Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

Evolution of Linoleic Acid Biosynthesis Paved the Way for Ecological Success of Termites.

Termites are dominant animals of tropical terrestrial ecosystems. Their success is due to their eusocial organization as well as their ability to digest dead plant tissues. While being extremely abundant, the termite diet is poor in crucial nutrients, such as fatty acids. Linoleic acid (LA) is a precursor for many vital biomolecules, and most animals depend on its dietary supply. Termites count among the exceptions known to produce LA de novo, presumably via the action of an unknown Δ12 fatty acyl desaturase (FAD) introducing the second double bond into monounsaturated oleic acid. Here, we search for the evolutionary origin of LA biosynthesis in termites. To this end, we compile the repertoire of FAD homologs from 57 species of termites and their closest relatives, the cockroaches, analyze FAD phylogeny, and identify a potential Δ12 FAD branch, which arose through duplication of a likely Δ9 FAD. We functionally characterize both paralogs and identify the Δ9 activity in the ancestral FAD-A1a and the Δ12 activity responsible for LA biosynthesis in FAD-A1b. Through the combination of homology modeling and site-directed mutagenesis, we pinpoint structural features possibly contributing to the distinct functions, regiospecificities, and substrate preferences of the two enzymes. We confirm the presence of both paralogs in all 36 studied species of the Blattoidea lineage (Blattidae, Lamproblattidae, Cryptocercidae, and termites) and conclude that we identified an evolutionary event important for the ecological success of termites, which took place in their cockroach ancestors roughly 160 My and remained conserved throughout termite diversification into 3,000 extant species.

Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies.

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus, we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

Pervasive relaxed selection in termite genomes.

Genetic changes that enabled the evolution of eusociality have long captivated biologists. More recently, attention has focussed on the consequences of eusociality on genome evolution. Studies have reported higher molecular evolutionary rates in eusocial hymenopteran insects compared with their solitary relatives. To investigate the genomic consequences of eusociality in termites, we analysed nine genomes, including newly sequenced genomes from three non-eusocial cockroaches. Using a phylogenomic approach, we found that termite genomes have experienced lower rates of synonymous substitutions than those of cockroaches, possibly as a result of longer generation times. We identified higher rates of non-synonymous substitutions in termite genomes than in cockroach genomes, and identified pervasive relaxed selection in the former (24-31% of the genes analysed) compared with the latter (2-4%). We infer that this is due to reductions in effective population size, rather than gene-specific effects (e.g. indirect selection of caste-biased genes). We found no obvious signature of increased genetic load in termites, and postulate efficient purging of deleterious alleles at the colony level. Additionally, we identified genomic adaptations that may underpin caste differentiation, such as genes involved in post-translational modifications. Our results provide insights into the evolution of termites and the genomic consequences of eusociality more broadly.

The influence of first desaturase subfamily genes on fatty acid synthesis, desiccation tolerance and inter-caste nutrient transfer in the termite Coptotermes formosanus.

Desaturase enzymes play an essential role in the biosynthesis of unsaturated fatty acids (UFAs). In this study, we identified seven "first desaturase" subfamily genes (Cfor-desatA1, Cfor-desatA2-a, Cfor-desatA2-b, Cfor-desatB-a, Cfor-desatB-b, Cfor-desatD and Cfor-desatE) from the Formosan subterranean termite Coptotermes formosanus. These desaturases were highly expressed in the cuticle and fat body of C. formosanus. Inhibition of either the Cfor-desatA2-a or Cfor-desatA2-b gene resulted in a significant decrease in the contents of fatty acids (C16:0, C18:0, C18:1 and C18:2) in worker castes. Moreover, we observed that inhibition of most of desaturase genes identified in this study had a negative impact on the survival rate and desiccation tolerance of workers. Interestingly, when normal soldiers were reared together with dsCfor-desatA2-b-treated workers, they exhibited higher mortality, suggesting that desaturase had an impact on trophallaxis among C. formosanus castes. Our findings shed light on the novel roles of desaturase family genes in the eusocial termite C. formosanus.

Major changes in domain arrangements are associated with the evolution of termites.

