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Blackwell Science, LtdOxford, UKEREEcological Research1440-17032004 Ecological Society of Japan2004192141148Original ArticleCaste polyphenism in social insectsT. Miura

Ecological Research (2004) 19: 141–148

Special Submission from the Winner of the 7th Denzaburo Miyadi Award

Proximate mechanisms and evolution of caste polyphenism in social insects: From sociality to genes Toru MIURA* Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan

Evidence has accumulated over several decades to prove the kin selection theory of evolution of social insects, however, proximate mechanisms of social behavior, and/or caste differentiation remain obscure. Genes that regulate these mechanisms are apparently selected through kin selection, and organisms have consequently acquired sociality. Here, I will discuss several studies that were performed recently by Matsumoto Laboratory, University of Tokyo, Tokyo, Japan, in various social insects, such as termites and ants, in order to reveal the regulatory mechanisms of social behavior and the evolutionary processes of sociality. First, I will review the foraging behavior of the black marching termite Hospitalitermes medioflavus, where well-organized task allocation among castes is apparent. This suggests that regulation of postembryonic development is important in social behavior. Next, I will summarize recent progress in identifying caste-specific gene expression in the damp-wood termite Hodotermopsis sjostedti. This constitutes the basis for molecular mechanisms of caste differentiation, and moreover, the genes identified might be good markers for social evolution. Finally, the mechanism underlying winglessness in ant workers is reviewed. Apoptotic cell death was detected at the stage of pupation in wingless worker castes. Furthermore, the areas of study recently designated as ‘sociogenomics’ and ‘ecological developmental biology’ are discussed. Key words: caste differentiation; ecological developmental biology; polyphenism; social behavior; sociogenomics.

Introduction

Fig. 1. Dr T. Miura.

In eusocial insect colonies, there are several morphologically and behaviorally distinct types of individuals, which cooperate with each other in a nest, and to which tasks of the colony are allocated. These functional types of individuals, termed ‘castes’, are well-known in eusocial insects, and some of the castes are sterile and work as helpers for repro-

ductive individuals. These traits of sterile castes are thought to have evolved through the process of kin selection. Most of the studies in sociobiology in social insects have focused on the ultimate factors of social evolution since Hamilton’s rule was described (Hamilton 1964), however, little has been studied on the proximate factors regulating the caste differentiation systems. In this review, I will summarize the social behavior of a termite species, demonstrating elaborate task allocation and the relationship between the division of labor and caste developmental systems. Next, I will discuss intrinsic factors regulating caste differentiation, and introduce our trials identifying caste-specific gene expression. In addition, hormonal controls and

*Email: [email protected] Received 1 October 2003, Accepted 29 October 2003.

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morphogenesis engaged in the caste development will be discussed. Finally, I will discuss our study on the developmental reprogramming of wings during caste differentiation of ant workers.

Organization of social behavior in termites In the social behavior of some eusocial insects, there are elaborate systems of task allocation (Wilson 1971). In attine ants, for example, there are 29 tasks allocated to various morphological types of workers (Wilson 1980). The castes are primarily divided by morphological characteristics, but in some cases, especially in social hymenoptera, tasks are allocated according to the age of individuals; this is referred to as ‘age polyethism’ or ‘temporal polyethism’. In the case of termites, another group of major eusocial insects, age polyethism is more complicated because termites are hemimetabolous insects, and therefore immature individuals have a similar body form to that of adult insects, so that they can behave as ‘workers’ (terminology according to Thorne 1996). Therefore, the postembryonic developmental process of termites is very important in understanding the social behavior. However, there are few studies on the behavior of termites because most termite species live cryptically in nests, and therefore observations of termites under natural conditions are relatively difficult. In several species of termites, however, colony members forage in the open air, coming out of their nests, thereby facilitating observations of their natural foraging behavior. The termite genus Hospitalitermes (Termitidae, Nasutitermitinae) is one of the open-air foraging termite groups, and is distributed throughout Southeast Asia (Tho 1992). The foraging behavior usually starts in the evening. First, soldiers guarding the nest entrances come out of the nest and initiate the foraging activity. Soldiers play the role of scouts, leading the foraging column composed of large numbers of individuals to the foraging sites. Foraging sites are normally located at the surface of tree trunks or fallen logs. They feed on lichens spreading over the surface of those trees. Lichens are rich in nitrogen, and are therefore an important food source for this termite species (Miura & Matsumoto 1997). At the foraging sites, workers of this termite show conspicuous cooperative behavior. Most of the workers spread over the sites and gnaw food materials, while some workers stand still among those gnawing workers. Gnawing individuals intermittently pass small pieces of food material to the waiting workers, from mouth to mouth (Fig. 2a). When the amount of food material gathered by workers reaches an appropriate size, the waiting workers carry those food balls back to their nests. We collected gnawing individuals

