Viewed As an Adaptive Mechanism That Could Ensure

DIFFERENCES IN MALE SCENT IN THE TWO HOST ASSOCIATED STRAINS OF SPODOPTERA FRUGIPERDA AND EVIDENCE OF MATE DISCRIMINATION BY FEMALES

DISSERTATION

Presented in Partial Fulfillment of the Requirements of the Degree Doctor of Philosophy

in the Graduate School of The Ohio State University

Paulo Marques, M.S.

The Ohio State University 2004

Approved by

Dissertation Committee:

Professor Larry. Phelan, advisor Professor David Horn Adviser Professor Woodbridge Foster Department of Entomology Professor John Wenzel ABSTRACT

Spodoptera frugiperda (J. E. Smith), considered for a long time a generalist feeder, now is known to comprise two genetically differentiated host-associated strains, designated as corn strain (CS) and rice strain (RS). Previous studies have revealed that females attract males of both strains but mate almost exclusively with their own strain.

Males are known to possess two sets of hairpencils (potential pheromone-producing structures), but their role has not been previously assessed. The species is a good model to test Phelan’s (1992) Asymmetric Tracking model, which predicts that the sex with the greater parental investment will be more discriminating in their response to sexual signals, and that signals produced by the other sex will evolve to track this preference.

Courtship sequences of S. frugiperda were videorecorded in an arena under infrared light, and analyzed frame by frame. Individual behaviors were transcribed to three-dimension transitional matrices, which were submitted to Information Theory analysis for quantification of intra- and inter-individual communication.

Information analysis confirmed the existence of an asymmetry in the response between the sexes in S. frugiperda. In both intra- and inter-strain courtships, female-to- male information flow was lower than for male-to-female. Moreover, both RS and CS females were more likely to show a receptive response to male behaviors associated with hairpencil displays when courted by males of their own strain. These findings of a male-

ii signal-based female preference for assortative mating, particularly in the absence of mate discrimination by males are consistent with the predictions of the Asymmetric Tracking hypothesis (Phelan, 1992) and the more specific hypothesis of Phelan and Baker (1987) that many lepidopteran male pheromones may have evolved in the context of reproductive isolation through sexual selection.

Chemical characterization of each set of hairpencils was also conducted by solvent extraction and analysis by gas chromatography/mass spectrometry. Among the ca. 150 chemicals identified in the extracts, two stood out as showing clear differences between males of the two strains, 2,4-diphenyl-4-methyl-1-(E)-pentene and 2,4-diphenyl-

4-methyl-2-(Z)-pentene. These compounds had not previously been described in any

Lepidoptera.

iii

ACKNOWLEDGEMENTS

I thank my advisor Professor Larry Phelan for welcoming me at his laboratory

and for giving me the opportunity of doing research on such an interesting subject - the

evolution of sex pheromones. It was a hard and stimulating work on several aspects very

much as I had anticipated when I left Portugal to this new and exciting learning

experience in a US University. I thank him also for letting me share his valuable

experience, for all the support he provided me, and for all the discussions we had, that

made me think sharper and express my thoughts clearer and overall mature as scientist.

I am grateful to my committee (Professor David Horn, Professor Woodbridge

Foster and Professor John Wenzel) for their valuable suggestions.

I would also like to thank Professor Dorothy Prowell from the Louisiana State

University for her help in gathering the collections of Spodoptera frugiperda specimens with which I started both colonies at the OARDC-OSU. I also thank her for kindly letting me use her laboratory resources and for introducing me the necessary tools to assess S. frugiperda strain status.

My gratitude also goes to Dr. Margaret McMichael at LSU for the valuable time

she took to teach me the techniques for assessing S. frugiperda strain status, and in

solving all the problems associated with the method after I returned to OSU. I thank her

also for helping me in the field collections of specimens. iv

I am also grateful to Professor Sally Miller for letting me use her laboratory

resources when assessing the status of the S. frugiperda specimens brought from

Louisiana.

I thank Emeritus Professor Roy Rings for letting me “explore” and “damage”

some of the S. frugiperda specimens of his precious collections at the OARDC-OSU.

I would like to thank LeAnn Beanland, Seppo Salminen, and the other graduate

students and staff working at the OARDC. They all made me feel at home while I lived

and worked in the USA and I was privileged for the time and experiences we shared.

Here, I especially would like to thank my amigo Jim Mason for his friendship and his remarkably unselfish support. His long experience with lepidopteran colonies and his natural interest helped solve the small and big ordinary laboratory problems on a daily basis.

And last but not least I would like to thank my wife Augusta for believing in me

all the way, for her endless encouragement and support. Her extraordinary strength and

contagious optimism were vital to my accomplishment of this task.

Fundação para a Ciência e Tecnologia, of the Portuguese state office Minitério da

Ciência e do Ensino Superior, supported this research (Grant:Praxis XXI BD/9402/96)

VITA

November 08, 1963 Born, Lisbon, Portugal

1992 Finishing Undergraduate Coursework in Biology and obtaining the Degree Licenciatura, Faculdade de Ciências da Universidade de Lisboa (University of Lisbon's Faculty of Sciences, Portugal)

1992/1993 High School Biology Teacher, Escola Secundária das Olaias (Lisboa)

1993/1994 On a science research scholarship from Junta Nacional de Investigação Científica e Tecnológica) (National Board for Scientific Reasearch, Portugal).

1994/1995 High School Biology Teacher, Escola Secundária de Vendas Novas (Vendas Novas, Portugal)

1995/1996 High School Biology Teacher, Escola Secundária nº 1 de Loures (Loures, Portugal)

1996 M. S. in Ethology, Instituto Superior de Psicologia Aplicada (Sup. Institute of Applied Psychology, Portugal)

1996/1997 High School Biology Teacher, Escola C+S da Ericeira (Ericeira, Portugal)

2003 Invited TA at the Universidade de Évora (University of Evora, Portugal

FIELD OF STUDY

Major Field: Entomology

vii

TABLE OF CONTENTS Page

Abstract………………………………………………………………………….. ii

Acknowledgments……………………………………………………………….. iv

Vita………………………………………………………………………………. vi

List of Figures …………………………………………………………………... x

List of Plates…………………………………………………………………….. xi

List of Tables…………………………………………………………………….. xii

Prologue...... …... 1

Chapters:

Chapter 1………………………………………………………………………… 23

1. Introduction……………………………………………………. 23

2. Materials and Methods………………………………………… 33

3. Results…………………………………………………………... 38

3.1. Description of Spodoptera frugiperda Courtship………………. 38

3.2. Ethogram of Spodoptera frugiperda…………………………… 39

3.3. Courtship success within and between strains………………….. 51

3.4. Information Theory analysis…………………………………… 55

3.4.1. Intra-strain Courtship analysis……………….………………... 55

3.4.2. Inter-strain Courtship analysis……………….………………… 64

4. Discussion……………………………………………………... 68

viii

Chapter 2………………………………………………………………………… 77

1. Introduction………………………………………………….…. 77

2. Materials and Methods……………………………………….… 86

3. Results………………………………………………………….. 91

3.1 Genital hairpencils…………………………………………….... 91

3.2 Femoral hairpencils……………………………………….…….. 97

4. Discussion…………………………………………….………... 104

Epilogue ………..……………..…………………………………………. 111

References ……………………………………………………………….…. 113

Appendix A Protocol for host strain status determination of S. frugiperda….. 125

Appendix B Chemicals identified in all extracts obtained from male and

female S. frugiperda…………………………………………….. 130

LIST OF FIGURES

FIGURE PAGE

1. Dendogram for the genital brushes’ dataset 96

2. Dendogram for the femoral hair-pencils dataset 103

LIST OF PLATES

PLATE PAGE

1. Female calling (CAL) 43

2. A mating sequence in the corn associated strain of Spodoptera 49 Frugiperda species.

3. Male displaying genital hairpencils and femoral hairpencils to 54 female’s head (AbH).

LIST OF TABLES

TABLE PAGE

Table 1. Mating outcomes of pairings between female and males of the two 55 host-strain of Spodoptera frugiperda of Spodoptera frugiperda

Table 2. Information Theory parameters for intra-strain courtships. 57

Table 3. Male-to-female information transmission 59

Table 4 Female-to-male information transmission 61

Table 5 Information theory parameters for inter-strain courtships 65

Table 6 Genital hairpencils chemicals 93

Table 7 Femoral hairpencils chemicals 100

Table 8. Chemicals identified in all extracts of male and female 131

xii

PROLOGUE

Species and species-isolating mechanisms

Species of sexually reproducing animals remain distinct, primarily because they are genetically independent. Genes present within individuals of one species usually combine only, in future generations, with genes from other members of the same species.

Thus members of a species are said to "share a common gene pool" and one species is separated from another by "barriers to gene exchange," according to the Biological

Species Concept (Mayr, 1940; 1976). Since evolution is based on the accumulation of genetic modifications, and the boundaries of species impose limits on the spread of genetic variation, the concept of species has a central place in evolutionary biology.

Evolutionary forces that lead to changes in the species’ behavioral patterns, primarily those comprising mating signal systems, can greatly influence the formation of new barriers to gene exchange and thus speciation.

Habitat choice and timing of reproductive activity influence the chance of meeting potential sexual partners, and sexual signaling systems have a major role in the recognition of potential mating partners - in bringing partners together and in stimulating sexual receptivity. If divergence occurs between populations in any of these behavioral patterns, a barrier to gene exchange may arise.

Habitat choice

It has been suggested that divergence in host preference in geographically isolated populations may be the result of exposure to different frequencies of alternative hosts, through the development of efficient searching tactics for the most common species. If the host preferences become sufficiently distinct, these populations may coexist without gene exchange should they ever come to live in sympatry (Butlin and Ritchie, 1994).

However, divergence could also occur without geographic isolation (sympatric speciation) if a biological barrier to inbreeding arose within the confines of a panmictic population, without any spatial segregation of the incipient species. Many models of sympatric speciation (e.g., Maynard Smith, 1966, Dickinson and Antonovics, 1973,

Felsenstein, 1981) are based on disruptive selection, as when two homozygotes at one or more loci are adapted to different resources, and there is a multiple-niche polymorphism.

Felsenstein (1981) suggested that divergence would require that the population be monomorphic at the locus conferring host preference, that both loci (the one providing adaptation and the one providing the preference) be tightly linked, and that selection based on adaptation to different hosts should be quite strong, with little gene flow caused by factors such as environmentally induced variation in host or mate preference, in order to produce the combination of mating and laying eggs on the same host. In two species of lacewings (Chrysoperla) that are associated with different vegetation and emerge at somewhat different seasons, the difference in coloration that makes them cryptic in their

respective habitats is largely controlled by one locus, and the differences in emergence

time by two loci. Because the genetic control of these characters is simple, as required by models of sympatric speciation, Tauber and Tauber (1982) have proposed that these species arose sympatrically.

Many species of phytophagous insects are highly host specific. Hence, even if

different host races have similar mating signals, assortative mating can be promoted by

host-plant preferences. In some cases, males and females are both attracted to the

oviposition site, where they find each other using short-range signals. An example is

provided by Rhagoletis pomonella in which females are attracted to the host from long

distances by its shape and color, and then to the individual fruits by their size, shape,

color and surface chemicals. The males search for the fruits in similar ways and mating

takes place on the fruits (Bush, 1974). Differences in host preference coupled with host

phenology have been suggested as the basis for reproductive isolation in species of the

genus Rhagoletis (Feder et al., 1994; 1997). Therefore, sexually active males and females

of different species are unlikely to encounter one another.

In other cases, there is also long-distance mate attraction. In the small ermine

moth, Yponomeuta, divergence in host preference seems to be controlled primarily by the

oviposition behavior of females. Several species of this moth feed exclusively on

different host bushes (Prunus, Crataegus, and Euonymus). However, there is very little

3 difference in survival when the caterpillars of different species are artificially placed on hosts they do not normally use. The change in oviposition behavior may have played a key role in speciation, although Yponomeuta mating signals must also have diverged since females preferentially attract conspecific males from long distances with pheromones (Menken et al., 1992).

Time of reproductive activity

Individuals may also be isolated by means of allochronic isolation (the time of

breeding). For example, eggs of Enchenopa binotata hatch in response to the rise of sap

in the host (Wood, 1993), which occurs at different times in different host species.

Because the duration of development is similar in each host, the link to the host leads to

separation in the emergence time of adults. The shift to a new host may be an adaptive

response to parasitism of eggs or larvae, because the timing of emergence of adult

parasitoids is independent of the host-plant phenology and parasitoid oviposition is

dependent on locating their host’s odor from frass (Loke and Ashley, 1984;

Agelopoulos et al,. 1995), long-range host kairomones (Parra et al., 1996), or the odor

of their host’s preferred plant (Udayagiri and Jones, 1992). They can also respond to the

odor released by plants after damage caused by larval feeding (Turlings et al 1993;

1995; Tabkabayashi et al. 1995; Ngi-Song et al. 1996).

Allochrony can also occur in mating. Pheromones are crucial to mate finding in

moths. With few exceptions, females attract males by their scent, typically over long

distances. Calling females generally remain stationary, the scent being carried by air

currents from glands usually situated between the abdominal segments A7 and A8 or A8

and A9 (Scoble, 1992). The female odor triggers optomotor anemotaxis in males, who fly

upwind towards the scent via a programmed self-steered series of reversals, resulting in a

zigzagging pattern of flight (Kennedy et al. 1980; Kennedy 1982; 1983). An example of allochronic isolation is given by the closely related species, Platyptilia carduidactyla and

P. williamsii (Lepidoptera: Pterophoridae). These species use the same sex pheromone, but while P. carduidactyla calls during the first half of the night, P. williamsii calls during the second half (Haynes and Birch, 1986). Artificial synchronization of the calling periods results in cross-attraction and even attempted copulation. According to Haynes and Birch (1986), the temporal differences between the two species are the primary factor preventing interspecific cross-attraction and courtship.

Differences in pheromone and genital incompatibilities can combine with different activity cycles, preventing closely related species from interspecific crosses, as suggested for Spodoptera latifascia and S. descoinsi (Monti et al. 1995).

Selective forces in the evolution of sexual communication

Evolution of mate signaling systems for reproductive isolation

Among the evolutionary forces shaping new species are those leading to changes

in the mating system, namely those affecting mate recognition and sexual receptivity. For

years, differences in the mating signals of species were interpreted as functioning to

prevent interspecific mating mistakes, which were seen as a threat to species integrity.

The specific mating signals were viewed as an adaptive mechanism that could ensure

reproductive isolation, thus being crucial to the process of speciation. The rationale was

that when the two genetically differentiated populations reestablish contact, the adaptive

complexes are exposed to disintegration due to gene flow between them, and any

divergence between the populations would be lost (Dobzhansky, 1940). This correlation

between interspecific contact and divergence of mating signals was termed reinforcement

(Blair, 1955). Two important predictions were made by the Dobzhansky model for the

evolution of ethological isolation: (1) divergence in the signals should be more

accentuated in those species overlapping in geographic distribution with related species

than in those that do not, and (2) sympatric populations of two related species would

show greater differences in their sexual signals than would allopatric populations of these same species. This pattern has been clearly demonstrated in a few cases, although it is difficult to separate adequately from other possible sources of signal variation. In the

Australian frogs, Litoria ewingi and L. verreauxi, divergence in the characteristics of the

mating call is closely associated with sympatry (Littlejohn and Watson, 1985). This

evidence may, however, be a result of reinforcement, reproductive character

displacement (i.e., selection for divergence in mate signals after genetic incompatibility

has occurred), or simply divergence in allopatry, although the authors favored the second

choice. Other studies, e.g., with katydids and crickets (Walker, 1974) and with several

Australasian bird species (McKnaught and Owen, 2002), have not found mating signals

diverging in a direction consistent with reproductive displacement.

Incidental evolution of mate signaling systems

Both mating signalers and receivers are subject to a variety of selection pressures.

Divergence in mating signals can thus be a consequence of different environmental

adaptations that incidentally develop in isolated populations and that end up creating premating barriers to interbreeding when individuals from separate populations reestablish

contact. Mating signals can be both attenuated and degraded by the physical properties of

the environment. A signal that functions efficiently in one environment may not be ideal

in another environment. Therefore, populations of a species inhabiting different habitats

may diverge in important properties of their mating signals (Butlin and Ritchie, 1994).

In addition, receptors typically have functions other than the reception of mating

signals that may influence their evolution and signals may be received by parasites and

7 predators as well as by potential mates (Butlin and Ritchie 1994). One such classical example is that of sticklebacks (Gasterostereus aculeatus: Pisces). Male sticklebacks have bright red nuptial coloration except in certain populations that are sympatric with an unrelated fish that preys on young sticklebacks. In these populations, males, which guard the young, are black, perhaps thereby attracting fewer predators to the nest. Females from these populations mate readily with black males, whereas females from other populations discriminate strongly against black males in favor of red. The change in the nuptial coloration presumably to avoid predation, would come to constitute a pre-mating barrier to interbreeding if individuals from separate populations come to meet with each other

(McPhail, 1969).

Critics of the Dobzhansky model argue that a pattern of greater difference between related sympatric species (when compared to related allopatric species) could also be predicted even if the mating signals evolved in the absence of interspecific contact. If populations of a species become geographically isolated and diverge in their mating signals while isolated, then when contact is reestablished, individuals of the two populations will not recognize each other, preadapting the populations for sexual isolation. If, however, divergence has not occurred, then they either form an interbreeding population or one population will drive the other to extinction (Paterson, 1978). So, rather than viewing differences in mate-signaling systems as barriers to gene flow, Paterson

(1985) regards species as a population of individuals that share a "specific mate-

recognition system" (SMRS) to find, recognize, and stimulate mates. Paterson (1985)

named this new concept of species the Recognition Species Concept. According to the concept, merging of egg and sperm is so important that the mechanisms mediating the process (the SMRS) will be under strong selection to become increasingly efficient in whatever environment the population is found. Thus, it is this strong selection on the

SMRS in a new environment that brings about reproductively isolated populations, not selection for reproductive isolation per se.

Coordination of signal and response

A key question to understanding the evolution of sexual communication is how can signal and response be coordinated for species specificity, and still shift during speciation (Phelan 1992; 1996a). Signal-response coevolution, genetic linkage, and evolution via asymmetric tracking have been presented as alternatives to explain how the coordination between signal and response can be achieved.

As explained above, the Recognition Concept considers that SMRS to be under strong mutually stabilizing selection (Paterson, 1980). This implies that adaptive change in the sexual communication system can only occur in small steps with coadaptation between signal and response. Under this perspective, mate-signaling systems are viewed as homeostatic systems (Paterson, 1985; 1993; Lambert et al 1987). A coevolved signal- response is thus expected to show low variance both within and among populations. A

review of the literature on insect sex pheromones by Lambert et al (1987) seems to

support this notion. Also a study conducted by Haynes and Baker (1988) on the ratios and

amounts of ZZ-/ZE-7,11-hexadecenyl acetate produced by Pectinophora gossypiella

females from several world locations supported this idea, showing small variability

worldwide (57%±4.0 to 63.1±2.1 ZZ).

