Effect of a single cold stress exposure on the reproductive behavior of male crickets.

Cold stress is an important abiotic factor that can impact insect physiology, behavior, and overall fitness. Upon exposure to cold temperature, many insects enter a reversible state of immobility called chill coma. If the cold stress is brief and mild enough, insects can recover and regain full mobility upon return to warmer temperatures. However, the long-term impact of sublethal cold stress on insect behavior has been understudied. Here, sexually naïve adult male Acheta domesticus crickets were exposed to a single 0 °C cold stress for 6 h. One week later, the ability of these males to mate with a female was examined. For mating trials, a cold stressed male cricket was paired with a non-cold stressed, control female. Control pairs were comprised of a non-cold stressed control male and control female. Cold exposed males were less successful at mating than control males because most did not carry a spermatophore at the time of their mating trials. However, when these cold stressed males were allowed 1 h of chemosensory contact with a female, most produced a spermatophore. Males that produced spermatophores were given the opportunity to mate once with a female, and stressed males that successfully mated sired as many offspring as did control males. However, our results support that a single cold stress exposure can negatively impact the reproductive fitness of male crickets since it reduced their capacity to carry spermatophores and, as a consequence, to attract females.

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.

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.

Maturation of the immune system of the male house cricket, Acheta domesticus.

The immune system functions to counteract the wide range of pathogens an insect may encounter during its lifespan, ultimately maintaining fitness and increasing the likelihood of survival to reproductive maturity. In this study, we describe the maturation of the innate immune system of the male house cricket Acheta domesticus during the last two nymphal stages, and during early and late adulthood. Total hemolymph phenoloxidase enzyme activity, lysozyme-like enzyme activity, the number of circulating hemocytes, and encapsulation ability were all determined for each developmental stage or age examined. The number of circulating hemocytes and lysozyme-like enzyme activity were similar for all developmental stages examined. Nymphs and newly molted adult males, however, had significantly lower total phenoloxidase activity than later adult stages, yet nymphs were able to encapsulate a nylon thread just as well as adults. Encapsulation ability would thus appear to be independent of total phenoloxidase activity.

Agonistic behavior and electrical stimulation of the antennae induces Fos-like protein expression in the male cricket brain.

Immediate early genes (IEG) such as c-Fos and Fos-related antigens (FRA) have been used as markers of neuronal activation. In this study, we determined whether the expression of c-Fos/FRAs is increased in the brains of adult male Acheta domesticus crickets following agonistic interactions. We looked for c-Fos/FRA proteins in the brain of un-fought, control male crickets and of dominant and subordinate male crickets sacrificed at different time periods following an agonistic interaction. Using immunoblot analysis, we found four different c-Fos/FRA-like proteins in the adult cricket brain. Continuous agonistic interaction increased c-Fos/FRA protein expression in the brains of subordinate males compared to control and dominant males. In addition, direct electrical stimulation of the male cricket antennae increased c-Fos/FRA-like protein in the brain. We identified the specific brain regions that exhibit c-Fos/FRA-like immunoreactivity in crickets. We detected c-Fos/FRA-like cellular immunoreactivity in different functional regions of the adult brain including the pars intercerebralis, protocerebrum, deutocerebrum, and the cortex of the mushroom bodies.

Agonistic behavior enhances adult neurogenesis in male Acheta domesticus crickets.

We examined the effect of agonistic behavior on cell proliferation and neurogenesis in the central nervous system (CNS) of adult male Acheta domesticus crickets. We combined 5-bromo,2'deoxyuridine (BrdU)-labeling of dividing cells with immunocytochemical detection of the neuronal marker horseradish peroxidase to examine the proliferation of progenitor cells and the survival of newborn neurons. In crickets, the mushroom bodies of the brain contain clusters of proliferative cells that divide and generate new neurons in adulthood. Pairs of male crickets were allowed to fight and establish social rank and were then injected with BrdU. Proliferation of mushroom body neurogenic cluster cells was unaffected by agonistic interactions; 24 h after a fight, the number of BrdU positive cells in fought and un-fought males did not significantly differ. However, agonistic interactions did influence cell survival. Two weeks after an agonistic interaction, fought males had more newborn neurons than males that did not fight. There was also a rank-specific effect because dominant males had significantly more new neurons than subordinates. We also report for the first time that neurogenesis in adult crickets can occur in other regions of the brain and in other CNS ganglia, including the terminal abdominal ganglion (TAG). Agonistic interactions enhanced the proliferation of these distributed precursor cells but did not increase the survival of the newborn neurons generated by these cells.