Notes on the sand fly fauna (Diptera: Psychodidae) in a region of Brazil Notas sobre a fauna de flebotomíneos (Diptera: Psychodidae) em uma região do Brasil Notas sobre la fauna de flebótomos (Diptera: Psychodidae) en una región de Brasil

The family Psychodidae has a cosmopolitan distribution with members that occur in many habitats, mainly in humid environments, and is most diverse in the tropics. Subfamilies Sycoracinae and Phlebotominae have females with hematophagous habits and the latter studied more due to medical and veterinary interest, since it includes species that can transmit diseases to animals and humans. The knowledge about the sand fly fauna in a region is extremely important for adequate monitoring and control measures for leishmaniasis. Thus, the objective of this study was to characterize Psychodidae fauna in relation to richness, abundance and molecular identification of Leishmania spp. in sand flies in southern Santa Catarina, Brazil. The ollections were carried out between 2015 and 2016 in three cities in Santa Catarina, Brazil. Samples were taken near feeding places for domestic animals, urban forest and peridomicile areas. The insects were identified and female sand flies were submitted to molecular analysis to detect the presence of Leishmania spp.. A total of 4,200 insects were collected, 4,193 from the Sycoracinae subfamily and 7 Phlebotominae Research, Society and Development, v. 10, n. 14, e596101422480, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.22480 2 from the Nyssomyia neivai and Pintomyia fisheri species. Of the studied municipalities, sandflies were registered in Tubarão and Imaruí and the most frequent habitat was the peridomicile areas. No samples were positive for Leishmania spp. In conclusion, the work highlights the presence of two species of sandflies, which were recorded for the first time in southern Santa Catarina.


Introduction
The family Psychodidae comprises small Diptera that are characterized mainly by having a body covered with bristles (Sherlock, 2003). It has a cosmopolitan distribution with members that occur in many habitats, mainly in humid environments, and is most diverse in the tropics. The family has more than 3,000 described species subdivided into 6 subfamilies: Bruchomyiinae, Psychodinae, Trichomyiinae, Sycoracinae, Phlebotominae, and Horaiellinae (the last occurs only in East Asia) (Bejarano & Estrada, 2016;Pape et al., 2011).
Subfamilies Sycoracinae and Phlebotominae have females with hematophagous habits and the latter studied more due to medical and veterinary interest, since it includes species that can transmit different diseases (e.g., leishmaniasis) to animals and humans (Guimarães et al., 2014;Pape et al., 2011;Sherlock, 2003). According to Young & Duncan (1994) (Young & Duncan, 1994), the Leishmania spp. vectors in the Americas belong to Lutzomyia, but in a new classification by Galati (2003) (Galati, 2003) this genus was divided in to many genera, especially Lutzomyia, Migonemyia, Pintomyia, Bichronomyia, Psychodopygus, Nyssomyia and Trichophoromyia (Galati, 2003;Young & Duncan, 1994). In addition, the largest number of species of sandflies is found in the Americas, with Brazil being the country with the greatest diversity of species (Galati, 2003).
Leishmaniasis is caused by an infection with Leishmania spp. and is among the most neglected diseases in the world, affecting poor populations, especially in developing countries. Leishmaniasis can occur in cutaneous and mucocutaneous (American Tegumentary Leishmaniasis) and visceral (Visceral Leishmaniasis) forms, depending on the parasitic species involved in the infection. Visceral leishmaniasis is the most worrisome form of the disease, and when not diagnosed and treated early, the mortality rate can reach 10% (Alves, 2009;de Brito et al., 2014;World Health Organization, 2010).
According to the World Health Organization (WHO) (World Health Organization, 2010), almost two million new cases of leishmaniasis are reported world wide each year.
In Brazil, at 2015, 20.975 LTA cases (Ministério da Saúde, 2020a) and 3.558 LV cases were confirmed (Ministério da Saúde, 2020c). This country, which concentrates 95% of cases of VL in the Americas (Alvar et al., 2012), had the southern region considered free of the disease until 2009, but the number of cases has grown in recent years (Deboni et al., 2011 (Figueiredo et al., 2012). Further, there are many problems with mapping the leishmaniasis cases as there are a large number of unreported cases (Gontijo & Melo, 2004). Thus, were reported revealed a new endemic area in the northeastern part of the state, with two Leishmania species present, whereas in the southern part the infection cases are predominantly imported, except for three Leishmania (Leishmania) amazonensis cases (Marlow et al., 2013).
The sand fly habitat in an area may be influenced by several factors, such as food and the rainfall index. With increasing deforestation and urbanization, these insects are also developing in peri-urban and urban areas, indicating their ability to inhabit anthropic environments (Feitosa & Castellón, 2006;Ramos et al., 2014;Souza et al., 2010). Thus, knowing the insect fauna is important so it can be compared with other regions to establish an epidemiological profile, since each region, depending on its location, temperature and other variables, may have different species (in quantity and variety).
Therefore, knowledge about the sand fly fauna in a region is extremely important for adequate monitoring and control measures for leishmaniasis. In addition, the sand fly fauna in Santa Catarina is poorly described, with few species listed for the state, and until now, no study had been carried out in the southern region. Thus, the objective of this study was to characterize Psychodidae fauna in relation to richness, abundance and molecular identification of Leishmania spp. in sand flies of the subfamily Phlebotominae in southern Santa Catarina, Brazil.
The geolocations of the different points are in Table 1. The insects were captured using CDC luminous traps from April 2015 to March 2016. The collections were made monthly for approximately 12 hours (~ 18:00 pm to 06:00 am) per sampling period. The collected insects were screened, separating, and those with a morphology similar to Psychodidae were further identified. Identifications were made using keys by Galati (2003) (Galati, 2003) and Sycoracinae was identified to subfamily.
All insects selected were stored in 70% alcohol. Source: Authors.

