中蜂囊状幼虫病毒对蜜蜂肠道菌群的影响与枯草芽孢杆菌的抗病毒活性

Effect of Chinese sacbrood virus on gut microbiota of honeybees and antiviral activity of Bacillus subtilis

摘要:

目的 探究中华蜜蜂(Apis cerana cerana)和意大利蜜蜂(Apis mellifera ligustica)工蜂幼虫感染中蜂囊状幼虫病毒(Chinese sacbrood virus, CSBV)后肠道微生物的变化差异，并评估枯草芽孢杆菌(Bacillus subtilis)对CSBV的抗病毒活性。方法 从蜂群中分别移取2日龄的中蜂幼虫和意蜂幼虫，置于34 ℃、85%湿度(relative humidity, RH)的培养箱中进行人工饲养。待幼虫发育至3日龄时对其接种CSBV病毒，并分别在4日龄和7日龄时取样进行16S rRNA基因测序分析。此外，针对3日龄中蜂幼虫，在接种CSBV病毒的同时补充饲喂不同浓度的枯草芽孢杆菌。在7日龄时检测其抗氧化能力指标，包括丙二醛(malondialdehyde, MDA)含量、超氧化物歧化酶(superoxide dismutase, SOD)活性；同时，利用实时荧光定量PCR技术测定Abaecin、Apidaecin、Hymenoptaecin、Defensin这4个编码抗菌肽基因，以及CSBV基因的相对表达量。结果 感染CSBV后，蜜蜂幼虫肠道微生物数量显著减少。7日龄的中蜂幼虫肠道内芽孢杆菌属(Bacillus)的丰度显著降低；意蜂幼虫肠道内链霉菌属(Streptomyces)丰度显著下降，而短波单胞菌属(Brevundimonas)丰度显著上升。在4日龄时，感染CSBV的中蜂幼虫肠道内蜜蜂球菌属(Melissococcus)丰度显著高于意蜂幼虫；7日龄时，感染CSBV的中蜂幼虫Chao1指数显著低于对照组及7日龄意蜂幼虫，这表明中蜂幼虫肠道菌群多样性受CSBV影响更为显著。饲喂1×10⁴ CFU/kg枯草芽孢杆菌制剂的7日龄中蜂幼虫，其SOD活性显著升高，而其他剂量的枯草芽孢杆菌对幼虫SOD活性无显著影响。所有剂量组的枯草芽孢杆菌制剂均显著降低蜜蜂体内MDA含量。此外，经1×10⁴、1×10⁶和1×10⁸ CFU/kg 3个浓度处理后，蜜蜂体内CSBV相对表达水平均显著降低。其中，1×10⁶ CFU/kg组CSBV表达水平显著低于1×10⁸ CFU/kg组；然而，不同处理组编码抗菌肽的4个基因的表达水平并无显著差异。结论 中蜂幼虫肠道菌群多样性受CSBV感染的影响更为显著。枯草芽孢杆菌能够在一定程度上抑制CSBV的活性。

Abstract:

Objective To explore the changes and differences of gut microbiota of worker bee larvae of Apis cerana cerana and Apis mellifera ligustica after infection with Chinese sacbrood virus (CSBV) and evaluate the antiviral activity of Bacillus subtilis against CSBV.Methods Two-day-old larvae of A. c. cerana and A. m. ligustica were collected from colonies and artificially reared in an incubator (34 ℃, RH 85%). The 3-day-old larvae were inoculated with CSBV and samples were collected when the larvae were 4 and 7 days old for 16S rRNA gene sequencing. In addition, the 3-day-old larvae of A. c. cerana were fed with different concentrations of B. subtilis suspensions during CSBV inoculation. When the larvae were 7 days old, the antioxidant capacity indicators malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Additionally, the relative expression levels of four antimicrobial peptide genes (Abaecin, Apidaecin, Hymenoptaecin, and Defensin) and the CSBV gene were determined by real-time fluorescence quantitative PCR.Results The number of gut microorganisms in bee larvae significantly decreased after CSBV infection. The abundance of Bacillus in the gut of 7-day-old A. c. cerana larvae significantly decreased. The gut of A. m. ligustica larvae showed significantly decreased abundance of Streptomyces and significantly increased abundance of Brevundimonas. At 4 days old, CSBV-infected A. c. cerana larvae had significantly higher abundance of Melissococcus in the gut than the infected A. m. ligustica larvae. At 7 days old, CSBV-infected A. c. cerana larvae had significantly lower Chao1 index than the control group and the infected A. m. ligustica larvae, which indicated that the gut microbiota diversity of A. c. cerana larvae was more susceptible to CSBV. The SOD activity of 7-day-old A. c. cerana larvae fed with 1×10⁴ CFU/kg B. subtilis significantly increased, while other doses of B. subtilis had no significant effect on the SOD activity in the larvae. All doses of B. subtilis significantly reduced the MDA content in bees. The relative expression levels of CSBV in the three treatment groups of 1×10⁴, 1×10⁶, and 1×10⁸ CFU/kg were significantly down-regulated. Moreover, the expression level of CSBV in the 1×10⁶ CFU/kg group was the lowest and significantly lower than that in the 1×10⁸ CFU/kg group. However, there were no significant differences in the expression levels of the four antimicrobial peptide genes among different treatment groups.Conclusion CSBV infection has a more significant impact on the gut microbiota diversity of A. c. cerana larvae. B. subtilis has a certain inhibitory effect on CSBV.