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Mem. Inst. Oswaldo Cruz vol.97 no.8 Rio de Janeiro Dec. 2002
Vol. 97(8): 1117-11120, December 2002
Sabrina Cardozo Gonçalvez de Carvalho/*, Ademir de Jesus Martins Junior/*, José Bento Pereira Lima/*, Denise Valle/*/+
Laboratório de Transmissores de Hematozoários, Departamento de Entomologia, Instituto Oswaldo Cruz- Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil *Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, Brasil
Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26ºC, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 ± 2ºC, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21 ± 2ºC decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.
Key words: Anopheles albitarsis - Anopheles aquasalis - Anopheles rearing - malaria vector - temperature influence - embryonic development
Nowadays, despite control efforts, malaria affects around 400 million persons each year. This disease is typical in tropical and subtropical regions, where the elevated relative humidity and temperature, together with poor social and economic conditions, offer the ideal environment for the development and maintenance of its vectors (Butler et al. 1997, Morel 2000).
In Brazil, malaria is one of the major endemic diseases, the Amazon region being responsible for more than 99% of the cases (Passos & Fialho 1998). In this country, Anopheles (Nyssorhynchus) darlingi Root, 1926 and Anopheles (Nyssorhynchus) aquasalis Curry, 1932 are primary vectors. Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga, 1878, a complex of at least four species, is considered a secondary malaria vector (Rosa-Freitas et al. 1990, Consoli & Lourenço-de-Oliveira 1994, Wilkerson et al. 1995).
Although An. albitarsis and An. aquasalis have been reared in our laboratory as free-mating colonies since 1995, unexpected mortality events and mosquito density fluctuations have been hampering their use as laboratory models. Our present aim is to investigate some basic aspects concerning their biology, in order to optimize Ano-pheles colony maintenance.
Our previous empirical observations suggested that temperature can greatly influence the developmental kinetics and the longevity of these vectors. Accordingly, there are several reports dealing with temperature influence over the developmental kinetics of larvae and adults of Aedes aegypti (Rueda et al. 1990, Tun-Lin et al. 2000), Culex spp. (Rae 1990, Rueda et al. 1990, Reisen 1995)and An. sergentii (Beier et al. 1987). Influence of temperature on mosquito vectorial competence (Kay et al. 1989), induced cross-tolerance between temperature and an insecticide (Patil et al. 1996) and temperature effect on phenotypic characteristics and its implication to taxonomy (Le Sueur & Sharp 1991) have also been noted. However, there are few reports dealing specifically with temperature influence over mosquito embryogenesis (Trpi et al. 1973, Rayah & Groun 1983, Van der Linde et al. 1990).
As a first approach to optimize the maintenance of our colonies, we decided to specifically investigate An. albitarsis and An. aquasalis embryonic development under two different temperatures.
Mosquitoes - Free mating colonies of both An. albitarsis s. s. and An. aquasalis, maintained in the laboratory since 1995, were used. Rearing conditions were 26ºC ± 1ºC and 80% r.h. as described elsewhere (Horosko et al. 1997). Larvae kept in dechlorinated (An. albitarsis) or 10% seawater (An. aquasalis) were fed with powdered fish food (Tetramin®) twice a day. Adults had continuous access to a 10% sucrose solution, and females were fed on anesthesized guinea pigs in order to produce eggs.
Synchronous egglaying - Since neotropical Anopheles females lay eggs preferentially at dawn, an insectary was adapted with a 12 h dark:12 h light cycle (lights turned off from 9:00 a.m to 9:00 pm and turned on from 9:00 pm to 9:00 a.m of the following day). This procedure enabled the collection of synchronized eggs (periods of 1 h) during the day.
Cold anesthesized females were transferred to Petri dishes (6 cm diameter) covered internally with a filter paper. After recovery of the females at 26ºC, the filter paper was wetted with 600 µl of dechlorinated (An. albitarsis) or brackish (An. aquasalis) water to induce oviposition (Valencia et al. 1996).
Egg hatching monitoring - Oviposition was performed in the insectary at 26ºC and the resulting eggs were either kept in the insectary (control group) or placed in an incubator, at 22ºC (experimental group). Twenty-four hours later the eggs were split in aliquots. A total of 935 and 479 An. albitarsis eggs were used in each experimental group divided in, respectively, 15 and 9 replicates, ranging from 48 to 70 eggs. Control An. albitarsis group consisted of 160 eggs raised in pool. Each An. aquasalis experimental group was divided in seven replicates ranging from 48 to 92, in a total of 544 and 350 eggs, respectively. Control An. aquasalis group consisted of 10 replicates of 100 eggs. Hatching was scored daily at each temperature as well as maximal and minimal temperatures in both the insectary and in the incubator. The maximal temperature in the experimental condition did not exceed 22ºC or 23ºC, in the case of An. aquasalis and An. al-bitarsis respectively. The minimal temperature for An. albitarsis experiments was 20ºC while for An. aquasalis it attained 17ºC.
