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Mosquitoes After Infection with Dengue Virus Type 2 (D2-16681)

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Mosquitoes After Infection with Dengue Virus Type 2 (D2-16681) Appl. Entomol. Zool. 44 (2): 257–265 (2009) http://odokon.org/ Peritrophic membrane structure of Aedes aegypti (Diptera: Culicidae) mosquitoes after infection with dengue virus type 2 (D2-16681) Supattra SUWANMANEE,1,† Urai CHAISRI,1 Ladawan WASINPIYAMONGKOL2 and Natthanej LUPLERTLOP2,* 1 Department of Tropical Pathology, and 2 Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University; Bangkok 10400, Thailand (Received 24 September 2008; Accepted 3 December 2008) Abstract The peritrophic membrane (PM) is a non-cellular tissue involved in the protection of midgut epithelium from mechan- ical damage and insults from pathogens. This study was carried out to determine the involvement of PM in mosqui- toes after infection with dengue virus. Aedes aegypti (Diptera: Culicidae) mosquitoes were fed sucrose and human blood with and without dengue virus type 2 (D2-16681), and collected at 0.5, 1, 6, and 12 h, respectively. Specimens were prepared for examination under light and electron microscopy. The results showed that PM was produced only in the blood-fed mosquitoes. The infected blood meal induced the mosquitoes to produce PM in their midgut earlier and thicker than in mosquitoes with blood alone. The initial evidence of PM occurred at 1 h post-blood meal (PBM) as a matrix-like structure. By 6 h PBM, PM had become a layer, which persisted at 12 h. Among mosquitoes fed with blood alone, this structure was found only from 6 and 12 h PBM. Dengue virus type 2 induced different modifications of mosquito PM construction and structures, confirmed under an electron microscope. Key words: Flaviviridae; peritrophic matrix; ultrastructure mosquito is the first site of interaction between the INTRODUCTION mosquito and pathogen and plays an important role Dengue virus is one of the most important in vector competence (Beerntsen et al., 2000). resurging mosquito-borne diseases belonging to Midgut epithelial cells produce the peritrophic the Flaviviridae family of small enveloped viruses. membrane, sometimes called the peritrophic matrix It carries a single-stranded RNA virus, divided into (PM), a non-cellular material that separates the four serotypes: DEN1, DEN2, DEN3, and DEN4. midgut lumen containing the ingested food from Dengue virus infection is prevalent in tropical the midgut epithelium (Jacobs-Lorena and Oo, areas of over 100 countries, with 2.5 billion people 1996; Tellam et al., 1999; Ibrahim et al., 2000; at risk of acquiring the infection. Fifty million esti- Shao et al., 2001). The PM is composed of chitin, mated infections and 500,000 dengue hemorrhagic proteins, and proteoglycans (Wang and Granados, fever (DHF) cases occur annually (Gubler, 2002). 2001) divided into two types: Type I PM is found Dengue infection is caused by the ingestion of in adult hematophagous insects and forms a thick viremic blood containing the virus by Aedes mos- bag-like structure that completely surrounds the in- quitoes followed by passage to a second human gested blood meal. Type II PM is produced in lar- host. Aedes aegypti (Diptera: Culicidae) is the most val and adult (except hematophagous) insects, important vector for dengue transmission, which is forms a thin open-ended tube-like structure and is initiated when the female mosquito ingests an in- produced in a specialized region located at the fective blood meal (Gubler, 2002). The midgut of a junction between the foregut and midgut called the * To whom correspondence should be addressed at: E-mail: [email protected] † Present address: Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchadewee, Bangkok 10400, Thailand. DOI: 10.1303/aez.2009.257 257 258 S. SUWANMANEE et al. cardia (Jacobs-Lorena and Oo, 1996; Tellam et al., tion for 90 min, 5 ml MEM medium (Gibco, USA) 1999). Functions of the PM include protecting the containing 10% fetal bovine serum (FBS) (Gibco) midgut epithelium from mechanical damage, act- and 1% penicillin-streptomycin was added. The ing as a lubricant helping food pass through the culture flask was incubated at 28°C in a 5% CO2 gut, and preventing swelling and rupture of the incubator. Aliquots of the culture medium were midgut after the ingestion of high molecular- harvested seven days after infection and stored at weight foods exerting high osmotic pressure. PM Ϫ80°C until plaque titration and mosquito inocula- serves as a barrier against the entry of pathogens tion. and toxins, prevents the rapid excretion of digestive Viral plaque assay. LLC-MK2 cells were enzymes and also limits the rate of food digestion. seeded in six-well cell culture plates (Corning) at a Moreover, it plays a role in the prevention of non- density of 5.0ϫ105 cells/well and were incubated specific binding of