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The subterranean termite Reticulitermes aculabialis was firstly described from the soldier caste in 1977 and diagnose from the imago has been difficult due to the lack of description of alate. In this paper, a detailed description of alate for R. aculabialis is presented, including additional data to soldiers. The data are chiefly based on the external morphology of R. aculabialis imago, which has never been described before. All the samples of alates and soldiers were collected in Xi’an, Shaanxi province, China. All of the essential specific characteristics are illustrated for identification purposes.

Caste differentiation in termites is one of the most conspicuous examples of facultative polyphenism in animals. It is clear that specific cuticular formation occurs in hard exocuticles during caste differentiation. However, the developmental pattern of the soft endocuticle in the differentiation pathways of castes is unknown. To reveal whether the endocuticle is involved in caste differentiation, we compared the exocuticle and endocuticle thickness of individuals in 2 pathways (nymph line and worker line) of caste differentiation in the termite Reticulitermes aculabialis. The endocuticle protein genes were identified by transcriptome analysis and the expression patterns of these genes were confirmed in caste differentiation. We found that the endocuticle structure showed dynamic changes in 2 pathways, and the first difference in endocuticle structure occurred after larvae differentiation bifurcated into workers and nymphs. The thinning of the endocuticle was a significant event from nymphs developing into alates with the thickest exocuticle and thinnest endocuticle. The thickest endocuticle layers were found in the heads of the workers and the ultrastructure of the endocuticle in the heads was more complex than that in the thorax–abdomens. Six endocuticle protein genes were identified and annotated as endocuticle structural glycoproteins SgAbd-2, SgAbd-9, and Abd-5. The expression levels of endocuticle protein genes changed dramatically during caste development and the expression levels in neotenic reproductives (secondary reproductives) were significantly higher than those in alates (primary reproductives). These results reveal the roles of endocuticles in caste differentiation and adaptation to the environment.

The longevity phenomenon is entirely controlled by the insulin signaling pathway (IIS-pathway). Both vertebrates and invertebrates have IIS-pathways that are comparable to one another, though no one has previously described de novo transcriptome assembly of IIS-pathway-associated genes in termites. In this research, we analyzed the transcriptomes of both reproductive (primary kings “PK” and queens “PQ”, secondary worker reproductive kings “SWRK” and queens “SWRQ”) and non-reproductive (male “WM” and female “WF” workers) castes of the subterranean termite Reticulitermes chinensis. The goal was to identify the genes responsible for longevity in the reproductive and non-reproductive castes. Through transcriptome analysis, we annotated 103,589,264 sequence reads and 184,436 (7G) unigenes were assembled, GC performance was measured at 43.02%, and 64,046 sequences were reported as CDs sequences. Of which 35 IIS-pathway-associated genes were identified, among 35 genes, we focused on the phosphoinositide-dependent kinase-1 (Pdk1), protein kinase B2 (akt2-a), tuberous sclerosis-2 (Tsc2), mammalian target of rapamycin (mTOR), eukaryotic translation initiation factor 4E (EIF4E) and ribosomal protein S6 (RPS6) genes. Previously these genes (Pdk1, akt2-a,mTOR, EIF4E, and RPS6) were investigated in various organisms, that regulate physiological effects, growth factors, protein translation, cell survival, proliferation, protein synthesis, cell metabolism and survival, autophagy, fecundity rate, egg size, and follicle number, although the critical reason for longevity is still unclear in the termite castes. However, based on transcriptome profiling, the IIS-pathway-associated genes could prolong the reproductive caste lifespan and health span. Therefore, the transcriptomic shreds of evidence related to IIS-pathway genes provide new insights into the maintenance and relationships between biomolecular homeostasis and remarkable longevity. Finally, we propose a strategy for future research to decrypt the hidden costs associated with termite aging in reproductive and non-reproductive castes.

