Cross-species Infection Of Honeybee Virus And Nosema Ceranae On Bumblebees

Bumblebees play an important role in maintaining the sustainable development ofagriculture, the diversity of crops and improving the quality of agricultural productsand production as a kind of important pollinators. In recent years, due to the diseasescaused by viruses, parasites and other pathogens that may affect the bumblebeeecology, distribution and the application of commercial pollination. Theagro-ecosystems have resulted in a significant negative impact. Recent domestic andforeign scholars have found that BQCV, DWV etc and N.ceranae can cross-speciesinfect bumblebees. However, our pathological study of the honeybee pathogenscross-species infect bumblebees is almost blank. Therefore, this paper use RT-PCR,qRT-PCR, fluorescence microscopy, electron microscopy technology to performresearch around the bumblebee BQCV and DWV virus disease, detection andidentification of N.ceranae and the pathogenic mechanism of Bombus hypocriteinfected by N.ceranae. It’s mainly four parts as follows:1) We detect7kinds of common bee virus (ABPV、BQCV、CBPV、DWV、KBV、IAPV、SBV) in bumblebees, including: BQCV infection rate of Bombus hypocrite andBombus ignitus were19.50%and20%, DWV infection rate is relatively low,respectively5.5%and4%. The strand-specific RT-PCR for analysis of BQCV andDWV replication reveals high (positive rate of negative-strand is36%) positive rate ofBQCV replication in the larvae of Bombus hypocrite, and the DWV replication are moreactive (positive rate of negative-strand is12%) in the worker of Bombus hypocrite.Quantitative analysis on RT-PCR shows that BQCV have reached the highest level in the larvae ofBombus ignitus and lowest level in the worker of Bombus ignitus. However, the workerof Bombus ignitus is the highest and the male of Bombus ignitus is the lowest in theinfection of DWV compared with Bombus hypocrite.2) The external morphological of N.ceranae found to be oval and the surface was rough foldby scanning electron micrographs. The internal morphological of N.ceranae verified the spores have thicker wall and three-layer structure from the inside to the outside,polar tubes surround around the nucleus and have a four-story structure bytransmission electron micrographs. It is easy to detection and identification of live anddead N.ceranae from host cells, bacteria and virus by using Calcofluor White M2Rand Sytox Green stains in dual staining, the dead spores were in yellow-greenfluorescence and the live were in White-blue fluorescence.3）Bombus hypocrite infected by N.ceranae does not exhibit obvious external diseasesigns except that they eat less and move slowly; in the late period, infected beesshowed dispirited, weak, and unable to fly. Through the observation of lightmicroscope, few N.ceranae but a lot of bacteria were found in the midgut of bees.From the electron microscopy, N.ceranae mainly infects midgut of epithelial cellsthrough intestinal villus; nuclear enlarges and deforms, mitochondria become smallereven disintegrating and endoplasmic reticulum becomes disorders. However, thespores only infect the cytoplasm of the host instead of invading the nuclear. At last,the disintegration of the mitochondria and the repture of the cell lead to the death.Quantitative analysis on RT-PCR shows that N.ceranae has reached the highest levelin the midgut and fat body after being infected and other organizations are barelydetected.Conclusion：we found two honey bee viruses, BQCV and DWV, in the domesticrearing bumblebees for the first time, and detected live and dead of N.ceranae byfluorescence in dual staining. We have reached a primary conclusion that N. ceranaecould cross-species infected Bumblebees.