The plasticity of immune memory in invertebrates.

Whether specific immune protection after initial pathogen exposure (immune memory) occurs in invertebrates has long been uncertain. The absence of antibodies, B-cells and T-cells, and the short lifespans of invertebrates led to the hypothesis that immune memory does not occur in these organisms. However, research in the past two decades has supported the existence of immune memory in several invertebrate groups, including Ctenophora, Cnidaria, Nematoda, Mollusca and Arthropoda. Interestingly, some studies have demonstrated immune memory that is specific to the parasite strain. Nonetheless, other work does not provide support for immune memory in invertebrates or offers only partial support. Moreover, the expected biphasic immune response, a characteristic of adaptive immune memory in vertebrates, varies within and between invertebrate species. This variation may be attributed to the influence of biotic or abiotic factors, particularly parasites, on the outcome of immune memory. Despite its critical importance for survival, the role of phenotypic plasticity in immune memory has not been systematically examined in the past two decades. Additionally, the features of immune responses occurring in diverse environments have yet to be fully characterized.

Does juvenile hormone prompt oxidative stress in male damselflies?

In invertebrates, it has recently been reported that secondary sexual characteristics (SSCs) reflect the antioxidant defense of their bearers, but it is not known what physiological link maintains the honesty of those signals. Here, we used the damselfly Hetaerina americana to test whether juvenile hormone plays such a role. First, we analyzed whether oxidative damage is a real threat in natural damselfly populations by examining the accumulation of oxidized guanines as a function of age in males. Then, we injected paraquat (a pro-oxidant agent) and added the juvenile hormone analog methoprene (JHa) to the experimental group and the JHa vehicle (acetone) to the control group, to determine whether JHa increases the levels of pro-oxidants and antioxidants. We found that DNA oxidation increased with age, and that levels of hydrogen peroxide and superoxide dismutase, but not catalase or glutathione, were elevated in the JHa group compared with the control group. We propose that juvenile hormone is a mediator of the relationship between SSCs and antioxidant capacity and, based on the literature, we know that JHa suppresses the immune response. We therefore suggest that juvenile hormone is a molecular mediator of the general health of males, which is reflected in their SSCs.

The costs of the immune memory within generations.

Immune response is evolutionary costly, but it is not clear whether these costs affect energetic expenditure (short-term cost), growth (medium-term cost), or reproduction (long-term cost). We tested the costs of immune memory in Tenebrio molitor against Metarhizium brunneum. To do this, we used two groups of T. molitor larvae: (a) the control group, which was injected first with Tween solution and 10 days later with M. brunneum and (b) the memory group, which was first injected with M. brunneum and 10 days later with M. brunneum. Compared to controls, larvae of the memory group were more likely to survive, but they also had an increased metabolic rate (CO2 production), spent a long time before becoming pupae, and had a shorter time from pupae to adulthood. In the adult stage, control females preferred control males, but there was no significant difference in the preference of memory females. Finally, control and memory males preferred control females. These results confirm that immune memory has costs in terms of energetic expenditure, growth, and reproduction. To the best of our knowledge, this is the first experimental demonstration that immune memory in larvae is traded-off with adult sexual selection involving mate choice.

Pathogen-produced catalase affects immune priming: A potential pathogen strategy.

Immune priming in invertebrates occurs when the first contact with a pathogen/parasite enhances resistance after a second encounter with the same strain or species. Although the mechanisms are not well understood, there is evidence that priming the immune response of some hosts leads to greater pro-oxidant production. Parasites, in turn, might counteract the host attack with antioxidants. Virulent pathogen strains may therefore mask invertebrate immune priming. For example, different parasite species overexpress catalase as a virulence factor to resist host pro-oxidants, possibly impairing the immune priming response. The aim of this study was firstly to evaluate the specificity of immune priming in Tenebrio molitor when facing homologous and heterologous challenges. Secondly, homologous challenges were carried out with two Metarhizium anisopliae strains (Ma10 and CAT). The more virulent strain (CAT) overexpresses catalase, an antioxidant that perhaps impairs a host immune response mediated by reactive oxygen species (ROS). Indeed, T. molitor larvae exhibited better immune priming (survival) in response to the Ma10 than CAT homologous challenge. Moreover, the administration of paraquat, an ROS-promoting agent, favoured survival of the host upon exposure to each fungal strain. We propose that some pathogens likely overcome pro-oxidant-mediated immune priming defences by producing antioxidants such as catalase.

