Candidate target genes of the male-specific expressed Doublesex in the termite Reticulitermes speratus.

Eusocial insects such as termites, ants, bees, and wasps exhibit a reproductive division of labor. The developmental regulation of reproductive organ (ovaries and testes) is crucial for distinguishing between reproductive and sterile castes. The development of reproductive organ in insects is regulated by sex-determination pathways. The sex determination gene Doublesex (Dsx), encoding transcription factors, plays an important role in this pathway. Therefore, clarifying the function of Dsx in the developmental regulation of sexual traits is important to understand the social evolution of eusocial insects. However, no studies have reported the function of Dsx in hemimetabolous eusocial group termites. In this study, we searched for binding sites and candidate target genes of Dsx in species with available genome information as the first step in clarifying the function of Dsx in termites. First, we focused on the Reticulitermes speratus genome and identified 101 candidate target genes of Dsx. Using a similar method, we obtained 112, 39, and 76 candidate Dsx target genes in Reticulitermes lucifugus, Coptotermes formosanus, and Macrotermes natalensis, respectively. Second, we compared the candidate Dsx target genes between species and identified 37 common genes between R. speratus and R. lucifugus. These included several genes probably involved in spermatogenesis and longevity. However, only a few common target genes were identified between R. speratus and the other two species. Finally, Dsx dsRNA injection resulted in the differential expression of several target genes, including piwi-like protein and B-box type zinc finger protein ncl-1 in R. speratus. These results provide valuable resource data for future functional analyses of Dsx in termites.

Molecular unraveling of polysaccharide digestion in wood-feeding termites: A solid-state NMR perspective.

Termites are among the most efficient organisms utilizing polysaccharides from wood and play a significant role in global carbon recycling, especially within tropical and subtropical ecosystems. Yet, the molecular details in polysaccharide degradation by termites remain largely unexplored. In this work, we have elucidated the shared and distinct molecular details in polysaccharides digestion by the higher termite Nasutitermes on poplar and the lower termite Cryptotermes on pine using high resolution solid-state nuclear magnetic resonance spectroscopy. For the first time, structural polymers are partitioned into the minor mobile and dominant rigid phases for individual examination. The mobile polysaccharides receive less structural impacts and exhibit greater digestibility compared to the rigid counterparts. While both termites effectively degrade cellulose, Nasutitermes significantly outperforms Cryptotermes in hemicellulose breakdown. In the rigid phase, cellulose is comprehensively degraded into a fragmented and more dynamically consistent structure; As Nasutitermes breaks down hemicellulose in a similar manner to cellulose, Cryptotermes selectively digests hemicellulose at its interfaces with cellulose. Additionally, crystalline cellulose undergoes selective degradation, and the digestion of amorphous cellulose might involve sugar chain detachment within microfibrils. Overall, our findings offer significant advancements and fresh perspectives on the polysaccharide digestion strategies of different termite lineages.

A genetic toolkit underlying the queen phenotype in termites with totipotent workers.

Social insect castes (e.g., queens, workers) are prime examples of phenotypic plasticity (i.e., different phenotypes arising from the same genotype). Yet, the mechanisms that give rise to highly fertile, long-lived queens versus non-reproducing, short-lived workers are not well understood. Recently, a module of co-expressed genes has been identified that characterizes queens compared to workers of the termite Cryptotermes secundus (Kalotermitidae): the Queen Central Module (QCM). We tested whether the QCM is shared in termite species, in which queens gradually develop via early larval and late larval instars, the latter functioning as totipotent workers (linear development). Similar as in C. secundus, gene expression profiles revealed an enrichment of QCM genes in Zootermopsis angusticollis queens, a species from another termite family (Archotermopsidae). The expression of these QCM genes became gradually enriched during development from early larval instars via workers to queens. Thus, our results support the hypothesis of a conserved genetic toolkit that characterizes termite queens with gradual linear development. Our data also imply a strong caste-specific tissue specificity with the QCM signal being restricted to head-prothorax tissues in termite queens. This tissue-specific expression of key aging-related genes might have facilitated the evolution of a long lifespan in termite queens.

