et al. 2008). JH controls the expression of sexual
65
receptivity in the female German cockroach; the CA must be present and active for females
to become sexually receptive and for copulation to occur (Schal and Chiang 1995). Males,
however, become sexually receptive even if their CA are extirpated (allatectomised), but
sexual readiness is delayed; conversely, administration of JH appears to accelerate the
expression of sexual readiness in males (Schal and Chiang 1995). It is conceivable that the
influence of JH on sexual maturation in males might be mediated by social cues, as in
females. Sexual maturation in males can be measured by their ability to display characteristic
courtship behavior, to produce viable sperm and to successfully transfer a spermatophore
during copulation.
Here, we investigated the relative effects of social isolation and social interactions in
B. germanica males on (1) JH biosynthesis by their CA, (2) protein content of their ARG, (3)
the onset age of courtship readiness, (4) copulation readiness (latency to copulate), and (5)
sperm maturation, measured indirectly as the frequency of egg case abortion. We conclude
that group-living facilitates reproduction in adult males, as it does in adult females, but this
social facilitation is of considerably lower magnitude in males than in females.
MATERIALS AND METHODS
Insects
Adult male B. germanica were collected from a laboratory colony of insecticide-susceptible
German cockroaches (American Cyanamid strain, also called Orlando strain, obtained in
66
1989) reared at 27 ± 1ºC and 40–70% ambient relative humidity on a 12L: 12D regime, with
unlimited access to water and food pellets (LabDiet 5001 Rodent Diet, PMI Nutrition
International, Brentwood, MO, U.S.A.). Newly-emerged adult males (day–0) of similar size,
degree of sclerotisation and with intact wings were selected for all experiments. Individuals
were either isolated or randomly assigned to pairs or groups. Unless otherwise specified,
males were housed either individually or in pairs in plastic Petri dishes (90 mm diameter, 15
mm high, Fisher Scientific, Pittsburgh, PA, U.S.A.), provisioned with food and water ad
libitum, and maintained and observed under the environmental conditions described for the
colony above. We use “paired” to denote pair-housed males, i.e., a B. germanica male
housed in the same dish with another male.
JH-III biosynthesis by the CA
To assess the effect of male density on JH biosynthesis, isolated and grouped males (2, 4, 8,
12, 16, 20 males) were housed for 7 days in plastic Petri dishes (200 mm diameter, 20 mm
high, Fisher Scientific). JH biosynthesis was measured in vitro using a radiochemical assay
developed by Pratt and Tobe (1974), with modifications following Holbrook et al. (2000).
This assay is based on the stoichiometric incorporation of radiolabeled methyl group from
methionine into JH-III under equilibrium conditions. Briefly, the CA-corpora cardiaca
complexes were dissected from decapitated males and incubated in 6 x 50 mm glass culture
tubes in 0.1 mL modified methionine-free TC-199 medium (Specialty Media, Lavalette, NJ,
U.S.A.), supplemented with 100 µM L-[3H-methyl]-methionine (198 mCi/mmol; NEN,
67
Wilmington, DE, U.S.A.), 5 mM CaCl2, 25 mM HEPES, and 20 mg/ml Ficoll type 400. After
a 3-hr incubation the medium was extracted with 0.25 mL isooctane, an aliquot of which was
assayed with a liquid scintillation counter.
Bla g 4 protein content of the ARG
Males were cold-anaesthetised and the ARG complex was carefully removed, homogenised
in 0.25 mL PBS, centrifuged (10,000 rpm at 4ºC for 20 min), the pellet was re-homogenised
and centrifuged again in fresh PBS, and the two supernatants were combined and stored at –
80ºC. We used an indirect ELISA that we previously developed and optimised to quantify
Bla g 4 protein using recombinant Bla g 4 standards (Fan et al. 2005).
Quantification of cuticular hydrocarbons
Males were freeze-killed at various ages and extracted individually for 1 min in 0.5 mL nhexane containing 5 μg n-hexacosane as internal standard. The extract was slowly reduced to
0.1 mL under a gentle stream of N2 and 1 μL was introduced with an Agilent 7683B autoinjector into the split-splitless inlet (operated at 310°C in splitless mode) of an Agilent
7890A gas chromatograph (GC). CHC were separated on a DB-5 column (20 m × 0.18 mm ×
0.40 μm film thickness) with hydrogen as carrier gas at a linear velocity of 40 cm/sec, and
detected with a flame-ionization detector. Total CHC were calculated by comparing the
summed area of all integrated CHC peaks to the integrated area of the internal standard peak.
68
Courtship readiness
To determine if paired males attain courtship readiness (capability to court females) before
males maintained in social isolation, we used a modification of the “antenna on a stick” assay
developed by Roth and Willis (1952), as recently modified by Eliyahu et al. (2009). An
antenna of a 6-day-old virgin B. germanica adult female was carefully ablated with fine
scissors just distal to the scape, and inserted into a small mass of modelling clay at the end of
a glass Pasteur pipette. This antenna was used immediately to stimulate the antennae of test
males that were individually housed in small plastic cages. The antennae of each male were
gently stroked with the test antenna for up to 2 min, and a positive response and its latency
were recorded when the male exhibited the first courtship response. Courtship is an
unmistakable behavior that occurs only in a sexual context and consists of the male rotating
his body relative to the stimulus and raising his wings (Roth and Willis 1952). One female
antenna was used for three successive assays (<6 min total) with one isolated male, one
paired male (one male of the pair was discarded before the assay), and a control 13–14-day
old group-housed male. The order of assays was randomised, and all assays were conducted
during the second half of the scotophase, when copulation is most frequent (Liang and Schal
1993), avoiding the last 2 hrs of the scotophase. Sample size was 41–74 males per treatment.
69
Copulation and mating success
To determine whether paired males attain greater copulation success (mate at a younger age,
successfully inseminate and fertilise females) than isolated males, isolated and paired males
were separately transferred from Petri dishes into two plastic cages (185 x 130 mm, 105 mm
high, Althor Products, Windsor Locks, CT, U.S.A.) with twice as many sexually mature 6day old virgin females. We then monitored copulations for 2 h and removed copulating pairs
every 15 min and placed them in plastic Petri dishes. Because copulation lasts ~90 min in
this species (Schal and Chiang 1995), this design guaranteed that we captured all successful
copulations. The time to copulation (to within 15 min) was recorded. When the copulating
pair separated, males were discarded and females were given food and water and monitored
daily for oviposition, hatching of nymphs and egg case abortions. This experiment was
conducted several times with 1, 2, 3, 4, 5 and 6-day-old males with 18–156 males per
treatment.
Data analyses
Data were analysed with Student’s unpaired t-test to compare two-sample means and with
one-way analysis of variance (ANOVA) for more than two groups, followed by Tukey’s
studentised range test for multiple comparisons using SAS® 9.1.3 software (SAS Institute
Inc. 2002-2003, Cary, NC, U.S.A.). We used PROC GLM to test for the effects of social
conditions on JH synthesis rates as dependent variable at various male densities. PROC GLM
70
was used to get the residuals from adjusted model and homogeneity of variances was tested
using Brown-Forsythe test to check whether residuals hold the assumption of homogeneous
variances within each treatment. Since the data were unbalanced, we used LSMEANS and
LSD test to compare means. Chi-square logistic regression was used to test for differences in
the frequencies of mating and the frequencies of female abortion. For all tests, the level of
significance was set to P < 0.05. Variation around the mean is represented by the standard
error of the mean (SE).
RESULTS
Social facilitation of JH-III synthesis in paired B. germanica males
The CA of pair-housed males produced significantly more JH on day-7 than the CA of
isolated males (t = 3.963, P = 0.0001; Fig. 1). This effect was even more pronounced at
higher male densities with 4, 8, 12, 16, or 20 males per dish.
Because the social facilitation of JH production was already apparent in paired males,
we compared the time-course of social facilitation using only isolated and paired males.
Within 3 days of adult emergence, CA activity of paired males began to diverge from that of
socially isolated males, and by day 7 paired males exhibited significantly higher rates of JH
synthesis than isolated males (2.23 ± 0.10 pmol/hr/CA pair, N = 58 vs. 1.73 ± 0.08
pmol/hr/CA pair, N = 59; ANOVA: F6,232 = 11.77, P < 0.0001; Fig. 2). Interestingly, the
differences between paired and isolated males persisted through at least day 19. These results
71
indicate that the rates of JH biosynthesis are socially facilitated in adult males, as previously
demonstrated for females.
Social facilitation of ARG protein
Because JH release rates influence ARG protein production in males (Piulachs et al. 1992;
Vilaplana et al. 1996a; Fan et al. 2005), we examined the effect of social conditions on Bla g
4, a protein produced exclusively in the male ARG. Results showed that as little as 3 days of
social interaction with a conspecific male resulted in significantly higher amounts of Bla g 4
protein in paired males than in isolated males (Student’s t-test: t = 1.734, P = 0.0013; Fig. 3).
The Bla g 4 content of the ARG steadily increased, so the ARG of 10-day old males
contained 3.5–4–times more Bla g 4 than 7 days earlier (Student’s t-test: t = 1.746, P =
0.0088). The pattern of accumulation of Bla g 4 protein by the ARG confirms that its
expression is socially facilitated in adult males.
Accumulation of CHC in males is not socially facilitated
The CHC gradually increased in both isolated and paired males throughout the first 15 days
after adult emergence: CHC increased in isolated males from 77.56 ± 3.74 μg on day 1 to
123.75 ± 5.47 μg on day 15, and in paired males from 79.99 ± 2.42 μg on day 1 to 123.51 ±
5.98 μg on day 15 (Fig. 4). However, there were no significant differences in the quantities of
CHC between isolated and paired males on any of these days. Thus, social interactions with
72
conspecific males do not appear to influence the rate of hydrocarbon accumulation on the
cuticular surface.
Effects of social conditions on male courtship behavior
The “antenna-on-a-stick” elicited courtship behavior in 100% of 13–14-day old males within
12.7 ± 1.26 sec (N = 115; Fig. 5A,B). We used 2- and 3-day old males to assess the
maturation of their courtship response. A surprisingly large percentage of 2-day old males
responded to the female antenna, but responses were independent of the males’ social
conditions. Even more 3-day old males exhibited courtship behavior (Chi-square test: χ2 =
9.06, P = 0.0026), but also independently of their social conditions (Fig. 5A). Notably,
however, while 3-day old paired males responded to female antennae significantly faster than
similarly maintained 2-day old males (53.3% faster; Student’s t-test: t = 4.026, P = 0.0001),
the courtship response by 3-day old isolated males was only 8.7% faster than by 2-day old
males (P > 0.05; Fig. 5B). These results indicate that the courtship response in males matures
much earlier than we suspected, and the only significant social facilitation of this behavioral
response was the decline in its latency between days 2 and 3.
Social facilitation of copulation success in males
The percentage of males that copulated increased with age, with no apparent differences
between isolated and paired males. None of 91 tested males copulated on day 1, only 18.6%
73
of 2-day old males copulated (N = 311), 89.5% of the males mated on day 6 (N = 38), and
100% of 13–14-day old males mated (N = 47; Fig. 6A). We found significant differences
between 2-day old males and all older males (Chi-square test: χ2 = 93.87, P < 0.0001), but
not between isolated and paired males at any age (χ2 = 1.33, P = 0.25). We also examined the
latency of copulation over the 2 hrs of observation, and likewise found no significant
differences between socially paired and isolated males until day 6; in all groups, most of the
males that mated did so within 35 min after sexually mature females were introduced to the
arena. On day 6, however, the average latency to copulation in paired males was significantly
lower than in isolated males (17.8 ± 1.51 min vs. 28.3 ± 3.61 min; Student’s t-test: t = 1.694,
P = 0.0075). Overall, in paired males the average latency to copulation declined 47.3% from
33.8 ± 3.94 min on day 2 to 17.8 ± 1.51 min on day 6 (Student’s t-test: t = 1.676, P =
0.0054), whereas in isolated males the latency varied little, from 34.2 ± 3.41 min on day 2 to
28.3 ± 3.61 min on day 6 (t = 1.683, P = 0.1265; Fig. 6B). Thus, either the ability of males to
copulate, or acceptance of males by females, appeared to vary with male social conditions.
Unmated B. germanica females, as well as females that fail to receive sperm during
copulation, oviposit unfertilised eggs into an egg case and subsequently abort the egg case
(Roth and Stay 1962). Therefore, abortions provide an indirect but reliable measure of male
competence to transfer sperm to the female and produce viable progeny. Only 2 of 47 (4.3%)
females that mated with 13–14-day old males aborted their egg case, whereas >60% of egg
cases from females that mated with 2-day old males were aborted (Fig. 6C). Significantly
more females aborted the egg case when mated with 2-day old males (Chi-square test: χ2 =
38.51, P < 0.0001) and 3-day old males (χ2 = 5.00, P = 0.03) than when mated with older
74
males. The frequency of abortions was 33.6% higher in females mated with 2-day old
isolated males than with pair-housed males, but there were no significant differences between
these two treatments for males of any age (χ2 = 2.15, P = 0.14). Therefore, it appears that
male competence to successfully fertilize eggs might be socially facilitated, but these effects
are slight and are rapidly obscured by age-related sexual maturation.