Although it has been argued that the low intraspecific variance in moth sex

pheromone production and response is consistent with the Recognition Concept (Lambert

et al. (1987), this model fails to explain the evolution of pheromonal antagonists (Phelan

1992). In the Heliothinae, four closely related species share the same major component of

their pheromone, Z11-16:Ald, but those that live in sympatry - Heliothis virescens with

Helicoverpa zea and Heliothis armigera with Helicoverpa peltigera - are known to differ

in their second component. Heliothis virescens produces Z9-14:Ald, whereas Helicoverpa zea produces Z9-16:Ald. Similarly, Heliothis armigera produces Z9-16:Ald, while

Helicoverpa peltigera uses Z9-14:Ald (Mustaparta, 1997). The Z9-14:Ald interrupts the attraction of Helicoverpa zea males to Heliothis virescens females (Shaver et a.,l 1982), thus preventing cross-attraction between Helicoverpa zea and Heliothis virescens (Haile et al., 1973; Roach, 1975; Carpenter et al., 1984; Lopez and Witz, 1988). This same compound interrupts attraction of Heliothis armigera males towards Helicoverpa peltigera females, thus also preventing cross-attraction between these species

(Mustaparta, 1997). This phenomenon, known as interspecific interruption, has been

documented among Lepidoptera and bark beetles (for a review see Masson and

Mustaparta, 1990).

Constraints on change in a coadapted signal and response could be reduced if they

were genetically linked (Hoy et al., 1977). In Colias butterflies, a coadapted gene complex located on the X chromosome may be largely controlling both male sexual signals and female preference (Grula and Taylor 1979; 1980). On the other hand, the pheromone production and response of moths have been found to be controlled independently, involving both autosomes and sex chromosomes. Maini (1979) and

Roelofs et al. (1987) found that an autosomal gene with two alleles controls the female pheromone ratio in Ostrinia nubilalis, for which two pheromonal races are known in

Europe and the United States. Roelofs et al. (1987) also found that the male behavior was maternally inherited, although the inheritance of the antennal receptor sensitive to the female E and Z –11-tetradecenyl acetate blend was controlled by a single autosomal gene

with two alleles. Subsequently Lofstedt et al. (1989) determined that the autosomal

factors controlling female production and male response assorted independently.

A view that mating systems rarely will be under strong mutually stabilizing

selection and that selection on signal and response will be asymmetric is behind the

asymmetric tracking hypothesis proposed by Phelan (1992). This model is based on the

idea that mate-signalling is best explained in the framework of sexual selection, which

11 also can predict when other selection and non-selection processes will play an important role. Since parental investment is important in explaining variability in mating success, the model takes into consideration that differential parental investment by the two sexes potentially produces a conflict of interests. Differential selection that is inversely related to the sexual asymmetry in parental effort will thus be expected. From this model, four important predictions are derived: (1) females will usually assume the least costly role in mate-signaling/finding; (2) females will be under only weak stabilizing selection to produce the pheromone to which male response is maximized, thus in the absence of other selective forces, significant pheromonal variation may occur among females within a population; (3) although males will respond to the most common pheromone blend in the mating population, their response should not be so narrow as to exclude other blends that signal a viable mate, even if these females are from a different race. Males will only experience selection against responding to females of other populations if the fitness of those mating is low relative to the cost of finding a female of their own population; (4) in species where males produce pheromones, the pheromones will be under selection to track female response.

The asymmetric tracking hypothesis thus predicts that in the absence of strong stabilizing selection, female pheromones are free to vary and might diverge in geographically isolated populations. Male response will then track the pheromonal changes. In opposition to Lambert et al.’s (1987) conclusion, several studies have shown

that although the female sex pheromone of Lepidoptera species can show consistency in

the pheromone blends (e. g., the blend ratio of the two-component pheromones of P.

gossypiella (Haynes and Baker, 1988)), it can also be polymorphic as in the pheromonal phenotypes of the European corn borer, O. nubilalis (Kochansky et al. 1975), the larch

bud moth, Zeiraphera diniana (Priesner and Baltensweiler, 1987), the turnip moth,

Agrotis segetum (Lofstedt et al., 1986), and the dingy cutworm, Feltia jaculifera (Byers

et al., 1990).

Zeiraphera diniana has two discrete pheromonal types differing in their ratios of

two components (100:1 and 1:100 blends of E9-12:Ac and E11-14:Ac), but intermediate

phenotypes, presumably arising from hybridization and introgression, also exist (Priesner

and Baltensweiler, 1987). In F. jaculifera, four pheromonal types have been found based

on different proportions of the three-component blend of Z7-12:Ac, Z9-14:Ac, and Z11-

16:Ac. The sympatric populations, which appear to be reproductively isolated, differ in

their main component, whereas the allopatric populations share the same main

component, although differing in the ratios of the other two (Byers et al., 1990).

Among the European populations of A. segetum, there is variation in the three- component sex pheromone, (Z5-10:Ac, Z7-12:Ac, and Z9-14:Ac) (Lofstedt et al., 1986), but there are no reports of populations with different blends living sympatrically.

Ostrinia nubilalis has two pheromonal types, occurring in North America

(Kochansky et al. 1975) and Europe (Pena et al., 1988), with a 3:97 or a 97:3 blend of

Z11-14:Ac and E-11-14:Ac. The two strains, as well as hybrids, exist sympatrically in

Europe, south of the Alps (Anglade et al., 1984; Barbattini et al., 1985). However, in

addition to these two pheromone components, other minor compounds have been found:

14:Ac, a compound present in both strains, with a synergistic effect (Stocckel, 1980) and

E9-14:Ac, found in small amounts only in the Z strain, with an inhibitory effect on male

response (Klun et al., 1979).

The examples of F. jaculifera and A. segetum show how divergence in pheromone

can evolve either with or without pressure for reproductive isolation, which is consistent

with the asymmetric tracking hypothesis. Since it has been proposed, the asymmetric

tracking model has received support from the studies of Liu and Haynes (1994) on female

mutants of Trichoplusia ni and Roelofs et al. (2002) on O. nubilalis (European corn

borer) and O. furnacalisis (Asian corn borer).

Contrary to the normal Trichoplusia ni female pheromone, consisting of Z-12:Ac,

12:Ac, Z5-12:Ac, 11-12:Ac, Z7-14:Ac and Z9-14Ac (100:7:17:3:2:1) (Bjostad et al.,

1984), mutant females were discovered that emit the same six components, but in the ratio 100:17:2:4:2:5 (Haynes and Hunt, 1990). As in other pheromonal systems, the genetic control of female production was found to be independent of male response

(Haynes and Hunt, 1990). Following this finding, Todd et al. (1992) showed that the

component-specific antennal receptors in the mutant males had a similar distribution to

that of normal males. Liu and Haynes (1994) followed in the laboratory T. ni populations either composed by mutant females and normal males or normal females and normal males, for 49 generations. Although at the start of their study, males from normal and mutant populations were twice as responsive to the normal pheromone as to the mutant blend, by the end, males of the mutant colonies were responding with equal frequency to both blends, whereas normal males continued to show preference for the normal blend.

The female pheromone had, however, remained unchanged. These results were in accordance with the second and third predictions of Phelan’s (1992) asymmetric tracking model.

Roelofs et al. (2002) provided compelling evidence on how a departure from the normal female pheromone might have arisen through asymmetric tracking in O. nubilalis and O. furnicalis. Their results also suggest how male response shifted to the new pheromone via asymmetric tracking, similarly to that found by Liu and Haynes (1994) for

T. ni. Both O. nubilalis and O. furnicalis share two desaturase-gene transcripts, whose

products are responsible for the location of the double bond of their respective

pheromone. However, only one product is found in each species. Ostrinia furnicalis is thought to have derived from populations of O. nubilalis when a departure from the normal pheromone arose as the expression of a pseudogene that had been unexpressed for

millions of years in the genome of O. nubilalis. As a result, the location of the double bond changed in the pheromone and a new pheromone was produced. Very broadly tuned males in the laboratory populations of O. nubilalis responding both to O. nubilalis and O. furnicalis showed how this shift might have happened.

Male-produced pheromones in the Lepidoptera

In some species of Lepidoptera, males may also disseminate scent molecules, which generally happens close to the female during courtship (see Birch et al., 1990 for a review). In some cases, these pheromones have a long-range attraction such as in

Creatonotus gangis and C. transiens (Arctiidae), where males form leks and attract

females from a distance (Wunderer et al., 1986), or in T. ni (Noctuidae), where males adopt a stationary position and attract females from several meters away (Landolt and

Heath, 1989). Unlike female pheromone systems, the male disseminating structures show a high degree of heterogeneity (McColl, 1969; Boppré, 1984b) and their appearance across taxa is ephemeral, correlating poorly with presumed taxonomic relatedness

(Phelan 1992). A number of functions have been postulated for the evolution of male pheromones, such as predator repellent, mating deterrent against conspecific males, and species-specific signal to prevent interspecific mates (Birch, 1974).

Evidence that male pheromones may act as deterrents against conspecific males

have been presented for the male-released chemicals of the summerfruit tortix moth,

Adoxophyes orana (Tortricidae) (Bijpost et al., 1985) and for Pseudaletia unipuncta

(Hirai et al., 1978). Laboratory studies showed that A. orana males, perceiving the male

pheromone while flying upwind, decreased wing fluttering and increased the lateral

component of their search path (Bijpost et al., 1985). The two chemicals extracted from

the males were identified as palmitic acid and myristic acid, although only the former

evoked a male response identical to that observed towards males in the situation

described. Hirai et al. (1978) have presented similar results for Pseudaletia unipuncta

male scents, although other studies have dismissed their findings (Fitzpatrick et al.,

1988).

Direct evidence for male pheromone disseminating structures as elicitors of

receptive female behavior has been provided for Grapholita molesta (Tortricidae) (Baker

and Cardé, 1979), Ephestia elutella (Phelan and Baker, 1986; Phelan et al. 1986), and

Phragmatobia fuliginosa and Pyrrharctia isabella (Krasnoff and Roelofs, 1990). In

others, the relevance of the male scent structures in the mating behavior was indirectly

demonstrated (Birch, 1970): Phlogophora meticulosa (Grant et al. 1975), Plodia interpunctella and Cadra cautella (Ellis and Brimacombe, 1980), and Spodoptera littoralis and Pseudaletia unipuncta (Fitzpatrick and McNeil,1989).

Males of the tortricid G. molesta bear a pair of white tuff hairs (Baker and Cardé,

1979), which disseminate a blend of four chemicals: ethyl trans-cinnamate, (R)-(-)-

17 mellein, methyl jasmonate, and methyl 2-epijasmonate (Baker et al., 1981; Nishida et al.,

1982). Females respond to the male abdominal hairpencil display, approaching the male and touching his abdominal tip with their head, which evokes his copulatory attempt

(Baker and Cardé, 1979).

Ephestia ellutella males release a pheromone from a wing gland that causes female acceptance posture, which was characterized as (E)-phytol, gamma-decalactone, and gamma-undecalactone (Phelan and Baker, 1986; Phelan et al., 1986).

In the arctiids, sac-like structures, covered with scales that are known as coremata, have been observed between the 7th and 8th abdominal sternites or on the genital valves of males of several species. These structures vary in size and may depend on how much pyrrolizidine alkaloids (PAs) have been ingested during the larval phase

(Schneider et al. 1982, Boppré and Schneider 1985), from which the chemicals found in this family, hydroxydanaidal and danaidal, are derived. The corematal extrusion is known to cause female acceptance in the case of Utetheisa ornatrix (Conner et al, 1981; 1989), but in species where males form leks such as Creatonotus transiens and C. gangis

(Wunderer et al., 1986), the corematal extrusion induces the attraction of both conspecific females and males.

In the Noctuidae, male structures can be found on the legs, thorax, and abdomen, the later showing a remarkable complexity (Birch, 1970; 1972). Abdominal structures

can also vary in location, being present on the anterior abdomen (associated with the 2nd,

3rd, and 4th abdominal sternites), usually referred to as hairpencils (e.g., several

Hadeninae, some Noctuinae, Cuculliinae, and Amphipyrinae), on the posterior abdomen

(8th abdominal sternite) (e.g., some Pantheinae, and Catocalinae), or on the ninth

abdominal sternite of some Heliothinae (e.g., Heliothis virescens, Teal et al. 1981;

Cibrian-Tovar and Mitchell 1991) and some Amphipyrinae (e.g., Spodoptera frugiperda,

Eltringham 1927, and S. littoralis, Ellis and Brimacombe 1980). The latter structures are

usually referred to as hairbrushes (Birch et al. 1990). In this family, congeneric species

sometimes share secretion components, e.g., Leucania impura, L. conigera, and L.

pallens all produce benzaldehyde (Aplin and Birch, 1970), but chemical variation among

congeneric species is also observed, as in members of the Mamestra genus (Aplin and

Birch (1970). Evidence that hairpencil extrusion induces female acceptance to mating

exists in several noctuids: Phlogophora meticulosa, (Birch 1970), S. littoralis, (Ellis and

Brimacombe 1980), Heliothis virescens, (Teal et al 1981), Pseudaletia unipuncta

(Fitzpatrick and McNeil 1989), H. subflexa, (Cibrian-Tovar and Mitchell 1991), and M. brassicae (Poppy and Birch 1994).

Baker and Cardé (1979) were the first to envisage how female-choice sexual selection could be linked with the evolution of these male structures. If a fitness-related trait involving odor could become preferred by females, and if conditions promoted differential mating success between males, both the trait and the preference for it could be driven to extremes through runaway selection.

Phelan and Baker’s (1986) study of phycitine male odors led them to propose that sexual selection by female choice has driven the evolution of lepidopteran courtship pheromone systems, and Phelan and Baker (1987) showed that male scent-emitting structures are more likely to occur in moth species sharing a host plant with a closely related species than in species without host overlap, thus seeming to provide support to the hypothesis that these species-specific male signals evolved to prevent interspecific mating. These signals are expected to evolve whenever females risk making mistakes by accepting a non-viable male from a different population and are predicted to be lost whenever this pressure no longer exists, i.e., when males are no longer cross-attracted to females (Phelan 1992). The diversity of these structures in the Noctuidae and their partial loss in several species are consistent with the idea that these structures have arisen independently and have been lost many times (Birch 1972), which is also consistent with their role in producing reproductive isolation. Evidence suggesting a vestigial role for the male pheromones has been provided by Krasnoff and Roelofs (1990) studies of

Phragmatobia fuliginosa and Pyrrharctia Isabella, where although coremata and

respective pheromone are present, they seem to play no role in male attractiveness.

The noctuid species, Spodoptera frugiperda (J. E. Smith), known as the fall armyworm is a good model to test Phelan´s (1992; Phelan and Baker, 1987) predictions, due to several aspects of its ecology and morphology. This species is known to be composed of two genetically differentiated strains, with different host preferences and specializations, and evidence of disadvantage in inter-strain crosses (Pashley and Martin,

1987; Whitford et al., 1988). Populations of the two host strains are known to live both in

allopatry and sympatry, to have a partial allochronic isolation (Pashley, 1986; 1988b),

and there is some evidence of differential composition in the female sex pheromone

(Tumlinson et al., 1986; Pashley et al, 1992). Males of these species also possess

eversible structures, though their role had not been fully assessed before the current

study.

In the first chapter, the role of these structures during courtship is assessed along

with the relative importance of several male signals towards mating sucess. Evidence is

provided that the proximal function of hairpencils is eliciting and maintaining female

receptivity: incorporating the display of these structures in the courtship behavioral

sequence significantly increases mating success. As to the ultimate function of

hairpencils, the data suggests that their display mediates assortative mating, since

21 acceptance behavior during courtship by females of the same strain is associated with presentation of male scent structures.

The second chapter reports an investigation of the chemicals that are carried and presumably disseminated by hairpencils during courtship. Several compounds present in rice strain male hairpencils are missing or present in very small concentrations in corn strain males, whether wild caught or laboratory reared, thus suggesting a role in the male pheromone.

These differences may very well be the result of female sexual selection since females are under pressure to mate with same-strain males in the sympatric context of these two strains.

CHAPTER 1

MATE DISCRIMINATION BY Spodoptera frugiperda FEMALES

1. INTRODUCTION

The asymmetric tracking hypothesis was proposed by Phelan (1992) to explain

the evolution of mate signaling systems. It makes specific predictions for the

circumstances in which a mate-signaling system is likely to change and the selective pressure behind it. This model takes into account the differential parental investment by

the two sexes, which potentially produces a conflict of interests. So, the asymmetric

tracking model predicts that in the absence of strong stabilizing selection, female pheromones are free to vary and might diverge even in geographically isolated

populations. Male response will then track the pheromonal changes.

Four predictions follow from the asymmetric tracking model: (1) females will

usually assume the least costly role in mate-signaling/finding; (2) females will be under

only weak stabilizing selection to produce the pheromone to which male response is

maximized, thus in the absence of other selective forces, significant pheromonal variation

may occur among females within a population; (3) although males will respond to the

most common pheromone blend in the mating population, their response should not be so narrow as to exclude other blends that signal a viable mate, even if these females are

from a different race. Males will only experience selection against responding to females 23

of other populations if the fitness of those mating is low relative to the cost of finding a

female of their own population; (4) in species where males produce pheromones, the

pheromones will be under selection to track female response.

Since it has been proposed, the asymmetric tracking model has received support

from the studies of Liu and Haynes (1994) on female mutants of Trichoplusia ni and

Roelofs et al. (2002) on O. nubilalis (European corn borer) and O. furnacalis (Asian corn

borer). Contrary to the normal Trichoplusia ni female pheromone of Z-12:Ac, 12:Ac, Z5-

12:Ac, 11-12:Ac, Z7-14:Ac and Z9-14Ac (100:7:17:3:2:1) (Bjostad et al., 1984), mutant females have been discovered that emit the same six components but in the ratio

100:17:2:4:2:5 (Haynes and Hunt, 1990). As in other pheromonal systems, the genetic control of female production was found to be independent of male response (Haynes and

Hunt, 1990). Following this finding, Todd et al. (1992) showed that the component- specific antennal receptors in the mutant males had a similar distribution to that of normal males. Liu and Haynes (1994) followed in the laboratory T. ni populations either composed by mutant females and normal males or normal females and normal males, for

49 generations. Although at the start of their study, males from normal and mutant populations were twice as responsive to the normal pheromone as to the mutant blend, by the end, males of the mutant colonies were responding with equal frequency to both blends, whereas normal males continued to show preference for the normal blend. The

24 female pheromone had, however, remained unchanged. These results were in accordance with the second and third predictions of Phelan’s (1992) asymmetric tracking model.

The Roelofs et al. (2002) study on O. nubilalis and O. furnicalis provides compelling evidence on how a departure from the normal female pheromone might have arisen to initiate the asymmetric tracking. It also provides evidence on how ancestral males via asymmetric tracking accomplished the shift to the new pheromone, similar to that provided by Liu and Haynes (1994) for T. ni. Both O. nubilalis and O. furnicalis share two desaturase-gene transcripts, whose products are responsible for the location of the double bond of their respective pheromone. However, only one product is found in each species. O. furnicalis is thought to have derived from populations of O. nubilalis when a departure from the normal pheromone arose as the expression of a pseudogene that had been unexpressed for millions of years in the genome of O. nubilalis. As a result, the location of the double bond changed in the pheromone and a new pheromone was produced. Very broadly tuned males in the laboratory populations of O. nubilalis responding both to O. nubilalis and O. furnicalis showed how this shift might have happened.

