The influence of environmental management and animal shelters in vector control of Culicoides (Diptera, Ceratopogonidae) in northeastern Brazil

Vol. 42, no. 1 Journal of Vector Ecology 113 The influence of environmental management and animal shelters in vector control of Culicoides (Diptera, Ceratopogonidae) in northeastern Brazil Maria da C.A. Bandeira 1*, Gustavo A. Brito 1, Adriane da Penha 2, Ciro L.C. Santos 3, and José M.M. Rebêlo 2,4 1 Programa de Pós Graduação em Biodiversidade e Conservação, Universidade Federal do Maranhão, Avenida dos Portugueses 1966, Campus do Bacanga, 65080-805, São Luís, Maranhão, Brasil, mariza_bandeira@hotmail.com 2 Laboratório de Entomologia e Vetores, Universidade Federal do Maranhão, São Luís, Maranhão, Brasil 3 Programa de Pós-Graduação em Ecologia e Conservação da Biodiversidade, Universidade Federal do Mato Grosso, Cuiabá, Mato Grosso, Brasil 4 Departamento de Biologia, Universidade Federal do Maranhão, São Luís, Maranhão, Brasil Received 18 October 2016; Accepted 4 January 2017 ABSTRACT: We investigated whether biting midges in peridomestic environments are affected by environmental management practices and the presence of domestic animals. We used CDC light traps to collect midges in 112 residences across 24 locations along tourism routes of Maranhão, Brazil. The collection areas were characterized as follows: i) peridomestic area with domestic animals and without management (dirty); ii) peridomestic with domestic animals and management (clean); iii) peridomestic without animals and with management (clean); iv) peridomestic without animals and without management (dirty). The first two treatments had higher biting midge species richness and abundance, respectively. Generalized linear models indicated a positive correlation between the presence of domestic animals and midge abundance, with an approximate four-fold increase in Culicoides (Diptera: Ceratopogonidae) abundance in peridomestic areas with animals. The same model showed that domestic animals have no influence on richness. Environmental management does not appear to influence species richness or abundance of biting midges. Journal of Vector Ecology 42 (1): 113-119. 2017. Keyword Index: Biting midges, vector control, biological vector, entomological surveillance. INTRODUCTION The Ceratopogonidae family is a group of nematoceran flies that is highly diversified both ecologically and with respect to species richness (Courtney and Merritt 2008). There are currently about 6,180 species distributed among 111 genera (Borkent 2015a). The Ceratopogoninae subfamily is epidemiologically important because it contains the genus Culicoides Latreille 1809, in which several species are known or suspected vectors of parasites that cause diseases in vertebrates. Culicoides, also known as midges, mosquito pólvora, or mosquito-do-mangue, are very small insects with wing patterns consisting of gray and white spots as the most striking taxonomic characteristics (Felippe-Bauer 2003). This genus is one of the largest groups in the family, with approximately 1,400 known species worldwide (Borkent 2015b). They occur on all continents and are distributed from the coasts to the peaks of great mountain ranges, and are only absent in the polar regions of Antarctica and New Zealand (Trindade and Gorayeb 2010). The distribution of Culicoides species is associated with local environmental conditions such as temperature, humidity, wind speed, and precipitation, as well as the physical and chemical properties of the soil in which larval development occurs (Braverman 1994, Mellor et al. 2000, Borkent and Spinelli 2007). These species have medical and veterinary importance because females take blood meals from vertebrate hosts, inflicting a painful bite wound that can cause discomfort, insomnia, or irritability, especially when insect abundance is high. Bites may also trigger allergic reactions from injected salivary proteins and peptides and have the capability to transmit various types of pathogens, such as