Inbreeding alters context-dependent reproductive effort and immunity in male crickets.

Infection can cause hosts to drastically alter their investment in key life-history traits of reproduction and defence. Infected individuals are expected to increase investment in defence (e.g., by increasing immune function) and, due to trade-offs, investment in other traits (e.g., current reproduction) should decrease. However, the terminal investment hypothesis postulates that decreased lifespan due to infection and the associated reduction in the expectation for future offspring will favour increased investment towards current reproduction. Variation in intrinsic condition will likely influence shifts in reproductive investment post-infection, but this is often not considered in such assessments. For example, the extent of inbreeding can significantly impact an individual's lifetime fitness and may influence its reproductive behaviour following a threat of infection. Here, we investigated the effects of inbreeding status on an individual's reproductive investment upon infection, including the propensity to terminally invest. Male crickets (Gryllodes sigillatus) from four genetically distinct inbred lines and one outbred line were subjected to a treatment from an increasing spectrum of simulated infection cue intensities, using heat-killed bacteria. We then measured reproductive effort (calling effort), survival and immune function (antibacterial activity, circulating haemocytes and haemocyte microaggregations). Inbred and outbred males diverged in how they responded to a low-dose infection cue: relative to unmanipulated males, outbred males decreased calling effort, whereas inbred males increased calling effort. Moreover, we found that inbred males exhibited higher antibacterial activity and numbers of circulating haemocytes compared with outbred males. These results suggest that an individual's inbreeding status may have consequences for context-dependent shifts in reproductive strategies, such as those triggered by infection.

INFLUENCE OF MICROSPORIDIAN SPORES ON PHENOLOXIDASE ACTIVITY IN THE HAEMOCYTES OF GRYLLUS SPP. (INSECTA: ORTHOPTERA) IN VITRO.

After incubation with spores of microsporidia Paranosema spp. during 1­2 h, the quotes of Gryllus spp. haemolymph cells giving positive reaction in histochemical assay for phenoloxidase (PO) activity were significantly lower as compared to the control. Spores of microsporidia P. grylli caused 3- and 5.4-fold decrease of this index in haemocyte monolayers of Gryllus bimaculatus and G. argentinus, respectively. The ability of P. grylli spores to decrease the quote of PO-positive cells in G. bimaculatus haemocyte monolayers was stronger when monolayers and spores had been coincubated for a longer period (2 h) and when the spores used were more fresh (with higher level pf infectivity). Treatment of P. grylli spores with antibiotics resulted in diminishing their ability to decrease the quote of PO-positive cells. The highest level of decrease of this index, being 28.7-fold, was registered when G. bimaculatus haemocytes had been incubated with spores of P. locustae, the highly aggressive parasite which is able to infect orthopteran insect hosts belonging to more than 100 species and to preserve a high level of infectivity for years. Strong correlation has been revealed between infectivity of microsporidian spores and their ability to suppress PO system, which is a key factor in invertebrate immunity.

The effect of maternal and paternal immune challenge on offspring immunity and reproduction in a cricket.

Trans-generational immune priming is the transmission of enhanced immunity to offspring following a parental immune challenge. Although within-generation increased investment into immunity demonstrates clear costs on reproductive investment in a number of taxa, the potential for immune priming to impact on offspring reproductive investment has not been thoroughly investigated. We explored the reproductive costs of immune priming in a field cricket, Teleogryllus oceanicus. To assess the relative importance of maternal and paternal immune status, mothers and fathers were immune-challenged with live bacteria or a control solution and assigned to one of four treatments in which one parent, neither or both parents were immune-challenged. Families of offspring were reared to adulthood under a food-restricted diet, and approximately 10 offspring in each family were assayed for two measures of immunocompetence. We additionally quantified offspring reproductive investment using sperm viability for males and ovary mass for females. We demonstrate that parental immune challenge has significant consequences for the immunocompetence and, in turn, reproductive investment of their male offspring. A complex interaction between maternal and paternal immune status increased the antibacterial immune response of male offspring. This increased immune response was associated with a reduction in son's sperm viability, implicating a trans-generational resource trade-off between investment into immunocompetence and reproduction. Our data also show that these costs are sexually dimorphic, as daughters did not demonstrate a similar increase in immunity, despite showing a reduction in ovary mass.