The noctuid species, Spodoptera frugiperda (J. E. Smith), known as the fall armyworm (FAW) is a good model to test the predictions of both Phelan and Baker

(1987), and Phelan (1992) as well due to several aspects of its ecology and morphology:

it is comprised of two strains, with different host preferences and specializations, and

evidence of lower fitness in hybrid offspring (Pashley and Martin, 1987; Whitford et al.,

1988). Populations of the two host strains are known to live both in allopatry and

sympatry, they have a partial allochronic isolation, and there is evidence of possible

differences in female sex pheromone (Tumlinson et al., 1986; Pashley et al, 1992). Males of these species also possess eversible structures, though their role has yet to be fully assessed.

The existence of two genetically differentiated strains

Spodoptera frugiperda is a major insect pest, with a vast geographic distribution.

Various approaches have been developed to control it, including insecticides (e.g. Young

1979; Chandler and Sumner 1991; Guillebeau and All 1991), cultural practices (e.g.

Roberts and All 1993), natural enemies (e.g. Ashley 1979; 1983; Ashley et al. 1983;

Gross and Pair 1991; Riggin et al 1992), resistant host strains (e.g. Wiseman and Davis

1979; Hedin et al. 1990), and traps with semiochemicals (e.g. Mitchel 1979; Sparks

1980; Ward et al. 1980; Silvain 1986).

For a long time, FAW has been considered a generalist feeder, consuming wheat,

oats, corn, barley, grasses, purslane, and even spruce (Riley, 1869). It is thought to be a

major pest of corn, rice and forage grasses throughout the Western Hemisphere

(Luginbill 1928), with a tropical-subtropical origin in the Western Hemisphere. The

author also reports that more than 60 plant species are attacked by FAW, despite this

species' preference for crabgrass, corn, sorghum, and bermudagrass. However, recent

data questioned the lack of dietary specialization by the fall armyworm (Pashley, 1986,

1988a, 1988b; Lu et al. 1992, 1994, 1996). Through allozyme analyses, Pashley (1986) found that FAW is composed of two genetically differentiated strains, each strain exhibiting different host specificity. Collections from Bermuda grass in Louisiana and rice in Puerto Rico were found to be genetically identical at 11 polymorphic enzyme loci and significantly different from populations on corn in Puerto Rico and Louisiana at five loci. The differences found were not caused by feeding on the host plant but were inherent to the strains, then designated as rice (RS) and corn strain (CS). Subsequent investigations of the nuclear and mitochondrial genomes, using restriction fragment length polymorphism (RFLP) techniques, revealed significant differences between the two strains (Pashley, 1989, Lu et al. 1992). The discovery of a unique repeated DNA

sequence in the genome of RS individuals (Lu et al. 1994) provided further evidence of

the genetic separation of the strains. More recently, Lu et al. (1996) developed an easier

and faster technique, allowing the identification of mtDNA restriction fragments from

total DNA.

Host preferences and specializations

The larval host has greater impact on the development of RS than on that of CS,

with the former having a significantly higher growing rate, and enhanced surviving rate

in Bermudagrass and rice plants (and pastures in general) than in corn fields, while CS

does not show differential development (Pashley et al., 1995). These findings support

the previous view of CS as a more generalist feeder (Pashley, 1988b) and suggest that

larvae physiology might have been a factor of specialization in RS (Whitford et al.

1988; Pashley, 1988a), since growing faster allows RS larvae to shorten their period of

vulnerability to predators and parasites. There were almost no larvae (2%) from CS

(larvae exhibiting the corn mtDNA) in RS habitats (pastures), and 18% of RS larvae

(larvae exhibiting the rice mtDNA) were found in CS habitats (Pashley, 1989).

Altogether, these reports can be seen as indicators of adaptation to this particular plant.

Populations living in allopatry and sympatrically

The geographic distribution of the two strains is documented by Pashley (1986,

1988b), with RS occurring sympatrically with CS, often in neighboring fields, in Puerto

Rico, Southern Florida, Georgia, and Louisiana. Reported attacks on forage grasses throughout much of Latin America are likely due to the presence of RS (Pashley, 1988b).

Evidence of possible differences in female sex pheromone

Using laboratory bioassays, Sekul and Sparks (1967) first identified (Z)-9- tetradecen-1-ol acetate (Z9-14:Ac) as a pheromone component of FAW females. Almost a decade later, Sekul and Sparks (1976) isolated and identified a second compound in females, (Z)-9-dodecen-1-ol acetate (Z9-12:Ac). Later, a third compound was identified from female glands as (Z)-11-hexadecen-1-ol acetate (Z-11-16:Ac) (Descoins and co- workers, in Tumlinson et al.1986). The effectiveness of these three compounds alone or in combinations was tested by several authors, and that of Z9-12:Ac alone seemed to be higher than any blend of these three components (Mitchell et al. 1983). However,

Tumlinson et al. (1986) later concluded that Z9-12:Ac was not in fact a component of the female FAW sex pheromone, despite its attractiveness to males. They identified as components the following chemicals: dodecan-1-ol acetate, (Z)-7-dodecen-1-ol acetate,

11-dodecen-1-ol acetate, (Z)-9-tetradecenal, (Z)-9-tetradecen-1-ol acetate, (Z)-11- hexadecenal, and (Z)-11-hexadecen-1-ol acetate. Field tests revealed that for optimum activity, both (Z)-7-dodecen-1-ol acetate and (Z)-9-tetradecen-1-ol acetate were required and that this blend was more effective as a lure than either virgin females or 25mg of Z9-

12:Ac.

The majority of females tested by Tumlinson et al. (1986) were obtained from larvae reared in laboratory on artificial diet and although they do not indicate from which

host the founders of the colony were collected, later additions to the colony were

collected from sorghum plants. They studied FAW female sex pheromone using extracted

glands and volatile chemicals (collected from females placed in aeration chambers). In

gland extracts, they found considerable variability in both ratios and total quantities

between laboratory-reared and wild females and also among batches of wild females

collected in different locations. Pashley et al. (1992) conducted an experiment in the field

in which caged virgin females of each strain were used to attract males. Although roughly

60% of the attracted males belonged to the same strain as the female they approached,

this difference may not be principally due to different female pheromonal blends, since

the authors also found that female calling times in the laboratory differed between the

two strains.

Evidence of partial allochronic isolation

Indeed, Pashley et al. (1992) found a very narrow common mating period between the two strains, with CS copulating earlier in the night than RS. The authors suggested CS to be a more temperate-adapted population (occurring in highland regions of Central America and northward into Canada) and RS to be a tropical-adapted one, which has only been found in tropical, subtropical and southern temperate regions. The differences in diel periods of mating activity are consistent with this hypothesis. These different patterns might contribute to isolation of the two strains. However, and despite

30 the temporal asynchrony of nocturnal mating activities, a certain degree of overlap in mating period exists and could permit encounters between individuals of the two strains.

Moreover, the laboratory experiments of Pashley et al. (1992) suggested that males may be strongly influenced by female behavior and more flexible in their mating times.

Evidence of hybrid disadvantage in inter-strain crosses

There were but a few reports on the mating system of FAW under laboratory conditions, most without reference to the two strains, although their existence has been known since Pashley’s (1986) study. ; Pashley and Martin (1987) reported unidirectional pre-reproductive incompatibility between the two strains (i.e., CS females did not mate with RS males), whereas Whitford et al. (1988) did not find such incompatibility, and reported successful mating in 80-100% of crosses between CS females and RS males and

77-100% in the reciprocal cross). They also found a high percentage (ca. 90%) of eggs hatched in the interstrain crosses. However, in the F1 generation, the same authors found

45-73% fewer mating pairs in the crosses between F1 female (RS female x CS male) x F1 male (RS female x CS male), than in crosses between F1 female (CS female x RS male) x

F1 male (CS female x RS male). The hatching percentage was 74% and 96%, respectively.

Evidence of male-eversible structures

Other studies on the mating of FAW focused on frequency or duration of mating, and on the influence of variables such as temperature on its occurrence (Simmons and

Marti, 1992), or the impact of male sterilization techniques on male ability to compete with non-sterilized males (Young et al. 1968). None of these studies included a detailed description of the mating sequence, and although male scent structures have been long described for this species (Eltringham 1927), no studies have attempted to determine their possible role in courtship. An analysis of courtship and possible male pheromones is important given that females prefer to mate assortatively. Pashley et al. (1992) reported

that 85% of females chose to mate with males of the same strain), but the basis of this

female discrimination has yet to be determined. Finding that these male structures are

implicated in female choice would also provide evidence for an adaptive origin of male

pheromones, given the potential for cross-mating in the field between two strains that are

genetically compatible but differentially adapted in their host range.

In this study, I sought to determine the basis for female discrimination between males of the two strains through a detailed comparative analysis of intra- vs. inter-strain courtship sequences.

2. MATERIALS AND METHODS

Insect source

Individuals of S. frugiperda used in this study came from the corn- and rice-strain colonies, established at the Ohio Agricultural Research and Development Center, Ohio, during 1998. The larvae were reared on artificial diet (Southland Products Inc.,

Arkansas), segregated by sex at the pupal stage, and maintained as adults on a light cycle of 14:10 L: D, 26±1ºC, 70-80%, with synchronized calling periods, on a hydrated honey- beer mix solution, ascorbic acid enriched diet.

Ethogram construction of Spodoptera frugiperda courtship

Ad libitum observations were initially conducted to identify and describe male and female courtship behaviors, using individuals of both strains, to produce a courtship behavioral catalog for the species. These observations were later followed by systematic observations.

Males and females, 2-6 days old, were brought from their respective environmental rearing chambers 1 hour before the beginning of the experiment, and kept separately in individual 15 ml plastic containers, in different rooms, at 30±1°C, 80±10%

R.H. During the first hour of scotophase, a 25W yellow light bulb was on in both rooms to simulate evening, then switched to an equivalent red light.

Courtships were initiated by introducing a male in a glass cage arena with wired

mesh tops, containing a calling female. Laminar airflow of 0.08-0.15m/s was forced

through the arena by a fan connected to an activated charcoal filter and then evacuated from the building by an exhaust fan. Recordings were made with a Sony Digital 8® DCR-

TRV310 NTSC model video camera equipped with a Nightshot® function and a 360X

zoom. The first three courtship sequences of each pairing were analyzed frame by frame,

with behaviors transcribed using the catalog mentioned above. These sequences were

produced by coding the behavior of one sex, followed by the behavior of the other sex,

generating a male-female-male-etc. behavioral chain. Two- and three-dimensional

transitional matrices were constructed for each sex, which were submitted to Information

Theory analysis for quantification of intra- and intersexual communication (Losey, 1978).

A total of 130 courtships were recorded: 41 between CS females and CS males,

37 between RS females and RS males, 24 between CS females and RS males, and 28

between RS females and CS males.

Information Theory analysis

The basic unit of measure for Information Theory is the uncertainty or diversity of

certain events in a set of observations (Losey 1978). The more uncertain an act, the more

information that is transmitted when it is expressed (measured as bits per act). If there are

many events that are all common, uncertainty, diversity, and information content of the

set are high. If only a few events are common and the others are rare, then the

information content is low.

Information content is quantified by the Shannon and Weaver (1949) equation:

H(r) = log2 N – 1/N ∑ ni log2 ni , (1)

where H(r) is an estimate of the information contained in the system r, with N being the total number of events and ni the number of times the ith behavior was exhibited. H values can range from zero, which means the system has only one possibility (no information), to log2 ni were all n possibilities are equally likely.

The Information Theory approach in this study was similar to that of Phelan and

Baker (1990), which was based on the model of Van den Bercken and Cools (1980). In

this approach, the uncertainty of response in courtship sequences r was partitioned into

the effect due to preponse (p, the behavior of the individual prior to its response), the

effect due to the signal behavior of the other sex (s), a statistical interaction effect, and an

error variance component. Equation (1) was then transformed into a three-dimensional

function,

i j k

H(p, s, r) = log2 N – 1/N ∑ ∑ ∑ nijk log2 nijk , (2)

which estimates the uncertainty in the behavioral triad: p, s, and r, where nijk is the number of occurrences of the behavioral sequence ijk. The degree to which the preponse is correlated to the response can be measured according to the equation:

T(p: r) = H(p) + H(r) – H(p, r) , (3) where T(p:r), is an estimation of the information transmitted from p to r, and H(p,r) the joint variability in the behavioral dyad: p, r. T(p:r) is referred to as autocovariability, and represents the influence that an individual’s act has on its subsequent act.

Similarly, the influence an individual’s act has on the behavior of another individual, here referred to as crosscovariability, can be estimated as:

T(s:r) = H(s) + H(r) – H(s,r) . (4)

An estimate of the total sequential covariability, which is the amount of correlation between r and both p, and s can be obtained by

T(p,s:r) = H(r) + H(p,s) – H(p,s,r) . (5)

An interaction covariability component in the system can also be estimated by subtracting autocovariability and crosscovariability from total sequential covariability:

P(p,s:r) = T(p:r) + T(s:r) – T(p,s:r) (6)

A negative interaction covariability value indicates that knowledge of the particular p, s sequence reduces uncertainty of response more than the sum of these two effects, whereas a positive value indicates some correlation between the two, making it more difficult to determine which is responsible for the response. Given that both autocovariability and cross-covariability share the interaction component in their measure, subtracting it from both terms will give a non-confounded measure of the individual’s effect on its own behavior – partial autocovariabilty (P(p:r)), and a true measure of the communication between individuals – partial cross-covariability (P(s:r)), respectively:

P(p:r) = T(p:r) – P(p,s:r) (7)

P(s: r) = T(s:r) – P(p,s:r) (8)

In this study, all partial covariabilities were normalized to total sequential covariability to determine the relative contribution of each to total covariability; total sequential covariability itself was normalized to H(r). The portion of H(r) that could not be attributed to T(p,s:r) was the residual variance.

All covariabilities were statistically analyzed for deviation from zero using

Miller’s Chi-square (Losey 1978). Significant cross-covariabilities were subdivided to

determine each individual behavioral contribution of the emitting sex to the respondent

sex using the weighted-average method [p(x)J(x;Y)] of Steinberg (1977). This equation

represents the product between the probability of occurrence of the signal x (px) and the

overall probability of the response Y given the occurrence of the signal x (x;Y= x1;y1+ x1;y2+…x1;yn), with y1 to yn being the behavioral repertoire of respondent).

3. RESULTS

3.1. Description of Spodoptera frugiperda Courtship

The courtship of Spodoptera frugiperda began as the male approached a

pheromone-emitting female from behind, either flying or walking, while displaying the

genital hairpencils. When walking, the male kept fluttering the wings as if preparing to

take off. Once behind the female, the male touched the female abdominal tip or wings,

and moved forward and parallel to the female body, still fluttering the wings and

displaying the genital hairpencils, while touching the female body with the antennae and

the foreleg. The female usually responded to the male contact by lifting the wings and

lowering the abdomen while retracting the pheromonal gland. At this point, the CS x CS

courtship diverged from that of RS x RS courtship. In the former, the male usually bent

and lifted the abdomen above the female wings and brought it to her head and antennae

before attempting copulation. In the latter, the male usually went directly to touching and

grasping the female abdominal tip. In both strains, the males opposed the females after

grasping the female abdomen with the claspers. When the copulatory attempt was

unsuccessful, the courtship sequence was usually repeated.

3.2. Ethogram of Spodoptera frugiperda courtship

The catalog of behaviors expressed was the same for corn and rice strains.

Moreover, one-way ANOVA measured no significant difference between the two strains

in the frequency distribution of behaviors for either females (F1,12=0.121; p=0.734) or males (F1,18=0.456; p=0.508)

Behaviors displayed by both sexes

Fanning (FAN)

Several minutes after the red light was on, females were either resting perched on

the container surfaces or wandering inside it, walking and flying. When at rest, they were

sometimes observed vibrating their wings in quick motion, keeping them parallel to the substrate, in a way that resembles a fan. Fanning usually preceded a small displacement

or the onset of calling. Sometimes they walked while fanning and calling simultaneously.

Fanning behavior was also observed in males, though less often.

Behaviors displayed by females

Calling (CAL)

A pheromone-emitting female usually perched on a container vertical wall, with the head pointed up, antennae forward, wings slightly raised, laterally spread, or less frequently held above the body in a V shape, abdomen either parallel to the substrate or slightly bent down, the pheromone gland between the 8th and 9th abdominal segments, exposed ventrally.

Spread Wings (SPW)

As above, wings were spread laterally, eventually slightly lifted, the abdomen was either bent down or parallel to the ground and the pheromone gland was retracted.

V-shape position of the wings (VSW)

This posture involved two simultaneous movements from an initial calling posture. The distal wing margin was raised while the apical margin was lowered, bringing the upper external surfaces of the wings to a diagonal, thus giving the wings the appearance of a V. At the same time, the abdomen was lowered, bent forward, and the gland was retracted, producing a 45° angle with the wings. This posture could revert to either a calling posture (CAL) or wings spread with gland retracted (SPW).

Fluttering the wings (FLF)

This behavior was characterized by a continuous vibration of the wings as if preparing to take off, usually with retraction of the pheromone gland. It sometimes followed intromission by the male.

Moving away (AWA)

Female displacement away from the male could occur either by walking or taking flight, gland retracted, usually following a discrete and fast vertical movement of wings

(flicking). It could be exhibited as a response to a male pre-copulatory behavioral signal, following male approach, or to a male copulation attempt (sometimes females simply drop from the surface of the glass cage). In some cases after having their genitalia clasped, females responded by shaking the males off by walking away in the opposite direction dragging the male or flying away.

Plate 1. Female calling (CAL), antennae directed forward (top view), with wings spread laterally (bottom view, lateral perspective).

Plate 1

Lifting the body and moving the abdomen away from male genital contact

(LBO)

Females stretched the hind legs and lifted their bodies, while doing vertical movements with the abdomen in the direction opposite to that of the male genital movement. Usually the female wings were kept spread and parallel to the body.

Kicking the male abdomen (KICK)

The female kicks the male abdomen with the hind legs, a behavior often associated with LBO, usually during a male copulation attempt, or after intromission, when the male proceeded to the rotation that ended in a tail-to-tail posture.

Behaviors displayed by males

Following introduction to the arena, males oriented their antennae forward, vibrated the antennae, and periodically fluttered the wings. They then raised their bodies, and while protruding the claspers, displayed the genital hairpencils. With their wings spread, they clapped their valvae, and flexed the abdomen. Subsequently, males vibrated their wings continuously and took off.

Approach (ADH)

Male displacement towards a female (usually a calling female) occurred either by flying or walking, often with the genital hairpencils extruded. Males usually flew briefly upwind, often hovered over the females, and then landed close to them, usually from behind. When males approached females while walking and showing genital hairpencils, they often fluttered their wings as if preparing to take off.

Hovering and touching female wings (HTW)

Following an approach in flight, the male hovered above the female, usually displaying his genital hairpencils, and touching the female wings with his forelegs.

Moving away (AWA)

Males sometimes moved away from the female, either by walking or flying.

Standing behind female (STB)

Following approach from behind, males stood close to females, fluttering the wings, often displaying the genital hairpencils.

Touching the female abdominal tip or wings (TFB)

Fluttering the wings behind the female, usually displaying the genital hairpencils, the male stretched one foreleg, exposing the femoral hairpencils and touched the female abdominal tip or wings with it. This behavior generally followed antennation of the female abdomen and ventral surface of wings.

