Nutrition often shapes the outcome of host-parasite interactions, however understanding the mechanisms by which this occurs is often confounded by the intimate nature of the association and by the fact that the host and parasite may compete for the same limiting nutrients. One way of disentangling this interaction is to combine in vivo and in vitro approaches. Here, we explore the role of host nutrition in determining the outcome of infections using a model insect-bacterium system: the cotton leafworm Spodoptera littoralis and the blood-borne bacterium Xenorhabdus nematophila. Spodoptera littoralis larvae were reared on one of a series of 20 chemically-defined diets ranging in their protein: carbohydrate (P:C) ratio and caloric density. They were then challenged with either a fixed dose of X. nematophila cells (live or dead) or were sham-injected. Survivorship of larvae challenged with live bacterial cells was strongly dependent on the protein levels of the diet, with mortality being highest on low-protein diets. This trend was reflected in the bacterial growth rate in vivo, which peaked in larvae fed low-protein diets. To determine whether in vivo bacterial growth rates were driven by the direct effects of blood nutrients or by the indirect effects of the host immune response, we used 20 synthetic haemolymphs ('nutribloods') that mimicked the nutritional content of host blood. In vitro bacterial growth rate was negatively impacted by the protein content of the nutribloods, replicating the patterns seen in vivo and suggesting that nutrient availability and not host immunity was driving the interaction. By comparing standardized bacterial growth rates in vivo and in vitro, we conclude that the outcome of this host-parasite interaction is largely driven by the 'bottom-up' effects of nutrients on bacterial growth, rather than by the 'top-down' effects of nutrients on host-mediated immune responses. The outcome of host-parasite interactions is typically assumed to be strongly determined by the host immune response. The direct effects of nutrition have been underexplored and may have broad consequences for host-parasite interactions across taxa.

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.

Nutrition is vital to health, but while the link between diet and body nutritional composition is well explored in humans and other vertebrates, this information is not well understood in insects, despite the vital roles they play in ecosystems, and their increasing use as experimental models. Here we used Nutritional Geometry to explore the rapid physiological response to ingested nutrients in the haemolymph nutritional profile of Spodoptera littoralis caterpillars. We ask whether blood nutrients are maintained homeostatically in the face of variable nutritional intake, or if regulation is more flexible for some nutrients than others (allostasis), which allows animals to adapt to stress by responding in a way that prioritises efficiency of responses in the face of trade-offs. Caterpillars were placed on 1 of 20 diets, systematically varying in their nutrient ratios (protein: carbohydrate) and density (calorie content), and their consumption was measured. After 48 h, caterpillars were bled, and the macronutrient (protein, carbohydrates and lipids) and nutrient metabolite (amino acids and simple sugars) content of the haemolymph was measured. Proteins comprised 93% of the haemolymph macronutrient pool on average and their concentration increased with protein eaten. The amino acid (AA) pool was dominated by five AAs, and the total pool increased with total nutrient intake. However, the ratio of essential to non-essential AAs increased as the proportion of protein consumed increased. Carbohydrates were tightly controlled, increasing only on the most extreme carbohydrate intakes. Simple sugars were dominated by glucose and trehalose, and overall, the simple sugar pool showed high levels of homeostasis. Rather than strict homeostasis of blood nutritional properties, an allostatic model seemed to be a better fit for blood nutrient regulation in this generalist herbivore. This flexibility in response to the nutritional composition of the diet may, in part, explain how this species has evolved to extreme dietary generalism and may play a role in its worldwide pest status. Given the range of fitness-related processes affected by the haemolymph, future studies should examine the physiological impacts of blood nutrient variation on reproduction, growth and response to infection and the trade-offs between them.