Insulin is a protein hormone that controls the metabolism of sugar, fat and protein via signal transduction in cells, influencing growth and developmental processes such as reproduction and ageing. From nematodes to fruit flies, rodents and other animals, glucose signalling mechanisms are highly conserved. Reproductive termites (queens and kings) exhibit an extraordinarily long lifespan relative to non-reproductive individuals such as workers, despite being generated from the same genome, thus providing a unique model for the investigation of longevity. The key reason for this molecular mechanism, however, remains unclear. To clarify the molecular mechanism underlying this phenomenon, we sequenced the transcriptomes of the primary kings (PKs), primary queens (PQs), male (WMs) and female (WFs) workers of the lower subterranean termite Reticulitermes chinensis. We performed RNA sequencing and identified 33 insulin signalling pathway-related genes in R. chinensis. RT-qPCR analyses revealed that EIF4E and RPS6 genes were highly expressed in WMs and WFs workers, while mTOR expression was lower in PKs and PQs than in WMs and WFs. PQs and PKs exhibited lower expression of akt2-a than female workers. As the highly conserved insulin signalling pathway can significantly prolong the healthspan and lifespan, so we infer that the insulin signalling pathway regulates ageing in the subterranean termite R. chinensis. Further studies are recommended to reveal the biological function of insulin signalling pathway-related genes in the survival of termites to provide new insights into biomolecular homeostasis maintenance and its relationship to remarkable longevity.

Background The reproductive plasticity of termite workers provides colonies with tremendous flexibility to respond to environmental changes, which is the basis for evolutionary and ecological success. Although it is known that all colony members share the same genetic background and that differences in castes are caused by differences in gene expression, the pattern of the specific expression of genes involved in the differentiation of workers into reproductives remains unclear. In this study, the isolated workers of Reticulitermes labralis developed into reproductives, and then comparative transcriptomes were used for the first time to reveal the molecular mechanisms underlying the reproductive plasticity of workers. Results We identified 38,070 differentially expressed genes and found a pattern of gene expression involved in the differentiation of the workers into reproductives. 12, 543 genes were specifically upregulated in the isolated workers. Twenty-five signal transduction pathways classified into environmental information processing were related to the differentiation of workers into reproductives. Ras functions as a signalling switch regulates the reproductive plasticity of workers. The catalase gene which is related to longevity was up-regulated in reproductives. Conclusion We demonstrate that workers leaving the natal colony can induce the expression of stage-specific genes in the workers, which leads to the differentiation of workers into reproductives and suggests that the signal transduction along the Ras-MAPK pathway crucially controls the reproductive plasticity of the workers. This study also provides an important model for revealing the molecular mechanism of longevity changes.

Fitness-determining interactions with fungi have often been considered a by-product of social evolution in insects. In higher termites, the mutualistic association between the basidiomycete genus Termitomyces and Macrotermitinae is well known. However, whether and how lower termites use fungi is unclear. Here, we found a large amount of brown sclerotium-forming fungi in egg piles of the lower termite Reticulitermes labralis and identified the sclerotia as Fibulorhizoctonia sp. There was a significant difference in morphology between the sclerotia and the termite eggs. The workers of R. labralis and R. chinensis actively gathered the sclerotia into the egg piles within their nests, whereas the workers of R. aculabialis did not gather sclerotia outside their nests. None of the sclerotia in the egg piles germinated in the presence of workers. However, the sclerotia germinated in the absence of workers, and then the hyphae killed the termite eggs. The data from cellulase activity demonstrated that Fibulorhizoctonia sp. was able to exhaustively digest cellulose into glucose.We confirmed for the first time that the workers carrying the sclerotia into the piles of eggs is not due to mistaking the sclerotia for their eggs and that the workers of R. labralis may be able to select favourite fungi.

We report the complete mitochondrial genome (mtDNA) of the subterranean termite Reticulitermes flaviceps. The mtDNA is 16,485 bp long in size. The 13 protein-coding genes have typical ATN initiation codon. But the stop codons were TAA, TAG and an incomplete termination codon (T) abutting an adjacent tRNA gene. Twenty-two tRNA genes, in addition to tRNAser (AGN) replaced lacking of the DHU stem with a simple loop, showed the typical clover-leaf secondary structure. The A + T-rich region was 1677 bp in length with 70.3 % A + T content. Addition to the A + T-rich region, non-coding sequences of the mtDNA genome harbored 18 intergenic spacers. There were two complete repeats and one partial repeats of repeat A and repeat B in CR. Phylogenetic tree based on the 12 complete mitochondrial genome sequences of closely related termite species accords well with the morphological phylogenetic analysis.

