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.

An accurate olfactory system for recognizing semiochemicals and environmental chemical signals plays crucial roles in survival and reproduction of insects. Among all olfaction-related proteins, olfactory receptors (ORs) contribute to the conversion of chemical stimuli to electric signals and thereby are vital in odorant recognition. Olfactory receptor co-receptor (Orco), one of the most conserved ORs, is extremely essential in recognizing odorants through forming a ligand-gated ion channel complex with conventional ligand-binding odorant receptors. We have previously identified aggregation pheromone in Protaetia brevitarsis (Coleoptera: Scarabaeidae), a native agricultural and horticultural pest in East-Asia. However, to our best knowledge, its olfaction recognition mechanisms are still veiled. To illustrate how P. brevitarsis recognize aggregation pheromone and host plants, in the present study we cloned and sequenced the full-length Orco gene from P. brevitarsis antennae (named PbreOrco) and found that PbreOrco is highly conserved and similar to Orcos from other Coleoptera insects. Our real-time quantitative PCR (qRT-PCR) results showed that PbreOrco is mainly expressed in antenna. We also demonstrated that silencing PbreOrco using RNA interference through injecting dsOrco fragment significantly inhibited PbreOrco expression in comparison with injecting control dsGFP and subsequently revealed using electroantennogram and behavioral bioassays that decreasing PbreOrco transcript abundance significantly impaired the responses of P. brevitarsis to intraspecific aggregation pheromone and prolonged the time of P. brevitarsis spending on food seeking. Overall, our results demonstrated that PbreOrco is crucial in mediating odorant perception in P. brevitarsis.

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.

Intestinal symbiotic bacteria have formed an interdependent symbiotic relationship with many insect species after long-term coevolution, which plays a critical role in host growth and adaptation. Spodoptera frugiperda (J. E. Smith) is a worldwide significant migratory invasive pest. As a polyphagous pest, S. frugiperda can harm more than 350 plants and poses a severe threat to food security and agricultural production. In this study, 16S rRNA high-throughput sequencing technology was used to analyze the diversity and structure of the gut bacteria of this pest feeding on six diets (maize, wheat, rice, honeysuckle flowers, honeysuckle leaves, and Chinese yam). The results showed that the S. frugiperda fed on rice had the highest bacterial richness and diversity, whereas the larvae fed on honeysuckle flowers had the lowest abundance and diversity of gut bacterial communities. Firmicutes, Actinobacteriota, and Proteobacteria were the most dominant bacterial phyla. PICRUSt2 analysis indicated that most of the functional prediction categories were concentrated in metabolic bacteria. Our results confirmed that the gut bacterial diversity and community composition of S. frugiperda were affected significantly by host diets. This study provided a theoretical basis for clarifying the host adaptation mechanism of S. frugiperda, which also provided a new direction to improve polyphagous pest management strategies.

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Acknowledgments 10 We would like to thank the editor and anonymous reviewers for valuable comments 11 and suggestions. Abstract 22 To improve the laboratory mass-rearing and the winter greenhouse use of the 23 multicolored Asian lady beetle Harmonia axyridis (Pallas) (Coleoptera, 24 Downloaded by [UNSW Library] at 03:27 11 August 2015A c c e p t e d M a n u s c r i p t Coccinellidae), the effects of photoperiod on the development and fecundity of H. 25 axyridis were investigated at 20°C and photoperiods ranging from 0L:24D to 24L:0D. 26 We found photoperiod to significantly affect the pre-imaginal development and 27 reproduction of H. axyridis. The development of the pre-imaginal stage was 28 significantly faster at shorter photoperiods (0 to 14 h) than at longer ones (16 to 24 h). 29 The proportion of females laying eggs within the first 30 days after eclosion, the mean 30 number of eggs per ovipositing female, and the long axial length of the 1st oocyte 31 were all lower at shorter photoperiods (6 to 12 h) than at longer ones (14 to 24 h). 32 These results suggest that adult H. axyridis may encounter problems in preying and 33 reproducing in the winter conditions of unheated and unilluminated greenhouses in 34 areas with temperatures lower than 20 °C and day lengths shorter than 12 h. 35