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.

Trichoderma-Derived Pentapeptides from the Infected Nest Mycobiome of the Subterranean Termite Coptotermes testaceus.

Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.

Termites Are Associated with External Species-Specific Bacterial Communities.

All termites have established a wide range of associations with symbiotic microbes in their guts. Some termite species are also associated with microbes that grow in their nests, but the prevalence of these associations remains largely unknown. Here, we studied the bacterial communities associated with the termites and galleries of three wood-feeding termite species by using 16S rRNA gene amplicon sequencing. We found that the compositions of bacterial communities among termite bodies, termite galleries, and control wood fragments devoid of termite activities differ in a species-specific manner. Termite galleries were enriched in bacterial operational taxonomic units (OTUs) belonging to Rhizobiales and Actinobacteria, which were often shared by several termite species. The abundance of several bacterial OTUs, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus, was reduced in termite galleries. Our results demonstrate that both termite guts and termite galleries harbor unique bacterial communities.IMPORTANCE As is the case for all ecosystem engineers, termites impact their habitat by their activities, potentially affecting bacterial communities. Here, we studied three wood-feeding termite species and found that they influence the composition of the bacterial communities in their surrounding environment. Termite activities have positive effects on Rhizobiales and Actinobacteria abundance and negative effects on the abundance of several ubiquitous genera, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus Our results demonstrate that termite galleries harbor unique bacterial communities.

Courtship Behavior Confusion in Two Subterranean Termite Species that Evolved in Allopatry (Blattodea, Rhinotermitidae, Coptotermes).

Congeneric species that live in sympatry may have evolved various mechanisms that maintain reproductive isolation among species. However, with the spread of invasive organisms owing to increased global human activity, some species that evolved in allopatry can now be found outside their native range and may have the opportunity to interact, in the absence of mechanisms for reproductive isolation. In South Florida, where the Asian subterranean termite, Coptotermes gestroi (Wamann), and the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae) are invasive, the two species can engage in heterospecific mating behavior as their distribution range and their dispersal flight season both overlap. Termites rely on semiochemicals for many of their activities, including finding a mate after a dispersal flight. In this study, we showed that females of both species produce (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE) from their tergal glands as a shared sex pheromone. We suggest that both species primarily rely on an inundative dispersal flight strategy to find a mate, and that DTE is used as a short distance pheromone or contact pheromone to initiate and maintain the tandem between males and females. The preference of C. gestroi males for C. formosanus females during tandem resulted from the relatively high amount of DTE produced by tergal glands of C. formosanus females, when compared with those of C. gestroi females. This results in confusion of mating in the field during simultaneous dispersal flights, with a potential for hybridization. Such observations imply that no prezygotic barriers emerged while the two species evolved in allopatry for ~18 Ma.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

RNA interference unveils the importance of Pseudotrichonympha grassii cellobiohydrolase, a protozoan exoglucanase, in termite cellulose degradation.

Based on prior work, a cellulase from glycosyl hydrolase family 7 (GHF7) was identified and found to be expressed at a high level in Coptotermes formosanus. To determine the function of GHF7 family members in vivo, we used RNA interference (RNAi) to functionally analyse the exoglucanase gene Pseudotrichonympha grassii cellobiohydrolase gene (PgCBH), which was highly expressed in Pseudotrichonympha grassii, a flagellate found in the hindgut of C. formosanus. In this study, the expression level of PgCBH was down-regulated by RNAi, causing the death of P. grassii, but no effect was observed for other flagellates found in C. formosanus. RNAi also resulted in significantly reduced exoglucanase activity, and no effect was observed for endoglucanase and ß-glucosidase activities. This result demonstrated that the PgCBH gene plays a role in the protist lignocellulolytic process and is also important for host survival. PgCBH can be used as a target gene and has potential as a bioinsecticide for use against termites.

Chemical Interaction among Termite-Associated Microbes.

Bacteria and fungi in shared environments compete with one another for common substrates, and this competition typically involves microbially-produced small molecules. An investigation of one shared environmental niche, the carton material of the Formosan subterranean termite Coptotermes formosanus, identified the participants on one of these molecular exchanges. Molecular characterization of several termite-associated actinobacteria strains identified eleven known antimicrobial metabolites that may aid in protecting the C. formosanus colony from pathogenic fungal infections. One particular actinobacterial-derived small molecule, bafilomycin C1, elicited a strong chemical response from Trichoderma harzianum, a common soil saprophyte. Upon purification and structure elucidation, three major fungal metabolites were identified, t22-azaphilone, cryptenol, and homodimericin A. Both t22-azaphilone and homodimericin A are strongly upregulated, 123- and 38-fold, respectively, when exposed to bafilomycin C1, suggesting each play a role in defending T. harzianum from the toxic effect of bafilomycin C1.