DISCUSSION
Our study revealed that several aspects of male reproductive physiology and behavior are
profoundly socially facilitated in the German cockroach, while others are not. Although
social facilitation of male sexual maturation has been demonstrated in phase-polyphenic
species (e.g., locusts; review: Pener and Simpson 2009), to our knowledge, this is the first
demonstration of social facilitation of reproductive processes—specifically endocrine
function—in a non-polyphenic insect species. Similarly to German cockroach females, males
undergo an endocrine-regulated sexual maturation after adult emergence, and social
interactions facilitate this process by stimulating higher JH production at a younger age,
which in turn stimulates faster production of accessory reproductive secretions and more
rapid maturation of sexual receptivity and sexual readiness. Nevertheless, some associated
events, such as accumulation of hydrocarbons on the cuticular surface, appear not to respond
to social conditions and thus may be relatively more independent of JH.
JH regulates the rate of female reproduction in the German cockroach (Bellés et al.
1987; Gadot et al. 1989b; Burns et al. 1991; Schal and Chiang 1995; Schal et al. 1997) and
75
we recently showed that social interactions in B. germanica females can modulate the rate of
JH production (Uzsák and Schal 2012 (Chapter 1)). Our current results with adult males are
consistent with previous reports that JH release rates in males are considerably lower than in
females (Piulachs et al. 1992), but the patterns of CA activity in males suggest a role for JH
in male sexual maturation, as in females. Also similar to females, we showed that JH
synthesis reached higher rates in males that were allowed social interaction with other males
and this response was maximal at a density of 4–8 individuals per cage. With both sexes,
however, social facilitation of JH production was evident at densities as low as two paired
individuals. Still, two major differences are apparent between the responses of males and
females to interactions with conspecific individuals. First, JH production was suppressed in
females under crowded conditions (20 females per dish) (Uzsák and Schal 2012 (Chapter 1)),
whereas male CA appeared less responsive to crowding under the same conditions. Second,
although CA activity was lower in isolated females than in paired females, ultimately females
in both treatments oviposited infertile eggs and their CA reached similar activity levels
(Gadot et al. 1989b). In males, on the other hand, the differences in JH production persist for
at least 19 days, well beyond the period of sexual maturation. It is important to note that all
our study subjects were group-reared throughout their 35-day nymphal development, and
either isolated or paired only in the adult stage. It is possible that isolation during nymphal
development might further magnify the effects of isolation in the adult stage, and the results
suggest that differences between grouped and isolated males may persist and affect
physiological and behavioral events beyond the period of sexual maturation.
76
ARG proteins are involved in spermatophore formation, protection of sperm,
facilitating sperm transfer to the female, sperm storage within the spermatheca, sperm
competition, and in modulating female physiology and behavior (reviews: Happ 1984; Gillott
2003; Avila et al. 2011). JH regulates male ARG development during sexual maturation in
many insect species, although in some ARG development appears to be independent of JH
(reviews: Happ 1984; Gillott 2003). In male B. germanica ARG protein content increases
gradually following adult emergence, and this age-related production is modulated by JH
(Piulachs et al. 1992; Vilaplana et al. 1996a, b; Fan et al. 2005). We used Bla g 4, an adult
male ARG-specific protein that is incorporated into the spermatophore and transferred to the
female during copulation (Fan et al. 2005), as a measure of the influence of social conditions
on the male’s reproductive physiology. The results demonstrate a clear social facilitation of
ARG maturation: the Bla g 4 contents of pair-housed males were significantly higher than in
socially isolated males on days 3 and 10. Moreover, the Bla g 4 contents of the ARG
diverged more between isolated and paired males on day 10 than on day 3, reflecting the
greater difference in JH production that was evident after day 5. ARG proteins are depleted
during mating, and elevated JH synthesis rates after mating induce the accumulation of new
proteins (Vilaplana et al. 1996a). Therefore, it would be fascinating to know whether isolated
males, because they produce less JH, are also less capable of replenishing their ARG
secretions than socially grouped males, regardless of their ages.
We suspected that other aspects of sexual maturation in males would be socially
facilitated as well. Surprisingly, however, we found only scant evidence for the effects of
social conditions on mating behavior, because rapid age-related maturation within the first 1–
77
3 days after adult emergence appeared to dominate and possibly obscure the effects of social
conditions. In investigating the maturation of the male courtship response, we were also
surprised to find that by 3-days after adult emergence an exceptionally high proportion of the
males (~85%) were able to respond sexually to a pheromone-bearing detached antenna from
a sexually mature virgin female. These results were in contrast to results reported by Nishida
and Fukami (1983), where such high sexual responses did not mature until males were 7–8
days old, but these differences likely resulted from dissimilar rearing conditions. Overall, it
appears that male sexual responses mature unusually fast, faster than other physiological
processes such as ARG maturation and the male’s ability to successfully inseminate the
female and fertilize her oocytes (below). Isolated and grouped males did not differ in
courtship attempts and number of matings (Lihoreau et al. 2009), but the latency of the
courtship response suggests some limited social facilitation of courtship. Whereas the
courtship latency of isolated males changed little between days 2 and 3, in paired males the
latency declined by 53.3% in just one day, and pair-housed 3-day old males responded nearly
as fast as 13–14-day old males. Further analyses of these fine-scale changes in response to
social interactions will require greater sample sizes and possibly slowing the rate of sexual
maturation by placing males at cooler temperatures.
Successful courtship in B. germanica is followed by copulation that lasts ~90 min,
during which the male transfers a spermatophore to the female. B. germanica females may
obtain and store enough sperm from a single copulation to last their entire reproductive life
(Cochran 1979), and apparently this severely biases the operational sex ratio and constitutes
strong selective pressure on males to mature rapidly and express low courtship thresholds
78
(Eliyahu et al. 2009). This was apparent in our assessment of mating success and frequency
of egg case abortions. Young males were either incapable of mating or were rejected by
females, but the males’ ability to copulate increased with age to 100% success by 2 weeks.
However, the only differences we found between isolated and paired males were in the
latency of copulation, not in the percentage of males mating, and even these differences were
in age-related changes rather than absolute differences between isolated and paired males—
the average latency to copulation declined dramatically in socially paired males and much
less so in isolated males. Moreover, few females mated with 2-day old males, and most of
these females aborted their egg case, presumably because sperm transfer was ineffective or
sperm were inviable. Although the frequency of abortions was much higher in females that
mated with isolated males than with paired males, again, this difference was not statistically
significant. To unambiguously determine whether the male’s competence to successfully
mate is socially facilitated will require a larger sample size than the 311 two-day old males
we used.
Finally, we found no evidence of social facilitation of CHC. CHC have been
investigated as pheromone precursors and as mediators of kin recognition in the German
cockroach (Lihoreau and Rivault 2009; Blomquist et al. 2011). While there is no evidence in
males of JH-modulated production of CHC-derived pheromones, as shown in females
(review: Blomquist et al. 2011), we hypothesized that social conditions might affect the
display of hydrocarbons on the cuticle because of their role in kin recognition. Moreover, as
water-proofing agents, more CHC might be placed on the epicuticle of isolated males to
retard water loss. Although the amount of CHC gradually increased with age, both isolated
79
and paired males accumulated similar quantities of CHC. In females, much more CHC
accumulate in internal stores (mostly haemolymph and fat body) than on the cuticular surface
(Schal et al. 1994), so it might be instructive to consider whether social conditions influence
the internal accumulation of CHC in males. Interestingly, in D. melanogaster CHC sex
pheromones vary with the circadian clock and social conditions (grouped in single or mixed
genotypes) affect the pattern of CHC accumulation, in support of the idea that social context
can shape the relationship between an individual’s phenotype and genotype (Krupp et al.
2008).
All in all, our results show that social interactions facilitate some aspects of male
reproductive physiology, but not others. Maturation of the male’s courtship response appears
to be relatively independent of social conditions, although its latency as well as the latency to
copulation was shorter in paired males than in isolated males. Likewise, egg case abortion
was more common in females that mated with isolated males than those that mated with
paired males. On the other hand, JH biosynthesis and ARG protein production, two critical
constituents of male sexual readiness, were significantly facilitated by group-living and
repressed by isolation. Indeed, CA activity in isolated males remained lower than in grouped
males even beyond the males’ sexual maturation. This observation suggests that isolated
males may represent a cryptic “solitarious phase”, distinct from “gregarious” males. It will be
important to determine if these two phenotypes are readily reversible, as shown in females
(Uzsák and Schal 2012 (Chapter 1)) and if they differ in their expression of behavioral
syndromes, such as aggression, exploration, foraging, sociability and interactions with
potential mates. Lihoreau et al. (2009) documented such differences between group living
80
and solitary nymphs, but it is not known whether differences in the social conditions in the
adult stage can affect behavior. Unlike locusts, where male sexual maturation and mating
behavior completely depend on the CA and JH, and social conditions affect CA activity
(review: Pener and Simpson 2009), sexual maturation in B. germanica males is less
dependent on both social conditions and JH. The German cockroach is an exemplary colonist
species and an obligatory human commensal pest. Because populations of B. germanica
undergo severe bottlenecks and frequent boom-and-bust cycles, it is perhaps not surprising
that males of this long-lived species have evolved a sexual maturation system that is
relatively independent of social context, compared to females. Socially isolated new colonists
can rapidly mature sexually and mate soon after adult emergence, yet if receptive females are
not available males are ready for new receptive females or even for the next colonisation
event.
ACKNOWLEDGMENTS
We thank to Yasmin J. Cardoza, Jules Silverman, and Wes Watson for critical comments on
this manuscript. We also thank Isaac Warren and Chad Gore, who contributed to the Bla g 4
study, Jane Bachmann for contributions to the JH studies, and Consuelo Arellano
(Department of Statistics, North Carolina State University) for help with statistical analyses.
This project was supported in part by the National Institute of Food and Agriculture, U.S.
Department of Agriculture (award number 2009-35302-05303), the National Science
Foundation (award IOS-1052238), the Blanton J. Whitmire endowment at North Carolina
81
State University and scholarships from the North Carolina Pest Management Association and
the Structural Pest Management Training and Research Facility.
82
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Blattella germanica (L.): Characterization and relation to hemolymph titers of juvenile
hormone and hydrocarbons. Journal of Insect Physiology, 45, 431-441. doi: 10.1016/S00221910(98)00142-5.
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21, 738-749. doi: 10.1016/j.cub.2011.06.006.
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Treiblmayr, K., Pascual, N., Piulachs, M., Keller, T. and Belles, X. 2006. Juvenile
hormone titer versus juvenile hormone synthesis in female nymphs and adults of the German
cockroach, Blattella germanica. Journal of Insect Science, 6, 1-7.
Uzsák, A. and Schal, C. 2012. Differential physiological responses of the German
cockroach to social interactions during the ovarian cycle. Journal of Experimental Biology,
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Vilaplana, L., Piulachs, M. and Bellés, X. 1996b. The conglobate gland of Blattella
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formation of the spermatophore in Blattella germanica (L.) (Dictyoptera, Blattellidae).
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reared alone and in groups. Journal of Insect Physiology, 29, 665-668. doi: 10.1016/00221910(83)90040-9.
93
FIGURES
c
JH release rate (pmol/hr/CA pair)
3
2
c
bc
bc
b
b
59
58
19
41
20
22
20
1
2
4
8
12
16
20
a
1
0
Adult males per dish
Figure 1. The effect of B. germanica adult male density on JH-III biosynthesis. Males were
isolated or grouped for 7 days after adult emergence (day 0). JH-III release rates were
measured in vitro using a radiochemical assay, and each bar represents the mean ± SE.
Sample size is indicated within each bar. ANOVA: F6, 232 = 11.79, P < 0.0001. Means were
compared by LSD, and means not sharing a letter are significantly different (P < 0.05).
94
JH release rate (pmol/hr/CA pair)
3
Isolated-reared
Isolated-reared
Isolated
Pair-housed
Pair-housed
Paired
*** ***
**
2
**
1
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19
Adult male age (day)
Figure 2. In vitro JH-III release rates during the first 19 days of adult B. germanica males.
Males were isolated or paired for 1–19 days after adult emergence (day 0) and JH-III release
rate were measured using an in vitro radiochemical assay. Each value represents the mean ±
SE of 20–59 males, and asterisks represent significant differences between the two
treatments on the same day (Student’s unpaired t-test, **P < 0.01; ***P < 0.001).
95
*
1.4
Bla g 4 (mg per ARG)
1.2
Isolated-reared
Isolated
Pair-housed
Paired
1.0
0.8
0.6
*
0.4
0.2
10
10
9
9
0.0
day 3
day 10
Adult male age
Figure 3. Social facilitation of protein accumulation in B. germanica adult male accessory
reproductive glands. Males were isolated or paired for 3 or 10 days after adult emergence
(day 0), their ARG extracted, and Bla g 4 protein was measured with ELISA. Each bar
represents the mean ± SE, sample size is indicated within each bar, and asterisks represent
significant differences between treatments within day (Student’s unpaired t-test; day 3: t =
1.734, P = 0.0013; day 10: t = 1.746, P = 0.0088).
96
Cuticular hydrocarbons (μg)
150
Isolated-reared
Isolated
Pair-housed
Paired
125
100
75
50
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Adult male age (day)
Figure 4. Effects of social isolation and social interactions on the amount of hydrocarbons on
the cuticular surface of B. germanica adult males. Newly-emerged adult males (day 0) were
socially isolated or housed in pairs in Petri dishes. Male extracts were analysed by GC, and
the total CHC amounts were calculated and compared. Each point shows the mean ± SE of
10 males. No significant differences were found between the two treatments (Student’s
unpaired t-test, P > 0.05).