nematodes, protozoa, or viruses to the vertebrate host. Female Culicoides generally need blood for egg developent, but some species are autogenous, meaning that they can produce the initial batch of eggs without a blood meal (Borkent and Spinelli 2007). Culicoides are generalist feeders that pursue blood meals from synanthropic domestic animals and humans (Costa et al. 2013, Gusmão et al. 2014) and can become nuisance pests near beaches, forests, mountains, and mangroves. These insects are vectors of filariae of the genus Mansonella in the Americas and Africa. In Brazil, Culicoides paraensis transmits the Oropouche virus that probably infected more than 500,000 people in the Amazon from 1961 to 1996 (Pinheiro et al. 1962), and C. insignis transmits bluetongue virus as the main vector of this disease in South America. More than 100 Culicoides species are known in Brazil (Wirth and Blanton 1973, Wirth et al. 1988, Felippe-Bauer and Oliveira 2001, Aparício et al. 2011). Roughly 40 species occur in Maranhão state (Silva and Rebêlo 1999, Barros et al. 2007, Silva and Carvalho 2013, Costa et al. 2013), primarily found in peridomestic areas of the countryside. Culicoides spp. occurrence in rural peridomestic areas has been attributed to alterations of natural habitats, and favorable environmental conditions in the yard areas of residences (Costa et al. 2013, Gusmão et al. 2014). The inhabitants of rural towns surrounding the Lençóis Maranhenses National Park (LMNP), a major tourist hub of northeastern Brazil, have reported concerns about the presence of biting midges. Costa et al. (2013) found a variety of Culicoides species in several towns surrounding the LMNP. The park

114 Journal of Vector Ecology June 2017 routes where most tourist activities take place contain swamps, mangroves, rivers, and small streams. Farmers, fishermen, and tourists have reported attacks by clouds of midges at these sites. These findings highlight the relevance of this genus, not only in medical and veterinary context, but also as pests with potential to interrupt agricultural activities, raising of livestock, and tourism, which strongly benefit the economy of this region. The advent of tourism may have contributed to the reduction of native vegetation and the introduction of midges to rural town areas (Costa et al. 2013). In the last two decades, the rural town areas surrounding the LMNP have expanded, motivated by speculation due to tourism-related projects (Assunção Junior et al. 2009). Further, environmental conditions in residential peridomestic areas, such as animal shelters without proper sanitation, vegetable gardens fertilized with animal manure, and runoff of household water, favor the formation of breeding sites through increases in soil moisture and organic matter richness (Pereira Filho et al. 2015). Indeed, some Culicoides species are known to breed in standing water on saturated soil and wet domestic animal feces (Braverman 1994, Borkent and Spinelli 2007). Culicoides control has been a great challenge and several management methods have been employed. Dichlorodiphenyltrichloroethane (DDT), commonly used for malaria vector control, is also used to control sand fly and biting midge populations. However, this and other organochlorine and organophosphorus compounds are not authorized for this purpose due to high toxicity (Carpenter et al. 2008). Recently, other substances with low toxicity (and thus less harmful to the environment) have been used, including pyrethroids, which are derived from plant products. However, field studies by Satta et al. (2004) showed that cypermethrin, esbiothrin, piperonyl butoxide, and synergetic pyrethroid application did not reduce Culicoides abundance. Due to restrictions on the use of insecticides, environmental management practices have been adopted in areas with known insect vectors. The management process involves cleaning, removal of soil foliage, pruning trees to increase light penetration, removal of garbage, and reduction of organic matter input near the towns. This