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Involvement of glucose transporter 4 in ovarian development and reproductive maturation of Harmonia axyridis (Coleoptera: Coccinellidae).

ABSTRACT: Glucose is vital to embryogenesis, as are glucose transporters. Glucose transporter 4 (Glut4) is one of the glucose transporters, which is involved in rapid uptake of glucose by various cells and promotes glucose homeostasis. Although energy metabolism in insect reproduction is well known, the molecular mechanism of Glut4 in insect reproduction is poorly understood. We suspect that Glut4 is involved in maintaining glucose concentrations in the ovaries and affecting vitellogenesis, which is critical for subsequent oocyte maturation and insect fertility. Harmonia axyridis (Pallas) is a model organism for genetic research and a natural enemy of insect pests. We studied the influence of the Glut4 gene on the reproduction and development of H. axyridis using RNA interference technology. Reverse transcription quantitative polymerase chain reaction analysis revealed that HaGlut4 was most highly expressed in adults. Knockdown of the HaGlut4 gene reduced the transcript levels of HaGlut4, and the weight and number of eggs produced significantly decreased. In addition, the transcript levels of vitellogenin receptor and vitellogenin in the fat bodies and the ovaries of H. axyridis decreased after the interference of Glut4, and decreased the triglyceride, fatty acid, total amino acid and adenosine triphosphate content of H. axyridis. This resulted in severe blockage of ovary development and reduction of yolk formation; there was no development of ovarioles in the developing oocytes. These changes indicate that a lack of HaGlut4 can impair ovarian development and oocyte maturation and result in decreased fecundity.

SUBMITTER: Li Y

PROVIDER: S-EPMC9298200 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.

Project description:Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in H. axyridis based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions. However, there have been no studies on the stability of reference genes used in H. axyridis. In this study, we systematically investigated expression profiles of nine candidate reference genes from H. axyridis, including ?-actin (ACTIN); elongation factor 1 ? (EF1A); ribosomal proteins L10, L18, L28, S13, and S15 (RPL10, RPL18, RPL28, RPS13 and RPS15); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and superoxide dismutase (SOD). Four analytical methods (geNorm, BestKeeper, NormFinder, and the ?Ct method) were used to evaluate the suitability of these genes as internal reference genes for three biotic factors (developmental stage, tissue, and sex) and two abiotic treatments (temperature and photoperiod). RefFinder, a comprehensive evaluation platform integrating the four analytical methods, was used to rank the overall stability of these reference genes. Among the nine candidate genes, different reference genes were identified as having the most stable expression across biotic and abiotic factors. Genes encoding ribosomal proteins typically had the most stable expression, though EF1A was the most stable across developmental stages and photoperiods. To validate the suitability of these reference genes, heat shock protein 90 (HSP90) was chosen as a target. Significant up-regulation in HSP90 expression level in response to both low and high temperature was observed when using the most suitable reference genes but not when using an arbitrarily selected reference gene. The reference genes identified in this study will provide the basis for future functional genomics research in H. axyridis and will also facilitate the establishment of a standardized qRT-PCR program for other related insects.

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Involvement of glucose transporter 4 in ovarian development and reproductive maturation of Harmonia axyridis (Coleoptera: Coccinellidae).

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