Fig. 2. Foraging behavior and division of labor in Hospitalitermes medioflavus. (a) Cooperative behavior in making food balls can be seen among workers at the foraging sites. (b) Scanning electron microscopy of head capsules of a monomorphic soldier, and three types of workers, which participate in the foraging behavior. (c) The system of task allocation (division of labor) can be depicted as ‘sexual and temporal polyphenism’, in which caste developmental pathways are very important in termite social behavior.

(gnawers) and carrying workers (carriers) separately, and measured the head width in order to discriminate morphological differences between the two tasks. Consequently, three types of workers could be distinguished (i.e. major, medium and minor workers). Carriers comprised major and medium workers, while gnawers comprised medium and minor workers. Medium workers were apparently engaged in both tasks (Fig. 2b; Miura & Matsumoto 1995, 1998). The question then arose regarding the relationships among these three types of workers. Termites are hemimetabolous insects, and there are males and females in sterile castes (Noirot 1969; Roisin 2000). Therefore, we tried to discriminate instars and sexes of all castes in colonies of H. medioflavus. Head width of all castes and immature instars in colonies was measured, and the sexes of those individuals were also determined. In addition, the number of antennal segments and direct observations of molting individuals also contributed


strong evidence about caste developmental pathways (Miura et al. 1998). We concluded that the caste developmental scheme of this termite species could be described as illustrated in Fig. 2c. Both sexes have two successive larval instar stages, after which male individuals become minor workers. Then, the male minor workers differentiate into soldiers, via a presoldier stage. In contrast, female second instar larvae molt into medium workers, and then successively molt into major workers. This schema indicates that task allocation in foraging activity is performed according to developmental stage (instar) and sex of each individual. Therefore, the division of labor observed in this termite can be termed ‘temporal and sexual polyethism’. As seen in this system of social behavior, the regulation of postembryonic development of the colony members is very important for the social system of termites.

Mechanisms of caste polyphenism in termites Caste differentiation is one of the phenomena of ‘polyphenism’, which is a special form of phenotypic plasticity (Nijhout 1994, 1999). Polyphenism is sometimes visualized as a reaction norm with a threshold function, so that a switch of discrete phenotype takes place according to environmental stimuli, and then differential pathways of development result in different phenotypes. The switching mechanisms are thought to have evolved as the result of adaptations, such as seasonal polyphenism in butterflies and phase polyphenism in locusts (Nijhout 1999, 2003). Among many cases of insect polyphenism, caste polyphenism in social insects is remarkable because multiple phenotypes (i.e. castes) appear in the same generation and cooperate with each other. In addition, in terms of the switching trigger, not only environmental factors such as temperature and daylength, but also interactions among individuals (social interactions) affect the expression of those phenotypes (Noirot 1991). Feedback systems must exist that regulate the caste ratio in the colonies. Caste-specific morphogenesis As mentioned above regarding the caste developmental pathways of Hospitalitermes, there is a specific molting event in terms of dynamic morphological changes. In the termite species, male minor workers differentiate into soldiers, via a presoldier stage. At the time of molting from minor worker to presoldier, the morphology of the head capsule is greatly altered. Thus, dynamic morphogenesis should occur prior to the molt to pre-

