Juan Rondan Dueñas

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.

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.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area.

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.

Isolation of Yellow Fever Virus from Mosquitoes in Misiones Province, Argentina

Yellow fever (YF) is a viral hemorrhagic fever endemic to tropical regions of South America and Africa. From 2007 to 2009 an important epidemic/epizootic of YF was detected in different populations of howler monkeys (Alouatta species) in Misiones, a northeastern Argentinian province. Yellow fever virus (YFV) infection was researched and documented by laboratory tests in humans and in dead Alouatta carayá. The objective of that research was to investigate the circulation of YFV in mosquitoes, which could be implicated in the sylvatic transmission of YF in Argentina. The above-mentioned mosquitoes were captured in the same geographical region where the epizootic took place. A YFV strain was isolated in cell culture from pools of Sabethes albiprivus. This study is not only the first isolation of YFV from mosquitoes in Argentina, but it is also the first YFV isolation reported in the species Sabethes albiprivus, suggesting that this species might be playing a key role in sylvatic YF in Argentina.

Morphological and molecular characterisation of the entomoparasitic nematode Hammerschmidtiella diesingi (Nematoda, Oxyurida, Thelastomatidae)

A population of Hammerschmidtiella diesingi was detected in specimens of Periplaneta americana collected in Córdoba city (Argentina) was studied. Nematodes were characterized based on morphological, morphometric and genetic (D2–D3 expansion segment) analyses. New data are provided, especially on particular morphological characters that were a matter of controversy in previous studies. H. diesingi is reported in Argentina for the first time.

Assessment of genetic variability in populations of Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 (Nematoda: Pratylenchidae) from Argentina

Nacobbus aberrans produces severe damage to agriculture and is considered to be of quarantine importance. The levels of polymorphism and genetic differentiation among Argentine populations of this species from different hosts and origin were studied using Inter-Simple Sequence Repeat (ISSR) as molecular markers. Second-stage juveniles from nine populations associated with three different hosts (tomato, quinoa and potato) were analysed individually. Three primers were selected because they produced clear and 100% reproducible patterns; 37 fragments were considered for the analysis. The technique employed revealed high levels of polymorphisms, the highest proportion of genetic diversity being found within populations (95%). Populations showed a tendency to group together according to their original host, based on their pairwise FST. Low levels of genetic differentiation among populations were observed, suggesting an extensive gene flow among them. Passive dispersal of nematodes by natural means and anthropogenic activities would probably be, at least partially, responsible for the results observed. This work is the first study of genetic structure of N. aberrans populations at a macrogeographical level using ISSR markers.

Phylogenetic relationships among populations of the Nacobbus aberrans (Nematoda, Pratylenchidae) complex reveal the existence of cryptic species

The plant‐parasitic nematode Nacobbus aberrans sensu lato is an agricultural pest of quarantine importance. Due to the morphometric, physiological and genetic variability observed within the species, there is no agreement on the taxonomy of this nematode. The objective of this study was to analyse the ITS rDNA region and the D2–D3 expansion segments of 28S rDNA in 10 Argentine populations and one from Ecuador and to establish their phylogenetic relationship with other known sequences from South and North America. Phylogenetic trees of the ITS gene showed seven statistically well‐supported clades; the high and significant Fst values obtained among these groups confirmed this partitioning. The Argentine populations here considered were separated into three clades: one comprising a population from the Andean region and two grouping nematodes from lower altitudes. Three other clades were distinguished for South American populations, which included known sequences of individuals from Peru, Bolivia and north of Argentina. The other clade included sequences from Mexico, Ecuador and two Argentine populations of unknown origin. The important degree of genetic divergence observed among Andean populations suggests that the Andes may have played a crucial role in speciation of Nacobbus, which would have originated in this region. Although D2–D3 segments exhibited lower variation, they were useful for establishing phylogenetic relationships among the Argentine populations considered in this work. As there are no other GenBank sequences available for these segments, it was not possible to make comparisons with other populations from South and North America. The considerable genetic differentiation observed in ITS rDNA region among Nacobbus populations showed evidence of cryptic species within the N. aberrans s.l. complex. Integration of morphological and morphometric studies and molecular analyses considering other genes may aid in the identification of species and their phylogenetic relationships within this genus.

