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.     

X‐ray computed tomography reveals that intraspecific competition promotes soldier differentiation in a one‐piece nesting termite

Investment in soldier production in eusocial lineages involves a trade‐off between maintenance costs and defense benefits. Termites are eusocial insects that live in colonies organized into three castes: primary reproductives, soldiers, and workers or pseudergates. Neotermes chilensis (Blanchard) (Isoptera: Kalotermitidae) is a one‐piece nesting termite that nests and forages in a single piece of wood. Two scenarios may be of importance in a defense context of one‐piece nesting termites: during swarms, when colonies may be invaded by winged termites (alates) in search of a place to found a new colony, and when colonies of conspecifics are present within the same substrate. It was hypothesized that the ratio of soldiers to non‐soldiers would be higher at the onset of the swarming period and in substrates bearing more than one termite colony. A method based on X‐ray computed tomography (CT) was developed to study gallery connectivity in colonies of N. chilensis and caste composition within colonies. Computed tomography allowed the digital reconstruction of the galleries within the substrate, even when they belonged to different colonies, and was effective in distinguishing termites from substrate, and soldiers from reproductives and pseudergates. Using CT, the ratio of soldiers to non‐soldiers was shown to be highest in colonies within multicolonial scapes (i.e., neighboring colonies were present in the same substrate) during the swarming season, thus supporting our initial hypotheses. These results constitute a unique example of induced defenses arising from intraspecific interactions in termites.     

Testing the role of cuticular hydrocarbons on intercolonial agonism in two subterranean termite species (Coptotermes) and their hybrids

Recognition of nestmates is an important function in many social insects, as it maintains colony integrity by preventing outsiders from entering the colony. Agonism usually results from the interaction of con-specific non-nestmate individuals in termite colonies. Previous studies hypothesized that the cuticular hydrocarbon (CHC) profile of individuals had a role in nestmate recognition. However, contradictory results from previous studies in some subterranean termites raise questions on the validity of the cuticular hydrocarbon hypothesis. In the current study, Coptotermes gestroi (Wasmann), Coptotermes formosanus Shiraki and their hybrids were reared in identical conditions from colony foundation. This approach eliminates sources of variability in their cuticular hydrocarbon profiles aside from a genetic component. The parental species displayed dissimilar profiles of predominant alkanes and methyl alkanes, but both hybrid types displayed an overlapping, intermediate profile of these CHC. The mixture of the most abundant CHCs alone did not determine kin recognition; while the two hybrid types’ CHC profiles converged, the hybrids still showed strong agonism. One of the hybrid mating types easily merged with C. formosanus, despite only partial genetic similarity and dissimilar cuticular profiles for the common alkanes and methyl alkanes. This study suggests that in Coptotermes termites, the variable abundance of the major alkanes and methyl alkanes commonly found in most Coptotermes species does not explain agonistic patterns, and that other factors such as possibly more complex but less abundant CHC are likely to be involved in colonial recognition.

     

Association between caste and genotype in the termite Mastotermes darwiniensis Froggatt (Isoptera: Mastotermitidae)

Abstract Termite workers and soldiers differ markedly in their morphology and behaviour. We sought evidence for genetic influences on caste determination in the giant northern termite, Mastotermes darwiniensis , by investigating if workers and soldiers from the same colony differed genetically. The genotypes of 795 termites from 11 distinct colonies were assayed at six polymorphic microsatellite loci. We found that the multilocus genotypes of workers and soldiers from 8 of the 11 colonies did not differ significantly. Thus, the majority of the data provided no evidence for a genetic association with caste in workers and soldiers of M. darwiniensis . However, the genotype frequencies of workers and soldiers from three colonies differed, suggesting that genotype is occasionally associated with caste in this species. The genetic differentiation of castes within these colonies could reflect differences in the propensities of termites with distinct genotypes to develop into particular castes and provide a selective advantage to colonies headed by multiple reproductives.     

Burial behaviour by dealates of the termite <Emphasis Type="Italic">Pseudacanthotermes spiniger</Emphasis> (Termitidae,Macrotermitinae) induced by chemical signals from termite corpses

In order to maintain healthy colonies, termite workers dispose of the cadavers of dead nest mates by cannibalism, burial, or necrophoresis. However, when multiple reproductives found a new colony by pleometrosis, there are no worker castes at the early stages of the foundation to eliminate or isolate the corpses. In this study, we showed that in young pleometrotic colonies, reproductives of Pseudacanthotermes spiniger had the ability to perform this task. Because of the claustral conditions, and the potential inability of the dealates to feed on their own, their behaviour was restricted to the burial of the cadaver within the initial chamber. This burial behaviour, previously not reported in the reproductive caste of termites, appeared to be induced by chemical signals released by the corpses during decomposition, among which various fatty acids, indole and phenol were the most active. The burial finally resulted in the physical isolation of corpses, thus reducing the chances for opportunistic pathogens to spread among the rest of the individuals.     