Caste polyphenism in social insects 143

soldier. Indeed, we observed the process of morphogenesis under the cuticle of minor workers, and found that there was a specific apparatus at the site of the future frontal gland of this termite soldier (Fig. 3a,b; Miura & Matsumoto 2000). We termed the precursor of the frontal gland (nasus) the ‘nasus disc’, because this structure is similar to those of imaginal discs seen in holometabolous insects. Not only Hospitalitermes, but also almost all species of termites possess soldiers with conspicuous defensive morphology. Many of them are categorized as ‘biting soldiers’, having a pair of mandibles developed as weapons against predators (Koshikawa et al. 2002). Under the cuticle of mandibles in pseudergates of Hodotermopsis sjostedti, newly formed soldier mandibles are observed with numerous folding structures prepared for the dynamic morphogenesis from pseudergate to presoldier (Fig. 3c,d; Koshikawa et al. 2003). Juvenile hormone action in caste differentiation The dynamic morphogenesis in caste differentiation takes place according to extrinsic factors such as social interactions. Linking the extrinsic factors and the actual caste differentiation, however, there must be some physiological mechanism that converts environmental factors into the caste-specific developmental processes (Miura 2001). The most well-known physiological mediator that plays important roles in caste differentiation is juvenile hormone (JH). This insect hormone is known to regulate insect development, and there are many reports on its role in caste differentiation in social insects (Nijhout 1994; West-Eberhard 2003). In the case of termites, the JH titer of the previous larval stage determines the differentiation fate of the subsequent postembryonic stage (Nijhout & Wheeler 1982). In addition, soldier differentiation can be easily induced by application of JH and its analogs (Howard & Haverty 1979). In a previous study by our laboratory on caste induction in Zootermopsis nevadensis, soldier traits were induced by JH application, even from the developmental line to winged alates (Miura et al. 2003). The developmental program into alternative phenotypes should be controlled by the timing of hormonal secretion and sensitivity to it (Nijhout 2003). However, the detailed molecular mechanism of JH signaling is unclear, and some recent studies have suggested that the mode of JH action may involve a lipid signaling system (reviewed in Wheeler & Nijhout 2003). Differential gene expression With respect to the process of caste differentiation, there must be an array of gene expression that generates the caste-specific phenotypes. Specific gene expression

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Fig. 3. Morphogenesis in soldier formation. (a) Under the cuticle of a molting male minor worker of Hospitalitermes medioflavus, a folding structure of the future nasus can be seen. (b) At exuviation to the presoldier stage, the folding structure is expanded to form the soldier nasus. (c) During soldier differentiation in Hodotermopsis sjostedti, which has mandibulate soldiers, a similar folding structure is seen, especially in mandibles. (d) The most folded part of the mandibles explains the allometric changes during soldier differentiation.

that appears only in a certain caste can be identified by recent molecular techniques. We have identified several gene candidates required for caste differentiation and maintenance of caste systems in social insects. Figure 4a schematically shows gene expression during the process of soldier differentiation in our focal termite species H. sjostedti. In this species, soldiers are differentiated from 7th instar individuals, which play a worker role (Miura et al. 2000), and thus the caste is termed ‘pseudergate’ (i.e. false worker; Grassé & Noirot 1947). In this stage prior to caste differentiation, specific genes are thought to be expressed, which are repressed at the onset of soldier differentiation. The onset of differentiation is thought to be triggered by JH, and then genes are switched on and expressed. Some genes might respond quickly, while some downstream genes are expressed slowly. Specific morphogenesis then occurs to produce soldier-specific morphology such as welldeveloped mandibles or nasus. After completion of the soldier differentiation, a specific set of genes might be expressed to maintain the soldier caste, and/or to pursue the soldier function. In a previous study by our

