Genetic method for the preferential elimination of females of anopheles albimanus.

Recent field experiments demonstrated the possibility of using the sterile male method for the control of Anopheles albimanus Wiedemann, the most important vector of human malaria in Central America. Until now there was no practical method for excluding females from the releases of sterile males. A genetic method was developed for the preferential elimination of females during any of the four life stages. This genetic sexing system utilizes propoxur (o-isopropoxyphenyl methyl-carbamate) susceptibility as a recessive conditional lethal a T(Y:2R) translocation, and an In(2R)inversion. The propoxur resistance allele (dominant) was linked to the Y chromosome via a radiation-induced translocation, and genetic recombination was suppressed by inversions. In one of the strains produced, 99.7 percent of the females are eliminated when treated with propoxur, without male loss.

Species-specific repeat units in the intergenic spacer of the ribosomal RNA cistron of Anopheles aquasalis Curry.

A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.

Computer simulation of the effectiveness of male-linked translocations for the control of Anopheles albimanus Wiedemann.

A deterministic simulation model was used to establish the potential value of releasing male-linked translocation heterozygotes as a control measure for Anopheles albimanus Wiedemann. Theoretical population reductions exceeding 90% were obtained within 90 and 120 days after releases at initial ratios of 5 translocation males (TM): 1 normal male (NM) and 1 TM: 1 NM, respectively. Additional simulations emphasized the importance of the need for a method that would eliminate females from the release material. Releases containing 15% females were less effective than those with none. When a malaria subroutine was included in the model, the calculations showed that all the theoretical releases greatly reduced the number of malaria-infective females and therefore would have a profound effect on transmission of the disease. The number of malaria-infective females present was eliminated completely when only translocation males were released; however, a small number were present when the releases contained 15% females. Male-linked translocation males required longer periods of time to bring about population control than males that were completely sterile.

Mass rearing the genetically altered MACHO strain of Anopheles albimanus Wiedemann.

Technology was developed for mass rearing males of the genetically altered MACHO strain of Anopheles albimanus Wiedemann, which allowed for elimination of females by treating the eggs with propoxur (o-isopropoxyphenyl methylcarbamate). This made it possible to eliminate virtually all the females (potential malaria vectors) being released in the field, and also reduced the space previously devoted to larval rearing by 50%, since the females were eliminated in the egg stage. Also, the difficulties involved in separating the sexes with previous techniques were eliminated. Because there is some genetic recombination, about 0.2% of the MACHO males become susceptible and an equal number of females become resistant each generation. Thus after 6-12 months, the strain is purged to remove these contaminants. With this system an average of more than 1 million pupae per day was produced during 3 weeks of a 5-week period, and an average of 968.2 thousand per day during the entire period. The pupae produced were 99.9% males with an average adult emergence of 90%.

Genetic differentiation and diagnostic loci of Anopheles nuneztovari, An. trinkae, and An. rangeli (Diptera: Culicidae).

Samples of Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. trinkae Causey, and An. nuneztovari Gabaldon from Venezuela, Ecuador, Brazil, and Bolivia were analyzed for genetic variability at 24 enzyme loci. Estimates of genetic variability for An. rangeli and An. trinkae from Ecuador and for An. nuneztovari in Venezuela had the following ranges: 46-58% polymorphic loci, 1.7-2.0 (SEM = 0.1-0.3) mean number of alleles per locus, and 0.069-0.113 (SEM = 0.03-0.04) expected mean heterozygosity. Genetic variability estimates of An. rangeli from Bolivia were 20.8-29.2% polymorphic loci, 1.2-1.6 (SEM = 0.1-0.2) mean number of alleles per locus, and 0.037-0.054 (SEM = 0.02-0.03) expected mean heterozygosity. The estimated genetic distance between An. rangeli and An. trinkae ranged from 0.149 to 0.197. The genetic distance between these 2 species and An. nuneztovari ranged from 0.319 to 0.440. Although there were allele frequency differences at some loci between samples of An. nuneztovari sampled from either side of the Andes Mountains in Venezuela, there were no diagnostic loci and the estimated genetic distance was only 0.023. Seven enzyme loci were diagnostic between An. nuneztovari and one or both of its sister species: Acon-2, Ao, Hk-1, Idh-2, Me, Pgi, and Pgm. The diagnostic loci Hk-1 and Acon-2 were not polymorphic in any species. An. rangeli and An. trinkae can be distinguished by the diagnostic loci Ao, Idh-2, and Me-1, and with a 97% probability by Pgm. Distance Wagner and unweighted pair-group method with arithmetic averaging analyses support a close phylogenetic relationship between An. trinkae and An. rangeli.

Sequence of a DNA probe specific for Anopheles quadrimaculatus species A (Diptera: Culicidae).

The nucleotide sequence was determined for a portion of a 12-kb genomic DNA clone specific for Anopheles quadrimaculatus species A. Four short, internally repeated sequences were identified. Synthetic oligonucleotide probes were prepared based on these four repeats. The oligonucleotides are highly specific and can be reliably used to separate individuals of An. quadrimaculatus species A from members of other species of the complex.

