Embryonic Development of Parthenogenetic and Sexual Eggs in Lower Termites.

Worldwide, termites are one of few social insects. In this research, the stages of embryonic development in the parthenogenetic and sexual eggs of Reticulitermes aculabialis and R. flaviceps were observed and described. In R. flaviceps, the egg development of the FF and FM groups happened during the early phases of development, whereas in R. aculabialis, this appeared mainly during the late phase of development. The variance in the number of micropyles between the R. flaviceps FF colony type and the R. aculabialis FF colony type was statistically significant. Five stages of egg development were found in both types of R. aculabialis but only the sexual eggs of R. flaviceps. In R. flaviceps, 86% of the parthenogenetic eggs stopped growing during the blastoderm development, with the yolk cell assembling frequently in the center of the egg. According to the results of the single-cell transcriptome sequencing, we investigated the egg-to-larval expression level of genes (pka, map2k1, mapk1/3, hgk, mkp, and pax6) and indicated that the levels of essential gene expression in RaFF were considerably higher than in RfFF (p < 0.05). We also discovered that the oocyte cleavage rate in the FF colony type was considerably lower in R. flaviceps compared to R. aculabialis, which gave rise to a smaller number of mature oocytes in R. flaviceps. During ovulation in both species, oocytes underwent activation and one or two cleavage events, but the development of unfertilized eggs ceased in R. flaviceps. It was shown that termite oocyte and embryonic development were heavily influenced by genes with significant expressions. Results from the databases KEGG, COG, and GO unigenes revealed the control of numerous biological processes. This study is the first to complete a database of parthenogenetic and sexual eggs of R. flaviceps and R. aculabialis.

Elevated expression of immune and DNA repair genes in mated queens and kings of the Reticulitermes chinensis termites.

Studies have identified that mating induces a series of physiological changes in animals. In this period, males tending to invest more energy, immune peptides, and other substances to reduce the cost of living for females. This results in lower survival rates in later life than females. Meanwhile, both males and females shorten lifespans due to reproduction. However, the reasons why termites' queens and kings are both extremely long-lived and highly fecund are unclear. Therefore, this study aimed to examine the effects of mating on the expression of immune and DNA repair genes for lifespan extension in termite queens and kings. Here, we reported that mated queens show relatively higher expression of immune genes (phenoloxidase, denfensin, termicin, transferrin), antioxidant genes (CAT, SOD), detoxification genes (GST, CYP450) than virgin queens in the Reticulitermes chinensis. In addition, mated kings also highly expressed these genes, except for termicin, transferrin, GST, and CYP450. After mating, both queens and kings significantly upregulated the expression of DNA repair genes (MLH1, BRCA1, XRCC3, RAD54-like). Mismatch repair genes (MMR) MSH2, MSH4, MSH6 were considerably increased in mated queens, while MSH4, MSH5, MSH6 were upregulated in mated kings. Our results suggest that mating increases the expression of immune and DNA repair genes in the termite queens and kings, and thus possibly improving their survival during reproductive span due to the omnipresent pathogens.

De Novo Transcriptome Assembly and Analysis of Longevity Genes Using Subterranean Termite (Reticulitermes chinensis) Castes.

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.

Transcriptomic evidence that insulin signalling pathway regulates the ageing of subterranean termite 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.

Complete mitochondrial genome of the "floppy-wing" morph reproductive termite, Reticulitermes labralis (Isoptera: Rhinotermitidae).

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 complete mitochondrial genome of the subterranean termite, Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae).

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 development of adultoid reproductives and brachypterous neotenic reproductives from the last instar nymphs in Reticulitermes labralis (Isoptera: Rhinotermitidae): a comparative study.

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.

Testicular development and modes of apoptosis during spermatogenesis in various castes of the termite Reticulitermes labralis (Isoptera:Rhinotermitidae).

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.

[Immunocytochemical localization of estrogen receptor in the spermatogenesis of termites].

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.