laboratory detecting soldier specific gene expression by differential display, in which mRNA was compared between the two tissues using reverse transcriptionpolymerase chain reaction (RT-PCR) (Liang & Pardee 1992), specific gene expression was detected. As we predicted (Fig. 4a), some genes were expressed relatively early in the stage of soldier differentiation, while others were expressed later (Fig. 4b). These included genes related to the metabolism of physiologically important molecules (R. Cornette, 2003, personal communication), morphogenesis and insect growth (S. Koshikawa, 2003, personal communication). One gene, termed SOL1, was identified as the terminally expressed gene in mature soldiers. SOL1 is expressed only in mature soldiers, and not during the course of soldier differentiation, indicating that its product is specific for terminally differentiated soldiers (Miura et al. 1999). Further experiments indicated that this gene is expressed in the mandibular glands of soldiers, and encodes an ORF, which has slight sequence homology with the lipocalin protein family. Prediction of the 3D structure of the SOL1 protein strongly indi-



Fig. 4. Differential gene expression during soldier differentiation in termites. (a) A conceptual diagram showing gene expression in the course of differentiation. Genes can be discriminated by the timing of expression. (b) Actual gene expression detected by Northern blotting in soldier differentiation of Hodotermopsis sjostedti. The coracle gene, which is known as a member of the septate junction and plays an important role in Drosophila embryogenesis, is expressed at the early stage of soldier differentiation (b-i), while cuticle protein is expressed in the later stage (b-ii). The SOL1 gene is only expressed in the mature soldiers (b-iii), suggesting that this gene product has some function in mature soldiers. W, worker (pseudergate); 1 week, 1 week application of JHA; 2 week, 2 week application of JHA; PS, presoldier; S, soldier. Two-week application of JHA normally induces presoldiers from pseudergates.

Fig. 5. Mandibular glands develop through soldier differentiation (a, d: pseudergate; b, e: presoldier; c, f: soldier). Proteinaceous substance, which is stained with anti-SOL1 antibody, is secreted and preserved in the reservoir of the glands. The bar indicates 1 mm.

cated that it has the structure of lipocalin proteins, which contain a ligand binding pocket. We then made an antibody against a partial predicted peptide, which specifically identified the SOL1 protein of soldiers. Immunohistochemical observations revealed that the protein product was extensively produced and secreted from the mandibular glands, suggesting that this protein is one of the major components of the mandibular gland secretion (Fig. 5). Our preliminary results suggested that this protein product is transferred from soldiers to other colony members through trophallaxis. Therefore, we speculate that the SOL1 protein may

function as a pheromone-like molecule (T. Miura, 2003, unpublished data). Genome size of termites The genome size of termites is unknown, although genome sizes are estimated in some hemimetabolous insects. Termites belong to Dictyoptera (termites, cockroaches and mantids), and are related to orthopteran insects (Maekawa et al. 1999). The genomes of locusts (~5000 Mbp) and cockroaches (2000– 3000 Mbp) are very large (Animal Genome Size data-

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base, http://www.genomesize.com/), and therefore, the termite genome size is probably similar to them. Sequencing the entire genome of a termite species is difficult, however, expressed sequence tags (EST) analysis using microarray techniques is now applicable to various organisms including termites (e.g. Wu-Scharf et al. 2003), as gene expression profiles among termite castes were recently compared using gene array and quantitative RT-PCR methods (Scharf et al. 2003).

Caste development and winglessness in ant workers In the case of social hymenoptera, the mechanism of caste differentiation is largely different from that of termites. The major reason for this is that social hymenopterans are holometabolous. All of the castes, including sterile castes, are adult insects, which develop through larval and pupal stages as postembryonic development. Among the hymenopterans, ants (Family Formicidae) possess remarkably differentiated morphological castes, because workers basically lose their wings, while colonies can produce winged reproductives (alates) in most species. This indicates that ants possess the genetic potential to produce wings, although wing formation is suppressed in worker castes.