97
Figure 5. Effects of isolation and social interactions on courtship behaviour in B. germanica
males. Newly-emerged adult males (day 0) were housed in Petri dishes either isolated or in
pairs for 2 or 3 days. Male courtship responses were tested with an antenna detached from a
6-day old B. germanica adult female and attached to a glass pipette (“antenna on a stick”
assay). The males’ antennae were gently stroked with the female antenna and a positive
response (turning and wing-raising) within 2 min was recorded. A) Percentage of males that
exhibited a courtship response (logistic regression and Chi-square test). B) Average ± SE
latency of the courtship response (Student’s unpaired t-test). Sample sizes are shown within
each bar. Asterisks represent significant differences between ages within treatments: *P <
0.01, **P < 0.001; there were no significant differences between treatments.
98
*
A
*
100
Males responded (%)
Isolated
Isolated-reared
Paired
Pair-housed
75
Control
50
25
68
74
74
41
40
41
0
2
13-14
B
3
13-14
**
Average courtship latency (sec)
50
40
30
20
10
43
51
74
35
34
41
0
2
13-14
3
13-14
Adult male age (day)
99
Figure 6. Effects of social isolation and social interactions in B. germanica adult males on the
frequency and latency of copulation, and the frequency of egg case abortion. Newly-emerged
adult males (day 0) were reared in isolation or in pairs and exposed to 6-day old sexually
mature virgin females. Different males were used each day and compared to 13–14-day old
males. A) Mating was monitored every 15 min during a 2 h period, and the percentages of
males that copulated were computed. B) The latency to copulation was obtained for each
male to the nearest 15 min interval and mean ± SE was calculated for each treatment
(Student’s unpaired t-test, *P < 0.05; **P < 0.01; ***P < 0.001). C) Mated females were
monitored daily and the percentage that aborted their egg cases was determined for each male
treatment. Sample sizes are shown within each bar. Data in A and C were analysed by
logistic regression and Chi-square test for model fit. The asterisks in A and C represent
significant differences among different ages (P < 0.05); there were no significant differences
between treatments within day.
100
100
A
*
100
100
100
75
Males mated (%)
75
75
75
50
25
Isolated-reared
Isolated-reared
Isolated-reared
Isolated
Isolated-reared
Pair-housed
Pair-housed
Pair-housed
Paired
Pair-housed
Control
Control
Control
50
50
50
25
25
25
0
3
4
5
6
0
0
0
B
45 46
155 156
1
1
38
22
1
35
38
33
2
42
30
44
3
30
20
55
18
47
66
4
5
13-14
13-14
6
13-14
100
50
Average latency of copulation (min)
2
75
40
Isolated-reared
Isolated-reared
Isolated-reared
**
Pair-housed
Paired-housed
Paired-housed
Control
**
50
30
20
25
10
0
0
45146
25 235
21 317
20 420
22 517
18 616
1
2
3
4
5
6
13-14
47
13-14
C
*
100
*
100
75
Abortion (%)
1
Isolated-reared
Isolated-reared
Pair-housed
Pair-housed
75
Control
50
50
25
25
0
45 46
1
25
35
2
21
17
3
20
20
4
22
17
5
Adult male age (day)
18
47
16
6
13-14
1
2
0
3
4
5
101
6
CHAPTER 3.
Sensory cues involved in social facilitation of reproduction in Blattella germanica
females
(This work was accepted and is currently in print in PLoS ONE)
102
ABSTRACT
Cockroaches, like many other animal species, form aggregations in which social stimuli from
conspecifics can alter the physiology, morphology, or behavior of individuals. In adult
females of the German cockroach, Blattella germanica, social isolation slows oocyte
development, sexual maturation, and sexual receptivity, whereas social interactions as
minimal as between just two females accelerate reproduction; however, the sensory
modalities and pathways that mediate these physiological and behavioral changes are poorly
understood.
We explored the roles of visual, olfactory, and tactile cues in the reproductive physiology of
German cockroach females, and whether their effects are species-specific and related to
circadian time. Our results show that tactile cues are the primary sensory input associated
with social conditions—with no evidence for involvement of the visual and olfactory
systems—and that the antennae play an important role in the reception of these tactile cues.
This conclusion is supported by the observation that interactions with other insect species of
similar or larger size and with similar antennal morphology also stimulate oocyte
development in B. germanica. Social facilitation of reproduction is expected to be influenced
by the circadian timing system, as females engage in more social contact during the day
when they shelter in aggregations with conspecifics. Surprisingly, however, the female’s
reproductive rate was unresponsive to social interactions during the photophase, whereas social interactions as short as two hours during the scotophase were sufficient to induce faster
103
reproduction.
We discuss the adaptive significance of these sensory-neuroendocrine responses in the
German cockroach.
INTRODUCTION
Group-living in animals is a critical component of various adaptive behaviors including
foraging, food handling, avoiding predators, mate finding and mate choice, and reproduction
(Krause and Ruxton, 2002). In many species, group-living can alter the behavior,
morphology, or physiology of individuals, usually promoting survival and greater fitness
(Krause and Ruxton, 2002; Wilson, 1971). This phenomenon—known as “social facilitation”
or “grouping effect” (Grassé, 1946; Gervet, 1948)— is well described in many insect species,
and is a form of group-induced phenotypic plasticity. Thus, grouping can affect larval
development (Chauvin, 1946; Long, 1953; Wharton et al., 1968; Izutsu et al., 1970;
McFarlane and Alli, 1984; Weaver et al., 1990; Holbrook and Schal, 1998; Lihoreau and
Rivault, 2008), and larval or adult morphology (Johnson, 1965; Lees, 1967; Sutherland,
1969; Iwanaga and Tojo, 1986; Zera and Tiebel, 1988; Lester et al., 2005). In several
cockroach species, for example, grouped nymphs develop faster and reach the adult stage
sooner than those reared in isolation (Roth and Willis, 1960; Wharton et al., 1968; Izutsu et
al, 1970; Woodhead and Paulson, 1983; Lihoreau and Rivault, 2008). The sensory cues that
104
mediate group-induced phenotypic plasticity may derive from direct social interactions or
from perceiving the presence of conspecifics without direct contact.
The effects of social interactions on reproduction have been investigated as well,
mainly in orthopterans (Norris, 1950; Norris, 1952; Highnam and Haskell, 1964; Sutherland,
1969; Bradley, 1985; Maeno et al., 2011). For example, when solitarious desert locust adults
(Schistocerca gregaria (Frosk.) (Orthoptera: Acriidae)) interact socially, a phase change
occurs and they produce gregarious-form hatchlings that are larger and darker than those
produced by solitarious females (Maeno et al., 2011). The effects of social conditions on
reproduction have also been investigated in two cockroach species, the German cockroach,
Blattella germanica (L.) (Dictyoptera: Blattellidae) and the brownbanded cockroach, Supella
longipalpa (Fabricius) (Dictyoptera: Blattellidae). During copulation, physical contact with
males and transfer of a spermatophore induce faster oocyte development in females of both
species; however, so far the German cockroach appears to be the only cockroach species
where females also respond to non-copulatory social interactions, including with other
females, with faster sexual maturation and oocyte development (Gadot et al., 1989b, Chon et
al., 1990; Schal et al., 1997; Holbrook et al., 2000). Juvenile hormone III (JH), a major insect
gonadotropic hormone produced and released by the corpora allata (CA), regulates the rate of
female reproduction in B. germanica, including the synthesis and uptake of vitellogenin and
other female-specific proteins, onset of sexual receptivity, production of sexual signals,
mating, and the time-course of oviposition (Bellés et al., 1987; Gadot et al., 1989a; Burns et
al, 1991; Schal and Chiang, 1995; Schal et al., 1997). We recently showed that social
interactions with other females can modulate the rate of all JH-dependent events in the
105
female reproductive cycle, as assayed by JH production, oocyte development, and sexual
maturation (Uzsák and Schal, 2012 (Chapter 1)).
The sensory pathways through which social interactions modulate the reproductive
rate vary widely among animals, but can include various combinations of visual, tactile,
chemical (olfactory and/or gustatory) or auditory cues. For example, in some lizards, female
ovarian development is stimulated by the sight of displaying males (Crews, 1975). Similarly,
in flamingos, a positive relationship has been revealed between behavioral stimulation from
group displays and reproductive success: large flock size on the breeding ground facilitates
pair formation and stimulates nesting and breeding (Stevens, 1994). The importance of
chemical cues is probably best described in eusocial insects, where pheromones play a
central role in the coordination and integration of colony activities such as caste
differentiation, division of labor, and foraging (Robinson, 1992). In other insect species, like
S. gregaria, just a few hours of social interaction are sufficient to induce a behavioral phasechange from solitarious to gregarious individuals. Gregarization is triggered by both the sight
and smell of other locusts (Roessingh et al., 1998; Hägele and Simpson, 2000), but direct
contact with other locusts is the primary trigger of phase change, and touch-sensitive sensilla
on the outer surfaces of the hind femora have been identified as the critical sensory structure
for contact stimulation (Roessingh et al., 1998; Hägele and Simpson, 2000; Simpson et al.,
2001; Rogers et al., 2003). In addition, when isolated adult female locusts are placed in a
group, tactile stimulation perceived through their antennae causes these females to produce
gregarious offspring (Maeno et al., 2011).
In cockroaches, the sensory stimuli responsible for social facilitation of nymphal
106
development have been investigated in B. germanica and in the American cockroach,
Periplaneta americana (L.) (Dictyoptera: Blattidae), but they remain poorly understood.
Both species are nocturnal, and in both, visual, olfactory and gustatory cues from
conspecifics appear to have little or no effect on nymphal growth, but tactile stimulation is
sufficient to trigger faster development (Wharton et al., 1968; Izutsu et al., 1970; Nakai and
Tsubaki, 1986; Lihoreau and Rivault, 2008). The effect of social experience on nymphal
development is not species-specific, because nymphs of B. germanica grouped with other
cockroach species or even locusts grow faster than isolated nymphs (Izutsu et al., 1970;
Lihoreau and Rivault, 2008). Since different taxa presumably produce different chemical
cues, it would appear that chemoreception is less important than tactile stimuli in modulating
the developmental rate. This inference is supported by remarkable observations that contact
with a rotating bird feather also stimulated faster development in B. germanica nymphs
(Lihoreau and Rivault, 2008). It is not known which sensory stimuli facilitate faster
reproduction in B. germanica females.
A proposed model for social facilitation of reproduction in B. germanica is that
sensory cues associated with social interactions stimulate the central nervous system (CNS),
which then accelerates reproduction by lifting allatostatic inhibition of the CA (Gadot et al.,
1989b; Uzsák and Schal, 2012 (Chapter 1)). However, the sensory modalities and pathways
by which such sensory cues are transduced have not been elucidated. Also of interest is
whether the effects of the grouping stimuli are modulated by a circadian timing system: Is the
“grouping effect” constant or circadian phase-dependent and effective only at specific phases
of the photocycle? Social behaviors in cockroaches that are under circadian control include
107
the release of sex pheromones by females (Smith and Schal, 1991; Liang and Schal, 1993),
behavioral response to sex pheromone (Liang and Schal, 1990; Zhukovskaya, 1995), timing
of copulatory behavior (Schal and Chiang, 1995; Rymer et al., 2007), and aggressive
interactions in males (Knadler and Page, 2009). The German cockroach is night-active, and
nymphs and adults aggregate in resting sites during the photophase (Rust et al., 1995). We
suspected that because females have more opportunities to socially interact with aggregating
cockroaches during the photophase, social facilitation of reproduction would be more
effective during the photophase than scotophase.
Here, we report on the effects of visual, olfactory, gustatory and tactile stimuli on
social facilitation of female reproduction in the German cockroach. Because they are the
anterior-most sensory appendage and are involved in social interactions, we hypothesized
that antennal contact would be crucial for this “grouping effect”. In addition to
chemosensilla, the cockroach antennae house an array of mechanoreceptors (Seelinger and
Tobin, 1981). We hypothesized that tactile cues are most important in socially facilitating
reproduction, as has been shown for nymph development. We also investigated whether the
stimuli responsible for group effects are species-specific in adult females, and whether these
effects are linked to a circadian timing system.
108
MATERIALS AND METHODS
Insects
The Blattella germanica colony (American Cyanamid strain, also known as Orlando strain,
in lab culture since 1989) was maintained in incubators at 27 ± 1ºC, 40–70% relative
humidity and under a 12:12 LD cycle. Cockroaches were allowed continuous access to water
and dry rodent diet food pellets (LabDiet 5001 PMI Nutrition International, Brentwood, MO,
USA). Newly emerged virgin adult females were separated immediately after eclosion (day
0). Only females of similar size and degree of sclerotization with intact wings were used for
all behavioral assays, which were conducted under the same environmental conditions
described for colony rearing. We use “paired” to denote pair-housed females, i.e., a B.
germanica female housed in the same dish with either a conspecific female or another insect,
as described below.
Oocyte dissection and measurements
In B. germanica, a single basal (vitellogenic) oocyte matures synchronously in each of
approximately 40 ovarioles during the preoviposition period, and the length of the basal
oocytes is a reliable measure of the female’s reproductive stage because it is highly
correlated to JH biosynthesis and JH titer (Sevala et al., 1999; Treiblmayr et al., 2006). The
ovaries of cold-anesthetized females were dissected under cockroach saline (Kurtti and
109
Brooks, 1976) and 10 basal oocytes were randomly measured with the aid of an ocular
micrometer in the eyepiece of a dissecting microscope. The lengths of 10 oocytes were
averaged for each female and represented a single replicate.
