Infection risk by oral contamination does not induce immune priming in the mealworm beetle (Tenebrio molitor) but triggers behavioral and physiological responses.

In invertebrates, immune priming is the ability of individuals to enhance their immune response based on prior immunological experiences. This adaptive-like immunity likely evolved due to the risk of repeated infections by parasites in the host's natural habitat. The expression of immune priming varies across host and pathogen species, as well as infection routes (oral or wounds), reflecting finely tuned evolutionary adjustments. Evidence from the mealworm beetle (Tenebrio molitor) suggests that Gram-positive bacterial pathogens play a significant role in immune priming after systemic infection. Despite the likelihood of oral infections by natural bacterial pathogens in T. molitor, it remains debated whether ingestion of contaminated food leads to systemic infection, and whether oral immune priming is possible is currently unknown. We first attempted to induce immune priming in both T. molitor larvae and adults by exposing them to food contaminated with living or dead Gram-positive and Gram-negative bacterial pathogens. We found that oral ingestion of living bacteria did not kill them, but septic wounds caused rapid mortality. Intriguingly, the consumption of either dead or living bacteria did not protect against reinfection, contrasting with injury-induced priming. We further examined the effects of infecting food with various living bacterial pathogens on variables such as food consumption, mass gain, and feces production in larvae. We found that larvae exposed to Gram-positive bacteria in their food ingested less food, gained less mass and/or produced more feces than larvae exposed to contaminated food with Gram-negative bacteria or control food. This suggests that oral contamination with Gram-positive bacteria induced both behavioral responses and peristalsis defense mechanisms, even though no immune priming was observed here. Considering that the oral route of infection neither caused the death of the insects nor induced priming, we propose that immune priming in T. molitor may have primarily evolved as a response to the infection risk associated with wounds rather than oral ingestion.

Growth and longevity modulation through larval environment mediate immunosenescence and immune strategy of Tenebrio molitor.

BACKGROUND: The Disposable Soma Theory of aging suggests a trade-off between energy allocation for growth, reproduction and somatic maintenance, including immunity. While trade-offs between reproduction and immunity are well documented, those involving growth remain under-explored. Rapid growth might deplete resources, reducing investment in maintenance, potentially leading to earlier or faster senescence and a shorter lifespan. However, rapid growth could limit exposure to parasitism before reaching adulthood, decreasing immunity needs. The insect immunity's components (cellular, enzymatic, and antibacterial) vary in cost, effectiveness, and duration. Despite overall immunity decline (immunosenescence), its components seem to age differently. We hypothesize that investment in these immune components is adjusted based on the resource cost of growth, longevity, and the associated risk of parasitism. RESULTS: We tested this hypothesis using the mealworm beetle, Tenebrio molitor as our experimental subject. By manipulating the larval environment, including three different temperatures and three relative humidity levels, we achieved a wide range of growth durations and longevities. Our main focus was on the relationship between growth duration, longevity, and specific immune components: hemocyte count, phenoloxidase activity, and antibacterial activity. We measured these immune parameters both before and after exposing the individuals to a standard bacterial immune challenge, enabling us to assess immune responses. These measurements were taken in both young and older adult beetles. Upon altering growth duration and longevity by modifying larval temperature, we observed a more pronounced investment in cellular and antibacterial defenses among individuals with slow growth and extended lifespans. Intriguingly, slower-growing and long-lived beetles exhibited reduced enzymatic activity. Similar results were found when manipulating larval growth duration and adult longevity through variations in relative humidity, with a particular focus on antibacterial activity. CONCLUSION: The impact of growth manipulation on immune senescence varies by the specific immune parameter under consideration. Yet, in slow-growing T. molitor, a clear decline in cellular and antibacterial immune responses with age was observed. This decline can be linked to their initially stronger immune response in early life. Furthermore, our study suggests an immune strategy favoring enhanced antibacterial activity among slow-growing and long-lived T. molitor individuals.

Senescence of the immune defences and reproductive trade-offs in females of the mealworm beetle, Tenebrio molitor.

