Genetic structure of the genera Psorophora (Diptera: Culicidae) in Columbian and North American populations using isoenzymes and ITS2 sequences

Two, a priori, Psorophora columbiae and one, a priori, Ps. toltecum populations from Colombia were studied by means of eleven isoenzyme loci. The levels of genetic diversity, Hardy-Weinberg equilibrium, gene flow estimates, isolation by distance and genetic relationships among these three populations were studied and the results were as follows: 1. The gene diversity levels were high as well as very similar among the three populations indicating no differences between them. 2. Within each population and from a hierarchical standpoint several loci were not in Hardy-Weinberg equilibrium simultaneously by excess and defect of heterozygous. This could reflect that several natural selection forces are acting upon these mosquito populations. 3. The gene flow estimate for these populations showed the existence of this event between them. This agrees quite well with the fact that the three populations really belong to a single species. In addition, maximum parsimony analyses with 16 isoenzyme for several individuals from four Colombian and two United States Psorophora populations showed that effectively only one species was present in the Colombian area surveyed, and that this species is Ps. columbiae, which hardly contrast with that previously suggested by other authors.     

Psorophora columbiae and Psorophora toltecum (Diptera: Culicidae) Colombian populations cannot be differentiated by isoenzymes

Two populations of the mosquito Psorophora columbiae from the central Andean area of Colombia and one population of Ps. toltecum from the Atlantic coast of Colombia were analyzed for 11 isoenzyme markers. Psorophora columbiae and Ps. toltecum are two of the main vectors of Venezuelan equine encephalitis. We found no conspicuous genetic differences between the two species. The relatively high gene flow levels among these populations indicate that these are not two different species or that there has been recent divergence between these taxa. In addition, no global differential selection among the loci was detected, although the alpha-GDH locus showed significantly less genetic heterogeneity than the remaining loci, which could mean that homogenizing natural selection acts at this locus. No isolation by distance was detected among the populations, and a spatial population analysis showed opposite spatial trends among the 31 alleles analyzed. Multiregression analyses showed that both expected heterozygosity and the average number of alleles per locus were totally determined by three variables: altitude, temperature and size of the human population at the locality. Individually, the expected heterozygosity is more related to these three variables than to the average number of alleles.     

Cibarial morphology in Psorophora Robineau-Desvoidy subgenera (Diptera: Culicidae)

We describe the cibarial morphology in eight Psorophora species Robineau-Desvoidy: Ps. (Grabhamia) cingulata (Fabricius), Ps. (Gra.) confinnis (Lynch Arribálzaga), Ps. (Janthinosoma) ferox (Humboldt), Ps. (Jan.) albipes (Theobald), Ps. (Jan.) cyanescens (Coquillett), Ps. (Psorophora) lineata (Humboldt), Ps. (Pso.) cilipes (Fabricius), y Ps. (Pso.) ciliata (Fabricius). The species belonging to subgenus Grabhamia Theobald are characterized by palatal papillae in central position and the presence of cibarial armature. The teeth in Ps. cingulata are equal meanwhile in Ps. confinnis the internal row is spatulate with apex denticulate. In Janthinosoma Lynch Arribálzaga we observed six palatal papillae (the fore pair with less size to others) and armature absent, instead we observe small spicules toward posterior plate: abundant in Ps. ferox, and few in Ps. albipes and Ps. cyanescens. The subgenus Psorophora presents four equal size palatal papillae, cibarial armature absent, also distinctive number of trichoid sensilla (12-17), in comparison to other two subgenera (5-10). We suggest to include those diagnostic characters in the future taxonomic and systematic studies in the genus Psorophora.     

Comparison of ribosomal dna nucleotide sequences of the mosquito genera Aedes and Ochlerotatus (Diptera: Culicidae: Aedini)

Nucleotide rDNA sequences of 9 species of the mosquito genus Ochlerotatus and 2 species of the genus Aedes were obtained and compared. Variation both within and between these genera was characterized. The phylogenetic relationships among the studied species of Ochlerotatus generally correspond to the grouping of species based on morphological characters.     