Dichotomous electrophoretic taxonomic key for identification of sibling species A, B, and C of the Anopheles quadrimaculatus complex (Diptera: Culicidae).

Samples of 17 populations of Anopheles quadrimaculatus Say from Florida, Alabama, Arkansas, Louisiana, Mississippi, Tennessee, New York, and New Jersey were analyzed for genetic variability at 33 enzyme loci. Statistical analysis of electromorph frequency distributions indicated that sympatric sibling (morphologically indistinguishable) species occurred in about 59% of the populations tested. The association of polytene chromosome and electrophoretic patterns of individual field-collected females confirmed species-specific diagnostic allozymes, which were useful in identifying sibling species A, B, and C and in estimating the proportions of each species at the 17 collection sites. A dichotomous electrophoretic key is presented for the identification of sibling species of the An. quadrimaculatus complex. The electrophoretic method is better than the ovarian polytene chromosome method, because mosquitoes of both sexes and females irrespective of their gonotrophic condition can be identified.

The ovarian nurse cell polytene chromosomes of Anopheles quadrimaculatus, species A.

Nurse cells in the ovaries of adults of Anopheles quadrimaculatus, Species A, were used to prepare a polytene chromosome map. The chromosome quality is superior to that of salivary glands, and it is easier to use adults rather than larvae for cytological analysis of field populations. The most reliable homologies between the salivary and ovarian maps are located in the distal ends of the respective arms, and one homologous region is a prominent landmark in all of the members of the nearctic Maculipennis complex and related species. The left arm of chromosome 3 is uniquely dimorphic. The homokaryotype for 3L1 is synonymous with 3L of the published map of salivary gland polytenes. The 3L heterokaryotype is mostly asynaptic, except for two small homologous, synaptic areas, one of which is inverted. Each homokaryotype contains a unique, diffuse puff that is adjacent to the centromere.

Techniques for mitochondrial and ribosomal DNA analysis of anopheline mosquitoes.

Methods are described for the isolation of mitochondrial and total cellular DNA from mosquitoes. The mitochondrial and ribosomal DNA restriction patterns could be detected in the total DNA of an individual mosquito by the use of cloned probes. DNA restriction analysis may prove to be a useful alternative to isozyme electrophoresis for the study of insect population genetics.

In situ hybridization mapping of histone genes in Anopheles albimanus.

The histone genes of Anopheles albimanus were mapped by in situ hybridization to 6 bands in Region 34A on the right arm of chromosome 3. A genomic library was made by cloning fragments of 15 to 23 kb (derived from partial EcoRI digestion) into the phage vector, EMBL4, and probed with the histone gene repeat of Drosophila melanogaster. Thirty-two phages containing histone gene sequences were isolated from about 10(5) plaque-forming units (pfu). Complete EcoRI digestion of DNA from 5 of the 32 recombinant phages and the genomic DNA of An. albimanus yielded a single 3.84-kb fragment that contained sequences homologous to the 5 histone genes of D. melanogaster. This 3.84-kb unit of mosquito histone genes was subcloned into puc19 plasmid, and the resulting clone (palbi34A) was used for in situ hybridization to salivary gland chromosomes.

Hexane preserves biological activity of isozymes and DNA.

Live or frozen insects are required for using isozyme and DNA RFLP methods in studies on population structure, systematics and incrimination of sibling species. Difficulty in keeping insects alive or unavailability of liquid nitrogen or dry ice at regular intervals during extended collection trips poses a serious problem. We describe a method for preserving insects in hexane, under field conditions, for isozyme and DNA analysis.

Recessive lethal mutations in Anopheles albimanus.

Six recessive lethal mutants of Anopheles albimanus are described. Homozygotes for three of the autosomal mutants, viz., bar eye, dot eye and hairy, die during the last larval or early pupal stages; complete linkage data are lacking for bar eye and hairy but dot eye is tightly linked to red eye on the right arm of chromosome 2. Larval and pupal mortality is high for homozygotes of the other two autosomal mutants, diseased larva and lunate. Survival to the adult stage of the lunate type is about 60% for both sexes, and for diseased larva the rates differed according to sex, 15% for males and 3% for females. A general lack of vitality of the surviving adults has prevented the establishment of homozygous stocks. From analyses of linkage data, diseased larva and lunate were assigned to chromosome 2, most probably on the left arm. The mutant, bubble head, is on the X chromosome, and therefore is expressed only in the hemizygous male, which usually dies during the early pupal stage.

A new cytotype of Anopheles nuneztovari from western Venezuela and Colombia.

Cytogenetic analysis of the larval polytene chromosomes of Anopheles nuneztovari from 5 collection sites in Táchira and Zulia states northwest of the Andean Cordillera in western Venezuela and from 2 sites in the Department of Valle, western Colombia, revealed what appears to be a distinctive cytotype informally designated as An. nuneztovari C. Its chromosomes are homosequential with those of An. nuneztovari B from western Venezuela southeast of the Cordillera but differ in the presence of a well-defined chromocenter and unique inversion polymorphisms. The large complex inversion in western Venezuela, 2Lb, is present at a frequency of 0.263 and deviates significantly from Hardy-Weinberg equilibrium in 3 of the 5 sites. Two smaller inversions (2Lc and 2Ld) that are included in 2Lb are present in the Colombian samples at a frequency of 0.300.

