Cristina N. Gardenal

Current resistance status to temephos in Aedes aegypti from different regions of Argentina

In Argentina, more than 25,000 cases of dengue were reported in the summer of 2009, even in provinces where the disease was formerly absent. We analysed the susceptibility levels to the larvicide temephos in seven populations of Aedes aegypti, the primary vector of dengue, collected during summer 2007/2008, using the susceptible Rockefeller strain as a control. Although no control failures were observed during the experiment, a majority of the lethal concentration and resistance ratio values indicate an incipient resistance. An integrative program to monitor the resistance of Ae. aegypti to insecticides is needed in the country.

Laboratory Evaluation of Susceptibility of Natural Subpopulations of Aedes aegypti Larvae to Temephos

ABSTRACT Aedes aegypti showed the ability to develop resistance to different insecticides, including temephos, the most widely used larvicide. The objectives of this work were to 1) determine the resistance value of 4 natural subpopulations of Ae. aegypti, identified by their different haplotypes, to the insecticide temephos “Abate 1G (1%)”; 2) determine the lethal concentration (LC)50 and LC90 values by using the Rockefeller strain as control; and 3) estimate the resistance ratios. Mosquito samples were collected in Catamarca, Córdoba, and Posadas (Argentina) and in Yacuiba (Bolivia). Six insecticide concentrations were tested. The Rockefeller strain and the Posadas sample showed susceptibility to the diagnostic concentration (0.012 mg/liter), whereas the mortality in Catamarca was 87%. In the Yacuiba and Córdoba collections, mortality was 74% and 75%, respectively, indicating resistance. These results were coincident with those of the Probit analysis from which the highest resistance ratios were estimated for the last 2 subpopulations (5.2 and 4.9, respectively). Before this study, no information was available about the existence of resistance in natural populations of Ae. aegypti in the studied area.

Two Different Routes of Colonization of Aedes aegypti in Argentina from Neighboring Countries

ABSTRACT Aedes aegypti L. (Diptera, Culicidae) is the main vector of dengue and yellow fever. In Argentina, the species was apparently eradicated approximately in 1964; by 1986, it was reintroduced. To identify different gene pools in geographical populations of the species and to ascertain the possible routes of colonization, we analyzed the diversity of mitochondrial DNA haplotypes in 572 specimens from Argentina and neighboring countries. We found that the restriction fragment length polymorphism-polymerase chain reaction screening of a large DNA fragment including the A+T-rich region was the best strategy to reconstruct the colonization pattern of Ae. aegypti in Argentina. Twenty haplotypes were recognized; levels of genetic similarity varied among populations from different geographical locations. The haplotype network constructed on the basis of genetic distances showed three well differentiated groups. Two of them exhibited a well defined spatial distribution and populations in these groups presented an isolation-by-distance pattern. The persistence of relictual populations after the last eradication campaigns would explain the high levels of haplotype diversity and the presence of exclusive haplotypes in urban centers from northwestern Argentina. Eastern Argentine populations showed one prevalent haplotype, also predominant in Brazil and Paraguay. Our results highlight the need for efficient surveys and control campaigns, given the strong effect of land trade on genetic exchange among mosquito populations from Argentina and neighboring countries where dengue is endemic.

Genetic Relationships Among Aedes aegypti (Diptera: Culicidae) Populations from Argentina Using Random Amplified Polymorphic DNA Polymerase Chain Reaction Markers

Abstract Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) polymorphism was analyzed in five Aedes aegypti (L.) populations from Argentina and one from Puerto Rico to estimate levels of intraspecific polymorphism and genetic relatedness. Allele frequencies were estimated assuming that RAPD products segregate as dominants and that genotype frequencies at those loci are in Hardy–Weinberg equilibrium. Mean expected heterozygosity (He) was 0.350; FST values were significant at all loci except one, supporting the usefulness of the fragments used here to discriminate among populations. Rogers’ genetic similarity between samples ranged from 0.806 to 0.621. The population from Puerto Rico was the most different from the Argentina populations. Considering that Ae. aegypti eggs, larvae, and pupae can be transported easily, relationships among the Argentinian populations may reflect the routes and intensity of commercial transit.

