Uncovering the function of insulin receptor substrate in termites' immunity through active immunization.

Insulin receptor substrate (IRS) proteins are key mediators in insulin signaling pathway. In social insect lives, IRS proteins played important roles in caste differentiation and foraging, but there function in disease defenses such as active immunization has not been reported yet. To investigate the issue, we successfully suppressed the IRS gene 3 days after dsRNA injection. Suppressing IRS gene increased the contents of glucose, trehalose, glycogen, and triglyceride and decreased the content of pyruvate in termites, and led to the metabolic disorder of glucose and lipids. IRS suppressing significantly enhanced grooming behaviors of nestmates of fungus-contaminated termites and hence increased the conidial load in the guts of the nestmates. Additionally, IRS suppressing led to significant downregulation of the immune genes Gram-negative bacteria-binding protein2 (GNBP2) and termicin and upregulation of the apoptotic gene caspase8, and hence diminished antifungal activity of nestmates of fungus-contaminated termites. The above abnormal behavioral and physiological responses significantly decreased the survival rate of dsIRS-injected nestmates of the fungus-contaminated termites. These findings suggest that IRS is involved in regulation of active immunization in termites, providing a better understanding of the link between insulin signaling and the social immunity of termites.

Involvement of a catalase gene in lignin catalysis and immune defense against pathogenic fungus in Coptotermes formosanus: a potential new target for termite control.

BACKGROUND: Detoxifying enzymes are likely involved in lignin feeding and immune defense mechanisms within termites, rendering them potential targets for biological control. However, investigations into the dual functionality of termite detoxification enzymes in vivo have not been documented. RESULTS: In this study, the complete cDNA of the catalase gene (Cfcat) derived from Coptotermes formosanus Shiraki was amplified. CFCAT comprises an open reading frame spanning 1527 bp, encoding a 508-amino acid sequence. The highest expression was observed in the epidermal tissues (including the fat body and hemolymph) followed by the foregut/salivary gland. Furthermore, we confirmed the catalase activity of the recombinant Cfcat protein. Using RNA interference (RNAi) technology, the importance of Cfcat in the lignin-feeding of C. formosanus was demonstrated, and the role of Cfcat in innate immunity was investigated. Survival assays showed that Cfcat RNAi significantly increased the susceptibility of C. formosanus to Metarhizium anisopliae. Irrespective of the infection status, Cfcat inhibition had a significant impact on multiple factors of humoral and intestinal immunity in C. formosanus. Notably, Cfcat RNAi exhibited a more pronounced immunosuppressive effect on humoral immunity than on intestinal immunity. CONCLUSION: Cfcat plays an important role in the regulation of innate immunity and lignin feeding in C. formosanus. Cfcat RNAi can weaken the immune response of termites against M. anisopliae, which may aid the biocontrol efficiency of M. anisopliae against C. formosanus. This study provides a theoretical basis and technical reference for the development of a novel biocontrol strategy targeting detoxifying enzymes of termites. © 2024 Society of Chemical Industry.

Roles of selenoprotein T and transglutaminase in active immunization against entomopathogenic fungi in the termite Reticulitermes chinensis.

Active immunization can protect individuals from infectious diseases in social insects. It is well established that trace elements are essential to the host immune system, but the related gene functions in insect social immunity are unknown. Here, we found that the levels of three free elements (Se, Ca and Cr) and selenoprotein T (SELT) expression were significantly decreased in the termite Reticulitermes chinensis Snyder during active immunization against the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin. Thus, we further explored the role of the SELT gene in the active immunization of termites. After SELT was significantly silenced by RNAi, the nestmates of fungus-contaminated termites exhibited reduced antifungal activity and increased mortality, along with increased expression of the immune genes transglutaminase (TG) and transferrin (Tsf), indicating that the active immunization of termites was disrupted by SELT silencing. Moreover, the TG-knockdown nestmates of fungus-contaminated termites significantly decreased grooming behavior, antifungal activity and survival, despite the upregulation of SELT expression, also suggesting that the active immunization of termites was disrupted by the silencing of TG. These findings demonstrated that both SELT gene and TG gene play important roles in driving active immunization against the entomopathogenic fungus M. anisopliae in R. chinensis.

