Effect of Cricket Frass Fertilizer on growth and pod production of green beans (Phaseolus vulgaris L.).

Cricket Frass Fertilizer (CFF) was tested for its efficiency and potential as a fertilizer on the growth of green beans (Phaseolus vulgaris L.) in central Madagascar from April 2020 to October 2020. We grew green beans experimentally for 93 days with seven different fertilizer treatments: NPK 200 kg/ha (0.47 g of N/plant), GUANOMAD (guano from bat) 300 kg/ha (0.26 g of N/ plant), CFF 100 kg/ha (0.12 g of N/plant), CFF 200 kg/ha (0.24 g of N/plant), CFF 300 kg/ha (0.38 g of N/plant), CFF 400 kg/ha (0.52 g of N/plant), and no fertilizer (0 g of N/plant). Three plant traits were measured: survival proportion, vegetative biomass, and pod biomass. The survival proportion of plants treated with the highest dose of CFF (400 kg/ha, 88.1%), NPK (79.8%), and GUANOMAD (81.2%) were similar, but plants treated with the former yielded significantly higher vegetative (35.5 g/plant) and pod biomass (11 g/plant). These results suggest that fertilizing green beans with CFF at a 400 kg/ha dose is sufficient for plant survival and growth, and improves pod production. In Madagascar where soil quality is poor, dependence on imported chemical fertilizers (NPK) and other organic fertilizer (GUANOMAD) can be reduced. Cricket Frass Fertilizer can be used as an alternative sustainable fertilizer for beans.

Impact of temperature on the bionomics and geographical range margins of the two-spotted field cricket Gryllus bimaculatus in the world: Implications for its mass farming.

Gryllus bimaculatus (Orthoptera: Gryllidae) is widely considered an excellent nutrient source for food and feed. Despite its economic importance, there is limited information on the impact of temperature on the bionomics of this cricket to guide its effective and sustainable mass production in its geographical range. The biological parameters of G. bimaculatus were investigated at eight different temperatures ranging from 20-40˚C. The Insect Life-Cycle Modelling (ILCYM) program was used to fit linear and non-linear functions to the data to describe the influence of temperature on life history parameters and its farmability under the current and projected climate for 2050. Our results revealed that G. bimaculatus was able to complete its lifecycle in the temperature range of 20°C to 37°C with a maximum finite rate of population increase (= 1.14) at 35°C. The developmental time of G. bimaculatus decreased with increasing temperature. The least developmental time and mortality were attained at 32°C. The highest wet length and mass of G. bimaculatus occurred at 32°C. The lowest temperature threshold for G. bimaculatus egg and nymph development was approximated using linear regression functions to be at 15.9°C and 16.2°C with a temperature constant of 108.7 and 555.6 degree days. The maximum fecundity (2301.98 eggs per female), net reproductive rate (988.42 daughters/ generation), and intrinsic rate of natural increase (0.134 days) were recorded at 32°C and the shortest doubling of 5.2 days was observed at 35°C. Based on our findings G. bimaculatus can be farmed in countries with temperatures ranging between 20 and 37°C around the globe. These findings will help the cricket farmers understand and project the cricket population dynamics around the world as influenced by temperature, and as such, will contribute to more efficient farming.

The effects of high-monosaccharide diets on development and biochemical composition of white-eyed mutant strain of house cricket (Acheta domesticus).

Tryptophan (TRP) is one of the essential amino acids in the animal body. Its exogenicity and low concentrations mean that it can be regarded as one of the key regulatory molecules at the cellular as well as physiological level. It has been shown to have a number of essential functions, such as in the production of other biologically active molecules. The main objective of this project was to investigate the effects of a high monosaccharide diet (HMD) on a hemimetabolic insect-house cricket (Acheta domesticus) and a mutant strain with impaired visual pigment synthesis (closely related to the tryptophan and kynurenine (KYN) metabolic pathway)-white eye. This study was aimed at determining the effects of glucose and fructose on cricket development and biochemical composition. A parallel goal was to compare the response of both cricket strains to HMD. ELISA assays indicated dysfunction of the TRP-KYN pathway in white strain insects and an elevated KYN/TRP ratio. Biochemical analyses demonstrated the effects of HMD mainly on fat and glycogen content. A decrease in food intake was also observed in the groups on HMD. However, no changes in imago body weight and water content were observed. The results of the study indicate a stronger response of the white strain to HMD compared to the wild-type strain. At the same time, a stronger detrimental effect of fructose than of glucose was apparent. Sex was found to be a modulating factor in the response to HMD.

