We describe the isolation and cloning of two integral membrane protein antigens ofPlasmodiumfakiparum. The antigens were isolated by Triton X-114 temperaturedependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage Agtll-Amp3 cDNA Integral membrane proteins of Plasmodium falciparum sporozoites and merozoites are potential components of a malaria vaccine. One such protein is the precursor to the major merozoite surface antigens (PMMSA), which is proteolytically processed to generate three antigens on the surface of the merozoite (1, 2). Others antigens have been reported to be present on the merozoite surface. Two ofthese are apparently adsorbed as they lack structural features required for anchoring in the membrane (3-5). Other may be integral membrane proteins, although structural studies have yet to be reported (6-8). We describe here a novel approach to the selection of recombinant clones expressing P. falciparum integral membrane protein antigens. Integral membrane proteins were isolated by temperature-dependent phase separation using the nonionic detergent Triton X-114 and blotted onto nitrocellulose. Human MATERIALS AND METHODS Parasites. The origin of P. falciparum isolate FCQ27/PNG (FC27) has been described elsewhere (9). Parasites synchronized by sorbitol treatment were cultured at 0.25% hematocrit with parasitemias ranging from 2% to 7%, washed, and stored as 1-ml packed-cell aliquots at -70°C.Triton X-114 Solubilization and Phase Separation. Triton X-114 solubilization and temperature-dependent phase separation of parasite antigens were performed essentially as described by Bordier (10) and adapted in this instance as follows. Triton X-114 was precondensed in human tonicity phosphate-buffered saline (HTPBS; 137 mM NaCl/2.7 mM KCl/8.1 mM sodium phosphate, pH 7.2). A 1-ml aliquot of pelleted parasitized erythrocytes was solubilized in 15 ml of 0.5% Triton X-114 for 90 min on ice, with mild mixing at 10-min intervals. A 1-ml sample of the total material was removed and snap-frozen. The remaining 15 ml was then centrifuged at 10,000 x g for 15 min at 4°C to remove insoluble material. The supernatant was collected, and the centrifugation step was repeated. The insoluble pellet material was washed three times in 0.5% Triton X-114 and then frozen. The remaining 15 ml of detergent-soluble material was carefully layered over a cold 10-ml sucrose cushion (6% sucrose/0.06% Triton X-114) in a 50-ml tube with minimal disruption to the interface and placed in a 37°C warm room for 5 min. The tube was then transferred to a centrifuge in the warm room and spun at 500 x g for 5 min. After centrifugation, the 15-ml detergent-depleted upper layer was collected and chilled on ice. The 10-ml sucrose cushion was discarded, and the detergent-enriched pellet (1-2 ml) was resuspended on ice with 10 ml of cold HTPBS. The resuspended detergent-enriched phase wa...

BackgroundThe mosquito Aedes aegypti is the main vector of dengue, Zika, chikungunya and yellow fever viruses. This major disease vector is thought to have arisen when the African subspecies Ae. aegypti formosus evolved from being zoophilic and living in forest habitats into a form that specialises on humans and resides near human population centres. The resulting domestic subspecies, Ae. aegypti aegypti, is found throughout the tropics and largely blood-feeds on humans.ResultsTo understand this transition, we have sequenced the exomes of mosquitoes collected from five populations from around the world. We found that Ae. aegypti specimens from an urban population in Senegal in West Africa were more closely related to populations in Mexico and Sri Lanka than they were to a nearby forest population. We estimate that the populations in Senegal and Mexico split just a few hundred years ago, and we found no evidence of Ae. aegypti aegypti mosquitoes migrating back to Africa from elsewhere in the tropics. The out-of-Africa migration was accompanied by a dramatic reduction in effective population size, resulting in a loss of genetic diversity and rare genetic variants.ConclusionsWe conclude that a domestic population of Ae. aegypti in Senegal and domestic populations on other continents are more closely related to each other than to other African populations. This suggests that an ancestral population of Ae. aegypti evolved to become a human specialist in Africa, giving rise to the subspecies Ae. aegypti aegypti. The descendants of this population are still found in West Africa today, and the rest of the world was colonised when mosquitoes from this population migrated out of Africa. This is the first report of an African population of Ae. aegypti aegypti mosquitoes that is closely related to Asian and American populations. As the two subspecies differ in their ability to vector disease, their existence side by side in West Africa may have important implications for disease transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0351-0) contains supplementary material, which is available to authorized users.