Increased genetic diversity from colony merging in termites does not improve survival against a fungal pathogen.

In some species of social insects the increased genetic diversity from having multiple breeders in a colony has been shown to improve pathogen resistance. Termite species typically found colonies from single mated pairs and therefore may lack the flexibility to buffer pathogen pressure with increased genetic diversity by varying the initial number of reproductives. However, they can later increase group diversity through colony merging, resulting in a genetically diverse, yet cohesive, workforce. In this study, we investigate whether the increased group diversity from colony fusion benefits social immunity in the subterranean termite Reticulitermes flavipes. We confirm previous findings that colonies of R. flavipes will readily merge and we show that workers will equally groom nestmates and non-nestmates after merging. Despite this, the survival of these merged colonies was not improved after exposure to a fungal pathogen, but instead leveled to that of the more susceptible or the more resistant colony. Our study brings little support to the hypothesis that colony fusion may improve immunity through an increase of genetic diversity in R. flavipes. Instead, we find that following exposure to a lethal pathogen, one colony is heavily influential to the entire group's survival after merging.

The Influence of Allogrooming Behavior on Individual Innate Immunity in the Subterranean Termite Reticulitermes chinensis (Isoptera: Rhinotermitidae).

Insect societies have evolved a series of disease defenses against pathogens, including social sanitary behavior and individual innate immunity. However, whether sanitary behavior can affect individual innate immunity remains unknown. Here, we exposed the termite Reticulitermes chinensis Snyder to the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin(Ascomycota: Hypocreales), and then measured their allogrooming behavior, conidia load, infection mortality, antifungal activity and immune gene expressions . Our results showed that most of the fungal conidia were fast removed from the cuticles of the grouped termites by intensive allogrooming behavior, resulting in low mortality. The antifungal activity and immune gene expressions (termicin and transferrin) in grouped exposed termites were significantly lower than those in single exposed termite but not significantly different from those in unexposed treatments. These results suggest that allogrooming behavior can fast remove fungal conidia from termite cuticles and then decrease their physiological investment in individual innate immunity.

Hemocyte Morphology and Cellular Immune Response in Termite (Reticulitermes speratus).

Because termites (Reticulitermes speratus) are very small, it is difficult to conduct experiments involving pathogen injection and hemocyte collection. Therefore, to observe hemocyte-mediated immune responses against foreign substances, in vitro hemocyte culture is essential. After collecting about 3 µl of hemolymph, hemocytes were cultured for 7 d, during which the cells maintained full function. Four types of hemocyte were identified, namely, granulocytes, plasmatocytes, oenocytoids, and prohemocytes, among which granulocytes are the main immune hemocytes that fight invasion by foreign substances. Most hemocytes were alive and/or functioning after 7 d of culture, but then either died or lost function.

Termite soldiers contribute to social immunity by synthesizing potent oral secretions.

The importance of soldiers to termite society defence has long been recognized, but the contribution of soldiers to other societal functions, such as colony immunity, is less well understood. We explore this issue by examining the role of soldiers in protecting nestmates against pathogen infection. Even though they are unable to engage in grooming behaviour, we find that the presence of soldiers of the Darwin termite, Mastotermes darwiniensis, significantly improves the survival of nestmates following entomopathogenic infection. We also show that the copious exocrine oral secretions produced by Darwin termite soldiers contain a high concentration of proteins involved in digestion, chemical biosynthesis, and immunity. The oral secretions produced by soldiers are sufficient to protect nestmates against infection, and they have potent inhibitory activity against a broad spectrum of microbes. Our findings support the view that soldiers may play an important role in colony immunity, and broaden our understanding of the possible function of soldiers during the origin of soldier-first societies.

Social Immunity: Emergence and Evolution of Colony-Level Disease Protection.

