Linkage of a gene causing malaria refractoriness to Diphenol oxidase-A2 on chromosome 3 of Anopheles gambiae.

An inbred line of the African malaria vector Anopheles gambiae is refractory to development of malaria parasites. It is homozygous for a 4.3-kb Sal I restriction fragment at the Dox-A2 locus, whereas the parent population is polymorphic at this locus, and a susceptible line is homozygous for an alternate 3.85-kb fragment. The Dox-A2 locus is located in the middle of chromosome 3R, in division 33B, and is tightly linked to a cluster of genes including Dopa decarboxylase that are involved in the production of melanin. Because the refractoriness phenotype, melanotic encapsulation of ookinete/oocysts, might involve activation of or alteration in one or more of these genes, we performed genetic crosses to determine whether a previously identified Plasmodium cynomolgi Ceylon refractoriness gene, Pif-C, is linked to Dox-A2. Backcross mosquitoes fed on one infected monkey developed infections of < or = 100 oocysts. About 50% of these mosquitoes appeared phenotypically refractory, as expected for the backcross performed, but gave slight evidence of linkage between a refractoriness gene and Dox-A2. In contrast, females fed on a monkey that yielded higher infection levels, up to > 300 oocysts, showed clear evidence of linkage between a refractoriness gene and Dox-A2. We conclude that this Dox-A2-linked refractoriness gene is expressed under conditions particular to the higher infection levels, or that environmental factors obscured the genetic effect of this gene at lower infection levels.

Effects of heat shock on the survival of transgenic Anopheles gambiae (Diptera: Culicidae) under antibiotic selection.

A gene for neomycin resistance linked to a Drosophila hsp 70 heat shock promoter was introduced into the germ line of Anopheles gambiae Giles. Effects of heat shock at 37 and 41 degrees C on the subsequent survival of the transgenic mosquito subjected to selection by the antibiotic G418 were studied. Heat shock did not enhance the survival of untransformed mosquitoes but greatly increased the survival of the transgenic mosquitoes. Survival after heat shock at 41 degrees C was greater than after heat shock at 37 degrees C.

Method for in situ hybridization to polytene chromosomes from ovarian nurse cells of Anopheles gambiae (Diptera: Culicidae).

A procedure for in situ hybridization to polytene chromosomes from the ovarian nurse cells of Anopheles gambiae Giles has been developed. This procedure involves a modification of established methods for Drosophila larval salivary gland polytene chromosomes. Treatment of chromosome squashes with xylene followed by slow rehydration provides required resolution of chromosome banding patterns, possibly because fatty contaminants are removed from ovarian nurse cell preparations. Using this procedure, unique DNA sequences from a genomic library of An. gambiae have been mapped on adult mosquito polytene chromosomes. The ability to locate genetic markers on chromosomes will allow correlation of physical and genetic maps. Such maps will facilitate identification of genetic loci and expand our knowledge of the genomic organization of An. gambiae.

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

Genetic analysis of three new eye colour mutations in the mosquito, Anopheles stephensi.

Three new eye colour mutants, scarlet eye (wsca), red-spotted eye (prs) and pigmentless eye (p) were isolated in the malaria vector, Anopheles stephensi. Genetic analysis revealed that all three mutants are sex-linked and recessive to the wild type. Scarlet eye is allelic to white eye but is not to either red-spotted eye or pigmentless eye, which are allelic to each other; 5.7% recombination was observed between the white eye and pigmentless eye loci.

Homozygous chromosomal aberrations in Anopheles stephensi.

Homozygotes for six autosomal paracentric inversions, an inserted paracentric inversion, an autosomal translocation, and two X-chromosome-chromosome 3 translocations in Anopheles stephensi are described. Three of these aberrations are being maintained in pure strains without the necessity of selection.

Two new mutations and a linkage map of Anopheles stephensi.

Genetic analyses of two new mutations in Anopheles stephensi are presented. Spotless wings (sl) and 2nd-3rd costal spots fused (2-3f) have been mapped on chromosome 2, approximately 79.5 map units apart. A preliminary linkage map for this species also is presented.

Four new mutations and a linkage map of species A of Anopheles culicifacies.

Four new mutations, brown eye (bw), colorless eye (c), scarlet eye (sca), and abnormal eye (ae) have been mapped on chromosome 2 of species A of Anopheles culicifacies. The gene sequence is bw--c--sca--ae. A linkage map of the genetically analyzed loci in this species is presented.

