Alongkot Ponlawat

Blood Feeding Patterns of Aedes aegypti and Aedes albopictus in Thailand

Abstract Aedes aegypti (L.) and Aedes albopictus (Skuse) were collected with aspirators from Mae Sot, Nakhon Sawan, Nakhon Ratchasima, Surat Thani, and Phatthalung study sites in Thailand from July 2003 though April 2004. The sandwich-B enzyme-linked immunosorbent assay was used to analyze 1,021 blood-fed specimens. Ae. aegypti almost exclusively fed on humans (99%, 658/664) in single host species, and 97% (86/88) of multiple-host bloodmeals included at least one human host. A low frequency of other hosts, including bovine, swine, cat, rat, and chicken were detected, but they represented <1% of bloodmeals. An even higher percentage of human feeding was detected in Ae. albopictus. Hosts of Ae. albopictus collected from sites in southern Thailand were entirely human (100%, n = 105) from both single and mixed meals. In the small number of double-host meals from Ae. albopictus, we detected 3.8% as swine–human and <1% from dog–human and cat–human. Forage ratios for Ae. aegypti indicated that human, dog, and swine were preferred hosts in order of preference. In contrast, bovine and chicken were avoided hosts for this species in Thailand.

Insecticide Susceptibility of Aedes aegypti and Aedes albopictus across Thailand

Abstract Aedes aegypti (L.) and Aedes albopictus (Skuse), two important vectors of dengue fever and dengue hemorrhagic fever, were collected from Mae Sot, Nakhon Sawan, Nakhon Ratchasima, Surat Thani, and Phatthalung, Thailand, from July 2003 to April 2004. The patterns of insecticide susceptibility to temephos, malathion, and permethrin of both Ae. aegypti and Ae. albopictus larvae were determined. Ae. aegypti from all study sites were resistant to permethrin, they but were susceptible to malathion. Resistance to temephos was detected in all strains of Ae. aegypti, except those from Nakhon Ratchasima. Ae. albopictus larvae had low levels of resistance to all three insecticides, except Mae Sot and Phatthalung strains, which were resistant to permethrin.

Worldwide patterns of genetic differentiation imply multiple ‘domestications’ of Aedes aegypti, a major vector of human diseases

Understanding the processes by which species colonize and adapt to human habitats is particularly important in the case of disease-vectoring arthropods. The mosquito species Aedes aegypti, a major vector of dengue and yellow fever viruses, probably originated as a wild, zoophilic species in sub-Saharan Africa, where some populations still breed in tree holes in forested habitats. Many populations of the species, however, have evolved to thrive in human habitats and to bite humans. This includes some populations within Africa as well as almost all those outside Africa. It is not clear whether all domestic populations are genetically related and represent a single ‘domestication’ event, or whether association with human habitats has developed multiple times independently within the species. To test the hypotheses above, we screened 24 worldwide population samples of Ae. aegypti at 12 polymorphic microsatellite loci. We identified two distinct genetic clusters: one included all domestic populations outside of Africa and the other included both domestic and forest populations within Africa. This suggests that human association in Africa occurred independently from that in domestic populations across the rest of the world. Additionally, measures of genetic diversity support Ae. aegypti in Africa as the ancestral form of the species. Individuals from domestic populations outside Africa can reliably be assigned back to their population of origin, which will help determine the origins of new introductions of Ae. aegypti.

Infectivity of Asymptomatic Plasmodium-Infected Human Populations to Anopheles dirus Mosquitoes in Western Thailand

Abstract The infectivity of Plasmodium-infected humans in western Thailand was estimated by feeding laboratory-reared Anopheles dirus Peyton and Harrison mosquitoes on venous blood placed in a membrane-feeding apparatus. Between May 2000 and November 2001, a total of 6,494 blood films collected during an active malaria surveillance program were checked by microscopy for the presence of Plasmodium parasites: 3.3, 4.5, and 0.1% of slides were P. falciparum- (Pf), P. vivax- (Pv), and P. malariae (Pm)-positive. Venous blood was collected from 70, 52, 6, and 4 individuals infected with Pf, Pv, Pm, and mixed Pf/Pv, respectively, with 167 uninfected individuals serving as negative controls. Only 10% (7/70), 13% (7/52), and 0% (0/6) of membrane feeds conducted on Pf-, Pv-, and Pm-infected blood yielded infected mosquitoes. One percent (2/167) of microscope-negative samples infected mosquitoes; however, both samples were subsequently determined to be Pf-positive by polymerase chain reaction. Gametocytes were observed in only 29% (4/14) of the infectious samples. All infections resulted in low oocyst loads (average of 1.2 oocysts per positive mosquito). Only 4.5% (10/222) of mosquitoes fed on the seven infectious Pf samples developed oocysts, whereas 2.9% (9/311) of mosquitoes fed on the seven infectious Pv samples developed oocysts. The probability of a mosquito becoming infected with Pf or Pv after a blood meal on a member of the human population in Kong Mong Tha was estimated to be 1 in 6,700 and 1 in 5,700, respectively. The implications toward malaria transmission in western Thailand are discussed.