The occurrence of immune priming can be species-specific in entomopathogens.

Immune priming in invertebrates refers to an improved immune response (and therefore a better chance of survival) upon a second encounter with a specific pathogen. Although the existence of immune priming has been evaluated in invertebrate hosts, the ability of a particular entomopathogen species or strain to influence the occurrence of immune priming has not been thoroughly evaluated. The aim of the current study was to compare the occurrence of immune priming in Tenebrio molitor larvae after homologous challenges (a dual exposure to similar entomopathogens) with Serratia marcescens, Bacillus thuringiensis and Metarhizium anisopliae. Larvae presented more effective immune priming (measured as survival rates) when exposed to M. anisopliae or B. thuringiensis than when exposed to S. marcescens. We hypothesize that the toll pathway may help T. molitor survive these enemies and that the IMD pathway may be expressed to a lesser degree in this species, which may explain why they succumb to Gram-negative bacteria. This and other recent evidence suggest that the occurrence of immune priming in these organisms must not be ruled out until this phenomenon is tested with different entomopathogens.

Microbiome symbionts and diet diversity incur costs on the immune system of insect larvae.

Communities of symbiotic microorganisms that colonize the gastrointestinal tract play an important role in food digestion and protection against opportunistic microbes. Diet diversity increases the number of symbionts in the intestines, a benefit that is considered to impose no cost for the host organism. However, less is known about the possible immunological investments that hosts have to make in order to control the infections caused by symbiont populations that increase because of diet diversity. Using taxonomical composition analysis of the 16S rRNA V3 region, we show that enterococci are the dominating group of bacteria in the midgut of the larvae of the greater wax moth (Galleria mellonella). We found that the number of colony-forming units of enterococci and expressions of certain immunity-related antimicrobial peptide (AMP) genes such as Gallerimycin, Gloverin, 6-tox, Cecropin-D and Galiomicin increased in response to a more diverse diet, which in turn decreased the encapsulation response of the larvae. Treatment with antibiotics significantly lowered the expression of all AMP genes. Diet and antibiotic treatment interaction did not affect the expression of Gloverin and Galiomicin AMP genes, but significantly influenced the expression of Gallerimycin, 6-tox and Cecropin-D Taken together, our results suggest that diet diversity influences microbiome diversity and AMP gene expression, ultimately affecting an organism's capacity to mount an immune response. Elevated basal levels of immunity-related genes (Gloverin and Galiomicin) might act as a prophylactic against opportunistic infections and as a mechanism that controls the gut symbionts. This would indicate that a diverse diet imposes higher immunity costs on organisms.

Costs and benefits of vertical and horizontal transmission of dengue virus.

Parasites can be transmitted vertically and/or horizontally, but the costs or benefits for the host of infection have only been tested after horizontal transmission. Here, we report for the first time, to our knowledge, the survival, reproduction and infection of Aedes aegypti during vertical and horizontal transmission of dengue virus 2 (DENV-2). Females infected horizontally produced more eggs, with a sex ratio skewed towards males, compared with uninfected controls. However, there was no significant difference in the number of emerging adults or in survival of mothers. In contrast, dengue-infected female offspring (vertical transmission) had a shorter lifespan but there were no significant differences in the number of eggs or sex ratio, compared with controls. Finally, the corroboration of infection revealed that virus infected about 11.5% and 8.8% of pools of mothers and of daughters, respectively. These results suggest that the mode of infection and the contact with the virus has no reproductive costs to female mosquitoes, which may explain why both types of transmission are evolutionarily maintained. In addition, we suggest that more attention should be paid to the male contribution to virus dissemination within and among populations and as reservoirs of the infection for human diseases.

