Decrease of Mosquito Salivary Gland Proteins after a Blood Meal: An Implication for Pathogenesis of Mosquito Bite Allergy Padet Siriyasatien MD, PhD*, Kuntida Tangthongchaiwiriya MSc* Kanyarat Kraivichian MD*, Surang Nuchprayoon MD, PhD* Apiwat Tawatsin MAppl Sc**, Usavadee Thavara PhD** * Department of Parasitology, Faculty of Medicine, Chulalongkorn University ** National Institute of Health, Department of Medical Sciences, Nonthaburi SalivOlY gland protein profiles of Aedes aegypti (L) and Culex quinquefasciatus (Say) pre- and post- bloodfeeding were analyzed. SDS-PAGE studies before blood feeding of Ae. aegypti demonstrated 8 major polypeptide bands of 20, 35, 37, 42, 45, 47, 70 kDa and a high molecular weight band> 118 kDa, whereas those ofCx. quinquefasciatus demonstrated 9 major polypeptide bands of 20, 26, 36, 38, 45, 47, 49 kDa and 2 high molecular weight bands> 118 kDa. After a blood feeding, salivary gland polypeptides of Ae. aegypti at 35,37,45,47, 70 kDa and high molecular weight band> 118 kDa were depleted, while the polypeptide bands of 20, 26, 36, 38 kDa were depleted in Cx. quinquefasciatus. The presented study suggests that these major polypeptides were introduced into vertebrate hosts when a mosquito took a blood meal, Further investigation in molecular, biochemical and immunological aspects of these salivOlY gland polypeptides may provide information for better understanding in the role of these proteins in mosquito bite allergy. Keywords: Mosquito bite allergy, Aedes aegypti, Culex quinquefasciatus, Mosquito saliva!)' gland protein J Med Assoc Thai 2005; 88(Suppl 4): S255-9 Full text. e-Journal: http://www.medassocthai.orgljournal Dennal allergyto mosquitobites isa common salivary glands of several mosquito species have been problemworldwide. Although in most cases of mos- investigated(7,12o20),changes of salivary gland protein quito bites elicit mild symptoms such as cutaneous post blood feeing using SDS-PAGE was demonstrated reactions, systemic reactions including generalized only in Annigeres (Ar.) subalbatus (Coquillett) mos- urticaria and angioedema, rhinitis, conjunctivitis, quito(2O). asthmahave been documented(l.3).Anaphylactic shock In Thailand, Aedes (Ae.) aegypti (L.) and Culex following mosquito bites also has been reported(4). (Cx.) quinquefasciatus (Say) mosquitoes are the most These reactions are caused by proteins in the mos- important mosquito species distributed throughout the quito saliva and involved in IgE, IgG I and IgG4 country. Ae. aegypti is the most important endophagic, responses and lymphocyte proliferation(S,6). daylight-bite mosquito and plays a major role of dengue Mosquito saliva contains a-glucosidases and virus transmission. Cx. quinquefasciatus is exophagic, a-amylases that initiate the digestion of carbohydrates night-bite mosquito found mainly in urbanized areas. present in dietary carbohydrate sources and other Mosquito bite allergy is a common problem found in enzymes and peptides involved in blood feeding and clinical practice especially in children. Despite this, only ingestion such as anticoagulants, vasodilators, and a few reports in which modem laboratory techniques plateletaggregation inhibitors(7.8).The saliva also con- . have been applied to the study of mosquito allergy in tains molecules that provoke a humoral and cellular Thailand2I. In the present study the authors would like immune response in the vertebrate host<9oll).Although to detennine the major polypeptides which were related to blood feeding of Ae. aegypti and Cx, quinquefascia- Correspondence to : Siriyasatien P. Department of Parasito- logy, Faculty of Medicine, Chulalongkorn University, Bangkok tus by SDS-PAGE. This would provide crucial infonna- 10330, Thailand. tion for further investigation in mosquito bite allergy. J Med Assoc Thai Vol.88 Suppl.4 2005 S255 Material and Method demonstrated 8 major polypeptide bands of20, 35,37. Mosquito rearing 42,45,47, 70 kDa and a high molecular weight band Ae. aegypti and Cx. quinquefasciatus mos- > 118 kDa. After a blood meal, the depletion of major quitoes were raised in an insectary at the Experimental peptide bands of 35, 37, 45, 47, 70 kDa and high Animal Unit, Faculty of Medicine, Chulalongkorn molecular weight band> 118 kDa was observed (Fig. I). University. Briefly, after the emergence as adults, the Study in Cx. quinquefasciatus found 9 major mosquitoes were reared in insectariums at 28QC:J:1QC, polypeptide bands of20, 25, 36, 38, 45, 47, 49 ilia and 2 80% :J:5% relative humidity under 12/12 hours light! high molecular weight hands> 118 kDa, the polypep- dark photo-period. Adults were supplied with a damp tide bands of20,26, 36 and 38 kDs were depleted after a cotton wool pad which contained 10% sucrose solu- blood feeding (Fig. 2). tion as