Domains as functional protein units and their rearrangements along the phylogeny can shed light on the functional changes of proteomes associated with the evolution of complex traits like eusociality. This complex trait is associated with sterile soldiers and workers, and long-lived, highly fecund reproductives. Unlike in Hymenoptera (ants, bees, and wasps), the evolution of eusociality within Blattodea, where termites evolved from within cockroaches, was accompanied by a reduction in proteome size, raising the question of whether functional novelty was achieved with existing rather than novel proteins. To address this, we investigated the role of domain rearrangements during the evolution of termite eusociality. Analysing domain rearrangements in the proteomes of three solitary cockroaches and five eusocial termites, we inferred more than 5,000 rearrangements over the phylogeny of Blattodea. The 90 novel domain arrangements that emerged at the origin of termites were enriched for several functions related to longevity, such as protein homeostasis, DNA repair, mitochondrial activity, and nutrient sensing. Many domain rearrangements were related to changes in developmental pathways, important for the emergence of novel castes. Along with the elaboration of social complexity, including permanently sterile workers and larger, foraging colonies, we found 110 further domain arrangements with functions related to protein glycosylation and ion transport. We found an enrichment of caste-biased expression and splicing within rearranged genes, highlighting their importance for the evolution of castes. Furthermore, we found increased levels of DNA methylation among rearranged compared to non-rearranged genes suggesting fundamental differences in their regulation. Our findings indicate the importance of domain rearrangements in the generation of functional novelty necessary for termite eusociality to evolve.

Hybrids of two destructive subterranean termites established in the field, revealing a potential for gene flow between species.

Hybridization between invasive pest species may lead to significant genetic and economic impacts that require close monitoring. The two most invasive and destructive termite species worldwide, Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann), have the potential for hybridization in the field. A three-year field survey conducted during the dispersal flight season of Coptotermes in Taiwan identified alates with atypical morphology, which were confirmed as hybrids of the two Coptotermes species using microsatellite and mitochondrial analyses. Out of 27,601 alates collected over three years, 4.4% were confirmed as hybrid alates, and some advanced hybrids (>F1 generations) were identified. The hybrid alates had a dispersal flight season that overlapped with the two parental species 13 out of 15 times. Most of the hybrid alates were females, implying that mating opportunities beyond F1 may primarily be possible through female hybrids. However, the incipient colony growth results from all potential mating combinations suggest that only backcross colonies with hybrid males could sometimes lead to brood development. The observed asymmetrical viability and fertility of hybrid alates may critically reduce the probability of advanced-hybrid colonies being established in the field.

Enhanced heterozygosity from male meiotic chromosome chains is superseded by hybrid female asexuality in termites.

Although males are a ubiquitous feature of animals, they have been lost repeatedly in diverse lineages. The tendency for obligate asexuality to evolve is thought to be reduced in animals whose males play a critical role beyond the contribution of gametes, for example, via care of offspring or provision of nuptial gifts. To our knowledge, the evolution of obligate asexuality in such species is unknown. In some species that undergo frequent inbreeding, males are hypothesized to play a key role in maintaining genetic heterozygosity through the possession of neo-sex chromosomes, although empirical evidence for this is lacking. Because inbreeding is a key feature of the life cycle of termites, we investigated the potential role of males in promoting heterozygosity within populations through karyotyping and genome-wide single-nucleotide polymorphism analyses of the drywood termite Glyptotermes nakajimai We showed that males possess up to 15 out of 17 of their chromosomes as sex-linked (sex and neo-sex) chromosomes and that they maintain significantly higher levels of heterozygosity than do females. Furthermore, we showed that two obligately asexual lineages of this species-representing the only known all-female termite populations-arose independently via intraspecific hybridization between sexual lineages with differing diploid chromosome numbers. Importantly, these asexual females have markedly higher heterozygosity than their conspecific males and appear to have replaced the sexual lineages in some populations. Our results indicate that asexuality has enabled females to supplant a key role of males.

A comprehensive sampling of mitogenomes shows the utility to infer phylogeny of termites (Blattodea: Termitoidae).

The mitogenome sequence data have been widely used in inferring the phylogeny of insects. In this study, we determined the complete mitogenome for Macrotermes sp. (Termitidae, Macrotermitinae) using next-generation sequencing. Macrotermes sp. possesses a typical insect mitogenome, displaying an identical gene order and gene content to other existing termite mitogenomes. We present the first prediction of the secondary structure of ribosomal RNA genes in termites. The rRNA secondary structures of Macrotermes sp. exhibit similarities to closely related insects and also feature distinctive characteristics in their helical structures. Together with 321 published mitogenomes of termites as ingroups and 8 cockroach mitogenomes as outgroups, we compiled the most comprehensive mitogenome sequence matrix for Termitoidae to date. Phylogenetic analyses were conducted using datasets employing different data coding strategies and various inference methods. Robust relationships were recovered at the family or subfamily level, demonstrating the utility of comprehensive mitogenome sampling in resolving termite phylogenies. The results supported the monophyly of Termitoidae, and consistent relationships within this group were observed across different analyses. Mastotermitidae was consistently recovered as the sister group to all other termite families. The families Hodotermitidae, Stolotermitidae, and Archotermopsidae formed the second diverging clade, followed by the Kalotermitidae. The Neoisoptera was consistently supported with strong node support, with Stylotermitidae being sister to the remaining families. Rhinotermitidae was found to be non-monophyletic, and Serritermitidae nested within the basal clades of Rhinotermitidae and was sister to Psammotermitinae. Overall, our phylogenetic results are largely consistent with earlier mitogenome studies.

