Endocrine control of cuticular hydrocarbon profiles during worker-to-soldier differentiation in the termite Reticulitermes flavipes

The social organization of termites, unlike that of other social insects, is characterized by a highly plastic caste system. With the exception of the alates, all other individuals in a colony remain at an immature stage of development. Workers in particular remain developmentally flexible; they can switch castes to become soldiers or neotenics. Juvenile hormone (JH) is known to play a key role in turning workers into soldiers. In this study, we analyzed differences in cuticular hydrocarbon (CHC) profiles among castes, paying particular attention to the transition of workers to soldiers, in the subterranean termite species Reticulitermes flavipes. CHCs have a fundamental function in social insects as they serve as cues in inter- and intraspecific recognition. We showed that (1) the CHC profiles of the different castes (workers, soldiers, nymphs and neotenics) are different and (2) when workers were experimentally exposed to a JH analog and thus induced to become soldiers, their CHC profiles were modified before and after the worker-presoldier molt and before and after the presoldier-soldier molt.     

Caste-biased locomotor activities in isolated termites

Eusocial insects are characterized by a well-developed division of labour among castes. Although the successful division of labour should stem from behavioural differentiation depending on caste identity, caste-specific intrinsic behavioural characteristics might be masked by social interactions within colonies. The present study explores caste-specific intrinsic locomotive activities of termites by quantifying them in isolation. We track individual movement trajectories of the damp-wood termite Hodotermopsis sjostedti over 30 min and extract individual locomotion parameters. Multivariate statistical analyses reveal significant differences among castes: soldiers move more actively than workers and neotenic reproductives. The morphometric data of test individuals indicate that locomotor activities reflected caste identity more strongly compared with quantitative morphological variations among individuals. We find that the different locomotor activities of soldiers compared with those of neotenics and workers probably reflect their physiological differentiation. The present study provides a basis for a deeper understanding of the roles of individual locomotor activities in social behaviours.     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several “key genes” were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.     

Soldier‐biased gene expression in a subterranean termite implies functional specialization of the defensive caste

In a termite colony, reproduction is typically monopolized by a small number of sexuals that are supported by reproductively altruistic soldiers and workers. We expect caste differentiation to be associated with clear‐cut differences in gene expression, and for these differences to reflect caste function and development. Here, we use RNA‐Sequencing to compare the gene expression profiles of sexual nymphs and two non‐reproductive helper castes (i.e., workers and soldiers) of the Eastern subterranean termite Reticulitermes flavipes. We found that of n = 93 genes that are strictly expressed as a function of caste, a majority (78%) show a soldier‐specific pattern. This conspicuous soldier‐bias in genome‐wide expression suggests that this defensively specialized caste is functionally well‐differentiated from both the reproductive and the other non‐reproductive caste of this species, despite a shared developmental program with workers. Gene ontology analysis supports the notion of functional specialization by soldiers, as soldier‐biased gene sets are enriched for novel biological processes. Whether this pattern reflects ancient or more recent bouts of selection for caste novelty at the gene‐regulatory level is not known, but because soldiers are sterile and thus have no direct fitness, any selection for novelty must have been mediated indirectly, through reproducing relatives.     

DNA methylation in the termite <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

Social insects are key examples of organisms that display polyphenism. Their genomes encode instructions for the development of multiple phenotypes, known as castes, which typically have highly divergent morphology, physiology and behaviour. DNA methylation, an epigenetic mechanism associated with modulation of gene expression in various eukaryotes, has recently been shown to provide a key link between environmental cues and caste-specific gene expression in honey bees (Hymenoptera). In termites—a major social insect group phylogenetically distant from Hymenoptera—the existence of DNA methylation has not, to our knowledge, been reported to date. Since genes encoding key DNA methylation enzymes are known to be absent in the genomes of a number of insect species, we sought to test whether termites are able to methylate their DNA, and, if so, whether caste-specific patterns of DNA methylation exist. We performed methylation-specific amplified fragment length polymorphism on the termite Coptotermes lacteus, and found evidence for DNA methylation. However, a comparison of methylation levels in different castes did not reveal any significant differences in methylation levels. The demonstration of DNA methylation in termites sets the stage for future epigenetic studies in these important social insects.     