Endogenous synchronization of the chemical signature of Reticulitermes (Isoptera: Rhinotermitidae) worker termites

Termites of the genus Reticulitermes are characteristic of temperate regions. Their colonies comprise various castes, the most numerous being that of workers which can develop into soldiers or secondary reproductives (neotenics). Each caste has a mixture of hydrocarbons (HCs) on the cuticle forming a chemical signature. The primary aim of this study was to compare the changes in the chemical signature of a population of worker termites fed on paper with juvenile hormone to differentiate them into soldiers with a control population of termites fed only on paper or wood for one month. Gas chromatography was used to analyze the cuticular profiles of Reticulitermes flavipes termites to determine whether they changed, and, if so, when and how. The data collected over one month showed that the workers fed with JH did not differentiate into soldiers but that there were progressive changes in the hydrocarbon profile independent of the treatment. These results indicate that the differentiation of the chemical signature of the worker caste is a dynamic process, depending only on time and not on colony membership, confirming that, for these termites, this signature has a lesser role in colony membership than caste membership, unlike the chemical signatures of other social insects. The temporal process of this cuticular change is also associated with a change in the alkene/methyl-branched alkane ratio.     

When predator odour makes groups stronger: effects on behavioural and chemical adaptations in two termite species

1. Being able to detect a predator before any physical contact is crucial for individual survival. Predator presence can be detected thanks to several types of signal, such as chemical cues. Chemical signals are produced by predators for their protection against desiccation, for their communication, or possibly as a side‐effect of their activity. In insects, chemical communication plays a key role in diverse biological processes, including prey‐predator or plant‐insect interactions, courtship behaviour, and kin or species recognition. 2. Cuticular hydrocarbons (CHCs) are specifically involved in recognition processes and social organisation (division of labour, caste ratios) in social insects. Here, the questions raised are whether termites can detect a predator with their cuticular compounds and whether the predator‐produced compounds can influence their prey. 3. The responses of termites Reticulitermes grassei (Clément, 1978) and Reticulitermes flavipes (Kollar, 1837) to the presence of the cuticular compounds produced by a predator, the ant species Lasius niger (Linnaeus, 1758), were investigated. More specifically, the study quantified termite traits such as caste ratios, mortality rates, CHC profile homogeneity and aggressiveness of workers after 2 months' exposure to predator‐produced compounds. 4. The results show that the predator odour did affect the aggressiveness of the native species R. grassei but not of the invasive R. flavipes. The caste ratios and the mortality rates were not affected for both species. 5. Differences between species are discussed around the native or invasive status of each species, along with the role played by chemical cues on behavioural and chemical adaptations.     

Soldiers are differentiated from male larval stages in incipient colonies of <Emphasis Type="Italic">Nasutitermes takasagoensis</Emphasis> (Isoptera: Termitidae)

In phylogenetically ancestral taxa of termites (the so-called lower termites), at least one soldier emerges and is maintained longitudinally in each incipient colony. However, in apical taxa (the so-called higher termites), the developmental pathway and regulation of soldiers in incipient colonies currently remain unknown. We therefore examined soldier and worker development in incipient colonies of higher termites (Nasutitermes takasagoensis Shiraki). Developmental stages and castes were successfully discriminated by head width in incipient colonies 4 months after colony foundation. Furthermore, differences were observed in the number of bristles on antennae between first- and second-instar larvae. In N. takasagoensis, there was more than one soldier in each incipient colony 4 months after its foundation. Presoldiers in the incipient colonies were differentiated from an earlier instar (male second-instar larvae), whereas, in mature colonies, they were differentiated from male third instars (= minor workers). The developmental period of the former (7 days) was markedly shorter than that of the latter (14 days). All female second-instar larvae molted into workers. The developmental processes shown here are useful for obtaining a clearer understanding of the mechanisms of soldier/worker differentiation in higher termites.     