Females suffer a reduction in the viability of stored sperm following an immune challenge.

Despite the ubiquitous nature of sperm storage in invertebrates, relatively little is known about its costs, or the impact that immune activation can have on a female's ability to maintain viable sperm stores. We explored the effects of an immune challenge on sperm storage under food-limited and ad libitum conditions in the field cricket, Teleogryllus oceanicus, by injecting mated adult females with either a LD5 dose of live bacteria or a nonpathogenic immune elicitor [lipopolysaccharide (LPS)] and then scoring the viability of their stored sperm. Females that were infected with bacteria showed a reduction in the viability of stored sperm 48 h after infection; interestingly, this pattern was not evident when females were injected with LPS. Reduction in sperm viability post-infection may reflect a reproductive trade-off between immune function and sperm store maintenance, as only females injected with bacteria showed an elevated antibacterial immune (lytic) response. Alternatively, bacteria may act directly on sperm quality. Dietary manipulations showed that lytic activity in females is condition dependent, irrespective of their immune challenge treatment. Diet affected the ability of females to maintain the viability of stored sperm, suggesting that sperm storage is condition dependent. That bacterial infection associated with a reduction in stored sperm quality has potentially important implications for the outcomes of sperm competition in T. oceanicus and in other species in which females store sperm between matings.

Sex-specific life history responses to nymphal diet quality and immune status in a field cricket.

Individual fitness is expected to benefit from earlier maturation at a larger body size and higher body condition. However, poor nutritional quality or high prevalence of disease make this difficult because individuals either cannot acquire sufficient resources or must divert resources to other fitness-related traits such as immunity. Under such conditions, individuals are expected to mature later at a smaller body size and in poorer body condition. Moreover, the juvenile environment can also produce longer-term effects on adult fitness by causing shifts in resource allocation strategies that could alter investment in immune function and affect adult lifespan. We manipulated diet quality and immune status of juvenile Texas field crickets, Gryllus texensis, to investigate how poor developmental conditions affect sex-specific investment in fitness-related traits. As predicted, a poor juvenile diet was related to smaller mass and body size at eclosion in both sexes. However, our results also reveal sexually dimorphic responses to different facets of the rearing environment: female life history decisions are affected more by diet quality, whereas males are affected more by immune status. We suggest that females respond to decreased nutritional income because this threatens their ability to achieve a large adult body size, whereas male fitness is more dependent on reaching adulthood and so they invest in immunity and survival to eclosion.

Granulocyte dynamics: a key player in the immune priming effects of crickets.

This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the killed Bt-primed group, suggesting a correlation between the heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the killed Bt-primed group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the PBS buffer-primed group exhibited extracellular DNA trap cell death (ETosis), while in the killed Bt-primed group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, the heat-killed Bt-primed group, the heat-killed E. coli-primed group, and the PBS-primed group were re-injected with live Bt 2 and 9 days post priming. Two days later, only the PBS-primed group displayed low survival rates. After injecting live Bt 9 days later, the heat-killed E. coli-primed group surprisingly showed a similarly low survival rate, while the heat-killed Bt-primed group exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.

The Allergen Profile of Two Edible Insect Species-Acheta domesticus and Hermetia illucens.

SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects.

