Mosquitoes of Northwestern Uganda.

Despite evidence of arbovirus activity in northwestern Uganda (West Nile Sub-region), there is very limited information on the mosquito fauna of this region. The only published study reported 52 mosquito species in northwestern Uganda but this study took place in 1950 and the information has not been updated for more than 60 yr. In January and June 2011, CO2 baited-light traps were used to collect 49,231 mosquitoes from four different locations, Paraa (9,487), Chobe (20,025), Sunguru (759), and Rhino Camp (18,960). Overall, 72 mosquito species representing 11 genera were collected. The largest number of distinct species was collected at Chobe (43 species), followed by Paraa (40), Sunguru (34), and Rhino Camp (25). Only eight of the 72 species (11.1%) were collected from all four sites: Aedes (Stegomyia) aegypti formosus (Walker), Anopheles (Cellia) funestus group, Culex (Culex) decens group, Cx. (Culex) neavei Theobald, Cx. (Culex) univittatus Theobald, Cx. (Culiciomyia) cinereus Theobald, Cx. (Oculeomyia) poicilipes (Theobald), and Mansonia (Mansonoides) uniformis (Theobald). Fifty-four species were detected in northwestern Uganda for the first time; however, these species have been detected elsewhere in Uganda and do not represent new introductions to the country. Thirty-three species collected during this study have previously been implicated in the transmission of arboviruses of public health importance.

Mosquitoes of Zika Forest, Uganda: species composition and relative abundance.

Mosquito collections were conducted in Zika Forest near Entebbe, Uganda, from July 2009 through June 2010 using CO2-baited light traps, ovitraps, and human-baited catches. In total, 163,790 adult mosquitoes belonging to 12 genera and 58 species were captured. Of these, 22 species (38%) were captured in Zika Forest for the first time. All the new records found in the forest in this study had previously been captured in other regions of Uganda, implying that they are native to the country and do not represent new introductions. More than 20 species previously collected in Zika Forest were not detected in our collections, and this may suggest a change in the mosquito fauna during the past 40 yr or variation in species composition from year to year. Arboviruses of public health importance have previously been isolated from >50% of the 58 mosquito species captured in Zika Forest, which suggests ahigh potential for transmission and maintenance of a wide range of arboviruses in Zika Forest.

Mosquitoes of western Uganda.

The mosquito fauna in many areas of western Uganda has never been studied and is currently unknown. One area, Bwamba County, has been previously studied and documented but the species lists have not been updated for >40 yr. This paucity of data makes it difficult to determine which arthropod-borne viruses pose a risk to human or animal populations. Using CO2 baited-light traps, from 2008 through 2010, 67,731 mosquitoes were captured at five locations in western Uganda including Mweya, Sempaya, Maramagambo, Bwindi (BINP), and Kibale (KNP). Overall, 88 mosquito species, 7 subspecies, and 7 species groups in 10 genera were collected. The largest number of species was collected at Sempaya (65 species), followed by Maramagambo (45), Mweya (34), BINP (33), and KNP (22). However, species diversity was highest in BINP (Simpson's Diversity Index 1-D = 0.85), followed by KNP (0.80), Maramagambo (0.79), Sempaya (0.67), and Mweya (0.56). Only six species Aedes (Aedimorphus) cumminsii (Theobald), Aedes (Neomelaniconion) circumluteolus (Theobald), Culex (Culex) antennatus (Becker), Culex (Culex) decens group, Culex (Lutzia) tigripes De Grandpre and De Charmoy, and Culex (Oculeomyia) annulioris (Theobald), were collected from all five sites suggesting large differences in species composition among sites. Four species (Aedes (Stegomyia) metallicus (Edwards), Anopheles (Cellia) rivulorum Leeson, Uranotaenia (Uranotaenia) chorleyi (Edwards), and Uranotaenia (Uranotaenia) pallidocephala (Theobald) and one subspecies (Aedes (Stegomyia) aegypti formosus (Walker)) were collected in Bwamba County for the first time. This study represents the first description of the mosquito species composition of Mweya, Maramagambo, BINP, and KNP. A number of morphological variations were noted regarding the postspiracular scales, hind tibia, and sternites that make Culex (Culex) neavei (Theobald) challenging to identify. At least 50 species collected in this study have previously been implicated in the transmission of arboviruses of public health importance suggesting a high potential for maintenance and transmission of a wide variety of arboviruses in western Uganda.