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.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

Cuticular hydrocarbons as a basis for chemosensory self‐referencing in crickets: a potentially universal mechanism facilitating polyandry in insects

Females of many species obtain benefits by mating polyandrously, and often prefer novel males over previous mates. However, how do females recognise previous mates, particularly in the face of cognitive constraints? Female crickets appear to have evolved a simple but effective solution: females imbue males with their own cuticular hydrocarbons (CHCs) at mating and utilise chemosensory self‐referencing to recognise recent mates. Female CHC profiles exhibited significant additive genetic variation, demonstrating that genetically unique chemical cues are available to support chemosensory self‐referencing. CHC profiles of males became more similar to those of females after mating, indicating physical transfer of CHCs between individuals during copulation. Experimental perfuming of males with female CHCs resulted in a female aversion to males bearing chemical cues similar to their own. Chemosensory self‐referencing, therefore, could be a widespread mechanism by which females increase the diversity of their mating partners.     

Transcriptomic analysis of epigenetic modification genes in the termite Reticulitermes speratus

Eusocial insects display a caste system in which different castes are morpho-logically and physiologically specialized for different tasks.Recent studies have revealed that epigenetic modifications,including DNA methylation and histone modification,me-diate caste determination and differentiation,longevity,and polyethism in eusocial insects.Although there has been a growing interest in the relationship between epigenetic mech-anisms and phenotypic plasticity in termites,there is ltte information about differential expression levels among castes and expression sites for these genes in termites.Here we show royaltissuc-specific expression of epigenetic modification genes in the termite Reticulitermes speratus.Using RNA-seq,we identified 74 genes,including three DNA methyltransferases,seven sirtuins,48 Trithorax group proteins,and 16 Polycomb group proteins.Among these genes,15 showed king-specific expression,and 52 showed age-dependent differential expression in kings and queens.Quantitative real-time PCR revealed that DNA methyltransferase 3 is expressed specifically in the king's testis and fat body,whereas some histone modification genes are remarkably expressed in the king's testis and queen's ovary.These findings imply that epigenetic modification plays important roles in the gamete production process in termite kings and queens.     

Post‐eclosion temperature effects on insect cuticular hydrocarbon profiles

The insect cuticle is the interface between internal homeostasis and the often harsh external environment. Cuticular hydrocarbons (CHCs) are key constituents of this hard cuticle and are associated with a variety of functions including stress response and communication. CHC production and deposition on the insect cuticle vary among natural populations and are affected by developmental temperature; however, little is known about CHC plasticity in response to the environment experienced following eclosion, during which time the insect cuticle undergoes several crucial changes. We targeted this crucial to important phase and studied post‐eclosion temperature effects on CHC profiles in two natural populations of Drosophila melanogaster. A forty‐eight hour post‐eclosion exposure to three different temperatures (18, 25, and 30°C) significantly affected CHCs in both ancestral African and more recently derived North American populations of D. melanogaster. A clear shift from shorter to longer CHCs chain length was observed with increasing temperature, and the effects of post‐eclosion temperature varied across populations and between sexes. The quantitative differences in CHCs were associated with variation in desiccation tolerance among populations. Surprisingly, we did not detect any significant differences in water loss rate between African and North American populations. Overall, our results demonstrate strong genetic and plasticity effects in CHC profiles in response to environmental temperatures experienced at the adult stage as well as associations with desiccation tolerance, which is crucial in understanding holometabolan responses to stress.     

Caste ratios affect the reproductive output of social trematode colonies

Intraspecific phenotypic diversification in social organisms often leads to formation of physical castes which are morphologically specialized for particular tasks within the colony. The optimal caste allocation theory argues that specialized morphological castes are efficient at specific tasks, and hence different caste ratios should affect the ergonomic efficiency, hence reproductive output of the colony. However, the reproductive output of different caste ratios has been documented in few species of insects with equivocal support for the theory. This study investigated whether the ratios of nonreproductive and reproductive morphs affect the reproductive output of a recently discovered social trematode, Philophthalmus sp., in which the nonreproductive members are hypothesized to be defensive specialists. A census of natural infections and a manipulative in vitro experiment demonstrated a positive association between the reproductive output of trematode colonies and the ratio of nonreproductive to reproductive morphs in the presence of an intra‐host trematode competitor, Maritrema novaezealandensis. On the contrary, without the competitor, reproductive output was negatively associated with the proportion of nonreproductive castes in colonies. Our findings demonstrate for the first time a clear fitness benefit associated with the nonreproductive castes in the presence of a competitor while illustrating the cost of maintaining such morphs in noncompetitive situations. Although the proximate mechanisms controlling caste ratio remain unclear in this trematode system, this study supports the prediction that the fitness of colonies is influenced by the composition of specialized functional morphs in social organisms, suggesting a potential for adaptive shifts of caste ratios over evolutionary time.     