Immunomarking reveals food flow and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Trophallaxis and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar), were examined using a novel marking technique, rabbit IgG protein coupled with an enzyme linked immunosorbent assay (ELISA) to detect the marker. Transfer experiments in small dishes evaluated the trophallactic transfer of the marker from donor workers fed IgG-treated paper to recipient workers or larvae. Worker donors rapidly acquired the marker, and 100% of donors tested positive within 24 h. Trophallactic transfer from donors to recipients was relatively inefficient, and 51 +/- 2% of recipient workers and 31 +/- 2% of recipient larvae tested positive at 72 h. Based on the mean optical density counts, approximately 27% of marker ingested by the donors was passed on to the recipient workers in the first 24 h, 14% to recipient larvae, and 26% to recipient soldiers. The ability of soldiers to feed independently of workers was examined in dish assays. Soldiers showed no significant uptake of the marker when isolated from the workers, and uptake increased significantly when workers were present. The distribution of the marker was further studied in larger colony fragments composed of workers, soldiers, nymphs, and larvae. Marker acquisition by the different castes/developmental stages was highly variable, with workers and nymphs acquiring the marker at a faster rate than soldiers and larvae. The results of this study contribute to our understanding of the foraging ecology and social behavior in R. flavipes. In addition, they may help design improved control programs for subterranean termites based on baits.     

Soldier-specific modification of the mandibular motor neurons in termites

Social insects exhibit a variety of caste-specific behavioral tendencies that constitute the basis of division of labor within the colony. In termites, the soldier caste display distinctive defense behaviors, such as aggressively attacking enemies with well-developed mandibles, while the other castes retreat into the colony without exhibiting any aggressive response. It is thus likely that some form of soldier-specific neuronal modification exists in termites. In this study, the authors compared the brain (cerebral ganglion) and the suboesophageal ganglion (SOG) of soldiers and pseudergates (workers) in the damp-wood termite, Hodotermopsis sjostedti. The size of the SOG was significantly larger in soldiers than in pseudergates, but no difference in brain size was apparent between castes. Furthermore, mandibular nerves were thicker in soldiers than in pseudergates. Retrograde staining revealed that the somata sizes of the mandibular motor neurons (MdMNs) in soldiers were more than twice as large as those of pseudergates. The enlargement of MdMNs was also observed in individuals treated with a juvenile hormone analogue (JHA), indicating that MdMNs become enlarged in response to juvenile hormone (JH) action during soldier differentiation. This enlargement is likely to have two functions: a behavioral function in which soldier termites will be able to defend more effectively through relatively faster and stronger mandibular movements, and a developmental function that associates with the development of soldier-specific mandibular muscle morphogenesis in termite head. The soldier-specific enlargement of mandibular motor neurons was observed in all examined species in five termite families that have different mechanisms of defense, suggesting that such neuronal modification was already present in the common ancestor of termites and is significant for soldier function.     

Identification of a juvenile hormone binding protein in the castes of the termite,Reticulitermes flavipes,by photoaffinity labeling

Hemolymph proteins of the Eastern subterranean termite, Reticulitermes flavipes (Isoptera, Rhinotermitidae, Rhinotermitinae) were examined from sterile and reproductive castes using native and denaturing polyacrylamide gel electrophoresis (PAGE). A high-mass protein (ca. 700 kDa) exhibited specific, JH III-displaceable photoaffinity labeling with [³H]EFDA, a diazoacetate analog of JH III. This protein was present in each termite caste, and had the characteristics of a glycosylated lipoprotein, i.e. a lipophorin. The JH-binding subunit of this protein showed a molecular size of 230 kDa using SDS-PAGE. The differences in the hemolymph proteins present in the soldiers, workers, larvae, nymphs, and replacement reproductives of this rhinotermitid are discussed.     

Developmental regulation of caste-specific characters in social-insect polyphenism

Phenotypes of organisms are not determined completely genetically, but vary according to environmental factors (phenotypic plasticity). Some organisms express several discrete adaptive phenotypes (polyphenism). Social insects possess a few types of individuals (castes) in their colonies, to which specific tasks are allocated. Here, I review studies on caste polyphenism in ants and termites, in terms of the developmental mechanisms of caste-specific characters, such as alate wings and soldier mandibles. In ants, the developmental fate of caste is probably determined by the pattern-formation genes in the early stage of postembryonic development, but apoptotic degeneration occurs in the wing primordia of future workers. As apoptotic wing degeneration has been observed in two phylogenetically distant groups of ants, this phenomenon is suggested to be conserved in many ant species. On the other hand, all termite species possess distinct sterile soldiers with specific morphologies suitable for defense. Recent studies using molecular techniques isolated genes related to soldier differentiation and analyzed the expression profiles of those genes in order to understand the mechanism of caste differentiation and the link between molecular and social evolution. In this review, I focus on these studies, in terms of the alteration of body plan in response to environmental signals, and discuss the evolutionary process of the interaction between ontogeny and environment.     

