A temporal observational study on culicid entomofauna was conducted in a region characterized as a fragment of the Atlantic Forest that forms the Tinguá Biological Reserve in the State of Rio de Janeiro. This investigation was performed with the aim of analyzing the influence of climatic factors (temperature and relative air humidity) on the activity levels at different times of the day among mosquito species within the ecosystems that form the Tinguá Biological Reserve. The abundance index and dominance coefficient were calculated in relation to 61 mosquito species that were caught at four sampling sites, in the mornings, afternoons, and evenings. The results revealed that culicid species were distributed with greater incidence during the two diurnal periods and that their preference for times of the day was directly influenced by the climatic variables analyzed. The latter acted as limiting factors for occurrences of mosquito species.
Knowledge of the distribution and abundance of culicids in forested areas that form remnants of the typical primary Atlantic Forest of Brazil is of great importance in relation to implementing eco-epidemiological analyses on the vector entomofauna. The biome of the Atlantic Forest presents great diversity both in its flora and fauna of vertebrates and invertebrates, which makes it possible for a multiplicity of niches to be available for culicid development. Although investigations on the ecological characteristics of culicid fauna have been conducted in various areas of Brazilian territory, in the State of Rio de Janeiro they have always been conducted because of the presence of certain transmitters of infectious agents within urban environments.
However, it should not be forgotten that, within natural environments, insect populations in any given biogeographical area are distributed in ecologically stable communities. These occur in a nonstatic form, and the relationships between populations are subject to the constant fluctuations on the environment. In these spaces, environmental factors influence how activities are carried out, whether new procreation areas are established, or the extent of geographical distribution that the species will have in these new areas. Thus, the behavior of the culicid fauna in natural ecotopes with a history of intensive use and subsequent conservation, as is the case of the Atlantic Forest in the State of Rio de Janeiro, needs to be monitored with periodic evaluations of the diversity and habits of the biological vectors, to improve the understanding of the impact of human interventions and their epidemiological implications.
The Tinguá Biological Reserve is surrounded by municipalities with high population density, and this gives rise to intensive human action such as unregulated tourism, poaching, plant extraction, and housing construction in the region. These factors, together with the hot and humid climate, arouse special interest with regard to gaining deeper knowledge of the hematophagous activity of the mosquito fauna that are vectors for agents with pathogenic action toward humans and domestic animals.
Lopes et al. 1999 reported the occurrence of ≈71 species of mosquitoes. Among these were Haemagogus leucocelaenus (Dyar and Shannon), Haemagogus capricornii Lutz, Haemagogus janthinomys Dyar, and Sabethes chloropterus (Von Humbold). These species have epidemiological significance as participants in the maintenance cycle of different arboviruses, including yellow fever. Other arboviruses, such as Wyeomyia, Ilhéus, Maguari, Tucunduba, and Una, have been isolated from Haemagogus species (Karabatsos 1985, Hervé et al. 1986). Also among the mosquitoes occurring in this region are certain species of anophelines. The species that have been correlated with transmission of the agent that causes malaria are Anopheles albitarsis Lynch-Arribalzaga, Anopheles darlingi Root (Deane et al. 1971), and Anopheles cruzii Dyar and Knab. These stand out because of their ability to migrate from forest environments to modified environments.
The aim of this study was to ascertain, evaluate, and correlate the hematophagous activity of the culicid fauna at four previously selected sampling sites in the Tinguá Biological Reserve, at three different times of the day.
Materials and Methods
The Tinguá Biological Reserve, which was created by Decree-Law 97,780 in 1989, is located between the municipalities of Nova Iguaçu, Duque de Caxias, Miguel Pereira, Petrópolis, and Japeri, between the coordinates 22°28′-22°39′S and 43°13′- 43°34′W. It is in the Serra do Mar, the coastal mountain range of the State of Rio de Janeiro, and forms part of the geomorphological unit of the Serra dos Órgãos, within the Serra do Mar system. The Tinguá Biological Reserve presents a vegetation cover typical of dense montane and submontane ombrophilic forest (Rizzini 1986), with a tropical wet climate without a defined dry season. The mean monthly temperatures range from 22.8°C to 25.2°C, and the relative air humidity ranges from 76 to 88%.
