Ecological aspects of mosquitoes ( Diptera : Culicidae ) in the gallery forest of Brasília National Park , Brazil , with an emphasis on potential vectors of yellow fever

Introduction: We analyzed the vertical and monthly distributions of culicid species in the gallery forest of Brasília National Park, with an emphasis on the potential vectors of yellow fever (YF). Methods: Between September 2010 and August 2011, mosquitoes were captured on the ground and in the canopy of the forest for fi ve consecutive days per month, from nine to 15 hours. The mosquitoes were examined to verify natural infection with fl aviviruses by isolation in Aedes albopictus Skuse, 1864 cells followed by indirect immunofl uorescence. Results: We identifi ed 2,677 culicids distributed in 29 species. Most of the mosquitoes were captured at ground level (69%) during the rainy season (86%). The most abundant species were Sabethes (Sabethes) albiprivus Theobald, 1903; Limatus durhamii Theobald, 1901; Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon, 1924; Haemagogus (Haemagogus) janthinomys Dyar, 1921; Aedes (Ochlerotatus) scapularis Rondani, 1848; Psorophora (Janthinosoma) ferox Von Humboldt, 1819; and Aedes (Ochlerotatus) serratus Theobald, 1901. Haemagogus janthinomys, Limatus durhamii, Psorophora ferox, Aedes scapularis and Aedes serratus showed signifi cant differences (p<0.05) in their habitat use. Haemagogus janthinomys was found more often in the canopy, unlike the other species. During the rainy season, the most abundant species were Sa. albiprivus, Haemagogus leucocelaenus and Haemagogus janthinomys. During the dry season, the potential YF vectors exhibited a very low frequency and abundance, except Aedes scapularis and Aedes serratus. No fl avivirus was detected in the 2,677 examined mosquitoes. Conclusions: We recommend continued and systematic entomological monitoring in areas vulnerable to the transmission of YF in the Federal District of Brazil.

Mosquitoes have been widely studied because of their role in the transmission of tropical diseases, such as malaria, dengue and yellow fever 1 .According to Mathers et al. 2 , infectious diseases transmitted by mosquitoes have a high impact on public health and are responsible for approximately 15% of all disability adjusted life years (DALYs) attributed to infectious and parasitic diseases throughout the world.
Yellow fever (YF) is an acute febrile illness that results from infection by an arbovirus of the genus Flavivirus 3 that remains endemic or enzootic in Africa and South and Central America 1,4,5 .In the urban cycle of YF, humans are the only known vertebrate host of the virus, and it is transmitted by the vector Aedes (Stegomyia) aegypti Linnaeus, 1762 6 .In contrast, sylvatic YF is primarily associated with nonhuman primates (NHP), and its vectors are female mosquitoes of the genus Haemagogus Williston, 1876, Sabethes Robineau-Desvoidy 1827 and Aedes Meigen 1818 in South and Central America 7,8 .
According to Tauil 9 , Brazil is the country with the largest endemic area of sylvatic YF in the world, at approximately 5 million km 2 .Between the years 2000 and 2009, a total of 320 human cases of YF, including 152 deaths, were recorded in 15 states.Between 2007 and 2009, outbreaks of sylvatic YF and epidemics in the NHP population occurred in several Brazilian states, primarily in the extra-Amazonian region 10,11 .
During the last outbreak (2007-2008) in the Federal District (FD), 90 human cases of YF were suspected, 15 of which were confi rmed to be sylvatic YF: two were in 2007 (one was autochthonous) (SINAN-net), and 13 were in 2008 (six were autochthonous) 12 .During the same period, the deaths of several METHODS NHP cases were reported, three of which were attributed to YF 11 .Entomological investigations discovered mosquitoes related to the transmission of this arbovirus, and the YF virus was isolated from Haemagogus (Haemagogus) janthinomys Dyar, 1921 and Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon, 1924 13 .
The Brasilia National Park (BNP) is a tourist destination that receives approximately 260,000 visitors a year.This preservation area contains populations of NHPs 14 and mosquitoes of the genus Sabethes and Haemagogus 13,15 .It is noteworthy that during the outbreak of sylvatic YF that occurred between 2007 and 2008 in the FD, multiple NHPs died in the BNP 13,15 .Therefore, it is important to understand the ecological aspects of the mosquito species in the BNP, with an emphasis on the YF potential vectors.Moreover, the detection of fl aviviruses in mosquitoes captured in this conservation unit is important information for the surveillance of this arbovirus in the FD.
In this context, the objectives of the present study were the following: I) to analyze the richness and abundance of the mosquitoes captured during different seasons in different strata of the gallery forest of the BNP, with an emphasis on potential YF vectors; and II) to determine the percentage of the captured mosquitoes that are infected with a fl avivirus.