Age and Body Size Influence Male Sperm Capacity of the Dengue Vector Aedes aegypti (Diptera: Culicidae)

Abstract Understanding mosquito mating biology is essential for studies of mosquito behavior, gene flow, population structure, and genetic control. In the current study, we examine the effect of age and body size on spermatozoa number in two laboratory strains of the dengue vector, Aedes aegypti (L.), Thailand and Rockefeller (ROCK), and in wild-collected mosquitoes from Thailand. Body size was a major predictor of total spermatozoa number, with significantly greater sperm numbers in large (2.27-mm wing length) versus small males (1.85-mm wing length) within the same age group. Total sperm capacity also varied by male age. Spermatozoa numbers in virgin Ae. aegypti males increased significantly up to 10 d after emergence and then leveled off until 20 d. Significant variations in sperm number were detected among Ae. aegypti strains, with wild-collected mosquitoes having the greatest total number of sperm. Our study provides the first evidence of spermatogenesis in adult mosquitoes and indicates high rates of spermatogenesis in male mosquitoes up to 10 d of age (3.3 degree-days). Our results emphasize the potential role of body size and age on the mating capacity of this important vector of dengue and yellow fever viruses.

Dengue-1 Virus Clade Replacement in Thailand Associated with Enhanced Mosquito Transmission

ABSTRACT Dengue viruses (DENV) are characterized by extensive genetic diversity and can be organized in multiple, genetically distinct lineages that arise and die out on a regular basis in regions where dengue is endemic. A fundamental question for understanding DENV evolution is the relative extent to which stochastic processes (genetic drift) and natural selection acting on fitness differences among lineages contribute to lineage diversity and turnover. Here, we used a set of recently collected and archived low-passage DENV-1 isolates from Thailand to examine the role of mosquito vector-virus interactions in DENV evolution. By comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to be transmitted by a present-day Aedes aegypti population from Thailand, we found that a major clade replacement event in the mid-1990s was associated with virus isolates exhibiting increased titers in the vectors hemocoel, which is predicted to result in a higher probability of transmission. This finding is consistent with the hypothesis that selection for enhanced transmission by mosquitoes is a possible mechanism underlying major DENV clade replacement events. There was significant variation in transmission potential among isolates within each clade, indicating that in addition to vector-driven selection, other evolutionary forces act to maintain viral genetic diversity. We conclude that occasional adaptive processes involving the mosquito vector can drive major DENV lineage replacement events.

Genetic Mapping of Specific Interactions between Aedes aegypti Mosquitoes and Dengue Viruses

Specific interactions between host genotypes and pathogen genotypes (G×G interactions) are commonly observed in invertebrate systems. Such specificity challenges our current understanding of invertebrate defenses against pathogens because it contrasts the limited discriminatory power of known invertebrate immune responses. Lack of a mechanistic explanation, however, has questioned the nature of host factors underlying G×G interactions. In this study, we aimed to determine whether G×G interactions observed between dengue viruses and their Aedes aegypti vectors in nature can be mapped to discrete loci in the mosquito genome and to document their genetic architecture. We developed an innovative genetic mapping strategy to survey G×G interactions using outbred mosquito families that were experimentally exposed to genetically distinct isolates of two dengue virus serotypes derived from human patients. Genetic loci associated with vector competence indices were detected in multiple regions of the mosquito genome. Importantly, correlation between genotype and phenotype was virus isolate-specific at several of these loci, indicating G×G interactions. The relatively high percentage of phenotypic variation explained by the markers associated with G×G interactions (ranging from 7.8% to 16.5%) is consistent with large-effect host genetic factors. Our data demonstrate that G×G interactions between dengue viruses and mosquito vectors can be assigned to physical regions of the mosquito genome, some of which have a large effect on the phenotype. This finding establishes the existence of tangible host genetic factors underlying specific interactions between invertebrates and their pathogens in a natural system. Fine mapping of the uncovered genetic loci will elucidate the molecular mechanisms of mosquito-virus specificity.

Genetic Characterization of Spondweni and Zika Viruses and Susceptibility of Geographically Distinct Strains of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae) to Spondweni Virus.

