Characteristics Of Artificial Rearing And Degradation Mechanism Of Bumblebee Bombus Lantschouensis (Hymenoptera: Apidae)

Bumblebees are important pollinators of many wild plants and crops,which play an important role in maintaining natural ecological balance,biodiversity and global agricultural production.As an important supporting technology of facility agriculture,bumblebee pollination has been widely used in many countries.China has the richest resources of bumblebee in the world,but we still need to import bumblebee to meet the needs of facility crop pollination,which increases the risk and challenge of biological invasion.As the dominant species of bumblebee in China,Bombus lantschouensis has the potential advantage of factory rearing.In the process of annual breeding of B.lantschouensis,there were some problems,such as isolation of rearing population from the outside world and the generation of diploid males by inbreeding,which resulted in the subgeneration degradation of bumblebee colonies and restricted the rearing and pollination application of B.lantschouensis.In this study,the native species B.lantschouensis in China was selected as the research object.By studying the rearing characteristics of bumblebee colonies,the reproductive biology of males and queens,the effects of males’ kin relationship on bumblebee annual breeding were clarified.The effects of reproductive indexes of males on colony subgeneration degradation were revealed.And the chemical qualitative and quantitative analysis of CLGS of males which virgin queen can recognize and locate males were carried out,and the molecular mechanism of spermatheca of the queen which is regulated by mating the was preliminarily explored.The results are as follows:1.Research on the annual rearing of Bombus lantschouensisThe effects of males’ kin relationship on queen mating latency,mating time,mating success rate and queen weight were observed.The results showed that the mating latency of queens in unrelated-male groups was significantly shorter than related-male groups(P < 0.05).There was no mating preference in mix groups(P > 0.05).The males from multiple colonies significantly increased the mating success rate(P < 0.05).The average weight of queens in the unrelated-male groups after diapause was significantly lower than that in related-male groups(P < 0.05).The queen could not recognize the related males.Inbreeding significantly prolonged the mating latency and affected queen weight after diapause.The effects of inbreeding on annual rearing of colonies were investigated by observing the rearing characteristics of colonies.The results showed that the queen egg laying time and emergence time of the first male in inbred groups were significantly longer than that of outbred groups(P < 0.05).And the number of first workers and first males,total number of workers and males in inbred groups were significantly lower than in outbred groups.All results showed that inbred was not conducive to the reproduction of B.lantschouensis colony.The effects of three induction methods of bumblebee workers,bumblebee male pupae and honeybee workers on the reproductive characteristics of bumblebee queens were studied.The results showed that three methods significantly shorten the emergence time of first workers and increased the number of workers(P < 0.05).There were significant differences in the queen egg laying time,the emergence time of the first workers,the number of first workers and total number of workers(P < 0.05)among different induction methods.Therefore,all three induction methods were conducive to the development of B.lantschouensis colonies.The effects of different diapause durations on the rearing characteristics of colonies were studied.The results showed that compared with the 2-month,4-month and 6-month diapause duration,the queen egg laying time and emergence time of first workers of the 3-month diapause groups were significantly shorter than other groups(P < 0.05).And the number of first workers was significantly larger than other groups(P < 0.05).It was preliminarily confirmed that 3-month diapause duration for the B.lantschouensis queen was suitable for colony reproduction.2.Reproductive biology of Bombus lantschouensis malesThe indexes of sperm transfer,sperm maturation,the quantity and viability of sperm were measured.The results showed that the sperm transfer from testis to accessory testis began at 3 d.Males sexually mature at 12 d-15 d,and the number of mature sperm in accessory testis was the largest(P < 0.05),indicating that the optimal mating period of males was 12 d-15 d.The quantity and viability of sperm of males were significantly lower in second-generation than those in first-generation(P < 0.05).It was significantly higher in the sperm viability of indoor rearing males than that of wild males(P < 0.05).In addition,the quantity of sperm in queen spermathecae in outbred groups were significantly higher than that in inbred groups(P < 0.05).The quantity and viability of sperm might be one of the factors that affect the reproduction degradation of B.lantschouensis.GC-MS was used to analyze the main components and relative contents of the CLGS of male in different generation.The results showed that there were 16 components in CLGS.Of those components,the contents of 6 components in first generation males were significantly higher than those in second generation and wild males(P < 0.05).They were Dodecanoic acid and ethyl ester;Ethyl 9-tetradecenoate;1,4-cyclohexanedimethanol;1,8,11-Heptadecatriene;(E)-9-Octadecenoic acid ethyl and Heneicosane.The results suggested that CLGS of males affected by generation,which may reduce the attractiveness of males to the queen.3.Molecular mechanism of spermatheca regulation of queens on matingTranscriptome sequencing and real-time quantitative PCR were used to study the differentially expressed genes in spermophore of queens before and after mating.In total,the expression of 11069 and 10862 genes were identified in virgins and mated queens,respectively.Of the 176 differentially expressed genes,110(62.5%)genes were up-regulated and 66(37.5%)genes were down-regulated in mated queens.RT-q PCR showed most of the differentially expressed genes were concentrated on immune response [LRRC70(35.8-fold),PO2(41.9-fold)and defensin(4.9-fold)]and sperm storage [TIL(6.2-fold),Tret1(1.7-,1.9-,2.4-,and 2.4-fold),and hn RNP(1.2-and 2.6-fold)] functions in the spermathecae of mated queens.In conclusion,sperm transfer into spermathecae led to significantly upregulation of immune response gene expression in bumblebees.