Study area
The FD is located in the Central-West region of Brazil between the parallels 15º 30' and 16º 03' south latitude and the meridians 47º 25' and 48º 12' west longitude, within the Cerrado biome.The average annual temperature ranges from 18ºC to 22ºC.The average annual rainfall varies between 1,200mm and 1,700mm 16 , but it is unevenly distributed over the year, with a well-defi ned dry season.The monthly distribution of rainfall accumulated in the FD can be grouped into four quarterly periods.The months between June and August register an average rainfall of zero (the dry period).In April, May and September, there is an average rainfall of 70mm (the intermediate period).Between October and December, an average rainfall of 216mm is observed (the fi rst quarter of rainfall), and between January and March, an average rainfall of 220mm is observed (the second quarter of rainfall) 17 .
The BNP, which is administered by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), is an approximately 42,389-hectare environmental conservation area that contains a great diversity of fl ora and fauna.This park is also a leisure option for residents of the City of Brasilia, and its pools serve as the main attractions.The BNP is situated in the northwestern portion of the FD, approximately 10 km from the city center 14 and just 2km from the Setor Habitacional Noroeste (SHN), whose construction began at the end of 2009 18 .
Two capture areas in the gallery forest near the old pool of the BNP were chosen, with a distance of 200m from one another (Area 1 coordinates: latitude 15° 44'13.5"Sand longitude 47° 55'37.6"W;Area 2 coordinates: latitude 15° 44 '18.6"S and longitude 47° 55'41.6"W).The choice of these areas was based on the presence of suitable trees for the construction of platforms.In each area, a platform was built 6m above the ground.

Mosquito capture
Mosquitoes were captured using entomological netting and manual aspirators 19 .From September 2010 to August 2011, adult mosquitoes were captured each month for fi ve consecutive days from 9a.m. to 3p.m. with a one hour interval between noon and 1p.m., for a total capture effort of 60 days.Entomological capture is a routine activity of the Diretoria de Vigilância Ambiental em Saúde do Distrito Federal (DIVAL) and was performed by health agents who were properly immunized and trained for this purpose.Using proper safety procedures and personal protective equipment, two individuals worked simultaneously in each sampling area; one on the ground and another on the platform 6m above the ground.

Mosquito identifi cation
At the end of the daily activities, the captured specimens were taken to the DIVAL's entomology laboratory and anesthetized by cooling to -6°C.The specimens were transferred to labeled tubes and kept in liquid nitrogen.
The captured mosquitoes were identifi ed with the aid of the dichotomous keys available from Consoli and Lourençode-Oliveira 20 and Forattini 8 .The identifi cation occurred on a cold table at -26°C with the aid of a stereoscopic microscope (Olympus Optical CO., Tokyo, Japan) .After identifi cation, the mosquitoes were divided into 302 batches according to species and capture area and stored in liquid nitrogen.Subsequently, the mosquitoes were sent to the Laboratório Central de Saúde Pública (LACEN) of the FD for fl avivirus isolation.