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.

A genetic linkage map and improved genome assembly of the termite symbiont Termitomyces cryptogamus.

BACKGROUND: The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect. METHODS: To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle. RESULTS: Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi. CONCLUSIONS: The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.

The remoulding of dietary effects on the fecundity / longevity trade-off in a social insect.

BACKGROUND: In many organisms increased reproductive effort is associated with a shortened life span. This trade-off is reflected in conserved molecular pathways that link nutrient-sensing with fecundity and longevity. Social insect queens apparently defy the fecundity / longevity trade-off as they are both, extremely long-lived and highly fecund. Here, we have examined the effects of a protein-enriched diet on these life-history traits and on tissue-specific gene expression in a termite species of low social complexity. RESULTS: On a colony level, we did not observe reduced lifespan and increased fecundity, effects typically seen in solitary model organisms, after protein enrichment. Instead, on the individual level mortality was reduced in queens that consumed more of the protein-enriched diet - and partially also in workers - while fecundity seemed unaffected. Our transcriptome analyses supported our life-history results. Consistent with life span extension, the expression of IIS (insulin/insulin-like growth factor 1 signalling) components was reduced in fat bodies after protein enrichment. Interestingly, however, genes involved in reproductive physiology (e.g., vitellogenin) were largely unaffected in fat body and head transcriptomes. CONCLUSION: These results suggest that IIS is decoupled from downstream fecundity-associated pathways, which can contribute to the remoulding of the fecundity/longevity trade-off in termites as compared to solitary insects.

A holobiont approach towards polysaccharide degradation by the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis.

BACKGROUND: Termites are among the most successful insects on Earth and can feed on a broad range of organic matter at various stages of decomposition. The termite gut system is often referred to as a micro-reactor and is a complex structure consisting of several components. It includes the host, its gut microbiome and fungal gardens, in the case of fungi-growing higher termites. The digestive tract of soil-feeding higher termites is characterised by radial and axial gradients of physicochemical parameters (e.g. pH, O2 and H2 partial pressure), and also differs in the density and structure of residing microbial communities. Although soil-feeding termites account for 60% of the known termite species, their biomass degradation strategies are far less known compared to their wood-feeding counterparts. RESULTS: In this work, we applied an integrative multi-omics approach for the first time at the holobiont level to study the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis. We relied on 16S rRNA gene community profiling, metagenomics and (meta)transcriptomics to uncover the distribution of functional roles, in particular those related to carbohydrate hydrolysis, across different gut compartments and among the members of the bacterial community and the host itself. We showed that the Labiotermes gut was dominated by members of the Firmicutes phylum, whose abundance gradually decreased towards the posterior segments of the hindgut, in favour of Bacteroidetes, Proteobacteria and Verrucomicrobia. Contrary to expectations, we observed that L. labralis gut microbes expressed a high diversity of carbohydrate active enzymes involved in cellulose and hemicelluloses degradation, making the soil-feeding termite gut a unique reservoir of lignocellulolytic enzymes with considerable biotechnological potential. We also evidenced that the host cellulases have different phylogenetic origins and structures, which is possibly translated into their different specificities towards cellulose. From an ecological perspective, we could speculate that the capacity to feed on distinct polymorphs of cellulose retained in soil might have enabled this termite species to widely colonise the different habitats of the Amazon basin. CONCLUSIONS: Our study provides interesting insights into the distribution of the hydrolytic potential of the highly compartmentalised higher termite gut. The large number of expressed enzymes targeting the different lignocellulose components make the Labiotermes worker gut a relevant lignocellulose-valorising model to mimic by biomass conversion industries.

Molecular Phylogeny Reveals the Past Transoceanic Voyages of Drywood Termites (Isoptera, Kalotermitidae).