Identification of potential natural products derived from fungus growing termite, inhibiting Pseudomonas aeruginosa quorum sensing protein LasR using molecular docking and molecular dynamics simulation approach.

Pseudomonas aeruginosa, the most common opportunistic pathogen, is becoming antibiotic-resistant worldwide. The fate of P. aeruginosa, a multidrug-resistant strain, can be determined by multidrug efflux pumps, enzyme synthesis, outer membrane protein depletion, and target alterations. Microbial niches have long used quorum sensing (QS) to synchronize virulence gene expression. Computational methods can aid in the development of novel P. aeruginosa drug-resistant treatments. The tripartite symbiosis in termites that grow fungus may help special microbes find new antimicrobial drugs. To find anti-quorum sensing natural products that could be used as alternative therapies, a library of 376 fungal-growing termite-associated natural products (NPs) was screened for their physicochemical properties, pharmacokinetics, and drug-likeness. Using GOLD, the top 74 NPs were docked to the QS transcriptional regulator LasR protein. The five lead NPs with the highest gold score and drug-like properties were chosen for a 200-ns molecular dynamics simulation to test the competitive activity of different compounds against negative catechin. Fridamycin and Daidzein had stable conformations, with mean RMSDs of 2.48 and 3.67 Å, respectively, which were similar to Catechin's 3.22 Å. Fridamycin and Daidzein had absolute binding energies of -71.186 and -52.013 kcal/mol, respectively, which were higher than the control's -42.75 kcal/mol. All the compounds within the active site of the LasR protein were kept intact by Trp54, Arg55, Asp67, and Ser123. These findings indicate that termite gut and fungus-associated NPs, specifically Fridamycin and Daidzein, are potent QS antagonists that can be used to treat P. aeruginosa's multidrug resistance.Communicated by Ramaswamy H. Sarma.

The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites.

Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo-acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60-80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non-reducing end of the cellulose-binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms.

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.

Characterization of chitinolytic bacteria newly isolated from the termite Microcerotermes sp. and their biocontrol potential against plant pathogenic fungi.

Chitinolytic bacteria were isolated from guts and shells of the termite Microcerotermes sp. Among the nineteen morphologically different chitinolytic isolates, three isolates with highest extracellular chitinase production ratio (≥2.26) were selected. Based on molecular identification of 16S rRNA gene sequences and biochemical characterizations using API test kits and MALDI-TOF MS, these isolates were closely related to Bacillus thuringiensis (Mc_E02) and Paenibacillus species (Mc_E07 and Mc_G06). Isolate Mc_E02 exhibited the highest chitinase-specific activity (2.45 U/mg protein) at 96 h of cultivation, and the enzyme activity was optimized at pH 7.0 and 45 °C. The isolate showed highest and broad-spectrum inhibitory effect against three phytopathogenic fungi (Curvularia lunata, Colletotrichum capsici, and Fusarium oxysporum). Its 36-kDa chitinase exhibited the biomass reduction and mycelium inhibition against all fungi, with highest effects to Curvularia lunata. This research provides novel information about termite chitinolytic bacteria and their effective chitinase, with potential use as biocontrol tool.

Reproductives signature revealed by protein profiling and behavioral bioassays in termite.

Proteins are known to be social interaction signals in many species in the animal kingdom. Common mediators in mammals and aquatic species, they have seldom been identified as such in insects' behaviors. Yet, they could represent an important component to support social signals in social insects, as the numerous physical contacts between individuals would tend to favor the use of contact compounds in their interactions. However, their role in social interactions is largely unexplored: are they rare or simply underestimated? In this preliminary study, we show that, in the termite Reticulitermes flavipes, polar extracts from reproductives trigger body-shaking of workers (a vibratory behavior involved in reproductives recognition) while extracts from workers do not. Molecular profiling of these cuticular extracts using MALDI-TOF mass spectrometry reveals higher protein diversity in reproductives than in workers and a sex-specific composition exclusive to reproductives. While the effects observed with extracts are not as strong as with live termites, these results open up the intriguing possibility that social signaling may not be limited to cuticular hydrocarbons or other non-polar, volatile chemicals as classically accepted. Our results suggest that polar compounds, in particular some of the Cuticular Protein Compounds (CPCs) shown here by MALDI to be specific to reproductives, could play a significant role in insect societies. While this study is preliminary and further comprehensive molecular characterization is needed to correlate the body-shaking triggering effects with a given set of polar compounds, this exploratory study opens new perspectives for understanding the role of polar compounds such as proteins in caste discrimination, fertility signaling, or interspecific insect communication.