Genetic variability estimated with RAPD-PCR markers in two populations of Heterodera glycines Ichinohe, 1952 (Nematoda: Heteroderidae) from Argentina

Knowledge of genetic variability within and between phytophagous nematode populations is important for the selection of suitable control strategies. The nematode Heterodera glycines is the most important pathogen of soybean in many producer countries, causing significant economic losses. The levels of variability were analysed in two populations of the nematode from Argentina, using Random Amplified Polymorphic DNA markers in order to evaluate the genetic population structure of this species. DNA was extracted from single females of race 1 and race 3 populations. Five primers were selected and 50 consistent fragments were considered for further analysis. Populations studied showed high levels of genetic diversity. Most of the total variation detected in allele frequencies was attributed to variability among individuals of the same population (83%) rather than inter-population differences (17%). None of the populations demonstrated exclusive bands. However, 68% of fragments showed significant differences in their allele frequencies. This study revealed an important degree of genetic differentiation between both populations, probably as a consequence of limited gene flow between them or because each population was under different management practices at its site of origin. Results herein represent the first analysis of genetic structure in H. glycines populations using RAPD-PCR technique.

Effects of insect cadavers infected by Heterorhabditis bacteriophora and Steinernema diaprepesi on Meloidogyne incognita parasitism in pepper and summer squash plants

E. E. Del Valle, P. Lax, J. Rondan Duenas, and M. E. Doucet. 2013. Effects of insect cadavers infected by Heterorhabditis bacteriophora and Steinernema diaprepesi on Meloidogyne incognita parasitism in pepper and summer squash plants. Cien. Inv. Agr. 40(1):109-118. The effects of insect cadavers infected with three isolates of Heterorhabditis bacteriophora and one isolate of Steinernema diaprepesi on a population of Meloidogyne incognita in pepper (Capsicum annuum) and summer squash (Cucurbita maxima) were evaluated in greenhouse experiments carried out in Santa Fe (Argentina). Insect cadavers were obtained for the experiments from last instar larvae of Galleria mellonella and Tenebrio molitor that had been infected with entomopathogenic nematodes. Two six-day-old insect cadavers per pot were placed below the soil surface, and the soil was inoculated with 100 second-stage juveniles of M. incognita. Sixty days after inoculation, the following parameters were recorded for each plant: number of leaves; dry weight of aerial parts; numbers of galls, egg masses and eggs; and numbers of galls, egg masses and eggs g -1 of root fresh matter. In pepper, the only variable affected by the infected cadavers with respect to control was the number of eggs in the treatment involving T. molitor cadavers infected with the H. bacteriophora isolate Rama Caida. In summer squash, several treatments using infected cadavers resulted in a decrease in the numbers of galls and egg masses. Only the treatment involving G. mellonella cadavers infected with the H. bacteriophora isolate Rama Caida proved to be efficient in reducing the number of M. incognita eggs. Our results indicated that the application of insect cadavers infected with the entomopathogenic nematodes studied might reduce M. incognita damage in pepper and summer squash plants.

Ecological aspects of an isolate of Steinernema diaprepesi (Rhabditida: Steinernematidae) from Argentina

Ecological aspects of Steinernema diaprepesi isolate SRC were studied to evaluate the species potential as biological control agent of insect pests. Under laboratory conditions, the following aspects were determined: the nematode life cycle, pathogenicity to several arthropods, reproductive capacity, tolerance to desiccation, effect of temperature on survival and infectivity of infective juveniles (IJs), and influence of soil texture and soil water potential on the isolate. The parasitic cycle on last-instar larvae of Galleria mellonella at 25°C was completed 8 days after infection. The nematode showed high virulence to lepidopteran larvae, being limited or nil in the remaining orders of arthropods evaluated. An acceptable offspring production of S. diaprepesi was confirmed in the species G. mellonella and S. frugiperda, suggesting that the isolate would have potential for control of lepidopteran larvae. Optimum temperature for reproduction was 20–25°C. IJs survived exposure to a range of temperatures between 10 and 40°C, with a significant reduction in the number of live IJs at 40°C. The nematodes remained infective at 20–40°C. IJ mortality was 100% on day 6 of exposure to 85% RH. The movement of IJs observed in the soil column experiments revealed that the isolate uses a cruiser-type search strategy. Soil texture and water potential significantly influenced IJ movement, search and penetration of G. mellonella larvae. The efficacy of this isolate was found to be favoured in sandy soils, regardless of the soil water potential.