Three types of replacement reproductives exist within termite colonies: nymphoids developed from nymphs, ergatoids from workers, and adultoids from alates. To date, the only two types of replacement reproductives described in Reticulitermes are nymphoids and ergatoids. We found that in addition to alates, the sixth stage nymphs (N6 nymphs) of R. labralis could also undergo eclosion and become one of two secondary reproductive types: (1) nymphoids or (2) floppy wing form reproductives (FFR). The FFRs would become replacement reproductives within their natal colony after wing shedding. These reproductives have a light pigmentation, with slight sclerotization of their floppy wings, and are capable of producing offspring within their natal colony. FFRs differed in morphology to the alates after eclosion. We found that oogenesis had completed yolk accumulation and reached the vitellogenesis stage in alates. By contrast, the vitellogenic oocytes were absent at the end of ovarioles in FFRs, nymphoids, and N6 nymphs, showing that oogenesis did not reach the vitellogenesis stage. In other words, the ovarian status of FFRs would more likely resemble nymphoids rather than primary reproductives. Microsatellite analyses of four loci indicated that FFR individuals, workers, and alates were all produced by sexual reproduction. Thus, FFRs can be classified as another type of secondary reproductives in addition to the nymphoid and ergatoid morphs in R. labralis. The R. labralis FFR is similar to the adultoids in higher termites except for its larval characteristics, which adds to the evidence that R. labralis is a transitional species between the lower and higher termite.

Reproductive systems of termite colonies may involve the number of individuals in the reproductive caste and the copulatory selectivity of reproductive individuals (i.e., polyandry or polygamy), both of which directly impact the fertility and genetic diversity of the colony. Polygamy is widespread in the lower termites, whereas polyandry appears to be mostly absent in termites. In this paper, the differentiation of male and female neotenics were observed in orphaned experimental colonies of the subterranean termite Reticulitermes labralis. The artificial orphaned colonies began to produce neotenics a week after colony establishing, with more neotenics appearing in the same group over time. Finally, each experimental group reserved multi-neotenics that consisted of male and female neotenic individuals. Our results demonstrated that these neotenic individuals retained in the colony participated in reproduction. A genetic analysis at four microsatellite loci showed that in addition to the conspicuous morphologically male reproductives, there were inconspicuous males or workers that had copulated with the females in the orphaned colony. Multiple male and female reproductive individuals existed together in a single colony, and one female neotenic could mate with several male reproductives in a short time. Thus, multiple male and female reproductive systems and a polyandric mating system are present in R. labralis.

The separation of primary reproductive and secondary reproductive roles based on the differentiation of alate adults and neotenic reproductives is the most prominent characteristic of termites. To clarify the mechanism underlying this differentiation, we sequenced the transcriptomes of alate adults (ARs), brachypterous neotenics (BNs) and adultoid reproductives (ANs) from the last instar nymphs of Reticulitermes labralis. A total of 404,152,188 clean sequencing reads was obtained and 61,953 unigenes were assembled. Of the 54 identified cuticular protein (CP) genes of the reproductives, 22 were classified into the CPR family and 7 were classified into the CPG family. qRT-PCR analyses of the 6 CP genes revealed that the CP genes involved in exocuticle sclerotization were highly expressed in the ARs and RR-1 involved in soft endocuticle was highly expressed in the ARs and ANs. These results suggest that the alate adults might increase cuticular component deposition to adapt to new or changing environments and that the development of reproductive individuals into primary or secondary reproductives is controlled by the expression of cuticular protein genes involved in the hardening of the exocuticle. In addition, the AN caste is a transitional type between the BN and AR castes in the process of evolution.

