A Century of Synergy in Termite Symbiosis Research: Linking the Past with New Genomic Insights.

Termites have long been studied for their symbiotic associations with gut microbes. In the late nineteenth century, this relationship was poorly understood and captured the interest of parasitologists such as Joseph Leidy; this research led to that of twentieth-century biologists and entomologists including Cleveland, Hungate, Trager, and Lüscher. Early insights came via microscopy, organismal, and defaunation studies, which led to descriptions of microbes present, descriptions of the roles of symbionts in lignocellulose digestion, and early insights into energy gas utilization by the host termite. Focus then progressed to culture-dependent microbiology and biochemical studies of host-symbiont complementarity, which revealed specific microhabitat requirements for symbionts and noncellulosic mechanisms of symbiosis (e.g., N2 fixation). Today, knowledge on termite symbiosis has accrued exponentially thanks to omic technologies that reveal symbiont identities, functions, and interdependence, as well as intricacies of host-symbiont complementarity. Moving forward, the merging of classical twentieth-century approaches with evolving omic tools should provide even deeper insights into host-symbiont interplay.

Mild thermal stress affects Steinernema carpocapsae infective juvenile survival but not protein content.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles.

Fitness costs of symbiont switching using entomopathogenic nematodes as a model.

BACKGROUND: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. RESULTS: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. CONCLUSIONS: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

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.

Microbiome toxicology - bacterial activation and detoxification of insecticidal compounds.

Insect gut bacteria have been implicated in a myriad of physiological processes from nutrient supplementation to pathogen protection. In fact, symbiont-mediated insecticide degradation has helped explain sudden control failure in the field to a range of active ingredients. The mechanisms behind the loss of susceptibility are varied based on host, symbiont, and insecticide identity. However, while some symbionts directly break down pesticides, others modulate endogenous host detoxification pathways or involve reciprocal degradation of insecticidal and bactericidal compounds both inspiring new questions and requiring the reexamination of past conclusions. Good steward of the chemical pesticide arsenal requires consideration of these ecological interactions from development to deployment.

In vitro assays reveal inherently insecticide-tolerant termite symbionts.

Introduction: Termite symbionts are well known for conferring a myriad of benefits to their hosts. Bacterial symbionts are repeatedly associated with increased fitness, nutritional supplementation, pathogen protection, and proper development across insect taxa. In addition, several recent studies link bacterial symbionts to reduced insecticide efficacy. This has important implications both in pest control management and environmental bioremediation efforts. Insects' guts may be a valuable resource for microbes with broad application given their unique niches and metabolic diversity. Though insecticide resistance in termites is considered unlikely due to their life history, the close association of termites with a multitude of bacteria raises the question: is there potential for symbiont-mediated pesticide tolerance in termites? Methods and results: We identified a candidate that could grow in minimal medium containing formulated pesticide. This bacterial isolate was then subjected to continuous culture and subsequently demonstrated improved performance in the presence of pesticide. Isolates subjected to continuous culture were then grown at a range of concentrations from 1-10X the formulation rate. After constant exposure for several generations, isolates grew significantly better. Conclusion: Here we demonstrate that naïve insect hosts can harbor symbionts with inherent insecticide tolerance capable of rapid adaptation to increasing insecticide concentrations overtime. This has broad implications for both pest control and environmental cleanup of residual pesticides.

Symbiont-Mediated Insecticide Detoxification as an Emerging Problem in Insect Pests.

Pesticide use is prevalent with applications from the backyard gardener to large-scale agriculture and combatting pests in homes and industrial settings. Alongside the need to control unwanted pests comes the selective pressure generated by sustained pesticide use has become a concern leading to environmental contamination, pest resistance, and, thus, reduced pesticide efficacy. Despite efforts to improve the environmental impact and reduce off-target effects, chemical pesticides are relied on and control failures are costly. Though pesticide resistance mechanisms vary, one pattern that has recently emerged is symbiont-mediated detoxification within insect pests. The localization within the insect host, the identity of the symbiotic partner, and the stability of the associations across different systems vary. The diversity of insects and ecological settings linked to this phenomenon are broad. In this mini-review, we summarize the recent trend of insecticide detoxification modulated by symbiotic associations between bacteria and insects, as well as highlight the implications for pesticide development, pest management strategies, and pesticide bioremediation.

Ecological characterization of Heterorhabditis sonorensis (Caborca strain) (Nematoda: Heterorhabditidae), an entomopathogenic nematode from the Sonoran Desert.

Heterorhabditis nematodes are parasites of a wide range of soil-dwelling insect species. Although these nematodes have been exploited as biological control agents since the last half of the 20th century, much research remains to be done to understand how these organisms function in agricultural and other ecosystems. In this study, we present some ecological traits of Heterorhabditis sonorensis, a natural parasite of the cicada Diceroprocta ornea, from the Sonoran Desert. Specifically, we evaluated its infectivity across a diverse panel of insect groups and assessed its fitness (infectivity and reproduction) considering different temperatures, and soil moisture levels. Three other Heterorhabditis species served as points of comparison for temperature and soil moisture assays. Host range experiments indicate that H. sonorensis, although isolated from seasonal cicada nymphs, is more virulent and reproductively fit in the lepidopteran hosts tested. This nematode has an optimum temperature range at 25-30 °C but can also successfully reproduce at temperatures ranging from 15 to 35 °C. Additionally, this nematode is adapted to a variety of soil moisture conditions with successful infections across the tested moisture range (3%-20%). Finally, we demonstrate that H. sonorensis infective juveniles have a high survival rate (over 80%) at various storage temperatures (10-25 °C) after 24 weeks of storage and remain infective as revealed by the post-storage infection assays.