Aedes aegypti (Linnaeus) and Aedes albopictus Skuse mosquitoes transmit serious human arboviral diseases including yellow fever, dengue and chikungunya in many tropical and sub-tropical countries. Females of the two species have adapted to undergo preimaginal development in natural or artificial collections of freshwater near human habitations and feed on human blood. While there is an effective vaccine against yellow fever, the control of dengue and chikungunya is mainly dependent on reducing freshwater preimaginal development habitats of the two vectors. We show here that Ae. aegypti and Ae. albopictus lay eggs and their larvae survive to emerge as adults in brackish water (water with <0.5 ppt or parts per thousand, 0.5–30 ppt and >30 ppt salt are termed fresh, brackish and saline respectively). Brackish water with salinity of 2 to 15 ppt in discarded plastic and glass containers, abandoned fishing boats and unused wells in coastal peri-urban environment were found to contain Ae. aegypti and Ae. albopictus larvae. Relatively high incidence of dengue in Jaffna city, Sri Lanka was observed in the vicinity of brackish water habitats containing Ae. aegypti larvae. These observations raise the possibility that brackish water-adapted Ae. aegypti and Ae. albopictus may play a hitherto unrecognized role in transmitting dengue, chikungunya and yellow fever in coastal urban areas. National and international health authorities therefore need to take the findings into consideration and extend their vector control efforts, which are presently focused on urban freshwater habitats, to include brackish water larval development habitats.

Global climate change can potentially increase the transmission of mosquito vector-borne diseases such as malaria, lymphatic filariasis, and dengue in many parts of the world. These predictions are based on the effects of changing temperature, rainfall, and humidity on mosquito breeding and survival, the more rapid development of ingested pathogens in mosquitoes and the more frequent blood feeds at moderately higher ambient temperatures. An expansion of saline and brackish water bodies (water with <0.5 ppt or parts per thousand, 0.5–30 ppt and >30 ppt salt are termed fresh, brackish, and saline respectively) will also take place as a result of global warming causing a rise in sea levels in coastal zones. Its possible impact on the transmission of mosquito-borne diseases has, however, not been adequately appreciated. The relevant impacts of global climate change on the transmission of mosquito-borne diseases in coastal zones are discussed with reference to the Ross–McDonald equation and modeling studies. Evidence is presented to show that an expansion of brackish water bodies in coastal zones can increase the densities of salinity-tolerant mosquitoes like Anopheles sundaicus and Culex sitiens, and lead to the adaptation of fresh water mosquito vectors like Anopheles culicifacies, Anopheles stephensi, Aedes aegypti, and Aedes albopictus to salinity. Rising sea levels may therefore act synergistically with global climate change to increase the transmission of mosquito-borne diseases in coastal zones. Greater attention therefore needs to be devoted to monitoring disease incidence and preimaginal development of vector mosquitoes in artificial and natural coastal brackish/saline habitats. It is important that national and international health agencies are aware of the increased risk of mosquito-borne diseases in coastal zones and develop preventive and mitigating strategies. Application of appropriate counter measures can greatly reduce the potential for increased coastal transmission of mosquito-borne diseases consequent to climate change and a rise in sea levels. It is proposed that the Jaffna peninsula in Sri Lanka may be a useful case study for the impact of rising sea levels on mosquito vectors in tropical coasts.

The four main Plasmodium species that cause human malaria, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, are transmitted between humans by mosquito vectors belonging to the genus Anopheles. It has recently become evident that Plasmodium knowlesi, a parasite that typically infects forest macaque monkeys, can be transmitted by anophelines to cause malaria in humans in Southeast Asia. Plasmodium knowlesi infections are frequently misdiagnosed microscopically as P. malariae. Direct human to human transmission of P. knowlesi by anophelines has not yet been established to occur in nature. Knowlesi malaria must therefore be presently considered a zoonotic disease. Polymerase chain reaction is now the definitive method for differentiating P. knowlesi from P. malariae and other human malaria parasites. The origin of P. falciparum and P. vivax in African apes are examples of ancient zoonoses that may be continuing at the present time with at least P. vivax, and possibly P. malariae and P. ovale. Other non-human primate malaria species, e.g., Plasmodium cynomolgi in Southeast Asia and Plasmodium brasilianum and Plasmodium simium in South America, can be transmitted to humans by mosquito vectors further emphasizing the potential for continuing zoonoses. The potential for zoonosis is influenced by human habitation and behavior as well as the adaptive capabilities of parasites and vectors. There is insufficient knowledge of the bionomics of Anopheles vector populations relevant to the cross-species transfer of malaria parasites and the real extent of malaria zoonoses. Appropriate strategies, based on more research, need to be developed for the prevention, diagnosis, and treatment of zoonotic malaria.