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D W Severson

Publications and source records attributed to D W Severson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

Animals↗
PubMed1996

Mosquito dopa decarboxylase cDNA characterization and blood-meal-induced ovarian expression.

Dopa decarboxylase (DDC) functions in insect catecholamine biochemistry to produce materials essential for cross-linking reactions that result in tanning and/or melanization, include tanning of the mosquito egg chorion and encapsulation of parasites. We have cloned Ddc from the mosquito, Aedes aegypti, and studied its expression in response to blood-feeding, which initiates events necessary for egg maturation in mosquitoes. The Ae. aegypti Ddc cDNA was isolated via heterologous screening using a clone from Drosophila melanogaster. A resulting 1.87 kilobase (kb) clone was sequenced to reveal an open reading frame of 1464 bp, as well as 5'- and 3'-untranslated segments. The inferred amino acid sequence of this clone shares 81% identity with the published Drosophila Ddc cDNA, including complete identity with twenty-four contiguous amino acids encompassing the pyridoxal-5-phosphate cofactor binding domain. Analysis of an F2 intercross population derived from a parental cross between two Ae. aegypti strains (Hamburg and Moyo-In-Dry) allowed us to map Ddc to a locus on linkage group 2. Expression studies demonstrated the presence of a 2.1 kb message, the majority of which occurs in the ovaries where Ddc-specific mRNA is up-regulated in response to ingestion of a blood meal. The potential for egg-tanning in anautogenous mosquitoes as a model for understanding specific genetic events in the regulation of catecholamine metabolism is addressed.

Aedes↗
PubMed1996

Generation of expressed sequence tags and sequence-tagged sites as physical landmarks in the mosquito, Aedes aegypti, genome.

Twenty-three clones were developed as expressed sequence tags (ESTs) and sequence-tagged sites (STSs) that provide broad coverage of the mosquito, Aedes aegypti, genome at an average spacing of about 7.2 cM. Each of these clones had been mapped previously as a restriction fragment length polymorphism (RFLP) marker. Nineteen of these clones represent anonymous cDNAs and 4 clones represent known genes. Forty-seven percent of the anonymous cDNAs showed significant deduced amino acid sequence similarity to previously described sequences from a wide variety of organisms. STSs developed from RFLPs will provide effective tools for rapid screening of YAC and cosmid libraries, and will therefore, provide anchor points for contig construction for any region in the A. aegypti genome. Also, they facilitate the simultaneous integration of physical and genetic mapping data for A. aegypti.

Aedes↗
PubMed1995

Aedes aegypti: a quantitative trait locus (QTL) influencing filarial worm intensity is linked to QTL for susceptibility to other mosquito-borne pathogens.

Because intensity of infection was significantly increased in a substrain of Aedes aegypti selected for susceptibility to the filarial worm, Brugia malayi, experiments were designed to assess numbers of microfilariae (mf) ingested and midgut penetration by mf in this susceptible substrain as compared to a refractory substrain selected from the same parental stock. Refractory mosquitoes ingested significantly fewer mf than susceptible mosquitoes and significantly fewer numbers of mf penetrated through refractory midguts as compared to susceptible midguts. In 16.7% of the refractory midguts, no mf were able to penetrate the midgut and in three refractory mosquitoes over 250 mf were ingested, but no mf penetrated the midgut. These results indicate that permissiveness of the midgut for penetration by microfilariae can determine not only parasite intensity, but also prevalence of infection. The genetic basis for ingestion of mf and midgut penetration was assessed using restriction fragment length polymorphism markers and quantitative trait loci (QTL) mapping. This mapping identified a QTL on chromosome 2, idb[2,LF181] (idb, intensity determinant for Brugia), that seems to influence ingestion ability. This QTL is linked to a previously identified QTL for susceptibility to B. malayi, fsb[2,LF98], as well as to loci for susceptibility to the malaria parasite, Plasmodium gallinaceum, and yellow fever virus. These results suggest that this region of chromosome 2 contains one or more genes that influence susceptibility of A. aegypti to several mosquito-transmitted pathogens.

Aedes↗
PubMed1995

Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti.

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F2 populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.

Aedes↗
PubMed1995

Comparative linkage maps for the mosquitoes, Aedes albopictus and Ae. aegypti, based on common RFLP loci.

Aedes albopictus and Aedes aegypti are members of the mosquito family Culicidae and share a haploid chromosome complement of three. Although a genetic linkage map based on restriction fragment length polymorphism (RFLP), markers exists for Ae. aegypti, the extent of synteny and linkage order conservation between the two species was unknown. A comparative linkage map for Ae. albopictus was constructed based mainly on cDNA clones from Ae. aegypti. Nearly all Ae. aegypti probes hybridized to Ae. albopictus DNA at high stringency. For eighteen RFLP markers tested, the linkage group and linear order appears to be identical for the two species. 78% of the loci tested exhibited significant deviations from the expected segregation ratio in at least one of the test crosses. An excess of heterozygote genotypes was recovered with most loci. This probably reflects the effects of lethal loci on survival of F2 progeny homozygous for the parental genotypes. These results demonstrate that comparative linkage maps based on common DNA markers provide a basis for rapidly developing linkage maps for various mosquito species, and the opportunity to examine the significance and function of orthologous quantitative trait loci associated with mosquito vector competence for disease transmission.

