Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study.

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.

Crickets in the spotlight: exploring the impact of light on circadian behavior.

Crickets serve as a well-established model organism in biological research spanning various fields, such as behavior, physiology, neurobiology, and ecology. Cricket circadian behavior was first reported over a century ago and prompted a wealth of studies delving into their chronobiology. Circadian rhythms have been described in relation to fundamental cricket behaviors, encompassing stridulation and locomotion, but also in hormonal secretion and gene expression. Here we review how changes in illumination patterns and light intensity differentially impact the different cricket behaviors as well as circadian gene expression. We further describe the cricket's circadian pacemaker. Ample anatomical manipulations support the location of a major circadian pacemaker in the cricket optic lobes and another in the central brain, possibly interconnected via signaling of the neuropeptide PDF. The cricket circadian machinery comprises a molecular cascade based on two major transcriptional/translational negative feedback loops, deviating somewhat from the canonical model of Drosophila and emphasizing the significance of exploring alternative models. Finally, the nocturnal nature of crickets has provided a unique avenue for investigating the repercussions of artificial light at night on cricket behavior and ecology, underscoring the critical role played by natural light cycles in synchronizing cricket behaviors and populations, further supporting the use of the cricket model in the study of the effects of light on insects. Some gaps in our knowledge and challenges for future studies are discussed.

Heatwaves inflict reproductive but not survival costs to male insects.

Climate change is having a dramatic effect on the environment, with rising global temperatures and more frequent extreme climatic events, such as heatwaves, that can hamper organisms' biological functions. Although it is clear that sudden and extreme temperatures can damage reproductive processes, there is limited understanding of the effects of heatwaves on male mating behaviour and reproductive success. We tested for the effects of heat stress induced by ecologically relevant heatwaves (33°C and 39°C for five consecutive days) on the mating behaviour, reproductive success, body mass and survival of male field crickets Gryllus bimaculatus, paired with untreated females. We predicted life-history and reproductive costs would increase with increasing heatwave intensity. Consistent with our expectations, males exposed to the highest heatwave temperature produced the fewest offspring, while having to increase courtship effort to successfully mate. Males also gained relatively more weight following heatwave exposure. Given that we found no difference in lifetime survival, our results suggest a potential trade-off in resource allocation between somatic maintenance and reproductive investment. Taken together, our findings indicate that sublethal effects of heatwaves could reduce the growth and persistence of animal populations by negatively impacting reproductive rates. These findings highlight the need for considering thermal ecologies, life history and behaviour to better understand the consequences of extreme climatic events on individuals and populations.

Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus.

Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.

Involvement of the scalloped gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect Gryllus bimaculatus.

The acquisition of wings was a key event in insect evolution. As hemimetabolous insects were the first group to acquire functional wings, establishing the mechanisms of wing formation in this group could provide useful insights into their evolution. In this study, we aimed to elucidate the expression and function of the gene scalloped (sd), which is involved in wing formation in Drosophila melanogaster, and in Gryllus bimaculatus mainly during postembryonic development. Expression analysis showed that sd is expressed in the tergal edge, legs, antennae, labrum, and cerci during embryogenesis and in the distal margin of the wing pads from at least the sixth instar in the mid to late stages. Because sd knockout caused early lethality, nymphal RNA interference experiments were performed. Malformations were observed in the wings, ovipositor, and antennae. By analyzing the effects on wing morphology, it was revealed that sd is mainly involved in the formation of the margin, possibly through the regulation of cell proliferation. In conclusion, sd might regulate the local growth of wing pads and influence wing margin morphology in Gryllus.

Pre- and post-copulatory sexual selection increase offspring quality but impose survival costs to female field crickets.

Whether sexual selection increases or decreases fitness is under ongoing debate. Sexual selection operates before and after mating. Yet, the effects of each episode of selection on individual reproductive success remain largely unexplored. We ask how disentangled pre- and post-copulatory sexual selection contribute to fitness of field crickets Gryllus bimaculatus. Treatments allowed exclusively for (i) pre-copulatory selection, with males fighting and courting one female, and the resulting pair breeding monogamously, (ii) post-copulatory selection, with females mating consecutively to multiple males and (iii) relaxed selection, with enforced pair monogamy. While standardizing the number of matings, we estimated a number of fitness traits across treatments and show that females experiencing sexual selection were more likely to reproduce, their offspring hatched sooner, developed faster and had higher body mass at adulthood, but females suffered survival costs. Interestingly, we found no differences in fitness of females or their offspring from pre- and post-copulatory sexual selection treatments. Our findings highlight the potential for sexual selection in enhancing indirect female fitness while concurrently imposing direct survival costs. By potentially outweighing these costs, increased offspring quality could lead to beneficial population-level consequences of sexual selection.