In ants, imaginal wing discs are typically found in both worker and queen larvae (Dewitz 1878; Wheeler & Nijhout 1981). Wing discs in queens develop synchronously with other imaginal discs through the prepupal stage, but disappear in workers before the pupal stage. In addition to the differentiation between workers and alates, some species have multiple discrete worker castes (subcastes) (e.g. major and minor workers). In such cases, there are apparently some switching mechanisms regulating the development of these caste phenotypes. In some ant genera, Abouheif and Wray (2002) demonstrated the caste-specific expression of several genes that regulate wing formation. With regard to these points of caste regulation, we focused on differential wing development among castes (including subcastes) in the ant species Pheidole magacephala, which has dimorphic workers (Fig. 6a). The determination between queen and workers occurs during oogenesis, where the juvenile hormone titer in the egg is the key to determining the caste fate between the two (Passera & Suzzoni 1979). The developmental switch between two worker castes occurs during postembryogenesis at the final instar. The larvae, which are provided protein-rich diets, maintain a high JH titer, which makes them continue to grow and transform into major workers (Passera & Suzzoni 1979; Wheeler & Nijhout 1983).

Fig. 6. (a) Dimorphic worker castes in Pheidole megacephala (drawn by M. Abe), and (b) a diagram of differential wing formation/ degeneration.



We performed extensive histological observations showing that both dimorphic workers possessed the mesothoracic wing discs (for forewings) as seen in the queen larvae, although they lose the metathoracic wing discs (Fig. 6b). In the final instar larvae of queens, wing discs gradually grow to form the sack-like shape. At the time of pupation, the wing epithelia evaginated outside of the wing sack and elongated to form the wing bud. The intercellular structures are then formed during the prepupal stage. In major workers, the mesothoracic wing disc grows like the queen wing discs, although they are smaller than those of queens. During the prepupal stage, evagination of the wing disc occurs, but the following structural formation does not occur, and thus the wing buds degenerate. In degenerating wing buds, hemocytes were observed in the evagination cavity. During this degenerating process, apoptotic cell death was detected by the TUNEL method, which detects DNA fragments produced in the course of apoptosis (S. Sameshima, 2003, unpublished data). In minor worker larvae, neither growth nor evagination of the wing discs occurs in prepupal stage. In this study on the degeneration process of wings, we showed the differential processes of wing formation according to various castes (Fig. 6b). Although JH switching, differential gene expression and the degeneration process responding to ecdysteroid titer are thought to be responsible for the developmental reprogramming, the relationship among them remains to be elucidated. Further studies integrating these components will enhance our understanding of the caste differentiation involved in wing polyphenism in ants.

Sociogenomics and ecological developmental biology In the study of social insects, new techniques analyzing molecular and genome levels have recently been applied to elucidate the ‘sociality’ of these insects. This indicates the integration of behavioral ecology and neurobiology, and this new study area has been termed ‘sociogenomics’ (Robinson 1999). As mentioned above regarding genetic analysis in termites, studies using EST in honeybees have been commenced (Whitfield et al. 2002). In addition, the genome project in honeybees is underway and will be completed by the end of 2003 (Robinson 2002). Using such genomic information, many problems in social insects that are currently obscure will be solved in the future. In the development of social insects, individuals can change their developmental pathways depending on environmental factors such as social interaction. Social insects and many other organisms can alter their developmental program to express alternative phenotypes

depending on environmental conditions. For example, some animals can change their phenotypes responding to chemicals released by their predators. They can develop abnormal structures for defense against those predators, which are thus termed predator-induced polyphenisms (Gilbert 2000). Thus, developmental processes in any organisms are determined not only by genetic information, but also by environmental information. In developmental biology, therefore, there is some movement to incorporate ecological aspects, and this new study area has recently been termed ‘ecological developmental biology’ (Eco-Devo; Gilbert 2001; Gilbert & Bolker 2003). The study on caste differentiation in social insects that I have described here can be also considered as part of this study area. Hence, such interdisciplinary studies on social insects will likely provide us new insights into the mechanisms and evolution of social systems in many social insects.