Four sampling sites were established, as follows: site A, forest close to the administration center of the reserve, with intensive human action and a rich flora of ornamental plants typical of the region; site B, forest with modified original vegetation and recomposed vegetation consisting predominantly of bamboo monoculture; site C, secondary forest with flora recomposition similar to the original biocenotic structure, cut by paths leading to the aqueduct dating from the end of the 19th century that is responsible for the water supply for the communities surrounding the reserve; and site D, forest similar to that at site C, but directly influenced by the river and waterfalls that form the small reservoir that supplies the aqueduct (Fig. 1). At each of these points, mosquito catching was performed at ground level every fortnight, at three different times over a consecutive 24-h period, as follows: 1000–1200 h, 1400–1600 h, and 1800–2000 h.
At each of these times of the day, members of the team from the Diptera Laboratory (Instituto Oswaldo Cruz/Fundaçao Oswaldo Cruz) went to collect mosquito specimens that had been caught in Shannon traps and to collect any specimens that were present on the surrounding vegetation, along with any specimens that would possibly be attracted by the investigators, with the aid of suction tubes. During the evening period, at the same time as catching mosquitoes using this method, sampling was also performed using illuminated Shannon traps. After catching the mosquitoes, they were transferred to standardized collection containers and were taken to the laboratory for screening and identification. They were then incorporated into the Entomological Collection of the Oswaldo Cruz Institute, under the title “Atlantic Forest Collection.” Temperature and relative air humidity data were recorded every hour, using combined thermometer/hygrometers (Weather Monitor II, David Instruments Corp., Haywood, CA). These were set up 1 meter above ground level at the capture sites.
The culicid species were identified by means of direct observation of the morphological characteristics that could be seen under a stereoscopic microscope. The identifications were based on the dichotomous keys prepared by Lane (1953a, b), Consoli and Lourenço-de-Oliveira (1994), and Forattini (2002). The abbreviations of the genus and subgenus names followed the proposals of Reinert (2001).
The field data were analyzed such that certain aspects of the ecological relationships between the culicid populations and the distribution over the times of the day could be expressed, thereby enabling population analysis in relation to the capture times. The comparisons between the numbers of mosquito species that made up the diversity encountered at the different times of the day were analyzed by means of the dominance coefficient (DC).
The DC was used to measure the dominance pattern among the species in a given habitat, in relation to the whole community analyzed. The DC was calculated in accordance with Serra-Freire (2002), such that DC = (Σxi/Σti) × 100, where Σxi = sum of the number of mosquitoes of a given species found in each geographical region and Σti = sum of the number of mosquitoes of all the species found in each geographical region.
To quantitatively analyze the degree of association between the samples in relation to altitude and abundance, a correlation coefficient was used, as follows: rj = Σ(dxj × dyj)/(n × sx × sy), where: rj = correlation coefficient, dxj = deviation of altitude values in relation to mean altitude, dyj = deviation of the number of insects in relation to the mean number of insects, sx = standard deviation for the altitudes, sy = standard deviation for the number of mosquitoes, and n = number of observations.
To evaluate the proportions of each mosquito species in relation to the total number of samples analyzed, and to compare these with the expected values from known data in the literature, the χ2 test was used (adherence test). The significance level was set at 10% for a type I error (α = 10%).
Over the 214 h of sampling, 2,177 mosquito specimens were collected, and these belonged to 61 species (Table 1). Some of these species have frequently been incriminated as vectors for the agents that cause diseases among humans (Tables 1, 2, and 3). Among the species of anophelines that were caught, the presence of An. cruzii stood out. This is an important vector for malaria in Brazil. The following other mosquito species that have traditionally been implicated in the transmission of diseases to humans and/or animals, caused by different types of arbovirus, were also found: Culex quinquefasciatus Say, Culex nigripalpus Theobald, Coquillettidia venezuelensis Theobald, Aedes scapularis (Rondani), Hg. capricornii, Hg. leucocelaenus, Sa. chloropterus, and Aedes serratus (Theobald).
The differences between the quantities of species that made up the diversity of mosquitoes in each of the three sampling periods at the four sampling sites were not significant. Consequently, it can be accepted that the mosquito capture periods at the four sites did not differ between each other (Table 4). However, we observed significant variations in the total number of species in the three sampling periods together, between the four sampling sites (Table 4). The forest at a low altitude on the path to the aqueduct (site C) presented greatest diversity. Qualitatively, it was seen that the diversity of species caught at the different times was dissimilar (Table 4).
The diversity of mosquito species present in a single collection period was significantly greater than the diversity of species present for two periods or for three periods. This indicates that there was a more tightly defined circadian period for two or more times the number of species that were present in the environment during more than one period (Table 5). The period from 0800 to 1000 h was, by a significant amount, the one with least hematophagous activity among the mosquito species, even considering that the dominant species differed between the times studied (Table 5). However, this preference was not shown among the species that sought a blood meal during all three periods investigated (Table 5).
