| Chinese sacbrood disease is most prevalent and important diseases in the Chinese honey bee(Apis cerana cerana),which is widespread and cause devastating lost in native beekeeping all over China,The causative pathogen,Chinese sacbrood virus(CSBV),is highly virulent to the larvae of A.c.cerana,can kill most of the brood and prepupae and quickly collapse the colonies.CSBV can infect the honeybee adult workers also though without discernible sypmtom.Researches about CSBV mainly focused on the larval stage,limited information about the CSBV infection progress in the adult life is available.Beside the CSBV,Nosema ceranae(Nc),another widespread microsporidan pathogen in A.C.cerana adult workers,can significantly shorten the life spans,dramatically reduce the production.As we all known,adult worker bees are most important work force in the colony,including rearing and foraging etc.The health of adult workers is really matter to a colony.Most of studies have been done on either one of two pathogen,rarely take them into account at the same time.However,multiple infection in one colony or even one worker is normal in beekeeping.Therefore,in order to elucidate the dual infection effects of CSBV and Nc on adult workers of A.c.cerana we firstly investigate the infection progress of CSBV in the workers,and investigate the dual infection popularity in a apiary,then challenged the workers indoor with both pathogens,measured the effects of infection by molecular technique.The main results are as following:(1)Three dose of CSBV,9.25×1011,1.85×1011,3.70×1010 copies(correspondingly abbreviation for 1/2?1/10?1/50 group)were individually inoculated to 0-day(less than 24 h after emergence)and 4-day old workers of A.c.cerana and A.m.ligustica.Then kept in incubator.After ten days feeding in the incubator,the mortality of 1/50 group was significant higher mortality compared to the 1/10 group(P=0.032)and the control(P=0.005)10 days post infection,.No significant differences were found among the rest groups.For the 4-day old worker bees,the mortality of 1/10 group was significant lower than that of 1/2 group(P=0.033).On 10th day post infection,the mortality of 1/10 group was significant lower than those of 1/50 and 1/2 group(P<0.01).the mortality of 1/50 group was significant lower than those of 1/50 and 1/2 group(P<0.01)and no significant difference compared with the control group(P>0.05).The western honey bee workers were also tested,the results showed that,the mortality of 1/50 group was significant lower than the control,1/10,and 1/2 groups at 5 day and 10 day post infection.For the 4-day old A.m.ligustica bees there was no significant differences among any groups.These indicated the mortality of worker bees of both species were not mainly affected by CSBVwithin 10 days investigation,the reason behind still needs to be elucidated(2)After two days,the virus quantity(copy number)in midguts increased significantly when 0-and 4-day old workers of both species were challenged with CSBV(P<0.01),the quantity stayed steady thereafter(2 to 10 day).For 4-day old workers the CSBV quantity increased in similar way for 1/2 and 1/10 group(P<0.05).These indicated that two days post infection maybe the main multiplication period for CSBV in the midgut of both honeybee species.(3)In order to investigate the proliferation of CSBV in bees midgut,A.c.cerana and A.mellifera worker bees werre inoculated at 0-day and 4-day old,and were measured at 2,6,10 day after inoculation.The results showed that the proliferation of CSBV in the midgut was correlated to the age of the A.c.cerana workers,the younger the easier and quicker.For the western honey bee workers,only the virus copies of 1/10 group had significantly increased(P<0.05),and 1/2 group after 6 days p.i.There was on significant increase in 1/50 group during 2 to 10 days p.i.(P>0.05),For the 1/10 and 1/2 group,the virus copies significantly increase in 0 day old western honey bee workers compared with 4 day old ones,Thus implied that CSBV only above a certain threshold showed the same proliferation pattern as in the Chinese honey bee workers.in another words,CSBV proliferated slower in western honeybee compared with Chinese honey bee,the long existing host of CSBV.(4)By measuring the CSBV proliferation in the midgut,it showed that 0 day old bees had higher tendency to proliferate more virus than 4 day old workers(P<0.05).For 10 day old workers,it would have higher virus volume when Oday old workers were inoculated compared with 4 day old workers.As for the 10 day old workers,it's similar case.In brief,CSBV proliferated differently in A.c.cerana and A.mellifera.(5)In order to know the status of dual infection in Chinese honey bee,20 colonies of A.c.cerana were investigated.They were classified into two groups:one with apparent symptoms of Chinese sacbrood disease(symptomatic group)and another without apparent symptom of CSBV(asymptomatic)group.Each group had 10 colonies,totally 200 bees were sampled and tested for Nc and CSBV both,The results showed:under the microscope detection,Nc infection in symptomatic group was significantly higher than asymptomatic group,However,by using qPCR techniques,there was no difference between them.By microscopy the overall Nc infection rate was 20%,and by qPCR the infection rate was 98.5%.CSBV infection rate was 90.1%,dual infection rate was as high as 89%,it meant that these Chinese honey bee workrs were severely infected by both Nc and CSBV,(6)Indoor experiments showed that,the survival of dual infection by Nc and CSBV with different doses were significantly lower(P<0.05)than those of single pathogen and control group within 23 days observation period.the survival of 5000 Nc group was higher than that of(5000 Nc+1/2 CSBV)group.Data showed these two pathogens can interfered each other proliferation in the midgut.(7)Respectively determined the absolutely proliferation of CSBV in intestine after worker bees inoculation 0,6,12 d,the results showed that the bee in the presence of double pathogens,after inoculation 6 d,Nc did not affect the virus multiplication in the midgut,the proliferation rate is only associated with the concentrations of the virus increased with the increasing of concentration of the virus.The proliferation of Nc may inhibit CSBV multiplication in the gut after Nc reached a threshold.(8)The expression level of two genes,def-1 and Vg,in the workers(midgut and head excluded)after CSBV or Nc inoculation were measured in 0,6,12 d bee worker bees respectively,The result showed that:CSBV was the main factor which influenced the expression of Vg compared with Nc,However,Nc could decrease Vg expression level by inhibiting CSBV proliferation when 10000 Nc and 1/2 CSBV dual infection happened.def-1 expression level would increased significantly after these pathogens inoculation.Nc could more easily induced def-1 expression compared with CSBV.These indicated Nc and CSBV may affected each other at some stage. |
PDF Full Text Request