Metatranscriptomic Techniques for Identifying Cellulases in Termites and their Symbionts.

Characterizing symbiotic communities, like that of the termite hindgut, is essential for understanding their functionality and capabilities. However, the same complexity that allows termites to digest wood so efficiently also makes them difficult to study. With the expansion in technology and sequencing strategies the feasibility of sequencing entire consortiums or microecosystems is now possible. Here we present an adapted library preparation strategy which allows for the detection and measurement of expressed genes from all three domains of life in a single sample simultaneously. This technique effectively captures the transcriptome contributions by the various members of the consortium regardless of their taxonomic identity, which can then be annotated using custom-built databases and reciprocal BLASTing. Joining the universality of this library prep strategy with the power of bioinformatics allows for the identification of cellulases and other genes encoding carbohydrate active enzymes from complex communities using metatranscriptomics.

Screening of 57 Candidate Double-Stranded RNAs for Insecticidal Activity Against the Pest Termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

RNA interference insecticides have received increasing attention in recent years due to their classification as a reduced-risk biopesticide and their proposed faster path to registration compared with conventional synthetic insecticides. The goal of this study was to synthesize and compare efficacy of 62 double-stranded RNAs (dsRNAs) from 31 target genes against the pest termite species, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae). Fifty-seven dsRNAs of ~125 base pairs each were successfully synthesized. First-tier screens using a combination immersion/feeding assay revealed 10 top candidates and also that dsRNAs coming from synthesis reactions with 80-90× yields were the most effective. Follow-up studies using uptake enhancers in combination with top candidate dsRNAs were unsuccessful. Subsequent concentration range feeding assays on the top candidates revealed two lead termiticidal dsRNAs (3' Hexamerin-2 and 3' Glycosyl Hydrolase Family [GHF] 9-2 cellulase) and another that enhanced feeding (5' GHF9-2 cellulase). Testing a matrix of combinations of these three dsRNAs revealed ultimately that the most consistently effective dsRNA combination was the 3' Hexamerin-2 + 3' GHF9-2 cellulase dsRNA combination. These results provide new information on candidate termiticidal dsRNAs and some apparent factors that have a bearing on their efficacy. Despite these successes, further research and development will be necessary to move dsRNA termiticides from pest management theory to real-world application.

Lower Termite Associations with Microbes: Synergy, Protection, and Interplay.

Lower-termites are one of the best studied symbiotic systems in insects. Their ability to feed on a nitrogen-poor, wood-based diet with help from symbiotic microbes has been under investigation for almost a century. A unique microbial consortium living in the guts of lower termites is essential for wood-feeding. Host and symbiont cellulolytic enzymes synergize each other in the termite gut to increase digestive efficiency. Because of their critical role in digestion, gut microbiota are driving forces in all aspects of termite biology. Social living also comes with risks for termites. The combination of group living and a microbe-rich habitat makes termites potentially vulnerable to pathogenic infections. However, the use of entomopathogens for termite control has been largely unsuccessful. One mechanism for this failure may be symbiotic collaboration; i.e., one of the very reasons termites have thrived in the first place. Symbiont contributions are thought to neutralize fungal spores as they pass through the termite gut. Also, when the symbiont community is disrupted pathogen susceptibility increases. These recent discoveries have shed light on novel interactions for symbiotic microbes both within the termite host and with pathogenic invaders. Lower termite biology is therefore tightly linked to symbiotic associations and their resulting physiological collaborations.

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

Quantification of symbiotic contributions to lower termite lignocellulose digestion using antimicrobial treatments.

Animal-microbe co-evolution and symbiosis are broadly distributed across the animal kingdom. Insects form a myriad of associations with microbes ranging from vectoring of pathogens to intracellular, mutualistic relationships. Lower termites are key models for insect-microbe symbiosis because of the diversity, complexity and functionality of their unique tripartite symbiosis. This collaboration allows termites to live on a diet of nitrogen-poor lignocellulose. Recent functional investigations of lignocellulose digestion in lower termites have primarily focused on the contributions of the eukaryotic members of the termite holobiont (termite and protist). Here, using multiple antimicrobial treatments, we induced differing degrees of dysbiosis in the termite gut, leading to variably altered symbiont abundance and diversity, and lignocellulolytic capacity. Although protists are clearly affected by antimicrobial treatments, our findings provide novel evidence that the removal of distinct groups of bacteria partially reduces, but does not abolish, the saccharolytic potential of the termite gut holobiont. This is specifically manifested by reductions of 23-47% and 30-52% in glucose and xylose yields respectively from complex lignocellulose. Thus, all members of the lower termite holobiont (termite, protist and prokaryotes) are involved in the process of efficient, sustained lignocellulase activity. This unprecedented quantification of the relative importance of prokaryotes in this system emphasizes the collaborative nature of the termite holobiont, and the relevance of lower termites as models for inter-domain symbioses.

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.

A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.