Diel periodicity of the reproductive response to social interactions
The German cockroach is active at night, and nymphs and adults aggregate in resting sites
during the photophase (Rust et al., 1995). Because females would be more likely to interact
with other cockroaches during the photophase, in aggregations, we hypothesized that
interactions during this period would be more effective at stimulating the grouping-mediated
acceleration of reproduction. Conversely, we suspected that less frequent social encounters
would occur in the scotophase, during foraging activities. To investigate the effects of
transient pairing in otherwise socially isolated females on oocyte maturation, cohorts of
newly-eclosed females (day-0) were reared in isolation, but paired daily in small plastic
cages (95 x 95mm, 90 mm high, Althor Products, Windsor Locks, CT, USA) for various
periods (1, 2, and 6 h) either during the middle of their scotophase or photophase. These
females were dissected and their oocytes were measured on day 6. Sample size was 19−22
females per treatment.
110
The effects of visual cues on reproductive rate
Because the German cockroach is nocturnal, we did not expect vision to be a major sensory
modality through which social interactions would accelerate reproduction. Newly-eclosed
females were either socially isolated or pair-housed with a conspecific female in a plastic
Petri dish (90 mm diameter, 15 mm high, Fisher Scientific, Pittsburgh, PA, USA)
provisioned with food and water. Some isolated females were allowed an unobstructed view
of other females through the tops and bottoms of the plastic dishes, whereas opaque dividers
between and around the dishes prevented other isolated females from the same cohort from
seeing conspecifics. The eyes of another set of females were painted with opaque nail polish
to mask their vision and these females were paired with an intact conspecific female in a
Petri dish; newly-eclosed intact control females were either isolated or paired. On day 6,
females were dissected and their basal oocytes were measured. Sample size was 18−23
females.
The effects of chemical cues on reproductive rate
We hypothesized that the presence of conspecific chemical cues would be sufficient to
accelerate oocyte development in socially isolated females. Individual newly-eclosed females
were socially isolated in Petri dishes. Chemical cues consisted of either cockroachconditioned filter papers or live cockroaches separated from our subjects by a fine-mesh
screen that prevented physical contact. Conditioned filter papers were obtained by placing
111
clean filter papers (Whatman#1, 90 mm diameter) for 7 days in dishes with either (1) 10
German cockroach nymphs or (2) 10 German cockroach females. Volatile chemical cues
were provided for 6 days by (1) using fine-mesh mosquito screen between dishes (15 mm
high Petri dishes) of isolated females, and (2) placing a screen between a socially isolated
female and a group of 15 conspecific females; this design prevented contact between
cockroaches. The length of the basal oocytes was measured on day 6. Sample size was 14−20
females per treatment.
Species-specificity of the “grouping effect” in B. germanica
To test whether females respond to social interaction with other insect species, newly-eclosed
individual females were placed in Petri dishes with food and water ad libitum and pairhoused for 6 days with one of the following insects: ant worker (Camponotus
pennsylvanicus), beetle larva (Tenebrio molitor), beetle adult (Tenebrio molitor), stink bug
adult (Chinavia hilaris), smokybrown cockroach nymph (Periplaneta fuliginosa), adult fly
(Musca domestica), katydid adult (Conocephalus strictus), brownbanded cockroach adult
female (Supella longipalpa), and camel cricket adult (Ceuthophilus maculatus). The basal
oocytes of B. germanica females were measured on day 6 and sample size was 17−22
females per treatment.
112
The effects of tactile cues on the reproductive response
In addition to chemosensilla, the cockroach antennae house an array of mechanoreceptors
(Seelinger and Tobin, 1981). Because they are the anterior-most sensory appendage and are
involved in social interactions, we hypothesized that antennal contact stimulation might be
sufficient to accelerate oocyte growth. Newly-eclosed females were socially isolated in Petri
dishes with food and water ad libitum under five experimental conditions. First we tested the
importance of motion by placing a freshly freeze-killed conspecific female into the dish;
dead females were replaced daily. In subsequent experiments, mechanical stimuli were
provided by glass beads (9 small, 4 mm diameter beads and 1 larger, 5 mm diameter bead;
Fisher Scientific) placed in each dish with one female. The dish was placed on a rotary
shaker (The Waver, VWR Scientific, pitch 6º, speed 4, 22 rpm) so that beads slowly
contacted the female in a random fashion. Contact with conspecific females was provided by
tethering the wings of a live female and either placing the tethered female inside the dish
with the test female or introducing only her antennae and a small portion of the head through
a small hole in the side of the Petri dish. The latter treatment was repeated with tethered
American cockroach females (P. americana). Each experiment lasted for 6 days, when B.
germanica females were dissected and their basal oocytes were measured. Sample size was
19−22 females per treatment.
In order to differentiate chemical cues from contact cues, we also devised a bioassay
for evaluating the effects of antennal contact cues alone. P. americana females were selected
to provide the contact stimulus. A live P. americana female was placed into a 15 ml plastic
113
tube, with only the antennae protruding through a small hole in the tube (see Fig. S1). The
head was covered with parafilm so that only the antennae extended through a hole into a Petri
dish (60 mm diameter, 15 mm high, Fisher Scientific), where a newly-eclosed B. germanica
female was placed. Three additional treatments included (a) P. americana females whose
antennae were extracted with hexane to eliminate chemical cues; we washed the antennae
sequentially in three vials containing hexane for 30 s each, and then allowed them to dry; (b)
P. americana females whose antennae were extracted, as described above, but we reapplied
on each extracted antenna a lipid extract from P. americana females that also contained
cuticular hydrocarbons (CHC; see below), and (c) P. americana females whose antennae
were carefully ablated with irridectomy scissors just distal to the scape and an artificial
microfibett (1 mm diameter, 40 mm long, smooth surface, Spirit River Inc., Roseburg, OR,
USA) was glued onto each scape supported by a plastic sleeve (Fig. S2). P. americana
females were replaced every other day during the 6-day-long experiment. On day 6, the
oocyte length of B. germanica females was measured. Sample size was 14−34 females per
treatment.
The re-application of cuticular lipids on the antennae (b above) was conducted as
follows. Fifteen freshly-killed P. americana females were extracted for 5 min in 75 mL of
pentane. The pentane was reduced to ~2 mL under a gentle stream of N2 and filtered through
silanized glass wool in a Pasteur pipet to remove particulates. The filtrate was then reduced
to dryness and redissolved in 1 mL hexane. A 1 µL aliquot of this solution was combined
with 0.1 mL of hexane containing 10 µg of n-dotriacontane as an internal standard, and the
concentration of the CHC in this solution was determined by gas chromatography. The CHC
114
solution (without the internal standard) was then adjusted to 16 µg of total CHC per 1 µL
hexane. We applied 80 µg total CHC (5 µL) to each antenna; this amount was determined by
gas chromatography to be the average amount of CHC extracted in 5 min from the antennal
cuticular surface of P. americana females.
Function of the antennae in receiving cues that stimulate reproduction
The antennae likely are essential in receiving sensory input that ultimately modulates the
reproductive rate in B. germanica. Therefore we conducted bilateral antennectomies to
eliminate these inputs. Females were briefly anesthetized with carbon-dioxide, placed on ice
and the flagellum of each antenna was cut with irridectomy scissors just distal to the scape.
Females were immediately put into one of five treatment groups: (1) isolated-housed intact
female (negative control); (2) isolated-housed antennectomized female; (3) an intact female
paired with an antennectomized female; (4) two antennectomized females; and (5) pairhoused intact females (positive control). Females were housed in plastic Petri dishes (60 mm
diameter, 15 mm high) provisioned with food and water ad libitum and their oocytes were
measured on day 6.
A complementary, less traumatic treatment involved separating two intact females
with a wide-mesh metal screen (2 mm mesh opening) which permitted some antennal contact
but prevented body contact between the two females.
115
Statistical analyses
Data were analyzed with one-way ANOVA for multiple comparisons using SAS® 9.1.3
software (SAS Institute Inc. 2002-2003, Cary, North Carolina). We used PROC GLM to test
for the effects of social conditions on oocyte length and JH synthesis as dependent variables.
PROC GLM was also used to get the residuals from adjusted model and test whether
residuals hold the assumption of homogeneous variances within each treatment. Since data
were unbalanced, we used LSMEANS and LSD test was used to compare means at 0.05
significance level. Variation around the mean is represented by the standard error of the
mean (SE).
RESULTS
Social facilitation of reproduction is more effective in the scotophase
Contrary to our expectations, social interactions for 6 h daily only during the photophase did
not accelerate the reproductive rate (oocyte length 1.20 ± 0.06 mm, N = 19) relative to social
isolation for the entire 6 days (negative control; 1.19 ± 0.06 mm, N = 24) (Fig. 1). In contrast,
the presence of another female for the same duration, but during the scotophase, significantly
enhanced oocyte maturation (1.61 ± 0.06 mm, N = 20) to the level of females paired with
another female for the entire 6 days (positive controls; 1.66 ± 0.03 mm, N = 20) (Fig. 1). A
dose (time)-response (oocyte length) experiment revealed that while daily social interaction
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for just 1 h during the middle of scotophase did not affect oocyte maturation, 2 h of daily
social interaction was sufficient to significantly accelerate oocyte maturation in B. germanica
females (Fig. 1).
Chemical and visual cues appear to not play a role in social facilitation of reproduction
We found no evidence of an effect of either contact or volatile chemical stimuli on the pace
of oocyte maturation (Fig. 2). In all treatments involving social isolation and exposure to
contact or volatile chemicals, the length of the basal oocytes was not significantly different
from the negative controls (females socially isolated during the entire experiment), indicating
a lack of response to social interaction via chemical communication. On the other hand, even
limited antennal contact through a wide-mesh screen stimulated oocyte maturation relative to
solitary females (1.15 ± 0.05 mm, N = 18 vs. 0.98 ± 0.05 mm, N = 18; P = 0.0562),
suggesting that antennal contact is pivotal to the grouping effect.
Similarly, visual stimuli also appeared to be unimportant because isolated females
exhibited slow oocyte growth whether their view of conspecifics was masked or not. The
reproductive rate was the same in isolated females that had visual contact with other females
and isolated females that were prevented from seeing other females by an opaque divider
between the dishes (Fig. 2). Likewise, when females with eyes painted were pair-housed with
another female for the 6-day-long experiment, the length of their basal oocytes was
approximately the same as in females with intact eyes that were also socially paired (positive
controls) (Fig. 2). These results suggest that the suppression of oocyte development in
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isolated females cannot be overcome by chemical or visual cues from other conspecific
females or nymphs.
Effects of social interactions with different insect species on reproductive rate
Contact with various insect species accelerated the reproductive rate of B. germanica
females. In the presence of one heterospecific insect of approximately similar or smaller size
than females of the German cockroach, females exhibited moderately faster oocyte
maturation than those reared in isolation (P < 0.05; Fig. 3). This effect was even more
pronounced when females were housed either with a katydid, brownbanded cockroach
female, or camel cricket, all of which have longer antennae than B. germanica females.
Interestingly, when females were housed with a beetle larva or with an ant, oocyte maturation
was slower (Fig. 3), possibly because of dramatically divergent antennal and body
morphology (beetle) or aggressive behavior (ant).
Antennal contact mediates the social facilitation of reproduction in B. germanica
The quality of contact stimuli is important, because contact with moving glass beads failed to
stimulate oocyte maturation in isolated females, whereas contact with moving antennae did
(Fig. 4). Moreover, interaction with a freshly-killed conspecific female had no positive effect
on oocyte maturation, indicating that movement is an important feature of the contact cue.
We found that the degree of social facilitation of reproduction by live conspecific females
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was related to the amount of their bodies exposed to the test female. Thus, interaction with
the antennae and body of a female that was tethered inside the dish stimulated significantly
faster oocyte maturation (1.54 ± 0.04 mm, N = 20) than interaction only with the antennae of
a conspecific female whose body was tethered outside the dish (1.32 ± 0.06 mm, N = 21)
(ANOVA, P < 0.0001). Notably, however, interaction with the antennae of P. americana
females (body tethered outside the dish) was as effective as interaction with the whole body
of B. germanica (1.55 ± 0.04 mm, N = 22) (ANOVA, P < 0.0001) (Fig. 4). These results
suggest that the quality and the quantity of social stimuli are important in the social
facilitation of reproduction in German cockroach females. The antennae appear to be
especially effective in this context, and longer and thicker antennae appeared to be more
effective than short antennae and non-filiform antennae.
Live antennae present to the test female both mechanosensory and chemical cues. We
uncoupled these cues by extracting the antennae of live females with hexane and reapplying a
cuticular lipid extract onto the antennae of some females. Females in the control group
(group 1; allowed to contact only the antennae of a tethered P. americana female; Fig. S1)
matured their oocytes significantly faster than socially isolated females (1.29 ± 0.03 mm, N =
34 vs. 0.89 ± 0.05 mm, N = 28; respectively) (ANOVA, P < 0.0001) (Fig. 5). Similar results
were obtained when we removed the cuticular lipids, including CHC, from the P. americana
antennae (group 2), and also when we reapplied the lipids onto the extracted antennae of P.
americana females (group 3); both treatments resulted in significantly faster oocyte growth
than in the isolated females (1.22 ± 0.06 mm, N = 14 and 1.22 ± 0.06 mm, N = 14;
respectively) (ANOVA, P < 0.0001) (Fig. 5). A slightly smaller effect was observed when
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we ablated the antennae of P. americana females and replaced them with artificial
(prosthetic) microfibetts (group 4, see Fig. S2; 1.11 ± 0.05 mm, N = 19); however, these
results were still significantly different from the negative control. Because all our treatments
for antennal contact using P. americana females showed nearly equivalent results, and
because even an artificial material replacing the antenna could induce faster oocyte growth,
these results provide compelling evidence that social interactions accelerate the reproductive
rate in B. germanica females mainly via mechanosensory rather than chemical cues.