Is there life after parasitism? Survival, longevity, and oogenesis in Acheta domesticus (Orthoptera: Gryllidae) infected with the hairworm, Paragordius varius (Phylum: Nematomorpha).

The costs parasites impose on hosts can lead to reductions in survival and fecundity, but few studies have evaluated the impacts after infection. Hairworms are parasites of terrestrial arthropods that are free-living in aquatic systems as adults. As parasitic juveniles, hairworms acquire nutrients from their definitive hosts, shifting resources away from host development to parasite growth. However, until now, only one study has examined survivorship of naturally infected hosts with hairworms. Using a different hairworm and host system, we conducted experimental infections to examine growth, survivorship, and egg production in virgin female Acheta domesticus infected with the hairworm, Paragordius varius. We found that infected crickets grew significantly less during hairworm development compared to sham-infected control crickets. After releasing their worms, infected crickets survived for 73 ± 32 days but had significantly shorter life spans by an average of 13 days compared to sham-infected control crickets. However, we found that 50% of previously infected crickets produced eggs after releasing their worms. Taken together, these observations suggest that female crickets infected with hairworms may experience less mortality than previous anecdotal evidence suggests. Finally, we discuss the definition of parasitoid and how it relates to nematomorphs, and we suggest that more field and laboratory research is required before suggesting hairworms are parasitoids.

Sex-specific associations between life-history traits and a novel reproductive polymorphism in the Pacific field cricket.

Associations between heritable polymorphisms and life-history traits, such as development time or reproductive investment, may play an underappreciated role in maintaining polymorphic systems. This is because selection acting on a particular morph could be bolstered or disrupted by correlated changes in life history or vice versa. In a Hawaiian population of the Pacific field cricket (Teleogryllus oceanicus), a novel mutation (flatwing) on the X-chromosome is responsible for a heritable polymorphism in male wing structure. We used laboratory cricket colonies fixed for male wing morph to investigate whether males and females bearing the flatwing or normal-wing (wild-type) allele differed in their life-history traits. We found that flatwing males developed faster and had heavier testes than normal-wings, whereas flatwing homozygous females developed slower and had lighter reproductive tissues than normal-wing homozygous females. Our results advance our understanding of the evolution of polymorphisms by demonstrating that the genetic change responsible for a reproductive polymorphism can also have consequences for fundamental life-history traits in both males and females.

High-Stakes Decision-Making by Female Crickets (Gryllus texensis): When to Trade In Wing Muscles for Eggs.

AbstractResource-intensive traits, such as dispersal and reproduction, can be difficult to express simultaneously because of resource limitations. One solution is to switch between resource-intensive behaviors. Such phenotypic plasticity is one strategy that organisms use to funnel resources from one expensive trait to another. In crickets (Gryllus texensis), the development and maintenance of flight muscles reduce resource availability for reproduction, leading to physiological trade-offs between the two traits. Long-winged female G. texensis can histolyze their wing muscles, resulting in increased egg production, but they can then no longer fly. Using a diet that mimics food availability in the field, we found that long-winged females adopted one of the three following strategies: early reproduction, intermediate reproduction, and late reproduction. Some late reproducers maintained their flight capability until the end of their natural life span and laid few eggs. If females lost the ability to fly (i.e., their hind wings are removed), they laid eggs earlier, leading to increased reproductive output. However, other environmental cues (e.g., an increased number of mates, increased oviposition substrate quality, or a bout of dispersal flight) had no effect. Late-reproducing females laid 96% fewer eggs than early reproducers, suggesting that late reproduction exacts a huge fitness cost. Nevertheless, some females maintain their flight muscles to the end of their natural life span in both the lab and the field. We suggest that the ability to fly allows for bet hedging against an environmental catastrophe (e.g., drought or flood). This benefit may help explain the persistence of late-reproducing long-winged females, despite the cost of this choice. As climate change increases drought and flood in Texas, late dispersal may be one factor that helps this species survive in the future. An increased understanding of factors that maintain seemingly low fitness strategies can help us predict the resilience of species under climate change.