Acknowledgements I am grateful to Professor T. Matsumoto for his valuable comments throughout these studies. Thanks are also due to Dr R. Cornette, S. Sameshima, and S. Koshikawa for their comments on the manuscript, Professor T. Kubo for his instructions on molecular techniques, and M. Abe for her illustrations. The results presented here were obtained by collaborative work with T. Matsumoto, T. Kubo, S. Koshikawa, and S. Sameshima. These studies were supported by a Grantin-Aid for Scientific Research (Nos. 11691174, 13440228, 13740435 and 15687001) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Grant-in-Aid from the Research for the Future Program of the Japan Society for the Promotion of Sciences (JSPS).

References Abouheif E. & Wray G. A. (2002) Evolution of the gene network underlying wing polyphenism in ants. Science 297: 249–252. Dewitz H. (1878) Beiträge zur postembryonalen Giliedmassenbildung bei den Insekten. Zeitschrift für Wissenschaftliche Zoologie 30: 78–105 (in German). Gilbert S. F. (2000) Developmental Biology, 6th edn. Sinauer Associates, Massachusetts. Gilbert S. F. (2001) Ecological developmental biology: Developmental biology meets the real world. Developmental Biology 233: 1–12. Gilbert S. F. & Bolker J. A. (2003) Ecological developmental biology: preface to the symposium. Evolution and Development 5: 3–8. Grassé P. P. & Noirot C. (1947) Le polymorphisme social du termite à cou jaune (Calotermes flavicollis F.). Les faux-ouvriers ou pseudergates et les mues régressives. Comptes Rendus de l’Académie Des Sciences 224: 219–221 (in French).

148 T. Miura Hamilton W. D. (1964) The genetical evolution of social behaviour, I, II. Journal of Theoretical Biology 7: 1–52. Howard R. & Haverty M. I. (1979) Termites and juvenile hormone analogues: a review of methodology and observed effects. Sociobiology 4: 269–278. Koshikawa S., Matsumoto T. & Miura T. (2002) Morphometric changes during soldier differentiation of the damp-wood termite Hodotermopsis japonica (Isoptera: Termopsidae). Insectes Sociaux 49: 245–250. Koshikawa S., Matsumoto T. & Miura T. (2003) Mandibular morphogenesis during soldier differentiation in the damp-wood termite Hodotermopsis sjoestedti (Isoptera: Termopsidae). Naturwissenschaften 90: 180–184. Liang P. & Pardee A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967–971. Maekawa K., Kitade O. & Matsumoto T. (1999) Molecular phylogeny of orthopteroid insects based on the mitochondrial cytochrome oxidase II gene. Zoological Science 16: 175–184. Miura T. (2001) Morphogenesis and gene expression in the soldier-caste differentiation of termites. Insectes Sociaux 48: 216– 223. Miura T. & Matsumoto T. (1995) Worker polymorphism and division of labor in the foraging behavior of the black marching termite Hospitalitermes medioflavus, on Borneo Island. Naturwissenschaften 82: 564–567. Miura T. & Matsumoto T. (1997) Diet and nest material of the processional termite Hospitalitermes, and cohabitation of Termes (Isoptera: Termitidae) on Borneo Island. Insectes Sociaux 44: 267–275. Miura T. & Matsumoto T. (1998) Foraging organization of the open-air processional lichen-feeding termite Hospitalitermes (Isoptera, Termitidae) in Borneo. Insectes Sociaux 45: 17–32. Miura T. & Matsumoto T. (2000) Soldier morphogenesis in a nasute termite: discovery of a disk-like structure forming a soldier nasus. Proceedings of the Royal Society London B 267: 1185–1189. Miura T., Hirono Y., MacHida M., Kitade O. & Matsumoto T. (2000) Caste developmental system of the Japanese dampwood termite Hodotermopsis japonica (Isoptera: Termopsidae). Ecological Research 15: 83–92. Miura T., Kamikouchi A., Sawata M., Takeuchi H., Natori S., Kubo T. & Matsumoto T. (1999) Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae). Proceedings of the National Academy of Sciences USA 96: 13874–13879. Miura T., Koshikawa S. & Matsumoto T. (2003) Winged presoldiers induced by a juvenile hormone analogue in Zootermopsis nevadensis: implications for plasticity and evolution of caste differentiation in termites. Journal of Morphology 257: 22– 32. Miura T., Roisin Y. & Matsumoto T. (1998) Developmental pathways and polyethism of neuter castes in the processional nasute termite Hospitalitermes medioflavus (Isoptera: Termitidae). Zoological Science 15: 843–848. Nijhout H. F. (1994) Insect Hormones. Princeton University Press, Princeton, New Jersey.

