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.

Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus.

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-ß-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Changes in mitochondrial function parallel life history transitions between flight and reproduction in wing polymorphic field crickets.

Mitochondria serve as critical producers of both cellular energy and metabolic precursors for biosynthesis required for organismal growth, activity, somatic maintenance, and reproduction. Consequently, variation in mitochondrial function is commonly associated with variation in life histories both within and across species. For instance, flight-capable, long-winged crickets have mitochondria with larger bioenergetic capacities than flightless, short-winged crickets investing in early lifetime fecundity instead of flight. However, we do not know whether differences in mitochondrial function associated with life history are fixed or result from flexible changes in metabolism throughout the life cycle. We measured mitochondrial function of fat body tissue across early adulthood of long-winged and short-winged crickets from two species of wing-polymorphic field crickets (Gryllus firmus and Gryllus lineaticeps). Fat body is a multifunctional organ that supports both flight and reproduction in insects. Consistent with flexibility in mitochondrial function specific for alternative life histories, the capacity for oxidative phosphorylation increases in mitochondria throughout early adulthood in the fat body of long-winged but not short-winged crickets. Furthermore, fat body mitochondrial oxidative phosphorylation capacities declined rapidly when long-wing crickets degraded their flight muscles and initiated large-scale oogenesis. This finding suggests that shifts in tissue function require a concurrent shift in mitochondrial function and that tissue-specific functional constraints may underpin the flight-oogenesis trade-off. In conclusion, changes in mitochondrial bioenergetics form a component of alternative life histories, indicating that mitochondrial function is dynamic and set to a level that matches current and future energy demands and biosynthetic requirements of life history.

Influence of protein source (cricket, pea, whey) on amino acid bioavailability and activation of the mTORC1 signaling pathway after resistance exercise in healthy young males.

PURPOSE: New dietary proteins are currently introduced to replace traditional animal protein sources. However, not much is known about their bioaccessibility and ability to stimulate muscle protein synthesis compared to the traditional protein sources. We aimed to compare effects of ingesting a protein bolus (0.25 g/kg fat free mass) of either cricket (insect), pea, or whey protein on circulating amino acid levels and activation of the mTORC1 signaling pathway in the skeletal muscle at rest and after exercise. METHODS: In a randomized parallel controlled trial, young males (n = 50) performed a one-legged resistance exercise followed by ingestion of one of the three protein sources. Blood samples were collected before and in the following 4 h after exercise. Muscle biopsies were obtained at baseline and after 3 h from the non-exercised and exercised leg. RESULTS: Analysis of blood serum showed a significantly higher concentration of amino acids after ingestion of whey protein compared to cricket and pea protein. No difference between protein sources in activation of the mTORC1 signaling pathway was observed either at rest or after exercise. CONCLUSION: Amino acid blood concentration after protein ingestion was higher for whey than pea and cricket protein, whereas activation of mTORC1 signaling pathway at rest and after exercise did not differ between protein sources. TRIAL REGISTRATION NUMBER: Clinicaltrials.org ID NCT04633694.

Changes in purine and uric acid content in edible insects during culinary processing.

Recently, edible insects were proposed to be promising alternative foods combining nutritional, environmental, and economic benefits. While composition of the basic nutrients of insects is quite well known, little is known about other compounds contained in them such as purines. From this point of view, raw insects were reported to belong among purine-rich foods. However, they are generally consumed after culinary processing, which is known to affect nutritional composition of foods. Therefore, we aimed to analyze the effect of culinary processing (including various combinations of boiling, roasting, blanching, baking, and oven drying) on purine (adenine, guanine, xanthine, and hypoxanthine) contents and their metabolite (uric acid) in three insects (Tenebrio molitor, Gryllus assimilis, and Acheta domesticus) fit for human consumption using RP-HPLC with UV detection. According to obtained data, boiling for 15 min significantly reduced the purine content in T. molitor but did not affect the purine levels in A. domesticus and G. assimilis. In contrast, the purine content increased in all insects after baking (especially at 220 °C). The information this study provides can help people suffering from gout interested in entomophagy to choose the best culinary treatment of insects to help prevent gout symptoms.

Constant Light, Pdp1, and Tim Exert Influence on Free-Running Period of Locomotor Rhythms in the Cricket Gryllus bimaculatus.

