Peroxiredoxin 1 transfer during mating protects eupyrene sperm against oxdative stress in Grapholita molesta.

BACKGROUND: Each Grapholita molesta female only copulates once during its lifetime and thus must maintain the viability of stored eupyrene sperm for male reproductive success. The male ejaculate comprises abundant accessory gland proteins produced by the male accessory gland (AG), and many of which are major effectors for sperm storage and maintenance. RESULTS: Here, we reported that an antioxidant protein, peroxiredoxin 1 (GmolPrx1), secreted by the male AG, is essential for protecting eupyrene sperm from oxidative stress and maintaining their quality during storage in the female bursa copulatrix (BC). Our data showed that GmolPrx1 is highly expressed in the AG of sexually mature males. The GmolPrx1 protein is localized to the cytoplasm of AG cells and delivered to the female BC during mating. Knockdown of GmolPrx1 strongly decreased the fertility of mated females. Additionally, we evaluated oxidative status in the spermatophore of females and found that the content of hydrogen peroxide increased significantly after mating with GmolPrx1 knockdown males. Finally, the quality assessment of eupyrene sperm demonstrated that the plasma membrane integrity, acrosome integrity, and DNA integrity were all severely impaired in the spermatophore of females after mating with GmolPrx1 knockdown males, which may contribute to the fertility decline in males. CONCLUSION: Our current data demonstrated that activities of eupyrene sperm stored in females can be significantly impaired by enhanced oxidative stress through knocking down of GmolPrx1 in males. Our finding thus may further lay new foundations for the control of G. molesta through suppressing their populations by manipulating male reproductive genes. © 2023 Society of Chemical Industry.

Molecular Identification of Two Thioredoxin Genes From Grapholita molesta and Their Function in Resistance to Emamectin Benzoate.

Thioredoxins (Trxs), a member of the thioredoxin system, play crucial roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative stress. In this study, we cloned and characterized two genes, GmTrx2 and GmTrx-like1, from Grapholita molesta. Sequence analysis showed that GmTrx2 and GmTrx-like1 had highly conserved active sites CGPC and CXXC motif, respectively, and shared high sequence identity with selected insect species. The quantitative real-time polymerase chain reaction results revealed that GmTrx2 was mainly detected at first instar, whereas GmTrx-like1 was highly concentrated at prepupa day. The transcripts of GmTrx2 and GmTrx-like1 were both highly expressed in the head and salivary glands. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides (emamectin benzoate). We further detected interference efficiency of GmTrx2 and GmTrx-like1 in G. molesta larvae and found that peroxidase capacity, hydrogen peroxide content, and ascorbate content all increased after knockdown of GmTrx2 or GmTrx-like1. Furthermore, the hydrogen peroxide concentration was increased by emamectin benzoate and the sensitivity for larvae to emamectin benzoate was improved after GmTrx2 or GmTrx-like1 was silenced. Our results indicated that GmTrx2 and GmTrx-like1 played vital roles in protecting G. molesta against oxidative damage and also provided the theoretical basis for understanding the antioxidant defense mechanisms of the Trx system in insects.

Functional analysis by RNAi of an glutaredoxin gene in Helicoverpa armigera.