Sand fly DNA extraction
DNA extraction was performed with a Gentra Puregene® QIAGEN tissue kit following the protocol modified by Quaresma et al (2011) (Quaresma et al., 2011: females were macerated in 100μl of cell lysis solution and 1μl of proteinase K was added to the mixture. The solution was homogenized by tube inversion and incubated at 55°C overnight.
After incubation, 1μl of RNA se was added and homogenized by inversion and incubated for 30 minutes at 37°C. After this, the samples were incubated for 3 minutes on ice and 100μl of protein precipitation solution was added.
The tubes were vortexed for 20 seconds and then centrifuged for 5 minutes at 1,400 rpm. The supernatant was placed in a new tube, 300μl of isopropanol was added by inverting the remaining sample, the sequence another centrifugation at 1400 rpm for 5minutes. The supernatant was discarded, and the tube was inverted and left to dryon absorbent paper. Subsequently, 70% alcohol was added to dry inverting it several times followed by centrifugation at 1400 rpm for 5 minutes. The supernatant was discarded, and the tube was inverted and left to dryon absorbent paper. Finally, 25μl of ultrapure H20 was added for DNA hydration.

PCR identification
To determine the presence of parasites of the collected sand fly females, the extracted DNA samples were analyzed based on a PCR technique used for Leishmania ITS1 (Tail et al., 2000). For amplification, the pair of LITSR primers was used: 5 'CTGGATCATTTTCCGATG 3' and L5.8S: 5 'TGATACCACTTATCGCACTT 3'.
DNA amplification was conducted with an Eppendorf® Mastercycler Gradient automatic cycler using the following: initial denaturation for 2 minutes at 95°C, sequencing 35 denaturation replicates for 30 seconds at 95°C, annealing for 60 seconds at 53°C and extension for 60 seconds at 72°C. The final extension was at 72°C for 10 minutes.
Twenty nanograms of DNA extracted from Leishmania braziliensis (MHOM / BR / 75 / M2903) was used as the positive control and sterile distilled water was used as the negative control. The results were observed on 2.0% agarose gel stained with ethidium bromide and viewed under ultraviolet (UV) light.