Reversing sodium differentials between the hemolymph and hindgut speeds chill coma recovery but reduces survival in the fall field cricket, Gryllus pennsylvanicus.

Chill-susceptible insects enter the reversible state of chill coma at their critical thermal minimum (CTmin). During chill coma, movement of Na+ and water from the hemolymph to the gut lumen disrupt ion and water balance. Recovery from cold exposure requires re-establishment of this balance, and failure to do so results in chilling injury or death. We hypothesized that the passive leak of Na+ and consequently water during cold exposure is driven by the [Na+] differential between the gut and hemolymph. To determine the extent to which this [Na+] differential affects cold tolerance, we used artificial diets to load the guts of fall field crickets (Gryllus pennsylvanicus) with various concentrations of Na+. Manipulating [Na+] differentials had no effect on the CTmin, agreeing with recent studies demonstrating that chill coma onset precedes loss of ion balance in the cold. A high [Na+] diet reversed the direction of the [Na+] differential between the gut and hemolymph. Crickets fed a high [Na+] diet recovered from 12 h of chill coma nearly twice as fast as those fed low [Na+] diets. However, the high [Na+] diet was detrimental to survival after prolonged cold exposure (three days at 0 °C). Therefore, while a reduced [Na+] differential helps crickets recover from short-term cold exposure, an increased gut Na+ load itself appears to carry longer-term costs and promotes irreversible chilling injury.

Effect of Diet on the Growth Performance, Feed Conversion, and Nutrient Content of the House Cricket.

The house crickets, Acheta domesticus, are sustainable and nutritious future sources of food, due to their nutritional benefits, particular high protein content and potential in solving global malnutrition. Different diets, particularly protein content, can influence the growth and nutritional value of crickets. The aim of this present study was to evaluate the effects of commercial diets and other formulated diets on the nutritional composition and growth parameters of the house crickets, being a major challenge to cricket's farmers in Thailand. Feed conversion ratio were 1.50, 1.50, and 1.51 for fed crickets on a blend of 22% protein and dry pulp pumpkin powder, fed 22% protein plus fresh pumpkin pulp, and fed 22% protein alone, indicated that these groups are high feed convertors and represented the quality of these diets compared to 1.73 and 1.81 for fed crickets on a blend of 22% and 16% protein, and those fed on 16% protein alone. Fed crickets on 22% protein had the highest amount of protein (76%), the lowest (48%) in those fed on 22% protein and fresh pumpkin pulp inclusion. The group on 22% protein diet also had the highest amount of phosphorus, potassium, calcium, and sodium. Fed 22% protein and either dry pulp pumpkin powder or fresh pumpkin pulp condition have shown improvement in vitamin B content. Crickets can effectively be produced on 22% protein diet to improve yield output and several minerals such as phosphorus, potassium, calcium, and sodium. In contrast, the supplementation of 22% protein diet with pumpkin (Cucurbita maxima) will improve vitamin B content.

Photoperiod and temperature separately regulate nymphal development through JH and insulin/TOR signaling pathways in an insect.

Insects living in the temperate zone enter a physiological state of arrested or slowed development to overcome an adverse season, such as winter. Developmental arrest, called diapause, occurs at a species-specific developmental stage, and embryonic and pupal diapauses have been extensively studied in mostly holometabolous insects. Some other insects overwinter in the nymphal stage with slow growth for which the mechanism is poorly understood. Here, we show that this nymphal period of slow growth is regulated by temperature and photoperiod through separate pathways in the cricket Modicogryllus siamensis The former regulates the growth rate, at least in part, through the insulin / target of rapamycin (TOR) signaling pathway. Lower temperature down-regulates the expression of insulin-like peptide (Ms'Ilp) and Target of rapamycin (Ms'Tor) genes to slow down the growth rate without affecting the number of molts. The latter regulates the number of molts independent of temperature. Short days increase the number of molts through activation of the juvenile hormone (JH) pathway and down-regulation of myoglianin (Ms'myo), a member of the TGFß family, which induces adult metamorphosis. In contrast, long days regulate Ms'myo expression to increase during the fifth to sixth instar to initiate adult metamorphosis. When Ms'myo expression is suppressed, juvenile hormone O-methyl transferase (Ms'jhamt) was up-regulated and increased molts to prolong the nymphal period even under long-day conditions. The present findings suggested that the photoperiod regulated Ms'myo, and the JH signaling pathway and the temperature-controlled insulin/TOR pathway cooperated to regulate nymphal development for overwintering to achieve seasonal adaptation of the life cycle in M. siamensis.

