Diversity of biting midges Culicoides (Diptera: Ceratopogonidae), potential vectors of disease, in different environments in an Amazonian rural settlement, Brazil

Abstract INTRODUCTION: The Culicoides transmit a variety of pathogens. Our aim was to survey the Culicoides species occurring in an Amazonian rural settlement, comparing abundance, richness, and diversity in different environments. METHODS: Culicoides were captured using CDC light traps. The Shannon-Wiener (H’) and Rényi indices were used to compare species diversity and evenness between environments, the equitability (J’) index was used to calculate the uniformity of distribution among species, and similarity was estimated using the Jaccard similarity index. A permutational multivariate analysis of variance was applied to assess the influence of environment on species composition. A non-metric dimensional scale was used to represent the diversity profiles of each environment in a multidimensional space. RESULTS: 6.078 Culicoides were captured, representing 84 species (45 valid species/39 morphotypes). H’ values showed the following gradient: forest > capoeira > peridomicile > forest edge. The equitability J’ was greater in capoeira and forests compared to peridomiciles and the forest edge. The population compositions of each environment differed statistically, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness. Species of medical and veterinary importance were found primarily in peridomiciles: C. paraensis, vector of Oropouche virus; C. insignis and C. pusillus, vectors of Bluetongue virus; C. filariferus, C. flavivenula, C. foxi, and C. ignacioi, found carrying Leishmania DNA. CONCLUSIONS: This study indicates that diversity was higher in natural environments than in anthropized environments, while abundance and richness were highest in the most anthropized environment. These findings suggest that strictly wild Culicoides can adapt to anthropized environments.


INTRODUCTION
Tropical forest ecosystems host two thirds of the Earth's terrestrial biodiversity and provide significant benefits to the biosphere and the global economy 1 . According to Steege et al. (2015) 2 , approximately 40% of the original Amazon forest will be lost by 2050 if historical rates of deforestation continue. Anthropization is the primary cause of environmental change and the degradation of tropical ecosystems.
Environmental changes caused by anthropogenic interference are associated with increased health risks. Vectors and pathogens previously found only in forests have been observed in human settlements. According to Gottdenker et al. (2014) 3 , disease transmission is affected land-use changes that include: deforestation, forest and habitat fragmentation, agricultural development, irrigation, urbanization, and suburbanization. In the Amazon, these changes may be responsible for the increased incidence of Hematophagous biting midge populations are affected by anthropogenic interference. In the Brazilian Amazon, Castellón (1990) 4 observed a greater abundance of biting midges in capoeira and clearings than in primary forest. In urban and rural areas in Maranhão State, Silva and Carvalho (2013) 5 found both a greater richness and abundance of biting midges in peridomiciles. According to Cazorla and Campos (2018) 6 , anthropization impacts ceratopogonid communities by decreasing biodiversity and favoring species that best adapt to altered environments.
This study was conducted in the settlement of Rio Pardo, Presidente Figueiredo Municipality, Amazonas State, Brazil. Until recently, human activity in Rio Pardo was limited to subsistence farming, raising livestock, hunting, fishing, and gathering wood for local use, but newly expanded branches of forestry and fish farming have had a significant impact on the natural environment. Disease transmission dynamics in the region may have been altered by the degradation of natural habitats causing increased human exposure to forests, and to disease vectors and their hosts. The increased supply of blood meal sources around peridomiciles and a lack of basic sanitation offer favorable conditions for insect vectors to flourish.
The genus Culicoides is comprised of 1.368 species worldwide; 299 of these species occur in the Neotropics and 122 species occur in the Brazilian Amazon Basin 7-8 . Certain Culicoides species transmit viruses such as African horse sickness virus (AHSV), Bluetongue virus (BTV) which infects domestic and wild ruminants, and Oropouche virus (OROV) which infect humans 9 . Oropouche virus is one of the most common arboviruses in Brazil; it has affected an estimated 500,000 people since it was first isolated in 1955 10 . Biting midges and simulids have been implicated in the transmission of some species of filariae to humans, including: Mansonella ozzardi, M. perstans and M. streptocerca 11 . Culicoides may also be involved in the transmission of Leishmania 12 .
Identifying possible disease vectors is therefore of significant epidemiological importance. The aim of this study was to survey the Culicoides species that occur in a typical Amazonian rural settlement, and to compare the abundance, richness, and diversity of Culicoides populations present in different environments.

METHODS
The rural settlement of Rio Pardo (1°49'02.3''S 60°19'03.5''W), Presidente Figueiredo Municipality, Amazonas State, Brazil (Figure 1), was founded in 1996 and has approximately 700 inhabitants (ILMD/FIOCRUZ). In 2002, approximately 95% of its total area, about 28,000 hectares, was composed of preserved Culicoides capture was performed in June, July, and August 2010 on four consecutive nights per month. Four different environments within the household buffer zone were sampled: peridomicile, areas with enclosures for livestock; capoeira, successional areas that have regenerated naturally from both functioning and abandoned agroecosystems; forest edge, areas of transition between capoeira and forest; and forest, comprised primarily of ombrophilous forest or "terra firme" (upland Amazonian forest that never floods) and river margins. Twenty-four household buffers were sampled. Four Centers for Disease Control (CDC) light traps per household were used to capture Culicoides, totaling 96 traps. These were installed 1.5 m above the ground and remained in the field from 6:00 p.m. to 6:00 a.m.
Biting midges were transported in 70% ethanol and slidemounted in phenol-balsam, as described by Wirth and Marston (1968) 14 . Species identification was performed following: Wirth and Blanton (1959) 15  Many specimens had morphological variations in common. Unidentified specimens were therefore grouped into morphotypes and treated as valid species in the diversity and richness analyses. The Shannon-Wiener (H') and Rényi indexes were used to compare species diversity and evenness between the four environments. The Pielou equitability index (J') was used to calculate the uniformity of distribution of individuals among species, and similarity was calculated using the Jaccard similarity index (Cj), which qualitatively compares species similarity along an environmental gradient 19 . A permutational multivariate analysis of variance (PERMANOVA) was applied to assess the influence of each environment on species composition. A non-metric dimensional scale (NMDS) was used to represent the diversity profiles of each environment in a multidimensional space. All analyses were carried using the statistical package R version 3.4.2 20 . The level of significance considered for all tests was 95%.