Statistical analysis
The following data were analyzed: richness, abundance, sex, infected females, and seasons of the year. Data meeting the prerequisite for normality were subjected to an analysis of variance (ANOVA). Tukey's post-hoc test was used when significant differences were detected (p < 0.05). Non-parametric data were analyzed using the Kruskal-Wallis test by rank and median. A Mann-Whitney pairwise test was used when significant differences were detected (p < 0.05) (Sokal & Rohlf, 2011).The correlations between climatic variables (temperature and air humidity) and population density of Psychodidae were Research, Society and Development, v. 10, n. 14, e596101422480, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.22480 5 evaluated by means of Spearman's coefficient (p <0.05). All analyses were performed using the statistical software PAST v3.07 (Hammer et al., 2001).

Results
In our studies, from April 2015 to March 2016, it was possible to distinguish four seasons of the year, when 4,200 insects of family Psychodidae were collected, including two subfamilies: Sycoracinae and Phlebotominae. Subfamily Sycoracinae was represented by 4,193 specimens and subfamily Phlebotominae by 7 specimens of two species: Nyssomyia neivai (=Lutzomyia neivai) and Pintomyia fischeri (=Lutzomyia fischeri). The highest Psychodidae abundance was observed in Imaruí (57.2%), followed by Criciúma (30.6%) and Tubarão (12.2%). An analysis of Psychodidae abundance revealed a significant difference between the number of specimens collected in the different cities (F= 27.1; p < 0.05). Psychodidae abundance was significantly different between the three collection sites, considering the three cities (F = 7.2; p< 0.05), with the highest abundance near feeding places for domestic animals (53.5%). Subfamily Phlebotominae was registered in Imaruí and Tubarão (Table 2). There was no significant difference in abundance between the different months, considering the three cities (H = 9.5; p > 0.05). In order to find other factors relevant to the presence of phlebotomine in the cities, we categorized the seasons of the year and observed that the family Psychodidae was constantly reported in the collections; the data showed a greater number in autumn and lower number in winter (Figure 1).