Comparison of nucleotide sequences of ribosomal DNA of the mosquito genera Aedes and Ochlerotatus (Diptera: Culicidae: Aedini)

Morphological study allowed identifying 9 species of mosquitoes of the genus Ochlerotatus and 2 species of the genus Aedes. Sequencing of the rDNA region was performed for all these speciemens. The sequences of rDNA were the following: A. cinereus -868 bp, A. vexans--889 bp, Och. cantans--803 bp, O. excrucians--801 bp, O. euedes--794 bp, Och. cyprius--777 bp, O. diantaeus--758 bp, O. intrudens--817 bp, Och. punctor--783 bp, O. dorsalis--748 bp, O. species--767 bp. On average, the size of Aedes rDNA fragments exceeds rDNA of Ochlerotatus by 90-100 bp. The sequences are characterized by a high number of insertions and deletions, and also by point substitutions of nucleotides. It is important to notice that interspecific differences include not only different regions of the internal transcribed spacers, but also the conservative site which is represented by the 5.8S gene. Among four substitutions in this gene, one (C/A) represents the difference between Aedes and Ochlerotatus, the next (T/A) distinguishes A. cinereus from Ochlerotatus speciment and A. vexans, and two substitutions (A/C, T/G) testify the similarity between O. dorsalis and O. species and specimens of Aedes. Besides, two more deletions are typical for O. dorsalis and O. species. One deletion is com- mon, it distinguishes them from the other examined species, and another one is typical only for O. dorsalis. When analyzing morphological characteristics and comparing nucleotide rDNA sequences of O. species with the database, the similarity to O. caspius has been revealed. On the whole, phylogenetic relationships among Ochlerotatus species correspond to subdivision into groups based on morphological characters. Probably, examination of the larger number of specimens will change the morphological division into groups.     

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.     

Effects of temperature and soil moisture on survival of eggs of the mosquito Psorophora columbiae (Diptera: Culicidae)

The results of laboratory tests indicated the average survival rates for Psorophora columbiae eggs remained quite high for all of the egg populations exposed to a temperature of 27°C (range 83.0–100.0% survival) after 96 days of exposure, except for the non‐diapausing eggs on dry soil (66.3%). In regard to the exposure of egg populations to moderately cold temperatures (i.e. 8°C, 4°C and ?2°C) for periods of up to 16 days, survival rates for egg populations exposed to 8°C continued to remain relatively high (average >85%) for the remainder of the experimental exposure period (i.e. 96 days). Diapausing Ps. columbiae eggs were more tolerant (82.0% survival) to low temperatures (?2°C) than non‐diapausing eggs (2.4% survival) for 64 days, particularly at temperatures of and below 4°C. Diapausing and non‐diapausing eggs were similar in their ability to survive under high temperatures (34°C and 38°C). High soil moisture (30–40%) or substrate moisture (95% relative humidity) content appeared to enhance the ability of the mosquito eggs to survive both low and high temperature extremes.     

Sequence and secondary structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae).

Sequences of the internal transcribed spacers (ITS1 and ITS2) of the mosquito Aedes aegypti, and the ITS2 of six related species, A. simpsoni, A. albopictus, A. vexans, A. triseriatus, Haemagogus mesodentatus, and Psorophora ferox are reported. Intraspecific variation in A. aegypti ITS1 is 1.07% among four clones from three individuals, and in the ITS2 is 1.17% among 15 clones from four individuals. In A. simpsoni, intraspecific ITS2 variation is 0.46% among 10 clones from a single individual. Alignment of the ITS2 sequence of the seven species reveals several homologous domains. Secondary structure predictions for the ITS2 region indicate that these domains base pair to form a core region central to several stem features. The sequence outside the ITS2 homologous domains tends to be GC-rich and characteristically slippage generated; these areas preserve or add to the stem length of the predicted secondary structures. These ITS2 intraspacer variable regions resemble previously described expansion segments of the 28S gene region. Evolutionary analysis of the ITS2 of these species, using both sequence and secondary structure information, leads to the prediction of divergence in the mosquito tribe Aedini that is not clearly reflected in current taxonomic designations.     

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.     

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.     

Phytogeny of genusGlossina (Diptera:Glossinidae) according to ITS2 sequences

The flies of genusGlossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from differentGlossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genusGlossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genusGlossina wherefusca group species formed the deepest branch followed bymorsitans andpalpalis groups, respectively. The placement ofGlossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest thatGlossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with themorsitans group species.     

