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In this study, we developed a SYBR Green I real-time PCR method for the rapid and sensitive detection of novel porcine parvovirus 7 (PPV7). Specific primers were designed based on the highly conserved region within the Capsid gene of PPV7. The established method was 1,000 times more sensitive than the conventional PCR method and had a detection limit of 35.6 copies. This method was specific and had no cross-reactions with PCV2, PCV3, PRV, PEDV, PPV1, and PPV6. Experiments testing the intra and interassay precision demonstrated a high reproducibility. Testing the newly established method with 200 clinical samples revealed a detection rate up to 17.5% higher than that of the conventional PCR assay. The established method could provide technical support for clinical diagnosis and epidemiological investigation of PPV7.

In this study, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed for rapid detection of porcine parvovirus (PPV) 6. Primer pairs targeting the conserved regions of PPV6 Capsid gene were designed. Sensitivity analyses revealed the lowest detection limit of the SYBR Green-based real-time PCR assay to be 47.8 copies/µL, which indicated it was 1000 times higher than that found in the conventional PCR investigations. This assay was specific and showed no cross-species amplification with other six porcine viruses. The assay demonstrated high repeatability and reproducibility; the intra-and inter-assay coefficients of variation were 0.79% and 0.42%, respectively. The positive detection rates of 180 clinical samples with SYBR Green-based real-time PCR and conventional PCR were 12.22% (22/180) and 4.44% (8/180), respectively. Our method is sensitive, specific, and reproducible. The use of SYBR Green-based real-time PCR may be suitable for the clinical detection and epidemiological investigation of PPV6.

Neonicotinoid insecticides are now the most widely used insecticides in the world. 10Previous studies have indicated that sublethal doses of neonicotinoids impair learning, memory 11 capacity, foraging and immunocompetence in honeybees (Apis mellifera). Despite this, few 12 studies have been carried out on the molecular effects of neonicotinoids. In this study, we focus on 13 the second-generation neonicotinoid thiamethoxam, which is currently widely used in agriculture 14 to protect crops. Using high-throughput RNA-Seq, we investigated the transcriptome profile of 15 honeybees after subchronic exposure to thiamethoxam (10 ppb) over 10 days. In total, 609 16 differentially-expressed genes (DEGs) were identified, of which 225 were up-regulated and 384 17were down-regulated. The functions of some DEGs were identified, and GO enrichment analysis 18 showed that the enriched DEGs were mainly linked to metabolism, biosynthesis and translation. 19 KEGG pathway analysis showed that thiamethoxam affected biological processes including 20 ribosomes, the oxidative phosphorylation pathway, tyrosine metabolism pathway, pentose and 21 glucuronate interconversions and drug metabolism. Overall, our results provide a basis for 22 understanding the molecular mechanisms of the complex interactions between neonicotinoid 23 insecticides and honeybees. 24 Summary statement: NR1, Cyp6as5, nAChRa9 and nAChRβ2 were up-regulated in honeybees 26 exposed to thiamethoxam, while CSP3, Obp21, defensin-1, Mrjp1, Mrjp3 and Mrjp4 were 27 down-regulated. 28 29 2 production (Breeze et al., 2011). In recent years, attention has been paid to the large decrease to 33 global apiculture (Neumann and Carreck, 2010; Potts et al., 2010; Van Engelsdorp et al., 2010; 34 Chauzat et al., 2013) but the reasons are still poorly understood. Recent studies have however 35suggested that the decrease could be due to the widespread use of insecticides (Johnson et al., 36 2010; Goulson et al., 2015; Schmuck and Lewis, 2016). 37Recently, there have been far-reaching changes in the insecticide market. Many of the 38 traditional insecticides, e.g. organophosphorus and pyrethroids, have been replaced by systemic 39 insecticides, especially neonicotinoids. Neonicotinoids act on the insect nervous system mainly 40 through agonistic action on nicotinic acetylcholine receptors (nAChRs) (Brown et al., 2006), and 41 since they have low mammalian toxicity (Tomizawa and Casida, 2005) they are widely used for 42 controlling insect pests. Neonicotinoids are commonly applied as seed coatings or as foliar sprays 43 on crops. Once absorbed into the plant, neonicotinoids can translocate to dew drops, nectar and 44 pollen of crops during florescence (Krupke et al., 2012; Stoner and Eitzer, 2012). The 45 contaminated nectar and pollen may be consumed by foragers (Goulson, 2013) or taken to the nest 46 for long-term storage where they are eaten by the young adults and larvae (DeGrandi-Hoffman et 47