As an accurate and convenient technique, the qRT-PCR is always used in the quantitative expression analysis of functional genes. Normalization of the data relies on stable reference genes. The fall armyworm Spodoptera frugiperda (J. E. Smith) is an important invasive and migratory pest that seriously threatens corn production around the world. In this paper, we selected 10 candidate reference genes (18S, AK, RPL10, RPS24, 28S, SOD, ATP, GAPDH, ACT, and a-TUB) and determined their expression levels under different conditions (different developmental stages, various tissues, mating status, hormones, diets, and temperatures). Subsequently, the stability of reference genes was evaluated by four algorithms (Delta Ct method, geNorm, NormFinder, BestKeeper). The optimal combination of reference genes for each treatment was obtained by geNorm. Finally, the comprehensive ranks were determined by the online tool RefFinder. Results showed that the most stable reference genes were SOD, RPL10, and RPS24 for developmental stages, α-TUB, RPL10, and ATP for different tissues, AK, RPL10, and 18S for mating status, 18S and AK under hormone treatment, 18S, RPL10, and SOD under diet treatment, RPL10, 18S, and RPS24 under temperature treatment. This study confirmed recent data on a few reference genes and provided an evaluation of a number of additional reference genes of S. frugiperda under various conditions.

Vitellogenin receptors (VgRs) have vital roles in reproduction by mediating endocytosis of the vitellogenin (Vg) in oviparous insects. Same as most insect species that possess only one VgR, in this study, a single VgR mRNA (HmVgR) was identified in an important natural enemy ladybeetle, Harmonia axyridis (Pallas). The open reading frame of HmVgR was 5,340 bases encoding a protein of 1,779 amino acids. Bioinformatic analyses showed that HmVgR had conserved domain motifs of low-density lipoprotein receptor family. Based on phylogenetic analysis, HmVgR had highly homologous within the Coleoptera. The transcriptional level of HmVgR was initially detected in the newly emerged female adults, gradually increased from day 3 to day 9, peaked on day 13, and then sharply decreased on day 15. Similar to most insects, HmVgR was expressed specifically in ovarian tissue. Moreover, RNA interference (RNAi) clearly decreased the transcription levels of HmVgR, which blocked the deposition of yolk protein in the ovaries, shortened the ovarian length, and led to negative impacts on reproductive-related parameters (i.e., prolonged preoviposition periods, reduced spawning and depressed hatchability). In sum, these results indicated that HmVgR may be critical for yolk protein deposition of oocytes and can play a key role in reproduction of female adults of H. axyridis. Our results provide conclusive proof for the important roles of HmVgR in fecundity, and establish a basis for further research on its interaction with vitellogenin.

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Juvenile hormone (JH) plays a key role in regulating insect reproductive processes. Methoprene-tolerant (Met), as a putative JH receptor, transduces JH signals by activating the transcription factor krüppel homolog 1 (Kr-h1). To understand the effects of Met and Kr-h1 genes on female reproduction of natural enemy insects, the Met and Kr-h1 were identified and analyzed from Harmonia axyridis Pallas (HmMet and HmKr-h1). The HmMet protein belonged to the bHLH-PAS family with bHLH domain, PAS domains, and PAC domain. HmMet mRNA was detected in all developmental stages, and the highest expression was found in the ovaries of female adults. The HmKr-h1 protein had eight C2H2-type zinc finger domains. HmKr-h1 mRNA was highly expressed from day 7 to day 9 of female adults. The tissue expression showed that HmKr-h1 was highly expressed in its wing, leg, and fat body. Knockdown of HmMet and HmKr-h1 substantially reduced the transcription of HmVg1 and HmVg2, inhibited yolk protein deposition, and reduced fecundity using RNA interference. In addition, the preoviposition period was significantly prolonged after dsMet-injection, but there was no significant difference after dsKr-h1-silencing. However, the effect on hatchability results was the opposite. Therefore, we infer that both HmMet and HmKr-h1 are involved in female reproduction of H. axyridis, and their specific functions are different in certain physiological processes. In several continents, H. axyridis are not only beneficial insects, but also invasive pests. This report will provide basis for applying or controlling the H. axyridis.

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