Secondary reproductives develop primarily from nymphs. However, they have been rarely studied; in particular, the development of adultoid reproductives (AR) with floppy wings is still unclear. In this study, the change in juvenile hormone (JH) levels, vitellogenin gene expression, and oogenesis during the development of AR and brachypterous neotenic reproductives (BN) from the last instar nymphs of Reticulitermes labralis are investigated and compared. The results showed that the AR derived from the last instar nymphs by molting, and they were more similar to neotenic reproductives in morphology. In addition, the paired AR were not able to survive in the absence of workers. In R. labralis, the process of the last instar nymphs developing into AR and BN took an increase in JH level as a starting point. The JH level of the last instar nymphs molting into BN was approximately 1.5-fold higher than that of the AR. Additionally, The JHIII level of BN peaked on day 5, and that of AR peaked on day 10, which induced the onset of vitellogenesis in BN and AR, respectively. After molting, the vitellogenin gene expression levels of both BN and AR initially increased and then declined, and the expression levels in the BN were significantly higher than those in the AR. In addition, the oocytes of BN matured earlier than those of the AR, and the number of eggs laid by the BN was higher than the number laid by the AR. Our results demonstrate that, in R. labralis, the last instar nymphs can develop into AR, which are significantly different from BN in their development.

The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites. However, little is known about the mechanism of male reproductive division that underlies caste differentiation. In the present study, testicular development and stage-specific apoptotic patterns were investigated and compared during spermatogenesis in reproductives, workers and soldiers of the termite Reticulitermes labralis. The results showed that male workers were divided into two types, the workers with spermatozoa (WS) and the workers without spermatozoa (WN). Spermatogenesis in WN and soldiers arrested at the spermatocyte stage. Moreover, there were significant differences in testicular size and spermatogenesis among the various castes. The mode of apoptosis in late instar WS was similar to the reproductives, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis. First, the majority of apoptotic cells were spermatogonia, and the spermatogonia of both late instar WS and reproductives exhibited lower apoptotic rates compared with late instar WN and soldiers. Second, the spermatocytes and spermatids showed very little apoptosis in the late instar WS and reproductives, and no TUNEL signal was detected in any of the examined spermatozoa. Our findings suggest that the male workers undergo a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers are able to provide normal spermatozoa to queens, or (ii) the neuter lineage, in which the male workers lose reproductive options. The level of testicular development may explain the significant discrepancies in reproductive capacity among the reproductives, workers and soldiers and reveal the reproductive division in male workers. These differences are controlled by apoptosis during early spermatogenesis.

We have determined the complete mitochondrial genome of Reticulitermes chinensis Snyder. The total length of the R. chinensis is 15,925 bp with 65.27% A + T content. It consists of 13 protein-coding, 22 tRNA, 2 rRNA genes and an A + T-rich control region. All the protein-coding genes used ATN as start codon. But the stop codons were TAA, TAG, and an incomplete termination codon (T) abutting an adjacent tRNA gene. The A + T-rich control region was 1125 bp in length with 67.02% A + T content.

The complete mitochondrial genome of Reticulitermes labralis (Isoptera: Rhinotermitidae) was determined for its nucleotide sequence of 16 113 bp. Its gene content and organization were identical with other Reticulitermes species. The 13 protein-coding genes (PCGs) have typical ATN initiation codon. But, the stop codons were TAA, TAG and an incomplete termination codon (T) abutting an adjacent tRNA gene. Twenty-two tRNA genes, in addition to tRNASer(AGN) replaced lacking of the DHU stem with a simple loop, showed the typical clover-leaf secondary structure. The A + T-rich region was 1311 bp in length with 65.98% A + T content. In addition to the A + T-rich region, non-coding sequences of the mtDNA genome harbored 17 intergenic spacers. There were three complete repeats of repeat A in CR, which were not discovered in other termite species. Phylogenetic tree based on the 11 complete mitochondrial genome sequences of closely related termite species accords well with morphological phylogenetic analysis.

The available information indicates that estrogen receptor(ER) play a physiological role in the regulation of spermatogenesis in vertebrates. However, the cellular distribution of ER in the testis is poorly understood in invertebrates. The aim of this study was to determine the presence and cellular distribution of ER in the spermatogenesis of termite (Reticulitermes aculabialis). Immunocytochemical analysis showed ER was present in the nucleus of the primary spermatocytes, and the expression of ER was relatively stronger in the primary spermatocytes of the swarming termites. Previous studies have demonstrated the procerebrum of the swarming male termites could strongly secrete FSH (Follicle Stimulating Hormone) and LH (Luteinizing Hormone) which stimulated estrogen secreting. In conclusion, we demonstrated here for the first time that ER might be an important factor in the regulation of the spermatogenesis of termites, and play an important role for starting and maintaining the meiosis cell division of spermatocytes.