In the theory of ageing, it has been assumed that ageing is associated with a decline in somatic defences, including the immune system, as a consequence of a trade-off with reproduction. While overall immunity suffers from age-related deterioration (immune senescence), the different components of the immune response appear to age differently. It is also likely that investment among the many arms of the immune system and reproduction with age is finely adjusted to the organisms' reproductive strategy. We investigated this possibility in females of Tenebrio molitor, a species of long-lived insect with reproductive strategies similar to those of long-lived mammals. We specifically tested the effects of immunological challenges imposed early or late in adult life on immune pathway activation as well as fertility early and late in life. We found complex patterns of changes in immune defences with age and age-specific immune challenges with contrasted relationships with female reproduction. While cellular and enzymatic defences showed signs of ageing, they did not trade-off with reproduction. By contrast, the induced antibacterial immune response was found to be unaffected by age and to be highly connected to female fecundity. These findings suggest that these immunological pathways have different functions with regard to female ageing in this insect species.

Age-specific fecundity under pathogenic threat in an insect: Terminal investment versus reproductive restraint.

The terminal investment hypothesis predicts that as an organism's prospects for survival decrease, through age or when exposed to a pathogenic infection, it will invest more in reproduction, which should trade-off against somatic maintenance (including immunity) and therefore future survival. Attempts to test this hypothesis have produced mixed results, which, in addition, mainly rely on the assessment of changes in reproductive effort and often overlooking its impact on somatic defences and survival. Alternatively, animals may restrain current reproduction to sustain somatic protection, increasing the chance of surviving for additional reproductive opportunities. We tested both of these hypotheses in females of the yellow mealworm beetle, Tenebrio molitor, an iteroparous insect with reproductive tactics similar to that of long-lived organisms. To achieve this, we mimicked pathogenic bacterial infections early or late in the life of breeding females by injecting them with a suspension of inactivated Bacillus cereus, a known natural pathogen of T. molitor, and measured female age-specific fecundity, survival, body mass and immunity. Inconsistent with a terminal investment, females given either an early or late-life immune challenge did not exhibit reduced survival or enhance their reproductive output. Female fecundity declined with age and was reduced by the early but not the late immune challenge. Both early and late-life fecundity correlated positively with life expectancy. Finally, young and old females exhibited similar antibacterial immune responses, suggesting that they both restrained reproduction to sustain immunity. Our results clearly demonstrate that age-specific reproduction of T. molitor females under pathogenic threat is inconsistent with a terminal investment. In contrast, our results instead suggest that females used a reproductive restraint strategy to sustain immunity and therefore subsequent reproductive opportunities. However, as infections were mimicked only, the fitness benefit of this reproductive restraint could not be shown.

Late-life reproduction in an insect: Terminal investment, reproductive restraint or senescence.

The terminal investment, reproductive restraint or senescence theories may explain individual late-life patterns of reproduction. The terminal investment hypothesis predicts that individuals increase reproductive allocation late in life as prospects for future survival decrease. The other two hypotheses predict reduced reproduction late in life, but for different reasons. Under the Reproductive Restraint hypothesis, individuals restrain their reproductive effort to sustain future survival and gain more time for reproducing, whereas under the Senescence process, reproduction is constrained because of somatic deterioration. While these hypotheses imply that reproduction is costly, they should have contrasted implications in terms of survival after late reproduction and somatic maintenance. Testing these hypotheses requires proper consideration of the effects of age-dependent reproductive effort on post-reproduction survival and age-related somatic functions. We experimentally tested these three hypotheses in females of the mealworm beetle, Tenebrio molitor, an iteroparous and income breeder insect. We manipulated their age-specific allocation into reproduction and observed the effects of this manipulation on their late-life fecundity, post-reproduction survival and immunocompetence as a measurement of somatic protection. We found that females exhibit age-related decline in fecundity and that this reproductive senescence is accelerated by a cost of early reproduction. The cost of reproduction had no significant effect on female longevity and their ability to survive a bacterial infection, despite that some immune cells were depleted by reproduction. We found that female post-infection survival deteriorated with age, which could be partly explained by a decline in some immune parameters. Importantly, females did not increase their reproductive effort late in life at the expense of their late-life post-reproduction survival. Late-life reproduction in T. molitor females is senescing and not consistent with a terminal investment strategy. Rather, our results suggest that females allocate resources according to a priority scheme favouring longevity at the expense of reproduction, which is in line with the reproductive restraint hypothesis. Such a priority scheme also shows that a relatively short-lived insect can evolve life-history strategies hitherto known only in long-lived animals. This puts in perspective the role of longevity in the evolution of life-history strategies.