Biodegradation of willow sawdust by novel cellulase-producing bacterial consortium from wood-feeding termites for enhancing methane production.

This study was designed to develop a cellulase-producing bacterial consortium (CBC) from wood-feeding termites that could effectively degrade willow sawdust (WSD) and consequently enhance methane production. The bacterial strains Shewanella sp. SSA-1557, Bacillus cereus SSA-1558, and Pseudomonas mosselii SSA-1568 exhibited significant cellulolytic activity. Their CBC consortium showed positive effects on cellulose bioconversion, resulting in accelerated WSD degradation. After nine days of pretreatment, the WSD had lost 63%, 50%, and 28% of its cellulose, hemicellulose, and lignin, respectively. The hydrolysis rate of treated WSD (352 mg/g) was much higher than that of untreated WSD (15.2 mg/g). The highest biogas production (66.1 NL/kg VS) with 66% methane was observed in the anaerobic digester M-2, which contained a combination of pretreated WSD and cattle dung in a 50/50 ratio. The findings will enrich knowledge for the development of cellulolytic bacterial consortia from termite guts for biological wood pretreatment in lignocellulosic anaerobic digestion biorefineries.

Gene expression profiles of chemosensory genes of termite soldier and worker antennae.

Termites have an elaborate social system that involves cooperation and division of labour among colony members. Although this social system is regulated by chemical signals produced in the colony, it remains unclear how these signals are perceived by other members. Signal transduction is well known to be triggered by the reception of odorant molecules by some binding proteins in the antennae, after which, a signal is transmitted to chemosensory receptors. However, there is insufficient information on the role of chemosensory genes involved in signal transduction in termites. Here, we identified the genes involved in chemosensory reception in the termite Reticulitermes speratus and performed a genome-wide comparative transcriptome analysis of worker and soldier antennae. First, we identified 31 odorant-binding proteins (OBPs), and three chemosensory protein A (CheA) from the genome data. Thereafter, we performed RNA sequencing to compare the expression levels of OBPs, CheAs, and previously identified chemosensory receptor genes between worker and soldier antennae. There were no receptor genes with significant differences in expression between castes. However, the expression levels of three non-receptor odorant-detection/binding proteins (OBP, CheA, and Sensory neuron membrane protein) were significantly different between castes. Real-time qPCR (RT-qPCR) analysis using antennae and other head parts confirmed that these genes were highly expressed in soldier antennae. Finally, independent RT-qPCR analysis showed that the expression patterns of these genes were altered in soldiers from different social contexts. Present results suggest that gene expression levels of some non-receptors are affected by both castes and behavioural interactions among colony members in termites.

Genome-scale community modeling for deciphering the inter-microbial metabolic interactions in fungus-farming termite gut microbiome.