The origins and radiation of Australian Coptotermes termites: from rainforest to desert dwellers.

The termite genus Coptotermes (Rhinotermitidae) is found in Asia, Africa, Central/South America and Australia, with greatest diversity in Asia. Some Coptotermes species are amongst the world's most damaging invasive termites, but the genus is also significant for containing the most sophisticated mound-building termites outside the family Termitidae. These mound-building Coptotermes occur only in Australia. Despite its economic and evolutionary significance, the biogeographic history of the genus has not been well investigated, nor has the evolution of the Australian mound-building species. We present here the first phylogeny of the Australian Coptotermes to include representatives from all described species. We combined our new data with previously generated data to estimate the first phylogeny to include representatives from all continents where the genus is found. We also present the first estimation of divergence dates during the evolution of the genus. We found the Australian Coptotermes to be monophyletic and most closely related to the Asian Coptotermes, with considerable genetic diversity in some Australian taxa possibly representing undescribed species. The Australian mound-building species did not form a monophyletic clade. Our ancestral state reconstruction analysis indicated that the ancestral Australian Coptotermes was likely to have been a tree nester, and that mound-building behaviour has arisen multiple times. The Australian Coptotermes were found to have diversified ∼13million years ago, which plausibly matches with the narrowing of the Arafura Sea allowing Asian taxa to cross into Australia. The first diverging Coptotermes group was found to be African, casting doubt on the previously raised hypothesis that the genus has an Asian origin.

Olfactory Cues from Pathogenic Fungus Affect the Direction of Motion of Termites, Coptotermes formosanus.

Formosan subterranean termites, Coptotermes formosanus, tend to avoid pathogen odors when tested in Y-tube olfactometers, but approach and groom exposed nestmates to remove pathogens from their cuticle and maintain a healthy population. To better understand their differential reaction to pathogens and their odors, the relationship between odor cues and direction of motion was examined with the fungus Isaria fumosorosea K3 strain. The results indicate that nestmate odor was strongly attractive only in tests where fungal odors were present in both branches of the olfactometer. Termites generally avoid fungal odors when offered a choice without fungal odor. We also tested termite aversion to 3-octanone and 1-octen-3-ol, major surface chemical compounds of I. fumosorosea K3, and estimated the total mass of these compounds present on the conidial surface by direct extraction method. The total quantity of these chemicals on the surface of fungal conidia was estimated to be approximately 0.01 ng per 10(7) conidia. This study demonstrates a context dependent behavioral change in termites in response to the odors of pathogenic fungi.

Foraging Activities of Coptotermes formosanus in Subtropical Areas in China.

The foraging activities, including foraging range and seasonal fluctuation of the Formosan subterranean termite, Coptotermes formosanus Shiraki, were investigated in subtropical areas in China. Six mature C. formosanus colonies were selected for this study. Foraging distance and area were conducted in Anqing1, Hengyang, and Wuxi1 colonies, while the seasonal fluctuation was conducted in Wuxi2, Wuwei, and Anqing2 colonies. Mark-release-recapture method analysis showed that the Formosan termites foraged at least 11.5, 28.7, and 56.8 m away from the main nest and covered 98.1, 543.7, and 671.9 m2 of foraging area at Anqing1, Hengyang, and Wuxi1 site, respectively. The seasonal fluctuation in termite colony activity showed an "M shape" pattern according to the wood damages caused by termites at monitoring stations. Peak colony activity at Wuxi2, Wuwei, and Anqing2 occurred in July and October, June and September, July and October, respectively. This study provides critical information for the integrated management of C. formosanus, including baiting application in the subtropical regions of China, where it constitutes the most destructive pest for household structures.

Chemical composition, antifeedant, repellent, and toxicity activities of the rhizomes of galangal, Alpinia galanga against Asian subterranean termites, Coptotermes gestroi and Coptotermes curvignathus (Isoptera: Rhinotermitidae).

