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.     

Morphological and developmental analysis of peripheral antennal chemosensory sensilla and central olfactory glomeruli in worker castes of Camponotus compressus (Fabricius, 1787)

The antennal lobes of different castes of the ant species Camponotus compressus show a marked diversity in the organization of their olfactory glomeruli. Notably, there is a significant difference in the number and size of glomeruli between the reproductives and the workers and among the different worker castes. In this report, we investigate the notion that these caste-specific differences in glomerular number might be accounted for, at least in part, by the differences in numbers of olfactory sensilla that target the antennal lobe. For this, we examine the number of sensilla on the antennal flagella of all the individual castes of C. compressus. This analysis reveals a striking correlation between sensillar number and the number of antennal glomeruli in a given caste. As a first step in investigating the causal mechanisms that might give raise to this correlation, we carry out an initial characterization of olfactory system development in the minor workers of C. compressus. We analyze the temporal pattern of innervations of the developing antennal lobe by olfactory sensory neuron axons. We document the development of the olfactory glomeruli in the antennal lobe during this process, which occurs during early pupal stages. Our findings provide the basis for future manipulative developmental studies on the role of sensory afferent number in glomerular development of different castes within the same species.     

Ecdysteroid titers in pupae of highly social bees relate to distinct modes of caste development

Modifications in endocrine programs are common mechanisms that generate alternative phenotypes. In order to understand how such changes may have evolved, we analyzed the pupal ecdysteroid titers in two closely related, highly social bees: the honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata. In both species, the ecdysteroid titers in queens reached their peak levels earlier than in workers. Titer levels at peak maxima did not differ for the honey bee castes, but in Melipona they were twofold higher in queens than in workers. During the second half of pupal development, when the ecdysteroid titers decrease and the cuticle progressively melanizes, the titer in honey bee queens remained higher than in workers, while the reverse situation was observed in Melipona. Application of the juvenile hormone analog Pyriproxyfen^® to spinning-stage larvae of Melipona induced queen development. Endocrinologically this was manifest in a queen-like profile of the pupal ecdysteroid titer. Comparing these data with previous results on preimaginal hormone titers in another stingless bee, we conclude that the timing and height of the pupal ecdysteroid peak may depend on the nature of the specific stimuli that initially trigger diverging queen/worker development. In contrast, the interspecific differences in the late pupal ecdysteroid titer profiles mainly seem to be related to caste-specific programs in tissue differentiation, including cuticle pigmentation.     

Replication of the Endosymbiotic Bacterium Blochmannia floridanus Is Correlated with the Developmental and Reproductive Stages of Its Ant Host

The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR. The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants). In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly. In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal. An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C. herculeanus and C. sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes.     

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.     

Caste specific modulation of juvenile hormone titers in Apis mellifera

The titers of JH III were studied in the larval and pupal stages of the two female honey bee castes, the queen and the worker. Whereas the early larval stages, L3 and L4, had to be pooled, all the last instar larvae, pupae, and newly hatched adults, were titered individually. The queen stages produce two-fold higher JH III titers in comparison with the worker stages. Both have relatively high titers during the early larval instars, decreasing from an average of 450 pmol/g at L3 to about 20 pmol/g in the queen and 75 pmol/g at L3 to 5 pmol/g at L5 in the worker. Both castes build up another JH III peak at the end of their spinning phase when entering the pharate pupa stage, with about 200 pmol/g in the queen and 60 pmol/g in the worker. No JH III was found in the pupal stage; the queen only develops a new JH III titer in the late pupal stage.     