Is Juvenile Hormone a potential mechanism that underlay the "branched Y-model"?

Trade-offs are a central tenet in the life-history evolution and the simplest model to understand it is the "Y" model: the investment of one arm will affect the investment of the other arm. However, this model is by far more complex, and a "branched Y-model" is proposed: trade-offs could exist within each arm of the Y, but the mechanistic link is unknown. Here we used Tenebrio molitor to test if Juvenile Hormone (JH) could be a mechanistic link behind the "branched Y-model". Larvae were assigned to one of the following experimental groups: (1) low, (2) medium and (3) high doses of methoprene (a Juvenile Hormone analogue, JHa), (4) acetone (methoprene diluents; control one) or (5) näive (handled in the same way as other groups; control two). The JHa lengthened the time of development from larvae to pupae and larvae to adults, resulting in adults with a larger size. Males with medium and long JHa treatment doses were favored with female choice, but had smaller testes and fewer viable sperm. There were no differences between groups in regard to the number of spermatozoa of males, or the number of ovarioles or eggs of females. This results suggest that JH: (i) is a mechanistic link of insects "branched Y model", (ii) is a double ended-sword because it may not only provide benefits on reproduction but could also impose costs, and (iii) has a differential effect on each sex, being males more affected than females.

Microbiota from Rhabditis regina may alter nematode entomopathogenicity.

Here we report the presence of the entomopathogenic nematode Rhabditis (Rhabditoides) regina affecting white grubs (Phyllophaga sp. and Anomala sp.) in Mexico and R. regina-associated bacteria. Bioassays were performed to test the entomopathogenic capacity of dauer and L2 and L3 (combined) larval stages. Furthermore, we determined the diversity of bacteria from laboratory nematodes cultivated for 2 years (dauer and L2-L3 larvae) and from field nematodes (dauer and L2-L3 larvae) in addition to the virulence in Galleria mellonella larvae of some bacterial species from both laboratory and field nematodes. Dauer and non-dauer larvae of R. regina killed G. mellonella. Bacteria such as Serratia sp. (isolated from field nematodes) and Klebsiella sp. (isolated from larvae of laboratory and field nematodes) may explain R. regina entomopathogenic capabilities. Different bacteria were found in nematodes after subculturing in the laboratory suggesting that R. regina may acquire bacteria in different environments. However, there were some consistently found bacteria from laboratory and field nematodes such as Pseudochrobactrum sp., Comamonas sp., Alcaligenes sp., Klebsiella sp., Acinetobacter sp., and Leucobacter sp. that may constitute the nematode microbiome. Results showed that some bacteria contributing to entomopathogenicity may be lost in the laboratory representing a disadvantage when nematodes are cultivated to be used for biological control.

Cross-cultural variation in men's preference for sexual dimorphism in women's faces.

Both attractiveness judgements and mate preferences vary considerably cross-culturally. We investigated whether men's preference for femininity in women's faces varies between 28 countries with diverse health conditions by analysing responses of 1972 heterosexual participants. Although men in all countries preferred feminized over masculinized female faces, we found substantial differences between countries in the magnitude of men's preferences. Using an average femininity preference for each country, we found men's facial femininity preferences correlated positively with the health of the nation, which explained 50.4% of the variation among countries. The weakest preferences for femininity were found in Nepal and strongest in Japan. As high femininity in women is associated with lower success in competition for resources and lower dominance, it is possible that in harsher environments, men prefer cues to resource holding potential over high fecundity.

To live free or being a parasite: The optimal foraging behavior may favor the evolution of entomopathogenic nematodes.