Species Delimitation in the Continental Forms of the Genus Epicrates (Serpentes, Boidae) Integrating Phylogenetics and Environmental Niche Models

Until recently, the genus Epicrates (Boidae) presented only one continental species, Epicrates cenchria, distributed in Central and South America, but after a taxonomic revision using morphologic characters five species were recognized: E. cenchria, E. crassus, E. maurus, E. assisi, and E. alvarezi. We analyzed two independent data sets, environmental niche models and phylogeny based on molecular information, to explore species delimitation in the continental species of this genus. Our results indicated that the environmental requirements of the species are different; therefore there are not evidences of ecological interchangeability among them. There is a clear correlation between species distributions and the major biogeographic regions of Central and South America. Their overall distribution reveals that allopatry or parapatry is the general pattern. These evidences suggest that habitat isolation prevents or limits gene exchange among them. The phylogenetic reconstruction showed that the continental Epicrates are monophyletic, being E. alvarezi the sister species for the remaining two clades: E. crassus - E. assisi, and E. maurus - E. cenchria. The clade grouping the continental Epicrates is the sister taxon of the genus Eunectes and not of the Caribbean Epicrates clade, indicating that the genus is paraphyletic. There is a non-consistent pattern in niche evolution among continental Epicrates. On the contrary, a high variation and abrupt shifts in environmental variables are shown when ancestral character states were reconstructed on the sequence-based tree. The degree of genetic and ecological divergence among continental Epicrates and the phylogenetic analyses support the elevation to full species of E. cenchria, E. crassus, E. maurus, E. assisi, and E. alvarezi.

Patterns of evolution in Graomys griseoflavus (Rodentia, Muridae). IV. A case of rapid speciation

The South American group of rodents known as Graomys griseoflavus comprises two sibling species differing only in diploid chromosomal complement: G. griseoflavus (2n = 36, 37 and 38) and G. centralis (2n=42). Reproductive barriers comprising postzygotic as well as precopulatory mechanisms prevent gene exchange between these species. We have studied genetic polymorphism in two populations of G. centralis and four of G. griseoflavus by means of gel electrophoresis of enzymes and other proteins giving information on a total of 30 loci. Values of interspecific genetic identity were similar to those obtained for conspecific populations, suggesting that fixation of Robertsonian fusions would have occurred without significant bottlenecks. It would also indicate that the cladogenetic process must be relatively recent. FIS values showed no evidence of inbreeding. Fixation indices (FST) for the ancestral species showed a tendency to form demes with very low gene flow among them, while in the derived species such tendency was not shown. However, because of the characteristics of the region they occupy, populations are of moderate size, and genic flow is low. Lack of correlation between gene flow levels and geographical distance between population pairs would indicate a recent and fast colonization of its distribution areas by the derived species. It is possible that fixation of Robertsonian fusions occurred in a marginal deme of the ancestral species, e.g. in a parapatric geographical context.

Molecular identification and phylogenetic relationships among the species of the genus Oligoryzomys (Rodentia, Cricetidae) present in Argentina, putative reservoirs of hantaviruses

The systematics and geographical distribution of the species of Oligoryzomys present in Argentina are poorly known. From some of the species different hantavirus genotypes have been recovered. In order to contribute to the accurate identification of those species and to infer their phylogenetic relationships, we analysed data of restriction sites and sequences of the mtDNA d‐loop region. The samples used represent almost all Oligoryzomys species known to occur in Argentina. The trees obtained with the two types of data were similar, showing high bootstrap values for the majority of the nodes. Our results support the idea that the specific name Oligoryzomys longicaudatus should be applied only to individuals from the south of Argentina and Chile and confirm that O. nigripes and O. delticola are conspecific. Specimens identified as O. flavescens conform three related clades, probably belonging to a species complex. This study also emphasises that the use of a DNA fragment characterised by a high evolutionary rate compare with other mitochondrial segments as the d‐loop, was appropriate to infer the phylogenetic relationships in a group originated by a rapid speciation process. We also suggest the following modifications in the rodent–hantavirus relationships: the only viral genotype associated to O. longicaudatus would be Andes Sout, while O. chacoensis would be the natural host of the genotype Oran. One of the forms of the O. flavescens complex would be the natural host of the genotype Bermejo. Our data are consistent with the hypothesis that each hantavirus genotype is associated with a specific rodent host.