[A YOUNG CHILD WITH ALLERGIC SYMPTOMS DUE TO INGESTION OF EDIBLE CRICKETS].

In recent years, the consumption of edible insects has gained attention, and the potential allergic risks associated with their ingestion have been pointed out, though there are limited case reports. A 3-year-old boy exhibited an immediate allergic reaction, showing symptoms of sneezing, nasal discharge, coughing, and eyelid edema after ingesting two cricket crackers. He had previously consumed shrimp but had never eaten edible insects. Given his lack of a history of allergic diseases, the onset of this allergy was unexpected. Subsequent prick tests and oral food challenge tests confirmed that the Two-spotted cricket (Gryllus bimaculatus) was the allergen responsible for his symptoms. The IgE inhibition test indicated that the cricket significantly suppressed the specific IgE levels for moth, shrimp, and mite (Dermatophagoides pteronyssinus). This incident marked the first time in the patient's life that he exhibited allergic symptoms, and it serves as a significant case highlighting the risks of allergies from edible insects. Known allergens in insects include tropomyosin and arginine kinase, which are common in arthropods, but there are reports of other allergens as well, suggesting potential sensitization from cross-reactions. As the consumption of insects becomes more widespread, the number of allergic cases may increase, and food labeling and preventive measures should be considered.

Hox genes limit germ cell formation in the short germ insect Gryllus bimaculatus.

Hox genes are conserved transcription factor-encoding genes that specify the identity of body regions in bilaterally symmetrical animals. In the cricket Gryllus bimaculatus, a member of the hemimetabolous insect group Orthoptera, the induction of a subset of mesodermal cells to form the primordial germ cells (PGCs) is restricted to the second through the fourth abdominal segments (A2 to A4). In numerous insect species, the Hox genes Sex-combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) jointly regulate the identities of middle and posterior body segments, suggesting that these genes may restrict PGC formation to specific abdominal segments in G. bimaculatus Here we show that reducing transcript levels of some or all of these Hox genes results in supernumerary and/or ectopic PGCs, either individually or in segment-specific combinations, suggesting that the role of these Hox genes is to limit PGC development with respect to their number, segmental location, or both. These data provide evidence of a role for this ancient group of genes in PGC development.

Road to The Red Carpet of Edible Crickets through Integration into the Human Food Chain with Biofunctions and Sustainability: A Review.

The Food and Agriculture Organization of the United Nations (FAO) estimates that more than 500 million people, especially in Asia and Africa, are suffering from malnutrition. Recently, livestock farming has increased to supply high-quality protein, with consequent impact on the global environment. Alternative food sources with high nutritive values that can substitute livestock demands are urgently required. Recently, edible crickets have been promoted by the FAO to ameliorate the food crisis. In this review, the distribution, nutritive values, health-promoting properties (antioxidant, anti-inflammatory, anti-diabetic and anti-obesity), safety, allergenicity as well as the potential hazards and risks for human consumption are summarized. Cricket farming may help to realize the United Nations sustainable development goal No. 2 Zero Hunger. The sustainability of cricket farming is also discussed in comparison with other livestock. The findings imply that edible crickets are safe for daily intake as a healthy alternative diet due to their high protein content and health-promoting properties. Appropriate use of edible crickets in the food and nutraceutical industries represents a global business potential. However, people who are allergic to shellfish should pay attention on cricket allergy. Thus, the objective of this review was to present in-depth and up-to-date information on edible crickets to advocate and enhance public perception of cricket-based food.

Tolerant pattern recognition: evidence from phonotactic responses in the cricket Gryllus bimaculatus (de Geer).