Facultative parasites can alternate between a free-living and a parasitic existence to complete their life cycle. Yet, it remains uncertain which lifestyle they prefer. The optimal foraging theory suggests that food preferences align with fitness benefits. To test this hypothesis, we investigated the facultative parasite nematode Rhabditis regina, assessing its host preference and the associated benefits. Two experiments were conducted using wild nematode populations collected from Phyllophaga polyphylla, their natural host. In the first experiment, we used a behavioral arena to assess host preference between the natural host and two experimental hosts: Spodoptera frugiperda which is an alternative host and dead Tenebrio molitor, which simulates a saprophytic environment. In the second experiment, we subjected wild nematodes to "experimental evolution" lasting 50 generations in S. frugiperda and 53 generations in T. molitor carcass. We then compared life history traits (the size, survival, number of larvae, and glycogen and triglycerides as energy reserves) of dauer larvae with those nematodes from P. polyphylla (control group). We found a significant preference for P. polyphylla, which correlated with higher values in the nematode's life history traits. In contrast, the preference for S. frugiperda and the saprophytic environment was lower, resulting in less efficient life history traits. These findings align with the optimal foraging theory, as the nematode's parasitic preferences are in line with maximizing fitness. This also indicates that R. regina exhibits specificity to P. polyphylla and is better adapted to a parasitic lifestyle than a free-living one, suggesting an evolutionary pathway towards parasitism.

Corrigendum: Development under predation risk increases serotonin-signaling, variability of turning behavior and survival in adult fruit flies Drosophila melanogaster.

[This corrects the article DOI: 10.3389/fnbeh.2023.1189301.].

Neural mechanisms involved in female mate choice in invertebrates.

Mate choice is a critical decision with direct implications for fitness. Although it has been recognized for over 150 years, our understanding of its underlying mechanisms is still limited. Most studies on mate choice focus on the evolutionary causes of behavior, with less attention given to the physiological and molecular mechanisms involved. This is especially true for invertebrates, where research on mate choice has largely focused on male behavior. This review summarizes the current state of knowledge on the neural, molecular and neurohormonal mechanisms of female choice in invertebrates, including behaviors before, during, and after copulation. We identify areas of research that have not been extensively explored in invertebrates, suggesting potential directions for future investigation. We hope that this review will stimulate further research in this area.

The immune response of the whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) when parasitized by Eretmocerus eremicus (Hymenoptera: Aphelinidae).

In insects, the innate immune system is subdivided into cellular and humoral defenses. When parasitoids attack insects, both reactions can be activated and notably, the phenoloxidase (PO) cascade and lytic activity are part of both cellular and humoral defenses. However, to our knowledge, no study has characterized any immune response of the whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) to the attack of Eretmocerus eremicus (Hymenoptera: Aphelinidae). Therefore, the first objective of the present study was to determine whether whitefly nymphs recently parasitized by E. eremicus exhibit any immune response. For this, we estimate the level of prophenoloxidase (proPO), phenoloxidase (PO), and lytic activity by colorimetric assays. A second objective was to assess whether the observed whitefly immune response could be related to a previously reported preference of the predator Geocoris punctipes (Hemiptera: Lygaeidae) for non-parasitized nymphs. We therefore offered non-parasitized and recently parasitized nymphs to the predator. Our results show that parasitism of whitefly nymphs by E. eremicus induced a highly estimated level of proPO and PO, and a lower level of lytic activity. In addition, we found that G. punctipes did not show a preference for non-parasitized over recently parasitized nymphs. The nymphs of T. vaporariorum activated the PO pathway against E. eremicus; however, the increase in proPO and PO levels was traded-off with decreased lytic activity. In addition, the previously reported preference for non-parasitized nymphs was not seen in our experiments, indicating that the induced immune response did not affect predator behavior by G. punctipes.

The Coincidental Evolution of Virulence Partially Explains the Virulence in a Generalist Entomopathogenic.