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.     

Intracolonial differences in gut bacterial community between worker and soldier castes of Coptotermes formosanus

Abstract The establishment of symbiotic relationships with intestinal microorganisms enables termites to thrive on recalcitrant substrates such as cellulose and wood. A termite colony is composed of several different castes which have distinct feeding habits. The soldiers, for example, cannot feed by themselves and depend on workers, who feed them with digested or semi‐digested foods. To investigate the influence of feeding habits on the bacterial symbionts, a comparative study of gut bacteria between worker and soldier castes of the termite Coptotermes formosanus was conducted. The bacterial communities of both castes were investigated using denaturing gradient gel electrophoresis (DGGE) and clonal analysis of 16S ribosomal DNA (rDNA). Both methods indicated Bacteroidetes was the common predominant group; the common dominant phylotype was affiliated with a reported uncultured Bacteroidetes phylotype (BCf1–03). There were significant differences in Bacteroidetes and Spirochaetes between two castes. Compared to the gut bacteria of workers, those of soldiers were lower in abundance and diversity of Bacteroidetes and slightly higher in Spirochaetes. Two phylotypes (W8, W11) affiliated to Bacteroidetes and two (W26, W29) affiliated to Spirochaetes were exclusively found in the DGGE profile of the worker caste. Bacteroidetes are assumed to be involved in fermentation of sugars and nitrogenous compounds as well as degradation of uric acid. Spirochaetes are supposed to aid in the functions of acetogenesis and N₂‐fixation. The different feeding habits between workers and soldiers of C. formosanus may explain the observed differences in the gut bacterial community.     

Colony structure and caste distribution in living trees of the Ryukyu drywood termite,Neotermes sugioi (Blattodea: Kalotermitidae) in Okinawa Island

Termites are major pests of houses and buildings, and also living plants such as agricultural crops, trees in forests, urban areas and gardens. However, in Japan, the basic ecology of termites nesting in living trees is not fully understood. In this study, we observed 255 colonies (nests) of the drywood termite Neotermes sugioi, collected in the field on Okinawa Island, and reported the frequency composition of the reproductive castes, the size of wood with termite gallery, the population size of colonies, and the relative position of the reproductive and non-reproductive castes within nests. Most colonies were headed by a primary queen and a primary king. However, colonies headed only by primary queens, primary kings, or neotenic kings, each accounted for approximately 5% of the colonies. A colony size of 1,000– 4,000 individuals (2058.2 ± 1695.0 [mean ± SD]) was common and the average length of colony branches was less than 100 cm. Queens and kings were found in the same or nearby nest areas, and more predominantly in the central or root side of nest wood areas. The termites may experience colony fragmentation and reproductive loss as a consequence of typhoons. Incipient colonies (i.e., colonies at an early stage of development) were found on 11.3% of branches of Leucaena leucocephala that did not show any obvious signs of infestation. In future research, it will be necessary to update the list of trees damaged by this termite species, compare the damage by tree species, and evaluate the economic impact.     

Release from prey preservation behavior via prey switch allowed diversification of cuticular hydrocarbon profiles in digger wasps

The cuticle of insects is covered by a layer of hydrocarbons (CHC), whose original function is the protection from desiccation and pathogens. However, in most insects CHC profiles are species specific. While this variability among species was largely linked to communication and recognition functions, additional selective forces may shape insect CHC profiles. Here, we show that in Philanthinae digger wasps (Crabronidae) the CHC profile coevolved with a peculiar brood‐care strategy. In particular, we found that the behavior to embalm prey stored in the nest with hydrocarbons is adaptive to protect larval food from fungi in those species hunting for Hymenoptera. The prey embalming secretion is identical in composition to the alkene‐dominated CHC profile in these species, suggesting that their profile is adaptively conserved for this purpose. In contrast, prey embalming is not required in those species that switched to Coleoptera as prey. Released from this chemical brood‐care strategy, Coleoptera‐hunting species considerably diversified their CHC profiles. Differential needs to successfully protect prey types used as larval food have thus driven the diversification of CHCs profiles of female Philanthinae wasps. To the best of our knowledge, this is the first evidence of a direct link between selection pressure for food preservation and CHC diversity.