Specialized microbial communities in the fungus-farming termite gut and fungal comb microbiome help maintain host nutrition through interactive biochemical activities of complex carbohydrate degradation. Numerous research studies have been focused on identifying the microbial species in the termite gut and fungal comb microbiota, but the community-wide metabolic interaction patterns remain obscure. The inter-microbial metabolic interactions in the community environment are essential for executing biochemical processes like complex carbohydrate degradation and maintaining the host's physicochemical homeostasis. Recent progress in high-throughput sequencing techniques and mathematical modeling provides suitable platforms for constructing multispecies genome-scale community metabolic models that can render sound knowledge about microbial metabolic interaction patterns. Here, we have implemented the genome-scale metabolic modeling strategy to map the relationship between genes, proteins, and reactions of 12 key bacterial species from fungal cultivating termite gut and fungal comb microbiota. The resulting individual genome-scale metabolic models (GEMs) have been analyzed using flux balance analysis (FBA) to optimize the metabolic flux distribution pattern. Further, these individual GEMs have been integrated into genome-scale community metabolic models where a heuristics-based computational procedure has been employed to track the inter-microbial metabolic interactions. Two separate genome-scale community metabolic models were reconstructed for the O. badius gut and fungal comb microbiome. Analysis of the community models showed up to ∼167% increased flux range in lignocellulose degradation, amino acid biosynthesis, and nucleotide metabolism pathways. The inter-microbial metabolic exchange of amino acids, SCFAs, and small sugars was also upregulated in the multispecies community for maximum biomass formation. The flux variability analysis (FVA) has also been performed to calculate the feasible flux range of metabolic reactions. Furthermore, based on the calculated metabolic flux values, newly defined parameters, i.e., pairwise metabolic assistance (PMA) and community metabolic assistance (CMA) showed that the microbial species are getting up to 15% higher metabolic benefits in the multispecies community compared to pairwise growth. Assessment of the inter-microbial metabolic interaction patterns through pairwise growth support index (PGSI) indicated an increased mutualistic interaction in the termite gut environment compared to the fungal comb. Thus, this genome-scale community modeling study provides a systematic methodology to understand the inter-microbial interaction patterns with several newly defined parameters like PMA, CMA, and PGSI. The microbial metabolic assistance and interaction patterns derived from this computational approach will enhance the understanding of combinatorial microbial activities and may help develop effective synergistic microcosms to utilize complex plant polymers.

Biochemical characterization of the ß-glucosidase Glu1B from Coptotermes formosanus produced in Pichia pastoris.

ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.

Evolution and functionalization of vitellogenin genes in the termite Reticulitermes speratus.

Eusociality has been commonly observed in distinct animal lineages. The reproductive division of labor is a particular feature, achieved by the coordination between fertile and sterile castes within the same nest. The sociogenomic approach in social hymenopteran insects indicates that vitellogenin (Vg) has undergone neo-functionalization in sterile castes. Here, to know whether Vgs have distinct roles in nonreproductive castes in termites, we investigated the unique characteristics of Vgs in the rhinotermitid termite Reticulitermes speratus. The four Vgs were identified from R. speratus (RsVg1-4), and RsVg3 sequences were newly identified using the RACE method. Molecular phylogenetic analysis supported the monophyly of the four termite Vgs. Moreover, the termites Vg1-3 and Vg4 were positioned in two different clades. The  dN/dS ratios indicated that the branch leading to the common ancestor of termite Vg4 was under weak purifying selection. Expression analyses among castes (reproductives, workers, and soldiers) and females (nymphs, winged alates, and queens) showed that RsVg1-3 was highly expressed in fertile queens. In contrast, RsVg4 was highly expressed in workers and female nonreproductives (nymphs and winged adults). Localization of RsVg4 messenger RNA was confirmed in the fat body of worker heads and abdomens. These results suggest that Vg genes are functionalized after gene duplication during termite eusocial transition and that Vg4 is involved in nonreproductive roles in termites.

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.

Combining OSMAC, metabolomic and genomic methods for the production and annotation of halogenated azaphilones and ilicicolins in termite symbiotic fungi.

We gathered a collection of termite mutualistic strains from French Guiana to explore the metabolites of symbiotic microorganisms. Molecular networks reconstructed from a metabolomic analysis using LC-ESI-MS/MS methodology led us to identify two families of chlorinated polyketides, i.e., azaphilones from Penicillium sclerotiorum and ilicicolins from Neonectria discophora. To define the biosynthetic pathways related to these two types of scaffolds, we used a whole genome sequencing approach followed by hybrid assembly from short and long reads. We found two biosynthetic gene clusters, including two FAD-dependent halogenases. To exploit the enzymatic promiscuity of the two identified FAD halogenases, we sought to biosynthesize novel halogenated metabolites. An OSMAC strategy was used and resulted in the production of brominated analogs of ilicicolins and azaphilones as well as iodinated analogs of azaphilones.

Tripartite Symbiotic Digestion of Lignocellulose in the Digestive System of a Fungus-Growing Termite.