Most insects show circadian rhythms of which the free-running period changes in a light-dependent manner and is generally longer under constant light (LL) than under constant dark conditions in nocturnal animals. However, the mechanism underlying this LL-dependent period change remains unclear. Here, using the cricket Gryllus bimaculatus, we examined the effects of long-term LL exposure on the free-running period of locomotor rhythms. Initially, the free-running period was considerably longer than 24 h but it gradually became shorter during long-term exposure to LL. The initial lengthening and ensuing gradual shortening under long-term LL exposure were observed even after unilateral removal of the optic lobe. Thus, these changes in the free-running period could be attributable to a single optic lobe clock. RNA interference (RNAi)-mediated silencing of the clock genes Par domain protein 1 (Pdp1) and timeless (tim) revealed that the treatments eliminated the initial period lengthening by LL without reducing circadian photoreceptor gene expression. However, they did not affect the period shortening during long-term LL exposure. The slopes of the regression line for the period change during long-term LL for Pdp1RNAi-treated and timRNAi-treated crickets were not different from that of the dsDsRed2-treated control. These results suggest that the initial period lengthening after transfer to LL requires tim and Pdp1, while the ensuing period shortening during long-term LL exposure is caused by a mechanism independent of tim and Pdp1.

Assessing Potential Perfluoroalkyl Substances Trophic Transfer to Crickets (Acheta domesticus).

Although many studies have assessed the bioaccumulation of perfluoroalkyl substances (PFAS) in plant tissues, to date there has been minimal research on the bioaccumulation of PFAS in soil invertebrates that results from consuming PFAS-contaminated media. The present study focused on two different consumption pathways in a population of crickets: individuals consuming PFAS-contaminated alfalfa and individuals consuming PFAS-spiked drinking water. Alfalfa was grown in a greenhouse and irrigated with PFAS-spiked water (∼1 ppm) containing seven unique PFAS. The alfalfa was then harvested and fed to crickets. Another population of crickets was supplied with PFAS-spiked drinking water at similar concentrations to irrigation water for direct consumption. Alfalfa accumulation of PFAS and subsequent consumption by the crickets resulted in overall similar tissue concentrations in the crickets who consumed PFAS-spiked water directly. This indicates that source concentration (water) may be an important factor in assessing the bioaccumulation of PFAS in organisms. To our knowledge, ours is the first study not only to assess the direct trophic transfer of PFAS from contaminated vegetation to invertebrates, but also to highlight the similarities in bioaccumulation regardless of ingestion pathway. Environ Toxicol Chem 2022;41:2981-2992. © 2022 SETAC.

Chitin Isolation and Chitosan Production from House Crickets (Acheta domesticus) by Environmentally Friendly Methods.

Alternative methods were evaluated for chitin isolation from Acheta domesticus. Chemical demineralization was compared to fermentation with Lactococcus lactis, citric acid treatment, and microwave treatment, leading to a degree of demineralization of 91.1 ± 0.3, 97.3 ± 0.8, 70.5 ± 3.5, and 85.8 ± 1.3%, respectively. Fermentation with Bacillus subtilis, a deep eutectic solvent, and enzymatic digestion were tested for chitin isolation, generating materials with less than half the chitin content when compared to alkaline deproteinization. Chitosan was produced on a large scale by deacetylation of the chitinous material obtained from two selected processes: the chemical treatment and an alternative process combining L. lactis fermentation with bromelain deproteinization. The chemical and alternative processes resulted in similar chitosan content (81.9 and 88.0%), antioxidant activity (59 and 49%), and degree of deacetylation (66.6 and 62.9%), respectively. The chitosan products had comparable physical properties. Therefore, the alternative process is appropriate to replace the chemical process of chitin isolation for industrial applications.

Flight capacity drives circadian patterns of metabolic rate and alters resource dynamics.