Moving forward to parallel female (MFS)

Usually following TFB, the male moved forward and parallel to the female body, displaying the hairpencils, and laterally touching the female abdomen with the antennae or inner foreleg, and the ventral surface of the female wings with the antennae.

Standing on female side, while displaying the hairpencils (STL)

The male might stand side by side with female while displaying the hairpencils

(femoral and/or genital).

Pushing up female wings with abdomen (AbU)

While siding with the female and fluttering, the male bends the abdomen laterally, elevating it above the female abdomen and below her wings while pushing the wings up.

This behavior involves displaying the genital hairpencils, and frequently the femoral hairpencils.

Plate 2. A mating sequence in the corn strain of Spodoptera frugiperda.

A-Male approaching female flying and displaying genital brushes (ADH);

B-Male standing behind female fluttering and continuing to display the genital brushes (STB);

C-Male moving forward and touching female from behind (TFB), while displaying the genital brushes, and the female showing wings lifted in a V-shape fashion (VSW), with gland retraction;

D-Male moving forward to parallel the female (MFS), keeping genital brushes out;

E-Male standing on female side still displaying (STL);

F Male siding with female and displaying genital brushes and femoral hairpencils to female’s head (AbH);

G-Male attempting copulation, while keeping the femoral hairpencils above female head (CAT);

H-Male opposing female (TT) and achieving the tail-to-tail posture.

Plate 2

Moving the abdomen above the female wings while displaying the hairpencils

(AbW)

While siding with the female and fluttering, the male displays his genital hairpencils and moves the abdomen above the female wings, sliding it back and forth.

Often this behavior involves raising the foreleg showing the femoral hairpencils.

Bring the genital hairpencils to female head or antennae (AbH)

Still fluttering and following AbW, the male bends the abdomen forward and with a sudden movement strikes the female head and/or antennae with the genital hairpencils.

Often the male keeps the inner foreleg raised, exposing the femoral hairpencil.

Male attempt to copulate (CAT)

Still siding with the female, fluttering and displaying the genital hairpencils, the male moves the abdomen towards the female abdomen, touching the tip and attempting intromission. If in the course of displaying the genital hairpencils, the male also had the foreleg raised, at this final stage he has already lowered it.

Male opposing (TT)

Following a copulation attempt, the male moved to the typical lepidopteran mating position, with heads in opposite direction. A mating attempt is considered

successful when following intromission (in the tail-to tail position), the couple remains

in that posture from 1 minute up to several hours. Successfully attaining the tail-to-tail

position thus is not equivalent to a successful mating – unless the position is maintained

for at least one minute, since those couples that stayed this long are generally those that

also stay for hours and a copula of about 30 minutes seems to be the minimum necessary

for FAW males to effectively pass sperm (Martin et al., 1989).

3.3. Courtship success within and between strains

Analysis of courtship success in intra-strain (CS female x CS male or RS female x

RS male) and inter-strain (CS female x RS male or RS female x CS male) pairings

showed that only a small percentage (8%) of RS males succeeded in their attempts to

mate with CS females. This success rate contrasted with that of CS males (75%) that

mated with RS females. A Chi-square analysis of these courtships also revealed

associations between some pairing types and mating success (Table 1) (X2(6df; N=130) =

32.4; p<0.001).

Considering adjusted standard residuals >2 as significant row x column associations (after Everitt, 1977), analysis showed that: 1) the frequency of pairs in which the males failed to oppose females was significantly higher than expected for within-CS pairings and CS females x RS males, but significantly lower than expected for RS females x CS males; 2) the frequency of pairs in which the tail-to-tail lasted less than 1

51 minute was lower than expected for within-CS pairings, but higher than expected for CS females x RS males; and 3) the frequency of pairs in which the tail-to-tail lasted at least 1 minute was higher than expected for RS females x CS males, and lower than expected for

CS females x RS males. These differences in courtship success in the four pairing types showed that although the two strains share the same behavioral repertoire, at least some of these behaviors must have different signal value in the two strains.

Plate 3. Male siding with female, fluttering and displaying genital hairpencils and femoral hairpencils to female head (AbH).

Plate 3

Unsuccessful Courtships Successful Courtships

Male failed to Tail-to-tail lasted Tail-to-tail lasted at least 1 min. Pairing oppose female <1 min. (frequency) (frequency) (frequency)

CS X CS 39% (16)** 10% (4)* 51% (21)

CS X RS 46% (11)** 46% (11)** 8% (2)*

RS X RS 22% (8) 32% (12) 46% (21)

RS X CS 4% (1)* 21% (6) 75% (21)**

Table 1. Mating outcomes of pairings between females and males of the two host strains of Spodoptera frugiperda. **/* Frequency higher/lower than expected, considering |adjusted standard residuals| > 2 as significant row x column associations (after Everitt 1977).

3.4. Information Theory analysis

3.4.1. Intrastrain Courtship Analysis

CS x CS Courtships

The three-dimensional information-theory model successfully partitioned the components of variance in CS x CS courtships; knowledge of the preponse and signal brought about a 45% reduction in the uncertainty of female behavior (Table 2). Female response was significantly influenced by both the preponse and the signal although

55 autocovariability was substantially greater than cross-covariability [67 vs 33% of the total sequential covariability- T(p,s:r)]. With CS males, a similar reduction in H(r) was accounted for by these two factors; however, male-to-female information transmission was greater than female-to-male transmission. Of the total covariance in male courtship behavior, 78% was due to autocovariability, and only 21% was due to the effects of female behavior on the male. The male-to-female information transmission also was greater than female-to-male transmission in CS x RS, RS x RS, and RS x CS pairings.

Responder Courtships N H(r) T(p,s:r) P(p: r) P(s: r) P(p,s: r) Corn Strain 41 774 Female 2.28 1.02* 0.69* 0.34* 0.00ns (45%)b (67%)c (33%) Male 3.21 1.49* 1.17* 0.31* 0.01ns (47%) (78%) (21%) Rice Strain 37 888 Female 2.32 0.92* 0.53* 0.45* -0.05ns (40%) (58%) (48%) Male 3.00 1.18* 0.87* 0.39* -0.08ns (39%) (73%) (33%)

Table 2. Information theory parameters measured from pooled intraspecific courtships of Spodoptera frugiperda corn strain (CS) females and CS males, and rice strain (RS) females and RS males using the triad: preponse (p), signal (s), and response (r) a. Transmission values marked with an ‘*’ are significant by Miller’s chi-square (p<0.05). aN = number of behavioral dyads, H(r) = individual behavioral variance of responder. T(p,s:r) = total sequential covariance. P(p:r) = partial auto-covariability (intraindividual). P(s:r) = partial cross-covariability (interindividual). P(p,s:r) = interaction covariability. bTotal sequential covariability normalized with respect to response variance. cAll partial covariabilities normalized with respect to total sequential covariability.

Subdividing cross-covariability for female response indicated that the copulation attempt had the greatest signal value, contributing more (32%) than any other male behavior to female response (Table 3). Females had a 0.29 probability of responding to this behavior by fluttering the wings and 0.19 probability of spreading the wings.

Females had only a small probability of 0.06 of responding to the copulation attempt by lifting their body and moving the abdomen away from male genital contact.

Moving the abdomen above the female wings while displaying the hairpencils was the fourth greatest contributor (12%) to cross-covariability for female response in CS x CS courtships. Forty-nine percent of the time the male moved the abdomen above the female wings while displaying the hairpencils the female responded by spreading the wings. Females had also a 0.39 probability of responding to this male behavior by keeping the V-shape position of the wings.

Female-to-male communication was dominated by the V-shape position of the wings with 31% of male cross-covariability due to this signal (Table 4). Males had a 0.20 probability of responding to this female behavior by moving the abdomen above the female wings while displaying the hairpencils, and a 0.18 probability of responding by moving parallel to the females while displaying the hairpencils and touching the female body laterally. Fourteen percent of the time that the female held the wings in a V-shape position, the male responded by bringing the hairpencils to the female head. Males also had a 0.15 probability of responding by attempting copulation.

Female response a b c Courtship Male signal AWA CAL FLF KICK LBO SPW VSW ALL Px Lx=PxJx;Y %=Lx/T(s:r) d CS x CS CAT 0.20 0.04 0.29 0.20 0.06 0.19 0.01 1.00 0.10 0.14 32 ADH 0.03 0.57 0.01 0.00 0.00 0.38 0.00 1.00 0.12 0.07 16 STB 0.06 0.44 0.02 0.00 0.00 0.44 0.03 1.00 0.18 0.05 13 AbW 0.00 0.08 0.04 0.00 0.00 0.49 0.39 1.00 0.07 0.05 12 AbU 0.06 0.04 0.11 0.00 0.02 0.40 0.36 1.00 0.07 0.04 9 AbH 0.00 0.04 0.04 0.00 0.00 0.46 0.46 1.00 0.04 0.03 8

CS x RS CAT 0.12 0.09 0.19 0.17 0.36 0.03 0.05 1.00 0.17 0.15 41 ADH 0.00 0.76 0.00 0.00 0.00 0.24 0.00 1.00 0.08 0.07 20 STB 0.10 0.56 0.01 0.00 0.01 0.27 0.04 1.00 0.12 0.04 12 AbW 0.14 0.43 0.00 0.00 0.00 0.14 0.29 1.00 0.01 0.01 2 AbU 0.11 0.25 0.14 0.02 0.03 0.19 0.25 1.00 0.11 0.02 7 AbH 0.00 0.50 0.50 0.00 0.00 0.00 0.00 1.00 0.00 0.00 1

RS x RS CAT 0.17 0.02 0.18 0.15 0.09 0.36 0.04 1.00 0.19 0.13 32 ADH 0.03 0.72 0.01 0.00 0.00 0.24 0.00 1.00 0.11 0.11 29 STB 0.03 0.46 0.00 0.00 0.00 0.44 0.07 1.00 0.07 0.03 8 AbW 0.03 0.17 0.06 0.00 0.03 0.19 0.53 1.00 0.04 0.02 5 AbU 0.08 0.00 0.00 0.00 0.00 0.12 0.81 1.00 0.03 0.04 11 AbH 0.08 0.17 0.00 0.00 0.08 0.42 0.25 1.00 0.01 0.00 1

RS x CS CAT 0.14 0.04 0.27 0.10 0.27 0.18 0.01 1.00 0.09 0.08 30 ADH 0.00 0.79 0.02 0.00 0.00 0.19 0.00 1.00 0.07 0.06 21 STB 0.12 0.50 0.02 0.00 0.01 0.32 0.02 1.00 0.16 0.05 19 AbW 0.09 0.09 0.06 0.00 0.22 0.41 0.13 1.00 0.04 0.01 5 AbU 0.06 0.25 0.13 0.01 0.10 0.27 0.18 1.00 0.10 0.01 4 AbH 0.09 0.00 0.27 0.00 0.18 0.45 0.00 1.00 0.01 0.01 4

Table 3. Male-to-female information transmission. Male’s behaviors showing greater contribution to female response. aPx - Observed probability of occurrence of male behavior on row x. bLx=PxJx;Y – Partialized information: contribution of male signal on row x to male-to-female information transmission. c%=Lx/T(s:r) - Partialized information normalized to male-to-female information transmission. See text for explanations. d Probability of occurrence of female response given the occurrence of male signal on row x.

Calling was the second most important behavior affecting male response, with

23% of male cross-covariability due to this signal. Thirty-one percent of the time when the female was calling, the male responded by touching the abdomen or the wings with the antennae and forelegs while displaying the genital hairpencils. Males had a 0.22 probability of responding to a calling female by standing behind her fluttering the wings and displaying the genital hairpencils, and a 0.17 probability of responding by moving parallel to the female while displaying the hairpencils and touching the female body laterally.

RS x RS Courtships

The three-dimensional information-theory model successfully partitioned the components of variance in RS x RS courtships as well; knowledge of the preponse and signal reduced the uncertainty of female behavior by 40% (Table 2). Female response was significantly influenced by both the preponse and the signal, but the effects of these factors on RS female response differed from those observed on CS female response. The preponse influenced the female response less [58 % of T(p,s:r)], whereas the effect of

Male response Courtship Fem Signal AbH AbU AbW ADH AWA CAT HTW MFS STB STL TFB TT ALL P(x)a Lx=PxJx b %=Lx/Ht c CS x CS VSW 0.14 d 0.11 0.20 0.00 0.00 0.15 0.00 0.18 0.07 0.13 0.02 0.00 1.00 0.14 0.10 31 CAL 0.00 0.00 0.01 0.00 0.06 0.06 0.06 0.17 0.22 0.10 0.31 0.00 1.00 0.26 0.07 23 AWA 0.00 0.01 0.01 0.00 0.21 0.06 0.01 0.08 0.31 0.17 0.13 0.00 1.00 0.10 0.05 14 FLF 0.02 0.02 0.03 0.00 0.06 0.08 0.00 0.11 0.16 0.24 0.08 0.19 1.00 0.08 0.04 12 KICK 0.00 0.00 0.00 0.00 0.04 0.07 0.00 0.04 0.19 0.33 0.07 0.26 1.00 0.03 0.04 11 LBO 0.00 0.17 0.00 0.00 0.00 0.17 0.00 0.00 0.17 0.50 0.00 0.00 1.00 0.01 0.01 3

CS x RS VSW 0.02 0.21 0.04 0.00 0.00 0.27 0.00 0.14 0.09 0.11 0.13 0.00 1.00 0.09 0.03 10 CAL 0.00 0.09 0.01 0.01 0.04 0.13 0.02 0.17 0.13 0.16 0.22 0.00 1.00 0.36 0.03 11 AWA 0.00 0.07 0.00 0.00 0.16 0.05 0.00 0.13 0.25 0.27 0.04 0.02 1.00 0.09 0.04 14 FLF 0.00 0.08 0.01 0.00 0.06 0.20 0.00 0.08 0.23 0.27 0.06 0.01 1.00 0.12 0.02 6 KICK 0.00 0.00 0.00 0.00 0.05 0.05 0.00 0.00 0.09 0.41 0.05 0.36 1.00 0.04 0.06 22 LBO 0.00 0.06 0.02 0.00 0.00 0.45 0.00 0.05 0.02 0.37 0.03 0.02 1.00 0.11 0.07 25

RS x RS VSW 0.02 0.12 0.08 0.00 0.02 0.21 0.01 0.25 0.05 0.20 0.05 0.00 1.00 0.19 0.06 18 CAL 0.01 0.00 0.03 0.02 0.02 0.10 0.07 0.20 0.12 0.12 0.31 0.00 1.00 0.20 0.07 23 AWA 0.00 0.00 0.01 0.00 0.04 0.09 0.00 0.12 0.41 0.28 0.01 0.03 1.00 0.08 0.06 19 FLF 0.00 0.00 0.00 0.00 0.05 0.37 0.00 0.07 0.05 0.39 0.03 0.03 1.00 0.07 0.04 11 KICK 0.00 0.00 0.00 0.00 0.14 0.09 0.00 0.03 0.09 0.31 0.03 0.31 1.00 0.04 0.05 16 LBO 0.00 0.00 0.08 0.00 0.00 0.29 0.00 0.04 0.00 0.54 0.04 0.00 1.00 0.03 0.02 7

RS x CS VSW 0.03 0.26 0.12 0.00 0.00 0.04 0.00 0.12 0.06 0.24 0.13 0.00 1.00 0.08 0.04 15 CAL 0.00 0.08 0.02 0.00 0.01 0.04 0.02 0.19 0.15 0.08 0.41 0.00 1.00 0.33 0.07 27 AWA 0.00 0.03 0.01 0.00 0.09 0.09 0.00 0.06 0.43 0.11 0.13 0.05 1.00 0.09 0.04 16 FLF 0.02 0.06 0.02 0.00 0.02 0.11 0.00 0.06 0.27 0.27 0.06 0.11 1.00 0.12 0.05 18 KICK 0.00 0.00 0.00 0.00 0.07 0.33 0.00 0.00 0.13 0.40 0.00 0.07 1.00 0.02 0.02 9 LBO 0.01 0.12 0.08 0.00 0.01 0.25 0.00 0.07 0.10 0.23 0.08 0.04 1.00 0.08 0.03 12

Table 4. Female-to-male information transmission. Female’s behaviors showing greater contribution to male response. aPx - Observed probability of occurrence of female behavior on row x. bLx=PxJx;Y – Partialized information: contribution of female signal on row x to female-to- male information transmission. c%=Lx/T(s:r) - Partialized information normalized to female-to-male information transmission. See text for explanations. d Probability of occurrence of male response given the occurrence of female signal on row x.

male behavior on the female response [48% of T(p,s:r)] was greater than that observed for CS x CS courtships (Table 2). With RS males, a reduction in H(r) was accounted for by these two factors as well. Similar to CS x CS courtships, a larger percentage of the total covariance in male courtship behavior (73%) was due to autocovariability, and a smaller percentage was due to the effects of female behavior on the male response (33% of total covariance). However, the effect of female behavior on male response was higher in RS x RS courtships than in CS x CS courtships.

As in CS x CS courtships, male-to-female communication in RS x RS courtships was dominated by the copulation attempt, with 32% of female cross-covariability due to this signal (Table 3). Thirty-six percent of the time that the male attempted copulation, the female responded by spreading the wings, twice as likely as fluttering the wings

(18%). As in CS x CS courtships, RS females also responded to the copulation attempt by lifting their body and moving the abdomen away from male genital contact with a small probability of 0.09.

In RS x RS courtships, pushing up the female wings with the abdomen while displaying the hairpencils was the third greatest contributor to cross-covariability for female response (11%). Females had a 0.81 probability of responding to this male behavior by keeping the V-shape position of the wings, and a 0.12 probability of spreading the wings. 62

Subdividing cross-covariability for male response indicated that calling had the

greatest signal value, contributing more than the V-shape position of the wings (23 vs

18%) (Table 4). Males had a 0.31 probability of responding to calling females by

touching their abdomen or wings with the antennae and forelegs while displaying the

genital hairpencils. Twenty percent of the time that the female was calling, the male

responded by moving parallel to the female while displaying the hairpencils and touching

the female body laterally. The male had only a 0.12 probability of responding to this

behavior by standing behind the female fluttering the wings and displaying the genital hairpencils (a probability lower than that observed for CS x CS courtships).

RS males differed from CS males in their response to the female V-shape

position. Twenty-five percent of the time that the female held the wings in a V-shape

position, the RS male responded by moving parallel to the female while displaying the

hairpencils and touching the female body laterally. Males also responded to this female

behavior by attempting copulation (0.21) and by standing on female side fluttering the

wings and displaying the genital hairpencils (0.20). Only 8% of the time did the male

respond by moving the abdomen above the female wings while displaying the

hairpencils.

3.4.2. Interstrain Courtship Analysis

CS x RS Courtships

As in intraspecific courtships, the behavioral variability of CS females could be partitioned to both autocovariability and cross-covariability (Table 5). The total sequential covariability of CS females courted by RS males was similar to that observed in intraspecific courtships and a similar percentage of it was due to autocovariability.

However, cross-covariability in CS females courted by RS males was greater than that seen in CS x CS courtships.