The antennae receive cues that modulate the reproductive rate in B. germanica
Bilateral antennectomies of both pair-housed females completely eliminated the “grouping
effect” compared to intact pair-housed females (0.76 ± 0.05 mm, N = 20 vs. 1.50 ± 0.05 mm,
N = 26) (ANOVA, P < 0.0001) (Fig. 6), suggesting that the antennae play a pivotal function
in receiving cues from social interactions. Both socially isolated antennectomized females
and paired antennectomized females failed to develop their oocytes even to the level of intact
isolated females (0.93 ± 0.05 mm, N = 20), suggesting that complete ablation of the antennal
flagella might have side-effects related to loss of sensory input. Although surgical ablation of
the antennae likely eliminated not only the “grouping effect” but other important sensorybased behaviors as well (e.g., feeding, drinking, oriented movement), when an
antennectomized female was pair-housed with an intact female the oocytes of the
antennectomized female grew significantly (1.16 ± 0.07 mm, N = 21) compared to the intact
female whose oocytes matured maximally (1.58 ± 0.03 mm, N = 21). Therefore, the antennae
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on the female providing social stimuli (intact female) appeared to be important for the
antennectomized test female. Nevertheless, these results demonstrate that it is difficult to
disentangle the processes of “giving” social stimuli from “receiving” social stimuli because
the antennae are used for both.
DISCUSSION
Pair-housed B. germanica females experience robust socially-mediated facilitation of
reproduction, an example of phenotypic plasticity, where environmental cues direct
development along divergent trajectories, leading to different phenotypes. Our investigations
address three major questions regarding the social-facilitation of reproduction in B.
germanica: (1) What are the major sensory inputs and features of sensory stimuli, and how
species-specific are they? (2) How are sensory stimuli received by the female? and (3) What
are the sensory-CNS-neuroendocrine transduction pathways that mediate the social
facilitation of reproduction? Our results shed light on the influence of socially-derived visual,
olfactory, and tactile stimuli on the pace of reproduction in B. germanica females. We
demonstrated that visual and chemical stimuli are ineffective in eliciting faster oocyte
growth, but tactile stimulation alone was as effective as housing a female with a conspecific
female. The quality of the tactile stimulation is important too, because contact with longer
and thicker heterospecific filiform antennae was even more effective than stimulation with
conspecific B. germanica antennae. Surprisingly, modulation of the reproductive rate through
121
social interactions operates only during the scotophase when females are active, and not
during the photophase when females are in shelters in close contact with conspecifics.
Sensory inputs that facilitate reproduction in B. germanica
We designed assays to differentiate among several sensory stimuli from other individuals that
might modulate the reproductive rate of female B. germanica. Vision was excluded as an
important sensory modality in this context because socially isolated females exhibited
equally slow oocyte maturation whether they were allowed to see other females or not, and
whether their vision was blocked or remained intact. Moreover, in pair-housed females,
social interactions equally stimulated oocyte development whether the female’s vision was
obstructed or not. Thus, vision does not appear to be important in the social modulation of
the rate of oocyte growth, although it is possible that visual stimuli from conspecifics may
play a minor modulatory function in concert with other sensory modalities. This conclusion
was not unexpected because B. germanica is a nocturnal insect and most behavioral
interactions occur in dark places (Rust et al., 1995).
In contrast to vision, chemical communication is widely used in the German
cockroach in many contexts including aggregation (Ishii and Kuwahara, 1967; Wileyto et al.,
1984; Dambach et al, 1995), long- and short-range mate attraction, courtship behavior
leading to mate choice, and pre- and post-copulatory nuptial exchanges exchanges (Liang
and Schal, 1994; Nojima et al., 1999; Gemeno and Schal, 2004; Nojima et al., 2005; Eliyahu
et al., 2008). It is surprising, therefore, that the strong effects of social interactions on the
122
reproductive rate do not involve chemical stimuli: neither contact nor volatile chemical cues
from conspecifics were sufficient to induce changes in the reproductive rate of isolatedreared females. Because tactile stimulation with whole insects or just with the antennae of
tethered insects also necessarily involves chemical cues, we separated these two modalities
by extracting lipids from the antennae of P. americana and replacing them in some insects
with a lipid extract of the body; the antennae and body contain similar cuticular
hydrocarbons. Female B. germanica developed their oocytes at comparable rates whether
they interacted with lipid-free or lipid-fortified antennae, as long as they experienced tactile
stimulation. These results further confirm that chemical cues are not involved in social
mediation of oocyte maturation and that the appropriate tactile stimuli alone can induce a
reproductive response in B. germanica females.
Recent evidence suggests that group size-dependent auditory signals from wingfanning behavior mediate sonotactic orientation and group joining in B. germanica
(Wijenberg et al. 2008). Although we did not directly investigate whether acoustic cues can
modulate the female reproductive rate, three lines of indirect evidence argue against this
proposition. First, when an isolated female was separated by a screen from other females,
auditory cues, if produced, failed to stimulate faster oocyte maturation. Second, in
experiments where a female was tethered outside the arena, with only her antennae
interacting with the test female, acoustic signaling was not possible, yet oocyte maturation
was induced. And third, when other insect species were pair-housed with B. germanica
females, it is unlikely that these insects could produce auditory cues that were relevant to the
German cockroach females, yet the latter experienced a robust “grouping effect”.
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Nevertheless, auditory stimuli, however unlikely, need to be more explicitly considered as
potential facilitators of reproduction in the German cockroach.
Earlier studies with locusts differed in which social stimuli induce gregarization
(Roessingh et al., 1998; Hägele and Simpson, 2000); however, recent studies agree that
tactile stimuli are the main sensory modality responsible for socially-induced developmental
changes in nymphs (Roessingh et al., 1998; Hägele and Simpson, 2000; Simpson et al., 2001;
Rogers et al., 2003), and similar conclusions have been reached with cockroach nymphs
(Izutsu et al., 1970; Ishii, 1971; Lihoreau and Rivault, 2008). Therefore, it is not surprising
that tactile stimulation is also the main mechanism through which social facilitation of
reproduction occurs in adult females of the German cockroach. Our results showing that
interaction with moving glass beads failed to stimulate oocyte maturation in German
cockroach females suggest that qualitative features of the tactile stimuli are important.
Moreover, interactions with a freshly-killed female also failed to stimulate oocyte growth,
suggesting that movement may be an important feature of the tactile stimuli. Pair-housing
females with several different insect species suggested that in addition to movement,
characteristics such as body size and morphology, antenna type and texture, activity period
(nocturnal vs. diurnal), and behavior might affect the reproductive rate of B. germanica
females. For example, interactions with ants, which exhibit aggressive behavior and have
short antennae, suppressed oocyte growth in cockroaches. Pair-housed beetle larvae, which
have rudimentary antennae and differ dramatically from cockroaches in body morphology,
also failed to stimulate oocyte growth in B. germanica. In contrast, various species with more
prominent antennae significantly stimulated oocyte development in the German cockroach.
124
Insects with slightly smaller or similar body size to the German cockroach had moderate
effects (beetle adult, stink bug adult, fly adult, smokybrown cockroach nymph), whereas
larger insects with longer antennae (katydid adult, brownbanded cockroach female, and
camel cricket adult) had an even more pronounced effect when pair-housed with a B.
germanica female. These results suggest that the length, activity level, and general
accessibility of the antennae during social interactions may be important in reproduction, as
they appear to be in promoting nymphal development in B. germanica (Lihoreau and Rivault,
2008).
Our experiment with B. germanica females tethered either within the experimental
arena (i.e., contact with all body parts) or outside the experimental arena (i.e., contact only
with the antennae) suggests that tactile stimuli originate mainly from antennae, and to a
lesser degree from the rest of the body. In support of this notion, contact only with the active
antennae of P. americana was as effective as being pair-housed with a conspecific female.
Furthermore, contact stimulation with an artificial “antenna”—a microfibett grafted to the
antennal scape of P. americana as a prosthetic “antenna” —produced a partial grouping
effect. We therefore conclude that antennal morphology and activity are integral components
of the tactile stimuli, and that the female antennae are the main receiver of these social cues.
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How do females receive the mechanosensory information relevant to the “grouping
effect”?
Mechanoreceptive sensilla are broadly distributed throughout the body surface of insects,
with particularly high density on the legs and on sensory appendages associated with the
head and cerci. In S. gregaria the development of gregarious behavior in nymphs is elicited
through tactile input to the antennae (Heifetz et al., 1996), but Simpson et al. (2001) also
identified specialized mechanoresponsive sensilla on the outside surface of the hind femur as
the primary site of tactile input (see also Rogers et al., 2003; Anstey et al., 2009; Pener and
Simpson, 2009). Even in a relatively narrow clade of closely related insect species, however,
morphological and reproductive phase change can be triggered by tactile stimulation of
markedly different sensory organs, because in the Australian plague locust (Chortoicetes
terminifera) the antennae are the sole organ receiving tactile stimulation (Cullen et al., 2010),
and the antennae appear to be the main organ involved in adult phase change in S. gregaria
(Maeno et al, 2011).
In addition to mechanoreceptive cells that are housed in many antennal sensilla, the
cockroach antenna also possesses hair plates, campaniform sensilla and chordotonal organs
that respond to tactile stimulation (Seelinger and Tobin, 1981). We attempted to elucidate
whether the antennae play a pivotal role in modulating the reproductive rate in B. germanica
using antennectomy. Indeed, intact females experienced significantly faster oocyte
maturation than antennectomized females that were pair-housed with them. However, this
surgical manipulation proved to exert traumatic side-effects because isolated,
126
antennectomized females developed their oocytes even slower than similarly isolated intact
females, and when both paired females were antennectomized their oocyte growth over the 6
day experiment was negligible. It is possible that loss of sensory input in antennectomized
females caused them to move less, have fewer social encounters with other females, and eat
and drink less. Since food intake is a major regulator of the reproductive cycle (Schal et al.,
1993), this surgical manipulation likely confounded the “grouping effect” with other
important regulators of reproduction.
A more revealing experiment allowed solitary females to interact with other females
through a wide-mesh screen using only their antennae. We confirmed for B. germanica adult
females that the antennae alone are sufficient to receive the tactile stimuli that accelerate the
reproductive rate.
What sensory-CNS-neuroendocrine pathways are involved in social facilitation of
reproduction?
Most cockroach species are nocturnal and they rest in shelters during the day (Saunders et al.,
2002). B. germanica nymphs and adults also exhibit distinct phases of foraging and sexual
activity at night and they are relatively inactive during the day (Rust et al., 2005; Liang and
Schal, 1993). We therefore predicted that females would respond to social cues when they
aggregate at their resting sites during the day. Our results, however, provided no evidence for
social facilitation of reproduction during the photophase; on the contrary, social interactions
stimulated oocyte growth only during the scotophase, and as little as 2 hours of daily
127
interactions with another female in the middle of the scotophase was sufficient to induce
oocyte maturation. Thus, counter-intuitively, intermittent social interactions during foraging
accelerate the reproductive rate, whereas constant contact with conspecifics in shelters does
not. We suspect that the mechanisms underlying these observations relate to the circadian
phase-related coupling and uncoupling of sensory inputs with the neuroendocrine system.
In B. germanica females, JH-III regulates the reproductive rate (Bellés et al., 1987;
Gadot et al., 1989a). The brain integrates a multitude of extrinsic and intrinsic stimuli,
including those related to group-housing, and paces the activity of the CA, dictating the rate
and magnitude of all JH-dependent events (Schal and Chiang, 1995; Schal et al., 1997). We
recently showed that social interaction with other females elevates the rate of JH production,
leading to faster oocyte development and earlier onset of sexual receptivity (Uzsák and
Schal, 2012 (Chapter 1)). Interestingly, sexual receptivity, production of sex pheromone, and
mating—behaviors regulated by JH—are known to be expressed in a diel manner during the
scotophase, consistent with our observation that socially facilitated reproduction is also
expressed only during the scotophase. Therefore, we propose that two major mechanisms
could account for the lack of a “grouping effect” during the photophase: (a) Uncoupling of
the sensory input or lower brain sensitivity to sensory/tactile stimuli during the photophase,
and (b) Lower CA sensitivity to brain directives during the photophase. The latter idea, that
CA activity in the cockroach is modulated on a circadian basis, would suggest that “grouping
effects” are expressed only when the CA can respond to brain directives during the
scotophase. There is no evidence thus far of diel periodicity in CA activity in the cockroach.
However, this mechanism is appealing because it has been convincingly shown in the cricket
128
Gryllus firmus that the long-winged flight-capable morph shows a robust circadian cycle in
JH titer, whereas the short-winged flightless morph does not; the brain-directed release of the
neuropeptide allatostatin into the CA appears to play a major role in JH regulation (Stay and
Zera, 2010). The titers of some other insect hormones also cycle in a circadian manner
(review: Vafopoulou and Steel, 2005), so it is plausible that JH in B. germanica also might
be under circadian control.
How tactile stimuli get transduced into neuronal and neuroendocrine signals that
regulate CA activity and JH titer in B. germanica is unknown. In S. gregaria tactile
stimulation of specialized mechanoreceptive sensilla on the hind legs causes an increase in
serotonin levels in the metathoracic ganglion, which appears to mediate the process of phase
transition and behavioral gregarization (Rogers et al., 2003; Rogers et al., 2004; Anstey et al.,
2009). It will be particularly interesting to reveal a connection between tactile stimulation,
biogenic amines and JH because the actions of allatotropins and allatostatins are known to be
regulated and tuned by other neuropeptides, and biogenic amines are also known to be
involved in circadian gating of behavior.