Presence of a given species only at a single capture site was significantly more frequent than presence of species at more than one site (Table 6). The DCs for one or two species per site did not differ significantly from each other, although the dominant species may have been different (Table 6). However, the difference was significant with regard to the presence of three or four species per capture site (Table 6).
Among the times of the day analyzed and the climatic conditions at the capture sites, site B between 1800 and 2000 h presented a significantly higher temperature (Table 7). At site A alone, the temperatures during the three capture periods differed significantly from each other, with the highest temperature between 1400 and 1600 h. At site B, the period with the lowest temperature (0800–1000 h) differed significantly from the other two periods. There were significant differences in relative air humidity percentages between the different capture sites, with greater air humidity shown during the earlier times of the day, and at capture sites A and B (Table 7).
From the sampling carried out using illuminated Shannon traps, it could be seen that these presented lower population densities, compared with the observations made when the targets were members of the laboratory team. There was no significant difference regarding the diversity of mosquito species caught. Culex (Melanoconion) sp. dominated among the samples (Table 8).
Capture site A presented the highest population density, considering both types of trap that were used. Approximately 40% of the total number of mosquitoes was caught during the evening period (1800 to 2000 h).
The forested area that presented widespread bamboo vegetation, where capture site B was set up, was the one that presented the highest capture rate from the sampling carried out on members of the laboratory team during the diurnal period (Table 4). The prevalent species were Sabethes identicus Dyar and Knab (0800 to 1000 h) and Wyeomyia theobaldi Lane and Cerqueira (1400 to 1600 h). Capture site D was located at a higher elevation, at ~1,000 m above sea level, with little oscillation in relative air humidity (Table 7), and its vegetation consisted of closed high altitude forest. This was the site with the highest total capture during the afternoon period (1400 to 1600 h), especially of Wy. theobaldi.
During the sampling in the evenings using the illuminated Shannon trap, 880 mosquitoes were caught (Table 2), with predominance of the genus Culex (754 specimens). The most abundant species were Cx. (Melanoconion) spp. (24.70%) and Cx. nigripalpus (23.80%), and there was no difference in percentage of occurrence between them. The other species with abundance greater than or equal to 1% were as follows: Cx. quinquefasciatus (20.80%), Culex spp. (11.60%), An. cruzii (5.90%), Culex declarator Dyar and Knab (4.10%), and Ae. scapularis (1.00%).
Members of the team from the Diptera Laboratory (Instituto Oswaldo Cruz) collected mosquitoes resting on vegetation and any specimen that possibly was attracted by the presence of humans. From these sources, 953 specimens were caught during the three sampling periods. Wy. theobaldi presented the highest population density (17.21%), followed by Sa. identicus Dyar and Knab (11.44%), An. cruzii (10.60%), and Cx. nigripalpus (4.93%) (Table 1).
The results from the four capture sites, for the three capture periods, in relation to members of the laboratory team at ground level, demonstrated that there was greater preference for the evening and afternoon periods (Tables 1 and 2), during which the mean temperature and humidity ranges were 19.9°C to 27.8°C and 61.2 to 89.9%, respectively (Table 7).
Sabethini species, which are typically diurnal, were the most abundant group over the course of the studies conducted in the Tinguá Biological Reserve. Among the species encountered, Runchomyia frontosa Theobald, Wyeomyia aporonoma Dyar and Knab, Wyeomyia luteoventralis Theobald, Sa. identicus, Wyeomyia pilicauda Root, and Wyeomyia flabelata Lane and Cerqueira were distributed almost exclusively during the morning and afternoon periods, with rare captures in the evenings. However, the only species that were absent from collections in the evenings were Wy. theobaldi and Wyeomyia palmata Lane and Cerqueira.
It was observed that Wy. aporonoma Dyar and Knab appeared in all the capture periods, in considerable numbers, but demonstrated a slight tendency toward performing hematophagy in the evenings, thus showing a certain degree of eclecticism (Table 1).
Among the species of the genus Haemagogus, Hg. capricornii had a clear preference for activity during the afternoons, whereas Hg. leucocelaenus was eclectic regarding the time of the day to perform hematophagy (Table 1).