小头钩白蚁巢结构研究

【目的】了解小头钩白蚁Ancistrotermes dimorphus Tsai et Chen的蚁巢结构、各品级在腔室中的分布和巢群个体数量,为有效防控该种白蚁提供科学依据。【方法】采用蚁巢定位和巢体结构解剖法研究蚁巢结构;采用统计法确定各品级在腔室中的分布和巢群个体数量。【结果】小头钩白蚁巢由腔室、菌圃、蚁路、王室、候飞室和分飞孔六部分组成;巢群个体数量为12 255~24 230头(含卵粒);卵、幼蚁、若蚁、工蚁、兵蚁和有翅成虫分散分布在蚁巢腔室中,并不集中于某一特定区域。【结论】小头钩白蚁巢属于极端分散类型,巢群个体数量较少。     

尖唇散白蚁兵蚁和工蚁卵巢和卵子形态特征的比较

兵蚁和工蚁是白蚁中的非生殖品级,兵蚁是由工蚁分化产生。为了探讨兵蚁品级性腺不育的原因以及兵蚁和工蚁性腺发育的差异,采用组织学染色观察与测量方法对尖唇散白蚁Reticulitermes aculabialis Tsai et Hwang的兵蚁和工蚁的卵子发生各阶段进行比较和分析。结果显示:两者性腺发育大小呈极显著性差异(P<0.01),兵蚁与工蚁卵巢横切面面积之比约为1∶7;兵蚁的卵子发生与工蚁相比,仅有卵母细胞的分化期,没有生长期;兵蚁的卵母细胞比工蚁的小,两者的分化期卵母细胞体积之比约为1∶16。该结果表明工蚁向兵蚁转化过程中性腺进一步退化,兵蚁性腺极度退化使其丧失了成为补充繁殖蚁的可能性。该研究结果为兵蚁不能生殖提供组织学上的证据,同时又揭示了兵蚁和工蚁在潜在生殖能力差异方面的组织学基础。     

Caste differences in feeding and trophallaxis in the western drywood termite, Incisitermes minor (Hagen) (Isoptera, Kalotermitidae)

Summary: Differences in feeding and trophallaxis among castes of the western drywood termite, Incisitermes minor (Hagen), were determined using rubidium (Rb) as a tracer. Both 5th- and 6th-instar nymphs and 3rd- and 4th-instar larvae fed directly on Rb-treated paper and acted as both donors and recipients in trophallactic exchanges with other larvae and nymphs, and as donors for soldiers and alates. Soldiers and alates did not feed on Rb-treated paper suggesting that they do not feed directly on wood and are completely dependent on nymphs for their nutritional needs. Larvae paired with Rb-fed nymphs received the greatest amount of Rb suggesting that there may be a hierarchy of trophallaxis in drywood termites. Transfer efficiency, the percentage of the total Rb intake of donors that is passed to the recipients, ranged from 1.1 % (nymphal donors to alate recipients) to 16.6 % (larval donors to nymphal recipients).     

Effects of Caste on the Expression of Genes Associated with Septic Injury and Xenobiotic Exposure in the Formosan Subterranean Termite

As social insects, termites live in densely populated colonies with specialized castes under conditions conducive to microbial growth and transmission. Furthermore, termites are exposed to xenobiotics in soil and their lignocellulose diet. Therefore, termites are valuable models for studying gene expression involved in response to septic injury, immunity and detoxification in relation to caste membership. In this study, workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus, were challenged by bacterial injection or by no-choice feeding with a sublethal concentration (0.5%) of phenobarbital. Constitutive and induced expression of six putative immune response genes (two encoding for lectin-like proteins, one for a ficolin-precursor, one for the Down syndrome cell adhesion molecule, one for a chitin binding protein, and one for the gram-negative binding protein 2) and four putative detoxification genes (two encoding for cytochrome P450s, one for glutathione S-transferase, and one for the multi antimicrobial extrusion protein), were measured via quantitative real time polymerase chain reaction and compared within and among 1) colonies, 2) treatment types and 3) castes via ANOVA. Eight genes were inducible by septic injury, feeding with phenobarbital or both. Colony origin had no effect on inducibility or differential gene expression. However, treatment type showed significant effects on the expression of the eight inducible genes. Caste effects on expression levels were significant in five of the eight inducible genes with constitutive and induced expression of most target genes being higher in workers than in soldiers.     

CYP4AB1, CYP4AB2, and Gp-9 gene overexpression associated with workers of the red imported fire ant, Solenopsis invicta Buren