Transcriptome analysis of life stages of the house cricket, Acheta domesticus, to improve insect crop production.

To develop genetic resources for the improvement of insects as food, we sequenced transcripts from embryos, one-day hatchlings, three nymphal stages, and male and female adults of the house cricket, Acheta domesticus. A draft transcriptome was assembled from more than 138 million sequences combined from all life stages and sexes. The draft transcriptome assembly contained 45,866 contigs, and more than half were similar to sequences at NCBI (e value < e-3). The highest sequence identity was found in sequences from the termites Cryptotermes secundus and Zootermopsis nevadensis. Sequences with identity to Gregarina niphandrodes suggest that these crickets carry the parasite. Among all life stages, there were 5,042 genes with differential expression between life stages (significant at p < 0.05). An enrichment analysis of gene ontology terms from each life stage or sex highlighted genes that were important to biological processes in cricket development. We further characterized genes that may be important in future studies of genetically modified crickets for improved food production, including those involved in RNA interference, and those encoding prolixicin and hexamerins. The data represent an important first step in our efforts to provide genetically improved crickets for human consumption and livestock feed.

Emergence phenology and temperature effect on the post-diapause egg development in the bush cricket Barbitistes vicetinus (Orthoptera, Tettigoniidae).

The tettigoniid Barbitistes vicetinus Galvagni & Fontana was described in 1993 as an endemic and rare bush-cricket of north-east Italy. Since 2008, this species has become a pest, causing repeated outbreaks with severe defoliations in broadleaf forests and neighbouring crops. Few data are currently available on ecology and life-cycle of this species, in particular about how temperature regulates egg-diapause and hatching phenology. The present work reports a field study regarding hatching phenology of B. vicetinus, surveyed with 84 emergence traps over four consecutive years (2013-2016). Moreover, the effect of temperature on the hatching was tested in the laboratory, exposing eggs to different temperatures. Field observations showed that hatching occurred between the end of March and beginning of April. In warmer years, hatching started early in the spring and lasted longer, while in colder years, hatching started later and was concentrated in a few days. Moreover, a significant effect of both elevation and exposure on the hatching start was observed. Results obtained from laboratory suggested the ability of the species to develop in the post-final diapause in a wide range of thermal conditions. After the diapause (terminated by a prolonged common exposure to low temperature) B. vicetinus was able to hatch from 6 to 23°C although, due to prolonged post-diapause development, hatching took place progressively later at colder temperatures. To manage B. vicetinus outbreaks, forecasting the seasonal phenology of egg hatching and its duration is important for an effective pest control.

Characterization of plexinA and two distinct semaphorin1a transcripts in the developing and adult cricket Gryllus bimaculatus.

Guidance cues act during development to guide growth cones to their proper targets in both the central and peripheral nervous systems. Experiments in many species indicate that guidance molecules also play important roles after development, though less is understood about their functions in the adult. The Semaphorin family of guidance cues, signaling through Plexin receptors, influences the development of both axons and dendrites in invertebrates. Semaphorin functions have been extensively explored in Drosophila melanogaster and some other Dipteran species, but little is known about their function in hemimetabolous insects. Here, we characterize sema1a and plexA in the cricket Gryllus bimaculatus. In fact, we found two distinct predicted Sema1a proteins in this species, Sema1a.1 and Sema1a.2, which shared only 48% identity at the amino acid level. We include a phylogenetic analysis that predicted that many other insect species, both holometabolous and hemimetabolous, express two Sema1a proteins as well. Finally, we used in situ hybridization to show that sema1a.1 and sema1a.2 expression patterns were spatially distinct in the embryo, and both roughly overlap with plexA. All three transcripts were also expressed in the adult brain, mainly in the mushroom bodies, though sema1a.2 was expressed most robustly. sema1a.2 was also expressed strongly in the adult thoracic ganglia while sema1a.1 was only weakly expressed and plexA was undetectable.

Regulatory mechanisms underlying the specification of the pupal-homologous stage in a hemimetabolous insect.