PURPOSE: The parasites' virulence is labile after jumping to a new host species, and it might derivate in gaining virulence against a new host as a side effect of living in a non-host environment (coincidental evolution of virulence hypothesis). METHODS: To test this hypothesis, we monitored the experimental evolution of the Rhabditis regina nematode for over 290 generations (4 years) in three environments (strains): (1) the natural host, Phyllophaga polyphylla, (2) an alternate host, Tenebrio molitor, and (3) saprophytic medium (beef; the food that may provide evidence for the coincidental evolution of virulence). Each strain was exposed to P. polyphylla, T. molitor, or Galleria mellonella. We compared the host survival and immune response (proPO, PO, and lytic activity) of infected versus uninfected hosts. RESULTS: The saprophytic nematodes gained virulence only against G. mellonella. However, the P. polyphylla strain was more effective in killing P. polyphylla than T. molitor, and the T. molitor strain was more effective against T. molitor than P. polyphylla. Additionally, one dauer larva was sufficient to kill the hosts. Finally, the immune response did not differ between the challenged and control groups. CONCLUSION: The coincidental evolution of virulence partially explains our results, but they might also support the short-sighted hypothesis. Additionally, we found evidence for immunomodulation because nematodes passed unnoticed to the immune response. It is crucial to analyze the virulence of entomopathogens from the point of view of the evolution of virulence to be aware of potential scenarios that might limit biological control.

The honey bees immune memory.

Invertebrates' immune priming or innate immune memory is an analogous response to the vertebrates' adaptive memory. We investigated if honey bees have immune memory. We compared survival and immune response between bees that were: 1) manipulated (Naïve), 2) challenged twice with the same pathogen Escherichia coli (Memory), 3) challenged twice with different pathogens (Staphylococcus aureus versus E. coli, Micrococcus lysodeikticus versus E. coli), or 4) with PBS (the diluent of bacteria) versus E. coli (heterologous challenge; Control). Results indicate better survival in the Memory than the Control group, and the Memory group showed a similar survival than Naïve insects. The Memory group had higher lytic activity but lower prophenoloxidase, phenoloxidase activity, and hemocyte count than the Control and Naïve groups. No differences were found in relative expression of defensin-1. This first demonstration of immune memory opens the questions about its molecular mechanisms and whether, immune memory could be used against natural parasites that affect honey bees, hence, if they could be "vaccinated" against some natural parasites.

Effect of juvenile hormone on phenoloxidase and hemocyte number: The role of age, sex, and immune challenge.

Hormones are key factors in determining the response of organisms to their environment. For example, the juvenile hormone (JH) coordinates the insects' development, reproduction, and survival. However, it is still unclear how the impact of juvenile hormone on insect immunity varies depending on the sex and reproductive state of the individual, as well as the type of the immune challenge (i.e., Gram-positive or Gram-negative bacteria). We used Tenebrio molitor and methoprene, a JH analog (JHa) to explore these relationships. We tested the effect of methoprene on phenoloxidase activity (PO), an important component of humoral immunity in insects, and hemocyte number. Lyophilized Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli were injected for the immune challenge. The results suggest that JH did not affect the proPO, PO activity, or hemocyte number of larvae. JH and immune challenge affected the immune response and consequently, affected adult developmental stage and sex. We propose that the influence of JH on the immune response depends on age, sex, the immune response parameter, and the immune challenge, which may explain the contrasting results about the role of JH in the insect immune response.

Genomics of the relict species Baronia brevicornis sheds light on its demographic history and genome size evolution across swallowtail butterflies.