ABSTRACTNutrition often shapes the outcome of host-parasite interactions, however understanding the mechanisms by which this occurs is confounded by the intimate nature of the association and by the fact that the host and parasite may compete for the same limiting nutrients. One way of disentangling this interaction is to combinein vivoandin vitroapproaches. Here, we explore the role of host nutrition in determining the outcome of infections using a model insect-bacterium system: the cotton leafwormSpodoptera littoralisand the blood-borne bacteriumXenorhabdus nematophila.Spodoptera littoralislarvae were reared on one of a series of 20 chemically-defined diets ranging in their protein: carbohydrate (P:C) ratio and caloric density. They were then challenged with either a fixed dose ofX. nematophilacells (live or dead) or were sham-injected. Survivorship of larvae challenged with live bacterial cells was strongly dependent on the protein levels of the diet, with mortality being highest on low-protein diets. This trend was reflected in the bacterial growth ratein vivo, which peaked in larvae fed low-protein diets.To determine whetherin vivobacterial growth rates were driven by blood nutrients, rather than an enhanced host immune response, we generated 20 synthetic haemolymphs (‘nutribloods’) that mimicked the nutritional content of host blood. Bacterial growth rate in the nutribloods was also negatively impacted by their protein content suggesting that nutrient availability and not host immunity was driving the interaction. By comparing standardized bacterial growth ratesin vivoandin vitro, we conclude that the outcome of this host-parasite interaction is largely driven by ‘bottom-up’ effects of nutrients on bacterial growth, rather than by ‘top-down’ effects of nutrients on host-mediated immune responses. The outcome of host-parasite interactions is typically assumed to be strongly determined by the host immune response. The direct effects of nutrition have been underexplored and may have broad consequences for host-parasite interactions across taxa.

Background Color vision and phototactic behavior based on opsins are important for the fitness of insects because of their roles in foraging and mate choice. Related topics, including the duplication and loss of opsin genes, have been well investigated in insect orders such as Coleoptera, Lepidoptera, Hymenoptera, Odonata and Orthoptera, and the findings have been used to develop pest management strategies involving light trapping. Mirid bugs of Hemiptera, which are pests that cause heavy economic losses, show capacity for color discrimination and phototaxis. However, the opsins in mirid bugs remain uncharacterized. Herein, we examined five species to investigate the evolution of opsins in the family Miridae. Results Using RNA-seq, we identified several contigs showing high identity with opsins, including four contigs in Apolygus lucorum and three contigs each in Adelphocoris suturalis, Adelphocoris fasciaticollis, Adelphocoris lineolatus and Nesidiocoris tenuis. Phylogenetic analyses indicated that one of these genes clustered with ultraviolet-sensitive (UV) opsins and that the others clustered with long-wavelength (LW) opsins, suggesting that duplication of LW opsins and loss of blue light-sensitive (B) opsins occurred in mirid bugs. The existence of introns in the LW opsins of mirid bugs suggested that the duplication events were DNA based. Both LW1 and LW2 opsins of mirid bugs were found to be under strong purifying selection. The LW1 opsins were significantly more highly expressed than the LW2 and UV opsins. Conclusions We identified the opsins of mirid bugs using five selected mirid species as a representative sample. Phylogenetic analyses clustered one of the genes with UV opsins and the others with LW opsins, suggesting the occurrence of LW opsin duplication and B opsin loss during the evolution of mirid bugs. Intron detection suggested that the identified duplication event was DNA based. The evidence of strong purifying selection and the relatively high expression levels suggested that these opsins exhibit fundamental functions in mirid bugs.

Recent research has suggested that the outcome of host-parasite interactions is dependent on the diet of the host, but most previous studies have focused on "top-down'' mechanisms, i.e., how the host's diet improves the host immune response to drive down the parasite population and improve host fitness. In contrast, the direct impacts of host nutrition on parasite fitness and the mechanisms underpinning these effects are relatively unexplored. Here, using a model host-pathogen system ( Spodoptera littoralis caterpillars and Xenorhabdus nematophila, an extracellular bacterial blood parasite), we explore the effects of host dietary macronutrient balance on pathogen growth rates both in vivo and in vitro, allowing us to compare pathogen growth rates both in the presence and absence of the host immune response. In vivo, high dietary protein resulted in lower rates of bacterial establishment, slower bacterial growth, higher host survival, and slower speed of host death; in contrast, the energy content and amount of carbohydrate in the diet explained little variation in any measure of pathogen or host fitness. In vitro, we show that these effects are largely driven by the impact of host dietary protein on host hemolymph (blood) osmolality (i.e., its concentration of solutes), with bacterial growth being slower in protein-rich, high-osmolality hemolymphs, highlighting a novel "bottom-up'' mechanism by which host diet can impact both pathogen and host fitness.