Isoenzimatic analysis of four Anopheles (Kerteszia) cruzii (Diptera: Culicidae) populations of Brazil

Anopheles cruzii is a small sylvatic mosquito and primary human Plasmodium vector in Southern Brazil. The distribution of this bromeliad-breeding mosquito follows the Atlantic forest coastal distribution, where bromeliads are abundant. Morphological, genetic, and molecular polymorphisms among different populations have been reported and it has recently been suggested that An. cruzii is a complex of cryptic species. The aim of this work is to analyze the gene flow between different populations of An. cruzii collected in four localities within the geographic distribution range of the species, and to examine if An. cruzii is a complex of cryptic species. The genetic distances show that populations of the states of Santa Catarina, Sao Paulo, and Rio de Janeiro are genetically closer (0.032 to 0.083) than populations of Bahia (0.364 to 0.853) based on profiles from 10 distinct isoenzyme loci. The Fst was lower (0.077) when the Bahia population was excluded than when it was included (0.300) in the analyses. The inferred number of migrants per generation was 2.99 individuals among populations from the states of Santa Catarina, Sao Paulo, and Rio de Janeiro and 0.58 migrants per generation among all populations. Results suggest that An. cruzii is a complex of species and that the specimens of state of Bahia can be considered as belonging to a species that is distinct from other three closely-related populations studied.     

Genetic mapping and characterization of aldehyde oxidase of Anopheles albimanus (Diptera: Culicidae)

Aldehyde oxidase (Ao) of Anopheles albimanus Wiedemann was mapped on chromosome 3. The sequence is hexokinase-1--19.2 +/- 1.8--stripe--28.3 +/- 2.2--beta-hydroxy acid dehydrogenase--3.6 +/- 0.3--aldehyde oxidase--2.6 +/- 0.4--esterase-8--6.1 +/- 1.9--esterase-4--?--esterase-6 (phosphoglucomutase). Aldehyde oxidase is 26.1 +/- 2.5 from phosphoglucomutase and 27.2 +/- 1.6 from esterase-6. The one-band electromorph of Ao in homozygotes and the three-band type in heterozygotes suggest that the enzyme is a dimer. The isoelectric points of slow and fast allozymes are 5.5 and 4.8, respectively. A variety of electrophoretic techniques was used to determine if the allozymes of Ao can be differentiated on a basis other than mobility. The slow, fast, and hybrid genotypes were analyzed for differences in thermostability, reactivity to thiol reagent, susceptibility to urea denaturation, substrate specificities, and response to chelating agents. The relative effect of p]H on allozymes was tested by varying the pH of the staining buffer over a range of 4-12. No significant differences were detected among allozymes and no additional allelic variations were observed.     

Molecular differentiation in natural populations of Anopheles oswaldoi sensu lato (Diptera: Culicidae) from the Brazilian Amazon, using sequences of the COI gene from mitochondrial DNA

Anopheles (Nyssorhynchus) oswaldoi (Peryassú, 1922) s. l., which has been incriminated as a potential human malaria vector in Western Brazilian Amazon, may constitute a cryptic species complex. However, the most recent study with isozymes indicated high similarity among samples from the States of Acre, Amazonas and Rond?nia in the Brazilian Amazon. In the present study, 45 individuals were sequenced from Sena Madureira (State of Acre), Coari (State of Amazonas), S?o Miguel (State of Rond?nia), and Moju (State of Pará), using the cytochrome oxidase I gene from mitochondrial DNA. Twenty-five haplotypes were identified in the four localities, and no haplotype was shared among them. The lowest haplotype number was detected in the Coari sample. The dendrogram based on maximum parsimony analysis yielded four groups: I) haplotypes 1, 2, 3, 4, and 5 from Sena Madureira and haplotypes 17 and 18 from S?o Miguel; II) haplotypes 13 to 16 and 19 to 22 from S?o Miguel; III) haplotypes 23 to 25 from Moju, and IV) haplotypes 6 to 9 from Sena Madureira and haplotypes 10 to 12 from Coari. The genetic distance (uncorrected p) obtained among the four groups ranged from 0.08 to 5.3%, whereas the highest values (4.97 to 5.3%) were found between groups I (Sena Madureira) and III (Moju). Based on male genitalia identification, it was suggested that group I may be A. oswaldoi s. s. whereas group IV may be A. konderi. Groups II and III could constitute other lineages or species within A. oswaldoi s. l., whose taxonomic status remains to be clarified. These results suggest that additional studies are necessary using samples of A. oswaldoi s. l. from a larger geographic area.     

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.