method serves to reduce or eliminate potential breeding areas, reducing insect harassment of residents (Oliveira et al. 2010). Environmental management is an inexpensive and effective process that can and should be conducted by the local inhabitants of focal areas (Wermelinger and Ferreira 2013). This vector control strategy is encouraged by the Ministry of Health along with epidemiological surveillance and entomological (vector) and chemical control. However, all control measures must be implemented cautiously and require integration of efforts by various government sectors (e.g., Secretaries of the Environment, Agriculture, and Education and Health), universities, and members of the affected population. The abundant presence of several midge species in peridomestic rural areas may be partially due to the attraction of these species to domestic animals such as birds, pigs, horses, cattle, and goats (Costa et al. 2013, Gusmão et al. 2014), animal breeding by locals, or environmental conditions favorable for insect development (Costa et al. 2013). This raises the hypothesis that the presence of domestic animals and animal shelters, and lack of environmental management (cleaning) may contribute to greater Culicoides richness and abundance. We employed a factorial experimental design to collect midges in peridomestic environments with or without domestic animals, and with or without environmental management. This is the first study in Brazil using this approach for refining target areas for control of Culicoides. MATERIALS AND METHODS Study area The study was conducted in the municipalities of Barreirinhas (2 45 S, 42 5 W) and Santo Amaro (2 30 S, 43 15 W), located on the eastern coast of Maranhão, Brazil. Barreirinhas has an area of 3,111 km 2, a population of 56,000 and is located 266 km from the state capital of São Luis. Santo Amaro has an area of 1,601 km 2, 14,000 inhabitants, and is located 243 km from São Luis (IBGE 2011). These municipalities together include much of the LMNP area (Figure 1). The climate in this region is sub-humid megathermal with annual rainfall ranging between 1,800-2,000 mm (DNPM 1973). There is a marked rainy season from January to June and a dry season from July to December. The dominant vegetation is restinga (IBGE 1984), a Brazilian biome that occurs in the study area as a mosaic of herbaceous, shrub, and tree formations; restinga has great ecological diversity and is characterized by unique soil conditions and maritime influence (Thomazi et al. 2013). The towns located further south within the study area exist in an ecotonal area with cerrado and seasonal forest vegetation. Field collections Residences were selected for sampling in different towns according to peridomestic characteristics. All residences were geo-referenced, and geographical coordinates were recorded using a GPS device. All towns were located along tourist routes and had histories of Culicoides infestation (Costa et al. 2013). Insects were collected in April, May (rainy season), and August (dry season) of 2015, with the intention of registering the biting midge in the two seasons of the year. One collection was carried out at each residence in an area surrounding the home. Residence peridomestic areas were classified into groups as follows: 56 contained domestic animals (chickens, pigs, or horses) and shelters (coops, sties, and stables); of these, 28 sites were maintained prior to the collection (cleaned, garbage and ground foliage removed from peridomestic areas) and the other 28 were maintained prior to collection (dirty). Another 56 residences were chosen without domestic animals or their shelters; of these, 28 were maintained prior to collection and another 28 were not. The 112 houses were randomly distributed in the respective localities, so that the group classification was for the purpose of analysis only. We thus employed a factorial design with two-level treatment groups as follows: i) with management, with animals (M+A+), ii) without management, with animals (M-A+), iii) with management, without animals (M+A-); and iv) without management, without animals (M-A-). CDC light traps were installed in the 112 peridomestic areas at 1.5 m height either in animal shelters, or if no shelters were present, in tree branches or other supportive structures in the vicinity of dwellings. Traps ran uninterrupted from 18:00 to 06:00, for a total capture effort of 112 traps x 12 h = 1,344 h. Captured insects were killed in ethyl acetate and placed in labeled