Fungus-growing termites are efficient in degrading and digesting plant substrates, achieved through the engagement of symbiotic gut microbiota and lignocellulolytic Termitomyces fungi cultivated for protein-rich food. Insights into where specific plant biomass components are targeted during the decomposition process are sparse. In this study, we performed several analytical approaches on the fate of plant biomass components and did amplicon sequencing of the 16S rRNA gene to investigate the lignocellulose digestion in the symbiotic system of the fungus-growing termite Odontotermes formosanus (Shiraki) and to compare bacterial communities across the different stages in the degradation process. We observed a gradual reduction of lignocellulose components throughout the process. Our findings support that the digestive tract of young workers initiates the degradation of lignocellulose but leaves most of the lignin, hemicellulose, and cellulose, which enters the fresh fungus comb, where decomposition primarily occurs. We found a high diversity and quantity of monomeric sugars in older parts of the fungus comb, indicating that the decomposition of lignocellulose enriches the old comb with sugars that can be utilized by Termitomyces and termite workers. Amplicon sequencing of the 16S rRNA gene showed clear differences in community composition associated with the different stages of plant biomass decomposition which could work synergistically with Termitomyces to shape the digestion process. IMPORTANCE Fungus-farming termites have a mutualist association with fungi of the genus Termitomyces and gut microbiota to support the nearly complete decomposition of lignocellulose to gain access to nutrients. This elaborate strategy of plant biomass digestion makes them ecologically successful dominant decomposers in (sub)tropical Old World ecosystems. We employed acid detergent fiber analysis, high-performance anion-exchange chromatography (HPAEC), high-performance liquid chromatography (HPLC), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), pyrolysis gas chromatography-mass spectrometry (Py-GC-MS), and amplicon sequencing of the 16S rRNA gene to examine which lignocellulose components were digested and which bacteria were abundant throughout the decomposition process. Our findings suggest that although the first gut passage initiates lignocellulose digestion, the most prominent decomposition occurs within the fungus comb. Moreover, distinct bacterial communities were associated with different stages of decomposition, potentially contributing to the breakdown of particular plant components.

Bacterial species metabolic interaction network for deciphering the lignocellulolytic system in fungal cultivating termite gut microbiota.

Fungus-cultivating termite Odontotermes badius developed a mutualistic association with Termitomyces fungi for the plant material decomposition and providing a food source for the host survival. The mutualistic relationship sifted the microbiome composition of the termite gut and Termitomyces fungal comb. Symbiotic bacterial communities in the O. badius gut and fungal comb have been studied extensively to identify abundant bacteria and their lignocellulose degradation capabilities. Despite several metagenomic studies, the species-wide metabolic interaction patterns of bacterial communities in termite gut and fungal comb remains unclear. The bacterial species metabolic interaction network (BSMIN) has been constructed with 230 bacteria identified from the O. badius gut and fungal comb microbiota. The network portrayed the metabolic map of the entire microbiota and highlighted several inter-species biochemical interactions like cross-feeding, metabolic interdependency, and competition. Further, the reconstruction and analysis of the bacterial influence network (BIN) quantified the positive and negative pairwise influences in the termite gut and fungal comb microbial communities. Several key macromolecule degraders and fermentative microbial entities have been identified by analyzing the BIN. The mechanistic interplay between these influential microbial groups and the crucial glycoside hydrolases (GH) enzymes produced by the macromolecule degraders execute the community-wide functionality of lignocellulose degradation and subsequent fermentation. The metabolic interaction pattern between the nine influential microbial species has been determined by considering them growing in a synthetic microbial community. Competition (30%), parasitism (47%), and mutualism (17%) were predicted to be the major mode of metabolic interaction in this synthetic microbial community. Further, the antagonistic metabolic effect was found to be very high in the metabolic-deprived condition, which may disrupt the community functionality. Thus, metabolic interactions of the crucial bacterial species and their GH enzyme cocktail identified from the O. badius gut and fungal comb microbiota may provide essential knowledge for developing a synthetic microcosm with efficient lignocellulolytic machinery.

Comparison of gene expression profiles among caste differentiations in the termite Reticulitermes speratus.