Vector competence of Culex restuans (Diptera: Culicidae) from two regions of Chicago with low and high prevalence of West Nile virus human infections.

Vector competence studies for West Nile virus (WNV) were conducted for two Culex (Culex) restuans Theobald populations Edison Park (EP) and Illinois Medical District (IMD), in Chicago, IL. The aim was to determine if there were differences between mosquito populations that contributed to the observed differences in the prevalence of WNV. Percentages of orally infected, disseminated, and transmitting mosquitoes were estimated using a generalized linear mixed effects model including a random effect for family to account for anticipated within-family correlation. Analysis indicated that percentages of infected, disseminated, and transmitting mosquitoes were not significantly different between EP and IMD. The within-family correlation was 0.46 (95% CI 0.28, 0.67), indicating reasonably strong tendency for WNV titers of bodies, saliva, and legs within families to be similar. Overall, our results show that vector competence of Cx. restuans for WNV is not a contributing factor to the observed differences in WNV human cases between the EP and IMD areas of Chicago.

Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus.

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

Phylogenetic and evolutionary relationships among yellow fever virus isolates in Africa.

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.

Breeding structure of the sand fly Lutzomyia longipalpis (Lutz & Neiva) in Brazil.

Eleven populations of Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of Leishmania chagasi, from different areas of Brazil were analyzed for genetic variation at 16 enzyme loci. In this region, the prevalence of visceral leishmaniasis (VL) caused by L. chagasi is spotty and reproductive isolation among populations of Lu. longipalpis has been reported. It is thought that morphologically similar cryptic species with varying vectorial capacity may be responsible for the discontinuous distribution of VL. The aim was to study the genetic structure of populations within this region and to identify demes that may represent sibling species. Genotypic frequencies within populations were in close compliance to Hardy-Weinberg expectations, suggesting there are no sympatric species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.03), consistent with local populations within a single sand fly species. When genotypic frequency data for all populations were pooled, 9 of the 13 polymorphic loci deviated from Hardy-Weinberg expectations, indicating some degree of genetic substructuring. Estimates of effective migration rates (N(e)m) among all populations were low, 2.73, suggesting that gene flow is restricted among populations, which is probably the reason for the observed genetic substructuring.

Metaphase karyotypes and G-banding in sandflies of the Lutzomyia longipalpis complex.

Mitotic metaphase chromosomes (2n = 8) from brain cells of fourth instar sandfly larvae of four geographical strains of the Lutzomyia longipaplis complex were examined microscopically, with bright-field illumination, after staining by a new G-banding technique involving exposure of air-dried chromosome preparations to quinacrine and ultraviolet light. Differences of G-banding and/or position of the centromere on chromosome 4 (the smallest chromosome pair) distinguished four putative sibling species from Costa Rica, Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves). The karyotype of the population from Jacobina, Brazil, showed an apparently plesiomorphic pattern of G-banding. On the basis of their recognizably different mitotic karyotypes, cytogenetic identification of separate taxa in the L. longipalpis complex should be useful for specific female vector competence and ecology studies.

Genetic variation among natural and laboratory colony populations of Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera: Psychodidae) from Colombia.

Genetic diversity among three field populations of Lutzomyia longipalpis in Colombia was studied using isozyme analysis. Study sites were as much as 598 km apart and included populations separated by the eastern Cordillera of the Andes. Genetic variability among populations, estimated by heterozygosity, was within values typical for insects in general (8.1%). Heterozygosity for field populations were compared with a laboratory colony from Colombia (Melgar colony) and were only slightly lower. These results suggest that establishment and long term maintenance of the Melgar colony has had little effect on the level of isozyme variability it carries. Genetic divergences between populations was evaluated using estimates of genetic distance. Genetic divergence among the three field populations was low (D = 0.021), suggesting they represent local populations within a single species. Genetic distance between field populations and the Melgar colony was also low (D = 0.016), suggesting that this colony population does not depart significantly from natural populations. Finally, comparisons were made between Colombian populations and colonies from Brazil and Costa Rica. Genetic distance values were high between Colombian and both Brazil and Costa Rica colony populations (D = 0.199 and 0.098 respectively) providing additional support for our earlier report that populations from the three countries represent distinct species.