Vol. 42, no. 1 Journal of Vector Ecology 115 Figure 1. Map of Maranhão State (Brazil) showing study areas in the Barreirinhas and Santo Amaro municipalities near the Lençóis Maranhenses National Park. polyethylene containers for later identification at the Laboratório de Entomologia e Vetores (LEV) at the Universidade Federal do Maranhão. Laboratory procedures Culicoides were separated from other insects using a dissecting microscope and identified using dichotomous keys by Wirth and Blanton (1973), Wirth et al. (1988) and Spinelli et al. (2005). The incomplete specimens without taxonomic identification conditions were grouped as Culicoides sp. Specimens were later fixed in 70% ethanol and deposited in the entomological collection of the Department of Biology, Federal University of Maranhão. Statistical analysis Culicoides species rank abundances were determined using the Kato index (Kato et al. 1952), in which dominant species are those with a lower confidence limit that is greater than the upper confidence limit for absent species. We used a factorial analysis of variance (ANOVA) to analyze differences in abundance and species richness by treatment using a significance level of p = 0.05. Species richness at each site was estimated by calculating the sum of the number of species obtained. We used the Shannon-Wiener index to evaluate diversity (Pileou 1975) with a Jackknife estimator (Zahl 1977). Pielou s index was used to evaluate evenness. Generalized linear models (GLM) with negative binomial distributions were used to analyze the association between abundance and richness of Culicoides as the predictor variables (presence of animals and the presence of environmental management). These GLMs were also used to evaluate the relationship of the presence and abundance of peridomestic animals (poultry, cattle, horses, dogs, and pigs) with the abundance and richness of Culicoides. The best models taking into account the 0.05 significance level were selected. All statistical analyses were performed with R software 3.2 (R Development Core Team 2016) using the vegan package (Oksanen et al. 2016) and MASS (Venables and Ripley 2002). RESULTS Species composition, richness, and relative abundance We captured 1,439 specimens in total, including 15 Culicoides species. The most abundant species were C. insignis Lutz 1913 (44.1%), C. leopoldoi Ortiz 1951(38,1%), C. limai Barreto 1944 (3.3%), C. ignacioi Forattini 1957 (3,0%), C. foxi Ortiz 1950 (2.8%), C. ruizi Forattini 1954 (2.6%), C. paucienfuscatus Barbosa 1947 (1.6%), Culicoides sp. (1.5%), and C. boliviensis Spinelli and Wirth 1984 (1.4%), which together accounted for 98.0% of the total sample. C. flavivenula Costa Lima 1937, C. guyanensis Floch & Abonnenc 1942, C. filariferus Hoffman 1939, C. travassosi Forattini 1957, C. furens Poey 1853, and C. debilipalpis Lutz 1913 contributed less than 1% each, together accounting for about

116 Journal of Vector Ecology June 2017 2% of the total sample (Table 1). According to the Kato index (1952), the dominant species were the first nine when ranked by abundance (Table 1) and C. boliviensis had the lowest dominance rank (contributing 1.9%; Figure 2). Species richness and abundance The M-A+ treatment had the greatest abundance with 784 individuals (54.5% of the total collection) and the highest richness (13 species). The M+A- treatment yielded 509 individuals (35.3%) from 12 species. The M-A- treatment yielded 78 individuals (5.4%) from seven species, and the M+A- treatment had the lowest abundance with 68 individuals (4.7%) from ten species. C. insignis and C. leopoldoi were the most abundant species at all sites. The treatment with the greatest species diversity was M+A- (H = 1.78), which was significantly higher than other treatments (M-A+: H = 1.20; M-A-: H = 1.40; M+A+: H = 1.45). M+A- also had the highest evenness value (E = 0.77), followed by M-A- (E = 0.72). Other treatments had relatively low evenness values (M+A+: E = 0.58; M-A+: E = 0.46), indicating unequal representation of species. In the GLM analysis, the abundance of Culicoides was associated with the presence of peridomicile animals (p <0.001) of about four-fold higher. However, despite the presence of animals increasing about two-fold the number of species, this association was not significant (p = 0.065). Peridomicile management was not associated with abundance (p = 0.364) and richness (p = 0.065) of midges. Furthermore, it was demonstrated through the GLM that the number of peridomestic birds was associated with midges (p <0.001) and may be up to three-fold greater in peridomestic areas with an average of 11 chickens. The species richness was not associated with the type of animal that was found in homes. DISCUSSION We demonstrated a link between the presence of domestic animals and Culicoides populations in peridomestic environments and showed that animal presence in peridomestic areas influenced midge abundance and richness more than environmental management practices. This is supported by Costa et al. (2013), as female midges were found in the same area with blood from domestic animals, including birds, dogs, pigs, and horses. In addition, in the present study we found the ornithophilic C. paucienfuscatus only in residences with birds, corroborating the observations of Castellón (1990) and Gusmão et al. (2014). Table 1. Culicoides abundance by treatment in Barreirinhas, Maranhão, Brazil in April, May, and August of 2015. Species Environments With animals (A+) Without management (M-) Without animals (A-) Total With animals (A+) With management (M+) Without Animals (A-) Total no. % C. insignis 396 25 421 192 21 213 634 44.1 C. leopoldoi 296 34 330 209 9 218 548 38.1 C. limai 7 6 13 35 0 35 48 3.3 C. ignacioi 4 8 12 10 21 31 43 3.0 C. foxi 19 2 21 16 3 19 40 2.8 C. ruizi 22 0 22 13 2 15 37 2.6 C. paucienfuscatus 0 0 0 23 0 23 23 1.6 Culicoides sp. 17 1 18 3 1 4 22 1.5 C. boliviensis 12 2 14 1 5 6 20 1.4 C. flavivenula 3 0 3 3 1 4 7 0.5 C. guyanensis 2 0 2 1 4 5 7 0.5 C. filariferus 2 0 2 0 1 1 3 0.2 C. travassosi 0 0 0 3 0 3 3 0.2 C. furens 2 0 2 0 0 0 2 0.1 C. debilipalpis 2 0 2 0 0 0 2 0.1 Numbers of individuals 784 78 862 509 68 577 1,439 - Percentage numbers 54.4 5.4 59.9 35.3 4.7 40.1-100 Species richness 13 7 13 12 10 13 15 -

Vol. 42, no. 1 Journal of Vector Ecology 117 50 45 40 Relative abundance (%) 35 30 25 20 15 10 5 0 Species Figure 2. Dominance rank of Culicoides species collected in Barreirinhas, Maranhão, Brazil in April, May, and August, 2015. The other species interacted with a variety of animals that could function as a blood source (Costa et al. 2013). GLMs showed no significant differences in the abundance of midges collected from environments with or without management, suggesting that environmental management has little effect on midge populations. This may be attributed to several factors, including the proximity of homes to the forest. Because residences are within walking distance (±50 m), midges may migrate from the forest to peridomestic areas to feed on domestic animals in shelters. This is a feasible scenario, as midges are known to travel distances up to 2 km (Lillie et al. 1981). After blood feeding, the midges likely return to the original ecotope for reproduction, away from the peridomestic area. Midge breeding areas may thus occur outside of the physical space of the peridomestic area and animal shelters, at least in the current study areas. If this occurs, Culicoides drawn from more distant ecotypes probably inhabit moist environments and marginal water bodies (streams and bogs) bordering the