Comparison of transmission parameters between Anopheles argyritarsis and Anopheles pseudopunctipennis in two ecologically different localities of Bolivia.

BACKGROUND: Anopheles (Anopheles) pseudopunctipennis is a recognized malaria vector in the slopes of the Andes of Bolivia. There, other species might be involved in malaria transmission and one candidate could be Anopheles argyritarsis. Although it is generally admitted that this species is not a malaria vector in the neotropical region, its potential role in transmission is still controversial and this situation has to be cleared, at least for Bolivia. Comparing the vectorial efficiency of An. pseudopunctipennis with that of An. argyritarsis could solve the question. METHODS: The two species were sampled throughout Bolivia to estimate their degree of co-existence in their distribution range. Vectorial efficiencies of the two species were compared in two ecologically different localities where the species were sympatric by analysing their vectorial capacities and components (i e, human biting rates, human biting index, survival, durations of the gonotrophic cycle and extrinsic cycle), and the entomological inoculation rates (EIR). Mosquitoes were sampled monthly during more than one year in the two localities. A monthly sample consisted in hourly captures in four houses (inside and outside) in each locality, during four consecutive nights. Climatic variables (temperature, humidity, potential evapo-transpiration and precipitations) were recorded to better understand variability in the entomological parameters. Relationships were analysed using multivariate methods. RESULTS: Anopheles pseudopunctipennis and An. argyritarsis are "altitude" species, sharing the same geographical distribution range in the Andes of Bolivia. No Plasmodium parasite was identified in An. argyritarsis and estimates of the vectorial capacity indicated that it is not a malaria vector in the two studied localities, unlike An. pseudopunctipennis which showed positive EIRs. This latter species, although not a very good malaria vector, exhibited better life traits values and better behavioural characteristics in favour of transmission as compared to An. argyritarsis. CONCLUSIONS: In the Andes of Bolivia, above 1000 m of altitude, An. pseudopunctipennis is likely to be the only malaria vector. There, it is present almost everywhere and priority control effort should be directed toward this species. Below 1000 m of altitude, vector incrimination should also be focused on other sympatric species (likely not An. argyritarsis) that might be locally important. From the present study, candidates would be among Anopheles rangeli, Anopheles triannulatus s.l., Anopheles trinkae, Anopheles nuneztovari s.l., Anopheles oswaldoi s.l. and Anopheles benarrochi s.l.

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae).

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.

A physiological time analysis of the duration of the gonotrophic cycle of Anopheles pseudopunctipennis and its implications for malaria transmission in Bolivia.

BACKGROUND: The length of the gonotrophic cycle varies the vectorial capacity of a mosquito vector and therefore its exact estimation is important in epidemiological modelling. Because the gonotrophic cycle length depends on temperature, its estimation can be satisfactorily computed by means of physiological time analysis. METHODS: A model of physiological time was developed and calibrated for Anopheles pseudopunctipennis, one of the main malaria vectors in South America, using data from laboratory temperature controlled experiments. The model was validated under varying temperatures and could predict the time elapsed from blood engorgement to oviposition according to the temperature. RESULTS: In laboratory experiments, a batch of An. pseudopunctipennis fed at the same time may lay eggs during several consecutive nights (2-3 at high temperature and > 10 at low temperature). The model took into account such pattern and was used to predict the range of the gonotrophic cycle duration of An. pseudopunctipennis in four characteristic sites of Bolivia. It showed that the predicted cycle duration for An. pseudopunctipennis exhibited a seasonal pattern, with higher variances where climatic conditions were less stable. Predicted mean values of the (minimum) duration ranged from 3.3 days up to > 10 days, depending on the season and the geographical location. The analysis of ovaries development stages of field collected biting mosquitoes indicated that the phase 1 of Beklemishev might be of significant duration for An. pseudopunctipennis. The gonotrophic cycle length of An. pseudopunctipennis correlates with malaria transmission patterns observed in Bolivia which depend on locations and seasons. CONCLUSION: A new presentation of cycle length results taking into account the number of ovipositing nights and the proportion of mosquitoes laying eggs is suggested. The present approach using physiological time analysis might serve as an outline to other similar studies and allows the inclusion of temperature effects on the gonotrophic cycle in transmission models. However, to better explore the effects of temperature on malaria transmission, the others parameters of the vectorial capacity should be included in the analysis and modelled accordingly.

Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies.

Without an adequate DNA extraction protocol, the identification of Plasmodium species in whole mosquitoes by PCR is difficult because of the presence of reaction inhibitors from the insects. In this study, eight DNA extraction protocols were tested, from which a chelex-based protocol was selected. Then a semi-nested multiplex PCR technique that detects and distinguishes among the four human Plasmodium species in single mosquitoes and in pools of up to 100 mosquitoes was optimized. The technique was used to detect P. vivax in wild-caught Anopheles pseudopunctipennis from a village in the Andean valleys of Bolivia in May 2003. The prevalence of infection was 0.9%. This is the first direct evidence of P. vivax transmission by this vector in this country. The extraction and PCR technique presented here can be useful to: (1) estimate Plasmodium prevalence in Anopheles populations in low prevalence areas where large numbers of individual mosquitoes would need to be processed to obtain a reliable estimate; (2) incriminate Anopheles species as malaria vectors; (3) identify all the circulating Plasmodium species in vectors from an area; (4) detect mixed infections in mosquitoes; and (5) detect mosquitoes with low-level parasite infections.

Laboratory colonization of Anopheles pseudopunctipennis (Diptera: Culicidae) without forced mating.

Anopheles pseudopunctipennis is one of the main malaria vectors in the Andean regions of South America. Few experimental data exist on this species because it is not very available in laboratories due to its eurygamic status that makes colony maintenance difficult. Indeed, individuals do not mate in the confined space of insectary cages. To avoid this problem, forced artificial mating can be used. However, this technique is time consuming, requires a well-trained technician, and is inadequate for easy mass production, which is sometimes necessary for certain experimental works. This study presents a technique based on exposure of adult mosquitoes to a blue stroboscopic light for 20 min during several nights, which encourages them to copulate naturally under laboratory conditions. After some generations, a self-free-mating strain was obtained. The technique is simple, inexpensive and is probably effective whatever the An. pseudopunctipennis strain considered.