a carbohydrate source until used. Discussion Mosquito blood feeding Morphology of Ae. aegypti and Cx. quin- Female mosquitoes were allowed to feed on quefasciatus from the present study is similar to the anaesthetized mice for 30 minutes. Groups of mosqui- pattern described for Ae. aegypti(I3.14),Ae. albopictus toes were reared simultaneously from the same cohort (Skuse)Cl31,Aetogoi(TheobaldYI9), Cx. pipiens (L),Ae. of eggs. Adult mosquitoes aged 4 to 5 days after emer- caspius (PallasY1S),and Ar. subalbatus(2O).The female gence were used. gland is composed of two identical lateral lobes and a shorter and wider median lobe. The lateral lobes could Mosquito salivary gland extraction be further divided into two regions, proximal aDddistal. Mosquito salivary gland extracts were pre- The salivary gland protein profile of Ae. pared from 5 days old femalemosquitoes. Mosquitoes aegypti and Cx. quinquefasciatus mosquito showed a were anaesthetized on ice and salivary gland dissec- tion was performed as in the method described by Suwan et al. (20v2)OSJ.Mosquitosalivary glands were M 1 2 then transferred to a microcentrifugetube containing a kDa ~inal1volume of PBS (phosphate buffer saline solu- tion) and kept at -70QCuntil used. 118 85 SDS-PA GE Analysis SDS-PAGE was performed according to 47 Laemmli (1970)(22)and the proteins were stained using a Coomassie Brilliant Blue (phastGelTMBlue R) accord- ing to the manufacturer's instruction. Twenty pairs of mosquito salivary glands were used for each sample. 36 and each experiment was repeated three times. 26 Results Morphology of mosquito salivary glands 20 The salivary glands offemaleAe. aegypri and Cx. quinquefasciatus are paired organs, located in the Fig.! Protein electrophoretic profile of salivary thorax. The gland is composed of two identical lateral glands ofAedes aegypti mosquitoes. Proteins lobes and a shorter and wider median lobe. The lateral were separated on a 12% SDS-PAGEgeland lobes could be further divided into two regions, proxi- Commasse Brilliant Blue stained. Lane I, mal and distal. Salivary glands of these two mosquito twenty pairs of salivary glands of female species are undistinguishable morphologically (data mosquitoes at day 5 after emergence (sugar not shown). feeding); Lane 2, twenty pairs of salivary glands of female mosquitoes dissected SDS-PAGEAnalysis immediatelyaftera bloodmeal; M: Molecular SDS-PAGEanalysisofsalivaryglandproteins weights markers of sizes (kDa) indicatedon of female Ae. aegypti mosquito pre-blood feeding the left side of the picture S256 J Med Assoc Thai VoL88 SuppL4 2005 1 2 was shared by all five Aedes species and also Cx. quinquefasciatus mosquitolS). In the present study the authors also found the 37 kDa salivary gland protein in 118 Aedes aegypti mosquito and this protein was depleted after blood feeding. 85, . N~ciomentoet a1.(2000)andMalafronteet a1.(2003) demonstrated that salivary gland proteins of 47 Cx. quinquefasciatus mosquito had 2 major polypep- tide bands of28.3 and 35.7 kDa, which induced immune response in mice(lI.16).In the present study the authors 36 also showed 36 kDa polypeptide band that related to blood feeding. But were unable to demonstrate deple- tion ofthe 28.3 kDa polypeptide protein in the present study. At present, laboratory diagnosis of mosquito bite allergy using the commercial mosquito extracts Fig.2 Protein electrophoretic profile of salivary prepared from whole mosquitoes are not standardized glands of Culex quinquefasuJtus mosqui- for diagnosis of mosquito allergy<2S).In order to improve toes. Proteins were separated on a 12% SDS- the precision of diagnosis of mosquito allergy, purified PAGEgelandCommasseBrilliantBluestained. mosquito saliva :>hould be developed. The present Lane 1, twenty pairs of salivary glands of study provides data of salivary gland proteins, which female mosquitoes at day 5 after emergence related to blood feeding. Therefore, these proteins may (sugar feeding); Lane 2, twenty pairs of sali- be related to mosquito bite allergy. Further study of vary glands of female mosquitoes dissected these purified or recombinant salivary gland proteins immediately after a blood meal; Molecular would help physicians to diagnose mosquito bite. weights markers of sizes (kDa) indicated on allergy more accurately. the left side of the picture Acknowledgements different pattern. The different protein. profiles are The authors wish to thank all the staff of the foundnot only in different species but also in the same . Department of Parasitology, Faculty of Medicine, mosquitospecies.Study by Moreria et a1.demonstrated Chulalongkorn University. This work was supported thatAnophelesdarlingi (Root) mosquito collected from by the Molecular Biology Project, Faculty of Medicine, differentgeographical