Chromosome-scale assembly of the yellow mealworm genome.

Background: The yellow mealworm beetle, Tenebrio molitor, is a promising alternative protein source for animal and human nutrition and its farming involves relatively low environmental costs. For these reasons, its industrial scale production started this century. However, to optimize and breed sustainable new T. molitor lines, the access to its genome remains essential. Methods: By combining Oxford Nanopore and Illumina Hi-C data, we constructed a high-quality chromosome-scale assembly of T. molitor. Then, we combined RNA-seq data and available coleoptera proteomes for gene prediction with GMOVE. Results: We produced a high-quality genome with a N50 = 21.9Mb with a completeness of 99.5% and predicted 21,435 genes with a median size of 1,780 bp. Gene orthology between T. molitor and Tribolium castaneum showed a highly conserved synteny between the two coleoptera and paralogs search revealed an expansion of histones in the T. molitor genome. Conclusions: The present genome will greatly help fundamental and applied research such as genetic breeding and will contribute to the sustainable production of the yellow mealworm.

Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor.

In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a "signal" (such as fragments of infecting bacteria), mRNA and/or protein/peptide effectors to protect their eggs against two natural bacterial pathogens, namely the Gram-positive Bacillus thuringiensis and the Gram-negative Serratia entomophila. By taking the mealworm beetle Tenebrio molitor as a biological model, our results suggest that eggs are mainly protected by an active direct transfer of a restricted number of immune proteins and of antimicrobial peptides. In contrast, the present data do not support the involvement of mRNA transfer while the transmission of a "signal", if it happens, is marginal and only occurs within 24h after maternal exposure to bacteria. This work exemplifies how combining global approaches helps to disentangle the different scenarios of a complex trait, providing a comprehensive characterization of TGIP mechanisms in T. molitor. It also paves the way for future alike studies focusing on TGIP in a wide range of invertebrates and vertebrates to identify additional candidates that could be specific to TGIP and to investigate whether the TGIP mechanisms found herein are specific or common to all insect species.

Origin of the natural variation in the storage of dietary carotenoids in freshwater amphipod crustaceans.

Carotenoids are diverse lipophilic natural pigments which are stored in variable amounts by animals. Given the multiple biological functions of carotenoids, such variation may have strong implications in evolutionary biology. Crustaceans such as Gammarus amphipods store large amounts of these pigments and inter-population variation occurs. While differences in parasite selective pressure have been proposed to explain this variation, the contribution of other factors such as genetic differences in the gammarid ability to assimilate and/or store pigments, and the environmental availability of carotenoids cannot be dismissed. This study investigates the relative contributions of the gammarid genotype and of the environmental availability of carotenoids in the natural variability in carotenoid storage. It further explores the link of this natural variability in carotenoid storage with major crustacean immune parameters. We addressed these aspects using the cryptic diversity in the amphipod crustacean Gammarus fossarum and a diet supplementation protocol in the laboratory. Our results suggest that natural variation in G. fossarum storage of dietary carotenoids results from both the availability of the pigments in the environment and the genetically-based ability of the gammarids to assimilate and/or store them, which is associated to levels of stimulation of cellular immune defences. While our results may support the hypothesis that carotenoids storage in this crustacean may evolve in response to parasitic pressure, a better understanding of the specific roles of this large pigment storage in the crustacean physiology is needed.