Dual choice bioassays were used to evaluate the antifeedant property of essential oil and methanolic extract of Alpinia galanga (L.) (locally known as lengkuas) against two species of termites, Coptotermes gestroi (Wasmann) and Coptotermes curvignathus (Holmgren) (Isoptera: Rhinotermitidae). A 4-cm-diameter paper disc treated with A. galanga essential oil and another treated with either methanol or hexane as control were placed in a petri dish with 10 termites. Mean consumption of paper discs (miligram) treated with 2,000 ppm of essential oil by C. gestroi was 3.30 ± 0.24 mg and by C. curvignathus was 3.32 ± 0.24 mg. A. galanga essential oil showed significant difference in antifeedant effect, 2,000 ppm of A. galanga essential oil was considered to be the optimum concentration that gave maximum antifeedant effect. The essential oil composition was determined using gas chromatography-mass spectrometry. The major component of the essential oil was 1,8-cineol (61.9%). Antifeedant bioassay using 500 ppm of 1,8-cineol showed significant reduction in paper consumption by both termite species. Thus, the bioactive agent in A. galangal essential oil causing antifeeding activity was identified as 1,8-cineol. Repellent activity shows that 250 ppm of 1,8-cineol caused 50.00 ± 4.47% repellency for C. gestroi, whereas for C. curvignathus 750 ppm of 1,8-cineol was needed to cause similar repellent activity (56.67 ± 3.33%). C. curvignathus is more susceptible compare to C. gestroi in Contact Toxicity study, the lethal dose (LD50) of C. curvignathus was 945 mg/kg, whereas LD50 value for C. gestroi was 1,102 mg/kg. Hence 1,8-cineol may be developed as an alternative control against termite in sustainable agriculture practices.

myo-Inositol and Phytate Are Toxic to Formosan Subterranean Termites (Isoptera: Rhinotermitidae).

Several rare and common monosaccharides were screened for toxic effects on the Formosan subterranean termite, Coptotermes formosanus Shiraki, with the aim of identifying environmentally friendly termiticides. myo-Inositol and phytic acid, which are nontoxic to mammals, were identified as potential termite control compounds. Feeding bioassays with termite workers, where both compounds were supplied on filter paper in concentrations from 160.2 to 1,281.7 µg/mm(3), showed concentration-dependent toxicity within 2 wk. Interestingly myo-inositol was nontoxic when administered to termites in agar (40 mg/ml) in the absence of a cellulosic food source, an unexplained phenomenon. In addition, decreased populations of termite hindgut protozoa were observed upon feeding on myo-inositol but not phytate-spiked filter paper. Radiotracer feeding studies using myo-inositol-[2-(3)H] with worker termites showed no metabolism after ingestion over a 2-d feeding period, ruling out metabolites responsible for the selective toxicity.

Toxicity and transmission of thiamethoxam in the Asian subterranean termite Coptotermes gestroi (Isoptera: Rhinotermitidae).

The toxicity and horizontal transmission of thiamethoxam was evaluated in the workers of the Asian subterranean termite Coptotermes gestroi Wasmann (Isoptera: Rhinotermitidae). Brief exposure to sand treated with thiamethoxam at concentration ranging from 0.25 to 50 µg/ml resulted in a dose-dependent mortality in C. gestroi. Sand treated with 50 µg/ml thiamethoxam resulted in very high mortality within 30-60 min of exposure. Termites exposed to sand treated with 0.25-25 µg/ml exhibited delayed toxicity and nonrepellency in C. gestroi. A horizontal transmission study using 25 µg/ml of thiamethoxam at donor-recipient ratio of at least 2:5 (treated:untreated) indicated that thiamethoxam can be transferred between exposed and unexposed workers, resulting in significant termite mortality in unexposed termites within 1-3 d post exposure.

Death zone minimizes the impact of fipronil-treated soils on subterranean termite colonies by negating transfer effects.