towns and within their range of distribution. The study area has sandy soil, which may have influenced midge abundance and richness. Unlike other areas with swamp and wetland formations, clay soil is rich in organic matter and provided favorable habitats for these insects. However, some Culicoides species are known to develop in puddles, animal dung, tree holes, and decomposing plant tissue (Kruger et al. 1990, Felippe-Bauer and Sternheim 2008), all common components of peridomestic environments. Although the statistical model we used did not detect significant effects of environmental management on midge populations, we cannot exclude the possibility that management is relevant in other areas with different environmental characteristics. We did find a trend toward lower Culicoides abundance in managed peridomestic areas (clean). This result suggests that environmental management practices should not be dismissed as inconsequential for vector control, but rather that such strategies should be integrated with other management practices for effective reduction of insect populations. The Culicoides fauna found in this study was diverse, and included 14 valid species and morphospecies. This result is consistent with previous entomological surveys carried out in other regions of Maranhão (Silva and Rebelo 1999, Barros et al., 2007, Costa et al. 2013, Silva and Carvalho 2013, Carvalho and Silva 2013, Bandeira et al. 2016), which found 17 or more species. Our species composition was similar to that found by Costa et. al. (2013), with C. furens being the only species not yet recorded in the Maranhenses National Park area. However, some species found by Costa et. al. (2013) were not found in the current study. The most abundant species in this study were C. insignis and C. leopoldoi, and similar results were found for surveys conducted

118 Journal of Vector Ecology June 2017 by Silva and Carvalho (2013) in the Chapadinha municipality and by Costa et al. (2013) in the Lençóis Maranhenses National Park. In the case of C. insignis, dominance is particularly important because it is the primary vector of the blue tongue virus, which has been detected in Brazil (Clavijo 2002, Konrad et al. 2003, Dorneles et al. 2012). In summary, we found a rich and abundant Culicoides fauna in peridomestic environments associated mainly with the presence of domestic animals. These results should be considered when implementing control measures for these insects in Barreirinhas. We note that while environmental management practices did not appear to significantly influence midge abundance, positive effects of sanitation cannot be ruled out. Several factors may have confounded our interpretation of the effectiveness of this strategy, including the availability of properties containing animals, site proximity to peridomestic areas with no animals, forest proximity to the residences, and water bodies in the area. In addition to environmental factors, inherent ecological characteristics of vectors, such as dispersal ability, may have impacted results. Further investigations should be carried out in order to better understand the ecology of biting midges in these areas and to establish adequate measures for the control of populations. Acknowledgments We thank the Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) for the extension grant to M.C.A.B. and A.P., the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scientific initiation scholarship granted to G.A.B. and A.P., and the productivity grant to J.M.M.R. REFERENCES CITED Aparício A.A.S., G.E. Castellón, and R.F.O. Fonseca. 2011. Distribuição de Culicoides (Dipetra: Ceratopogonidae) na Amazônia Legal através de técnicas de geoprocessamento. Rev Colombiana Ciencia Anim. 3: 283-299. Assunção Junior A.N., O. Silva., J.L.P. Moraes., F.R.F. Nascimento., Y.N.O. Pereira., J.M.L. Costa, and J.M.M. Rebêlo. 2009. Foco emergente de leishmaniose tegumentar no entorno do Parque Nacional dos Lençóis Maranhenses, Nordeste, Brasil. Gaz. Méd. Bahia 79: 103-109. Bandeira, M.C.A., A. Da Penha, J.L. Moraes, G.A. Brito, and J.M.M. Rebêlo. 