Dung beetles (Coleoptera: Scarabaeidae) perform essential ecological functions in pastures, such as dung removal, nutrient recycling and parasite control. However, the patterns of alimentary use by dung beetles in introduced Brazilian pastures are poorly known. Here, we compared dung beetle species richness, abundance and species composition in cattle and sheep dung, and identified the dung beetle species preference by each dung type. In January 2019, dung beetles were sampled with pitfall traps baited with cattle and sheep dung in 12 introduced pastures (Urochloa spp.), in Aquidauana, Mato Grosso do Sul, Brazil. A total of 592 individuals belonging to 14 species of dung beetles were collected. Of the 14 species sampled, nine were recorded in both dung types, five were found exclusively in sheep dung and no species was exclusive to cattle dung. Species richness and abundance were higher in sheep dung. Species composition was different between the dung types. Dichotomius bos (Blanchard), Genieridium bidens (Balthasar), Onthophagus aeneus Blanchard and Trichillum externepunctatum Preudhomme de Borre were associated with sheep dung. Our results provide evidence that sheep dung is more attractive to dung beetles with a distinct community species between the two dung types, although the studied pastures have never been used before for sheep breeding. Thus, our data shows that the introduction of a new alimentary resource (e.g. sheep dung) can be an important strategy to help to obtain a more diverse dung beetle assemblage in introduced Brazilian pastures.

Grazing by large herbivorous mammals is still a structuring force in tropical grassy ecosystems, and cattle grazing is one of the main economic activities carried out in these ecosystems in modern times. Therefore, understanding the impacts of cattle grazing removal on biodiversity may be a key step for conservation of this ecosystem. Here, we studied the successional trajectory of dung beetle communities in a tropical grassy ecosystem after cattle removal. For this, we assessed the patterns of dung beetle taxonomic and functional diversity of 14 natural grasslands with distinct cattle grazing removal ages (from 3 months to 22 years) along a chronosequence, applying the space-for-time substitution method. Our results show a strong decrease in dung beetle abundance (93 times) and species richness (6 times) in the first ten years of cattle removal. However, after ten years there is an increase in dung beetle abundance (73 times) and species richness (5 times). Taxonomic composition was also influenced by cattle removal time demonstrating the importance of cattle in the structuring of dung beetle communities in natural grasslands. In contrast, functional composition and diversity were not affected by cattle grazing removal, indicating these metrics are less sensitive to cattle absence than taxonomic diversity and composition. Our results provide evidence that cattle grazing removal, at least in the short term (10 years), may be an inefficient management tool for restoration and conservation of tropical grassy ecosystems. However, we highlight the need to investigate the reintroduction of cattle grazing after different removal times to provide complimentary information to livestock management able to integrate human use and conservation of tropical grassy ecosystems.

BackgroudHorizontal gene transfer and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms. Previously, polygalacturonase genes (PGs) were independently horizontally transferred and underwent multiple duplications in insects (e.g., mirid bugs and beetles). Here, we chose three phytozoophagous mirid bugs (Adelphocoris suturalis, A. fasciaticollis, A. lineolatus) and one zoophytophagous mirid bug (Nesidiocoris tenuis) to detect whether the duplication, molecular evolution, and expression levels of PGs were related to host range expansion in mirid bugs.ResultsBy RNA-seq, we reported 30, 20, 19 and 8 PGs in A. suturalis, A. fasciaticollis, A. lineolatus and N. tenuis, respectively. Interestingly, the number of PGs was significantly positive correlation to the number of host plants (P=0.0339) in mirid bugs. Most PGs (>17) were highly expressed in the three phytozoophagous mirid bugs, while only one PG was relatively highly expressed in the zoophytophagous mirid bug. Natural selection analysis clearly showed that a significant relaxation of selection pressure acted on the PGs in zoophytophagous mirid bugs (K=0.546, P=0.0158) rather than in phytozoophagous mirid bugs (K=1, P=0.92), suggesting a function constraint of PGs in phytozoophagous mirid bugs.ConclusionTaken together with gene duplication, molecular evolution, and expression levels, our results suggest that PGs are more strictly required by phytozoophagous than by zoophytophagous mirid bugs and that the duplication of PGs is associated with the expansion of host plant ranges in mirid bugs.