When the amplitude modulation of species-specific acoustic signals is distorted in the transmission channel, signals become difficult to recognize by the receiver. Tolerant auditory pattern recognition systems, which after having perceived the correct species-specific signal transiently broaden their acceptance of signals, would be advantageous for animals as an adaptation to the constraints of the environment. Using a well-studied cricket species, Gryllus bimaculatus, we analysed tolerance in auditory steering responses to 'Odd' chirps, mimicking a signal distorted by the transmission channel, and control 'Silent' chirps by employing a fine-scale open-loop trackball system. Odd chirps on their own did not elicit a phonotactic response. However, when inserted into a calling song pattern with attractive Normal chirps, the females' phonotactic response toward these patterns was significantly larger than to patterns with Silent chirps. Moreover, females actively steered toward Odd chirps when these were presented within a sequence of attractive chirps. Our results suggest that crickets employ a tolerant pattern recognition system that, once activated, transiently allows responses to distorted sound patterns, as long as sufficient natural chirps are present. As pattern recognition modulates how crickets process non-attractive acoustic signals, the finding is also relevant for the interpretation of two-choice behavioural experiments.

Does the choosiness of female crickets change as they age?

For crickets, which approach singing males by phonotaxis, the female choosiness hypothesis postulates that young females should be more selective of male calling song patterns than older individuals. However, there is no information about the behavioural preferences of females over their complete adulthood. We analysed phonotaxis in female Gryllus bimaculatus throughout their entire adult lifetime and measured the impact of sound amplitude, carrier frequency and the temporal pattern of test songs on their auditory response. Females of all ages demonstrated their best responses to male calling songs with a pulse period of 34-42 ms, a carrier frequency of 4.5 kHz and a sound pressure level of 75 dB. The response profile to somewhat less optimal song types did vary with age, but not in a manner consistent with a simple loosening of selectiveness in older females. Age, however, had an effect on the overall strength of phonotaxis, as very old females showed an overall diminishing response to all song types. Our data suggest that although there are minor changes in the relative preferences of crickets to individual song elements as they age, the breadth of song patterns to which they will perform phonotaxis remains similar across age groups.

Different transcriptional levels of Corazonin, Elevenin, and PDF according to the body color of the two-spotted cricket, Gryllus bimaculatus.

Body color in insects changes according to the living environment and physiological stresses possibly involved in endocrine factors. To date, 3 predominant bioactive peptides, Corazonin, Elevenin, and pigment-dispersing factor (PDF), have been illuminated to be involved in the body color in insects and crustaceans. Here, we examined the possibilities that these 3 factors would contribute to body color changes via melanization in the two-spotted cricket, Gryllus bimaculatus, whose body color changes according to population density drastically. Quantitative analyses revealed the higher transcriptional levels of Corazonin and Elevenin in the crowded-conditioned crickets, whereas the transcriptional level of PDF was higher in the isolated-conditioned crickets. However, the body color was not changed by knockdown of Corazonin, Elevenin, and PDF by RNA interference. The present data indicated that coloration mechanisms in G. bimaculatus is differently controlled from the previous observation in Locusta migratoria, a closely related orthopteran species.

Internal state transition to switch behavioral strategies in cricket phonotaxis.

Animals employ multiple behavioral strategies for exploring food and mating partners based on both their internal state and external environment. Here, we examined how cricket phonotaxis, which was considered an innate reactive behavior of females to approach the calling song of conspecific males, depended on these internal and external conditions. Our observation revealed that the phonotaxis process consisted of two distinctive phases: wandering and approaching. In the latter phase, crickets moved directly towards the sound source. The transition into this phase, referred to as the 'approach phase', was based on changes in the animal's internal state. Moreover, retention of the approach phase required recognition of the calling song, while song loss downregulated cricket mobility and induced frequent stopping. This is a typical movement in local search behaviors. Our results indicate that phonotaxis is not only a reactive response but a complicated process including multiple behavioral strategies.

(±)-Kituramides A and B, pairs of enantiomeric dopamine dimers from the two-spotted cricket Gryllus bimaculatus.