Virus isolation
The technique used to isolate the fl aviviruses from the mosquitoes in LACEN/DF is based on the sensitivity of Aedes albopictus Skuse, 1864 cells (clone C6/36) to several flaviviruses 21 .To cultivate these cells, cell culture bottles containing Leibovitz L-15 ® medium were used.
Each batch contained between 1 and 20 specimens of a single mosquito species.Each batch was homogenized in Eppendorf tubes with an antibiotic solution containing penicillin, streptomycin and amphotericin B and then individually inoculated into culture tubes containing C6/36 cells, which were maintained in an incubator at 24°C to 25°C for approximately eight days.Once grown, the inoculated cells were observed daily using an inverted optical microscope to visualize any possible cytopathic effects.Confi rmation of viral infection was obtained by indirect immunofl uorescence with polyclonal fl avivirus antibodies 22 .

Data analysis
To verify sampling suffi ciency, the species accumulation curve was analysed 23,24 .The species constancy during the sampling was assessed by the formula C%=(p/N).100,where p=the number of sampling occasions in which a species was
To determine the categories of dominance, the classifi cation established by Friebe 26 was determined using the formula D%=(i/t).100,where i=the total number of individuals in the species and t=the total number of individuals captured.Thus, the established categories were eudominant (D>10%), dominant (D>5-10%), subdominant (D 2-5%), eventual (D 1-2%) and rare (D<1%).
To measure the mosquito diversity patterns, the indices of Shannon-Wiener diversity, Shannon-Wiener evenness and Berger Parker dominance were used.Data analysis was performed using the program Diversidade de Espécies (DivEs), version 2.0 27 .
The variation in the number of mosquitoes caught between rainfall periods (dry, intermediate, fi rst rainfall quarter and second rainfall quarter) was analyzed using the Kruskal-Wallis test.In this analysis, we considered the daily abundance of seven species captured in the study to be the dependent variable.The daily occurrence of these species on the ground and in the canopy over the 60-day period was compared using chi-squared tests.The tests were performed in the Statistica® program, with signifi cance set at 5%.
According to the species accumulation curve (Figure 1), the overall sampling effort (60 days) was satisfactory to represent the species richness, indicating a trend towards stabilization of the number of accumulated species from the 54 th 2).
The seven most abundant species were also classified as constant.Five species were classifi ed as accessories, and 17 others were classifi ed as accidental.It was also observed that, in terms of dominance, only three species were eudominant (Sa.albiprivus, Li. durhamii and Hg.leucocelaenus).Another three were dominant (Hg.janthinomys, Ae. scapularis and Ps.ferox), four were subdominant (Ae.serratus, Cq. arribalzagae, Anopheles kompi and Sa.belisarioi), two were classifi ed as eventual and all of the others were considered rare (Table 1).
With respect to the monthly distribution, the mosquito abundance was higher during the fi rst and second quarter of rainfall.The largest number of mosquitoes at both the canopy and ground levels was captured during the fi rst quarter of rainfall (Table 2).
The highest diversity (H'=0.961),evenness (J=0.671) and richness (N=27) of mosquito species were detected at ground level.The Berger-Parker dominance index was similar for the ground (D bp =0.034) and the canopy (D bp =0.035).For the different rainfall classes, we observed a greater richness during the second quarter of rainfall (N=21).The greatest evenness was observed during the intermediate period (J=0.820), and the highest level of dominance was observed during the dry season (D bp =0.147).
None of the 302 batches of mosquitoes (N=2,677) sent to the Virology Laboratory-LACEN/DF for arbovirus isolation tested positive for fl aviviruses.