Two cytochrome P450 genes, CYP4AB1 and CYP4AB2, and the Gp-9 gene were identified as being specifically overexpressed in workers of the red imported fire ant using PCR-selected subtractive hybridization and cDNA array techniques. Full-length CYP4AB1 and CYP4AB2 were cloned and sequenced. The cDNA sequences of CYP4AB1 and CYP4AB2 have open reading frames of 1389 and 1533 nucleotides encoding proteins of 463 and 511 amino acid residues, respectively. Northern blot analysis was performed to compare expression levels of CYP4AB1, CYP4AB2, and Gp-9 for different developmental stages and castes of fire ants. We demonstrate that the expression of these three genes is developmentally and caste specifically regulated in red imported fire ants. Levels of CYP4AB1 mRNA were undetectable in 3rd+4th instars, worker pupae, and alate (mixed sex) pupae; readily detectable in male and female alates; increased (approximately 3-fold) in the queens; and rose to a maximum (13-fold) in workers. Similarly, the expression of CYP4AB2 mRNA was undetectable in 3rd+4th instars, worker pupae, and alate pupae; low in male and female alates and queens; and increased (approximately 7-fold) in workers. Levels of Gp-9 mRNA were readily detectable in male alates; increased (approximately 3-fold) in female alates; and reached a maximum (approximately 12-fold) in workers. Their caste-specific overexpression suggests the functional importance of CYP4AB1, CYP4AB2, and Gp-9 in workers of the red imported fire ant.     

Intra- and interspecific agonistic behavior of the subterranean termite Microcerotermes crassus (Isoptera: Termitidae)

The aim of our study was to investigate the intra- and interspecific agonistic behaviors exhibited by the worker and soldier castes of the subterranean termite Microcerotermes crassus Snyder (Isoptera: Termitidae). Aggression between M. crassus colonies from different field locations and also against three termite species--Coptotermes gestroi (Wasmann), Globitermnes sulphureus Haviland, and Odontotermes sp.--were observed in the laboratory. Termite responses were tested in paired combination of castes (soldiers versus soldiers, soldiers versus workers, and workers versus workers) consisting of 10 individuals each. Significant agonistic behaviors were observed only in encounters between pairings of different termite species. M. crassus was aggressive toward individuals from different species but not toward individuals from different M. crassus colonies. Mortality of M. crassus reached 100% in most of the interspecific encounters. However, no or low mortality was recorded in the intraspecific pairings.     

Response to orphaning in two Neotropical termites: shape Armitermes euamignathus and shape Embiratermes festivellus

In this paper we examine the potential of the termites Armitermes euamignathus Silvestri: 1901 and Embiratermes festivellus (Silvestri, 1901) (Isoptera, Termitidae, Nasutitermitinae) to produce neotenics experimentally. Three nests of the mound-building termite A. euamignathus, from the Brazilian cerrado, had their primary queens removed in August 1994. After 12 months, only one mound survived; it had a normal appearance. In this healthy, orphaned colony we found the primary king, six physogastric nymphoid female replacement reproductives, two ergatoid female replacement reproductives, 46 nymphs, several presoldiers, soldiers, workers, larvae and many eggs. These data show that neotenics in A. euamignathus may originate from both workers and nymphs, but nymphoids are produced in larger numbers. The biometric study of nymphs and nymphoids suggests that these brachypterous neotenics were derived from third instar nymphs after a single moult or from four instar nymphs after a reduction of wing bud length. A piece of an E. festivellus nest with some third instar nymphs, soldiers and workers was kept under laboratory conditions. After 12 months, the whole experimental subcolony was examined and appeared to contain two pigmented nymphoid females, two pigmented nymphoid males, only one larva, seven nymphs of the same instar, 148 workers, five soldiers and many eggs. These results also indicate the capacity of the termite E. festivellus to produce nymphoid neotenics. These neotenic females were laying eggs, but they were not physogastric after a year, unlike some nymphoids of the same species collected from natural colonies.     

Caste morphology and development in Termitogeton nr. planus (Insecta,Isoptera, Rhinotermitidae)

The termite family Rhinotermitidae displays a wide diversity in its patterns of social organization in castes. The genus Termitogeton probably branched off early in the evolution of this family. We studied the developmental pathways of a putative undescribed species from New Guinea, Termitogeton nr planus. The development begins with two white inactive instars (larvae), the second of which possesses small wing buds. These are followed by a relatively homogenous group of active immature stages (pseudergates) among which a biometric study revealed the presence of four instars. The first of these instars possesses wing buds that regress at subsequent molts. The external morphology of older instars resembles that of higher termite workers. Older pseudergates can differentiate into presoldiers and then into soldiers, but they are also able to molt into a unique stage with long wing pads (nymph), preceding the imago. Colony maturity can be reached with about 10(3) individuals. The sex ratio is near 1:1 in all castes except soldiers, among which females are more numerous than males. The caste pattern of T. nr. planus is reminiscent of those of the Kalotermitidae and Termopsidae, and of Prorhinotermes among the Rhinotermitidae. None of these taxa possess a true worker caste, permanently diverted from imaginal development: social tasks are done by unspecialized immatures that retain a full array of developmental options, including that of proceeding to the imago. The most remarkable trait of T. nr. planus is the presence in all second-instar individuals of wing buds that later regress to reappear in the single nymphal stage. We suggest that the traditional definitions of the terms larvae, nymphs, and pseudergates should be revised because they cannot be satisfactorily applied to the castes of Termitogeton.