RESULTS
A total of 6.078 Culicoides (96.38% females and 3.62% males) were collected, representing 84 species, comprised of 45 valid species and 39 morphotypes.
Richness between environments varied from 22 to 58 species/morphotypes. Abundance varied significantly between environments, with values ranging from 85 to 3.725 individuals ( Table 1). Species diversity was highest in forests (H'= 2.55) and lowest at the forest edge (H'=1.71). H' values showed the following gradient: forest > capoeira > peridomicile > forest edge. The equitability (J') index was greater in capoeira and forest than in peridomicile and the forest edge ( Table 1), which indicates that Culicoides individuals are more equitably distributed among different species in capoeira and forest environments.
Similarity was greater between the forest and capoeira than between capoeira and peridomiciles. The population compositions of each environment differed statistically (PERMANOVA S.S=1.61, PSEUDO-F=1.96, p=0.04) (Figure 2 and Figure 3). Richness differed between environments, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness (Table 1 and Figure 4).  Species of medical and veterinary importance were found primarily in peridomiciles, these include: Culicoides paraensis, which is a vector of OROV in humans; C. insignis and C. pusillus, which are vectors of BTV in domestic and wild ruminants; C. filariferus and C. flavivenula, which have been found carrying Leishmania amazonensis DNA; and C. foxi and C. ignacioi which have been found carrying Le. braziliensis DNA 9, 26 . All of these species were found in this study, but some in low abundance. This is probably due to the collection method. In entomological inventories, it is common for some species to appear with low frequency because bait type, capture effort, collection environment, and the presence of animals favors the capture of other species 27 . This low frequency may also be related to the fact that some species are diurnal while others are nocturnal [28][29] .
Abundance was greatest in peridomiciles, followed by the forest edge, forest, and capoeira. Culicoides fusipalpis has been observed in high abundance in anthropic environments where it feeds on a variety of blood meal sources, including humans, other mammals and birds 34 . Species richness was greatest in peridomiciles, followed by forest, forest edge, and capoeira. The concentration of a variety of blood meal sources and the presence of suitable breeding sites may be attracting Culicoides to peridomiciles. Santiago-Alarcon et al. (2013) 33 surveyed Culicoides feeding behavior in households near an urban forest in Germany where the dominant Culicoides species were known to be ornithophilous and found that blood ingested by these midges contained the mitochondrial DNA of mammals such as cows and humans. Generalist species are able to tolerate a broad set of environmental conditions and make use of a wide range of resources, which allows them to become both widespread and locally abundant (Brown, 1984) 35 .
In Rio Pardo, species diversity was highest in forests, while richness and abundance was highest in peridomiciles. In a study conducted in Rondônia, Carvalho et al. (2016) 36 found that species diversity was higher in forests than in pasture. In a study conducted in rural and urban areas in Maranhão, Silva and Carvalho (2013) 5 found that species diversity was highest in the Cerrado (savanna) and in gallery forests, while richness and abundance were highest in peridomiciles.
Equitability was highest in capoeira environments, where the number of individuals per species ranged from 1 to 27, and equitability was lowest in the forest edge environments, where the number of individuals per species ranged from 1 to 745 ( Table 1). The high equitability observed in capoeira environments is the result of low abundance and a relatively homogeneous distribution of species. The low equitability observed in forest edge environments is likely due to an uneven distribution of species abundance caused by the predominance of C. dasyophrus, which comprised 47.76% of all individuals collected at the forest edge. These findings demonstrate that the local diversity index may decrease in environments where one species is highly dominant.
Species similarity between environments was low (> 50%), which is likely due to the different degrees of anthropic interference present in each environment. Capoeira-forest edge and forest-forest edge exhibited the highest similarity. These findings suggest that strictly wild Culicoides fauna tends to adapt to anthropized environments.
We observed that richness differed between environments, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness. This is probably due to the three-month capture effort occurring during the dry season, a period that the abundance of general hematophagous dipteria are low 13 , which may have interfered with the results, indicating the need for more captures and in different seasons.
The data obtained in this study indicate that diversity was higher in natural environments (forest) than in anthropized environments (capoeira), while abundance and richness were both highest in the most anthropized environment (peridomiciles). It is likely that the concentration of a variety of food sources and the presence of suitable breeding grounds in peridomiciles favors the establishment of certain species.
In settled areas, the presence of domestic and wild animals provides vectors with a rich variety of food sources and this fosters their adaptation to new environments. This behavior may alter pathogen transmission dynamics and increase the risk of disease transmission by Culicoides.

AUTHORS' CONTRIBUTION
EF designed the study, slide-mounted and identified specimens, and wrote the manuscript; JAF slide-mounted specimens and wrote the manuscript; JWPS and LSC performed the statistical analysis and wrote the manuscript; SLBL, CMRV and FACP: conceived and designed the study, and wrote the manuscript; all authors read and approved the final version of the manuscript.