Genetic structure of natural populations of Anopheles albimanus in Colombia.

Electrophoretic and cytogenetic studies were undertaken on the population structure of Anopheles albimanus from 11 localities in Colombia, 3 from northern (Atlantic coast) and 8 from southern (Pacific coast) regions. Of the 25 allozyme loci examined, significant allele frequency differences were observed at 4 loci: hydroxy acid dehydrogenase (Had-1) and 3 esterases (Est-2, Est-4 and Est-6). The northern populations had higher variability, with 55% polymorphic loci, a mean heterozygosity of 20.4% and a mean of 3.0 alleles per locus. These values for southern populations were 24%, 9.1% and 1.5%, respectively. There were neither diagnostic loci nor clinal effect on frequencies of allozymes. Except for a small inversion on the X chromosome in low frequency in certain populations, all populations were homosequential in chromosomal banding patterns. Hybrids from matings between natural populations and the Gainesville laboratory strain were fully fertile. Estimates of genetic similarities (0.95-0.97 among southern and 0.99-1.00 among northern populations) suggest a lack of significant genetic differentiation among distant populations in this species. Based on the chromosomal, hybridization and electrophoretic data, we concluded that mosquitoes from the 11 collections were conspecific populations of An. albimanus.

Heat-shock mortality and induced thermotolerance in larvae of the mosquito Anopheles albimanus.

Temperature effects on Anopheles albimanus larval survival were investigated. Larvae were exposed to 30 min heat shocks at various temperatures. Almost no mortality was observed at 40 degrees C, but was complete at 43 degrees C. Increased larval thermotolerance could be induced by higher rearing temperature or by a 30 min exposure to 37 degrees C.

Quick blots and nonradioactive detection of DNA probes for the identification of mosquitoes.

The quick blot protocol is an improved technique for preparing crude insect homogenates for hybridization to nucleic acid probes. Individual insects are ground in wells of a microtiter plate and transferred to a dot blot manifold. This allows preparation of multiple filters and provides uniformity and an orderly arrangement of samples. The high background detection resulting from use of crude insect homogenates with nonradioactive detection systems was eliminated by incubating quick blot filters in a laundry stain remover containing proteases. We used mosquito species-specific DNA probes to demonstrate the effectiveness of nonradioactive DNA labeling systems with quick blots.

A genetic sexing strain of Anopheles quadrimaculatus, species A.

A genetic sexing strain of a mosquito, Anopheles quadrimaculatus, Species A, was synthesized for the preferential elimination of females during the egg stage. Malathion susceptibility was used as a conditional lethal, and the dominant malathion-resistance allele was linked to the Y chromosome via a radiation-induced reciprocal translocation involving the terminal end of the right arm of chromosome 3 and the Y chromosome. Examination of mitotic chromosomes and salivary polytene chromosomes revealed the precise nature of the translocation. Genetic leakage, through recombination, in the strain was very low (0.02%).

Identification of species D, a new member of the Anopheles quadrimaculatus species complex: a biochemical key.

Sibling species D, a new member of the Anopheles quadrimaculatus species complex was identified in collections from Pickwick Lake, Tishomingo County, Mississippi and Choctawhatchee, Bay County, in West Florida. This species occurred sympatrically with the previously described species, A, B and C. Evidence for identification of species D includes diagnostic allozymes, a lack of polytene chromosomes in the ovarian nurse cells, and inviability of F1 progeny and lack of sperm transfer in hybridization crosses. An electrophoretic taxonomic key for distinguishing species D from A, B and C is presented.

Analysis of the Anopheles (Anopheles) quadrimaculatus complex of sibling species (Diptera: Culicidae) using morphological, cytological, molecular, genetic, biochemical, and ecological techniques in an integrated approach.

The Anopheles quadrimaculatus complex of 5 cryptic species (i.e., An. diluvialis Reinert, new species; An. inundatus Reinert, new species; An. maverlius Reinert, new species; An. quadrimaculatus Say; An. smaragdinus Reinert, new species) is analyzed using multiple techniques, including morphological, cytological, molecular, genetic, biochemical, and ecological procedures. All life stages (egg, 4th-instar larva, pupa, and female and male adults) are described using morphological features, and pertinent stages or structures are illustrated. A neotype for An. quadrimaculatus is designated, and the synonymy of An. annulimanus Van der Wulp is confirmed. Several new morphological features are described. New and summarized data from published literature on hybridization, cytological, electrophoretic, molecular, and cuticular hydrocarbon studies are included. Immature and adult bionomics are given. The geographic distribution for each species is listed and shown on maps. Procedures for collecting, processing, and rearing specimens are described. Keys using morphological characters are included for the eggs, 4th-instar larvae, pupae, adult females, and male genitalia. Also, a biochemical key for the 5 species is included. Color and pattern variations of larvae and pupae are discussed.