Animals must acquire, use, and allocate resources, and this balancing act may be influenced by the circadian clock and life-history strategy. Field (Gryllus) crickets exhibit two distinct life-history strategies during early adulthood-flight-capable females invest in flight muscle at a cost to ovary mass, whereas flight-incapable females instead invest solely into ovaries. In female Gryllus lineaticeps, I investigated the role of life-history strategy in resource (food) acquisition and allocation, and in circadian patterns of energy use. Flight capacity increased the standard metabolic rate (SMR) due to greater late-day SMR and flight-capable crickets exhibited greater circadian rhythmicity in SMR. Flight-capable crickets also ate less food and were less efficient at converting ingested food into body or ovary mass. Thus, investment into flight capacity reduced fecundity and the amount of resources available for allocation to other life-history traits. Given the increasing uncertainty of food availability in many global regions, work in Gryllus may clarify the important roles of food and circadian patterns in life-history evolution in a changing world.

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.

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.

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.

Effect of Three Defatting Solvents on the Techno-Functional Properties of an Edible Insect (Gryllus bimaculatus) Protein Concentrate.

Edible insects have received global attention as an alternative protein-rich food. However, their structural characteristics make them difficult to digest. To overcome this obstacle, we assessed the techno-functional properties of three protein concentrates from the cricket Gryllus bimaculatus. Freeze-dried G. bimaculatus powder was defatted using ethanol, hexene, or acetone as solvents, and the techno-functional properties (protein solubility, water and oil holding capacity, foaming properties, emulsion capacity, and gel formation) of the protein concentrates were determined. Freeze-dried G. bimaculatus powder comprised approximately 17.3% crude fat and 51.3% crude protein based on dry weight. Ethanol was the most effective solvent for reducing the fat content (from 17.30% to 0.73%) and increasing the protein content (from 51.3% to 62.5%) of the concentrate. Techno-functionality properties drastically differed according to the defatting solvent used and foaming properties were most affected. Thus, the techno-functional and whole properties must be considered for proper application of edible insects to achieve global food sustainability.

Low-Field NMR Study of Shortcake Biscuits with Cricket Powder, and Their Nutritional and Physical Characteristics.

The growing human population renders challenges for the future supply of food products with high nutritional value. Here, we enhanced the functional and nutritional value of biscuits, a popular sweet snack, by replacing the wheat flour with 2%, 6%, or 10% (w/w) cricket powder. Consumer acceptance ratings for reference and 2% augmented cookies were comparable, whereas the higher levels of enhancement received inferior consumer scores. This relatively small change in biscuit recipe provided significant and nutritionally desirable enhancements in the biscuits, observed in a series of analyses. An increase in the protein content was observed, including essential amino acids, as well as minerals and fat. This conversion also affected the physical properties of the biscuits, including hardness, and water molecular dynamics measured by 1H NMR. Cricket powder-augmented biscuits join the line of enhanced, functionally superior food products. This and similar food augmentation provide a viable scenario to meet the human food demands in the future.

Food Limitation but Not Enhanced Rates of Ejaculate Production Imposes Reproductive and Survival Costs to Male Crickets.

Estimating costs of ejaculate production is challenging. Metabolic investment in ejaculates may come at the expense of other physiological functions and may negatively affect future reproduction and/or survival. These trade-offs are especially likely to occur under constrained resource pools (e.g., poor nutrition). Here, we investigated costs of ejaculate production via trade-offs in the field cricket Gryllus bimaculatus. We experimentally increased rates of ejaculate production, while keeping an unmanipulated group, in adult males kept at high and low feeding regimes and tested the effects of our treatments on (i) somatic maintenance (i.e., changes in male body mass), (ii) future reproduction (i.e., the likelihood of producing a spermatophore and the viability of its sperm), and (iii) lifetime survival and longevity. We predicted investment in ejaculates to impinge upon all measured responses, especially in low-fed individuals. Instead, we only found negative effects of food limitation, suggesting low or undetectable costs of spermatophore production. High mating rates may select for males to maximize their capacity of ejaculate production, making ejaculate traits less prone to trade-offs with other fitness-related life history traits. Nevertheless, males were impaired due to nutrient deficiency in producing viable ejaculates, suggesting condition-dependent costs for ejaculate production.

Expression of 20-hydroxyecdysone-related genes during gonadal development of Teleogryllus emma (Orthoptera: Gryllidae).

Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.

Adaptation of codon and amino acid use for translational functions in highly expressed cricket genes.