To improve thermoregulation in colder environments, insects are expected to darken their cuticles with melanin via the phenoloxidase cascade, a phenomenon predicted by the thermal melanin hypothesis. However, the phenoloxidase cascade also plays a significant role in insect immunity, leading to the additional hypothesis that the thermal environment indirectly shapes immune function via direct selection on cuticle color. Support for the latter hypothesis comes from the cricket Allonemobius socius, where cuticle darkness and immune-related phenoloxidase activity increase with latitude. However, thermal environments vary seasonally as well as geographically, suggesting that seasonal plasticity in immunity may also exist. Although seasonal fluctuations in vertebrate immune function are common (because of flux in breeding or resource abundance), seasonality in invertebrate immunity has not been widely explored. We addressed this possibility by rearing crickets in simulated summer and fall environments and assayed their cuticle color and immune function. Prior to estimating immunity, crickets were placed in a common environment to minimize metabolic rate differences. Individuals reared under fall-like conditions exhibited darker cuticles, greater phenoloxidase activity and greater resistance to the bacteria Serratia marcescens. These data support the hypothesis that changes in the thermal environment modify cuticle color, which indirectly shapes immune investment through pleiotropy. This hypothesis may represent a widespread mechanism governing immunity in numerous systems, considering that most insects operate in seasonally and geographically variable thermal environments.

Dominance is not always an honest signal of male quality, but females may be able to detect the dishonesty.

Females prefer dominant males as mating partners in numerous species. Male dominance rank is considered as an honest signal of male quality, because only healthy males in good condition are thought to be able to win fights with other males. Here, we tested whether activation of the immune system influences the success of males in male-male competition and mating in the field cricket, Gryllus integer. We activated the immune system of males with a nylon monofilament (to mimic a parasitoid larva), and arranged fights between male pairs to assess male dominance and associated mating success. Activation of the immune system with nylon monofilament substantially enhanced the fighting success of males during male-male competition but had no effect on mating success. However, sham-manipulation (a wound only) did not have any effect on fighting success although females mated more often with dominant males. Our study suggests that when male crickets meet an apparent survival threat they may behave more dominantly, probably owing to terminal investment. Male success during male-male competition is not always an honest signal of males' quality, but females may be able to detect this dishonesty.

Context dependency and generality of fever in insects.

Fever can reduce mortality in infected animals. Yet, despite its fitness-enhancing qualities, fever often varies among animals. We used several approaches to examine this variation in insects. Texas field crickets (Gryllus texensis) exhibited a modest fever (1 °C increase in preferred body temperature, T pref) after injection of prostaglandin, which putatively mediates fever in both vertebrates and invertebrates, but they did not exhibit fever during chronic exposure to heat-killed bacteria. Further, chronic food limitation and mating status did not affect T pref or the expression of behavioural fever, suggesting limited context dependency of fever in G. texensis. Our meta-analysis of behavioural fever studies indicated that behavioural fever occurs in many insects, but it is not ubiquitous. Thus, both empirical and meta-analytical results suggest that the fever response in insects 'is widespread, although certainly not inevitable' (Moore 2002). We highlight the need for future work focusing on standardizing an experimental protocol to measure behavioural fever, understanding the specific mechanism(s) underlying fever in insects, and examining whether ecological or physiological costs often outweigh the benefits of fever and can explain the sporadic nature of fever in insects.

Quantitative genetics of immunity and life history under different photoperiods.

Insects with complex life-cycles should optimize age and size at maturity during larval development. When inhabiting seasonal environments, organisms have limited reproductive periods and face fundamental decisions: individuals that reach maturity late in season have to either reproduce at a small size or increase their growth rates. Increasing growth rates is costly in insects because of higher juvenile mortality, decreased adult survival or increased susceptibility to parasitism by bacteria and viruses via compromised immune function. Environmental changes such as seasonality can also alter the quantitative genetic architecture. Here, we explore the quantitative genetics of life history and immunity traits under two experimentally induced seasonal environments in the cricket Gryllus bimaculatus. Seasonality affected the life history but not the immune phenotypes. Individuals under decreasing day length developed slower and grew to a bigger size. We found ample additive genetic variance and heritability for components of immunity (haemocyte densities, proPhenoloxidase activity, resistance against Serratia marcescens), and for the life history traits, age and size at maturity. Despite genetic covariance among traits, the structure of G was inconsistent with genetically based trade-off between life history and immune traits (for example, a strong positive genetic correlation between growth rate and haemocyte density was estimated). However, conditional evolvabilities support the idea that genetic covariance structure limits the capacity of individual traits to evolve independently. We found no evidence for G × E interactions arising from the experimentally induced seasonality.

Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket Gryllus texensis.

In many parts of the world, climate change is increasing the frequency and severity of heat waves. How do heat waves impact short-lived poikilotherms such as insects? In the cricket, Gryllus texensis, 6 days of elevated temperatures (i.e. 7°C above the average field temperature and 5°C above their preferred temperature) resulted in increased egg laying, faster egg development and greater mass gain. The increased temperature also increased activity of phenoloxidase and lysozyme-like enzymes, two immune-related enzymes, and enhanced resistance to the Gram-negative bacterium Serratia marcescens. When given a sublethal S. marcescens infection, G. texensis maintained increased reproductive output at the elevated temperature (33°C). These data suggest that heat waves could result in more numerous, disease resistant, crickets. However, resistance to the Gram-positive bacterium, Bacillus cereus was lower at temperatures above or below the average field temperature (26°C). A sublethal infection with B. cereus reduced egg laying at all temperatures and suppressed the increase in egg laying induced by higher temperatures. These results suggest that for some species-pathogen interactions, increased temperatures can induce trade-offs between reproduction and disease resistance. This result may partly explain why G. texensis prefers temperatures lower than those that produce maximal reproductive output and enhanced immune function.

Exposure to sexual signals during rearing increases immune defence in adult field crickets.

Increased investment in immunity is expected to be beneficial under crowded conditions because of the greater risk of pathogen and parasite transmission, but the evolution of this facultative response relies on the ability to accurately assess social cues in the environment and adjust immune defences accordingly. Because of their highly conspicuous nature, long-range sexual signals are prime candidates to be used in evaluating the social conditions likely to be experienced upon adulthood in continuously breeding species; however, their role in mediating immune responses is unknown. We tested whether exposure to acoustic sexual signals in the field cricket Teleogryllus oceanicus affects immunity by manipulating male juvenile experience of acoustic signals, and measuring the effect on adult immunity. Adult males exposed to song during rearing showed stronger immune responses than males reared in silence: they were better able to encapsulate artificial nylon implants and showed higher levels of antimicrobial lysozyme-like activity in their haemolymph. Experience of sexual signals thus translates into increased immunity, which suggests that such signals may play a role in conveying information about population demography and shaping density-dependent responses in unintended receivers.

Cellular immunosenescence in adult male crickets, Gryllus assimilis.

Ecological immunity studies in invertebrates, particularly insects, have generated new insights into trade-offs between immune functions and other physiological parameters. These studies document physiologically directed reallocations of immune costs to other high-cost areas of physiology. Immunosenescence, recognized as the age-related deterioration of immune functions, is another mechanism of radically altering immune systems. We investigated the hypothesis that aging brings on immunosenescence in adult males of the cricket, Gryllus assimilis. Our data show that the intensity of melanotic nodule formation decreased with adult age from after 3-week post-adult emergence. Circulating hemocyte populations similarly decreased from about 5,000 hemocytes/µl hemolymph to about 1,000 hemocytes/µl hemolymph. The numbers of damaged hemocytes in circulation increased from less than 10% at 1-week post-adult emergence to approximately 60% by 3-week post-adult emergence. The composition of hemocyte types changed with age, with increasing proportions of granulocytes and decreasing proportions of plasmatocytes. The declines in nodule formation were not linked to the adult age of sexual behaviors, which begin shortly after entering adulthood in this species. We infer that age-related senescence, rather than cost reallocations, may account for observed declines in various parameters of immune functions in insects, as seen in other animals.

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.

Allergenicity assessment of the edible cricket Acheta domesticus in terms of thermal and gastrointestinal processing and IgE cross-reactivity with shrimp.

The allergenic potency of the cricket Acheta domesticus, a promising edible insect, has never been assessed. This work aims to study the immunoreactivity of Acheta domesticus, and its cross-reactivity with the shrimp Litopenaeus vannamei, assessing the effect of cooking and gastrointestinal digestion on their allergenic properties. Different cricket proteins were detected by immunoblotting with shrimp-allergic patients' sera. Tropomyosin was identified as the most relevant IgE-binding protein, and its cross-reactivity with shrimp tropomyosin was demonstrated by ELISA. While shrimp tropomyosin showed scarce stability to gastric digestion, cricket tropomyosin withstood the whole digestion process. The sarcoplasmic calcium-binding protein, specifically detected in shrimp, showed exceptional stability to gastrointestinal digestion. IgE-binding proteins in a model of enriched baked products were partially protected from proteolysis. In conclusion, the ingestion of A. domesticus proteins poses serious concerns to the Crustacean-allergic population. The high stability of tropomyosin may represent a risk of primary sensitization and clinical cross-reactivity.

Give 'til it hurts: trade-offs between immunity and male reproductive effort in the decorated cricket, Gryllodes sigillatus.

Trade-offs between life-history variables can be manifested at either the phenotypic or genetic level, with vastly different evolutionary consequences. Here, we examined whether male decorated crickets (Gryllodes sigillatus) from eight inbred lines and the outbred founder population from which they were derived, trade-off immune effort [lytic activity, phenoloxidase (PO) activity or encapsulation] to produce spermatophylaxes: costly nuptial food gifts essential for successful sperm transfer. Canonical correlation analysis of the outbred population revealed a trade-off between spermatophylax mass and lytic activity. Analysis of our inbred lines, however, revealed that although PO activity, encapsulation, body mass, spermatophylax mass and ampulla (sperm capsule) mass were all highly heritable, lytic activity was not, and there was, therefore, no negative genetic correlation between lytic activity and spermatophylax mass. Thus, males showed a phenotypic but not a genetic trade-off between spermatophylax mass and lytic activity, suggesting that this trade-off is mediated largely by environmental factors.

Why should an immune response activate the stress response? Insights from the insects (the cricket Gryllus texensis).

Mediators of the stress response (e.g. glucocorticoids and norepinephrine) can be immunosuppressive. Nevertheless, immune challenge leads to the release of these compounds in vertebrates. To resolve this paradox, it has been suggested that stress hormones help restore immune homeostasis, preventing self-damage. A comparative approach may provide additional hypotheses as to why an immune challenge induces the release of stress hormones/neurohormones. Octopamine, a neurohormonal mediator of the stress response in the cricket Gryllus texensis, increased in concentration in the hemolymph during an immune challenge. Therefore, the release of stress hormones during an immune response occurs in animals across phyla. Octopamine induced an increase in lipid concentration in the hemolymph. After an acute stress (flying or running) the total number of hemocytes in the hemolymph increased. Injections of octopamine had the same effect, suggesting that it may enhance hemocyte-dependent immune functions. On the other hand, octopamine decreased lysozyme-like activity in vitro, suggesting that it inhibits some immune functions. However, lysozyme-like activity was increased by the presence of heat-killed bacteria in vitro and this increase was significantly augmented by the presence of octopamine. Therefore, the effect of octopamine on immune function differed depending on the presence of pathogens. Stress hormones may help shift immune function into the most optimal configuration depending on the physiological context.

Inbred decorated crickets exhibit higher measures of macroparasitic immunity than outbred individuals.

Inbreeding is assumed to have negative effects on fitness, including the reduced ability to withstand immune challenges. We examined the immunological consequences of inbreeding in decorated crickets, Gryllodes sigillatus, by comparing lytic activity, phenoloxidase (PO) activity, and encapsulation ability of crickets from eight inbred lines with that of crickets from the outbred founder population. Surprisingly, crickets from inbred lines had a greater encapsulation ability compared with crickets from the outbred population. We suggest that because inbred crickets have reduced reproductive effort, they may, therefore, have the option of devoting more resources to this form of immunity than outbred individuals. We also found that both inbred and outbred females had higher immunity than males in PO activity and implant darkness. This result supports the hypothesis that females should devote more effort to somatic maintenance and immunity than males. PO activity and implant darkness were heritable in both males and females, but lytic activity was only heritable in females. Males and females differed in the heritability of, and genetic correlations among, immune traits, suggesting that differences in selective pressures on males and females may have resulted in a sexual conflict over optimal immune trait values.