Juvenile hormones and the genetic interaction between the transcription factors Krüppel homologue 1 (Kr-h1) and Broad (Br) regulate the transformation of insects from immature to adult forms in both types of metamorphosis (holometaboly with a pupal stage versus hemimetaboly with no pupal stage); however, knowledge about the exact instar in which this occurs is limited. Using the hemimetabolous cricket Gryllus bimaculatus (Gb), we demonstrate that a genetic interaction occurs among Gb'Kr-h1, Gb'Br and the adult-specifier transcription factor Gb'E93 from the sixth to final (eighth) nymphal instar. Gb'Kr-h1 and Gb'Br mRNAs were strongly expressed in the abdominal tissues of sixth instar nymphs, with precocious adult moults being induced by Gb'Kr-h1 or Gb'Br knockdown in the sixth instar. The depletion of Gb'Kr-h1 or Gb'Br upregulates Gb'E93 in the sixth instar. By contrast, Gb'E93 knockdown at the sixth instar prevents nymphs transitioning to adults, instead producing supernumerary nymphs. Gb'E93 also represses Gb'Kr-h1 and Gb'Br expression in the penultimate nymphal instar, demonstrating its important role in adult differentiation. Our results suggest that the regulatory mechanisms underlying the pupal transition in holometabolous insects are evolutionarily conserved in hemimetabolous G. bimaculatus, with the penultimate and final nymphal periods being equivalent to the pupal stage. This article is part of the theme issue 'The evolution of complete metamorphosis'.

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'.

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.

The plant-based by-product diets for the mass-rearing of Acheta domesticus and Gryllus bimaculatus.

Edible insect rearing could provide one alternative for protein production by having a smaller environmental impact than traditional livestock farming due to insects' ability to convert organic side streams. Currently, the insect rearing industry utilizes soybeans as a major source of protein in the feeds. Protein-rich by-products of food industry could be used to replace them in insect feeds, but it is not known if they also meet the insects' nutritional requirements. Our study evaluated the growth performance of two widely used edible cricket species, Acheta domesticus and Gryllus bimaculatus (Orthoptera: Gryllidae), on 18 experimental diets. The experimental diets included commercial chicken feeds and cricket diets, where soybean was partly and completely replaced with by-products from food industry: potato protein, barley mash, barley feed, compressed leftover of turnip rape and mix of broad bean and pea on three levels of protein. We found that the high- and medium-protein turnip rape and barley mash diets produced the highest yield and an increase in all performance variables. Overall, the high- and medium-protein diets produced the highest yield, growth and fastest development. Our results showed that by-products of food industry could be utilized as a part of the cricket feeds and thus advance the goals of circular economy.

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.

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 µm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect.

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis. Cold-acclimated G. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis. Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo, with implications for choosing new molecules for cryopreservation.

Involvement of heat shock protein 40 in the wing dimorphism of the house cricket Acheta domesticus.

Wing dimorphism is a common phenomenon in a wide range of insect taxa. In most insects, the two morphs are macropterous and micropterous, in extreme cases of the latter, wing shedding can occur. Wing dimorphism contributes significantly to the ecological success of many insect species. However, the molecular basis of wing dimorphism is not fully understood, especially for wing-shed. Here, differentially expressed genes over eight developmental stages of the house cricket Acheta domesticus, which undergoes wing-shed dimorphism, were studied. The results revealed a wing-shed peak during adult development in which many DEGs were highly upregulated and it's influenced by cricket population density. A weighted correlation network analysis (WGCNA) grouped 21,922 DEGs among 141,456 unigenes into 18 modules of different expression patterns. The module in which the gene expression pattern correlated with the wing-shed phenotypic data was selected for further analyses with STEM and Cytoscape, and three candidate genes (AdomHSP40: Heat shock protein 40, AdomCFDP: Craniofacial development protein, AdomDIS3L: DIS3 Like 3'-5' Exoribonuclease) were identified by gene network analysis as the DEGs most relevant to wing-shed occurrence. The RNA interference of these genes together with an insulin receptor and Nylanderia fulva virus showed that the silencing of AdomHSP40 significantly decreased wing-shed occurrence, whereas the silencing of other candidate genes did not, suggesting that AdomHSP40 plays a crucial role in the wing-shed of Acheta domesticus. These findings provide insights into the molecular mechanisms underlying wing dimorphism in the house crickets, which differ from those found in other insects such as the planthopper.