Two-spotted cricket Gryllus bimaculatus is one of many cricket species, and it is widely used as a food source for insectivorous animals. Moreover, this species is one of the edible insects approved by the Korea Food and Drug Administration (KFDA). (±)-Kituramides A (1) and B (2), which are pairs of novel enantiomeric dopamine dimers possessing a formamide group, were isolated from the two-spotted cricket, together with four other known biosynthetically related compounds (3-6). The chemical structures of 1 and 2 were elucidated using a combination of 1D and 2D NMR spectroscopic experiments and HR-ESIMS data. Compounds 1 and 2 were identified as racemic mixtures; the enantiomers (+)-1/2 and (-)-1/2 were successfully separated by utilizing a chiral HPLC column. The absolute configurations of (±)-1 and (±)-2 were unambiguously delineated by the application of quantum chemical ECD calculations. Further, these insect-derived substances were evaluated to understand their effects on cytokine expression in adipocytes. Treatment with (-)-1, (+)-2, and (-)-2 during adipocyte differentiation significantly promoted the expression of Leptin and IL-6, which resembles the actions of dopamine.

The transcriptional repressor Blimp-1 acts downstream of BMP signaling to generate primordial germ cells in the cricket Gryllus bimaculatus.

Segregation of the germ line from the soma is an essential event for transmission of genetic information across generations in all sexually reproducing animals. Although some well-studied systems such as Drosophila and Xenopus use maternally inherited germ determinants to specify germ cells, most animals, including mice, appear to utilize zygotic inductive cell signals to specify germ cells during later embryogenesis. Such inductive germ cell specification is thought to be an ancestral trait of Bilateria, but major questions remain as to the nature of an ancestral mechanism to induce germ cells, and how that mechanism evolved. We previously reported that BMP signaling-based germ cell induction is conserved in both the mouse Mus musculus and the cricket Gryllus bimaculatus, which is an emerging model organism for functional studies of induction-based germ cell formation. In order to gain further insight into the functional evolution of germ cell specification, here we examined the Gryllus ortholog of the transcription factor Blimp-1 (also known as Prdm1), which is a widely conserved bilaterian gene known to play a crucial role in the specification of germ cells in mice. Our functional analyses of the Gryllus Blimp-1 ortholog revealed that it is essential for Gryllus primordial germ cell development, and is regulated by upstream input from the BMP signaling pathway. This functional conservation of the epistatic relationship between BMP signaling and Blimp-1 in inductive germ cell specification between mouse and cricket supports the hypothesis that this molecular mechanism regulated primordial germ cell specification in a last common bilaterian ancestor.

Evolution of the neural sex-determination system in insects: does fruitless homologue regulate neural sexual dimorphism in basal insects?

In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex-specific gene products under the control of the sex-specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex-specific gene products of fruitless homologues have been identified in other holometabolous insects such as mosquitoes and a parasitic wasp, suggesting the fruitless-dependent neural sex-determination system is widely conserved amongst holometabolous insects. However, it remains obscure whether the fruitless-dependent neural sex-determination system is present in basal hemimetabolous insects. To address this issue, identification, characterization, and expression analyses of the fruitless homologue were conducted in the two-spotted cricket, Gryllus bimaculatus, as a model hemimetabolous insect. The Gryllus fruitless gene encodes multiple isoforms with a unique zinc finger domain, and does not encode a sex-specific gene product. The Gryllus Fruitless protein is broadly expressed in the neurones and glial cells in the brain, and there was no prominent sex-related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex-determination in the cricket brain.

Function of myosuppressin in regulating digestive function in the two-spotted cricket, Gryllus bimaculatus.

Myosuppressin is one of essential peptides controlling biological processes including feeding behavior. Here we identified and characterized the cDNAs that encode myosuppressin precursor and its receptor in the two-spotted cricket Gryllus bimaculatus. The presence of the mature peptide (Grybi-MS) was confirmed by direct measurement of adult brain. RT-PCR revealed the tissue distribution of these transcripts; myosuppressin is expressed predominantly in the brain and central nervous system, whereas its receptor is ubiquitously expressed in the cricket body. To address the function of Grybi-MS, we performed several bioassays to test concerning feeding behavior and digestive function upon exposure to Grybi-MS. Administration of synthetic Grybi-MS resulted in increased feeding motivation, accompanied by an increase in food intake. Meanwhile, the hemolymph lipid and carbohydrate titers were both elevated after Grybi-MS injection. As the intestinal contraction is significantly inhibited by the exposure to Grybi-MS, the upregulating feeding index might be complicated in the cricket body. The current data indicate that Grybi-MS modulates feeding behavior to control the physiological processes in the cricket.

TGF-ß signaling in insects regulates metamorphosis via juvenile hormone biosynthesis.

Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.

Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus.

Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression.