To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

Background: Microbial acquisition and development of the gut microbiota impact the establishment of a healthy host-microbes symbiosis. Compared with other animals, the eusocial bumblebees and honeybees possess a simple, recurring, and similar set of gut microbiota. However, all bee gut phylotypes have high strain-level diversity. Gut communities of different bee species are composed of host-specific groups of strains. The variable genomic regions among strains of the same species often confer critical functional differences, such as carbon source utilization, essential for the natural selection of specific strains. The annual bumblebee colony founded by solitary queens enables tracking the transmission routes of gut bacteria during development stages.Results: Here, we first showed the changes in the microbiome of individual bumblebees across their holometabolous life cycle. Some core gut bacteria persist throughout different stages of development. Gut microbiota of newly emerged workers always resembles those of their queens, suggesting a vertical transmission of strains from queens to the newborn workers. We then follow the dynamic changes in the gut community by comparing strain-level metagenomic profiles of queen-worker pairs longitudinally collected across different stages of the nest development. Species composition of both queen and worker shifts with the colony’s growth, and the queen-to-worker vertical inheritance of specific strains was identified. Finally, comparative metagenome analysis showed clear host-specificity for microbes across different bee hosts. Species from honeybees often possess a higher level of strain variation, and they also exhibited more complex gene repertoires linked to polysaccharide digestion. Our results demonstrate bacterial transmission events in bumblebee, highlighting the role of social interactions in driving the microbiota composition.Conclusions: By the community-wide metagenomic analysis based on the custom genomic database of bee gut bacteria, we reveal strain transmission events at high resolution and the dynamic changes in community structure along with the colony development. The social annual life cycle of bumblebees is key for the acquisition and development of the gut microbiota. Further studies using the bumblebee model will advance our understanding of the microbiome transmission and the underlying mechanisms, such as strain competition and niche selection.

Diapause is a complex physiological adaptation phenotype, and the transcription factor Forkhead-box O (FoxO) is a prime candidate for activating many of its diverse regulatory signaling pathways. Hormone signaling regulates nymphal diapause in Laodelphax striatellus. Here, the function of the FoxO gene isolated from L. striatellus was investigated. After knocking-down LsFoxO in diapausal nymphs using RNA interference, the titers of juvenile hormone III and some cold-tolerance substances decreased significantly, and the duration of the nymphal developmental period was severely shorted to 25.5 days at 20°C under short day-length (10 L:14 D). To determine how LsFoxO affects nymphal diapause, analyses of RNA-sequencing transcriptome data after treatment with LsFoxO–RNA interference was performed. The differentially expressed genes affected carbohydrate, amino acid and fatty acid metabolism, and phosphatidylinositol 3-kinase/protein kinase B signaling pathway. Thus, LsFoxO acts on L. striatellus nymphal diapause and is, therefore, a potential target gene for pest control. This study may lead to new information on the regulation of nymphal diapause in this important pest.