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.

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.

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.

Innate immune system function following systemic RNA-interference of the Fragile X Mental Retardation 1 gene in the cricket Acheta domesticus.

Fragile X syndrome (FXS), caused by a mutation in the Fragile X Mental Retardation 1 (FMR1) gene, is a common form of inherited mental retardation. Mutation of the gene leads to a loss of the gene product Fragile X Mental Retardation Protein (FMRP). While a loss of FMRP has been primarily associated with neural and cognitive deficits, it has also been reported to lead to immune system dysfunction in both humans and flies. We used the Acheta domesticus transcriptome to identify a highly conserved cricket ortholog of FMR1 (adfmr1). We cloned a partial cDNA of adfmr1, used systemic RNA interference (RNAi) to knockdown adfmr1 expression, and examined the impact of this knockdown (KD) on the cellular and humoral responses of the insect innate immune system. Following RNAi, both male and female crickets exhibited an increase in the number of circulating hemocytes, a decrease in total hemolymph phenoloxidase (PO) activity, and an increase in fat body lysozyme expression. Despite similar changes in these immune parameters in both sexes, male and female crickets responded differently to an immune challenge. Most KD males failed to survive an intra-abdominal injection of bacterial lipopolysaccharide, while KD females were just as likely as control females to survive this challenge. Our results support that decreased fmr1 expression can alter the cellular and humoral defenses of the insect innate immune system, and may lead to a decrease in male, but not female, immunocompetence.

Life History and Immune Challenge Influence Metabolic Plasticity to Food Availability and Acclimation Temperature.

Animals vary in their rates of energy expenditure for self-maintenance (standard metabolic rate [SMR]). Yet we still lack a thorough understanding of the determinants of SMR, potentially because of complex interactions among environmental, life-history, and physiological factors. Thus, we used a factorial design in female sand field crickets (Gryllus firmus) to investigate the independent and interactive effects of food availability (unlimited or limited access), acclimation temperature (control or simulated heat wave), life-history strategy (flight-capable or flight-incapable wing morphology), and immune status (control or chronic immune activation) on SMR (CO2 production rate) measured at 28°C. Both environmental factors independently affected SMR where heat wave and food limitation reduced SMR. Furthermore, wing morphology and immune status mediated the plasticity of SMR to food and temperature. For example, the hypermetabolic effect of food availability was greater in flight-capable crickets and reduced in immune-challenged crickets. Therefore, although SMR was directly affected by food availability and acclimation temperature, interactive effects on SMR were more common, meaning several factors (e.g., life history and immune status) influenced metabolic plasticity to food and temperature. We encourage continued use of factorial experiments to reveal interaction dynamics, which are critical to understanding emergent physiological processes.

Hemocyte-hemocyte adhesion by granulocytes is associated with cellular immunity in the cricket, Gryllus bimaculatus.

In this study, more than 1,000 cricket (Gryllus bimaculatus) hemocytes were classified based on their size and morphology. These hemocytes were classified into six types: granulocytes, plasmatocytes, prohemocytes, spherulocytes, coagulocytes, and oenocytoids. Hemocyte cultures was observed in real time to determine which hemocytes were associated with cellular immune responses against potential pathogens. Granulocytes were identified as the professional immune cell that mediates nodulation, encapsulation, and phagocytosis of pathogens. Granulocytes have been shown to actively produce various sticky nets (amoeba-like hairs and extracellular traps) from their plasma membranes that they use to gather other hemocytes and to implement cellular immune responses. The activation of lysosomes in granulocytes started at 4 h, peaked at 12 h, and returned to baseline by 24 h post-infection. At 48 h post-infection, cells could be found within the cytoplasm of granulocytes and reactivated lysosomes surrounding these cells were visible. This result seems to reflect a phenomenon in which necrotic granulocytes are removed by other healthy granulocytes. This unique mechanism of cellular immunity is therefore a way to efficiently and effectively remove pathogens and simultaneously maintain healthy hemocytes.

Immune gene regulation in the gut during metamorphosis in a holo- versus a hemimetabolous insect.

During metamorphosis, holometabolous insects completely replace the larval gut and must control the microbiota to avoid septicaemia. Rapid induction of bactericidal activity in the insect gut at the onset of pupation has been described in numerous orders of the Holometabola and is best-studied in the Lepidoptera where it is under control of the 20-hydroxyecdysone (20E) moulting pathway. Here, using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in a holometabolous (Galleria mellonella) and a hemimetabolous insect (Gryllus bimaculatus). We find that in G. mellonella, the expression of numerous immune effectors and the transcription factor GmEts are upregulated, with peak expression of three antimicrobial peptides (AMPs) and a lysozyme coinciding with delamination of the larval gut. By contrast, no such upregulation was detectable in the hemimetabolous Gr. bimaculatus. These findings support the idea that the upregulation of immune effectors at the onset of complete metamorphosis is an adaptive response, which controls the microbiota during gut replacement. This article is part of the theme issue 'The evolution of complete metamorphosis'.

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.

Sex and life history shape the strength of cellular and humoral immune responses in a wing dimorphic cricket.

Immune function is a complex collection of responses that often trade-off with one another and with other life history traits, because of the high costs of mounting and maintaining immune responses. Animals, even those from the same populations, may emphasize different aspects of immune function depending on their habitat and phenotype. For example, host population density mediates the threat from density-dependent parasites. Animals at high densities may emphasize fast-acting humoral responses, while those at low densities may favor slower, but more specific, cellular responses. However, these predictions may be dependent on other life history traits, like sex, which is associated with variation in many immune responses. We used wing dimorphic Gryllus firmus crickets to test humoral responses, measured by lysozyme and phenoloxidase activities, and cellular immune responses, measured by encapsulation, between morphs and sex. We found that both morphs and sexes differed in aspects of immune function. Long wing morphs had stronger encapsulation responses than short winged morphs. Additionally, females exhibited higher PO activity than males, and by contrast, males had higher lysozyme activity than females. Our study suggests that G. firmus morphs prioritize different immune responses that may reflect a balancing between the costs of immunity and differing pathogen threats. Male and female crickets exhibit differences in humoral immune responses that may reflect their different life history demands.

Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy.

Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.

Limited cross reactivity among arginine kinase allergens from mealworm and cricket edible insects.

Insects are seen as a solution to the increasing demand for protein sources for food. However, entomophagy has unfortunately been linked to allergic reactions in Europe with people with professional contacts. As mealworms (Tenebrio molitor) and crickets (Acheta domesticus) have recently become commercially available (both whole or in food formulation) in several European countries, this research assessed the cross allergenicity of arginine kinase (AK). Based on the collection of sera from a entomology laboratory staff, oven cooked insects but also purified AK fractions were tested. Immunoblotting against the protein extracts revealed different Immunoglobulin E reactivity of sera according to the insect target species: two bands (40 and 14 kDa) for crickets and a pattern including light responses at 17, 25 and 37 kDa for mealworms. Focusing on AK, low specific allergenicity was here illustrated and discussed in relation to the development of a safe edible insect consumption by humans.

Age-dependent variation in the terminal investment threshold in male crickets.

The terminal investment hypothesis proposes that decreased expectation of future reproduction (e.g., arising from a threat to survival) should precipitate increased investment in current reproduction. The level at which a cue of decreased survival is sufficient to trigger terminal investment (i.e., the terminal investment threshold) may vary according to other factors that influence expectation for future reproduction. We test whether the terminal investment threshold varies with age in male crickets, using heat-killed bacteria to simulate an immune-inducing infection. We measured calling effort (a behavior essential for mating) and hemolymph antimicrobial activity in young and old males across a gradient of increasing infection cue intensity. There was a significant interaction between the infection cue and age in their effect on calling effort, confirming the existence of a dynamic terminal investment threshold: young males reduced effort at all infection levels, whereas old males increased effort at the highest levels relative to naïve individuals. A lack of a corresponding decrease in antibacterial activity suggests that altered reproductive effort is not traded against investment in this component of immunity. Collectively, these results support the existence of a dynamic terminal investment threshold, perhaps accounting for some of the conflicting evidence in support of terminal investment.

Immune function trade-offs in response to parasite threats.

Immune function is often involved in physiological trade-offs because of the energetic costs of maintaining constitutive immunity and mounting responses to infection. However, immune function is a collection of discrete immunity factors and animals should allocate towards factors that combat the parasite threat with the highest fitness cost. For example, animals on dispersal fronts of expanding population may be released from density-dependent diseases. The costs of immunity, however, and life history trade-offs in general, are often context dependent. Trade-offs are often most apparent under conditions of unusually limited resources or when animals are particularly stressed, because the stress response can shift priorities. In this study we tested how humoral and cellular immune factors vary between phenotypes of a wing dimorphic cricket and how physiological stress influences these immune factors. We measured constitutive lysozyme activity, a humoral immune factor, and encapsulation response, a cellular immune factor. We also stressed the crickets with a sham predator in a full factorial design. We found that immune strategy could be explained by the selective pressures encountered by each morph and that stress decreased encapsulation, but not lysozyme activity. These results suggest a possible trade-off between humoral and cellular immunity. Given limited resources and the expense of immune factors, parasite pressures could play a key factor in maintaining insect polyphenism via disruptive selection.

Females gain survival benefits from immune-boosting ejaculates.

Females in many animal taxa incur significant costs from mating in the form of injury or infection, which can drastically reduce survival. Therefore, immune function during reproduction can be important in determining lifetime fitness. Trade-offs between reproduction and immunity have been extensively studied, yet a growing number of studies demonstrate that mated females have a stronger immune response than virgins. Here, we use the Texas field cricket, Gryllus texensis, to test multiple hypotheses proposed to explain this postmating increase in immune function. Using host-resistance tests, we found that courtship, copulation, and accessory fluids alone do not affect female immunity; rather, only females that acquire intact ejaculates containing testes-derived components exhibit significant increases in survival after exposure to bacterial pathogens. Our data suggest that male-derived components originating from an intact ejaculate and transferred to females during sex are required for the increased immune function characteristic of mated female crickets to occur.

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.

Response of the insect immune system to three different immune challenges.

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD50 dose of LPS from Escherichia coli elicited a more rapid response than an LD50 dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2hpi, followed by an increase by 7dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system's response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.

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.

No Detectable Trade-Offs Among Immune Function, Fecundity, and Survival via a Juvenile Hormone Analog in the House Cricket.

Hormones are key regulators of resource allocation among functions and thus play an important role in resource-based trade-offs. The juvenile hormone (JH) is an insect hormone that mediates resource allocation between immunity and life history components. Here, we have tested whether this is the case using the house cricket. We investigated whether increased levels of JH (using methoprene, a JH analog) enable an enhanced survival and fecundity (via egg number) at the cost of reduced hemocyte number (a trait that is associated with immune response in insects) in the house cricket, Acheta domesticus L. We had three groups of adult crickets of both sexes: experimental (methoprene and acetone), positive control (methoprene), and negative control (no manipulation). Prior to and after experimental treatments, we counted the number of hemocytes (for the case of both sexes) and recorded the number of eggs laid and survival of females after the manipulation. There was no difference in hemocyte number, egg number, and survival. These results do not support a JH-mediated trade-off among immune ability, survival, and fecundity. We provide arguments to explain the lack of JH-mediated trade-offs in the house cricket.