Social insect colonies have evolved many collectively performed adaptations that reduce the impact of infectious disease and that are expected to maximize their fitness. This colony-level protection is termed social immunity, and it enhances the health and survival of the colony. In this review, we address how social immunity emerges from its mechanistic components to produce colony-level disease avoidance, resistance, and tolerance. To understand the evolutionary causes and consequences of social immunity, we highlight the need for studies that evaluate the effects of social immunity on colony fitness. We discuss the roles that host life history and ecology have on predicted eco-evolutionary dynamics, which differ among the social insect lineages. Throughout the review, we highlight current gaps in our knowledge and promising avenues for future research, which we hope will bring us closer to an integrated understanding of socio-eco-evo-immunology.

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies.

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus.

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

Metatranscriptome analysis reveals bacterial symbiont contributions to lower termite physiology and potential immune functions.

BACKGROUND: Symbioses throughout the animal kingdom are known to extend physiological and ecological capabilities to hosts. Insect-microbe associations are extremely common and are often related to novel niche exploitation, fitness advantages, and even speciation events. These phenomena include expansions in host diet, detoxification of insecticides and toxins, and increased defense against pathogens. However, dissecting the contributions of individual groups of symbionts at the molecular level is often underexplored due to methodological and analytical limitations. Termites are one of the best studied systems for physiological collaborations between host and symbiota; however, most work in lower termites (those with bacterial and protist symbionts) focuses on the eukaryotic members of this symbiotic consortium. Here we present a metatranscriptomic analysis which provides novel insights into bacterial contributions to the holobiont of the eastern subterranean termite, Reticulitermes flavipes, in the presence and absence of a fungal pathogen. RESULTS: Using a customized ribodepletion strategy, a metatranscriptome assembly was obtained representing the host termite as well as bacterial and protist symbiota. Sequence data provide new insights into biosynthesis, catabolism, and transport of major organic molecules and ions by the gut consortium, and corroborate previous findings suggesting that bacteria play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion. With regard to fungal pathogen challenge, a total of 563 differentially expressed candidate host and symbiont contigs were identified (162 up- and 401 downregulated; α/FDR = 0.05) including an upregulated bacterial amidohydrolase. CONCLUSIONS: This study presents the most complete bacterial metatranscriptome from a lower termite and provides a framework on which to build a more complete model of termite-symbiont interactions including, but not limited to, digestion and pathogen defense.

Phenoloxidase activity in the infraorder Isoptera: unraveling life-history correlates of immune investment.

Within the area of ecological immunology, the quantification of phenoloxidase (PO) activity has been used as a proxy for estimating immune investment. Because termites have unique life-history traits and significant inter-specific differences exist regarding their nesting and foraging habits, comparative studies on PO activity can shed light on the general principles influencing immune investment against the backdrop of sociality, reproductive potential, and gender. We quantified PO activity across four termite species ranging from the phylogenetically basal to the most derived, each with their particular nesting/foraging strategies. Our data indicate that PO activity varies across species, with soil-dwelling termites exhibiting significantly higher PO levels than the above-ground wood nester species which in turn have higher PO levels than arboreal species. Moreover, our comparative approach suggests that pathogenic risks can override reproductive potential as a more important driver of immune investment. No gender-based differences in PO activities were recorded. Although termite PO activity levels vary in accordance with a priori predictions made from life-history theory, our data indicate that nesting and foraging strategies (and their resulting pathogenic pressures) can supersede reproductive potential and other life-history traits in influencing investment in PO. Termites, within the eusocial insects, provide a unique perspective for inferring how different ecological pressures may have influenced immune function in general and their levels of PO activity, in particular.

Experimental verification and molecular basis of active immunization against fungal pathogens in termites.