The use of nonrepellent liquid termiticides against subterranean termites has long relied on the assumption that foraging termites in soils could transfer toxicants to nestmates to achieve population control. However, their dose-dependent lethal time can lead to rapid termite mortality in proximity of the treatment, triggering secondary repellency. The current study characterizes the dynamic nature of the "death zone," i.e., the area adjacent to soil termiticides that termites would avoid owing the accumulation of cadavers. Using whole subterranean termite laboratory colonies of Coptotermes gestroi (Wasmann) with 3 × 15 m foraging distances, fipronil was implemented at 1.5 m, 7.5 m, or 12.5 m away from colony central nests, emulating a corrective action against an termite structural infestation. For treatments at 7.5 m and 12.5 m, the death zone stabilized at an average of ~2.56 m away from the treatment after 40 d post-treatment, and colonies suffered as little as 1.5% mortality by 200 d post-treatment. Colonies located 1.5 m away from the treatment minimized the death zone to ~1.1 m and suffered as little as 23.5% mortality. Mortality only occurred within the first few days of treatment from initial exposure, as the rapid emergence of the death zone negated further transfer effects among nestmates over time. In some cases, foraging termites were trapped within the infested structure. While technically nonrepellent, fipronil becomes functionally repellent from the rapid mortality onset near the treatment. Even if diligently implemented to successfully protect structures, surrounding termite colonies are minimally impacted by fipronil soil treatments.

Oleic acid triggers burial behavior in a termite population through an odorant binding protein.

Social insects maintain hygienic conditions through their social immunity behaviors. Among these behaviors, burial behavior of termites is central for protecting healthy individuals from corpses. Many factors trigger burial behavior, and it is generally believed that chemicals released by corpses, such as oleic acid, are the most important cues for triggering burial behavior in termites. However, the contribution of the olfactory system to this behavior remains unclear. Here we report an odorant binding protein (OBP) that transports oleic acid and triggers burial behavior in Coptotermes formosanus Shiraki. We demonstrated that CforOBP7 is highly expressed in the antennae of workers. Fluorescent competition binding experiments exhibited that CforOBP7 has a strong affinity for oleic acid. Furthermore, the antennal response to oleic acid was significantly reduced, and oleic acid-triggered burial behavior was also inhibited in CforOBP7-silenced termites. We conclude that CforOBP7 governs the burial behavior of C. formosanus triggered by oleic acid.

Phylogenetic relationship of subterranean termite Coptotermes gestroi (Blattodea: Rhinotermitidae) inhabiting urban and natural habitats.

Coptotermes termites were collected from forestry habitats at University Sains Malaysia, Penang, while urban samples were collected from residentials from Penang and Kedah, Malaysia. Mitochondrial DNA markers, Cytochrome Oxidase 1 (CO1), and 16S ribosomal RNA (16S rRNA) genes were amplified and sequenced to confirm the species of the termite samples as Coptotermes gestroi. Through Blastn, all 25 CO1 and 16S rRNA sequences, respectively from urban and natural habitats were found to be 99.54-100.00 % similar to C. gestroi reference sequences from previous studies in Peninsular Malaysia. The phylogenetic trees constructed using Neighboring-joining (NJ) and Maximum Likelihood (ML) methods resulted in CO1 sequences clustering in two clades and 16S rRNA sequences clustering in a single clade. The overall mean distance was low for the C. gestroi populations from natural habitats and urban settings (FST = 0.004). Analysis of natural habitat populations using CO1 sequences revealed two haplotypes within the population, with a haplotype diversity (Hd) of 0.045 ± 0.005, while the urban population shared a common haplotype with the natural habitat populations and there was no haplotype diversity recorded between the populations. Urban and natural habitats included only one haplotype for 16S rRNA sequences, indicating a lack of nucleotide diversity. Based on the findings, a non-significant difference between the natural habitat and urban population suggests C. gestroi inhabiting both regions likely originated from a similar source and underwent population homogeneity in different settings facilitated by anthropogenic dispersal.

Equivalent Colony Growth of Hybrids of Two Invasive Coptotermes Species Can Threaten Urban Areas.

Coptotermes formosanus Shiraki and C. gestroi (Wasmann) are economically important structural pests in urban areas. Due to anthropogenic activity, both species have been introduced into the United States, with their respective invasive ranges now overlapping in Florida, and the two species have the capability to hybridize. The potential for structural damage from subterranean termite colonies primarily depends on colony size. However, long-term colony growth and wood consumption capabilities of hybrid Coptotermes colonies remain to be investigated, to determine the potential pest status of field-established hybrid colonies. In this study, we investigated long-term colony development over four years to determine if aging hybrid colonies display vigor in terms of colony growth. In addition, we compared wood consumption rate of hybrid colonies to compare their potential impact as structural pests with the two parental species. In aging colonies (four-year-old), both hybrid mating types displayed a colony growth equivalent to C. formosanus. However, the wood consumption rates of four-year-old colonies of the two parental Coptotermes species and their hybrids were similar, indicating equal damaging potential. We also found multiple secondary reproductives in hybrid colonies, even in the presence of primary reproductives, which may favor their potential establishment and spread. Although hybrid colonies or hybrid alates have yet to be detected in the field, our results suggest that such hybrid colonies would be an additional termite threat in the future if they were established in the field.