Subdividing cross-covariability for female response indicated that the copulation attempt contributed far more (41%) than any other male behavior (Table 3). Females had a 0.36 probability of responding to the copulation attempt by lifting the body and moving the abdomen away from male genital contact. This probability was 6x higher than that observed for CS females courted by CS males. CS females courted by RS males had also a smaller probability of responding to the copulation attempt by spreading the wings than

CS females courted by CS males (0.3 vs 0.19).

In CS x RS courtships, male movement of the abdomen above the female wings while displaying the hairpencils contributed less to cross-covariability for female response than in CS x CS courtships (2 vs 12%). Forty-three percent of the time thatRS males moved the abdomen above the female wings while displaying the hairpencils the 64

Responder Courtships N H(r) T(p,s:r) P(p: r) P(s: r) P(p,s: r) CS x RS 24 597 Female 2.51 1.03* 0.67* 0.47* -0.11ns (41%)b (65%)c (46%) Male 2.93 1.24* 0.96* 0.41* -0.13ns (42%) (77%) (33%) RS x CS 28 881 Female 2.40 0.88* 0.62* 0.40* -0.14ns (37%) (70%) (46%) Male 2.99 1.38* 1.11* 0.31* -0.04ns (46%) (80%) (22%)

Table 5. Information theory parameters measured from pooled interspecific courtships of Spodoptera frugiperda corn strain (CS) females and RS males, and rice strain (RS) females and CS males using the triad: preponse (p), signal (s), and response (r) a. Transmission values marked with an ‘*’ are significant by Miller’s chi-square (p<0.05). aN = number of behavioral dyads, H(r) = individual behavioral variance of responder. T(p,s:r) = total sequential covariance. P(p:r) = partial auto-covariability (intraindividual). P(s:r) = partial cross-covariability (interindividual). P(p,s:r) = interaction covariability. bTotal sequential covariability normalized with respect to response variance. cAll partial covariabilities normalized with respect to total sequential covariability.

CS females responded by resuming calling. In CS x CS courtships, females never resumed calling when the males moved the abdomen above the female wings while displaying the hairpencils.

Female lifting of the body and moving the abdomen away from male genital contact dominated female-to-male communication in CS x RS courtships (Table 4).

Twenty-five percent of male cross-covariability was due to this female signal. This

65 female behavior had less signal value in CS x CS courtships contributing only 3% to male cross-covariability. In CS x RS courtships, RS males had a 0.45 probability of responding to this female behavior by attempting to copulate and a 0.37 probability of responding by standing on female side fluttering the wings and displaying the genital hairpencils. In CS x CS courtships, CS males had a 0.17 probability of responding to this behavior by attempting to copulate, and a 0.50 probability of responding by standing on female side fluttering the wings and displaying the genital hairpencils.

RS x CS Courtships

As in intraspecific courtships, the behavioral variability of RS females partitioned to both autocovariability and cross-covariability (Table 5). The total sequential covariability of RS females courted by CS males was similar to that observed in intraspecific courtships. Although a similar percentage of it was accounted for by cross- covariability, autocovariability in RS females courted by CS males was greater than that seen in RS x RS courtships (Table 2).

Again the copulation attempt contributed more (30%) than any other male behavior to female response (Table 3). Females had a 0.27 probability of responding to the copulation attempt by lifting the body and moving the abdomen away from male genital contact, 3x more likely than that observed in RS x RS courtships. RS females

66 courted by CS males also were less likely to respond to the male copulatory attempt by spreading the wings than RS females in intra-strain courtships (0.18 vs 0.36).

Pushing up the female wings with the abdomen contributed less to cross-female covariabilty in RS x CS courtships than in RX x RS courtships (4 vs 11%). RS females were far less likely to respond to this male behavior by keeping the V-shape position of the wings when courted by CS males than when courted by RS males (0.18 vs 0.81). RS females courted by CS males also had a 0.25 probability of responding to this male behavior by resuming calling, a response never observed in RS females courted by RS males.

Subdividing cross-covariability for male response, calling contributed more

(27%) than any other female behavior (Table 4). Forty-one percent of the time that the female was calling, the male responded by touching the female abdomen or wings with the antennae and forelegs while displaying the genital hairpencils. Males had a 0.19 probability of responding to a calling female by moving parallel to female while displaying the hairpencils and touching the female body laterally. The male had also a

0.15 probability of responding to a calling female by standing behind the female fluttering the wings and displaying the genital hairpencils.

4. DISCUSSION

In both strains of S. frugiperda, females adopted a calling posture similar to that of S.

littoralis (Ellis and Brimacombe, 1980), Plodia interpunctella (Hubner) (Grant, 1976),

and Heliothis virescens (Teal et al., 1981). Their wings were slightly raised or less frequently held above the body in a V shape, with the pheromone gland exposed ventrally. Occasionally, females fanned while calling, a behavior also described for

Helicoverpa. zea (Boddie) (Agee, 1969) and Anticarsia gemmatalis (Hubner) (Leppa et al., 1987).

In the current study, as in other species, the primary releaser of male FAW reproductive behaviors was the female sex pheromone. In both strains, males responded to calling females in a way similar to that of S. exempta males, as described by

Khasimuddin (1978). Shortly after being introduced in the cages, S. frugiperda males oriented their body towards females, with their antennae forward and vibrating, periodically fluttering the wings. Then, while raising their body and protruding the claspers, they exposed the genital hairpencils at their base, clapped the valvae, and flexed the abdomen. Wing fanning is a common pre-flight response to sex pheromone among moths. Examples include: Agryrotaenia velutiana, (Baker et al., 1976), Pectinophora gossypiella (Collins and Cardé, 1989), H. zea (Agee, 1969), Phlogophora meticulosa

(Birch 1970), Prodenia eridania (Redfern et al. 1970), Heliothis virescens (Teal et al.,

1981), Pseudaletia unipuncta (Turgen et al., 1983), Phragmatobia fuliginosa and 68

Pyrrharctia isabella (Krasnoff and Roelofs, 1990). However, the extension of male claspers and exposure of hairpencils is not common to all species in which males have these structures. For example, H. virescens males do not extend or sway their abdomen

prior to take to the flight, nor do they expose the hairpencils at this time (Teal et al.,

1981). On the other hand, members of the stored-product moths (Pyralidae: Phycitinae) that possess wing glands display them as they fan the wings (Phelan and Baker, 1987).

After these pre-flight behaviors in response to the female pheromone, S. frugiperda

males vibrated their wings and took flight, approaching females from downwind, with the

genital hairpencils extended, as S. littoralis males do (Ellis and Brimacombe, 1980). This

hairpencil display differs from reports on other species in which these structures are

exposed only in close proximity of the female (e.g., Phlogophora meticulosa (Birch,

1970) and H. virescens (Teal et al, 1981)). Spodoptera frugiperda males seldom found

females at first attempt, often alighting while exposing the genital hairpencils before

taking off again or simply walking towards females, while fluttering and continuing to

display.

It has been suggested that the exposure of male hairpencils prior to landing

reinforces female quiescence, either by inhibiting female flight, such as in P. meticulosa

(Birch 1970), by inducing an overt response on the female (e.g. Plodia interpunctella

(Phelan and Baker, 1987)), or both (e.g. Mamestra brassicae (Poppy and Birch, 1994)),

where females respond by remaining stationary and curving their abdomen towards the

male genital brush-bearing claspers. In several Noctuidae, an overt female response

consists of lifting the wings in a V shape and retracting the pheromone gland (e.g., H.

virescens (Teal et al. 1981), H. subflexa (Cibrian-Tovar and Mitchell, 1991), and S.

littoralis (Ellis and Brimacombe, 1980)). Despite the male hairpencil display during

flight and before landing, S. frugiperda females responded only when touched. This

response differs markedly from that of S. littoralis females, whose response is elicited

simply by the male genital hairpencil display, while hovering above the female head and

thorax (Ellis and Brimacombe, 1980). Showing readiness to mate by raising the wings in

response to antennation on the female abdominal tip is also known outside the

Lepidoptera, as in Agapetus fuscipes (Trichoptera: Glossosomatidae) (Ivanov and

Rupprecht, 1992).

In H. subflexa, where the female response can also be elicited without prior physical contact, touching the female abdomen with the antennae reinforces receptivity, eliciting further lifting of the wings and parallel alignment between the female abdomen and the male abdomen. Given that females reject most of the males that fail to do so, this male behavior is considered a critical behavioral step in mate recognition (Cibrian-Tovar and

Mitchell, 1991).

In S. frugiperda courtships, males were often observed pushing up the female wings with the abdomen while displaying both sets of hairpencils. A somewhat similar male behavior, with the male foreleg coming in close proximity to the female head (after the male positions himself parallel to the female), was observed in H. virescens (Teal et al.,

1981). In other instances, the males were seen moving the abdomen above the female wings while displaying the hairpencils and bringing the genital hairpencils close to the head and antennae of females (often both sets of hairpencils). The genital hairpencil display resembles that of hybrid males (from H. subflexa females X H. virescens males) towards H. subflexa females, and also the head-thump of Ephestia elutella males

(Pyralidae) (Phelan and Baker, 1990). In the latter, the male behavior induces a change in

female behavior that facilitates the male’s attempt to copulation, thus enhancing the male

probability of success.

The present study showed the existence of male mating differential success in intra- versus inter-strain courtships in S. frugiperda. Whitford et al. (1988) reported a 0.8-1.0

probability of success in inter-strain courtships. The present study found a similar

probability of success in RS x CS crossings (0.75), but a much smaller probability of

success was found in CS x RS crossings (0.08). These observations were more consistent

with those of Pashley and Martin (1987) who reported RS females mating with CS males,

but not CS females mating with RS males. Based solely on the outcome of courtships, the

present study confirmed a certain degree of assortative mating between CS females and

CS males, while RS females seemed to show a preference for CS males compared to

males of their own strain. In the later, however, the probability of success does not reflect

a behavioral preference by RS females, as success may have been greatly determined by

the male as one can infer from the detailed behavioral observations.

Premature breaking of genital contact was observed in S. frugiperda courtships in inter- as well as in intra-strain courtships. Premature breaking of genital contact also has been reported for S. littoralis in courtships with antennaless females (Ellis and

Brimacombe, 1980), and for Pseudaletia unipuncta in courtships with brushless males

(Fitzpatrick and McNeil, 1988). As reported for P. unipuncta, S. frugiperda females managed to free themselves from the grasp of males by walking away in the opposite direction, dragging the male, or even flying away.

The information analysis conducted in the present study confirmed the existence

of an asymmetry in the response between the sexes in S. frugiperda, consistent with the

predictions of Phelan’s (1992) asymmetric tracking model. As expected, in both intra-

and inter-strain courtships, the influence of female behavior on male response (female-to-

male information) was lower than the influence of male behavior on female response

(male-to-female information). Male autocovariability was also higher than female

autocovariability.

For both within- and between-strain courtships, male behaviors having greater

contributions to female cross-covariability were either at the beginning - approaching the female - or at the end of the courtship - attempting to copulate. Male attempt to copulate was both the greatest contributor and the most informative. In both inter-strain courtships, the females were more likely to respond by lifting the body and moving the abdomen away from male genitalia than in intra-strain courtships. This was particularly evident in

CS x RS pairings. This avoidance behavior was similar to that observed by Cibrian-Tovar and Mitchell (1991) in unsuccessful courtships of H. subflexa, where females would reject their suitors by curving the abdomen upward and adopting a resting posture (i.e.,

wings covering the abdomen).

In CS x RS crosses, female behaviors that had the greatest contributions to male

response were lifting the body and moving the abdomen away from male genitalia and kicking the male’s abdomen. RS male response to kicking was more often opposing than

keeping the attempt to copulate, contrasting with the opposite response of CS males

(more often maintaining attempts to copulate than opposing the female). This was

probably related to the outcome of these courtships - most of the time that males achieved

the tail-to-tail posture while being kicked, the courtships ended in a premature breaking

of genital contact. It is possible that the courtship success of CS males with RS females is

an artifact of the small observation arena used in this study. The CS males could be

inherently stronger than RS males, and thus better able to clasp the female genitalia and

overcome female resistance to mate. This would be consistent with the fact that CS larvae

and pupae are heavier on average than RS larvae and pupae, when reared in the same

type of host (Whitford et al., 1988). RS pupae and adults in the current study were also

visibly smaller than those of CS, although no measurements were made. Perhaps outside

such confinement, CS x RS courtships would be less likely to end in copulation.

Two male behavior patterns with lower contributions to female cross-covariability

may in fact have been crucial in maintaining female receptivity to copulation: pushing up

the female wings with the abdomen while displaying both set of hairpencils, and moving the abdomen above the female wings while displaying the hairpencils. CS females

showed a preference for the male movement of the abdomen above female wings while

displaying the hairpencils only when it was shown by CS males. Almost half the time that

RS males moved the abdomen above the female wings while displaying the hairpencils,

CS females responded by resuming calling. RS females, on the other hand, showed a

preference for the male pushing up of the female wings with the abdomen while

displaying both set of hairpencils. RS females were more likely to stay receptive when

this behavior was displayed by RS males than when exhibited by CS males.

Although there is evidence that evolution of a strong mate preference may have

led to speciation (even in the absence of hybrid inviability or sterility) in two closely

related butterfly species Heliconius erato and H. himera (low mate propensity of certain

hybrid males in some crosses being the only existing vestigial classical hybrid

incompatibility (McMillan et al., 1997) it is possible that in S. frugiperda, a female preference for within-strain matings is reinforced by the reduced probability of producing hybrids, which in this species have lower fitness, thus increasing the fitness of “choosy” females. Contrasting with CS larvae that develop equally well on a wide range of host plants, RS larvae show a higher development rate on its preferred host, indicating a degree of physiological adaptation (Pashley et al., 1995). Therefore, mating with a CS male may be less advantageous for a RS female if viability of hybrid progeny is lower on its preferred host. In addition, in each new generation, genetic incompatibilities could accumulate in hybrids. Although in the laboratory, hybrids have proven to be fertile, laboratory studies have shown the existence of a reduction in egg viability of F1 X F1 hybrids of the two strains (Whitford et al., 1988). This reduction was shown to be higher for (RS x CS F1) x (RS x CS F1) progenies (26%) than for (CS x RS F1) x (CS x RS F1) progenies (4%). Any of these reasons would constitute a pressure for the evolution of a male signal matching the female preference, signaling the male strain identity and thus assisting females in discriminating RS and CS males. Although the contribution of other cues was not ruled out by this study, the prominent display of two sets of male hairpencils during courtship suggests that male pheromones are the signals playing the lead role in this female choice.

The present comparative study of courtship behavior in two genetically compatible but host-differentiated strains of S. frugiperda provides evidence of assortative mating due to female choice. These findings of female preference for strain- based male signals, particularly in the absence of male mate discrimination are consistent with the predictions of the asymmetric tracking hypothesis (Phelan, 1992) and the more specific hypothesis of Phelan and Baker (1987) that many lepidopteran male pheromones may have evolved in the context of reproductive isolation through sexual selection. Given the possibility of attracting males of the other strain, females cue on the signals provided by males to mate assortatively. The lack of discrimination by males had been predicted given the low parental investment of males, whose strategy is mating with as many mates as possible as long as the cost of mating with a female outside their own strain is low when compared with the time and energy spent looking for a same strain female. Chapter

2 reports chemical investigations of S. frugiperda male hairpencils set to determine if chemical differences exist between males of the two strains that might mediate female preference for males of like strain.

CHAPTER 2

CHEMICAL ANALYSIS OF MALE SPODOPTERA FRUGIPERDA SCENT

STRUCTURES

1. INTRODUCTION

Male eversible structures in Lepidoptera

The existence of male eversible structures, or male scent disseminating structures,

is known in several families in the Lepidoptera. These structures clearly differ from that

of females in aspects such as the structural complexity and location of the disseminating

organs, as well as the chemical structure and amount of the chemicals released. With

some exceptions, these structures are displayed close to females, during courtship, and

release large quantities of chemicals compared to the amounts released by females. These

structures can be classified in six basic types (McColl 1969, Scoble 1992). In many

species, a behavioral response to the volatile chemicals collected from these male

structures has yet to be demonstrated, thus holding in question their designation as male

pheromones. In other species, a behavioral reaction to male secretions has been observed

in females (e.g. Grapholita molesta, Baker and Cardé 1979; Utetheisa ornatrix, Conner et al. 1981, Conner et al. 1989; Ephestia ellutella, Phelan and Baker 1986; Phelan et al.

1986) and in some cases, males (e.g. Bijpost et al. 1985).

Morphology and chemical characterization: probable role in courtship

Birch et al. (1990) provided a comprehensive review of male scent structures and scents in moths. Accounts of their presence and possible effects on the recipients are given in detail for the Tortricidae, Arctiidae, and Noctuidae, and some other families.

Tortricidae

Grapholita molesta is perhaps the best-studied species, for which morphological,

behavioral and chemical studies have been undertaken. Males in this species bear a pair

of white tuft hairs (Baker and Cardé 1979), which disseminate a blend of 4 chemicals:

ethyl trans-cinnamate, (R)-(-)-mellein, methyl jasmonate, and methyl 2-epijasmonate

(Baker et al 1981; Nishida et al. 1982). Females respond to the male abdominal hairpencil

display, approaching the male and touching his abdominal tip with their head, which

evoke his copulatory attempt (Baker and Cardé 1979). A rather different role for the

male-released chemicals in this family is known for the summerfruit tortix moth,

Adoxophyes orana. Laboratory studies showed that males perceiving the male pheromone while flying upwind decreased wing fluttering and increased the lateral component of

their search path (Bijpost et al. 1985). The two chemicals extracted from the males were identified as palmitic acid and myristic acid, although only the former evoked a male

response identical to that observed towards males in the situation described. 78

Pyralidae

Galleria mellonella males are known to produce a sex pheromone in glands located on their forewings (Barth 1937; Roller et al. 1968), which Leyer and Monroe

(1973) initially identified as a (7:3) mixture of nonanal and undecanal and found to be attractive to females. Later Romel et al. (1992) found two additional compounds, nonanol and undecanol.

Ephestia ellutella males also release a pheromone from a wing gland that causes a female acceptance posture, which was characterized as (E)-phytol, gamma-decalactone, and gamma-undecalactone (Phelan and Baker 1986; Phelan et al. 1986).

Arctiidae

Sac-like structures, covered with scales that are known as coremata, have been observed between the 7th and 8th abdominal sternite or on the genital valves of males of several species. These structures vary in size and may depend on how much pyrrolizidine alkaloids (PAs) have been ingested during the larval phase (Schneider et al. 1982, Boppré and Schneider 1985), from which the chemicals found in this family, hydroxydanaidal and danaidal, are derived. The corematal extrusion is known to cause female acceptance in the case of Utetheisa ornatrix (Conner et al. 1981, Conner et al. 1989), but in species

where males form leks such as Creatonotus transiens and C. gangis (Wunderer et al.

1986), the corematal extrusion induces attraction of both conspecific females and males.

Noctuidae

The male eversible structures in this family vary in morphology and location.