Termite castes express specialized phenotypes for their own tasks and are a good example of insect polyphenism. To understand the comprehensive gene expression profiles during caste differentiation, RNA-seq analysis based on the genome data was performed during the worker, presoldier, and nymphoid molts in Reticulitermes speratus. In this species, artificial induction methods for each molt have already been established, and the time scale has been clarified. Three different periods (before the gut purge (GP), during the GP, and after the molt) were discriminated in each molt, and two body parts (head and other body regions) were separately sampled. The results revealed that many differentially expressed genes (head: 2884, body: 2579) were identified in each molt. Based on the independent real-time quantitative PCR analysis, we confirmed the different expression patterns of seven out of eight genes in the presoldier molt. Based on the GO and KEGG enrichment analyses, the expressions of genes related to juvenile hormone titer changes (e.g., JH acid methyltransferase), nutrition status (e.g., Acyl-CoA Delta desaturase), and cell proliferation (e.g., insulin receptor), were shown to specifically fluctuate in each molt. These differences may have a crucial impact on caste differentiation. These data are important resources for future termite sociogenomics.

Hormone-related genes heterochronically and modularly regulate neotenic differentiation in termites.

Caste development in social insects requires the coordination of molting and metamorphosis during postembryonic development. In termites, i.e., hemimetabolous eusocial insects, caste fate is determined during postembryonic development. However, it is not fully understood how the mechanisms of molting/metamorphosis are regulated in the course of differentiation between reproductive and sterile castes. In termites, only reproductives derived from alates are imagos and other sterile castes (including developmentally-terminal soldier caste) are basically juveniles or nymphs. Furthermore, supplementary reproductives that appear when the original queens and kings die or become senescent, exhibit larval features such as winglessness, and are called neotenics. Therefore, the question of whether neotenics are larvae or imagos is still under debate. In this study, by inducing female neotenic differentiation in a damp-wood termite Hodotermopsis sjostedti, morphological investigations together with juvenile hormone (JH) quantification and expression/functional analyses of genes responsible for molting and/or metamorphosis were carried out. JH titer and expression of one of the downstream genes (Kr-h1) were shown to be temporarily lowered, but increased just prior to the molt into neotenics, while consistently lowered in imaginal molt (i.e., alate differentiation). In contrast, ecdysone-related genes (EcR and E93) were upregulated at both neotenic and alate differentiation, suggesting that the heterochronic actions of ecdysone and JH lead the neotenic differentiation. Moreover, expression analyses, supported by reverse genetic experiments, showed that EcR and E93 were specifically upregulated in genital sternites (EcR and E93) and ovaries (E93) and required for the development of imaginal characters. These results suggest that the resultant mosaic phenotype of female neotenics is due to modular responses of different body parts to hormonal actions.

Prophenoloxidase of Odontotermes formosanus (Shiraki) (Blattodea: Termitidae) Is a Key Gene in Melanization and Has a Defensive Role during Bacterial Infection.

Melanization mediated by the prophenoloxidase (PPO)-activating system is an important innate immunity to fight pathogens in insects. In this study, the in vitro time-dependent increase in the intensity of melanization and phenoloxidase (PO) activity from the hemolymph of Odontotermes formosanus (Shiraki) challenged by pathogenic bacteria was detected. PPO is one of the key genes in melanization pathway, whereas the molecular characteristics and functions of O. formosanus PPO are unclear. The OfPPO gene was cloned and characterized. The open reading frame of OfPPO is 2085 bp in length and encodes a 79.497 kDa protein with 694 amino acids. A BLASTx search and phylogenetic analyses revealed that OfPPO shares a high degree of homology to the Blattodea PPOs. Moreover, real-time fluorescent quantitative PCR analysis showed that OfPPO is ubiquitously expressed in all castes and tissues examined, with the highest expression in workers and variable expression patterns in tissues of different termite castes. Furthermore, the expression of OfPPO was significantly induced in O. formosanus infected by pathogenic bacteria. Intriguingly, in combination with silencing of OfPPO expression, pathogenic bacteria challenge caused greatly increased mortality of O. formosanus. These results suggest that OfPPO plays a role in defense against bacteria and highlight the novel termite control strategy combining pathogenic bacteria application with termite PPO silencing.