DISCUSSION
The present study shows that the abundance and species richness of mosquitoes in the gallery forest of the BNP are higher on the ground and during the rainy season.Even species with recognized acrodendrophilic habits, such as Hg.janthinomys, were captured on the ground; these results are similar to those of Alencar et al. 28 and Ramirez et al. 29 .These results indicate that precipitation is a crucial factor explaining the richness and abundance of Culicidae fauna in the gallery forests of the Cerrado, as has already been observed in other Brazilian biomes 8,30,31 .
Among the species identifi ed in the BNP, 11 have been previously found to be naturally infected with the YF virus: Ae. aegypti, Ae. scapularis, Ae. serratus, Ps. albipes, Ps. ferox, Hg. leucocelaenus, Hg. janthinomys, Hg. tropicalis, Sa. chloropterus, Sa. soperi and Sa.albiprivus 13,20,[32][33][34][35][36] .Moreover, the natural infection of Hg. janthinomys and Hg.leucocelaenus by the YF virus has been previously reported in the administrative region of São Sebastião, FD 13 .However, none of the 302 batches of 2,677 mosquitoes examined were infected with fl aviviruses.This may be because of a low rate of infection among primates in the BNP, as this work was conducted during the interepidemic period.Furthermore, the infection of mosquitoes by the YF virus tends to be low, even in areas of YF outbreaks.The results of Cardoso et al. 34 reinforce this hypothesis, showing that Hg. leucocelaenus and Ae.serratus in YF outbreak areas have minimum infection rates of 3.70 and 1.88, respectively.
Three specimens of Ae. aegypti were detected in the gallery forest of the BNP.The occurrence of this species in urban environments has been widely reported 37 ; however, the occurrence of Ae. aegypti in sylvatic environments is rare.Barbosa et al. 38 and Soares et al. 39 reported the presence of this species in a rural area in the states of Amazonas and Rio de Janeiro.Tauil 6 highlights the diffi culty of identifying whether some cases of YF are transmitted by sylvatic or urban vectors.Considering that the sampling areas were located near recreational areas in the BNP, it is possible that the specimens of Ae. aegypti captured in the gallery forest were from artifi cial breeding sites.Future monitoring studies of mosquitoes in gallery forests may better clarify the occurrence of Ae. aegypti in sylvatic environments.
Environments that are still considered sylvatic are strongly influenced by the rapid urbanization process, in which population and housing growth generate anthropogenic changes that contribute to the establishment of mosquito species that are better adapted to the human environment 40 .The FD has undergone considerable environmental changes caused by the expansion of its urban area, which can change the behavior of the mosquito species present in the BNP.The occurrence of species that are adapted to urban anthropogenic environments, such as Ae.aegypti and Culex (Culex) quinquefasciatus Say, 1823, could be attributed to ecological changes that occurred with the construction of the Setor Habitacional Noroeste.
Aedes albopictus was also found during the survey.The ability of this species to transmit arboviruses has already been demonstrated [41][42][43] .It is noteworthy that Ae. albopictus has been documented as naturally infected with the virus that causes eastern equine encephalitis 44,45 and with dengue virus during an outbreak that occurred in Mexico 46 .Some authors admit the possibility that Ae. albopictus may become a vector that  connects sylvatic and urban cycles of YF in Brazil 20,39,47 .Gomes et al. 41 and Albuquerque et al. 48point to the need to monitor this species in Brazil.
There are few studies related to the vertical distribution of mosquitoes in the Central-West Region of Brazil 49 .In the present study, we detected a higher occurrence of Hg. janthinomys in the canopy, reinforcing the known acrodendrophilic behavior of this species 29,50 .Specimens of Hg. leucocelaenus were very common on the ground.Forattini et al. 51 in São Paulo and Pinto et al. 50n Pará also reported the predominance of Hg. leucocelaenus on the ground.However, Guimarães et al. 52 showed that 74% of Hg. leucocelaenus specimens were captured in the canopy at the National Park of the Serra dos Orgãos, Rio de Janeiro.Ae. scapularis and Ae.serratus showed signifi cant differences in habitat use and were more abundant on the ground.The same preference for these species to occur at ground level has also been observed by Fe et al. 53 , Forattini et al. 51 , Guimaraes et al. 52 and Julião et al. 30 .Moreover, the highest occurrence of Ps. ferox at ground level was also observed by Forattini et al. 51 .