ACKNOWLEDGMENTS
We thank to Yasmin J. Cardoza, Jules Silverman, and Wes Watson (Department of
Entomology, North Carolina State University) for critical comments on this manuscript. We
also thank Katalin Böröczky for help with chemical analysis, Jules Silverman for suggesting
the microfibett as a prosthetic “antenna” and Consuelo Arellano (Department of Statistics,
129
North Carolina State University) for help with statistical analyses. We are grateful to Rick
Santangelo for technical assistance during the experiments. This project was supported in
part by the Blanton J. Whitmire endowment at North Carolina State University and
scholarships from the North Carolina Pest Management Association and the Structural Pest
Management Training and Research Facility.
130
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FIGURES
b
b
1.8
b
Oocyte length (mm)
1.6
a
a
1.4
a
1.2
1.0
0.8
0.6
Paired daily: 0 h
1h
2h
scotophase
1
6h
6h
24 h
photophase
Figure 1. Response of B. germanica females to transient social interaction during the
photophase and scotophase. Newly-eclosed females were reared either in social isolation
(negative control) or paired with a conspecific female (positive control) for the entire 6-daylong experiment. Other females were transiently paired 6 h daily either in the middle of the
photophase or the middle of the scotophase. Some females were transiently paired for 1 or 2
h daily in the middle of the scotophase. Mean basal oocyte length ± SE Different letters
above the bars indicate significant differences among treatments (ANOVA, LSD, F5, 121 =
13.69, P < 0.0001).
140
1.8
Contactand
and volatile
volatile chemical
chemical cues
Contact
Vision
Vision obscured
obscured
Oocyte length (mm)
b
b
1.6
1.4
a
1.2
1.0
a
a
a
a
a
0.8
0.6
(1)
(1)
(2)
clear
transparent
conditioned paper
divider
nymph
♀
nymphs
♀♀
(3)
1
(4)
odors of
of
odors
1♀
Isolated
Isolated
15
15 ♀♀
(5)
(6)
opaque
divider
eyes
painted
Paired
Paired
Figure 2. Effects of visual and chemical stimuli on the reproductive rate of B. germanica
females. Newly-eclosed females were socially isolated in stacked transparent Petri dishes
with unobstructed views of other isolated females above and below (isolated; negative
control), filter paper contaminated by 10 German cockroach nymphs (1) or 10 conspecific
females (2), the odors of one (3), or 15 conspecific females (4) through a fine-mesh screen,
or an opaque divider between dishes that prevented them from seeing each other (5). Females
paired with another female either had their eyes painted to obscure their vision (6), or left
untreated (paired; positive control). Variation around the mean is represented by the standard
error of the mean (SE). ANOVA: F7, 147 = 21.72, P = 0.0001. Means not sharing a letter are
significantly different (LSD, P < 0.05).
141
c
c
Beetle adult
Stink bug adult
1.4
d
c
bc
Ant
Isolated
mean
Katydid adult
a
0.8
Fly adult
1.0
Cockroach nymph
b
1.2
0.6
d
c
Beetle larva
Oocyte length (mm)
1.6
d
Cricket adult
cd
Cockroach female
1.8
Paired
Paired partner
Figure 3. Effects of social interaction with different insect species on the reproductive rate of
B. germanica females. Females socially isolated during the entire 6-day experiment represent
the negative control, and females that were socially paired during the entire experiment
represent the positive control. Grey bars represent social pairing for 6 days with different
insect species, including a worker ant (Camponotus pennsylvanicus), beetle larva (Tenebrio
molitor), adult beetle (Tenebrio molitor), adult stink bug (Chinavia hilaris), cockroach
nymph (Periplaneta fuliginosa), adult fly (Musca domestica), adult katydid (Conocephalus
strictus), adult female cockroach (Supella longipalpa), and adult camel cricket (Ceuthophilus
maculatus). Variation around the mean is represented by the standard error of the mean (SE).
ANOVA: F10, 205 = 15.02, P = 0.0001. Means not sharing a letter are significantly different
(LSD, P < 0.05).
142
Figure 4. Effects of contact stimuli on the reproductive rate of B. germanica females. Newlyeclosed females were either reared in isolation (negative control), allowed to interact with
glass beads that were moved to simulate social interaction (1), paired with a freshly-killed
conspecific female that was replaced daily (2), allowed to interact with the antennae of a live,
conspecific female whose body was tethered either outside the dish (3) or within the dish (4),
or allowed to interact with the antennae of an American cockroach female whose body was
outside the dish (5). Females paired with a conspecific female for the entire experiment
represented the positive control. Mean basal oocyte length ± SE ANOVA: F6, 136 = 25.97, P <
0.0001. Different letters above the bars indicate significant differences among treatments
(LSD, P < 0.05).
143
1.8
c
c
(4)
(5)
c
Oocyte length (mm)
1.6
b
1.4
1.2
a
a
(1)
(2)
a
1.0
0.8
0.6
Isolated
beads
(3)
1
B. germanica
dead
tethered,
contact
antennae
only
Paired
P. americana
tethered,
contact
whole
body
tethered,
contact
antennae
only
144
Figure 5. Effects of interaction with the antennae on the reproductive rate of B. germanica
females. Newly-eclosed females were either socially isolated (negative control), or socially
paired for the entire experiment (positive control). An American cockroach female was
placed in a plastic tube outside the dish with only its antennae protruding into the dish to
interact with the otherwise isolated female. Group 1 represents untreated P. americana
antennae, in group 2 the antennae were extracted with hexane to eliminate chemical cues, and
in group 3 the antennae were similarly extracted but a lipid extract from P. americana
females that also contained cuticular hydrocarbons (CHC) was reapplied on each extracted
antenna. In group 4 the P. americana antennae were carefully ablated and an artificial
microfibett was glued onto each antenna supported by a plastic sleeve. Mean basal oocyte
length ± SE ANOVA: F5, 137 = 25.41, P < 0.0001. Different letters above the bars indicate
significant differences among treatments (LSD, P < 0.05).
145
1.8
d
Oocyte length (mm)
1.6
1.4
c
bc
bc
b
1.2
a
1.0
0.8
0.6
Isolated
(1)
(2)
1
(3)
(4)
Paired
P. americana antennae
untreated
extracted
extracted,
lipids
reapplied
replaced
with
artificial
microfibett
146
1.8
intact
antennectomized
d
d
Oocyte length (mm)
1.6
1.4
c
1.2
b
1.0
a
a
0.8
0.6
1
Isolated
Paired
Figure 6. Function of the antennae in receiving social cues that stimulate reproduction.
Newly-eclosed females were ice-anesthetized and the flagellum of each antenna was ablated
just distal to the scape. Socially isolated intact females represent the negative control, and
intact females that were socially paired represent the positive control. Other females received
one of the following treatments: (1) isolated-housed antennectomized female; (2) an intact
female paired with an antennectomized female; and (3) pair-housed antennectomized
females. Mean basal oocyte length ± SE ANOVA: F5, 122 = 52.29, P < 0.0001. Different
letters above the bars indicate significant differences among treatments (LSD, P < 0.05).
147
CHAPTER 4.
Social facilitation of insect reproduction with motor-driven tactile stimuli
148
ABSTRACT
Tactile stimuli provide animals with important information about the environment, including
physical features such as obstacles, and biologically relevant features such as food, mates,
hosts and predators. The antennae, the principal sensory organs of insects, house an array of
sensory receptors for olfaction, gustation, audition, nociception, balance, stability,
graviception, static electric fields, and thermo-, hygro-, and mechanoreception. The antennae,
being the anterior-most sensory appendages, play a prominent role in social interactions with
conspecifics that involve primarily chemosensory and tactile stimuli. In the German
cockroach (Blattella germanica) antennal contact during social interactions modulates brain
regulated juvenile hormone production, ultimately accelerating the reproductive rate in
females. The primary sensory modality mediating this social facilitation of reproduction is
antennal mechanoreception. We investigated the key elements, or stimulus features, of
antennal contact that socially facilitate reproduction in B. germanica females. Using motordriven antenna mimics we assessed the physiological responses of females to artificial tactile
stimulation. Our results indicate that tactile stimulation with a broad array of artificial
materials, some deviating significantly from the native antennal morphology, can facilitate
female reproduction. However, none of the artificial stimuli matched the effects of social
interactions with a conspecific female.
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INTRODUCTION
Insects exploit multiple sensory modalities, including auditory, olfactory, gustatory and
tactile sensing, in a wide range of behavioral contexts such as locating resources, avoiding
predators, mate finding and mate choice (reviews: Hebets and Papaj, 2005; Partan and
Marler, 2005). Antennae are important multi-sensory organs in insects, largely because they
are located at the anterior-most position of the insect, they often extend up to several body
lengths and sweep and sample nearly 360o of space around the insect, and they can receive
information about smell, taste, sound, humidity, temperature, and various mechanosensory
cues (review: Staudacher et al., 2005). The antennae are also pivotal sensory organs in social
interactions, especially in nocturnal insects.
Mechanoreceptive sensilla are broadly distributed throughout the body surface of
insects, with particularly high density on sensory appendages, including the antennae
(Seelinger and Tobin, 1981; Staudacher et al., 2005). Antennal mechanosensors have been
studied most extensively in the contexts of obtaining position information and localization
and feature discrimination of obstacles (Harley et al., 2009). But antennal mechanoreception
is also important in courtship behavior, where positional information is integrated with
chemosensory signaling between the sexes, as shown for example in cockroaches, crickets
and moths (Nishida and Fukami, 1983; Schmiederwenzed and Schruft, 1990; Balakrishnan
and Pollack, 1997; Briceno and Eberhard, 2002; Khadka et al., 2011). In the behavioral phase
change of the Australian plague locust, Chortoicetes terminifera, tactile stimulation of the
antennae appears to be the sole mechanism that evokes a shift from solitarious to the
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gregarious state and ultimately swarming (Cullen et al., 2010). The antennae also appear to
be the main organ involved in adult phase change in the desert locust, Schistocerca gregaria
(Maeno et al., 2011). In addition, maternal determination of progeny characteristics has been
observed in S. gregaria, where tactile stimulation perceived by their antennae causes females
to produce gregarious offspring (Maeno et al., 2011).
In the German cockroach, Blattella germanica (L.) (Dictyoptera, Blattellidae), the
interchange of chemosensory and tactile signals during social interactions affects behavioral
and physiological responses of nymphs and adults. Lihoreau and Rivault (2009) showed that
nymphs can discriminate siblings from non-sibs and that this kin recognition ability requires
antennal contact between individuals. Social interactions also accelerate nymphal
development in the German cockroach, and tactile stimulation of the antennae appears to be
sufficient to trigger faster development in socially grouped than in solitary nymphs (Izutsu et
al., 1970; Ishii, 1971; Nakai and Tsubaki, 1986, Lihoreau and Rivault, 2008). Social
facilitation of reproduction has been shown in both females and males of B. germanica.
Females undergo an endocrine-regulated sexual maturation after adult emergence, and social
interactions facilitate this process by lifting brain-imposed allatostatic inhibition of the
corpora allata (CA), stimulating higher juvenile hormone (JH) production which in turn
stimulates reproduction. Social interactions thus indirectly modulate all JH-related activities,
including attainment of sexual receptivity, production of sexual signals, mating, and the timecourse of oviposition (Gadot et al., 1989a; Schal et al., 1997; Holbrook et al., 2000; Uzsák
and Schal, 2012 (Chapter 1)). Similarly, social interactions have a profound effect on certain
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aspects of male reproductive physiology and behavior, such as JH biosynthesis, protein
production in the accessory reproductive glands, and sexual maturation (Chapter 2).
Tactile cues are the primary sensory channel through which social conditions
stimulate or accelerate reproduction in adult B. germanica females—with no evidence for
involvement of the visual and olfactory systems—and the antennae play an important role in
the reception of these tactile cues (Chapter 3). Moreover, tactile stimulation of the antennae
with “prosthetic” antenna-like artificial materials in place of the antennal flagellum can also
accelerate oocyte growth (Chapter 3), confirming that social interactions accelerate
reproduction in B. germanica females via mechanosensory cues perceived mainly by the
antennae. Although some features of antennal tactile communication involved in social
facilitation have been described in phase-polyphenic locusts (Cullen et al., 2010; Maeno et
al., 2011), to our knowledge, the specific stimulus characters of tactile cues that facilitate
reproduction in other arthropod species have not been investigated.
Our aim in this study was to identify the specific elements of a social tactile stimulus
responsible for accelerating reproduction in B. germanica females. First, we evaluated the
role of antennal contact in a social context. Then, we established a system for testing
physiological responses of females to artificial, motor-driven tactile stimulation that
mimicked the cockroach antenna. Finally, we dissected the tactile cues focusing on features
such as speed of movement, duration of stimulation and morphology of the tactile stimulus.
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MATERIALS AND METHODS
Insects and rearing conditions
Blattella germanica cockroaches, from a colony originally obtained from American
Cyanamid in 1989 (also referred to as the Orlando strain), were kept at 27 ± 1ºC and 40–70%
ambient relative humidity, under a 12:12 L/D photoperiod with continuous access to dry
LabDiet rat chow (#5001; Rodent Diet, PMI Nutrition International, Brentwood, MO, USA)
and water. Newly eclosed females were selected from the colony on the day of adult
emergence (day 0). Only females of similar size and degree of sclerotization and with intact
wings were selected for each experiment and were maintained under the same conditions
described above.
Ovary dissection and oocyte measurements
Test females were ice-anesthetized and their ovaries were removed under cockroach saline
(Kurtti and Brooks, 1976). In B. germanica, only a single basal (vitellogenic) oocyte matures
in each ovariole and all basal oocytes mature synchronously in each of the approximately 40
ovarioles in the paired ovaries. A random sample of 10 basal oocytes of each female was
selected and oocyte lengths were averaged for each female. Measurements were done with an
ocular micrometer in the eyepiece of a dissecting microscope.
153
Effects of tactile cues on the reproductive response to social interactions
The antennae are essential in receiving sensory input, and contact with another female
provides the main stimuli that modulate the reproductive rate in B. germanica (Chapter 3).
Moreover, tactile cues – and not chemical cues – play a prominent role in the social
facilitation of reproduction in B. germanica females, and the tactile stimuli are not speciesspecific (Chapter 3). Therefore, Periplaneta americana females were used to provide the
tactile stimuli. A P. americana female was placed into a 15 ml plastic tube, with only the
antennae protruding through a small hole in the tube. We covered the head with parafilm so
that only the antennae extended through a hole into a Petri dish (60 mm diameter, 15 mm
high, Fisher Scientific, Pittsburgh, PA, USA), where a newly emerged B. germanica female
was placed with food and water ad libitum. In an additional treatment – using the same
bioassay design – we bilaterally ablated the P. americana antennae to eliminate tactile
stimuli by the antennae. P. americana females were briefly anesthetized with CO2, placed on
ice and the flagellum of each antenna was cut with fine scissors just distal to the pedicel. P.
americana females were replaced every other day during the 6-day-long experiment. On day
6, the oocyte lengths of 20−23 B. germanica females per treatment were measured.
Motorized tactile stimulation system
To investigate features of tactile stimuli that ultimately accelerate oocyte maturation, we
designed a motorized system that could stimulate isolated females with substrates that
154
mimicked cockroach antennae. The system was comprised of a controller and twenty parallel
stepper motors. The controller consisted of a microcontroller (PIC18F4520, Microchip
Technology, Chandler, AZ, USA), speed and direction controls, an LCD display showing the
motor angular velocity and ten microstepping bipolar stepper motor drivers (A4988, Pololu,
Las Vegas, NV, USA) (Fig. 1). Each motor driver controlled two stepper motors (ROB09238, SparkFun Electronics, Boulder, CO, USA). The output of the microcontroller could
generate angular velocities of 0.1 to 30 revolutions per min (rpm) and each motor was set to
rotate in a clockwise direction. Each motor was securely attached to the underside of a 6-mm
thick acrylic sheet that held 10 motors. A Petri dish (90 mm diameter, 15 mm high, Fisher
Scientific, Pittsburgh, PA, USA) was place on the acrylic sheet with a hole at its center
aligned with the hole in the acrylic sheet. The motor shaft thus penetrated into the Petri dish
and materials simulating insect antennae were attached onto the shaft. Although the acrylic
sheet effectively insulated the Petri dishes from the motors, a room fan was used to ensure
rapid dissipation of heat from the electronics.
Validation and use of the motorized system
First, we determined whether the rotating motors influenced oocyte growth by producing
heat, vibration, or other confounding stimuli. Newly-emerged females were either socially
isolated, or paired in Petri dishes and placed either on the rotating motors or beside them with
food and water ad libitum. Since social interactions stimulate oocyte growth only during the
scotophase, when females are active, and not during the photophase when females aggregate
155
in close contact with each other (Chapter 3), the motors were turned on during the entire
scotophase (12 h) at a maximum speed of 30 rpm and with a bare motor shaft. The basal
oocytes of 10 B. germanica females per treatment were measured on day 6.
Movement of the antennae is necessary to glean tactile information from the surroundings
(Krause and Dürr, 2004). The cockroach antennae are flexible and they regularly sweep
through space, vibrate, palpate rhythmically and get deflected when the insect comes in
contact with conspecifics, suggesting that movement is an important feature of the tactile
cue. To determine the most effective motor speed and the optimal duration of stimulation,
newly-eclosed females were socially isolated in Petri dishes and placed on the motors with
food and water ad libitum. Artificial tactile stimulation was provided by a commercially
sanitized natural duck feather (CDC201, Wapsi Fly Inc., Mountain Home, AR, USA)
attached to each motor shaft. Each set of females received stimulation for different durations
(3, 4.5, 6, 7.5, 12 h [scotophase only] and 24 h) and at different motor speeds (0, 1, 5, 10, and
30 rpm). Sample size was 16−20 females per treatment.
In addition to movement, some physical properties of the antenna such as flexibility,
thickness, and the structural complexity may influence the reproductive rate of B. germanica
females. Various artificial materials were attached to the shafts of the stepper-motors and
females received artificial tactile stimulation for 6 h daily in the middle of the scotophase
with an artificial substrate rotated at 1 rpm (based on preliminary results). Flexibility was
tested with 3 different substrates: small diameter PTFE tubing (least flexible; outer diameter
1.5 mm, Cole-Parmer Instrument Co., Vernon Hills, IL, USA), microfibett (flexible; 1 mm
diameter, 40 mm long, smooth surface, Spirit River Inc., Roseburg, OR, USA), and an
156
epoxy-fabricated bare “antenna” (most flexible; see below). Thickness was tested with PTFE
tubing with either 0.5 mm or 5 mm outer diameter (Cole-Parmer). Complexity of the
antennal morphology was tested with epoxy-fabricated “antennae” (0.1−0.6 mm thick from
tip to base, 45 mm long; based on the approximate dimensions of P. americana antenna) with
no “sensilla” (bare, least complex), with short “sensilla” (antenna-mimic, complex), and
feather-like “sensilla” (most complex). See below for fabrication procedures. Independently,
we also tested antennal complexity with either an intact duck feather, feather with its barbs
cut to 1 mm, or bare feather with its barbs completely removed. Newly-emerged females
from the same cohort as the experimental females were either socially isolated (negative
control) or paired for the entire experiment (positive control). Sample size was 10−26
females per treatment.
Fabrication of synthetic antennae
Various synthetic antenna structures were fabricated using ultraviolet light curable epoxy.
These structures varied in their base length and width as well as hair (“sensillum”) length,
width and density. The antenna fabrication procedure was carried out through a
photolithography process in a clean room free from any ambient UV source. A HybriWell
(Grace Bio-Labs, Bend, Oregon, USA) micro-chamber was formed on a microscope slide.
For thicker “antennae”, multiple HybriWell sheets were placed on top of each other. A UVcurable epoxy (Delo Katiobond 4670, Delo Industrial Adhesives, Sudbury, MA, USA) was
157
injected into this micro-chamber through the openings of the HybriWell and the openings
were sealed off with seal tabs after the injection.
To define various antennal structures, positive tone photo masks were designed using
Adobe Illustrator and printed at 3300 dpi resolution. Because of light refraction and
scattering in uncured epoxy resin, the fabricated structures had larger dimensions than the
patterns on the photo mask. Therefore, calibration bars were fabricated initially to
characterize the mismatch and the necessary adjustment was applied to the photomask
patterns to fabricate the desired antenna feature sizes. The micro-chamber was rotated upside
down (glass side on the top and HybriWell on the bottom) and the mask was placed on the
slide. A second larger glass slide was placed on the mask for contact lithography. The setup
was exposed to UV light for 5 min at an approximate intensity of 20 mW/cm2. The irradiated
setup was given a curing time of 6−12 h in the dark at room temperature. After curing, the
HybriWell was slowly peeled away from the slide and the cured antennal structure was
cleaned with isopropyl alcohol.
Statistical analyses
Data were analyzed with one-way ANOVA for multiple comparisons using SAS® 9.1.3
software (SAS Institute Inc. 2002-2003, Cary, NC, USA). We used PROC GLM to test for
the effects of social conditions on oocyte length as dependent variable. PROC GLM was also
used to get the residuals from adjusted model and test whether residuals held the assumption
of homogeneous variances within each treatment. Since data were unbalanced, LSMEANS
158
and LSD test was used to compare means at 0.05 significance level. Variation around the
mean is represented by the standard error of the mean (SEM).
RESULTS
Antennal contact mediates the social facilitation of reproduction
To confirm the importance of antennal contact in social facilitation of female reproduction,
each isolated B. germanica female was allowed to interact only with the antennae of a P.
americana female whose body was bound in a tube outside the dish; these B. germanica
females developed their oocytes significantly faster (1.29 ± 0.05 mm, n = 23) than females
that were fully isolated (0.99 ± 0.07 mm, n = 22) or females that interacted with a P.
americana female whose antennae were ablated (0.87 ± 0.06 mm, n = 20) (ANOVA, p <
0.0001) (Fig. 2). Therefore, the antennae are pivotal sensory appendages that receive sensory
stimuli as well as provide social stimuli. Because of this dual role in social facilitation of
reproduction, it is difficult to disentangle the relative significance of “giving” social stimuli
(touching) versus “receiving” social stimuli (being touched). Nevertheless, because
interaction with an immobile (freshly killed) female fails to stimulate reproduction and tactile
stimulation of the female with “prosthetic” antenna-like artificial materials in place of the
antennal flagellum also induces faster oocyte growth (Chapter 3), it appears that active tactile
sensing of social stimuli is crucial for reproductive maturation.
159
Validation of the motorized tactile stimulation system
We implemented artificial tactile stimulation with stepper motors, which have some intrinsic
features that might confound our results. For example, they might produce heat and vibration
that might influence the female’s reproductive rate. Social isolation significantly delayed
oocyte maturation in both sets of solitary females, both on and off the motors, whereas both
sets of paired females exhibited faster oocyte growth regardless of whether they were on or
beside the motors (ANOVA, p < 0.05) (Fig. 3). There were no significant differences
between isolated or paired females within each location, indicating that (a) the motors
themselves did not affect oocyte growth, (b) the rotating motor shaft was not an appropriate
tactile facilitator of reproduction in B. germanica, and (c) our motorized system was thus
suitable for testing physiological responses of females to artificial tactile stimulation.
Stimulus features related to artificial “antenna” movement
We recently found that two hours of daily social interactions with another female in the
middle of the scotophase were sufficient to induce faster reproduction in B. germanica
(Chapter 3). To determine the optimal conditions for artificial tactile stimulation, we fixed a
duck feather to each motor and assessed the effects of various motor speeds and durations of
daily stimulation on reproductive development in B. germanica females. The results again
confirmed that movement is an important stimulus feature because static feathers
continuously present in the Petri dishes for 6 days failed to stimulate oocyte growth (Fig. 4,
160
black bar). With dynamic tactile stimulation, oocyte maturation was inversely correlated with
both motor speed and duration of stimulation (Fig. 4). Rapid tactile stimulation at 30 rpm
failed to elicit oocyte maturation at all the stimulus durations that we tested (average oocyte
length across all stimulus durations: 0.80 ± 0.05 mm, n = 59); 0.95 ± 0.05 mm (n = 49) in
females that were isolated during the entire experiment. Decreasing motor speed to 1–5 rpm
or decreasing stimulation duration to 3−6 h resulted in maximal oocyte growth. In our 6-day
assays, females responded with the fastest oocyte development (1.28 ± 0.09 mm, n = 20) to 6
h stimulation at 1 rpm (Fig. 4, black arrow). This combination was used in subsequent
experiments.
Stimulus features related to artificial “antenna” morphology
In previous research, we showed that slowly rolling glass beads failed to stimulate oocyte
maturation in female B. germanica (Chapter 3), suggesting that movement alone is not
sufficient and that specific morphological characteristics of the stimulus are responsible for
socially facilitating oocyte growth in female cockroaches. To test for structural complexity,
we used feathers with 18 mm barbs representing super-normal “sensilla”, short 1 mm barbs
representing long “sensilla”, as well as a bare feather shaft (rachis) with no visible barbs.
Stimulation with a bare rachis stimulated oocyte growth, but not significantly more than in
isolated females. However, “sensilla”-bearing feathers elicited significantly faster oocyte
development than in the isolated control females (ANOVA, F4, 85 = 12.21, p < 0.0001; LSD,
p < 0.05) (Fig. 5).
161
Because it was difficult to alter the flexibility of the duck feather, we used artificial
materials of varying flexibility—a stiff PTFE tubing, a flexible microfibett, and a more
flexible epoxy-fabricated bare “antenna”. Each of the three substrates, rotating on the stepper
motors at 1 rpm for 6 hrs during the middle of the scotophase, significantly enhanced oocyte
maturation compared to females that were socially isolated during the entire experiment
(ANOVA, F5, 114 = 11.05, p < 0.0001; LSD, p < 0.05); however, there were no significant
differences among the three substrates (Fig. 6). Similar results were obtained when testing
for the effects of stimulus thickness. Both 0.5 mm and 5 mm OD PTFE tubes significantly
stimulated oocyte maturation relative to the isolated females, and surprisingly, the thicker
tube was slightly, but not significantly more effective (Fig. 6).
DISCUSSION
Numerous animal behavior studies have used fabricated models to delineate key elements
(stimulus characters) of relevant stimuli, dating back to Niko Tinbergen’s elegant and rather
straightforward studies, using cardboard models to define the relevant stimulus characters
that comprise sign stimuli that guide bird and insect behaviors (Tinbergen 1951). However,
such studies have been heavily biased in the visual, auditory, and chemosensory modalities.