Aedes↗
PubMed1994

Aedes aegypti: characterization of a hemolymph polypeptide expressed during melanotic encapsulation of filarial worms.

We report on the initial characterization of an 84-kDa polypeptide that is differentially expressed in Aedes aegypti during melanotic encapsulation immune reactions against filarial worms. [35S]Methionine-labeled hemolymph from mosquitoes inoculated with saline, parasites that are melanized, or parasites that are not melanized was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results show that the level of the 84-kDa polypeptide increases considerably in those mosquitoes undergoing encapsulation reactions against parasites but remains down-regulated in those mosquitoes exposed to parasites that are not melanized or are undergoing wound healing responses (saline-inoculated). Experiments involving glycosidase treatment of hemolymph samples indicate that this polypeptide is not heavily glycosylated. Amino acid microsequencing was performed and two internal sequence fragments (15 continuous amino acids and 12 noncontinuous amino acids) were obtained. Analysis of these sequences to known sequences in a protein database did not yield any conclusive information as to the identify of the 84-kDa polypeptide. Therefore, degenerate oligonucleotide primers were designed, based on the sequence of the 15-amino-acid fragment, and used with the polymerase chain reaction (PCR) to amplify from A. aegypti genomic DNA the region between the primers. The PCR product was cloned and sequenced to verify that the nucleic acid sequence matched the known protein sequence. Screening of an A. aegypti cDNA library with this small PCR-generated clone resulted in the selection of an approximately 540-bp clone. Northern analysis with this larger cDNA clone indicates that it hybridizes to an approximately 2.0-kb message that is differentially expressed in mosquitoes undergoing melanotic encapsulation reactions against filarial worms. Furthermore, sequencing of this approximately 540-bp clone showed that it contained the 15-amino-acid sequence that had been used to design the degenerate PCR primers, indicating that an appropriate clone was selected. However, sequence analysis of this clone at the protein and nucleic acid level did not provide any conclusive answers to the identity or function of the 84-kDa polypeptide.

Aedes↗
PubMed1994

Applications of molecular marker analysis to mosquito vector competence.

A rapidly expanding cadre of molecular biology techniques is being developed for human and plant genetics, including development of the technology to identify large numbers of genetic markers and to evaluate these markers relative to phenotypic observations. In this review, David Severson discusses applications of these techniques for the analysis of mosquito vector competence.

Journal Article↗
PubMed1994

Chromosomal mapping of two loci affecting filarial worm susceptibility in Aedes aegypti.

Two quantitative trait loci (QTL) affecting susceptibility of the mosquito Aedes aegypti to the filarial worm parasite Brugia malayi were identified using restriction fragment length polymorphism (RFLP) markers. The first locus, fsb[1,LF178], resides within a 10 cM interval on chromosome 1 and exhibits a recessive effect with respect to susceptibility. The second locus, fsb[2,LF98], resides within a 9 cM interval on chromosome 2 and exhibits an additive effect on susceptibility. Significant epistasis was detected between these loci, although the effect of fsb[2,LF98] was dependent on the genetic background of the mosquito strains. Suggestions for a standard QTL nomenclature are included.

Aedes↗
PubMed1994

The suitability of restriction fragment length polymorphism markers for evaluating genetic diversity among and synteny between mosquito species.

Restriction fragment length polymorphism (RFLP) markers derived from the yellow fever mosquito, Aedes aegypti, were used in hybridizations to genomic DNA of the following mosquito species: Ae. albopictus, Ae. togoi, Armigeres subalbatus, Culex pipiens, and Anopheles gambiae. Interspecific hybridization with Ae. aegypti probes varied from 50% (An. gambiae) to 100% (Ae. albopictus) under high stringency conditions. We demonstrated the usefulness of using RFLP profiles to examine genetic diversity between mosquito populations; Ae. aegypti RFLP markers were used to examine genetic relatedness between 10 laboratory strains of Ae. aegypti as well as between nine populations representing four Cx. pipiens subspecies. These results indicate that many Ae. aegypti RFLP markers should have direct applicability in gaining a better understanding of genome structure in other mosquito species, including RFLP linkage mapping and determinations of genetic relatedness among field populations.

Animals↗
PubMed1994

Reinterpretation of the genetics of susceptibility of Aedes aegypti to Plasmodium gallinaceum.

Several studies have demonstrated a genetic basis for variation in susceptibility of Aedes aegypti to Plasmodium gallinaceum. Although 25 yr ago it was reported that P. gallinaceum susceptibility in Ae. aegypti is determined primarily by a single autosomal dominant gene, evidence for additional genetic factors has emerged. Two sublines, 1 refractory and 1 of intermediate susceptibility to P. gallinaceum, have been selected from the Moyo-In-Dry strain (MOYO) of Ae. aegypti. Prior to selection, the MOYO population was 20.3% refractory. Genetic crosses of the highly susceptible Rockefeller strain (ROCK) and the 2 selected sublines of the MOYO strain provide evidence for a complex mode of inheritance of Plasmodium susceptibility in Ae. aegypti.

