Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers.

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito. Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A aygpti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.

Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.

Quantitative genetics of vector competence for dengue-2 virus in Aedes aegypti.

A quantitative genetic study of the ability of Aedes aegypti to propagate dengue-2 (DEN-2) virus in the midgut and in a disseminated infection in the head was conducted with a standard half-sib breeding design. Aedes aegypti aegypti and A. aegypti formosus differ markedly in oral susceptibility to DEN-2 virus. Mosquitoes were orally infected and, after an extrinsic incubation period of 14 days, virus titer (by tissue culture infectious dose, 50% endpoint) was determined in the midgut (MT) and head (HT). Body size as measured by wing length was not significantly different between infected and uninfected mosquitoes and was not correlated with MT or HT The heritability for MT in both subspecies was 0.41 and was 0.39 for HT in A. aegypti formosus. In A. aegypti aegypti, HT appeared to be controlled by dominant alleles. The MT was not correlated with HT nor did MT determine whether virus disseminated out of the midgut. These results suggest that it is the barriers to infection and dissemination, independent of virus titer, that determine vector competence for DEN-2 virus.

Variation in the efficiency of vertical transmission of dengue-1 virus by strains of Aedes albopictus (Diptera: Culicidae).

Five geographical strains of Aedes albopictus (Skuse) were compared for their ability to transmit vertically a dengue-1 isolate from Jamaica. The OAHU strain of Ae. albopictus and a strain of Aedes aegypti (L.) from the United States were included as controls. The offspring of orally infected females were assayed individually for vertical infection. Vertical transmission rates among strains ranged from 11 to 41%, and filial infection rates of strains ranged from 0.5 to 2.9%. Filial infection rates of individual positive families within strains ranged from 1.4 to 17.4%. These rates were higher than those previously recorded for Ae. albopictus. The observed differences in rates of vertical transmission among the strains were not statistically significant, because 95% of the measured variation was attributed to families within strains. The most significant source of variation in vertical transmission of dengue-1 by Ae. albopictus was at the individual level.

New state record for Culiseta morsitans in Colorado.

Culiseta (Culicella) morsitans is reported for the first time from Colorado. Collections from the Rocky Mountains in the northern part of the state extend the range of this species approximately 300 km south and east of previous records.

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.

Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes.

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leafhoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.