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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

(±)-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.

A Novel Head Capsule Labial Gland Lobe in the Black Field Cricket (Orthoptera: Gryllidae).

We describe a pair of labial gland lobes on either side of the retrocerebral complex in the head of the Australian black field cricket, Teleogryllus commodus Walker. As the retrocerebral complex includes the corpora cardiaca and corpora allata, hormones secreted by these glands can be absorbed by these lobes. These lobes of the labial gland are connected to the thoracic lobes via a relatively long duct that enters the main duct draining the thoracic lobes. Measurement of the flow rate of dye from head to thorax in the ducts is rapid, suggesting that these glands may serve as a transport system into the thoracic region. Both serotonin and adipokinetic hormone are shown to be present in the lobes near the retrocerebral complex and the ducts of the thoracic lobes, but whether this connection between the head and thorax acts as a hormone transporter is still unclear.

Temperature Entrainment of Circadian Locomotor and Transcriptional Rhythms in the Cricket, Gryllus bimaculatus.

Most animals exhibit circadian rhythms in various physiological and behavioral functions regulated by circadian clock that resides in brain and in many peripheral tissues. Temperature cycle is an important time cue for entrainment, even in mammals, since the daily change in body temperature is thought to be used for phase regulation of clocks in peripheral tissues. However, little is known about the mechanisms by which temperature resets the clock. In the present study, we investigated the effect of temperature on circadian activity rhythm and clock gene transcription by using the cricket, Gryllus bimaculatus. We show that temperature cycle can entrain both behavioral and transcriptional rhythms of clock genes, such as period, timeless, cryptochrome2 and cycle in the circadian pacemaker tissue, optic lobe. Under temperature cycle, phase of evening peak of locomotor activity occurred 1 h before the warm-to-cold phase transition, which is associated with earlier peaks of mRNA expression rhythm of the clock genes than that under light/dark cycles. When the temperature cycle was advanced by 6 h, behavioral rhythms re-entrained to newly phased temperature cycle after ∼16 transient cycles. The mRNA oscillation of period and timeless gained stable rhythm under phase advanced temperature cycles with a lesser number of transient cycles than cryptochrome2 and cycle. These results suggest that temperature cycle can entrain behavioral and molecular rhythms in cricket and clock genes vary in sensitivity to temperature. It is thus likely that clock genes play differential roles in resetting the clock with environmental temperature changes.

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.

Bone morphogenetic protein signaling in distal patterning and intercalation during leg regeneration of the cricket, Gryllus bimaculatus.

The cricket, Gryllus bimaculatus, is a classic model of leg regeneration following amputation. We previously demonstrated that Gryllus decapentaplegic (Gb'dpp) is expressed during leg regeneration, although it remains unclear whether it is essential for this process. In this study, double-stranded RNA targeting the Smad mathers-against-dpp homolog, Gb'mad, was used to examine the role of bone morphogenetic protein (BMP) signaling in the leg regeneration process of Gryllus bimaculatus. RNA interference (RNAi)-mediated knockdown of Gb'mad led to a loss of tarsus regeneration at the most distal region of regenerating leg segments. Moreover, we confirmed that the phenotype obtained by knockdown of Dpp type I receptor, Thick veins (Gb'tkv), closely resembled that observed for Gb'mad RNAi crickets, thereby suggesting that the BMP signaling pathway is indispensable for the initial stages of tarsus formation. Interestingly, knockdown of Gb'mad and Gb'tkv resulted in significant elongation of regenerating tibia along the proximodistal axis compared with normal legs. Moreover, our findings indicate that during the regeneration of tibia, the BMP signaling pathway interacts with Dachsous/Fat (Gb'Ds/Gb'Ft) signaling and dachshund (Gb'dac) to re-establish positional information and regulate determination of leg size. Based on these observations, we discuss possible roles for Gb'mad in the distal patterning and intercalation processes during leg regeneration in Gryllus bimaculatus.

Genotype-by-sex-by-diet interactions for nutritional preference, dietary consumption, and lipid deposition in a field cricket.

Changes in feeding behaviour, especially the overconsumption of calories, has led to a rise in the rates of obesity, diabetes, and other associated disorders in humans and a range of animals inhabiting human-influenced environments. However, understanding the relative contribution of genes, the nutritional environment, and their interaction to dietary intake and lipid deposition in the sexes still remains a major challenge. By combining nutritional geometry with quantitative genetics, we determined the effect of genes, the nutritional environment, and their interaction on the total nutritional preference (TP), total diet eaten (TE), and lipid mass (LM) of male and female black field crickets (Teleogryllus commodus) fed one of four diet pairs (DPs) differing in the ratio of protein to carbohydrate and total nutritional content. We found abundant additive genetic variance for TP, TE, and LM in both sexes and across all four DPs, with significant genetic correlations between TE and TP and between TP and LM in males. We also found significant genotype-by-DP and genotype-by-sex-by-DP interactions for each trait and significant genotype-by-sex interactions for TE and LM. Complex interactions between genes, sex, and the nutritional environment, therefore, play an important role in nutrient regulation and lipid deposition in T. commodus. This finding may also help explain the increasing rate of obesity and the maintenance of sex differences in obesity observed across many animal species, including humans.

Dietary antioxidants, but not courtship effort, affect oxidative balance in the testes and muscles of crickets.

Recent interest has focused on the role of reactive oxygen species (ROS) as universal constraints in life-history evolution. Empirical studies have examined the oxidative costs of reproduction for females, with little work conducted on males. The male germline is thought to be particularly susceptible to oxidative damage because the testes, and the sperm themselves, can be prolific producers of ROS. We tested the hypothesis that protection of the male germline from oxidative damage represents a cost of reproduction for males. We fed male crickets, Teleogryllus oceanicus, with one of two experimental diets in which we manipulated the availability of dietary antioxidants, and we induced variation in their expenditure on courtship effort by manipulating access to females. We measured the total antioxidant capacity, levels of ROS production and the amount of oxidative damage to proteins in both testis and thoracic muscle tissues. Dietary antioxidants contributed to positive oxidative balance in both tissue types. Although the testes had greater antioxidant defences than muscle tissue, they also produced considerably higher levels of ROS and sustained higher levels of oxidative damage. Courtship effort had no impact on any measure of oxidative balance. Our data confirm that the male germline is especially susceptible to oxidative stress and that dietary antioxidants can alleviate this oxidative cost of reproduction.