Background Zika virus (ZIKV) has extended its known geographic distribution to the New World and is now responsible for severe clinical complications in a subset of patients. While substantial genetic and vector susceptibility data exist for ZIKV, less is known for the closest related flavivirus, Spondweni virus (SPONV). Both ZIKV and SPONV have been known to circulate in Africa since the mid-1900s, but neither has been genetically characterized by gene and compared in parallel. Furthermore, the susceptibility of peridomestic mosquito species incriminated or suspected in the transmission of ZIKV to SPONV was unknown. Methodology/Principal Findings In this study, two geographically distinct strains of SPONV were genetically characterized and compared to nine genetically and geographically distinct ZIKV strains. Additionally, the susceptibility of both SPONV strains was determined in three mosquito species. The open reading frame (ORF) of the SPONV 1952 Nigerian Chuku strain, exhibited a nucleotide and amino acid identity of 97.8% and 99.2%, respectively, when compared to the SPONV 1954 prototype South African SA Ar 94 strain. The ORF of the SPONV Chuku strain exhibited a nucleotide and amino acid identity that ranged from 68.3% to 69.0% and 74.6% to 75.0%, respectively, when compared to nine geographically and genetically distinct strains of ZIKV. The ORF of the nine African and Asian lineage ZIKV strains exhibited limited nucleotide divergence. Aedes aegypti, Ae. albopictus and Culex quinquefasciatus susceptibility and dissemination was low or non-existent following artificial infectious blood feeding of moderate doses of both SPONV strains. Conclusions/Significance SPONV and ZIKV nucleotide and amino acid divergence coupled with differences in geographic distribution, ecology and vector species support previous reports that these viruses are separate species. Furthermore, the low degree of SPONV infection or dissemination in Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus following exposure to two geographically and genetically distinct virus strains suggest a low potential for these species to serve as vectors.

Differential Susceptibility of Two Field Aedes aegypti Populations to a Low Infectious Dose of Dengue Virus

Background The infectious dose required to infect mosquito vectors when they take a blood meal from a viremic person is a critical parameter underlying the probability of dengue virus (DENV) transmission. Because experimental vector competence studies typically examine the proportion of mosquitoes that become infected at intermediate or high DENV infectious doses in the blood meal, the minimum blood meal titer required to infect mosquitoes is poorly documented. Understanding the factors influencing the lower infectiousness threshold is epidemiologically significant because it determines the transmission potential of humans with a low DENV viremia, possibly including inapparent infections, and during the onset and resolution of the viremic period of acutely infected individuals. Methodology/Principal Findings We compared the susceptibility of two field-derived Aedes aegypti populations from Kamphaeng Phet, Thailand when they were orally exposed to low titers of six DENV-2 isolates derived from the serum of naturally infected humans living in the same region. The infectious dose, time-point post-blood feeding, viral isolate and mosquito population, were significant predictors of the proportion of mosquitoes that became infected. Importantly, the dose-response profile differed significantly between the two Ae. aegypti populations. Although both mosquito populations had a similar 50% oral infectious dose (OID50), the slope of the dose-response was shallower in one population, resulting in a markedly higher susceptibility at low blood meal titers. Conclusions/Significance Our results indicate that mosquitoes in nature vary in their infectious dose-response to DENV. Thus, different mosquito populations have a differential ability to acquire DENV infection at low viremia levels. Future studies on human-to-mosquito DENV transmission should not be limited to OID50 values, but rather they should be expanded to account for the shape of the dose-response profile across a range of virus titers.

Evaluation of the VecTest Malaria Antigen Panel Assay for the Detection of Plasmodium falciparum and P. vivax Circumsporozoite Protein in Anopheline Mosquitoes in Thailand

Abstract We evaluated the performance of the VecTest Malaria Antigen Panel (V-MAP) assay for the detection of Plasmodium falciparum and P. vivax (variants 210 and 247) circumsporozoite protein in anopheline mosquitoes in Thailand. The V-MAP assay is a rapid, one-step procedure using a ‘dipstick’ wicking test strip. The circumsporozoite (CS) ELISA was used as the reference standard. Mosquitoes evaluated in the study included field-collected specimens (n = 930) and laboratory-reared specimens that had been fed on blood collected from patients with and without Plasmodium gametocytes (n = 4,110) or on cultured P. falciparum gametocytes (n = 262). Field-collected mosquitoes were triturated individually or in pools of 2–5 and tested using 613 V-MAP assays. Laboratory-reared specimens were tested individually using 4,372 V-MAP assays. Assay performance depended on the species of Plasmodium and the number of sporozoites used as the cut-off. For P. falciparum, optimal performance was achieved using a cut-off of 150 sporozoites (sensitivity = 100%, specificity = 99.2%, and accuracy = 0.99). For P. vivax variant 210, optimal performance was also achieved using a cut-off of 150 sporozoites (sensitivity = 94.8%, specificity = 94.5%, and accuracy = 0.95). We were unable to develop a standard-curve for the CS-ELISA using P. vivax variant 247 because of a lack of sporozoites; however, using a cut-off of 30 pg P. vivax 247 antigen (mosquitoes with less than this amount of antigen were considered negative), assay performance (sensitivity = 94.3%, specificity = 99.2%, and accuracy = 0.99) was comparable to that achieved for P. falciparum and P. vivax 210. These results clearly demonstrate that the V-MAP assay performs at an acceptable level and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.