To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

Chemosensory proteins (CSPs) have been predicted to be involved in development; however, direct evidence for their involvement is lacking and genetic basis is largely unknown. To determine the function of the chemosensory protein 9 (Si-CSP9) gene inSolenopsis invicta,we used RNA interference to silenceSi-CSP9in 3rd-instar larvae. The 3rd-instar larvae failed to shed their cuticle after being fedSi-CSP9-directed siRNA and expression profiling of RNAi-treated and untreated control larvae showed that 375 genes were differentially expressed. Pathway enrichment analysis revealed that 4 pathways associated with larval development were significantly enriched. Blast analysis revealed that one fatty acid amide hydrolase (FAAH) gene was up-regulated and 4 fatty acid synthase (FAT) genes and one protein kinase DC2 gene (PKA) were down-regulated in the enriched pathways. Significantly higher expression of these genes was found in 4th-instar larvae and Pearson correlation analysis of the expression patterns revealed significant relationships amongSi-CSP9, PKA, FAAHandFAT1-4. Moreover, we confirmed that expression levels ofSi-CSP9, FAAH,andFAT1-4were significantly reduced and that the development of 3rd-instar larvae was halted withPKAsilencing. These results suggest thatSi-CSP9andPKAmay be involved in the network that contributes to development of 3rd-instar larvae.

Wolbachia has been shown to induce thelytokous parthenogenesis in Trichogramma species, which have been widely used as biological control agents around the world. Little is known about the changes of bacterial community after restoring arrhenotokous or bisexual reproduction in the T. pretiosum. Here, we investigate the emergence of males of T. pretiosum through curing experiments (antibiotics and high temperature), crossing experiments, and high-throughput 16S ribosomal RNA sequencing (rRNA-seq). The results of curing experiments showed that both antibiotics and high temperatures could cause the thelytokous T. pretiosum to produce male offspring. Wolbachia was dominant in the thelytokous T. pretiosum bacterial community with 99.01% relative abundance. With the relative abundance of Wolbachia being depleted by antibiotics, the diversity and relative content of other endosymbiotic bacteria increased, and the reproductive mode reverted from thelytoky to arrhenotoky in T. pretiosum. Although antibiotics did not eliminate Wolbachia in T. pretiosum, sulfadiazine showed an advantage in restoring entirely arrhenotokous and successive bisexual reproduction. This study was the first to demonstrate the bacterial communities in parthenogenetic Trichogramma before and after antibiotics or high-temperature treatment. Our findings supported the hypothesis that Wolbachia titer-dependence drives a reproduction switch in T. pretiosum between thelytoky and arrhenotoky.

Eocanthecona furcellata is an important predatory stinkbug that attacks many lepidopteran pests. For mass-rearing, artificial diets are used to rear this predator in the laboratory; however, the fitness of the predators is reduced, and little is known about the cause. Since gut microbiota plays vital roles in the digestion and development of many hosts and can consequently affect host fitness, an understanding of the microbial community composition of E. furcellata may help to solve this unresolved problem. We compared the development and reproduction of E. furcellata reared on an artificial diet, and a natural (Spodoptera litura) or semi-natural (Tenebrio molitor) diet, and then the midgut microbiota were assessed using high-throughput 16S rRNA. The results of the high-throughput 16S rRNA show that the bacterial richness and diversity in the artificial diet gut samples increased considerably compared with the other samples. Proteobacteria and Firmicutes were the dominant phyla in E. furcellata. At the genus level, Serratia (however, the relative abundance was lower in the artificial diet gut samples), Enterococcus, and an uncultured bacterium genus of family Enterobacteriaceae, were dominant. The midgut microbiota components significantly differed among the diets, indicating that the gut bacteria had a dietary association with E. furcellata. This study provides a better understanding of midgut microbiota and the artificial diets that might affect them in E. furcellata.

Spodoptera frugiperda is the preferred host of the parasitoid Telenomus remus. Cold storage can preserve a sufficient quantity of parasitoids and their hosts in a laboratory colony for mass release. First, the effects of cold storage on the reproductive capacity of T. remus reared on non-stored S. frugiperda eggs and the hatching rate of unparasitized S. frugiperda eggs were investigated. Further, the dual effects of cold storage and stored S. frugiperda eggs on the reproductive capacity of T. remus were studied to determine the optimal storage conditions and the maximal shelf life for both the host and the parasitoid. The emergence rate, the number of adults produced, and the female proportion of T. remus were affected by cold storage factors. Pupae stored at 13 °C for 15 days is optimum for T. remus reared on non-stored S. frugiperda eggs. Spodoptera frugiperda eggs could only be stored at 10 °C for five days to be suitable for rearing T. remus. The optimum cold storage conditions for T. remus parasitizing stored eggs were 7 °C for 5 days in the larval stage. The maximal shelf lives of T. remus parasitizing cold-stored S. frugiperda eggs were 10 days. Cold storage affected the hatching rate of S. frugiperda eggs, thereby further affecting the reproductive capacity of T. remus. The findings suggest that different storage conditions should be used when mass-rearing T. remus on stored and non-stored eggs. Telenomus remus should be reproduced using fresh laid S. frugiperda eggs for maximum shelf life.