Termites are major decomposers in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae form the second-largest termite family and are distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes of ∼120 species, about 27% of kalotermitid diversity, including representatives of 21 of the 23 kalotermitid genera. Our mitochondrial genome phylogenetic trees were corroborated by phylogenies inferred from nuclear ultraconserved elements derived from a subset of 28 species. We found that extant kalotermitids shared a common ancestor 84 Ma (75-93 Ma 95% highest posterior density), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the ∼40 disjunctions among biogeographic realms were dated at <50 Ma, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for critical taxonomic revision and future comparative analyses of Kalotermitidae.

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.

More effective transposon regulation in fertile, long-lived termite queens than in sterile workers.

Transposable elements (TEs) are mobile genetic sequences, which can cause the accumulation of genomic damage in the lifetime of an organism. The regulation of TEs, for instance via the piRNA-pathway, is an important mechanism to protect the integrity of genomes, especially in the germ-line where mutations can be transmitted to offspring. In eusocial insects, soma and germ-line are divided among worker and reproductive castes, so one may expect caste-specific differences in TE regulation to exist. To test this, we compared whole-genome levels of repeat element transcription in the fat body of female workers, kings and five different queen stages of the higher termite, Macrotermes natalensis. In this species, queens can live over 20 years, maintaining near maximum reproductive output, while sterile workers only live weeks. We found a strong, positive correlation between TE expression and the expression of neighbouring genes in all castes. However, we found substantially higher TE activity in workers than in reproductives. Furthermore, TE expression did not increase with age in queens, despite a sevenfold increase in overall gene expression, due to a significant upregulation of the piRNA-pathway in 20-year-old queens. Our results suggest a caste- and age-specific regulation of the piRNA-pathway has evolved in higher termites that is analogous to germ-line-specific activity in solitary organisms. In the fat body of these termite queens, an important metabolic tissue for maintaining their extreme longevity and reproductive output, an efficient regulation of TEs likely protects genome integrity, thus further promoting reproductive fitness even at high age.

Wood feeding and social living: Draft genome of the subterranean termite Reticulitermes lucifugus (Blattodea; Termitoidae).

Termites (Insecta, Blattodea, Termitoidae) are a widespread and diverse group of eusocial insects known for their ability to digest wood matter. Herein, we report the draft genome of the subterranean termite Reticulitermes lucifugus, an economically important species and among the most studied taxa with respect to eusocial organization and mating system. The final assembly (~813 Mb) covered up to 88% of the estimated genome size and, in agreement with the Asexual Queen Succession Mating System, it was found completely homozygous. We predicted 16,349 highly supported gene models and 42% of repetitive DNA content. Transposable elements of R. lucifugus show similar evolutionary dynamics compared to that of other termites, with two main peaks of activity localized at 25% and 8% of Kimura divergence driven by DNA, LINE and SINE elements. Gene family turnover analyses identified multiple instances of gene duplication associated with R. lucifugus diversification, with significant lineage-specific gene family expansions related to development, perception and nutrient metabolism pathways. Finally, we analysed P450 and odourant receptor gene repertoires in detail, highlighting the large diversity and dynamical evolutionary history of these proteins in the R. lucifugus genome. This newly assembled genome will provide a valuable resource for further understanding the molecular basis of termites biology as well as for pest control.

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.

Characterization of the complete mitochondrial genome of Cryptotermes domesticus (Blattodea: Kalotermitidae): Genome description and phylogenetic implications.

The complete mitochondrial genome of Cryptotermes domesticus (Haviland) was sequenced and annotated to study its characteristics and the phylogenetic relationship of C. domesticus to other termite species. The mitogenome of C. domesticus is a circular, close, and double-stranded molecule with a length of 15,655 bp. The sequenced mitogenome contains 37 typical genes, which are highly conserved in gene size, organization, and codon usage. Transfer RNA genes (tRNAs) also have typical secondary structures. All of the 13 protein-coding genes (PCGs) start with an ATN codon, except for nad4, which starts with GTG and terminates with the terminal codon TAA and TAG or the incomplete form T-- (cox2 and nad5). Most tRNAs have a typical cloverleaf structure, except for trnS1, in which this form is replaced by a simple loop and lacks the dihydrouridine (DHU) arm. The nucleotide diversity (Pi) and nonsynonymous (Ka)/synonymous (Ks) mutation rate ratios indicate that nad1, cox1, and cox3 are the most conserved genes, and that cox1 has the lowest rate of evolution. In addition, an 89 bp repeated sequence was found in the A + T-rich region. Phylogenetic analysis was performed using Bayesian inference (BI) and maximum likelihood (ML) methods based on 13 PCGs, and the monophyly of Kalotermitidae was supported.

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.