Ecological Research (2004) 19: 141–148 Nijhout H. F. (1999) Control mechanisms of polyphenic development in insects. Bioscience 49: 181–192. Nijhout H. F. (2003) Development and evolution of adaptive polyphenisms. Evolution and Development 5: 9–18. Nijhout H. F. & Wheeler D. E. (1982) Juvenile hormone and the physiological basis of insect polymorphisms. Quarterly Review of Biology 57: 109–133. Noirot C. (1969) Formation of castes in the higher termites. In: Biology of Termites, Vol. 1 (eds K. Krishna & F. M. Weesner), pp. 311–350. Academic Press, New York. Noirot C. (1991) Caste differentiation in Isoptera: basic features, role of pheromones. Ethology, Ecology and Evolution 1: 3–7. Passera L. & Suzzoni J. P. (1979) La role de la reine de Pheidole pallidula (Formicidae, Myrmicinae) dans la sexualisation du couvain après traitement par l’hormone juvénile. Insectes Sociaux 26: 343–353 (in French with English abstract). Robinson G. E. (1999) Integrative animal behaviour and sociogenomics. Trends in Ecolgy and Evolution 14: 202–205. Robinson G. E. (2002) Sociogenomics takes flight. Science 297: 204–205. Roisin Y. (2000) Diversity and evolution of caste patterns. In: Termites: Evolution, Sociality, Symbioses, Ecology (eds T. Abe, D. E. Bignell & M. Higashi), pp. 95–119. Kluwer Academic Publishers, Dordrecht. Scharf M. E. W. U.,-, Pittendrigh B. R. & Benett G. W. (2003) Caste- and development-associated gene expression in a lower termite. Genome Biology 4: R62. Tho Y. P. (1992) Termites of Peninsular Malaysia. Malayan Forest Records No. 36 (ed. L. G. Kirton). Forest Research Institute, Malaysia. Thorne B. (1996) Termite terminology. Sociobiology 28: 253–263. West-Eberhard M. J. (2003) Developmental Plasticity and Evolution. Oxford University Press, New York. Wheeler D. E. & Nijhout H. F. (1981) Imaginal wing discs in larvae of the soldier caste of Pheidole bicarinata (Hymenoptera: Formicidae). International Journal of Insect Morphology and Embryology 10: 131–139. Wheeler D. E. & Nijhout H. F. (1983) Soldier determination in Pheidole bicarinata: Effect of methoprene on caste and size within caste. Journal of Insect Physiology 29: 847–854. Wheeler D. E. & Nijhout H. F. (2003) A perspective for understanding the modes of juvenile hormone action as a lipid signaling system. Bioessays 25: 994–1001. Whitfield C. W., Band M. R., Bonald M. F., Kumar C. G., Liu L., Pardinas J. R., Robertson H. M., Bento Soares M. & Robinson G. E. (2002) Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee. Genome Research 12: 555–566. Wilson E. O. (1971) The Insect Societies. Harvard University Press, Cambridge, Massachusetts. Wilson E. O. (1980) Caste and division of labor in leaf-cutter ants (Hymenoptera: Formicidae: Atta). Behavioral Ecology and Sociobiology 7: 143–156. Wu-Scharf D., Scharf M. E., Pittendrigh B. R. & Bennett G. W. (2003) Expressed sequenced tags from a polyphenic Reticulitermes flavipes cDNA library. Sociobiology 41: 479–490.