These structures can be found on the legs, thorax and abdomen, the latter showing

remarkable complexity (Birch 1970; 1972). Abdominal structures can also vary in

location, being present on the anterior abdomen (associated with the 2nd, 3rd, and 4th abdominal sternites), usually referred to as hairpencils (e.g., several Hadeninae, some

Noctuinae, Cuculliinae, and Amphipyrinae), on the posterior abdomen (8th abdominal

sternite) (e.g., some Pantheinae, and Catocalinae), or on the ninth abdominal sternite of

some Heliothinae (e.g., Heliothis virescens, Teal et al. 1981; Cibrian-Tovar and Mitchell

1991) and some Amphipyrinae (e.g., Spodoptera frugiperda, Eltringham 1927; S.

littoralis, Ellis and Brimacombe 1980). The latter structures are usually referred to as

hairbrushes (see Birch et al. 1990).

In the Noctuidae, congeneric species sometimes share secretion components, e.g.,

Leucania impura, L. conigera, and L. pallens all produce benzaldehyde (Aplin and Birch,

1970), but chemical variation among congeneric species is also observed, as in members

of the Mamestra genus (Aplin and Birch (1970).

Evidence that hairpencil extrusion induces female acceptance to mating exists in

several noctuids: S. littoralis (Ellis and Brimacombe 1980), Heliothis virescens (Teal et al 1981), Pseudaletia unipuncta (Fitzpatrick and McNeil 1989), H. subflexa (Cibrian-

Tovar and Mitchell 1991), and M. brassicae (Poppy and Birch 1994).

The evolution of male pheromones through sexual selection

The tremendous range of male-scent disseminating structures throughout the

Lepidoptera suggests an important function(s) played by these organs. Moreover, the many examples of incomplete (probably vestigial) organs among the Noctuidae suggests that whatever this function(s), it has been lost many times, and that entirely new organs have evolve later (Birch, 1972, PART II).

Baker and Cardé (1979) were the first to envisage how female-choice sexual selection could be linked with the evolution of these male structures. If a fitness-related trait involving odor could become preferred by females, and if conditions were to promote differential mating success among males, both the trait and the preference for it could be driven to extremes through runaway selection.

Among the issues pertaining to the evolution of these structures and scents are what started the female preference in the first place and the nature of the trait itself (Birch

et al. 1990). Was the odor associated with a male investment in the form of a gift

transferred directly to females to increase fecundity or survival of the offspring?

Alternatively, was the odor itself the gift, handed down through the choosy mother to her

sons, which discriminating females of their generation would favor for mating? Evidence

exists to support both hypotheses.

There is direct evidence for sexual selection with a fitness advantage in E. elutella

females (Phelan and Baker 1986). These females preferentially mate with larger males,

which contain approximately twice as much pheromone as smaller males, and actively

reject smaller ones. Females that mate with larger males produce more offspring, have

larger sons that will be expected to be at a mating advantage, and also produce larger

daughters.

In Utetheisa ornatrix, males pass pyrrolizidine alkaloids (PA) to the female

during copulation. These alkaloids are used in defense against predators, and also are the

precursors to the male pheromone. It appears that females accept males for mating

through the evaluation of their pheromone titer presented during the courtship display

(Conner et al. 1990; Dussourd et al. 1991). There is evidence that PA content of males is positively correlated with pheromone level and that females can gain benefits by adding

PA’s from the male to their own, which they in turn allocate to their eggs (Dussourd et al.1988). There is also evidence that females gain benefits for themselves as well, as their

82 enhanced PA levels make them unacceptable to spiders the moment they uncouple, a protection that extends throughout their lives (González et al. 1999).

A different type of advantage associated with male pheromone may be present in the arctiid species, Pyrrharctia isabella, where the value of the PA transfer is not linked with defense, but with pheromonal competence of the sons (Krasnoff and Roelofs 1989).

This species is a facultative feeder on PA-containing plants, thus an adult female may lack PAs in her body tissues. If a PA-deprived female mates with a PA-laden male, the display of the coremata by her sons evokes the ultrasonic clicks of females, a behavior associated with receptivity, while offspring from PA-deprived parents do not evoke this female behavior. As in P. isabella, females of the oriental fruit fly, Bactrocera dorsalis, appear not to derive any direct benefit by selecting males producing high levels of the pheromone, trans-coniferyl alcohol. Males synthesize this compound through conversion of trans-3,4-dimethoxycinnamyl alcohol and trans-3,4-dimethoxycinnamaldehyde, compounds that they obtain as adults from the flowers of Fagrarea berteriana. However, there may be indirect benefit to the female, since selecting mates with high pheromone titers may increase the likelihood that their sons will successfully locate and collect these compounds and thereby enjoy a mating advantage (Nishida et al. 1997).

The above example of P. isabella has been used to illustrate how forces from within a population could explain both the male apparatus and the scent, as opposed to a

83 different view that favors the occurrence of these structures as an adaptation against inter- population mating mistakes. According to Krasnoff (1987), there has been a trend away from a primitive condition of host-plant specialization towards a more generalized feeding habit in the Arctiidae, which in P. isabella would explain why the use of these host-derived PA derivatives as courtship pheromones has atrophied with the shift in preference by females of this and other arctiid species away from PA-containing plants

(Krasnoff and Roelofs 1990). These authors claim that the male structures play no role in the isolation of the species. Thus its presence, and the odors that are disseminated can be seen as the vestiges of a preference for the host-plant odor in ancestral species.

Phelan and Baker (1986) suggest a different origin for many lepidopteran male pheromones, that sexual selection by female choice has driven the evolution of these pheromone systems, and Phelan and Baker (1987) presented evidence for an adaptive origin of the female preference. Using larval host-plants as a measure of the potential for interspecific contact and thus mating mistakes among congeners, these authors found a positive correlation between host-plant overlap and the presence of these structures in males among each of the five lepidopteran families studied.

Objectives

Direct behavioral evidence on the use of male hairpencils in elaborated courtship in the two genetically differentiated host-differentiated strains of S. frugiperda provided

84 evidence of assortative mating due to female choice (see previous chapter). The prominent display of the two sets of male hairpencils during courtship was significantly associated with elicitation and maintenance of female receptivity in intra-strain courtships.Failure of males to induce and maintain female receptivity in inter-strain courtships suggests that mate discrimination by females could be chemically mediated, with possible differences in the chemical blends driving the observed assortative mating.

A strong mate preference may be reinforcing the genetic divergence of these two differentially host-adapted populations of S. frugiperda. Demonstration that assortative mating in this species is due to female sexual selection on a male scent distributed during courtship would be consistent with the hypothesis of Phelan and Baker (1987) that many lepidopteran male pheromones have evolved through female choice as an adaptive response to mating mistakes. If mating between individuals from differentially adapted populations produces hybrids of lower fitness, there will be a selective pressure for females to prefer males of her own kind, as well as pressure on males to evolve an ability to advertise their identity effectively.

In this study, I sought evidence for chemical differences that might mediate female discrimination between males of the two strains, through an analysis of the compounds founding the male eversible structures of S. frugiperda.

2. MATERIALS AND METHODS

Confirmation of the existence of male Spodoptera frugiperda hairpencils

Based on the accounts of Eltringham (1927), specimens from the OARDC

Entomology Museum Collection gathered by the Emeritus Professor Rings, were dissected in water and observed through a stereoscopic microscope for confirmation of male scent structures. The male genitalia and the thorax/femur areas were dissected.

Although very dry, the specimens were complete and the presence of these structures could be confirmed. Spodoptera frugiperda males have two different sets of hairpencils: one set of dark brown structures, located at the base of the femurs of the prothoracic legs.

Each structure is normally concealed inside a thoracic pocket, and is exposed when the male stretches the leg during courtship (Chapter 1). A second set of lighter-colored hairpencils, referred by Eltringham (1927) as brushes, is located on the valvae, and only visible when the male extrudes the genitalia. These structures were later observed in live specimens from the two laboratory-reared strains, and from wild-captured males of S. frugiperda, and removed for extraction and analysis of their putative chemicals.

Live insects

Live individuals of S. frugiperda used in this study came from corn- and rice- strain colonies, established at the Ohio Agricultural Research and Development Center, during 1998, and from wild-captured males (unknown ages). The corn strain (CS) colony 86 was founded in May 1998 with neonates and third-instar larvae from a CS colony established at the Louisiana State University by Dorothy Prowell. The colony was supplemented with an additional 20 pupae in August 1998 from the LSU colony.

The rice strain (RS) colony was founded in August 1998, with larvae of various instars collected on grasses in a pasture near Baton Rouge, Louisiana. The time of the year, the locale, and the host plants were chosen to increase the chances of collecting rice strain individuals. After completing their development to adults, individuals were randomly selected to form pairs, and each pair initiated a lineage. Progeny were maintained in the colony only if their parents proved to be of the rice strain. The strain identity of parents was determined using a PCR protocol developed by Dr. Prowell

(personal communication) based on analysis of mitochondrial DNA (mtDNA). The full protocol is attached (see Appendix A), but a brief explanation of the method follows:

After removing the wings, legs, and abdomen, the head and thorax were homogenized for mtDNA extraction. After extraction and purification of the mtDNA, sequences of 600 base pairs were amplified, using the appropriate primers in a polymerase chain reaction, and an agar gel was used to confirm their presence. Digestion of these fractions was conducted using a restriction enzyme, HinFI, whose pattern was compared with that of the rice strain (a 550 base-pair sequence). From the fifty lineages initially created, 22 were positively identified as rice strain.

Live-captured wild males were collected at the OARDC arboretum, adjacent to a

corn field, and in a corn field near a residential area of Wooster, Ohio, using T-shape

PVC traps with a glass collecting jar at the bottom similar to that developed by Dowd et al. (1992). Traps were baited with virgin females, 2-6 days old. Field-collected (wild) males were identified according to Ring´s identification keys. The strain identity of these males was assumed since only CS S. frugiperda are known to migrate to this region. The

wild males were included in this study to assess the influence of artificial diet on the

production of male chemicals.

Collection of male Spodoptera frugiperda hairpencils

Males (2-6 days old) were anesthetized with a CO2 flux before the removal of both sets of hairpencils. These structures were collected under a stereomicroscope, using small forceps and dissecting scissors. Both instruments were cleaned with acetone after each hairpencil collection, to prevent extract contamination. Each femoral hairpencil was pulled out of the thorax pocket, by pulling apart the foreleg. The two femurs with their hairpencils were immediately placed in the extracting solvent. The removal of the genital hairpencils was performed while squeezing the abdomen and exposing the valvae and their hairpencils. These structures were pulled out with forceps at their point of insertion on the valvae.

The femurs and the remaining body parts of five laboratory-reared CS females were extracted separately. These extracts served as experimental controls to which male extracts were compared to distinguish compounds exclusive to males from general cuticular chemicals.

Chemical extraction of hairpencils

Each set of hairpencils was separately immersed in 200µl methylene chloride in a sealed glass vial for 24 hrs, at which time, extracts were filtered with glass wool. An identical volume of solvent was used to rinse the vial and added to the sample, after filtration, for a final 400µl volume. A similar procedure was used to extract the female femurs. For the remaining female body 500µl were used, and an equivalent volume was used to rinse the glass vial for a final volume of 1ml. Filtered extracts were subsequently concentrated under nitrogen and stored at –17ºC until analyses were performed.

Chemical analysis of extracts

Just prior to analysis, internal standards were added to extracts (200ng tridecanoic acid for femoral hairpencils and 200ng pentadecanoic acid for genital brushes), which were further concentrated under nitrogen to 2µl. Samples were then injected into a HP

5890 gas chromatograph and analyzed with a Hewlett Packard 5970 series mass selective detector. The GC column was DB1 0.25x30m (J&W Scientific, Folsom, CA) with a temperature program of 30-100°C, at 5°C/min, then increased to 220°C at 15°C/min with 89 a final hold time of 23 min. The injector was 210°C and the transfer line to the mass selective detector was 250°C. Solvents were also analyzed for contaminants.

Each peak found in a sample was tentatively identified by comparing its mass spectrum with the NIST mass spectral computer library and its retention time registered.

Peak retention times and identities were compiled in Excel 5.0 for comparison among samples, and SPSS 11.0 was used for all statistical analyses.

Two strain X chemical MANOVAs were performed to measure differences in chemical profiles for hairpencils of CS, RS, and wild corn (Wild C) males. When significant differences in chemical profiles were indicated, one-way ANOVA was applied to individual compounds to determine which contributed to profile differences.

Post-hoc comparisons between strains were run using Bonferroni and because the

MANOVA produced significant effects, the family error rate was kept at 0.05 (Johnson,

1998).

Chemicals that showed significant differences between strains by ANOVA were then submitted to a principal component analysis (PCA) to provide a multivariate characterization of femoral and genital hairpencils from different strains. The relationship between each compound and each new dimension generated by PCA was assessed using

Johnson´s (1998) recommendations: small (i.e. an absolute value between 0.2 and 0.4), substantial (0.4-0.7), marked (0.7-0.9), and very dependable (0.9-1.0).

The ability to assign males to strain based on these PCA profiles was tested by performing cluster analysis on the PCA scores of individuals (Johnson, 1998). Main components (new factors) that were submitted to cluster analysis were selected based on the cumulative percentage of variance explained and the scree plot interpretation method

(Johnson, 1998). The cluster analysis was performed using complete linkage and cosine of vector of values used. The interpretation of clusters was based on the relative importance of the chemicals characterizing each component submitted.

3. RESULTS

From a total of 174 chemicals that emerged from the analysis of extracts of femoral hairpencils and genital brushes, 116 chemicals were found to be present in both hairpencil extracts, 20 appeared to be present only in the femoral hairpencils, while 38 were exclusive to the genital hairpencils. However, 23 of these chemicals were also present on the female prothoracic legs or on other female body parts (see Appendix F).

3.1. Genital hairpencils

MANOVA revealed differences in chemical profiles among genital hairpencils of

RS, CS, and Wild C males (Roy’s Largest Root =404.164, F=24.495, df =33, p<0.05).

Tests of between-subject effects revealed significant differences (p<0.05) among the three groups (RS, CS, and Wild C) for 35 chemicals.

Five of these chemicals were found only in the genital hairpencils, and 30 were present in both genital and femoral hairpencils (although 6 out of these 30 were also found in the female body as well). Bonferroni post-hoc tests showed significant differences between RS and CS males, and between RS and Wild C males (Table 6).

When principal component analysis was performed to reduce the data to a smaller set of variables, three dimensions were found to explain 65.0% of the variance and were used in a cluster analysis to determine chemical similarity of individuals relative to host- adapted strain (Figure 1). From this cluster analysis, two main clusters emerged, clearly separating corn from rice individuals. A subdivision of the rice cluster also occurred.

Mean Conc.(ng) in Genital hairpencils

Ret time % % %

interval Putative Chemical RS of sample CS of sample Wild C of sample

5.15-5,46 Not identified 13.43 a 33% 0.00 b 0% 0,34 b 8% 7.30-7,39 4-Penten-2-ol / 2-Pentanol,4-methyl 21.30 ab 50% 7.82 b 50% 42,54 a 92% 10.05-10.28 p-Benzoquinone 6.14 a 33% 0.00 b 0% 0.00 b 0% 13.00-13.14 Phenol 949.84 a 92% 26.34 b 58% 0.00 b 0% 15.92-16.04 Heptanoic acid 11.46 b 50% 11.68 b 50% 41.04 a 83% 17.68-17.89d Octanoic acid 39.71 b 100% 60.46 b 100% 113.94 a 100% 18.41-18.46 Benzothiazole 21.90 a 83% 3.31 b 8% 0.00 b 0% 18.52-18.58 Cyclohexane,isothiocyanato 39.73 a 92% 4.23 b 8% 0.75 b 8% 19.30-19.38 4-Octene,2,3,6-trimethyl- /1-Undecyn-4-ol 8.13 b 42% 55.98 b 17% 861.19 a 50% 19.49-19.56d Formamide,N,N-dibutyl 15.59 a 83% 5.37 b 50% 20.64 a 92% 20.90-20.93d 2-Propenal,3- (2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)- 126.94 a 67% 40.45 b 67% 79.89 ab 67% 21.33-21.38d Undecanoic acid 0.00 a 0% 2.40 ab 25% 8.42 b 50% 21.52-21.58 2-Undecanol 24.55 a 92% 9.01 b 58% 15.53 ab 75% 21.85-21.91 Urea, N'-cyclooctyl-N,N-dimethyl 22.54 a 83% 1.43 b 8% 1.06 b 8% 22.90-22.94 Tetradecane? / Pentadecane 8.53 a 58% 0.00 b 0% 0.00 b 0% 23.07-23.13 Dibutyl 3-dydroxybutyl phosphate 53.89 a 75% 14.45 b 67% 16.24 b 67% 24.01-24.09 Tridecanal / Pentadecane /2-Dimethylphenol 69.68 a 83% 22.60 b 50% 10.76 b 42% 24.10-24.16d Tetradecanoic acid 8.00 a 33% 0.00 b 0% 0.00 b 0% 24.34-24.39 2,4-Diphenyl-4-methyl-2(Z)- pentene 50,52 a 67% 5.71 b 8% 0.00 b 0% 24.90-24.98 7-Heptadecyne,1-chloro- /2,4-Heptadienal,2,4-dimethyl- 16.54 a 33% 0.00 b 0% 0.00 b 0% 25.23-25.31 Not identified 49.13 a 42% 0.00 b 0% 0.00 b 0%

Continued

Table 6. Genital hairpencil chemicals whose concentrations varied among Spodoptera frugiperda host strains (Bonferroni Post Hoc tests at alpha = 0.05), and percentage of individuals bearing them in each sample.

Table 6 continued

25.33-25.39 2,4-Diphenyl-4-methyl-1 (E)-pentene 83.80 a 67% 10.67 b 8% 0.00 b 0% 25.82-25.89e Not identified 118.21 a 75% 12.35 b 8% 0.00 b 0% 26.93-27.06 4-t-Butyl-2-(dimethylbenzyl)phenol 52.20 a 75% 0.00 b 0% 6.00 b 8% 27.10-27.18d Nonacosane /Dodecane,5,8-diethyl- 32.89 a 42% 0.00 b 0% 0.00 b 0% 28.08-28.14 Hexadecanoic acid / 2,5-Octadecadiynoic acid, methyl ester 22.38 a 33% 0.00 b 0% 0.00 b 0% 28.83-28.89 1-Phenantrenecarboxilic acid,... 21.60 a 58% 0.00 b 0% 0.00 b 0% 29.36-29.39e Not identified 15.72 a 42% 0.00 b 0% 0.00 b 0% 29.40-29.62 2-t-Butyl-4-(dimethylbenzyl)phenol 71.36 a 100% 3.03 b 8% 0.00 b 0%

d- present also on female extracts e- exclusively present on genital brushes Means followed by the same letter are not significantly different at the 0.05 level (Bonferroni Post Hoc tests) RS- Laboratory reared rice strain CS- Laboratory reared corn strain Wild C- field collected corn strain

Figure 1. Similarity dendrogram of genital hairpencils from individual male Spodoptera frugiperda based on their chemical profiles. Cluster analysis used the complete linkage method of the cosine of the first three components from a principal component analysis of the chemicals extracted from the hairpencils.

2,4-Diphenyl-4-methyl-(E)-1-pentene and 2,4-diphenyl-4-methyl-(Z)-2-pentene had the highest coefficients for the first dimension (very dependable), but several others also had high coefficients. Peak 143 (not identified) showed a strong negative relationship with the second dimension. A substantial association with the third dimension was found for tetradecanoic acid, 1-chloro-7-heptadecyne (probably a misidentification or a solvent contaminant) 2,4-dimethyl-2,4-heptadienal, and for 4-t- butyl-2-(dimethylbenzyl)-phenol.