Geographic variation of caste structure among ant populations

Morphologically distinct worker castes of eusocial insects specialize in different tasks. The relative proportions of these castes and their body sizes represent the demography of a colony that is predicted to vary adaptively with environments. Despite strong theoretical foundations, there has been little empirical evidence for the evolution of colony demography in nature. We show that geographically distinct populations of the ant Pheidole morrisi differ in worker caste ratios and worker body sizes in a manner consistent with microevolutionary divergence. We further show that the developmental mechanism for caste determination accounts for the unique pattern of covariation observed in these two traits. Behavioral data reveal that the frequency of different tasks performed by workers changes in a caste-specific manner when caste ratios are altered and demonstrate the importance of the major caste in colony defense. The population-level variation documented here for P. morrisi colonies supports the predictions of adaptive demography theory and illustrates that developmental mechanisms can play a significant role in shaping the evolution of phenotype at the colony level.     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Caste and sex specific olfactory glomerular organization and brain architecture in two sympatric ant species Camponotus sericeus and Camponotus compressus (Fabricius, 1798)

We use monoclonal antibodies against synaptic proteins and anterograde tracing with neurobiotin to describe the architecture of the antennal lobes in different castes of two ant species – Camponotus sericeus and Camponotus compressus. The reproductives and worker classes are readily categorized based on size and external morphology. The overall organization of brain neuropile is comparable between castes with differences only in the visual ganglia. Males have a larger fraction of neuropile occupied by the medulla and lobula than females. In the diurnal species, C. sericeus these regions are more highly represented, than in the nocturnal species C. compressus. The most striking differences are in the antennal lobe where males possess a macroglomerulus, which is about ten times larger in volume than the other glomeruli; such a specialization is absent in females. Minor workers possess a significantly larger number of glomeruli than the majors despite the smaller overall volume of the lobe. These caste-specific differences occur mainly within glomerular clusters that receive input from sensory neurons that project in tracts – T4 and T5 – within the antennal nerve. The comparative anatomy of different castes of ants provides an entry point into a future systematic analysis of how divergent brain architectures can arise within a single species.     

LSD1/CoREST reversible opening-closing dynamics: discovery of a nanoscale clamp for chromatin and protein binding

LSD1 associated with its corepressor protein CoREST is an exceptionally relevant target for epigenetic drugs. Hypotheses for the role of LSD1/CoREST as a multidocking site for chromatin and protein binding would require significant molecular flexibility, and LSD1/CoREST large-amplitude conformational dynamics is currently unknown. Here, molecular dynamics simulation reveals that the LSD1/CoREST complex in solution functions as a reversible nanoscale binding clamp. We show that the H3 histone tail binding pocket is a potential allosteric site for regulation of the rotation of SWIRM/SANT2 domains around the Tower domain. Thus, targeting this site and including receptor flexibility are crucial strategies for future drug discovery.     

Caste‐biases in gene expression are specific to developmental stage in the ant Formica exsecta

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co‐opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of ‘toolkit’ genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA‐Seq to compare caste‐biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste‐biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste‐biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste‐biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste‐biased at least in some life stages in F. exsecta, and the caste‐biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.     

Differential expression of antioxidant system genes in honey bee (Apis mellifera L.) caste development mitigates ROS-mediated oxidative damage in queen larvae

The expression of morphological differences between the castes of social bees is triggered by dietary regimes that differentially activate nutrient-sensing pathways and the endocrine system, resulting in differential gene expression during larval development. In the honey bee, Apis mellifera, mitochondrial activity in the larval fat body has been postulated as a link that integrates nutrient-sensing via hypoxia signaling. To understand regulatory mechanisms in this link, we measured reactive oxygen species (ROS) levels, oxidative damage to proteins, the cellular redox environment, and the expression of genes encoding antioxidant factors in the fat body of queen and worker larvae. Despite higher mean H₂O₂ levels in queens, there were no differences in ROS-mediated protein carboxylation levels between the two castes. This can be explained by their higher expression of antioxidant genes (MnSOD, CuZnSOD, catalase, and Gst1) and the lower ratio between reduced and oxidized glutathione (GSH/GSSG). In worker larvae, the GSG/GSSH ratio is elevated and antioxidant gene expression is delayed. Hence, the higher ROS production resulting from the higher respiratory metabolism in queen larvae is effectively counterbalanced by the up-regulation of antioxidant genes, avoiding oxidative damage. In contrast, the delay in antioxidant gene expression in worker larvae may explain their endogenous hypoxia response.     