Discussion
This study collected two subfamilies of Psychodidae: Sycoracinae and Phlebotominae. In a similar study in the city of  Research, Society andDevelopment, v. 10, n. 14, e596101422480, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.22480 8 number of individuals collected, these authors collected 4 Lu neivai in peridomicile area, while in the present study 2 males and 2 females were collected in a peridomicile area in Imaruí, as well as 1 male and 1 female in an urban forest in Tubarão.
Beyond this point, the authors also observed a greater predominance of this species near residences. For Lu. fischeri, the authors reported the same number of individuals that were found in this study; for both studies, the presence of 1 female was observed for point near banana plants (Guimarães et al., 2014).
As found in the present study, for collections made in eastern Santa Catarina in 2000 (Marcondes et al., 2005), and in a study conducted in the northeast of state at 2008 (Grott et al., 2014), was observed Nyssomyia neivai as a predominant phlebotomine species. Similar results were also obtained in a study conducted during 2012 in Paraná State (dos Santos et al., 2016). This species of phlebotomine is often found in peridomiciliary regions (dos Santos et al., 2016;Grott et al., 2014;Marcondes et al., 2005).
Pintomyia fischeri has been recorded in the states of the South Region of Brazil and is essentially a wild species, although it has already been found in peridomestic rural habitats (Pita-Pereira et al., 2011). Although only one specimen of this species was collected in the present work, the habitat characteristics that Pita-Pereira et al. (2011) (Pita-Pereira et al., 2011 correlate with encountering P. fischeri correspond to the similarity of the collection point where the species was found. Considering the abundance of P. fischeri in deforested areas, it can be suggested that this species is well suited to live in the surroundings of forests and environments with anthropic changes (Pita-Pereira et al., 2011).
The geographic distribution of Psychodidae species can be influenced by physical barriers, rainfall, vegetation, luminosity and abundance of vertebrate hosts (Aguiar et al., 1985). It is also known that the presence of animals influences the density of sand flies near human dwellings and, consequently, increases the transmission risk of Leishmania species to humans (Arias et al., 1985;Lainson et al., 1983;Rangel & Lainson, 2009).
Individuals of family Psychodidae were collected in peridomicile, urban forest and animal feeding source areas; a greater quantity was collected in the city of Imaruí, followed by the cities of Criciúma and Tubarão. For subfamily Phlebotominae the records were from the cities of Imaruí and Tubarão. The greater number of individuals for both Psychodidae and Phlebotominae in Imaruí is probably related to the fact that this city has a large amount of vegetation, even in places near the peridomicile area. In Tubarão, the sand fly specimens were found in a region near urban forest, thus corroborating authors who noted that the majority of phlebotomine species are essentially wild, although they have been found in rural peridomestic habitats (Feitosa & Castellón, 2006;Guimarães et al., 2014;Marlow et al., 2013).
When the collection points were compared, we found a predominance of individuals close to the animal food source, possibly due to the diversity of ecotypes that comprise the sites selected as an animal food source and to the eclectic habits of the captured species, since in these areas there are domestic animals such as dogs, birds, pigs and cattle. On the other hand, the peridomiciliary environment has more human interference, which probably hinders the adaptation of some species of sand flies (Rêgo et al., 2014;Ximenes et al., 1999). It is important to mention that the peridomicile areas of the houses studied have mostly rural characteristics with the presence of domestic animals and plantations of fruit trees and grains that provide an environment with refuges and food for sand flies and the organic matter necessary for the development of immature forms of these insects. This may explain the occurrence of sand fly records in these areas, even in low numbers.
In Paraná State, researchers evaluated the seasonality of phlebotomines and demonstrated that in hot and humid seasons of the year these insects are captured in greater numbers (Teodoro et al., 1993(Teodoro et al., , 2001. The fact that southern Santa Catarina State has few sand flies may explain the low number of leishmaniasis cases, since this disease is not endemic to this region, although we know that there is a serious problem of unreported cases (Gontijo & Melo, 2004). Thus, constant monitoring of circulating species is important since, when an increase of species or specimens is recorded over years, control methods must be considered that can prevent this disease from becoming endemic to southern Santa Catarina.
All sand fly samples that were subjected to molecular analysis had negative results, which is probably related to the low number of samples. Another factor that contributes to the lack of protozoan detection is that in non-endemic areas the circulation of this protozoan is low. Dias et al. Therefore, should be taken into account that the epidemiology of leishmaniasis is conditioned the presence of vectors (phlebotomines) that can transmit the etiological agent Leishmania spp. and the presence of wild and synanthropic mammalian hosts and/or reservoirs (Souza et al., 2010). The great diversity of vectors, reservoirs and agents influence changes in the epidemiological patterns of leishmaniasis, and the influence of humans on the environment also makes it difficult to control the disease (de Brito et al., 2014).

Conclusion
Clinical, biological and epidemiological characterization studies are fundamental to the development of individualized action and prevention strategies, since transmission patterns, vector species, leishmaniasis species, and response to treatment can be very different in some regions. Thus, it is important that we better understand which species are circulating in our study area. The present study updated and expanded the knowledge of the geographic distribution of leishmaniasis vectors, which may assist both in the acquisition of knowledge about the behavior of species, as well as in the strategies of surveillance and control of this disease. The areas without confirmed records, but suitable for the occurrence of the species, should be verified in future studies of survey of phlebotomine fauna. We suggest that the monitoring of these species to be accomplished out periodically in other municipalities in the south of Santa Catarina in addition to those highlighted in this study so that you can have an overview of the recurrent species and make a comparison with those that have already been listed for the state.