Looking at the concentrations of these chemicals, the two main clusters were separated primarily by 2,4-diphenyl-4-methyl-(E)-1-pentene and 2,4-diphenyl-4-methyl-

(Z)-2-pentene, which were found in rice individuals (lower cluster) but absent in all but one corn individual. The subdivision of the large rice cluster into two smaller rice clusters seems to be largely based on the concentrations of the unidentified peak 143, tetradechanoic acid, 2,4-dimethyl-2,4-heptadienal, and 4-t-butyl-2-(dimethylbenzyl)- phenol.

3.2. Femoral hairpencils

MANOVA also revealed chemical profile differences in femoral hairpencil from the three groups (Roy’s Largest Root =1.6E+09, F=4.8E+07, df =34, p<<0.001).

Univariate analysis followed by mean separation of chemical concentration revealed significant differences (p = 0.05) among the strains for 44 chemicals, although six of these chemicals were also found on the female body (Table 7). As with the genital 97 hairpencils, Bonferroni post-hoc tests indicated these differences were primarily between

RS and either lab-reared or wild CS males (39 compounds). Lab-reared and wild CS males showed significant differences in concentration for six compounds.

Principal component analysis of the FHP chemicals produced three principal dimensions that explained 70% of variance in concentrations. Cluster analysis based on these three dimensions produced two main clusters – one subdividing into two other lower hierarchical level clusters and the other including mostly rice individuals. The former dividing cluster (above cluster) shows within it a rather clear-cut separation between corn and rice individuals: one of the subclusters is exclusively corn, whereas the other combines rice and corn individuals. Thus, separation of the strains based on femoral hairpencils is less clear than for genital hairpencils

Compounds showing the strongest association (very dependable) with the first dimension are: 2,4-diphenyl-4-methyl-(E)2-pentene, 2,4-diphenyl-4-methyl-(Z)-2- pentene, tridecane/pentanal/pentadecane/2-dimethylphenol, 2-t-butyl-4-

(dimethylbenzyl)phenol, tetradecane/pentadecane, nonacosane/5,8-diethyldodecane, 4-t- butyl-2-(dimethylbenzyl)phenol and podocarp-7-en-3À-ol,13,13-dimethyl. Compounds associated with the second dimension (substantial and negatively) are phenol, isothiocyanatocyclohexane and benzothiazole.

Compounds associated with the third dimension (substantial positive correlation)

are 4-hydroxybenzaldehyde, heptanoic acid, N,N-dibutylformamide, octanoic acid, 2,3,6-

trimethyl-4-octene /1-undecyn-4-ol, peak 143 and peak 193 (both unidentified).

The substances that explain the two second-level cluster split (corn cluster and 2nd rice cluster) are those correlated with the first dimension listed above – absent or with trace concentrations in corn individuals and with higher concentrations in rice individuals

(higher concentrations in RS than CS). The 2nd cluster rice individuals separate from the

higher-level rice cluster (Figure 2) based on their concentration of the third dimension

chemicals listed above.

Mean Conc.(ng) in Femoral hairpencils

Ret time Putative Chemical RS % CS % Wild C %

7.40-7.49 Propanoic acid, propyl ester 9.07 ab 75% 6.97 b 58% 19.53 a 67%

13.00-13.14 Phenol 623.76 a 100% 21.32 b 67% 12.30 b 50%

13.55-13.57 Cyclohexane, isocyanato 43.19 a 42% 0.00 b 0% 0.00 b 0%

15.92-16.04 Heptanoic acid 19.40 b 83% 25.32 ab 100% 46.76 a 92%

17.68-17.89d Octanoic acid 38.81 b 92% 54.17 b 100% 106.72 a 100%

18.41-18.46 Benzothiazole 18.08 a 92% 0.72 b 8% 0.00 b 0%

18.52-18.58 Cyclohexane,isothiocyanato 30.46 a 92% 3.06 b 8% 0.00 b 0%

18.98-19.03 Hidroquinone / 1-Butanamine, N-butyl-N-nitroso- 12.13 a 58% 0.00 b 0% 5.05 ab 25%

19.30-19.38 4-Octene,2,3,6-trimethyl- /1-Undecyn-4-ol 77,42 b 42% 171.37 ab 50% 783.11 a 50%

19.49-19.56d Formamide,N,N-dibutyl 14.26 ab 42% 8.91 b 50% 24.84 a 50%

19.82-19.91 Benzenemethanol,3-hydroxy 15.54 a 58% 4.38 ab 8% 0.00 b 0%

20.06-20.08 Benzaldehyde, 4-hydroxy 10.91 ab 67% 7.72 b 17% 39.62 a 67%

21.24-21.28 5,9-Undecadien-2-one,6,10-dimethyl-,(E) / (Z) 3.87 a 42% 0.00 b 0% 0.00 b 0%

21.85-21.91 Urea, N'-cyclooctyl-N,N-dimethyl 15.78 a 83% 1.62 b 8% 0.00 b 0%

22.00-22.26d 4-Methyl-5-decanol /3-Ethyl-4-nonanal 58.01 a 100% 17.30 b 75% 18.11 b 58%

22.44-22.49 Limonen-6-ol, t-butyrate 6.15 a 67% 0.00 b 0% 0.00 b 0%

22.81-22.88d Propanoic acid,2-methyl-1,-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl 56.29 a 100% 11.77 b 83% 14.18 b 92%

22.90-22.94 Tetradecane / Pentadecane 13.84 a 83% 0.95 b 8% 0.00 b 0%

23.07-23.13 Dibutyl 3-dydroxybutyl phosphate 45.65 a 100% 13.95 b 92% 17.05 b 92%

23.45-23.64d Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2-butynyl]-? 31.18 a 92% 1.61 b 17% 7.08 b 33%

23.80-23.85 Not identified 12.82 a 42% 0.00 b 0% 0.77 ab 8%

24.01-24.09 Tridecane / Pentanal / Pentadecane /2-Dimethylphenol 78.07 a 100% 13.98 b 75% 15.40 b 67%

24.17-24.22 Heptadecane, 2,6-dimethyl / Pentadecane 19.99 a 92% 0.82 b 8% 2.57 b 17%

24.34-24.39 2,4-Diphenyl-4-methyl-2(Z)- pentene 68,57 a 100% 3.91 b 8% 0.00 b 0% Continued

Table 7 Femoral hairpencil chemicals whose concentrations varied among Spodoptera frugiperda host strains (Bonferroni Post Hoc tests at alpha = 0.05), and percentage of individuals bearing them in each sample.

100

Table 7 continued

24.90-24.98 7-Heptadecyne,1-chloro- /2,4-Heptadienal,2,4-dimethyl- 8.28 a 25% 0.00 b 0% 0.00 b 0%

25.00-25.02 Cholestan-3-ol,2-methylene-,(3á,5à)- /Hexanedioic acid, bis(2-ethylhexyl)ester 25.26 a 75% 0.00 b 0% 0.00 b 0%

25.14-25.16 Not identified 19.48 a 58% 0.00 b 0% 0.00 b 0%

25.23-25.31 Not identified 78.29 ab 67% 49.57 b 42% 196.02 a 100%

25.33-25.39 2,4-Diphenyl-4-methyl-1 (E)-pentene 168.68 a 100% 12.71 b 8% 0.00 b 0%

25.66-25.75 Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2-butynyl]-? 19,00 a 50% 0,00 b 0% 0.00 b 0%

25.91-25.96 2,4-Diphenyl-4-methyl-2 (E)-pentene 129,36 a 100% 10.34 b 17% 0.42 b 8%

d- present also on female extracts Means followed by the same letter are not significantly different at the 0.05 level (Bonferroni Post Hoc tests) RS- Laboratory reared rice strain; CS- Laboratory reared corn strain; Wild C- field collected corn strain

101

Figure 2. Similarity dendrogram of femoral hairpencils from individual male Spodoptera frugiperda based on their chemical profiles. Cluster analysis used the complete linkage method of the cosine of the first three dimensions from a principal component analysis of the chemicals extracted from the hairpencils.

102

103

4. DISCUSSION

During their courtship displays, S. frugiperda males exposed to females both their

genital and femoral hairpencils. Comparative behavioral characterization (Chapter 1) of

intra- and interstrain courtships indicated the importance of these displays in mating

success, with a possible role in the assortative mating of the two host strains. Thus, it is

important to characterize the chemical composition of both structures and determine

qualitative and/or quantitative differences in chemical profiles between the strains that

might underlie the assortative pattern of female choice.

Over 100 chemicals were found to be unique to extracts of the eversible structures

of S. frugiperda males. Depending on mechanisms for release from these structures, a

number of these compounds are sufficiently volatile that they could be perceived as odors

during the male courtship display . Others of lower volatility might be only perceived

upon contact between the sexes. Attempts to develop a behavioral bioassay for S.

frugiperda male pheromone were unsuccessful since the crude hairpencil extracts failed

to elicit an overt response in females, as has been seen in other moth species (Phelan et al. 1986). Without behavioral evidence, we cannot yet conclude a pheromonal role for any of the compounds. Nevertheless, the finding of substantial differences in the concentrations of hairpencil components between the two host-adapted strains is consistent with the predictions that male pheromones may be partially mediating

104 assortative female mate choice. In addition, these chemical differences should guide future behavioral studies as to which compounds are responsible for pheromonal activity.

In this regard, chemical analysis showed clear differentiation in the chemical composition of both hairpencil sets between the two strains. In both cases, the distinction was due primarily to the absence of certain compounds in the CS males that are present in

RS males. Given the crucial importance of the hairpencils display in CS female acceptance (see Chapter 1), these compounds may act as deterrents for CS females, who actively reject RS males (Chapter 1), whereas for RS females these substances could enhance receptivity. Although we do not provide direct evidence in the present study that females respond to these chemicals, the prominence of hairpencil displays during several stages of courtship, the observations of active rejection behaviors by females during interstrain courtships, along with the chemical differences in those hairpencils all combine to suggest an important role. The high concentrations of the distinctive compounds of RS males could be the result of positive selection by RS females. If such is the case, assortative mating is taking place, thereby contributing to a growing divergence between the two strains.

Chemicals clearly distinctive in RS and CS males

Among the above mentioned substances, two compounds stand out as the ones that allow a clear RS vs CS strain distinction: (2,4-Diphenyl- 4-methyl-1-(E)-pentene and

105

2,4-Diphenyl- 4-methyl-2- (Z)-pentene. These compounds had not yet been described in this family or in any other in the Lepidoptera.

Diet can affect the abundance and ratios of male pheromone components

(Ponomarev et al. 1997). To determine the possible effects of diet on S. frugiperda hairpencil contents, field-collected corn-strain males were included in the present chemical analyses. Although univariate statistical analysis revealed quantitative differences for a few compounds in both hairpencil sets between field-collected and lab- reared CS males (e.g. 2,3,6-trimethyl-4-octene/1-undecyn-4-ol), cluster analysis based on the larger chemical set did not reveal clear differentiation. The clear chemical separation between strains evident in the current study is all the more interesting because the possibility that the differing compounds could be an artifact of the laboratory diet has been discarded. The RS vs CS chemical differences are robust because the cluster analysis showed strong overlap between lab and wild CS males, demonstrating that the laboratory diet did not produce differences in the hairpencil chemicals of males. This stands true for both genital and femoral hairpencils, since the same non-split pattern of lab and wild CS males was maintained by cluster analysis.

It is noteworthy that there is a striking difference in the concentration of 2,3,6- trimethyl-4-octene/1-undecyn-4-ol between CW males and both RS and CS males in FHP extracts. This is likely due to dietary differences since RS and CS were raised under the same diet. However, this difference does not stand as a major classifier to separate CW

106

from either CS or RS. Certainly this chemical would not stand alone to separate CW from

CS on the basis of diet – for instance octanoic acid also varies significantly between wild

and lab corn individuals. These differences seem however too unimportant compared

with other chemical differences in the global male sampled here – so Corn are never

separated even with these seemingly dietary-based differences.

The femoral hairpencils of RS males showed a larger heterogeneity than those of

CS, producing two distinct rice clusters that differed primarily in their levels of 2,3,6-

trimethyl-4-octene/1-undecyn-4-ol and the unidentified peak 143. A certain amount of

variability is not uncommon in male pheromonal blends: Mamestra brassicae (Jaquin et al., 1991) and Galleria mellonella (Ponomarev et al., 1997) males have been found to vary substantially in the levels of their pheromone components.

Phenol, isothiocyanatocyclohexane and benzothiazole also differed in concentration in femoral hairpencil extracts between the RS and CS males and contributed to separate the subcluster of RS from the one containing CS males. Phenol has been previously identified in Lepidoptera but as a minor component of the male pheromone blend of M. brassicae, by Jaquin et al. (1991), according to whom such minor blend components may be relevant for female discrimination, possibly acting towards the promotion of species isolation, given the similarity in the pheromonal blends of several

Noctuidae (e.g., members of the Hadeninae). The high concentration of phenol in RS males makes it worthy of further investigation as a possible pheromone component in S.

107

frugiperda. Finding phenol in another subfamily (Amphipyrinae) is not surprising, since

some compounds are known to be shared by species of different subfamilies of the

Noctuidae (e.g. 2-methylbutanoic acid is produced by Phlogophora meticulosa,

Amphipyrinae (Aplin and Birch 1970; Birch 1970), by Cucullia umbratica, Cucullinae

(Birch 1972), and Mamestra brassicae, Hadeninae (Jaquin et al. 1991).

Two other compounds commonly shared by different noctuid subfamilies are

benzaldehyde and benzyl alcohol. In the Hadeninae, benzaldehyde (as well as iso-butyric

acid) is found in both Mythima impura and M. conigera (Aplin and Birch, 1968; Aplin and Birch, 1970). Benzyl alcohol and 2-phenylethanol is shared by Polia nebulosa

(Birch, 1972) and Mamestra persicariea (Aplin and Birch, 1970; Bestmann et al. in Birch et al. 1990).

In the femoral hairpencils, two unidentified chemicals (peak 143 and 193) distinctive in RS and CS males are also worth investigating.

It is admissible that the chemical differences in male hairpencils here reported function as strain-based male signals through which females of the two host-strains discriminate between RS and CS males when males display these structures during courtship (see Chapter 1). In each strain, the existence of a clear female preference for their own strain male display (which, according to the present results, is delivering the distinctive male odor of that strain) is indicative that in S. frugiperda the male chemicals are currently, or have been, diverging through female sexual selection. This is consistent 108

with the predictions of Phelan and Baker (1986) that the evolution of male pheromones is

driven by sexual selection. In S. frugiperda, the female preference seems to be adaptive,

since in F1 the hybrid progeny from both crosses produced less viable eggs (Whitford et

al. 1988). Since S. frugiperda females are the sex with higher parental investment, choosing a mate from the other strain is more costly for females than for males, thus coupling odor information with mate viability is very relevant for females as predicted by

Phelan’s (1992) asymmetric tracking model. With their low parental investment on the other hand males can still be less selective in which females they choose to mate with until mating with a female of the other strain produces no progeny at all.

One could speculate that the preference of RS females for some of the compounds present almost exclusively on RS males - and its possible repellent effect on CS females - could result from a sequence of events, starting with a host shift. It is known that RS individuals develop faster in pastures than in corn, which might have allowed the individuals to decrease the parasitism and predatory pressure upon them (Pashley et al.,

1995). It is likely that the physiological changes promoting a better adaptation to the new host could have had an impact on male odor production. The physiological changes responsible for the male odor could be settled with increasing numbers of intra- population matings in the new host, given the tendency for the adults to mate on the larval host (Sparks, 1979) and with decreasing numbers of females mating with males from a nearby corn habitat due to asynchronic development.

109

Nevertheless a number of females developed from delayed postures living at the boundaries of the two habitats would still attract and mate with corn males. If hybrids (or the F2 progeny) from these crosses were less viable than the progeny resulting from intra- population mating, then the development by the female of a marked preference for population-based male signal would increase female fitness. Corn females using the corn habitat would benefit also from keeping the old preference, as mating with those males bearing the new odor would also decrease their fitness.

110

EPILOGUE

Minor differences in the female pheromone of RS and CS have been assumed to play a minor role in keeping the two strains separate in the field. In previous experiments,

CS and RS males showed 35% and 40% cross-attraction in the field, respectively to live females (used as baits) (Pashley et al., 1992). Laboratory studies have also shown that females have a strong preference for males of their own strain (Pashley et al., 1992), with only 15% of females choosing to mate with a male outside their strain. The combination of these results thus suggested that S. frugiperda females had more intrastrain fidelity in response to mating signals than did the males - this would be consistent with Phelan’s

(1992) asymmetric tracking model.

Although hybrid mating still produces viable offspring, hybrid egg viability from hybrid F1 pairings is lower than intrastrain egg viability particularly between hybrids of rice female x corn male Whitford et al. (1988). The pressure to avoid interstrain mating is therefore higher on female than on males. Selection for male discrimination of females is thus not predicted until rejection by interstrain females is extremely high, or hybrid fitness becomes very low (Hammerstein and Parker, 1987). In either case we should expect males to develop a more acute discrimination for a viable mate.

Eltringham´s (1927) study on the S. frugiperda male eversible structures had long been forgotten and a detailed behavior analysis of the species courtship had yet not been conducted. Neither has the use or possible role of these structures been referred in 111 relation to the species courtship. Therefore the basis for female discrimination had not yet been assessed when the current study began.

The present study presents evidence on the use by both srains of the eversible structures mentioned above and shows that significant differences exist in the composition of the chemicals that were present in both femoral hairpencils and genital hairpencils: phenol and other phenol derived compounds. Although the study does not provide evidence that these chemicals have a clear implication on the acceptance of females in intrastrain courtships, they may have an important role for two reasons: on the one hand, the male display of these structures in intrastrain courtships associates with mating success, and also with the elicitation and maintenance of a female response that has been described as a receptive female behavior in other species of Spodoptera (Ellis and Brimacombe, 1980). On the other hand, males failed to elicit that same behavior in females in interstrain courtships even in those where males were able to mate, eliciting instead several female rejection behaviors. The choosiness of females thus seems to be a response to the signal provided by males during courtship, which is consistent with the disadvantage of crossmating for females as stated above.

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APPENDIX A

PROTOCOL FOR HOST STRAIN STATUS DETERMINATION OF S. frugiperda

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PROTOCOL FOR HOST STRAIN STATUS DETERMINATION OF S. frugiperda

This is a procedure developed by Professor Prowell and her team, and is currently used at the LSU, Baton Rouge, Louisiana, for host strain status determination of S. frugiperda. It uses the species mithocondrial DNA, and it involves a) obtaining the

DNA, b) amplify it in a PCR machine, using two primers, and producing 600bp fragments, c) their digest with a restriction endonuclease HinfI that cuts only the rice strain 600bp fragments producing 550bp fragments, and d) running the samples on a

2% agarose gel. tDNA collection

Day one: Removal of Fall armyworm (FAW) adults from freezer. Individuals thaw a little to allow the removal of wings, legs, and abdomen. The head and thorax (1 sample) are immersed in 500µl of ice-cold citrate (0,025 M Sodium citrate) in a 15ml polypropylene blue-capped tube and kept on ice.