Restriction fragment-length polymorphism of the mtDNA A+T-Rich region as a genetic marker in Aedes Aegypti (Diptera: Culicidae)

Abstract The usefulness of the control region of mtDNA as a tool for the study of population structure in the mosquito Aedes aegypti (L.) was analyzed. Population samples were taken from several geographic areas of Argentina; one additional sample from the city of San Juan, Puerto Rico, was included for comparison. In individual mosquitoes, the A+T-rich and the COII-COIII regions were amplified by polymerase chain reaction using universal insect primers. Restriction fragment-length polymorphisms were investigated from amplification products. In the A+T region, three amplified fragments of different total length were obtained (of ≈2,500, 2,300, and 2,100 bp). The enzymes Ssp I, Dra I, Pac I, and Apo I produced polymorphic patterns. Including total fragment length as a variable, eight different haplotypes were resolved for Argentinian populations, some presenting a restricted geographic distribution. The coding COII-COIII region revealed very low polymorphism. Although 11 restriction enzymes were employed to analyze this region, only two different haplotypes were found, one of them shared by populations as distant as Buenos Aires and San Juan. We demonstrated that total length and restriction fragment-length polymorphisms within the noncoding A+T-rich region, but not the coding COII-COIII fragment, are informative to discriminate Aedes aegypti populations. Mean FST value (0.48, P < 0.001) indicates an important degree of differentiation among populations. Absence of an isolation by distance pattern was demonstrated.

Distribution and hybridization between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina

To better undesrtand the distribution of Culex pipiens and Cx. quinquefasciatus in Argentina, samples were collected from six localities situated in a North-South line from Castelli (Chaco Province) to Puerto Madryn (Chubut Province). Identification was based on the morphology of male genitalia. Only Cx. quinquefasciatus was found in Castelli and Esperanza, while in Rosario, 95.3% belonged to this species and 4.7% represented hybrid forms. Southern samples included only Cx. pipiens. With the purpose of verfying if Cx. pipiens and Cx. quinquefasciatus hybridize, different crosses between the two species were perfomed. All crosses produced viable egg rafts. Hatching ranged from 70 to 100%, except in one cross, female Cx. pipiens x male Cx. quinquefasciatus, where a high incompatibility was observed (11.1%hatch). The F1 hybrids obtained all crosses were fertile. The finding of hybrid forms in nature can be interpreted as evidence for subspecific status of Cx. pipiens and Cx. quinquefasciatus in Argentina.

A contribution to the subgeneric systematics of Oligoryzomys (Rodentia, Muridae) from Argentina by means of PCR–RFLP patterns of mitochondrial DNA

Subgeneric recognition of rodents of the genus Oligoryzomys from Argentina is specially difficult because morphologic and morphometric criteria to identify species are poorly defined. In order to contribute to the unequivocal identification and geographic distribution of Oligoryzomys species, we studied the restriction fragment length polymorphism (PCR-RFLP) patterns of the D-loop region of the mitochondrial DNA in specimens collected from different regions of Argentina. PCR amplification products of 59 individuals were digested with five restriction enzymes. Patterns obtained were used to produce two phylogenetic consensus trees, one obtained with PAUP and the other with MIX analyses. All specimens from the Patagonia (southern region) clustered together and would correspond to O. longicaudatus. Individuals from the North grouped in four clusters (two of them comprising one individual), supported by high bootstrap values. The one including the 82% of the rodents from the northern region would correspond to the species O. chacoensis.