Xylophagous insects often thrive on nutritionally suboptimal diets through symbiotic associations with microbes that supplement their nutritional requirements, particularly nitrogen. The wood-feeding cerambycid Anoplophora glabripennis (Motschulsky) feeds on living, healthy host trees and harbors a diverse gut microbial community. We investigated gut microbial contributions to larval nitrogen requirements through nitrogen fixing and recycling (urea hydrolysis) processes, using a combination of molecular, biochemical, and stable isotope approaches. Genes and transcripts of conserved regions of the urease operon (ureC) and nitrogen fixing (nif) regulon (nifH) were detected in A. glabripennis eggs and larvae from naturally infested logs and from larvae reared on artificial diet. Significant nitrogen fixation and recycling were documented in larvae using (15)N2 gas and (15)N-urea, respectively. Subsequent (15)N-routing of incorporated recycled nitrogen into larval essential and nonessential amino acids was shown for (15)N-urea diet-fed larvae. Results from this study show significant gut microbial contributions to this insect's metabolic nitrogen utilization through nitrogenous waste product recycling and nitrogen fixation.

Insects are unable to synthesize essential amino acids (EAAs) de novo, thus rely on dietary or symbiotic sources for them. Wood is a poor resource of nitrogen in general, and EAAs in particular. In this study, we investigated whether gut microbiota of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), a cerambycid that feeds in the heartwood of healthy host trees, serve as sources of EAAs to their host under different dietary conditions. δ(13)C-stable isotope analyses revealed significant δ(13)C-enrichment (3.4 ± 0.1‰; mean ± SEM) across five EAAs in wood-fed larvae relative to their woody diet. δ(13)C values for the consumers greater than 1‰ indicate significant contributions from non-dietary EAA sources (symbionts in this case). In contrast, δ(13)C-enrichment of artificial diet-fed larvae (controls) relative to their food source was markedly less (1.7 ± 0.1‰) than was observed in wood-fed larvae, yet still exceeded the threshold of 1‰. A predictive model based on δ(13)CEAA signatures of five EAAs from representative bacterial, fungal, and plant samples identified symbiotic bacteria and fungi as the likely supplementary sources of EAA in wood-fed larvae. Using the same model, but with an artificial diet as the dietary source, we identified minor supplementary bacterial sources of EAA in artificial diet-fed larvae. This study highlights how microbes associated with A. glabripennis can serve as a source of EAAs when fed on nutrient-limited diets, potentially circumventing the dietary limitations of feeding on woody substrates.

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Insect gut microbiota contribute significantly to host nutritional ecology. Disrupting insect gut microbial assemblages impacts nutrient provisioning functions, and can potentially affect host standard metabolic rate (SMR), a measure of host energy balance. In this study, we evaluated the effect of disrupting gut microbial assemblages on the SMR of female Periplaneta americana cockroaches fed dog food (DF, high protein/carbohydrate (p/c) ratio), and cellulose-amended dog food (CADF, 30% dog food, 70% cellulose, low p/c ratio) diets, supplemented with none, low, or high antibiotic doses. Bacterial loads decreased significantly between diet types (P = 0.04) and across antibiotic doses (P = 0.04). There was a significant diet type x antibiotic dose interaction on SMR of females on both diets (P = 0.05) by the end of the seven-day experimental period. In CADF-fed females, SMR decreased linearly with decreasing bacterial load. However, SMR of DF-fed females on the low dose was significantly higher than those in the control and high dose groups. This is interpreted as a diet-dependent response by low dose DF-fed females to the loss of nutritional services provided by gut bacteria. Severe reductions in bacterial load at high doses reduced SMR of females on both diet types. This study provides insights into the potential role of gut bacteria as modulators of host energy expenditure under varying dietary conditions.

Insect gut microbes have been shown to provide nutrients such as essential amino acids (EAAs) to their hosts. How this symbiotic nutrient provisioning tracks with the host’s demand is not well understood. In this study, we investigated microbial essential amino acid (EAA) provisioning in omnivorous American cockroaches (Periplaneta americana), fed low-quality (LQD) and comparatively higher-quality dog food (DF) diets using carbon stable isotope ratios of EAAs (δ13CEAA). We assessed non-dietary EAA input, quantified as isotopic offsets (Δ13C) between cockroach (δ13CCockroach EAA) and dietary (δ13CDietary EAA) EAAs, and subsequently determined biosynthetic origins of non-dietary EAAs in cockroaches using 13C-fingerprinting with dietary and representative bacterial and fungal δ13CEAA. Investigation of biosynthetic origins of de novo non-dietary EAAs indicated bacterial origins of EAA in cockroach appendage samples, and a mixture of fungal and bacterial EAA origins in gut filtrate samples for both LQD and DF-fed groups. We attribute the bacteria-derived EAAs in cockroach appendages to provisioning by the fat body residing obligate endosymbiont, Blattabacterium and gut-residing bacteria. The mixed signatures of gut filtrate samples are attributed to the presence of unassimilated dietary, as well as gut microbial (bacterial and fungal) EAAs. This study highlights the potential impacts of dietary quality on symbiotic EAA provisioning and the need for further studies investigating the interplay between host EAA demands, host dietary quality and symbiotic EAA provisioning in response to dietary sufficiency or deficiency.

Diet may be a significant determinant of insect gut microbiome composition. However, the extent to which dietary shifts shape both the composition and relevant functions of insect gut microbiomes, and ultimately impact host energy balance (i.e. metabolic phenotype), is not well understood. We investigated the impacts of diet switching on Diploptera punctata females maintained on a dog food (DF) diet relative to those fed a comparatively sub-optimal cellulose-amended dog food (CADF) diet for 4 weeks. After this period, dietary shift resulted in a significantly higher average mass-specific standard metabolic rate (SMR) in CADF-fed females compared with DF-fed females. We also uncovered significant 13 C-enrichment in DF-fed insect samples relative to CADF-fed insect samples and lowered bacterial essential amino acid (EAA) provisioning in CADF-fed samples. Differences in SMR and EAA provisioning were not accompanied by significant differences in overall microbiome composition between the two groups. However, cellulolytic and nitrogen-fixing bacterial families dominant in wild omnivorous cockroaches and wood-feeding termites were significantly enriched in CADF-fed females than in DF-fed females, at the end of the study. We propose that these changes in microbiome composition after dietary shifts are associated with changes in EAA provisioning and possibly SMR. Further studies are needed to comprehensively understand the relative importance of gut microbial functions among the complexity of factors known to underscore SMR responses in insects under varying dietary conditions.

Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ13CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following 13C stable isotope analysis. Termite carcass samples (−27.0 ± 0.4‰, mean ± s.e.) were significantly different from termite gut filtrate samples (−27.53 ± 0.5‰), but not the wood diet (−26.0 ± 0.5‰) (F(2,64) = 6, P < 0.0052). δ13CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ13CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function.

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