An ovarian chromosome map of Anopheles stephensi.

A polytene chromosome map of Anopheles stephensi has been prepared from the ovarian nurse cells of adult females. Many homologous regions can be recognized in comparisons between the ovarian and salivary gland chromosome maps but band for band homologies are not readily evident. The preparation of polytene chromosomes from ovarian nurse cells is easier than from the larval salivary glands and the results more consistent.

Inversion polymorphisms in natural populations of Anopheles stephensi.

A preliminary survey of natural populations of Anopheles stephensi Liston in Pakistan has uncovered the presence of 16 autosomal paracentric inversions. Twelve previously undescribed inversions were observed in the field populations and an additional new one was seen in a laboratory colony. A comparison of urban and rural populations showed striking differences in the kinds and frequencies of paracentric inversions.

Vector incrimination studies and observations on species A and B of the taxon Anopheles culicifacies in Pakistan.

Studies on anopheline mosquitoes in selected villages of Punjab Province, Pakistan have incriminated Anopheles culicifacies species A as the primary malaria vector. Although An. stephensi and An. subpictus showed higher immediate gut infection rates, estimations of relative abundance, age structure and survivorship, and observations of late gut and salivary gland infection rates suggested that neither species was a major vector in these villages. A survey of An. culicifacies populations detected the presence of both species A and B in several localities in Pakistan. Laboratory investigations demonstrated that both species are capable of supporting the extrinsic cycle of Plasmodium vivax. No recombination was observed between X-linked loci in species A/species B hybrid females; however, recombination was observed between loci in chromosome 2. Allelic tests between the X-linked white eye locus in species A and an X-linked white eye mutant in species B showed that the two loci are not allelic.

Induced chromosomal aberrations and linkage group-chromosome correlation in Anopheles stephensi.

Genetic and cytogenetic investigations of 14 strains of Anopheles stephensi with induced chromosomal aberrations have resulted in the assignment of the three linkage groups to their respective chromosomes. In addition, the correlation between the ovarian polytene chromosomes and the mitotic chromosomes was demonstrated. Indirect evidence for the presence of heterochromatic segments in the chromosomal complement was found.

Linkage group III in the malaria vector, Anopheles stephensi.

The linkage relationships among four loci in linkage group III of Anopheles stephensi Liston have been investigated. The data indicate that the sequence is sp-dp-Bl-Dl, and the observed recombination frequencies are as follows: sp-dp = 35.88 percent, dp-Bl = 19.48 percent, Bl-Dl = 21.77 percent, and sp-Dl = 77.13 percent.

Esterases in the malaria vector mosquito, Anopheles culicifacies. Genetic and linkage analyses of an alpha and beta polymorphism.

The genetic and linkage analyses of an alpha and beta esterase polymorphism in Anopheles culicifacies are presented. A survey of laboratory strains uncovered four electrophoretic variants for the alpha esterase and two for the beta esterase. Genetic analyses indicated that the variants of the alpha esterase are under the control of codominant alleles of a single locus and that this locus is linked to the locus controlling the expression of the codominant alleles of the beta esterases. The esterases are not linked either to sex (chromosome 1) or to maroon eye (chromosome 2) but to the chromosome 3 markers, dieldrin resistance and phosphoglucomutase. The gene sequence is Est-alpha--Dl--Pgm--Est-beta dnd the recombination frequencies are as follows: Est-alpha--Dl = 2.9 percent; Dl--Pgm = 5.8 percent; Pgm--Est-beta = 6.4 percent; Est-alpha--Est-beta = 15.1 percent, Est-alpha--Pgm = 8.7 percent and Dl--Est-beta = 12.2 percent.

Genetic sexing for a mosquito sterile-male release.

Two strains of Anopheles culicifacies have been synthesized in which the dieldrin resistance locus on chromosome 3 has been translocated to the Y chromosome. Both strains appear sufficiently stable for the preferential recovery of males for mass production of mosquitoes for field releases.

A phosphoglucomutase polymorphism in the mosquito, Anopheles culicifacies.

A survey of phosphoglucomutase (Pgm) among laboratory strains of Anopheles culicifacies has uncovered two electrophoretic variants. Detailed genetic analysis revealed that these variants are inherited as codominant alleles at a single locus. The Pgm locus has been assigned to linkage group III approximately 39 map units from Acph (acid phosphatase) and 8.5 map units from Dl (dieldrin resistance). The data indicate that the probable gene sequence is Acph-Dl-Pgm.