High-performance liquid chromatography (HLPC) measurement of catecholamines in single honeybee brains reveals caste-specific differences between worker bees and queens in apis mellifera

1. Catecholamines were determined by HPLC in honeybee brains using automatized extraction and on-line detection with an electrochemical detector.2. This method has high sensitivity in the range of fmols. Thus, catechols can be measured in single brains as well as in parts of brains.3. The comparison of brains of worker bees and queens shows a caste-specific content of eatecholamine concentrations in worker bees and queens.4. The amount of norepinephrine and dopamine are higher in queens than in worker bees.5. The differences are not caused by the different size of the respective brains, but reflect a difference in the two female castes.     

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies.     

Epigenetic inheritance and genome regulation: is DNA methylation linked to ploidy in haplodiploid insects?

Organisms show great variation in ploidy level. For example, chromosome copy number varies among cells, individuals and species. One particularly widespread example of ploidy variation is found in haplodiploid taxa, wherein males are typically haploid and females are typically diploid. Despite the prevalence of haplodiploidy, the regulatory consequences of having separate haploid and diploid genomes are poorly understood. In particular, it remains unknown whether epigenetic mechanisms contribute to regulatory compensation for genome dosage. To gain greater insights into the importance of epigenetic information to ploidy compensation, we examined DNA methylation differences among diploid queen, diploid worker, haploid male and diploid male Solenopsis invicta fire ants. Surprisingly, we found that morphologically dissimilar diploid males, queens and workers were more similar to one another in terms of DNA methylation than were morphologically similar haploid and diploid males. Moreover, methylation level was positively associated with gene expression for genes that were differentially methylated in haploid and diploid castes. These data demonstrate that intragenic DNA methylation levels differ among individuals of distinct ploidy and are positively associated with levels of gene expression. Thus, these results suggest that epigenetic information may be linked to ploidy compensation in haplodiploid insects. Overall, this study suggests that epigenetic mechanisms may be important to maintaining appropriate patterns of gene regulation in biological systems that differ in genome copy number.     

Characterization of gut bacterial community associated with worker and soldier castes of Globitermes sulphureus Haviland (Blattodea: Termitidae) using 16S rRNA metagenomic

Globitermes sulphureus is a major pest in coconut plantations and also a secondary pest in the urban, suburban and rural areas of building structures, as well as in tropical agriculture areas. Although the literature revealed microbiome in termite guts, the bacterial community in G. sulphureus intestinal tracts remain largely unidentified. Here, we aimed to characterize the bacterial community associated with the worker and soldier castes of G. sulphureus using 16S metagenomic. The Illumina HiSeq 2500 sequencing machine was used to amplify the V3 and V4 regions of the 16S rRNA gene. The sequencing output was analyzed using Qiime pipeline v1.9.1. The result of the analysis showed that Spirochaetes, Fibrobacteres, Firmicutes, Proteobacteria, and Bacteroidetes were the dominant phyla that reside in the guts of the worker and soldier castes. We found that the worker and soldier castes shared similar bacterial phyla in their guts but different bacterial genera. Our findings provided a baseline information on microbial community inhabiting G. sulphureus guts up to the genus level for some phyla.     

Wing disc development during caste differentiation in the ant Pheidole megacephala (Hymenoptera: Formicidae)

The genus Pheidole has three distinct castes in females: queen, major, and minor workers. It has been believed that the larvae of major workers have prominent mesothoracic wing discs, although the minor worker larvae lack them. Here we conducted histological examinations of wing discs during larval development in P. megacephala. We show that all three castes have mesothoracic wing discs, at least in their early stage of the final larval instar, and that the wings degenerate differently in the dimorphic worker castes. The minute wing discs of minor workers neither grow nor metamorphose but disappear during the prepupal stage. On the contrary, the wing discs of major workers evaginate at the onset of the prepupal stage but subsequently degenerate by apoptotic cell death. This apoptotic wing degeneration in the prepupal stage was contradistinguished from wing degeneration in some lepidopteran insects, in which apoptosis occurs in the pupal wing buds. Our results suggest that each worker caste shows a different degeneration process to express the wingless character and that apoptotic degeneration has been adopted in association with the evolution of worker dimorphism.