Termites are constantly exposed to many pathogens when they nest and forage in the field, so they employ various immune strategies to defend against pathogenic infections. Here, we demonstrate that the subterranean termite Reticulitermes chinensis employs active immunization to defend against the entomopathogen Metarhizium anisopliae. Our results showed that allogrooming frequency increased significantly between fungus-treated termites and their nestmates. Through active social contact, previously healthy nestmates only received small numbers of conidia from fungus-treated individuals. These nestmates experienced low-level fungal infections, resulting in low mortality and apparently improved antifungal defences. Moreover, infected nestmates promoted the activity of two antioxidant enzymes (SOD and CAT) and upregulated the expression of three immune genes (phenoloxidase, transferrin, and termicin). We found 20 differentially expressed proteins associated with active immunization in R. chinensis through iTRAQ proteomics, including 12 stress response proteins, six immune signalling proteins, and two immune effector molecules. Subsequently, two significantly upregulated (60S ribosomal protein L23 and isocitrate dehydrogenase) and three significantly downregulated (glutathione S-transferase D1, cuticle protein 19, and ubiquitin conjugating enzyme) candidate immune proteins were validated by MRM assays. These findings suggest that active immunization in termites may be regulated by different immune proteins.

Molecular signatures of nicotinoid-pathogen synergy in the termite gut.

Previous studies in lower termites revealed unexpected synergies between nicotinoid insecticides and fungal entomopathogens. The present study investigated molecular mechanisms of nicotinoid-pathogen synergy in the lower termite Reticulitermes flavipes, using the nicotinoid, imidacloprid, in combination with fungal and bacterial entomopathogens. Particular focus was placed on metatranscriptome composition and microbial dynamics in the symbiont-rich termite gut, which houses diverse mixes of protists and bacteria. cDNA microarrays containing a mix of host and protist symbiont oligonucleotides were used to simultaneously assess termite and protist gene expression. Five treatments were compared that included single challenges with sublethal doses of fungi (Metharizium anisopliae), bacteria (Serratia marcescens) or imidacloprid, and dual challenges with fungi + imidacloprid or bacteria + imidacloprid. Our findings point towards protist dysbiosis and compromised social behavior, rather than suppression of stereotypical immune defense mechanisms, as the dominant factors underlying nicotinoid-pathogen synergy in termites. Also, greater impacts observed for the fungal pathogen than for the bacterial pathogen suggest that the rich bacterial symbiont community in the R. flavipes gut (>5000 species-level phylotypes) exists in an ecological balance that effectively excludes exogenous bacterial pathogens. These findings significantly advance our understanding of antimicrobial defenses in this important eusocial insect group, as well as provide novel insights into how nicotinoids can exert deleterious effects on social insect colonies.

Bacterial microbiome of Coptotermes curvignathus (Isoptera: Rhinotermitidae) reflects the coevolution of species and dietary pattern.

Coptotermes curvignathus Holmgren is capable of feeding on living trees. This ability is attributed to their effective digestive system that is furnished by the termite's own cellulolytic enzymes and cooperative enzymes produced by their gut microbes. In this study, the identity of an array of diverse microbes residing in the gut of C. curvignathus was revealed by sequencing the near-full-length 16S rRNA genes. A total of 154 bacterial phylotypes were found. The Bacteroidetes was the most abundant phylum and accounted for about 65% of the gut microbial profile. This is followed by Firmicutes, Actinobacteria, Spirochetes, Proteobacteria, TM7, Deferribacteres, Planctomycetes, Verrucomicrobia, and Termite Group 1. Based on the phylogenetic study, this symbiosis can be a result of long coevolution of gut enterotypes with the phylogenic distribution, strong selection pressure in the gut, and other speculative pressures that determine bacterial biome to follow. The phylogenetic distribution of cloned rRNA genes in the bacterial domain that was considerably different from other termite reflects the strong selection pressures in the gut where a proportional composition of gut microbiome of C. curvignathus has established. The selection pressures could be linked to the unique diet preference of C. curvignathus that profoundly feeds on living trees. The delicate gut microbiome composition may provide available nutrients to the host as well as potential protection against opportunistic pathogen.

Immune-related transcriptome of Coptotermes formosanus Shiraki workers: the defense mechanism.