Sex-specific patterns of senescence in artificial insect populations varying in sex-ratio to manipulate reproductive effort.

BACKGROUND: The disposable soma theory of ageing assumes that organisms optimally trade-off limited resources between reproduction and longevity to maximize fitness. Early reproduction should especially trade-off against late reproduction and longevity because of reduced investment into somatic protection, including immunity. Moreover, as optimal reproductive strategies of males and females differ, sexually dimorphic patterns of senescence may evolve. In particular, as males gain fitness through mating success, sexual competition should be a major factor accelerating male senescence. In a single experiment, we examined these possibilities by establishing artificial populations of the mealworm beetle, Tenebrio molitor, in which we manipulated the sex-ratio to generate variable levels of investment into reproductive effort and sexual competition in males and females. RESULTS: As predicted, variation in sex-ratio affected male and female reproductive efforts, with contrasted sex-specific trade-offs between lifetime reproduction, survival and immunity. High effort of reproduction accelerated mortality in females, without affecting immunity, but high early reproductive success was observed only in balanced sex-ratio condition. Male reproduction was costly on longevity and immunity, mainly because of their investment into copulations rather than in sexual competition. CONCLUSIONS: Our results suggest that T. molitor males, like females, maximize fitness through enhanced longevity, partly explaining their comparable longevity.

Storage of Carotenoids in Crustaceans as an Adaptation to Modulate Immunopathology and Optimize Immunological and Life-History Strategies.

Why do some invertebrates store so much carotenoids in their tissues? Storage of carotenoids may not simply be passive and dependent on their environmental availability, as storage variation exists at various taxonomic scales, including among individuals within species. While the strong antioxidant and sometimes immune-stimulating properties of carotenoids may be beneficial enough to cause the evolution of features improving their assimilation and storage, they may also have fitness downsides explaining why massive carotenoid storage is not universal. Here, the functional and ecological implications of carotenoid storage for the evolution of invertebrate innate immune defenses are examined, especially in crustaceans, which massively store carotenoids for unclear reasons. Three testable hypotheses about the role of carotenoid storage in immunological (resistance and tolerance) and life-history strategies (with a focus on aging) are proposed, which may ultimately explain the storage of large amounts of these pigments in a context of host-pathogen interactions.

Trans-generational Immune Priming in Invertebrates: Current Knowledge and Future Prospects.

Trans-generational immune priming (TGIP) refers to the transfer of the parental immunological experience to its progeny. This may result in offspring protection from repeated encounters with pathogens that persist across generations. Although extensively studied in vertebrates for over a century, this phenomenon has only been identified 20 years ago in invertebrates. Since then, invertebrate TGIP has been the focus of an increasing interest, with half of studies published during the last few years. TGIP has now been tested in several invertebrate systems using various experimental approaches and measures to study it at both functional and evolutionary levels. However, drawing an overall picture of TGIP from available studies still appears to be a difficult task. Here, we provide a comprehensive review of TGIP in invertebrates with the objective of confronting all the data generated to date to highlight the main features and mechanisms identified in the context of its ecology and evolution. To this purpose, we describe all the articles reporting experimental investigation of TGIP in invertebrates and propose a critical analysis of the experimental procedures performed to study this phenomenon. We then investigate the outcome of TGIP in the offspring and its ecological and evolutionary relevance before reviewing the potential molecular mechanisms identified to date. In the light of this review, we build hypothetical scenarios of the mechanisms through which TGIP might be achieved and propose guidelines for future investigations.

Immune Defenses of a Beneficial Pest: The Mealworm Beetle, Tenebrio molitor.

The mealworm beetle, Tenebrio molitor, is currently considered as a pest when infesting stored grains or grain products. However, mealworms are now being promoted as a beneficial insect because their high nutrient content makes them a viable food source and because they are capable of degrading polystyrene and plastic waste. These attributes make T. molitor attractive for mass rearing, which may promote disease transmission within the insect colonies. Disease resistance is of paramount importance for both the control and the culture of mealworms, and several biotic and abiotic environmental factors affect the success of their anti-parasitic defenses, both positively and negatively. After providing a detailed description of T. molitor's anti-parasitic defenses, we review the main biotic and abiotic environmental factors that alter their presentation, and we discuss their implications for the purpose of controlling the development and health of this insect.

Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect.

Many organisms can improve their immune response as a function of their immunological experience or that of their parents. This phenomenon, called immune priming, has likely evolved from repetitive challenges by the same pathogens during the host lifetime or across generation. All pathogens may not expose host to the same probability of re-infection, and immune priming is expected to evolve from pathogens exposing the host to the greatest probability of re-infection. Under this hypothesis, the priming response to these pathogens should be specifically more efficient and less costly than to others. We examined the specificity of immune priming within and across generations in the mealworm beetle, Tenebrio molitor, by comparing survival of individuals to infection with bacteria according to their own immunological experience or that of their mother with these bacteria. We found that insects primed with Gram-positive bacteria became highly protected against both Gram-positive and Gram-negative bacterial infections, mainly due to an induced persistent antibacterial response, which did not exist in insects primed with Gram-negative bacteria. Insects primed with Gram-positive bacteria also exhibited enhanced concentration of haemocytes, but their implication in acquired resistance was not conclusive because of the persistent antibacterial activity in the haemolymph. Offspring maternally primed with Gram-positive and Gram-negative bacteria exhibited similarly improved immunity, whatever the bacteria used for the infection. Such maternal protection was costly in the larval development of offspring, but this cost was lower for offspring maternally primed with Gram-positive bacteria. While T. molitor can develop some levels of primed response to Gram-negative bacteria, the priming response to Gram-positive bacteria was more efficient and less costly. We concluded that Gram-positive bacterial pathogens were of paramount importance in the evolution of immune priming in this insect species.

Trans-generational immune priming in the mealworm beetle protects eggs through pathogen-dependent mechanisms imposing no immediate fitness cost for the offspring.

Immune-challenged mothers can improve their offspring immunity through trans-generational immune priming (TGIP). In insects, TGIP endows the offspring with lifetime immunity, including the eggs, which are likely exposed soon after maternal infection. Egg protection may rely on the transfer of maternal immune effectors to the egg or/and the induction of egg immune genes. These respective mechanisms are assumed to have early-life fitness costs of different magnitude for the offspring. We provide evidence in the mealworm beetle Tenebrio molitor that enhanced egg immunity following a maternal immune challenge is achieved by both of these mechanisms but in a pathogen-dependent manner. While previously found having late-life fitness costs for the offspring, TGIP here improved egg hatching success and early larval survival, in addition of improving offspring immunity. These results suggest that early-life of primed offspring is critical in the optimization of life history trajectory of this insect under trans-generational pathogenic threats.

A dietary carotenoid reduces immunopathology and enhances longevity through an immune depressive effect in an insect model.

Immunopathology corresponds to self-damage of the inflammatory response, resulting from oxidizing molecules produced when the immune system is activated. Immunopathology often contributes to age-related diseases and is believed to accelerate ageing. Prevention of immunopathology relies on endogenous antioxidant enzymes and the consumption of dietary antioxidants, including carotenoids such as astaxanthin. Astaxanthin currently raises considerable interest as a powerful antioxidant and for its potential in alleviating age-related diseases. Current in vitro and short-term in vivo studies provide promising results about immune-stimulating and antioxidant properties of astaxanthin. However, to what extent dietary supplementation with astaxanthin can prevent long-term adverse effects of immunopathology on longevity is unknown so far. Here, using the mealworm beetle, Tenebrio molitor, as biological model we tested the effect of lifetime dietary supplementation with astaxanthin on longevity when exposed to early life inflammation. While supplementation with astaxanthin was found to lessen immunopathology cost on larval survival and insect longevity, it was also found to reduce immunity, growth rate and the survival of non immune-challenged larvae. This study therefore reveals that astaxanthin prevents immunopathology through an immune depressive effect and can have adverse consequences on growth.