In a general manner, the Tinguá Biological Reserve can be classified as a preserved area of Atlantic Forest. It remains an unknown area with regard to the mosquito species that are considered to be vectors for agents with pathogenic action toward humans and wild animals. Although the four sampling sites of the current study belong to the same ecosystem, they present biocenotic structures with some variations. It was observed that these configurations caused changes to the profile of population dynamics, both in terms of species diversity and in terms of the number of specimens caught. The mosquito species in the Tinguá Biological Reserve that were analyzed regarding the activity time of their hematophagy were distributed with greater incidence during the two diurnal periods studied. The subfamilies Anophelinae and Culicinae appeared significantly more frequently in the evenings, with the exception of the Sabethini tribe. This corroborates the results found by Guimarães and Victorio (1986), in areas of the Serra dos Órgãos national Park, in the State of Rio de Janeiro, and by Dalla Bona (2008), in areas of the Palmito State Forest, in the municipality of Paranaguá, State of Paraná.
The incidence of culicid species caught in the Tinguá Biological Reserve was directly influenced by the plant coverage of the Atlantic Forest in the study area, and by the local factors of temperature and relative air humidity (Table 7).
According to the observations of Guimarães et al. (1984) and Guimarães et al. (2000), the main climatic factors that could influence the biocenotic structure of the culicid fauna would be the temperature and relative air humidity, which could cause mosquito species to disappear during the dry months of the year.
Climate changes could worsen the threat of infectious diseases. First, many diseases and their vectors could appear because of the higher temperatures; and second, greater pollution and the destruction of natural biomes could cause resurgence of both recent and old diseases. One prominent example of this is the way in which occurrences of yellow fever, dengue, and other diseases have been correlated with deforestation and its unpredicted effects (Martins 2008).
Mosquitoes in the genus Culex were potentially nocturnal, because capture during the diurnal periods was rare. Lourenço-de-Oliveira and Silva (1985), Guimarães and Victório (1986), and Dalla Bona (2008) made similar observations. Mosquitoes were also found at high densities close to human habitation in the evenings, and these rates were favored through human modifications to the environment surrounding dwellings, as affirmed by Klein et al. (1992).
The Sabethini species were confirmed as presenting typically diurnal behavior, and the ones with greatest density were Wy. theobaldi and Sa. identicus. It is important to take these into consideration regarding the risk of arbovirus infection, given that some species of the genus Sabethes have been found to be naturally infected by such pathogens to humans (Forattini 1965).
Guimarães et al. (1986) and Alencar et al. 2008 observed that Hg. capricornii increased significantly in density over the course of the day, as the luminosity and temperature increased, with consequent lowering of the relative air humidity. Similar behavior was observed in the Tinguá Biological Reserve for the species Wy. theobaldi, Wyeomyia fuscipes Edwards, Wy. pilicauda, and Wy. palmata. However, this was not shown for Sa. identicus, which followed a tendency toward inverse distribution, with greater population density in the mornings and a decrease over the course of the day. In the current study, only one specimen was caught in the evening.
Few specimens of An. cruzii were caught. This is the vector for the agent that causes wild and simian malaria, and it has hematophagous activity during the diurnal period, with greatest density during the early hours of the night. This behavior is in accordance with the findings of Consoli and Lourenço-de-Oliveira (1994), who reported that shaded, damp forested areas formed a good shelter for this species. In such areas, this species is found to present hematophagous activity both diurnally and nocturnally, but with sharply increased activity at dusk and during the early hours of the night.
During the sampling period in the Tinguá Biological Reserve, it was not possible to evaluate the degree of eclecticism of Ae. scapularis, in the way that was observed by Forattini et al. (1981) and Lourenço-de-Oliveira and Silva (1985). In the current study, this species was only caught in the evenings. Guimarães and Victório (1986) reported that Aedes terrens Walker and Ae. scapularis, species that are found at low density and with nocturnal preference, can be found performing hematophagy during the day. Hence, it may be considered that in the Tinguá Biological Reserve, the low density of Ae. terrens during the evenings and afternoons and the low density of Ae. serratus at all three times of the day confirmed the affirmations of Guimarães and Victório (1986).
In view of the incidence and abundance, in the Tinguá Biological Reserve, of species that are usually implicated in transmission of pathogenic agents, it may be considered this region is vulnerable to occurrences of diseases. Thus, entomological surveillance needs to be maintained.
We thank Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis for the licenses for investigations in a conservation unit; the managers of the Tinguá Biological Reserve (Maria L. Xavier and Luís H. Teixeira) for the opportunity to carry out fieldwork in the reserve and for the support provided; and the Research Funding Agency of Rio de Janeiro (FAPERJ).