An. aquasalis - Two trials at 20 ± 2ºC have been performed, with a total of 894 eggs. Hatching was monitored up to 10 days after egglaying. Results were similar in both experiments: control eggs, maintained at 26ºC, hatched mainly on the 2nd day after egglaying (Fig. 1A). Total eclosion rate was 75% for the control group. On the other hand, 66-70% of eggs from the experimental groups hatched and the eclosion peak was attained at the 5th day (Fig. 1B). No eclosion was observed from the 7th day after egglaying on.
An. albitarsis - Two trials at 21 ± 2ºC were also performed with this species, with a total of 1,414 eggs. Hatching was monitored up to 20 days after egglaying. Again in this case the results obtained were similar for both experiments. In the control condition (26ºC) the majority of the eggs hatched between the 2nd and the 3rd day and total hatching was 60%. In the experimental groups, however, total eclosion did not exceed 22% (Fig. 2A). In this situation, when daily percent of hatching eggs was plotted against time, two eclosion peaks were noted, the first at the 4th-5th day after oviposition and the second after the 8th day (Fig. 2B). In both trials a period of at least 24 h with no eclosion event at all was observed, around the 6th-7th day. It was also realized that 60-70% of An. albitarsis eggs hatched before this period of no eclosion while 30-40% eclosion was obtained after this period. No eclosion was observed from the 17th day after egglaying.
Studies regarding temperature influence over the embryonic development of Culicidae can help laboratory rearing. On the other hand, the precise knowledge of mosquito embryonic developmental kinetics would assist the improvement of transgenic production protocols: generation of stable transformed lineages depends on the injection of exogenous DNA in a precise stage during embryogenesis (Catteruccia et al. 2000), which, in turn, varies greatly with temperature (Clements 1992). However, in spite of its potential importance, little has been done concerning this subject. The great majority of data related to temperature influence over Culicidae development deals mainly with larvae and adults (Rueda et al. 1990, Rae 1990, Tun-Lin et al. 2000).
Analysis of temperature influence over Aedes sticticus embryonic development revealed that the minimal and maximal temperature thresholds are, respectively, 6-8°C and 33°C. It was also verified that the time span of embryogenesis is inversely related to temperature: first instar larvae eclosion took 11.3 days (272 h) at 15ºC and only 6 days (120 h) at 30ºC. The authors did not investigate the rate of eggs' viability under different temperatures (Trpi et al. 1973).
Cx. quinquefasciatus minimal and maximal temperature thresholds for embryonic development are 13ºC and 39ºC, respectively. In this species the hatching rate varies directly with temperature, up to 32ºC, after which eclosion rates drop gradually (Rayah & Groun 1983).
An. sergentii embryos kept at 34ºC do not hatch. Nevertheless, their viability at 17°C and 27ºC is equivalent (around 85%) although in this case, as temperature increases, the duration of embryogenesis is reduced: at 27ºC, 95% of hatching is obtained on the second day, while eggs kept at 17ºC only hatch on the 4th-5th day after egglaying (Beier et al. 1987).
We analyzed the time spent to the completion of embryonic development and the eclosion rates of two neotropical Anopheles species under two different temperatures. In both cases, our data point to important differences between An. aquasalis and An. albitarsis, although both species belong to the same subgenus. Similar to other Culicidae, the duration of embryogenesis varies inversely with temperature for both Anopheles species tested. However, a high decrease in viability was only observed for An. albitarsis. This is particularly significant if it is taken into account that temperature varied from 20-23ºC for An. albitarsis and only 17-22ºC for An. aquasalis (Table).
These data indicate that temperature can influence development in a differential manner even when related species are considered. An. albitarsis was shown to be much more susceptible to lower temperature than An. aquasalis, in terms of both kinetics and rate of eclosion. Additionally, the appearance of two eclosion peaks in An. albitarsis at the lower temperature suggests developmental variability in individuals of this species related to the temperature.
We are now analyzing the effect of a broader range of temperatures over the embryonic development of these species. Additionally, the recent description of An. albitarsis embryogenesis (Monnerat et al. 2002) will be taken into account to investigate whether temperature influence is exerted preferentially over any given developmental stage or if, alternatively, embryogenesis is affected as a whole.
To the support of Instituto de Biologia do Exército for laboratory facilities.
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This investigation received financial assistance from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Conselho Nacional de Desen-volvimento Científico e Tecnológico, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, and Fundação Oswaldo Cruz (Papes II Program).
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Received 7 February 2002
Accepted 12 September 2002