Each sample is homogenized using a tissue homogenizer with a ground glass tip specifically for microcentifuge tubes. The tip is cleaned with water and then 95% Ethanol before reused in next sample.

The tube is spinned down in microcentifuge at 6500 rpm at 4ºC, after which the supernatant is removed an discarded.

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The pellet is resuspended in STE (500µl) in a vortex mixer at room temperature. To each

sample is added 15iµl of 0,5 EDTA, 15µl of a 20% SDS, and 12,5µl of proteinase K

(cold) in this order. Inverting the tubes several times gently mixes the mixture.

The tubes incubate for 1 hour in a water-bath at 60ºC, then overnight at 42ºC, mixing while transferring between baths.

Day two: Spinning down overnight digests at room temperature for 10 minutes at

13,000rpm, and then transfer the supernatant with a pipette to a new tube (vol will be ca

400µl).

To each tube 250µl of TRIS-saturated phenol and 250µl of Chloroform: isoamyl (CLCl3)

(24:1) are added. The tubes are tightly closed, and rotated in a rocking platform a for 20 minutes, and then on a microcentrifuge at 13,000rpm, at room temperature.

The supernatant is transferred to a new tube. To each adding 500µl of CLCl3 and rotate

15 minutes and then spinning for 8 minutes. Transfer again the supernatant to a new tube.

Add to each tube 1000µl of 95% Ethanol at-20ºC. Closing tubes and inverting gently several times. Incubating overnight at –20ºC.

Day three: Pellet DNA by spinning samples in microcentifuge at 6500rpm for 10 minutes, at 4ºC. Remove carefully the Ethanol and and invert tubes for 15 minutes on a rack. Air dry pellet or use Savant Vacuum drier (=speed vac) for 10 minutes.

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Resuspend each pellet in 50µl of RNAse/TE pH7.5. Incubate for 30 minutes at 37ºC (or

3-4 hrs at room temperature, or overnight at 4ºC).

Heat inactivation of RNAse by placing samples in a 60ºC water-bath for 30 minutes.

Allow samples to cool to room temperature. Dilute samples (2µl of DNA sol in 38µl

10mM TRIS).

TDNA Amplification and electrophoresis

PCR protocol:

1-To ‘0’ tube: add 5µl H20, plus 15µl of a master mixture for a total volume of 20µl.

2-To each sample tube: add 4,5µl H20 plus 0,5 tDNA, plus 15µl of a master mixture for a total volume of 20µl.

Master mixture: 8,7µl H20 + 1,0µl of a 20xbuffer + 1,2µl MgCl2 (25mM) + 1,6µl of a dNTP mixture (dATP, dCTP, dGTP, and dTT at 2,5mM) + 1,0µl 16S primer (10µl stock)

+ 1,0µl ND1 primer (10µl stock) + 0,5µl Tfl (Thermo-stabile polymerase enzyme).

3-Add 25µl light mineral oil to each tube and place tubes in the rack of the PCR machine.

4-select the PCR program: step 1-93ºC for 3 min.; step 2-60ºC for 1 min.; step 3-72ºC for

1 min.; step 4-93ºC for 1 min.; step 5-60ºC for 1 min.; step 6-72ºC for 1 min.; step 7-29x to step 4; step 8-72ºC for 5 min.; step 9-4ºC hold; step 10-end.

5-Remove samples from machine

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6-add to each new tube 5µl of buffer: dye (violet coloration) and 5µl of sample; to ‘0’ tube add 5µl of buffer: dye to 5µl of its volume.

(buffer= tris acetate EDTA; 0,04M tris acetate/0,001M EDTA)

7-Run samples in the electrophoresis apparatus in a 2% agarose gel (2g/100ml tris actetate EDTA), selecting the voltage of 90V and the current intensity of 85nA.

129

APPENDIX B

CHEMICALS IDENTIFIED IN ALL EXTRACTS OBTAINED FROM MALE AND FEMALE S. Frugiperda

130

RS CS Wild C Ret. time interval Putative Chemical Male Female gb:hp gb:hp gb:hp 4,87-5,14 n-propyl acetate Gb, fhp absent/not known 5,15-5,46 Not identified Gb, fhp absent/not known 5,50-5,68 Ethane,1,1-diethoxy Gb, fhp absent/not known 5,77-6,65 Acetic acid,2-methylpropyl ester / 3-Pentanone,2-methyl Gb, fhp absent/not known 6,68-6,69 3-Hexane Gb, fhp absent/not known 6,71-6,79 3-Hexanone Gb, fhp absent/not known 6,80-6,85 Not identified fhp absent/not known 7,04-7,05 Not identified Gb absent/not known 7,11-7,21 1-Pentanol Gb, fhp absent/not known 7,22-7,29 Butanoic acid, ethyl ester Gb, fhp absent/not known 7,30-7,39 4-Penten-2-ol / 2-Pentanol,4-methyl Gb, fhp absent/not known 7,40-7,49 Propanoic acid, propyl ester Gb, fhp absent/not known 7,51-7,78 Acetic acid butyl ester Gb, fhp absent/not known 9,44- 3-Hexanone,2-methyl Gb, fhp absent/not known 9,61-9,71 1-Butanol,3-methyl-acetate Gb, fhp absent/not known 9,83-9,90 3-Hexanone,5-methyl Gb, fhp absent/not known 10,05-10,28 p-Benzoquinone Gb, fhp absent/not known 10,45-10,48 Pentanoic acid, ethyl ester Gb, fhp absent/not known 10,56-10,63 Ethanol,2-butoxy Gb, fhp absent/not known 11,01-11,06 3-Hexen-1-ol, formate, (z) Gb, fhp absent/not known 11,35-11,39 Benzene,(1-methylethyl)- Gb, fhp absent/not known 13,00-13,14 Phenol Gb, fhp absent/not known 1:0,66 1:0,81 0:1 13,21-13,54 Hexanoic acid Gb, fhp fpl 13,55-13,57 Cyclohexane, isocyanato Gb, fhp absent/not known 13,61-13,68 Not identified Gb, fhp absent/not known 13,77-13,85 Pentanoic acid,3-methyl-,ethyl ester Gb, fhp absent/not known 14,24-14,28 Acetic acid, hexyl ester Gb, fhp absent/not known 14,62-14,63 Heptanoic acid, methyl ester Gb, fhp absent/not known 14,73-14,86 Benzaldehyde,-2-hydroxy Gb, fhp absent/not known 14,98-15,06 D-Limonene Gb, fhp absent/not known 15,92-16,04 Heptanoic acid Gb, fhp absent/not known 16,25- Not identified fhp absent/not known 14,73-14,86 Benzaldehyde,-2-hydroxy Gb, fhp absent/not known 14,98-15,06 D-Limonene Gb, fhp absent/not known 15,92-16,04 Heptanoic acid Gb, fhp absent/not known 16,25- Not identified fhp absent/not known

Continued

Table 8. Chemicals identified in all extracts obtained from male and female S. Frugiperda

131

Table 8 continued

16,44-16,49 Nonanal gb, fhp absent/not known 16,61-16,67 Methyllisourea hydrogen sulfate gb, fhp absent/not known 16,87-16,93 Not identified gb, fhp absent/not known 17,09-17,15 Methyl-2-methoxypropeonate gb, fhp absent/not known 17,20-17,21 2,6-Dimethyl-6-nitro-2-hepten-4-one gb absent/not known 17,46- Not identified gb absent/not known 17,59-17,61 Pyrido[1,2-a]azepine-6,7,8,9-tetracarboxylic acid,10- (benzoyloxy)-6,7-dhydro-tetramethyl ester fhp Fbody 17,68-17,89 Octanoic acid gb, fhp fpl, fbody 17,90-17,93 Ethanol,2-(2-butoxyethoxy)- gb, fhp absent/not known 17,98-17,99 1H-Indene,1-methylene gb, fhp absent/not known 18,16-18,19 Not identified fhp absent/not known 18,23-18,28 Decanal gb, fhp fpl, fbody 18,41-18,46 Benzothiazole gb, fhp absent/not known 1:0,83 1:0,22 0:0 18,52-18,58 Cyclohexane,isothiocyanato gb, fhp absent/not known 1:0,77 1:0,72 1:0 18,66-18,68 Not identified gb absent/not known 18,66-18,68 Not identified gb absent/not known 18,73-18,79 Formamide, N-cyclohexyl gb, fhp absent/not known 18,85-18,92 2-Propanol,1,1-[1-methyl-1,2-ethanediyl) bis (oxy)]bis gb, fhp absent/not known 18,89-19,95 Salicyl Alcohol/1,2-Benzenediol,3-methyl gb, fhp absent/not known 18,98-19,03 Hidroquinone / 1-Butanamine, N-butyl-N-nitroso- gb, fhp fpl, fbody 19,12-19,29 Nonanoic acid gb, fhp 19,30-19,38 4-Octene,2,3,6-trimethyl- /1-Undecyn-4-ol gb, fhp absent/not known 19,49-19,56 Formamide,N,N-dibutyl gb, fhp fpl 19,76- Not identified gb absent/not known 19,82-19,91 Benzenemethanol,3-hydroxy gb, fhp absent/not known 20,06-20,08 Benzaldehyde, 4-hydroxy gb, fhp absent/not known 20,28-20,38 Decanoic acid gb, fhp fpl, fbody 20,49-20,54 Propanoic acid,2-methyl,-3-hydroxy-2,4,4-Trimethylpentyl ester gb, fhp Fpl 20,60- Not identified gb absent/not known 20,79- Not identified gb absent/not known 20,82-20,88 9,9-Dimethoxybicyclo[3.3.1]nona-2,4-dione gb, fhp absent/not known 20,90-20,93 2-Propenal,3- (2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)- gb, fhp fpl, fbody 21,10-21,16 Hexanedioic acid, bis (1-methylethyl)ester gb, fhp absent/not known 21,20-21,21 Naphtalene,1,2,3,5,6,7,8a-octahydro-1,8a-dimethyl-7- (1-methylethenyl)-,[is-(1à,7à,8à)]- fhp absent/not known 21,24-21,28 5,9-Undecadien-2-one,6,10-dimethyl-,(E) / (Z) gb, fhp absent/not known 21,33-21,38 Undecanoic acid gb, fhp fpl

continued

132

Table 8 continued

21,52-21,58 2-Undecanol gb, fhp absent/not known 21,85-21,91 Urea, N'-cyclooctyl-N,N-dimethyl gb, fhp absent/not known Formamide,N-[1-[1-cyano-2- 21,93-21,99 methylpropyl)hydroxyamino]ethyl]- gb absent/not known 22,00-22,26 4-Methyl-5-decanol /3-Ethyl-4-nonanal gb, fhp fpl 22,28-22,36 Dodecanoic acid gb, fhp fpl 22,40- Not identified gb absent/not known 22,44-22,49 Limonen-6-ol, t-butyrate gb, fhp absent/not known 22,70-22,74 Not identified gb absent/not known 22,75-22,79 11-Dodecen-2-one,7,7-dimethyl- gb, fhp absent/not known Propanoic acid,2-methyl-1,-(1,1-dimethylethyl)-2- 22,81-22,88 methyl-1,3- propanediyl ester gb, fhp fbody 0,6 22,90-22,94 Tetradecane / Pentadecane gb, fhp absent/not known 2:1 0:1 0:0 23,03-23,05 Not identified gb absent/not known 23,07-23,13 Dibutyl 3-dydroxybutyl phosphate gb, fhp absent/not known 23,14-23,19 Not identified gb, fhp absent/not known 23,20-23,29 Benzene,[3-(2-cyclohexylethyl)-6-cyclopentylhexyl]- gb, fhp absent/not known Acetamide, N-methyl-N-[4-[4-methoxy-1- 23,45-23,64 hexahydropyridyl]-2-butynyl]- gb, fhp fpl 23,73-23,78 2-Propenoic acid, octyl ester gb, fhp absent/not known 23,80-23,85 Not identified gb, fhp absent/not known 23,98-23,99 Not identified gb, fhp absent/not known 24,01-24,09 Tridecane / Pentanal / Pentadecane /2-Dimethylphenol gb, fhp absent/not known 24,10-24,16 Tetradecanoic acid? gb, fhp fpl, fbody 24,17-24,22 Heptadecane, 2,6-dimethyl / Pentadecane gb, fhp absent/not known 24,25-24,30 Oleic acid? gb absent/not known 0,7 24,34-24,39 2,4-Diphenyl-4-methyl-2(Z)- pentane gb, fhp absent/not known 4:1 1:0,68 0:0 24,47-24,54 Not identified gb absent/not known 24,55-24,61 Tetradecanoic acid? gb, fhp fbody 24,70- Not identified gb absent/not known 24,74-24,78 Cyclohexane,1,4-dimethyl-2-octadecyl gb, fhp absent/not known 2H-Benzocyclohepten-2-one, decahydro-9a-methyl- 24,80-24,86 ,trans gb, fhp absent/not known 24,90-24,98 7-Heptadecyne,1-chloro- /2,4-Heptadienal,2,4-dimethyl- gb, fhp absent/not known Cholestan-3-ol,2-methylene-,(3á,5à)- /Hexanedioic acid, 25,00-25,02 bis(2-ethylhexyl)ester gb, fhp absent/not known 25,04-25,13 Acetic acid, (2-benzothiazolythio)- gb absent/not known 25,14-25,16 Not identified gb, fhp absent/not known 25,23-25,31 Not identified gb, fhp absent/not known 0,5 25,33-25,39 2,4-Diphenyl-4-methyl-1 (E)-pentene gb, fhp absent/not known 0:1 0,84:1 0:0 25,42-25,50 Not identified gb, fhp absent/not known

Continued

133

Table 8 continued

25,58-25,64 Octadecane, 6-methyl gb, fhp absent/not known Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2- 25,66-25,75 butynyl]-? gb, fhp absent/not known 25,82-25,89 Not identified gb absent/not known 1:0 1:0 0:0 25,91-25,96 2,4-Diphenyl-4-methyl-2 (E)-pentene gb, fhp absent/not known 0,17:1 0:1 1:0,29 26,09-26,15 Phenol,4-(1-methyl-1-phenylethyl)- gb absent/not known 26,43-26,50 Benzene,1-(1,5-dimethyl-4-hexenyl)-4-methyl gb absent/not known 26,51-26,57 7-Heptadecene,1-chloro / 9,12,15-Octadecatrienal gb, fhp absent/not known 26,65- Not identified gb absent/not known 26,78-26,79 Not identified gb absent/not known 26,84-26,86 L-Serine,o-(phenylmethyl)- fhp absent/not known 26,93-27,06 4-t-Butyl-2-(dimethylbenzyl)phenol gb, fhp absent/not known 1:0,56 0:1 1:0 27,10-27,18 Nonacosane /Dodecane,5,8-diethyl- gb, fhp fbody 27,26-27,27 Not identified gb absent/not known 27,34-27,35 2,5-Octadecadiynoic acid, methyl ester gb, fhp absent/not known 27,38-27,41 Not identified gb absent/not known 27,42-27,50 1-Acetoxy-2-(dodecyloxy)ethane gb absent/not known 27,52-27,59 9-Hexadecenoic acid / Oleic acid gb, fhp fpl 27,60-27,61 Not identified gb absent/not known 27,80-27,89 Hexadecanoic acid / Caryophyllene oxide? gb, fhp fpl, fbody 27,90-28,03 Hexadecanoic acid? gb, fhp fbody 28,08-28,14 Hexadecanoic acid / 2,5-Octadecadiynoic acid, methyl ester gb, fhp absent/not known 28,17-28,23 Not identified gb, fhp absent/not known 28,39-28,46 Not identified gb absent/not known 28,51-28,54 Androstan-3-ol,(3á,5à) fhp absent/not known 28,78-28,79 Not identified gb, fhp absent/not known 28,83-28,89 1-Phenantrenecarboxilic acid,... gb, fhp absent/not known 28,90-28,95 Podocarp-7-en-3à-ol,13,13-dimethyl fhp absent/not known 28,97-29,06 Not identified gb absent/not known 29,11-29,16 Not identified fhp absent/not known 29,25-29,29 Nonadecane? gb fpl 29,36-29,39 Nonadecane? gb absent/not known 1:0,00 0:0 0:0 0,75: 29,40-29,62 2-t-Butyl-4-(dimethylbenzyl)phenol gb, fhp absent/not known 1:0,72 1 0:0 29,63-29,69 Not identified gb, fhp absent/not known 29,82-29,93 Not identified gb, fhp absent/not known 30,38-30,45 Not identified gb, fhp absent/not known 30,52-30,64 2(1H) Phenantrenone, 3,4,4a.... gb, fhp absent/not known 1:0,6 30,92-31,18 2,6-Bis(t-butyl)-4-(dimethylbenzyl) phenol gb, fhp absent/not known 1:0,50 3 0:0 31,23-31,29 Not identified gb, fhp absent/not known 31,36-31,38 Octadecadiynoic acid, methyl ester gb absent/not known

Continued

134

Table 8 continued

31,44-31,47 Not identified gb, fhp absent/not known 31,75-31,80 Not identified fhp absent/not known 31,87- 10-Heptadecen-8-ynoic acid, methyl ester (E)- fhp absent/not known 31,94-31,99 Retinol acetate gb, fhp absent/not known 32,02-32,10 Not identified gb absent/not known 1:0 0:0 1:0 32,14-32,19 Benzene,(1-ethyl-2-propenyl)- gb, fhp absent/not known 32,21-32,50 Nonadecane? gb, fhp absent/not known 32,54-32,59 Not identified gb absent/not known 32,66-32,67 Not identified gb absent/not known 32,71-32,79 9,12-Octadecadienoic acid (Z,Z)- gb, fhp absent/not known 32,80-32,90 8,11,14-Ericosatrienoic acid, (Z,Z,Z)- / 9,12,15-Octadecatrienal gb, fhp absent/not known 9,12,15-Octadecatrienoic acid methyl ester, (Z,Z,Z)- / (Z)6,(Z)9- 32,91-32,98 Pentadecadien-1-ol gb, fhp fpl 33,00-33,08 9,12-Octadecadienoyl chloride, (Z,Z)- gb, fhp absent/not known 33,09-33,14 Not identified gb, fhp absent/not known 33,45- Not identified fhp absent/not known 33,92-33,97 Oleic acid? fhp fpl 34,49- Not identified gb absent/not known 34,81-34,87 Not identified gb absent/not known 35,10-35,14 Not identified fhp absent/not known 35,17- Not identified fhp absent/not known 36,01-36,17 Pentadecane /Nonadecane? fhp absent/not known 36,25-36,47 Nonadecane? gb absent/not known 1:0 1:0 0:0 36,52-36,59 Not identified fhp absent/not known 37,33-37,41 Not identified gb absent/not known 37,64-37,69 Nonadecane? gb absent/not known 37,93-37,99 Not identified gb, fhp absent/not known 38,04-38,09 Not identified fhp absent/not known 38,12-38,16 Not identified fhp absent/not known 41,12- Not identified fhp absent/not known 41,66-41,73 Not identified gb, fhp absent/not known 41,84-41,88 Nonadecane? gb, fhp absent/not known 41,90-41,94 Not identified gb absent/not known 1:0 0:0 0:0

gb : genital hairpencils fhp : femoral hairpencils fpl : female prothoracic leg fbody : female body

135