2016. Infestation of Brazilian peridomiciliary areas by Culicoides (Diptera: Ceratopogonidade in humid and semi humid climates. J. Med. Entomol. 53: 1163-1168. Barros, V.L.L., R.M. Marinho, and J.M.M. Rebêlo. 2007. Ocorrência de espécies de CulicoidesLatreille (Diptera, Ceratopogonidae) na área metropolitana de São Luís, Maranhão, Brasil. Cad. Saúde Públ. 23: 2789 2790. Borkent, A. and G.R. Spinelli. 2007. Neotropical Ceratopogonidae (Diptera: Insecta). In J. Adis, J.R. Arias, G. Rueda-Delgado, and K.M. Wnatzen. Aquatic Biodiversity in Latin America (ABLA), Vol. 4, Pensoft, Sofia-Moscow, pp. 1 198. Borkent, A. 2015a. World species of biting midges (Diptera: Ceratopogonidae). Illinois Natural History Museum. http://www.inhs.illinois.edu/ research/ FLYTREE/ Borkent.html <Acessado em: outubro de 2016>. Borkent, A. 2015b. The subgeneric classification of species of Culicoides. Illinois Natural History Museum. http:// www. inhs.uiuc. edu/research/flytree /Culicoides Subgenera.pdf <Acessado em: setembro de 2016. Braverman, Y. 1994. Nematocera (Ceratopogonidae, Psychodidae, Simuliidae and Culicinae) and control methods. Review Sci. Tech. Off. Int. Epiz.13: 1175 1199. Carpenter, S., P.S. Mellor, and S.J. Torr. 2008. Control techniques for Culicoides biting midges and their application in the U.K. and northwestern Palaearctic. Med. Vet. Entomol. 22: 175 187. Carvalho, L.P.C and F.S. Silva. 2013. Seasonal abundance of livestock-associated Culicoides species in northeastern Brazil. Med. Vet. Entomol. doi: 10.1111/mve.12043. Castellón, E.G. 1990. Culicoides (Diptera: Ceratopogonidae) na Amazônia brasileira. II. Espécies coletadas na reserva florestal Ducke, aspectos ecológicos e distribuição geográfica. Acta Amazônica 20: 83 93. Clavijo, A., L. Sepulveda, J. Riva, M. Pessoa-Silva, A. Tailor-Ruthes, and J.W. Lopez. 2002. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South America. Vet. Rec. 151: 301-302. Costa, J.C., E.S. Lorosa., J.L.P. Moraes., and J.M.M. Rebêlo. 2013. Espécies de Culicoides (Diptera; Ceratopogonidae) e hospedeiros potenciais em área de ecoturismo do Parque Nacional dos Lençóis Maranhenses, Brasil. Rev. Pan-Amaz. Saúde 4: 11 18. Courtney, G.W. and R.W. Merritt. 2008. Capítulo 22. Aquatic Diptera, Part one, Larvae of aquatic Diptera, In: R.W. Merritt, K.W. Cummins, and M.B. Berg (eds.), An Introduction to the Aquatic Insects of North America, 4 th ed. pp. 687-722. Kendall/ Hunt Publishing Co, Dubuque, IA. Dorneles, E.M.S., F.C., Morcati, A.S., Guimarães, Z.I.P. Lobato, A.P. Lage, V.S.P. Gonçalves, A.M.G. Gouveia, and M.B. Heinemann. 2012. Prevalence of bluetongue virus antibodies in sheep from Distrito Federal, Brazil. Semina: Ciênc. Agrár. Londrina 33: 1521 1524. DNPM. Projeto RADAM. 1973. Mapas de geologia e geomorfologia. Folha SB-23 - Teresina e parte da Folha SB- 24- Jaguaribe, Folha SA-23- São Luís e parte da Folha SA-24- Fortaleza Rio de Janeiro.V.3. Felippe-Bauer, M.L. and S.J. Oliveira. 2001. Lista dos Exemplares Tipos de Ceratopogonidae (Diptera, Nematocera) Depositados na Coleção Entomológica do Instituto Oswaldo Cruz, Rio de Janeiro, Brasil. Mem. Inst. Osw. Cruz 96: 1109 1119. Felippe-Bauer, M.L. 2003. A importância do padrão das manchas das asas em Culicoides (Latreille, 1809) (Diptera: Ceratopogonidae): sua limitação. Entomol. Vectors 10: 595 600. Felippe-Bauer, M.L. and U.S. Sternheim. 2008. Culicoides paraensis (Diptera: Ceratopogonidae) infestations in cit-ies of the Itapocu River Valley, S. Brazil. Entomol. News 119: 185-192. Gusmão, G.M.C., E.S. Lorosa, G.A. Brito, L.S. Moraes, V.J.C. Bastos, and J.M.M. Rebêlo. 2014. Determinação das fontes de repasto sanguíneo de Culicoides Latreille (Diptera, Ceratopogonidae) em áreas rurais do norte do estado do

