The Functions And Mechanisms Underlying Vertical Transmission Of Hamiltonella In The Whitefly Bemisia Tabaci

The whitefly Bemisia tabaci is a species complex consisting of more than 36 cryptic species.Among them,Middle East-Asia Minor 1(MEAM1)and Mediterranean(MED)are globally important pests.Whiteflies in the Bemisia tabaci complex have a diversity of symbionts including the obligate symbiont Portiera and seven facultative symbionts Hamiltonella,Rickettsia,Arsenophonus,Cardinium,Wolbachia,Fritschea and Hemipteriphilus.Most of the symbionts are restricted to specialized cells known as bacteriocytes and transmitted vertically via the ovary.Various studies show that some symbionts play important roles in regulating the metabolic capabilities and fitness of whitefly.Nevertheless,the functions of most symbionts remain largely unknown and the mechanisms of their vertical transmission are obscure.In order to explore the interaction of symbionts and their whitefly host,we first surveyed the diversity of symbionts in field population of whitefly.We then investigated the roles of Hamiltonella in affecting the biology of its whitefly host.Further,we observed the vertical transmission of bacteriocyte associated symbionts.Finally,we analyzed the effects of heat stress on symbionts of different distribution patterns.The results are summarized as follow:(1)Diversity of symbionts in different species of the whitefly complexWe surveyed the diversity of symbionts in 7 B.tabaci species in 42 field populations.Hamiltonella were only detected in the invasive species MEAM1 and MED whiteflies,and the infection frequencies were 100%in 27 of all the 30 populations,and other the three populations had 88.9%,90.5%and 95%infection frequencies respectively.In addition,MEAM1 populations had high infection rate of Rickettsia ranging from 66.7%to 100%and MED populations were found to be infected by Cardinium with frequencies ranging from 0%to 85.7%and only one population was infected by Rickettsia with a frequency of 12.5%.None of five species indigenous to China in the 12 populations was infected by Hamiltonella.These indigenous species harboured mostly Wolbachia or Arsenophonus,and in some of the samples with low proportion of Rickettsia,Cardinium and/or Hemipteriphilus.(2)The symbiont Hamiltonella as determinant of the sex ratio in whiteflyTo investigate the significance of Hamiltonella to its whitefly host,this bacterium was eliminated from the insects by two complementary methods:heat treatment and a cocktail of selective antibiotics.The sex ratio of the progeny and subsequent generations of the Hamiltonella-free females was skewed from ca.1:1(male:female)to an excess of males,often exceeding 20:1 ratio,with no difference in egg viability and independent of the Hamiltonella status of males.Bemisia tabaci is haplodiploid,with diploid females derived from fertilized eggs and haploid males from unfertilized eggs.The Hamiltonella status of the insects did not affect copulation frequency and sperm reserve in the spermathecae,indicating that the male-biased sex ratio is likely to be associated with failure in fertilization or post-zygotic defects of Hamiltonella-cured female uinsects.In natural populations with high prevalence of Hamiltonella-positive insects,excess male production bya Hamiltonella-free female promotes the probability that her offspring mate with a Hamiltonella-ositive female,re-instating the Hamiltonella infection in her grand-daughters.This effect rescues the fitness of the maternally-inherited Hamiltonella and,if the insect benefits from the association,also the insect host.(3)Cellular mechanism of bacteriocyte for vertical transmission of symbiontsThrough monitoring the dynamics of bacteriocyte,we found that bacteriocyte is proliferous and remodelled at the developmental transition from nymph to adulthood.This transition involves the loss of cell-cell adhesion,shape change and onset of cell mobility,enabling the bacteriocytes to crawl to the ovaries.For the bacteriocyte associated symbionts Portiera and Hamiltonella,which were transferred to oocytes along with the intact bacteriocytes.This study demonstrated,for the first time,cell behaviours underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends.(4)The effects of heat stress on bacteriocytes and symbionts of whiteflyWe found that following continuous heat stress,the bacteriocyte symbionts Portiera and Hamiltonella were almost completely depleted in MEAM1 and MED whiteflies.Meanwhile,cell size of bacteriocytes was increased,while cell number was severely decreased leading to reduction of total volume of bacteriocytes in female adults.Moreover,bacteriocyte organelles and associated symbionts were lysed,and huge amount of electron-dense inclusions accumulated.Eventually,bactriocytes as well as Portiera and Hamiltonella failed to be transmitted to the next generation.In contrast,the scatter distributed Rickettsia was found to be reduced at a much slight level,and it could be successfully transmitted to eggs.The results suggest that the thermal sensitivity of bacteriocytes may limit thermal tolerance and vertical transmission of the associated symbionts,and consequently different patterns of distribution of symbionts in the host may affect their capacity to tolerate unfavourable temperatures and persistence in the host.Taken together,in this study we find Hamiltonella specifically infects MEAM1 and MED,and the symbiont plays a significant role in mediating host sex ratio.In addition,the specialized host cell bacteriocyte exhibits a series of cell behaviours that mediate the vertical transmission of its associated symbionts.However,the bacteriocyte was vulnerable to heat stress,which was likely to limit the thermal tolerance and vertical transmission of the associated symbionts under high temperature.Overall,our data provide further insight into the importance of symbionts and bacteriocytes to their hosts.Thus,symbionts and bacteriocytes may be conceived as new targets in the development of new technology for pest management through interrupting their mutualistic relationship with hosts.