In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well‐formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double‐strand RNA (dsRNA)‐expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA‐expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting “naked” dsRNA or dsRNA‐expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding “naked” dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont‐mediated dsRNA delivery, which we believe is the most convenient approach for large‐scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.

To examine how nutritional quality and resident gut bacteria interplay in improving the fitness of an oligophagous fruit fly, Bactrocera minax , artificial sucrose diets and full diets (sucrose, tryptone and yeast extract) were fed to flies with and without antibiotic supplementation. Furthermore, Klebsiella oxytoca and Citrobacter freundii were supplemented to sucrose-only diets. Flies were maintained in the laboratory and the fitness parameters, male and female longevity, number of copulations and female fecundity, were recorded. Full diet without bacterial depletion significantly increased fecundity and copulation. In the absence of gut bacteria, flies fed with full diets had significantly decreased mean fecundity and copulation rate. Flies that were fed with sucrose diet had a very low copulation rate and produced no eggs. Diet type and the presence of bacteria did not have any effect on the average longevity of male and female flies. Bacterial supplementation in sucrose diets did not improve any of the measured parameters. The results demonstrate that gut bacteria interact with diet to influence mating and reproduction in B. minax . Symbiotic bacteria significantly and positively impact reproduction in B. minax ; however, their impact can only be fully realized when the flies are fed with a nutritionally complete diet.

The gut pH plays crucial roles in diet preference, habitat choice, insect fitness, and insect-microbial relationships. It significantly impacts enzyme activity efficiency, as well as the internalisation and efficacy of pesticides. Without a comprehensive understanding of the gut environment, potential pest management strategies cannot be fully optimised. This study investigates the gut pH of the globally invasive pest insect Western flower thrips Frankliniella occidentalis , and the effect its Gram-negative symbiotic gut bacterium BFo2 has on pH modulation. Indicator dyes were fed to F. occidentalis and the gut pH was found to vary between 6 and 7. In general, the larval and adult guts appear to have a pH of between 6 and 6.5; however, the posterior gut of some adults appears to be closer to 7. This almost neutral pH offers a favourable environment for the neutrophilic symbiotic BFo2. The ability of BFo2 isolates to buffer pH towards neutral was also observed during in vitro culture using broths at different pH values. This paper also discusses the implications of this gut environment on dsRNAi delivery. By laying the foundation for understanding how gut pH can be leveraged to enhance current pest management strategies, this study particularly benefits research aimed at optimising the delivery of lethal dsRNA through symbiont-mediated RNAi to Western flower thrips in pest management programs.

Understanding the role of nutrients in microbial population dynamics relies on a sound appreciation of their nutritional environment and how this may vary in different habitats. For microbial pathogens and commensals, this can be especially challenging because the microbe may share nutritional resources with its host. Here we design a series of 20 synthetic cell‐free haemolymphs ( nutribloods ) that mimic haemolymph nutrient profiles of caterpillars fed on one of 20 chemically defined diets that vary in their protein: carbohydrate (P:C) ratio and caloric density. Using these, we are able to simulate the range of nutritional conditions that insect blood pathogens might face, providing a model system for understanding the role of nutrition in microbial growth. We tested this using the entomopathogen, Xenorhabdus nematophila , a Gram‐negative extracellular bacterium of insect hosts. This revealed that whilst bacterial fitness peaked in nutriblood nutrient space that was high in carbohydrates and low in proteins, levels of amino acids in the nutribloods also appear to be an important driving force for bacterial growth. Using synthetic haemolymphs that had average levels of all nutrients other than carbohydrate, protein or amino acids, we also established that bacterial growth is generally enhanced by carbohydrates and amino acids but reduced by proteins. Here, we have established a tractable model system for examining the role that nutrition plays in the growth of an entomopathogenic bacterium. In future work, this model host–pathogen system can be used to test a range of nutritionally driven processes, including competition during co‐infection and interactions with the host microbiome, as well as comparative studies of other entomopathogens.