BACKGROUND: For multicellular organisms, much remains unknown about the dynamics of synonymous codon and amino acid use in highly expressed genes, including whether their use varies with expression in different tissue types and sexes. Moreover, specific codons and amino acids may have translational functions in highly transcribed genes, that largely depend on their relationships to tRNA gene copies in the genome. However, these relationships and putative functions are poorly understood, particularly in multicellular systems. RESULTS: Here, we studied codon and amino acid use in highly expressed genes from reproductive and nervous system tissues (male and female gonad, somatic reproductive system, brain and ventral nerve cord, and male accessory glands) in the cricket Gryllus bimaculatus. We report an optimal codon, defined as the codon preferentially used in highly expressed genes, for each of the 18 amino acids with synonymous codons in this organism. The optimal codons were mostly shared among tissue types and both sexes. However, the frequency of optimal codons was highest in gonadal genes. Concordant with translational selection, a majority of the optimal codons had abundant matching tRNA gene copies in the genome, but sometimes obligately required wobble tRNAs. We suggest the latter may comprise a mechanism for slowing translation of abundant transcripts, particularly for cell-cycle genes. Non-optimal codons, defined as those least commonly used in highly transcribed genes, intriguingly often had abundant tRNAs, and had elevated use in a subset of genes with specialized functions (gametic and apoptosis genes), suggesting their use promotes the translational upregulation of particular mRNAs. In terms of amino acids, we found evidence suggesting that amino acid frequency, tRNA gene copy number, and amino acid biosynthetic costs (size/complexity) had all interdependently evolved in this insect model, potentially for translational optimization. CONCLUSIONS: Collectively, the results suggest a model whereby codon use in highly expressed genes, including optimal, wobble, and non-optimal codons, and their tRNA abundances, as well as amino acid use, have been influenced by adaptation for various functional roles in translation within this cricket. The effects of expression in different tissue types and the two sexes are discussed.

Biochemically identified neuropeptides in a caddisfly (Trichoptera) and a pygmy mole cricket (Orthoptera: Caelifera: Tridactyloidea).

One representative of the order Trichoptera, namely the caddisfly Chaetopteryx villosa, was investigated along with the pygmy mole cricket Xya capensis which is a representative of the most basal superfamily of the caeliferan Orthoptera, that is, the Tridactyloidea. From both clades neuropeptides have not been biochemically characterized before this study. Here, members of the adipokinetic hormone family (AKHs) are sequenced via liquid chromatography (LC)-ion trap mass spectrometry from methanolic extracts from the corpora cardiaca of respective species. The corpora cardiaca were dissected, methanolic extracts prepared, peptides separated by liquid chromatography (LC), and AKHs detected and sequenced by ion trap mass spectrometry. Both species investigated contain an octapeptide AKH: the trichopteran species has the peptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Ser-Trp amide; the ambiguity of the isobaric amino acids Leu and Ile at position two was solved by comparing retention times on LC and by co-elution with the synthetic Leu2 -form. This peptide is known as Aedae-AKH and found in certain dipteran species and in an alderfly (Megaloptera). The tridactyloid species contains the peptide with the sequence pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide which had first been identified in a member of the order Mantophasmatodea and is called Manto-CC. Comparisons are made between the AKH complements of the sister groups Trichoptera and Lepidoptera and their possible relatedness and, on the other hand, between the AKH of X. capensis with those of closely related caeliferan superfamilies. The biology of the two studied species is used to speculate about a possible function of the elucidated hormones. Lastly, the use of a larval stage as starting material for structural neuropeptide information is discussed.

Lipidome of cricket species used as food.

The variation in lipidome of house cricket, banded cricket, Jamaican field cricket and two-spotted cricket was studied using high-throughput screening techniques for fingerprinting (MALDI TOF MS, GC-MS and LC MS-MS) and well-stablished chromatographic techniques for quantification (HPLC-ELSD, GC- FID). Although the four cricket species were reared in identical conditions, two-spotted & banded crickets had a lipid content 1.5 fold higher than house cricket. The lipids were high in UFA (>63%) and unsaturated TAG (>98%) making them liquid at room temperature, thus an oil. Cholesterol and several phytosterols were profiled finding high cholesterol concentration which is a point of concern. Eight phospholipid types (211 species) were identified with no major differences among cricket species. Using high-throughput screening techniques we demonstrate the complexity of cricket lipidome. Information on the lipidome of these crickets with high commercial value is important to estimate its nutritional value and their potential food applications.