Genetic variability among populations of the sand fly Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) from Central America.

Eleven Central American populations of Lutzomyia longipalpis (Lutz & Neiva) were analyzed for genetic variation at 16 enzyme loci. The aim was to study the genetic structure among populations within this region and to identify demes that may represent different sibling species. Genotypic frequencies within populations agreed with Hardy-Weinberg expectations, indicating that there were no sympatric sibling species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.02). Some substructing was evident, because after genotypes of all populations mere pooled, 7 of the 16 enzyme loci deviated from Hardy-Weinberg expectations. Estimates of effective migration rates among populations (Nm) were low (3.7), indicating that gene flow was restricted. These data explained observed genetic substructuring when all genotypes were pooled.

Linkage map for the Asian tiger mosquito [Aedes (Stegomyia) albopictus] based on SSCP analysis of RAPD markers.

A linkage map of the Asian tiger mosquito [Aedes (Stegomyia) albopictus (Skuse)] was constructed in an F1 intercross by monitoring the segregation of randomly amplified polymorphic DNA (RAPD) markers analyzed for single-strand conformation polymorphisms (SSCP). We hypothesized that SSCP analysis would reveal point mutations in RAPD fragments that would then segregate as codominant rather than dominant markers which are typically revealed through routine RAPD analysis. Markers were mapped to individual chromosomes by testing for cosegregation with Sex (chromosome I) or a polymorphism at the a-GPD allozyme locus (chromosome II). All other markers that cosegregated were assigned to chromosome III. Six RAPD primers amplified 68 polymorphic markers that segregated in a Mendelian fashion and were mapped. Contrary to our hypothesis, no codominant SSCP polymorphisms were detected, but fractionation of RAPD products on polyacrylamide gels and detection through silver staining proved to be a sensitive technique that allowed us to identify more markers than the standard analysis of RAPD PCR products on agarose gels.

First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico.

Mosquito collections were conducted during a dengue outbreak in Reynosa, Tamaulipas, Mexico, July-December 1995. A total of 6694 adult mosquitoes (four genera and nine species) were captured, of which 2986 (78.3% females and 21.7% males) were Aedes albopictus and 2339 (39.7% females and 60.3% males) were Ae.aegypti. These two species comprised 84.2% of the total collection. Specimens were grouped into pools, nearly 50% of them processed for detection of virus by cythopathic effect in C6-36 and VERO cell cultures and by haemagglutination test. Five pools gave positive haemagglutination reactions and were examined by immunofluorescence using monoclonal antibodies to flavivirus and to dengue virus. One pool of ten Ae.albopictus males was positive for dengue virus: serotypes 2 and 3 were identified by serotype-specific monoclonal antibodies and confirmed by RT-PCR. This is the first report of Ae.albopictus naturally infected with dengue virus in America. Also, it is the very first time Ae.albopictus males have been found infected with dengue virus in the wild.

Reducing the overwintering ability of Aedes albopictus by male release.

Eggs of temperate Aedes albopictus populations are cold hardy and can diapause, but tropical populations are not cold hardy and cannot diapause. Heterozygotes possess intermediate diapause and cold hardiness. Males of a tropical strain from Malaysia with a distinctive genetic marker were released into an existing temperate population in East St. Louis, Illinois. Subsequent egg samples from the release site had genetic marker frequency of up to 24%. Reduced cold hardiness and decreased diapause incidence were also observed in the release site population. No such changes occurred at a nearby control site. The rank order of overwintering survival of eggs at the release site was: Aedes triseriatus > temperate Ae. albopictus > hybrid temperate/tropical Ae. albopictus > tropical Ae. albopictus. Eggs collected from the release population the next summer showed total absence of the genetic marker; presumably carriers were removed by the winter.

Isolation of eastern equine encephalitis virus from Aedes albopictus in Florida.

Fourteen strains of eastern equine encephalitis (EEE) virus were isolated from Aedes albopictus mosquitoes collected in Polk County, Florida. These are the first isolations of an arbovirus of proven public health and veterinary importance from naturally infected Ae. albopictus in the United States since established populations of this introduced mosquito were first discovered in 1985. The widespread distribution of Ae. albopictus in Florida and in other areas of the United States where EEE is endemic raises concern that this species may become an epizootic and epidemic vector of EEE virus.