Examples of contemporary investigations using this approach include computer animations
to explore the visual stimuli that guide mating behavior of jumping spiders (Clark and Uetz,
1990) and virtual computer-generated prey assemblages to explore the visual behavior of
predatory fish (Ioannou et al., 2012). Robots have also been used to understand visually and
162
chemically guided animal choices, including mate choice in grouse (Patricelli and Krakauer,
2010) and collective assembly in cockroaches (Halloy et al., 2007). Likewise, auditory
biologists have recorded and synthesized acoustic stimuli to understand key elements that
convey specific information to the receiver, and chemosensory biologists have similarly
conducted chemical structure-activity studies with synthetic analogs of odorants and tastants
to understand the respective olfactory and gustatory receptors and the behaviors they drive.
Near-range sounds and vibrational stimuli, which stimulate mechanoreceptors, have also
been investigated, especially in arthropods (review: Staudacher et al., 2005). Notably,
however, it has been exceptionally challenging to fabricate synthetic tactile stimuli mainly
because the insect antenna is a compound sensory organ in which touch is but one
component of a multi-sensory network, unlike the vertebrate whisker or vibrissa which is
strictly tactile and inert with receptors located in the follicle at its base (review: Diamond et
al. 2008). It has also been difficult to isolate and quantify the salient tactile features that elicit
behavior, including shape, fine structure, texture, friction, force and various spatio-temporal
features of antennal contact. Moreover, tactile cues often involve complex and contextdependent active movements of both the signaler’s and receiver’s head and antennae, and
unlike most communication messages that operate through a medium (air, water), the
signaler in tactile communication can also be the receiver, as for example in reciprocal
antennal fencing where the antenna both touches and is touched and processes both
proprioreceptive and exteroreceptive information (review: Staudacher et al., 2005). Finally,
the insect antenna is endowed with multiple types of mechanosensory structures (e.g., hairs)
that are differentially distributed across various antennal segments, and arranged as single
163
sensilla or as specialized arrays of mechanosensory units (e.g., hair plates, chordotonal
organs) (Seelinger and Tobin, 1981), making it even more difficult to determine where and
how socially relevant tactile stimuli are received.
Thus, it is not surprising that the relevance of most antennal mechanoreceptive sensilla in
social interactions remains largely unknown, as most investigations have focused on
relatively instantaneous non-social behaviors such as steering and course control, obstacle
navigation and escape responses (review: Staudacher et al., 2005). These behaviors depend
largely on object localization (i.e., where) and much less so on object discrimination and
identification (i.e., what). The effectiveness of tactile cues in a social context is profoundly
dependent on the receiver being able to identify the source of the stimulus (its shape, texture,
biomechanics), and endeavors to disentangle discrete social tactile stimulus characters are
therefore considerably more challenging than in other sensory modalities. The challenge is
substantially even more imposing in systems that require multiple days of in situ tactile
stimulation whose outcomes are physiological rather than behavioral, as in the slow contactmediated social facilitation of reproduction in B. germanica. While the behavioral outputs of
associative learning experiments can decode the tactile stimulus characters, more
sophisticated paradigms are required to unravel the primer-type stimuli that drive slow
physiological changes. This is apparent in B. germanica, where 24 hrs of social interactions
on any day during the 6-day preoviposition period of otherwise isolated females is
insufficient to elicit a “grouping effect”, and even 48 hrs of interactions on days 2–3
minimally facilitate reproduction in females (Chapter 3).
164
We established and validated a motorized system that enabled us to investigate the effects
of artificial tactile stimuli on the reproduction of female cockroaches. B. germanica females
respond to social interactions with conspecifics—and even with other insects—by elevating
their rate of JH production and accelerating the rate of oocyte maturation, a form of
phenotypic plasticity (Gadot et al., 1989a; Schal et al., 1997, Holbrook et al., 2000; Uzsák
and Schal, 2012 (Chapter 1)). In a previous study we established that visual and
chemosensory cues play little, if any, role in this social facilitation of reproduction, whereas
tactile stimuli alone can elicit faster reproduction (Chapter 3). In the current work, we
demonstrate that “antenna”-like structures, driven by stepper motors, can also accelerate the
rate of oocyte growth. Notably, however, all of our treatments were significantly inferior to
normal social interactions with a conspecific female, indicating that other prominent features
of this social facilitation system remain to be delineated. Since limited interaction only with
the antennae of either P. americana or B. germanica was not as effective as interaction with a
whole conspecific female (Chapter 3), we infer that non-antennal tactile stimuli are also
important facilitators of reproduction.
Nevertheless, an emerging picture from our studies is that antennal movement and
morphology are integral components of the tactile stimuli. In support of our in situ studies
demonstrating that social facilitation of reproduction is gated with the photocycle and can be
achieved with as little as 2 hrs of contact in the scotophase (Chapter 3), we now demonstrate
that the duration and magnitude of artificial tactile stimulation also impact oocyte growth.
The effect of duration of artificial stimulation on oocyte maturation represents an upsidedown (concave downward) parabola, with a peak at about 6 hrs of stimulation in the middle
165
of the scotophase. We suspect that as the quality of the tactile stimulus increases, the minimal
required duration of stimulation will diminish. We also demonstrated that oocyte maturation
was inversely related to the frequency of stimulation, represented by motor speed. Thus,
oocyte maturation was greatly suppressed by recurrent tactile stimulation at 30 rpm but
considerably stimulated with slow intermittent contact with a duck feather. Coupled with the
observations that reproduction in isolated females is not facilitated by an immobile feather or
by the presence of freshly killed (i.e., immobile) females (Chapter 3), these results indicate
that stimuli related to kinematics of the insect antennal motor system and biomechanics and
dynamic properties of the antennal flagellum play essential roles in facilitating reproduction.
The morphology of the artificial stimulus also had a bearing on oocyte maturation.
Although the bare shaft (rachis) of a denuded feather stimulated oocyte development, grater
structural complexity increased its effectiveness. Moreover, some exaggerated stimulus
characters appeared to act as super-normal stimuli, as feathers with long barbs were slightly
more effective than feathers with shorter barbs, and P. americana antennae were more
effective than the native B. germanica antennae (Chapter 3). Antennal shape thus appears to
be an important feature in tactile social stimulation, in support of our in vivo studies showing
that certain insects with non-flagellar antennal morphology were less effective at stimulating
female reproduction in B. germanica (Chapter 3). Further support for the importance of
antennal morphology, flexibility and texture comes from male courtship behavior in B.
germanica. The female-produced contact sex pheromone readily elicits male courtship when
placed on various flagellum-type antennae from a variety of insect species, but not on thick
or club-shaped antennae or smooth surfaces such as a nylon line or a human hair (Nishida
166
and Fukami, 1983), suggesting that an important feature of this sexual signal is the
integration of chemical and tactile stimuli. The importance of textural cues in determining
stimulus identity was also demonstrated in the cockroach P. americana, where cuticular
surface properties of a wolf spider trigger escape, whereas touching a conspecific does not,
even when both are solvent extracted to eliminate contact chemoreception (Comer et al.,
2003). The fine shape discrimination capabilities of the cockroach antennae remain to be
determined, but it would not be surprising if the diversity and spatial organization of
mechanosensory sensilla on the antenna enable feature discrimination comparable to that of
rodent whiskers (Diamond et al., 2008).
Our motorized tactile stimulation system represents an essential foundation for further
studies of stimulus characters that socially facilitate reproduction in B. germanica. Promising
lines of investigation include mounting more realistic engineered jointed “antennae” on
robots programmed to execute species-specific and context-dependent behaviors in multiple
axes. Decoding the tactile stimulus characters will further allow us to identify neuronal and
endocrine circuit elements in the transduction pathway through which mechanoreceptors
communicate relevant discrete tactile cues through the antennal nerve to sensory neuropils in
the deutocerebrum and to higher brain centers, which ultimately accelerate female
reproduction in B. germanica by disinhibiting the activity of the CA. This neuroendocrine
pathway has been best described in phase transition in S. gregaria, where tactile stimulation
of specific mechanoreceptive sensilla on the hind legs causes an increase in serotonin levels
in the metathoracic ganglion which then leads to gregarization (Rogers et al., 2003; Rogers et
al., 2004; Anstey et al., 2009). It will be fascinating to know whether biogenic amines also
167
respond to social antennal contact resulting in the social facilitation of reproduction in B.
germanica females.
ACKNOWLEDGMENTS
We thank Yasmin J. Cardoza, Jules Silverman, and Wes Watson (Department of
Entomology, North Carolina State University) for critical comments on this manuscript,
Alper Bozkurt, James Dieffenderfer and Tahmid Latif (Electrical and Computer Engineering
Dapertment, North Carolina State University) for assisting in the design and construction of
the stepper-motor system, and Consuelo Arellano (Department of Statistics, North Carolina
State University) for help with statistical analyses. We are grateful to Rick Santangelo for
technical assistance during the experiments. This project was supported by the Blanton J.
Whitmire endowment at North Carolina State University and scholarships from the North
Carolina Pest Management Association and the Structural Pest Management Training and
Research Facility.
168
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FIGURES
Figure 1. Motorized tactile stimulation system showing the power supply (right), controller
(center), and 20 Petri dishes each mounted on a stepper motor (top and bottom). The inset
shows an isolated B. germanica female in a Petri dish with a duck feather mounted on the
rotating motor shaft.
174
1.7
Ablated antennae
c
Oocyte length (mm)
Intact antennae
1.5
b
1.3
a
1.1
a
0.9
0.7
Isolated
P. americana
Paired
Figure 2. Effects of interaction with P. americana antennae only on the reproductive
maturation of B. germanica females. Newly-emerged adult females were either socially
isolated (negative control), or paired for the entire 6-day experiment (positive control) (grey
bars). An American cockroach female was bound in a plastic tube outside the dish with only
its antennae protruding into the dish to interact with the otherwise isolated B. germanica
female. In an additional treatment, the American cockroach female antennae were carefully
ablated eliminating antennal contact. Mean basal oocyte length ± SEM. Different letters
above the bars indicate significant differences among treatments (ANOVA, F3, 83 = 21.78, p <
0.0001; LSD, p < 0.05).
175
1.9
On motor
b
b
Off motor
Oocyte length (mm)
1.7
1.5
1.3
a
a
1.1
0.9
0.7
Isolated
Paired
Figure 3. Validation of the motorized tactile stimulation system. Newly-emerged adult
females were either socially isolated or paired in Petri dishes and placed either on the rotating
motors or beside them. The motors were powered during the entire 12 h scotophase at a
speed of 30 rpm and with no substrate attached to their shafts. The basal oocytes of B.
germanica females were measured on day 6 and sample size was 10 females per treatment.
Mean basal oocyte length ± SEM. Different letters above the bars indicate significant
differences among treatments (ANOVA, F3, 36 = 4.99, p = 0.0054; LSD, p < 0.05).
176
Figure 4. Dose-response histogram for the effects of motor speed and duration of stimulation
on the reproductive rate of B. germanica females. Newly-emerged adult females were
socially isolated in Petri dishes and placed on the motors. Artificial tactile stimulation was
provided by a duck feather attached to the shaft of each motor. Each set of females received
stimulation for different durations in the scotophase (3, 4.5, 6, 7.5, or 12 h) or for the entire
24 h day and at different motor speeds (0, 1, 5, 10, and 30 rpm). Black arrow corresponds to
the best combination of motor speed and stimulation duration (1 rpm for 6 h) and the black
bar indicates a “no movement” control treatment. Sample size was 16−20 females per
treatment.
177
1.7
Feather rachis
c
Oocyte length (mm)
1 mm barbs
1.5
ab
1.3
1.1
b
18 mm barbs
b
a
0.9
0.7
Isolated
Paired
Duck feather
Figure 5. Evaluation of the significance of the structural complexity of an artificial tactile
stimulus on the reproductive rate of B. germanica females. Newly-emerged adult females
were either socially isolated (negative control), or paired for the entire experiment (positive
control) (grey bars). Other newly-emerged females from the same cohort were isolated in
Petri dishes and placed on the motors. Each female received artificial tactile stimulation for 6
h at 1 rpm by a duck feather attached to the shaft of the motor. Stimulus complexity was
tested with either intact feathers with 18 mm barbs, feathers with short 1 mm barbs, or
feathers with the barbs completely removed leaving a bare feather rachis. Mean basal oocyte
length ± SEM. Different letters above the bars indicate significant differences among
treatments (ANOVA, F4, 85 = 12.21, p < 0.0001; LSD, p < 0.05).
178
1.7
Epoxy-fabricated shaft
Microfibett
c
Oocyte length (mm)
PTFE tubing OD 0.5 mm
PTFE tubing OD 5 mm
1.5
b
b
b
b
1.3
a
1.1
0.9
0.7
Isolated
Paired
More flexible
Thicker
Figure 6. Evaluation of the significance of the flexibility and thickness of artificial tactile
stimuli on the reproductive rate of B. germanica females. Newly-emerged adult females were
either socially isolated (negative control), or paired for the entire 6-day experiment (positive
control) (grey bars). Other newly-emerged females from the same cohort were isolated in
Petri dishes and placed on the motors. Each female received artificial tactile stimulation for 6
h at 1 rpm by an artificial stimulus attached to the shaft of the motor. Stimulus flexibility was
tested with stiff PTFE tubing, a flexible microfibett, and a more flexible epoxy-fabricated
bare “antenna”. Stimulus thickness was tested with 0.5 mm or 5 mm outer diameter PTFE
tubing. Mean basal oocyte length ± SEM. Different letters above the bars indicate significant
differences among treatments (ANOVA, F5, 114 = 11.05, p < 0.0001; LSD, p < 0.05).
179