Formosan subterranean termites, Coptotermes formosanus Shiraki, live socially in microbial-rich habitats. To understand the molecular mechanism by which termites combat pathogenic microbes, a full-length normalized cDNA library and four Suppression Subtractive Hybridization (SSH) libraries were constructed from termite workers infected with entomopathogenic fungi (Metarhizium anisopliae and Beauveria bassiana), Gram-positive Bacillus thuringiensis and Gram-negative Escherichia coli, and the libraries were analyzed. From the high quality normalized cDNA library, 439 immune-related sequences were identified. These sequences were categorized as pattern recognition receptors (47 sequences), signal modulators (52 sequences), signal transducers (137 sequences), effectors (39 sequences) and others (164 sequences). From the SSH libraries, 27, 17, 22 and 15 immune-related genes were identified from each SSH library treated with M. anisopliae, B. bassiana, B. thuringiensis and E. coli, respectively. When the normalized cDNA library was compared with the SSH libraries, 37 immune-related clusters were found in common; 56 clusters were identified in the SSH libraries, and 259 were identified in the normalized cDNA library. The immune-related gene expression pattern was further investigated using quantitative real time PCR (qPCR). Important immune-related genes were characterized, and their potential functions were discussed based on the integrated analysis of the results. We suggest that normalized cDNA and SSH libraries enable us to discover functional genes transcriptome. The results remarkably expand our knowledge about immune-inducible genes in C. formosanus Shiraki and enable the future development of novel control strategies for the management of Formosan subterranean termites.

Resource competition between two fungal parasites in subterranean termites.

Subterranean termites live in large groups in underground nests where the pathogenic pressure of the soil environment has led to the evolution of a complex interaction among individual and social immune mechanisms in the colonies. However, groups of termites under stress can show increased susceptibility to opportunistic parasites. In this study, an isolate of Aspergillus nomius Kurtzman, Horn & Hessltine was obtained from a collapsed termite laboratory colony. We determined that it was primarily a saprophyte and, secondarily, a facultative parasite if the termite immunity is undergoing a form of stress. This was determined by stressing individuals of the Formosan subterranean termite Coptotermes formosanus Shiraki via a co-exposure to the virulent fungal parasite Metarhizium anisopliae (Metch.) Sorokin. We also examined the dynamics of a mixed infection of A. nomius and M. anisopliae in a single termite host. The virulent parasite M. anisopliae debilitated the termite immune system, but the facultative, fast growing parasite A. nomius dominated the mixed infection process. The resource utilization strategy of A. nomius during the infection resulted in successful conidia production, while the chance for M. anisopliae to complete its life cycle was reduced. Our results also suggest that the occurrence of opportunistic parasites such as A. nomius in collapsing termite laboratory colonies is the consequence of a previous stress, not the cause of the stress.

Identification of mycosis-related genes in the eastern subterranean termite by suppression subtractive hybridization.

The Eastern subterranean termite Reticulitermes flavipes (Isoptera, Rhinotermitidae) is a cosmopolitan, structural pest that is the target of research into termite innate immunity. In this study, we use suppression subtractive hybridization to construct a normalized cDNA library of genes excessively expressed upon fungal infection. At 24 h postinfection with Metarhizium anisopliae, the library revealed 182 expressed sequence tag (EST) clones that potentially represent immune responsive genes. The nucleotide sequence from a majority (97%) of ESTs assembled into a small number (n = 13) of contiguous sequences, with the remainder (n = 6) representing singletons. Our screen therefore captured as many as 19 different mRNAs highly expressed in response to the fungal pathogen at this time. Primary sequencing of all loci revealed that approximately half (n = 10) contained open reading frames with significant similarity to known proteins. These clones represent nuclear and mitochondrial coding genes, as well as putative long noncoding RNA genes. Quantitative polymerase chain reaction analysis of coding genes on independently infected groups of worker termites confirms in each case that the transcripts identified from the library are up-regulated postfungal infection. The genes identified here are relevant to future studies on termite biocontrol and social insect immunity.