Vol. 42, no. 1 Journal of Vector Ecology 119 Maranhão, Brasil. Biotemas 28: 51 58. Instituto Brasileiro de Geografia e Estatística. 1984. Estado do Maranhão Vegetação. Rio de Janeiro: IBGE. Disponível em: <http://www.ibge.gov.br> (acessado em setembro de 2016). Instituto Brasileiro de Geografia e Estatística. 2011. Censo demográfico 2010. Características da população e dos domicílios: resultados do universo. Rio de Janeiro: IBGE. Disponível em: <http://www.ibge.gov.br> (acessado em setembro de 2016). Kato, M., T. Matsuda, and Z. Yamashita. 1952. Associative ecology of insects found in the paddy field cultivated by various planting forms. Sci. Rep. Tohoky Univ. IV, Biol 19: 291 301. Konrad, P.A., R.O. Rodrigues, A.C.P. Chagas, G.F. Paz, and R.C. Leite. 2003. Anticorpos contra o vírus da Língua Azul em bovinos leiteiros de Minas Gerais e Associações com problemas reprodutivos. Revist. FZVA 10: 117 125. Kruger, E.L., L.G. Pappas, and C.D. Pappas. 1990. Habitat and temporal partitioning of tree hole Culicoides (Diptera: Ceratopogonidae). J. Am. Mosq. Contr. Assoc. 6: 390-393. Lillie, T., W. Marquardt, and R. Jones. 1981. The flight range of Culicoides variipennis (Diptera: Ceratopogonidae). Canad. Entomol. 113: 419 426. Mellor, P.S., J. Boorman, and M. Baylis. 2000. Culicoides biting midges: their role as arbovirus vectors. Annu. Rev. Entomol. 45: 307 340. Oksanen, J, F.G. Blanchet, R. Kindt, P. Legendre, P.R. Minchin, R.B. O Hara, G.L. Simpson, P. Solymos, M.H.H. Stevens, and H. Wagner. 2016. Package vegan. Community ecology package, version, v. 2. Oliveira, G.M.G., E.A. Figueiró Filho, G.M.C. Andrade, L.A. Araújo and R.V. Cunha. 2010. Flebotomíneos (Diptera: Psychodidae: Phlebotominae) no município de Três Lagoas, área de transmissão intensa de Leishmaniose Visceral, Estado de Mato Grosso do Sul, Brasil. Rev. Pan-Amazônica Saúde 1: 83-94. Pereira Filho, A.A., M.C.A. Bandeira, R.S. Fonteles, J.L.P. Moraes, C.R.G. Lopes, M.N. Melo, and J.M.M. Rebêlo. 2015. An ecological study of sand flies (Diptera: Psychodidae) in the vicinity of Lençóis Maranhenses National Park, Maranhão, Brazil. Parasites Vectors 8: 1-8. Pielou, E.C. 1975. Ecological Diversity. Wiley InterScience, N.Y. 165 pp. Pinheiro, F.F., M. Pinheiro, G. Bensabath, O.R. Causey, and R. Shope. 1962. Epidemia de vírus Oropouche em Belém. (Nota Prévia) Rev. Serv. Espec. Saúde Públ. 12: 15-23. Satta, G., M. Goffredo, S. Sanna, L. Vento, G.P. Cubeddu, and E. Mascherpa. 2004. Field disinfestation trials against Culicoides in north-west Sardinia. Vet. Italiana 40: 329-335. Silva, F.S. and J.M.M. Rebêlo. 1999. Espécies de Culicoides Latreille (Diptera: Ceratopogonidae) da ilha de São Luís, Maranhão Brasil. Bol. Mus. Paraense Emílio Goeldi 15: 169 179. Silva, F.S. and L.P.C. Carvalho. 2013. A Population study of the Culicoides biting midges (Diptera: Ceratopogonidae) in urban, rural, and forested sites in a cerrado area of northeastern Brazil. Entomol. Soc. Am. 106: 463 470. Spinelli, G.R., M.M. Ronderos, F. Díaz, and P.I. Marino. 2005. The bloodsucking biting midges of Argentina (Diptera: Ceratopogonidae). Mem. Instit. Oswaldo Cruz 100: 137-150. Thomazi, D.R., T.R. Rocha, V.M. Oliveira, S.S. Bruno, and G.A. Silva. 2013. Um panorama da vegetação das restingas do Espírito Santo no contexto do litoral brasileiro. Natureza online 11: 1 6. Trindade, R.L. and I.S. Gorayeb. 2010. Maruins (Diptera: Ceratopogonidae: Culicoides), após a estação chuvosa, na Reserva de Desenvolvimento Sustentável Itatupã-Baquiá, Gurupá, Pará, Brasil. Rev. Pan-Amaz. Saude 1: 121 130. Venables, W.N. and B.D. Ripley. 2002. Modern Applied Statistics with S. 4 th ed. Springer, New York. ISBN 0-387-95457-0. Wermelinger, E.D. and A.P. Ferreira. 2013. Métodos de controle de insetos vetores: um estudo das classificações. Revista Pan- Amazônica Saúde 4: 49-54. Wirth W.W. and F.S. Blanton. 1973. A review of biting midges of the genus Culicoides (Diptera: Ceratopogonidae) in the Amazon Basin. Amazoniana 4: 405 470. Wirth W.W., A.L. Dyce, and G.R. Spinelli. 1988. An atlas of wing photographs, with a summary of the numerical characters of the neotropical species of Culicoides (Diptera: Ceratopogonidae). Contrib. Am. Entomol. Inst. 25: 1 72. Zahl, S. 1977. Jackknifing an index of diversity. Ecology 58: 907-913.