Effects of soil moisture on tunneling, survivorship, and food consumption of the Formosan and eastern subterranean termites (Blattodea: Rhinotermitidae).

Soil moisture is a critical environmental factor for the survival and behavior of subterranean termites (family Rhinotermitidae). The invasive Formosan subterranean termite, Coptotermes formosanus Shiraki, and the native eastern subterranean termite, Reticulitermes flavipes (Kollar), co-occur in the southeastern United States, while R. flavipes is distributed in a wider geoclimatic range. Previous studies showed that subterranean termites preferred higher soil moisture levels for tunneling and feeding; however, the impacts of constant moisture remained to be characterized to understand their moisture tolerance. In this study, we hypothesized that different soil moisture regimes can alter termite foraging and survival, and that the effects differ between the two species. The tunneling activity, survivorship, and food consumption of termites were documented for 28 days with different sand moisture conditions ranging from no moisture to full saturation (0%, 1%, 5%, 15%, 25%, and 30%). We found that there were no significant differences in the responses between C. formosanus and R. flavipes. In both species, termites did not survive or tunnel with 0% moisture. Termites performed tunneling with only 1% sand moisture, although they did not survive for 28 days. A minimal of 5% sand moisture was required for survival, and there were no significant differences in survivorship, tunneling activity, or food consumption among moisture contents of 5-30%. The results suggest that subterranean termites are resilient to moisture extremes. Colonies can tolerate low moisture conditions in their foraging environment for extended times, which may allow them to tunnel and find new moisture sources for colony survival.

Genome-wide identification of Coptotermes formosanus immune genes and their potential roles in termite control.

In recent years, the use of microbes to control termites has attracted increasing attention. It was found that pathogenic bacteria, nematodes, and fungi effectively control termites under laboratory conditions. However, their effects have not been replicated in the field, and one reason for this is the complex immune defense mechanisms of termites, which are mainly regulated by immune genes. Therefore, altering the expression of immune genes may have a positive influence on the biocontrol efficacy of termites. Coptotermes formosanus Shiraki is one of the most economically important termite pests worldwide. Currently, the large-scale identification of immune genes in C. formosanus is primarily based on cDNA library or transcriptome data rather than at the genomic level. In this study, we identified the immune genes of C. formosanus according to genome-wide analysis. In addition, our transcriptome analysis showed that immune genes were significantly downregulated when C. formosanus was exposed to the fungus Metarhizium anisopliae or nematodes. Finally, we found that injecting dsRNA to inhibit three immune genes (CfPGRP-SC1, CfSCRB3, and CfHemocytin), which recognize infectious microbes, significantly increased the lethal effect of M. anisopliae on termites. These immune genes show great potential for C. formosanus management based on RNAi. These results also increase the number of known immune genes in C. formosanus which will provide a more comprehensive insight into the molecular basis of immunity in termites.

Metagenomic 16S rRNA amplicon data of gut microbial diversity in three species of subterranean termites (Coptotermes gestroi, Globitermes sulphureus and Macrotermes gilvus).

In this paper, we present the metagenomic dataset of gut microbial DNA of the lower group of subterranean termites, i.e. Coptotermes gestroi, and the higher groups, i.e. Globitermes sulphureus and Macrotermes gilvus, in Penang, Malaysia. Two replicates of each species were sequenced using Next-Generation Sequencing (Illumina MiSeq) and analysed via QIIME2. The results returned with 210,248 sequences in C. gestroi, 224,972 in G. sulphureus, and 249,549 in M. gilvus. The sequence data were deposited in the NCBI Sequence Read Archive (SRA) under BioProject number of PRJNA896747. The community analysis showed that Bacteroidota is the most abundant phylum in C. gestroi and M. gilvus, while Spirochaetota is prevalent in G. sulphureus.