Nutrition is vital to health and the availability of resources has long been acknowledged as a key factor in the ability to fight off parasites, as investing in the immune system is costly. Resources have typically been considered as something of a “black box”, with the quantity of available food being used as a proxy for resource limitation. However, food is a complex mixture of macro- and micronutrients, the precise balance of which determines an animal's fitness. Here we use a state-space modelling approach, the Geometric Framework for Nutrition (GFN), to assess for the first time, how the balance and amount of nutrients affects an animal's ability to mount an immune response to a pathogenic infection. Spodoptera littoralis caterpillars were assigned to one of 20 diets that varied in the ratio of macronutrients (protein and carbohydrate) and their calorie content to cover a large region of nutrient space. Caterpillars were then handled or injected with either live or dead Xenorhabdus nematophila bacterial cells. The expression of nine genes (5 immune, 4 non-immune) was measured 20 h post immune challenge. For two of the immune genes (PPO and Lysozyme) we also measured the relevant functional immune response in the hemolymph. Gene expression and functional immune responses were then mapped against nutritional intake. The expression of all immune genes was up-regulated by injection with dead bacteria, but only those in the IMD pathway (Moricin and Relish) were substantially up-regulated by both dead and live bacterial challenge. Functional immune responses increased with the protein content of the diet but the expression of immune genes was much less predictable. Our results indicate that diet does play an important role in the ability of an animal to mount an adequate immune response, with the availability of protein being the most important predictor of the functional (physiological) immune response. Importantly, however, immune gene expression responds quite differently to functional immunity and we would caution against using gene expression as a proxy for immune investment, as it is unlikely to be reliable indicator of the immune response, except under specific dietary conditions.

The complete genome of a novel virus from Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10633 nucleotides (nt) in length, not including the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a polypeptide of 1320 amino acids (aa) with a predicted molecular mass of 147.92 kDa and theoretical isoelectric point (pI) of 6.96. ORF2 encodes a polypeptide of 1728 aa with a predicted molecular mass of 197.09 kDa and pI of 6.73. Phylogenetic analysis with the deduced aa sequences of the conserved RNA dependent RNA polymerase domain as well as whole genome nt sequences indicated that the virus clusters with viruses classified within the genus Iflavirus, with a high bootstrap value in the maximum-likelihood and neighbor-joining trees. However, this virus has a distinct genome structure with two ORFs, iflaviruses normally having one, suggesting the virus might be a prototype of a new genus. We named the virus isolate Nesidiocoris tenuis virus 1 (NtV-1). The prevalence of NtV-1 infection in wild samples of N. tenuis was at a low level (7.32%, 6 positive in 82 samples), suggesting a possible harmful effect to its host.

Psychoneuroimmunology studies the increasing number of connections between neurobiology, immunology and behaviour. We establish Drosophila melanogaster as a tractable model in this field by demonstrating the effects of the immune response on two fundamental behaviours: sleep and memory ability. We used the Geneswitch system to upregulate peptidoglycan receptor protein (PGRP) expression, thereby stimulating the immune system in the absence of infection. Geneswitch was activated by feeding the steroid RU486, to the flies. We used an aversive classical conditioning paradigm to quantify memory and measures of activity to infer sleep. Immune stimulated flies exhibited reduced levels of sleep, which could not be explained by a generalised increase in waking activity. The effects on sleep were more pronounced for day compared to night sleep. Immune stimulated flies also showed a reduction in memory abilities. These results establish Drosophila as a model for immune-neural interactions and suggest a possible role for sleep in the interplay between the immune response and memory.