No evidence of an immune adjustment in response to a parasitoid threat in Lobesia botrana larvae.

Immune function is a key determinant of an organism's fitness, and natural insect populations are highly variable for this trait, mainly due to environmental heterogeneity and pathogen diversity. We previously reported a positive correlation between infection prevalence by parasitoids and host immunity in natural populations of the vineyard pest Lobesia botrana. Here, we tested whether this correlation reflects a plastic adjustment of host immunity in response to the local presence of parasites. To this end, we measured immunity of non-parasitized L. botrana larvae exposed, respectively, to one of the two most common species of parasitoids in vineyards, over 6days. Larvae were able to sense the parasitoid through visual, chemical, or mechanical cues, but contact larvae-parasitoid were excluded. Contrary to our hypothesis, we found that L. botrana larvae did not increase their immune defenses in the presence of parasitoids, despite their ability to sense a potential threat. Our results therefore suggest that the positive correlation between infection prevalence by parasitoids and L. botrana immunity among natural populations may result from micro-evolutionary changes resulting from long-term local selection pressures imposed by parasitoids in wild populations rather than plastic adjustments of immunity.

Variation in the immune state of Gammarus pulex (Crustacea, Amphipoda) according to temperature: Are extreme temperatures a stress?

Temperature is known to impact host-parasite interactions in various ways. Such effects are often regarded as the consequence of the increased metabolism of parasites with increasing temperature. However, the effect of temperature on hosts' immune system could also be a determinant. Here we assessed the influence of temperature on the immunocompetence of the crustacean amphipod Gammarus pulex. Amphipods play a key ecological role in freshwater ecosystems that can be altered by several parasites. We investigated the consequences of three weeks of acclimatization at four temperatures (from 9 °C to 17 °C) on different immunological parameters. Temperature influenced both hemocyte concentration and active phenoloxidase enzymatic activity, with lower values at intermediate temperatures, while total phenoloxidase activity was not affected. In addition, the ability of gammarids to clear a bacterial infection was at the highest at intermediate temperatures. These results suggest a dysregulation of the immune system of gammarids in response to stress induced by extreme temperature.

Personality, immune response and reproductive success: an appraisal of the pace-of-life syndrome hypothesis.

The pace-of-life syndrome (POLS) hypothesis is an extended concept of the life-history theory that includes behavioural traits. The studies challenging the POLS hypothesis often focus on the relationships between a single personality trait and a physiological and/or life-history trait. While pathogens represent a major selective pressure, few studies have been interested in testing relationships between behavioural syndrome, and several fitness components including immunity. The aim of this study was to address this question in the mealworm beetle, Tenebrio molitor, a model species in immunity studies. The personality score was estimated from a multidimensional syndrome based of four repeatable behavioural traits. In a first experiment, we investigated its relationship with two measures of fitness (reproduction and survival) and three components of the innate immunity (haemocyte concentration, and levels of activity of the phenoloxidase including the total proenzyme and the naturally activated one) to challenge the POLS hypothesis in T. molitor. Overall, we found a relationship between behavioural syndrome and reproductive success in this species, thus supporting the POLS hypothesis. We also showed a sex-specific relationship between behavioural syndrome and basal immune parameters. In a second experiment, we tested whether this observed relationship with innate immunity could be confirmed in term of differential survival after challenging by entomopathogenic bacteria, Bacillus thuringiensis. In this case, no significant relationship was evidenced. We recommend that future researchers on the POLS should control for differences in evolutionary trajectory between sexes and to pay attention to the choice of the proxy used, especially when looking at immune traits.

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes.

A Novel Mechanism of Immune Memory Unveiled at the Invertebrate-Parasite Interface.

Pinaud et al. recently provided the first global investigation of the molecular processes underlying innate immune memory in an invertebrate species. They showed that the memory response of the snail Biomphalaria glabrata to Schistosoma mansoni infection is associated with a shift from cellular to humoral mechanisms.