Aedes↗
PubMed1990

Heat stress induced enhancement of heat shock protein gene activity in the honey bee (Apis mellifera).

We employed in vitro translation of mRNA and product separation using SDS-PAGE to examine the heat-shock response of the worker honey bee. Increases in the levels of 6 translatable RNA populations were observed following heat stress. The greatest response was observed among bees aged 9 days. Slight levels of induction of 70 and 82 kDa heat shock proteins were evident among bees taken directly from the colony.

Age Factors↗
PubMedDate not supplied

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti.

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.

Aedes↗
PubMedDate not supplied

Comparative linkage maps for the mosquitoes (Culex pipiens and Aedes aegypti) based on common RFLP loci.

We report construction of a comparative linkage map for the mosquito (Culex pipiens) based on restriction fragment length polymorphisms (RFLPs) using cDNA clones from Aedes aegypti as probes to Southern blots of Cx. pipiens genomic DNA. Seventy-one cDNA clones were screened for hybridization and genetic diversity among three Cx. pipiens strains. Fifty-two of 71 cDNA clones, isolated from and previously mapped in Ae. aegypti (73.2%), were hybridized under high-stringency conditions with Cx. pipiens genomic DNA. Thirty-four clones (47.9%) reflected strain-specific polymorphisms. The map consists of 21 cDNA markers that identify 22 loci covering 165.8 cM. The loci mapped in Cx. pipiens cover 7.1 cM on chromosome 1, 80.4 cM on chromosome 2, and 78.3 cM on chromosome 3. Linkage relationships of the RFLP markers for chromosome 1 in Cx. pipiens are the same as chromosome 1 in Ae. aegypti, indicating that chromosome 1 is highly conserved between the two species. The comparative RFLP linkage maps for chromosomes 2 and 3 in Cx. pipiens and Ae. aegypti reflect apparent whole-arm translocations. RFLP markers for chromosome 2 in Ae. aegypti identified homologous loci on one arm of chromosomes 2 and 3 in Cx. pipiens, and RFLP markers for chromosome 3 in Ae. aegypti identified homologous loci on the opposite arms of chromosomes 2 and 3 in Cx. pipiens.

Aedes↗
PubMedDate not supplied

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae.

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.

Aedes↗
PubMedDate not supplied

Comparisons of genetic variability and genome structure among mosquito strains selected for refractoriness to a malaria parasite.

Restriction fragment length polymorphism (RFLP) markers were used to evaluate Aedes aegypti genome structure and genetic variability within and between substrains selected for different levels of refractoriness to the malaria parasite, Plasmodium gallinaceum. The MOYO-R substrain was previously selected for complete refractoriness and the MOYO-IS substrain for intermediate susceptibility from the Moyo-In-Dry (MOYO) strain by selective inbreeding (F = 0.5). Eighteen mapped RFLP markers were used to provide coverage of the mosquito genome. The two substrains showed reduced genetic diversity compared with the MOYO strain, including significant reductions in mean heterozygosity, number of alleles per locus, and proportion of polymorphic loci. Genetic differentiation between the two substrains was statistically significant, as reflected by differences in allele frequencies. Significant pairwise linkage disequillbrium among the RFLP loci was detected in all three strains, most evidently in the MOYO strain. This is surprising because the RFLP loci examined are separated by large map distances, and therefore linkage disequilibrium should decay to zero after many generations of laboratory culture. Our hypothesis to explain this phenomena is that lack of recombination, or low recombination rates in some regions of the A. aegypti genome, is a result of chromosome inversions. Finally, we used graphical genotyping, wherein whole genome genotypic information for individual mosquitoes is represented in a simple graphic format, to illustrate genome structure and allelic variation within and among the mosquito strains. Our analysis revealed an apparent chromosomal deletion on chromosome 3 for some individuals in the MOYO strain and MOYO-IS substrain.

Aedes↗
PubMedDate not supplied

Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).

Several loci conferring insecticide resistance in the yellow fever mosquito (Aedes aegypti) have previously been mapped by simple recombinational mapping. Here we describe correlation of these resistance phenotypes with molecular gene probes for insecticide target sites by RFLP mapping. The para sodium channel gene homologue and the GABA receptor gene Resistance to dieldrin map to the same genome regions as the DDT/pyrethroid and cyclodiene resistance loci, respectively. Although the acetylcholinesterase (target site of organophosphorus and carbamate insecticides) gene Ace does not map to any known resistance locus, it maps very close to the sex-determining locus. We discuss the possibilities that, if identified, Ace-mediated resistance in A. aegypti will be sex linked or that, as suggested for anopheline mosquitoes, two independent Ace loci may exist, one of which is autosomal. These results support the importance of target site insensitivity as an insecticide resistance mechanism in mosquitoes.

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