Relict species, like coelacanth, gingko, tuatara, are the remnants of formerly more ecologically and taxonomically diverse lineages. It raises the questions of why they are currently species-poor, have restrained ecology, and are often vulnerable to extinction. Estimating heterozygosity level and demographic history can guide our understanding of the evolutionary history and conservation status of relict species. However, few studies have focused on relict invertebrates compared to vertebrates. We sequenced the genome of Baronia brevicornis (Lepidoptera: Papilionidae), which is an endangered species, the sister species of all swallowtail butterflies, and is the oldest lineage of all extant butterflies. From a dried specimen, we were able to generate both long-read and short-read data and assembled a genome of 406 Mb for Baronia. We found a fairly high level of heterozygosity (0.58%) compared to other swallowtail butterflies, which contrasts with its endangered and relict status. Taking into account the high ratio of recombination over mutation, demographic analyses indicated a sharp decline of the effective population size initiated in the last million years. Moreover, the Baronia genome was used to study genome size variation in Papilionidae. Genome sizes are mostly explained by transposable elements activities, suggesting that large genomes appear to be a derived feature in swallowtail butterflies as transposable elements activity is recent and involves different transposable elements classes among species. This first Baronia genome provides a resource for assisting conservation in a flagship and relict insect species as well as for understanding swallowtail genome evolution.

Development under predation risk increases serotonin-signaling, variability of turning behavior and survival in adult fruit flies Drosophila melanogaster.

The development of high-throughput behavioral assays, where numerous individual animals can be analyzed in various experimental conditions, has facilitated the study of animal personality. Previous research showed that isogenic Drosophila melanogaster flies exhibit striking individual non-heritable locomotor handedness. The variability of this trait, i.e., the predictability of left-right turn biases, varies across genotypes and under the influence of neural activity in specific circuits. This suggests that the brain can dynamically regulate the extent of animal personality. It has been recently shown that predators can induce changes in prey phenotypes via lethal or non-lethal effects affecting the serotonergic signaling system. In this study, we tested whether fruit flies grown with predators exhibit higher variability/lower predictability in their turning behavior and higher survival than those grown with no predators in their environment. We confirmed these predictions and found that both effects were blocked when flies were fed an inhibitor (αMW) of serotonin synthesis. The results of this study demonstrate a negative association between the unpredictability of turning behavior of fruit flies and the hunting success of their predators. We also show that the neurotransmitter serotonin controls predator-induced changes in the turning variability of fruit flies, regulating the dynamic control of behavioral predictability.

Incubation in shaded hatcheries biases sex-determination but preserves Lepidochelys olivacea hatchling physiology.

Some studies have associated ex situ conservation with cerebral and gonadal developmental delay, as well as decreased motor performance in Lepidochelys olivacea offspring. Ex situ management is also related to a more mature spleen and a differential leukocyte count in newly emerged Lepidochelys olivacea hatchlings. The physiological relevance of a more mature spleen is unknown in sea turtles, but studies in birds suggest an increased immune response. Because egg relocation to hatcheries is a common conservation practice, it is imperative to know its impact on hatchling physiology. Herein, plasma activity of superoxide dismutase, alkaline phosphatase and the alternative complement pathway, as well as total antioxidant capacity and hydrogen peroxide concentrations were quantified in hatchlings from in situ and ex situ nests under basal conditions at nest emergence. Toll-like receptor 4 (tlr4), heat shock proteins (hsp) 70 and hsp90 expression were quantified in the spleen and liver of the hatchlings. Hepatocyte density and nuclear area were quantified in histological sections of the liver and all turtles were sexed by histological sectioning of the gonads. Total antioxidant capacity and hydrogen peroxide concentrations in plasma were lower in turtles from ex situ nests, while tlr4 and hsp70 mRNA expression was higher in the spleen but not in the liver. Ex situ incubation produced 98% male hatchlings, whereas in situ incubation produced 100% females. There were no other differences in the attributes sampled between hatchlings emerging from ex situ and in situ treatments. The results suggest that ex situ relocated turtles may be less prone to oxidative stress than in situ incubated hatchlings and could have more mature splenic function. Together, the data suggest that ex situ relocation to shaded hatcheries biased sex determination but preserved the general physiological condition of sea turtle hatchlings.