Glutaredoxins play crucial roles in maintaining intracellular redox homeostasis via scavenging of excess reactive oxygen species. In this study, a glutaredoxin domain-containing cysteine-rich gene from Helicoverpa armigera, named HaGdccr, was characterized. Sequence analysis revealed that it contains a glutaredoxin domain and a conserved cysteine and shares high sequence identity with other insect genes. HaGdccr mRNA expression was highest in molting larvae of the 3rd instar and was mainly detected in the central nervous system of larvae and the wings of adults. Quantitative real-time PCR results revealed that the expression of HaGdccr was suppressed at 1 and 6 h and increased at 24 h after the larvae were treated with 4 °C and hydrogen peroxide. When the larvae were exposed to 20 °C, HaGdccr decreased at 1 h and was induced at 12 and 24 h. HaGdccr transcription level was downregulated at 2 and 12 h and upregulated at 24 h after the adults were exposed to 0 °C. However, transcript levels were increased by high temperature in both larvae and adults. After knockdown of HaGdccr by RNA interference, the expression of antioxidant genes, including thioredoxin-like (Trx-like), catalase (CAT), glutathione-S-transferase (GST), thioredoxin reductase (TrxR), and thioredoxin (Trx), was increased, whereas that of thioredoxin peroxidase (Tpx) was decreased. In addition, we found that HaGdccr knockdown enhanced the enzymatic activity of superoxide dismutase and the contents of hydrogen peroxide and ascorbate. Taken together, these results indicate that HaGdccr may play significant roles in protecting organisms against oxidative damage.

Molecular identification of three novel glutaredoxin genes that play important roles in antioxidant defense in Helicoverpa armigera.

Glutaredoxins (Grxs), also known as thioltransferases, play key roles in maintaining intracellular redox balance and protecting cells from oxidative damage in plants and mammals. We tested whether Grxs play important roles in antioxidant defense in insects using the moth, Helicoverpa armigera. We obtained the full-length cDNA sequences of three novel Grx genes, named HaGrx, HaGrx3, and HaGrx5. Sequence analysis indicated that HaGrx shared a high amino acid identity (58%-78%) and a CPYC motif of conserved redox activity with homologues from other selected insect species. In contrast, HaGrx3 and HaGrx5 both shared a CGF(S/G) motif, a conserved catalytic domain, with other orthologous genes. Quantitative real-time PCR results revealed that HaGrx, HaGrx3, and HaGrx5 exhibited temporally- and spatially-dependent patterns of expression. The mRNA expression of HaGrx, HaGrx3, and HaGrx5 was induced by various temperature stresses and H2O2 treatments. We further investigated the knockdown of HaGrx, HaGrx3, and HaGrx5 in H. armigera larvae and found that most of the selected antioxidant genes were up regulated. However, Tpx was down regulated, and further interpretation of the complementary functions of these antioxidant genes is still required. We also determined the effect of HaGrx, HaGrx3, and HaGrx5 knockdown on antioxidant enzymatic activity and metabolite content. The enzymatic activities of SOD, CAT, and POD, and the metabolite contents of hydrogen peroxide, ascorbate, protein carbonyl, and total GSH increased after RNAi mediated knockdown of HaGrx, HaGrx3, and HaGrx5. These results supported our hypothesis that HaGrx, HaGrx3, and HaGrx5 play important roles in antioxidant defense of Helicoverpa armigera and provided a theoretical basis for further in-depth study of physiological function in the insect glutaredoxin family genes.

Molecular identification of a pancreatic lipase-like gene involved in sex pheromone biosynthesis of Bombyx mori.

Cytoplasmic lipid droplet (LD) lipolysis is regulated by pheromone biosynthesis activating neuropeptide (PBAN) in Bombyx mori. To elucidate the molecular mechanism of cytoplasm LD lipolysis, the pancreatic lipase-like gene in B. mori pheromone glands (PGs), designated as B. mori pancreatic lipase-like gene (BmPLLG), was identified in this study. Spatial expression analysis revealed that BmPLLG is a ubiquitous gene present in all studied tissues, such as PGs, brain, epidermis, egg, midgut, flight muscle and fat body. Temporal expression analysis showed that the BmPLLG transcript begins to express 96 h before eclosion (-96 h), continues to increase, peaks in newly emerged females and steadily decreases after eclosion. Translational expression analysis of BmPLLG using a prepared antiserum demonstrated that BmPLLG was expressed in an age-dependent pattern at different development stages in B. mori. This finding was similar to the transcript expression pattern. Further RNA interference-mediated knockdown of BmPLLG significantly inhibited bombykol production. Overall, these results demonstrated that BmPLLG is involved in PBAN-induced sex pheromone biosynthesis and release.