undigested materials to midgut for three to four days at 37°C in 5% CO2 to pro- microvilli surfaces or binding to transport proteins duce a confluent monolayer. Cell monolayers were at the midgut surface (Zhuzhikov, 1964; Terra, inoculated with 10-fold serial dilutions of virus in 1990; Lehane, 1997; Villalon et al., 2003). In addi- a final volume of 0.2 ml. Viral adsorption (from ex- tion, it helps in heme detoxification (Pascoa et al., tracted samples: negative media alone, sucrose, in- 2002), and the assimilation and elimination of fected mosquito and infective blood meal) was al- toxic ammonium ions (Kato et al., 2002). Dengue lowed to proceed for 90 min at 37°C with plate virus can invade and replicate in the midgut epithe- rocking every 15 min. A 3 ml overlay of MEM, 5% lium of Ae. aegypti mosquitoes. The morphology FBS, and 0.6% SEQEM agar was added at the con- of the PM may change after the mosquitoes receive clusion of adsorption. The infected monolayers an insult from this virus. were incubated at 37°C in 5% CO2. Seven days The role of PM formation in dengue invasion of after infection, a second overlay, similar to the first the midgut has not been examined; therefore, the but with the addition of 1.5% neutral red (Sigma- purpose of this study was to examine the involve- Aldrich, UK), was added to the wells, and the ment of PM in midgut of Ae. aegypti mosquitoes plates were incubated at 37°C in 5% CO2 after infection with dengue virus type 2 (D2- overnight. The plaques were counted, and the viral 16681) at different time points. titer was calculated and expressed as pfu/ml. Infection of mosquitoes and verification of dengue infection. Insectary-maintained Ae. ae- MATERIALS AND METHODS gypti mosquitoes were infected by oral feeding Rearing mosquitoes. Ae. aegypti mosquitoes with dengue virus type 2 (D2-16681) at 106 pfu/ml. (white-eyed Liverpool strain) were maintained in Mosquitoes were maintained at 28°C and 70 to an insectary in the Department of Medical Ento- 80% relative humidity. Pools consisting of five mology (Faculty of Tropical Medicine, Mahidol mosquitoes were removed at different time points, University, Bangkok, Thailand). Eggs were hatched 0.5, 1, 6, 12 h post-infection, and frozen for RT- and transferred to the rear of a plastic tray which PCR analysis. Dengue virus in mosquitoes was de- was half-filled with well water and artificial food. tected by RT-PCR and serotype-specific primers by After hatching from the pupa, the adults were nested-PCR. maintained at 27°C with 80% relative humidity RNA extraction. Total RNA was extracted with under a 14 : 10 h light/dark cycle. Adult mosquitoes a QIAmp viral RNA kit (QIAGEN, USA), accord- were offered 10% sucrose. Three- to four-day-old ing to the manufacturer’s suggested protocol. RNA female mosquitoes were collected for the experi- was eluted twice in 40 ml nuclease free water. The ment. extracted solution was stored at Ϫ70°C until evalu- Virus propagation. Dengue virus type 2 strain ated. (D2-16681) was obtained from the Armed Forces Nested RT-PCR assay. Nested RT-PCR of Research Institute of Medical Sciences (AFRIMS) dengue virus RNA was carried out with dengue (Bangkok, Thailand). The virus had been routinely virus consensus and serotype-specific primers, as passaged in C6/36 cells culture performed in a described previously (Lanciotti et al., 1992). Modi- 25 cm2 culture flask (Corning, USA). After adsorp- fications to the procedure were as follows: 5 ml of PM Expression after DEN2 Infected Ae. aegypti 259 RNA in 50 ml reaction volume was used with the Microscopy Sciences, USA). Transverse sections QIAGEN OneStep RT-PCR kit (QIAGEN). RT- of semithin (1 mm thick) were cut by an ultramicro- PCR was carried out according to the manufac- tome, placed on a glass slide, stained with 1% tolu- turer’s instructions with 55°C annealing tempera- idine blue, and examined under a light microscope ture. The resultant PCR product was diluted to to study the entire tissue. When the area of interest 1 : 500 in water. Nested-PCR was carried out with was located, ultrathin sections were cut, picked up 5 ml of the diluted RT-PCR product in 50 ml reac- and placed on copper grids. They were then stained tion volume with the TaqPCR Master Mix kit (QI- with uranyl acetate and lead citrate before exami- AGEN). Initial denaturation of 10 min at 94°C was nation under a transmission electron microscope followed by 25 cycles, each consisting of 94°C for (Hitachi, model H-7000, Japan) at 75 kV. 30 s, 50°C for 30 s, and 72°C for 45 s, followed by Statistical analysis. PM thickness and the size a final extension step of 72°C for 10 min. Both the of midgut epithelial cells (in the anterior region of RT-PCR and nested-PCR products were analyzed the midgut) were accurately measured from micro- by gel electrophoresis on 2% agarose gel contain- graphs using the Image Frame Work program.
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