The Screening Of Aedes Albopictus With Deltamethrin And The Study Of Its Susceptibility To Dengue Virus

Aedes Albopicutos is an important vector for dengue virus,causing frequent outbreak of dengue fever due to the wide spreading in most areas of China.In the absence of advanced vaccine or drug,the control of the vector using chemical insectcides,such as the most used deltamethrin,in the aim of killing Aedes Albopicutos is the major method to prevent this disease from prevailing.The massive administration of chemical insecticide has present a screening pressure on the tolerance of Aedes Albopicutos to deltamethrin,which causes series changes physically,biochemically or genetically.It was reported that the resistant Anopheles gambiae can compromise the transmission of Plasmodium falciparum by affecting the development of its sporozoite.There is no report on the aspects of the infection or dissemination of dengue virus inside the resistant Aedes Albopictus.This study established an Aedes Albopictus resistant strain using deltamethrin in the laboratory and focused on the susceptibility of it to dengue virus.Objective:1.To establish a lab origined Aedes Albopictus resistant strain using deltamethrin 2.To determine the susceptibilities of resistant and susceptible Aedes Albopictus to type2 dengue virusMethods:1.Selection of resistant population Larvae immersion method were employed to determine LC50 of the larvae according to the recommendation of World Health Organization.This concentration was then applied to 1500 forth-instart larvae.The survived were collected to rear to adults,fed on mouse blood and allowed to lay eggs.This procedure was performed on every generation.Susceptibility of adults was tested with WHO tube test with 0.05%deltamethrin soaked filter paper,performed according to standard procedure.2.Enrich dengue virus 2 in C6/36 cell and infect resistant and susceptible Aedes Albopictus through oral feeding with a virus titer of 106.83 TCID50/mL.3.Use immunofluorescence to detect the existence of type2 dengue virus in tissues preliminarily.4.Dissect resistant and susceptible Aedes Albopictus on 0/4/7/10 dpi to get midguts、ovaries and salivary glands,followed by extraction of total RNA,reverse transcription and PCR.Using RT-qPCR to determin the quatity of virus.Results:1.After 9 generations of selection,the resistance of Aedes Albopictus larvae was 0.053mg/L and the resistance ratio(RR)was 10.The 13th,0.052mg/L.RR of the 9th to 13th generation remained between 10-11.2.The adults resistant level of the 9th resistant generation was 80%in mortality according to the tube testing.The 13th,67%.3.The green fluorescence was observed under the fluorescence microscope on 4/7/10 dpi in midguts、on 7/10 dpi in ovaries and salivary glands of both the resistant and susceptible Aedes Albopictus.4.On 0 dpi,100%of the midgut samples were positive for the test of dengue virus in resistant and susceptible Aedes Albopictus strains.The infection rate of midgut did not vary with the changing of time(P>0.05),with an infection rate between 92.75%～97.18%.The infection rates of ovaries was increased(P<0.05)from 36.11%on day 4 to 84.72%on day 10,the susceptible strain,and 38.89%on day 4 to 86.11%on day 10,the resistant strain.while the differences of each tissue at each time point between the two strains were not significant(P>0.05).The infection rates of salivary glands was 80.56%and 83.33%in the resistant and susceptible strain on day 7.Neither of them was increased on day 10.While the differences of infection rates of ovaries or salivary glands at each time point between the two strains showed no significance(P>0.05).5.The resistant and susceptible Aedes Albopictus strains has significant virus copy difference in the midguts on day 1 post infection and ovaries on day4/7/10 post infection(In midguts P=0.005;In ovaries 4dpi,P=0.012;7dpi,P=0.001;10dpi P=0.006)The increase of the quantities of virus were most distinct in the ovaries,in which the resistant strain had higher virus amount than the susceptible ones through all the tested time point.No significance was observed in salivary glands,though both of their virus numbers went up with time.Conclusion:1.the systematic application of deltamethrin could resulte in the increase of the resistance in larvae and adult.A stable moderate resistant strain was established in the laboratory.2.The infection rates increased differently in different tissue.No difference was observed between the resistant and susceptible Aedes Albopictus.3.The midgut of the resistant strain on dayO with a higher load of virus might due to a stronger obligation of blood ingestion.The higher amount of virus in the ovaries of the resistant strain indicated that this tissue might favor the prolification of virus.The susceptibility of salivary glands to dengue virus were not changed.