There is evidence that vertical distribution differences between and within species depends upon the type of vegetation cover.Forattini et al. 51 suggested that a vertical distribution difference is most evident in tropical rain forests, where the trees are tall and dense.In less dense forests and in places with long dry seasons, these differences tend to decrease, as observed in the present study.
Other important factors infl uencing the vertical distribution of mosquitoes are vertical mobility and daily activity.According to Service 54 , mosquitoes can be attracted to a feeding source at a distance of seven to 30 meters.Forattini et al. 51 and Vasconcelos et al. 55 showed that even species that were predominantly active in the higher levels of the canopy, such as Hg.janthinomys, often move to ground level when they detect the presence of a feeding source.Moreover, Guimarães et al. 52 showed differences in the vertical distribution of An. cruzii captured at different periods of the day, with the highest number of specimens found in the canopy at night.Therefore, the vertical movement of mosquitoes can weaken the evidence of vertical stratifi cation of species, and it obviously infl uences the dynamics of pathogen transmission 30 .
The effects of rainfall on the total abundance of mosquitoes captured were evident.There was a strong infl uence of rainfall on the annual cycle of the species of Sabethes and Haemagogus, which have eggs that are very resistant to desiccation and sometimes require repeated contact with water to hatch 20 .Similar situations related to these genera have been described in Mato Grosso 31 and Tocantins 56 .Studies in southern Brazil 10 and in Trinidad 57 also indicated a higher occurrence of Hg. leucocelaenus during the rainy months.The highest occurrence of A. scapularis, A. serratus and Ps.ferox in the rainy season is also in agreement with the literature 20 .
The present study increases the information available regarding the ecology of important YF virus vectors in Central Brazil.Although seroepidemiological surveys indicate that YF control efforts have achieved good results 58 , entomological monitoring is recommended in the BNP and in other areas that are susceptible to the transmission of the YF virus.We emphasize that the BNP is located 10km from the center of Brasília and 2km from the Setor Habitacional Noroeste, where many apartments are being constructed.Furthermore, are houses located at the edge of a roadway that is very close to the park.Because the fl ying range of YF vectors can reach 500m 59 , there is the possibility of sylvatic YF transmission to people who reside close to the BNP and to park visitors.This transmission would be more likely during periods of NHP epidemics, when the natural infection rate of mosquitoes tends to be higher 34 .Although no viral isolates were obtained from the specimens captured in this study, studies related to the ongoing natural infection of mosquitoes is of paramount importance as a means of predicting events related to YF and other arboviruses.Such information will contribute to a better understanding of the true role of the vector species that occur within Central Brazil.It is also recommended that the relevant agencies give special attention to the deaths of NHPs and to the vaccination programs against YF for visitors to the BNP.

FIGURE 1 -FIGURE 2 -
FIGURE 1 -Species richness curve of mosquitoes captured between September 2010 and August 2011 at Brasilia National Park, Federal District, Brazil.
Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Individuals

TABLE 2 -
Rainfall classes and number of mosquitoes captured at both the canopy and ground levels in the gallery forest of Brasilia National Park, Federal District, Brazil, from September 2010 to August 2011.Ps. ferox (Kruskal-Wallis H 3.60 =18.6, p < 0.01) and Ae.serratus (Kruskal-Wallis H 3.60 =17.5, p<0.01).species were more abundant during the rainy quarters.During the dry season, the potential YF vectors exhibited very low frequency and abundance, with the exception of Ae. scapularis and Ae.serratus.
Number of mosquitoes of species that are potential vectors of yellow fever in Brasilia National Park, Federal District, Brazil, on the ground and in the canopy between September 2010 and August 2011.Only the most abundant species (>100 individuals captured) are listed.Hg.: Haemagogus; Ae.: Aedes ; Sa.: Sabethes; Ps.: Psorophora.