| With global warming and frequent human activity, the whitefly Bemisia tabaci have caused extensive damage to agriculture due to their rapid and widespead invasions around the world in the past 30 years. The emergence of begomoviruses is closely associated with the increased prevalence of the whitefly B. tabaci and it has caused extensive crop losses to casava, tomato, cotton and tabacco. The begomovirus Papaya leaf curl China virus (PaLCuCNV) was a newly isolated monopartite begomovirus without satellite DNA in Guangxi Province of China. Studies have shown that different species of the B. tabaci complex may transmit a given begomovirus with various levels of efficiency or specificity. To study the role of whitefly vectors in the epidemiology of PaLCuCNV, we conducted laboratory experiments to compare the acquisition, retention and transmission of PaLCuCNV by four species of the B. tabaci complex. We also investigated the physiological and molecular mechanisms underlying these differences between MEAM1 and MED. The results are summarized as follows:(1) Comparison of transmission of PaLCuCNV among four cryptic species of the B. tabaci complex.PaLCuCNV DNA was detected in 10% of the tested MED adults with a 30 min acquisition access period (AAP) and in 10% of all the four whitefly species with a 1 h AAP. The acquisition of PaLCuCNV DNA in the whiteflies increased with the AAP. After a 24 h AAP for MEAM1, MED and Asia Ⅱ 7, and a 48 h AAP for Asia 1, all individuals acquired PaLCuCNV DNA. Nearly all of the viruliferous adults of each of the four species retained PaLCuCNV DNA for up to 4-5 days after transfering to cotton. Thereafter, the proportions of adults with virus DNA in MED, Asia 1 and Asia Ⅱ 7 declined steadily, down to zero by 25 days in Asia 1 and 50-60% by 40 days in both MED and Asia Ⅱ 7. In contrast, the proportion of MEAM1 individuals with virus DNA remained at 100% until 30 days post virus acquisition. For each of the four whitefly species, transmission of PaLCuCNV was achieved with a single adult per plant, and the efficiency of virus transmission increased when the number of adults per plant increased to 5 or 10. However, with a single adult per plant the levels of successful transmission were below 10% by both MED and Asia 1, only 33% by Asia Ⅱ 7, but reached 80% by MEAM1. Furthermore, the relative virus concentration was the highest in MEAMl, followed by Asia Ⅱ 7 and MED, and finally by Asia 1. Our data showed that all the four cryptic species of the B. tabaci complex tested in this study were able to acquire, retain and transmit PaLCuCNV, but the transmission efficiency varied substantially with the whitefly species. MEAM1 transmitted PaLCuCNV to tomato with substantially higher efficiency than did MED, Asia 1 and Asia Ⅱ 7, while Asia Ⅱ 7 was a slightly more efficient vector of this virus than MED and Asia 1. In addition, the data indicated that the differences in the ability of PaLCuCNV transmission between the whitefly species were positively associated with the amount of virus in their body.(2) The physiological and molecular mechanisms underlying the transmission differences between MEAM1 and MED.We examined the ability of MEAM1 and MED whiteflies continously feeding on virus-infected tomatoes for a week to transmit PaLCuCNV. Our data showed that the transmission efficiency was only 5% by MED, but reached 93% by MEAM1. Also, the amount of virus in MEAM1 was significantily higher than that in MED. In addition, more virus was detected in the honeydew of MED. These data suggested that the reason underlining the low efficiency of MED whiteflies to transmit PaLCuCNV was due to their low efficiency to acquire the virus from its digestive system rather than to ingest less virus from the pholem of the plant than MEAM1 did. So the higher amount of virus in MEAM1 may be due to the higher efficiency of PaLCuCNV to enter MEAM1 body from the digestive system. We next examined whether potential barriers inside the whitefly body restricted the PaLCuCNV passage by quantitive PCR and immunofluorescence staining of PaLCuCNV in different whitefly tissues along the virus transmission pathway. The results showed that the relative amount of PaLCuCNV in midgut, hemolymph and head plus thorax of MEAM1 were significantly higher than that in MED. This suggested that the efficiency for the virus to enter and cross the midgut of MED was much lower than that to enter and cross the midgut of MEAM1. To investigate whether viral CP is involved in the specificity of virus entering and crossing the midgut, we exchanged a partial CP sequence of TYLCV which has lower similarity with that of PaLCuCNV and examined the acquisition of recombinant virus in MEAM1 and MED whiteflies. After replacing part the PaLCuCNV CP by TYLCV CP, both MEAM1 and MED could efficiently acquire the mutant PaLCuCNV and the viral signal of mutant PaLCuCNV in the MED midgut was as strong as that in MEAM1. However, after replacing part the TYLCV CP by PaLCuCNV CP, MEAM1 acquired the mutant TYLCV more than MED did and the viral signal of mutant TYLCV in the MED midgut was much less than that in MEAM1. These results showed that the ability for PaLCuCNV to cross midgut of MED is weak and the specificity of begomovirus transmission is determined jointly by the whitefly midgut and PaLCuCNV CP.In summary, this study investigated the acquisition, retention and transmission ablities of PaLCuCNV by four species of the B. tabaci complex. In addition, the physiological and molecular mechanisms underlying the accumulation differences between MEAM1 and MED were elucidated. This could lead to the development of feasible strategies for controlling PaLCuCNV in the field and it also deepened our understanding of the interactions between whiteflies and begomoviruses. |
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