This longitudinal survey revealed, for the first time, 13 different sand fly species in Foz do Iguaçu. Previously, eight sand fly species [44] had been signaled in 1990 in the west region of the State of Paraná, with Lutzomyia longipalpis being observed for the first time in 2012 [22] and 11 species in 2014 in a cross-sectional study conducted in Foz do Iguaçu [13]. Lutzomyia longipalpis was present for 11 months (except July) in the present study, representing 71.5% of the populations and prevailing in the 20 peridomicile and intradomicile traps. In the cross-sectional survey mentioned before, the abundance of this species on the Brazilian side was 55.7%, representing 74.2 and 47.9% in Argentina and Paraguay, respectively [45, 46]. These results indicate that the vectors do not respect borders, and control measures must embrace all three countries. In Brazil, the abundance of Lu. longipalpis can vary from 25 to 97%, depending on the region [47–50], and males generally predominate over females [48, 49, 51, 52]. For example, in an area of active VL transmission in the Southeast region of the country, the ratio of males to females was 2.9:1 [48, 51]. In the Northeast region, the same ratio was 2:1 [48].
At the studied location, Ny. whitmani accounted for 19.9% of the sand fly fauna, whereas Ny. neivai accounted for only 1.9%, with the females of these species predominating in colder months. In the cross-sectional study, the abundance values were 16.4 and 9.4 for Ny. whitmani and Ny. neivai, respectively. In Argentina and Paraguay, the abundance of Ny. whitmani was 25% (Puerto Iguazú) and 38.8% (Alto Paraná Department), respectively. In Brazil, in the northern region of Paraná, research conducted in rural areas where CL is endemic has shown an alternate prevalence of Ny. whitmani/Nyssomyia intermedia s.l. In these areas, transmission of L. braziliensis is linked to forest remnants [53–56]. The predominance of Ny. whitmani could be reach rates up to 68% [57, 58]. In the northwest of the state (City of Japurá), Ny. intermedia s.l. is the dominant species, followed by Ny. whitmani [59, 60]. In south-eastern Brazil, these species have been found with a high density in endemic areas in the States of São Paulo, Minas Gerais, and Espírito Santo [61]. Their presence has been recorded in animal shelters and in the peridomicile and intradomicile.
In Foz do Iguaçu, Lu. longipalpis occurred mostly in the summer and autumn, especially in urbanized areas, and had a lower presence from June to August. Still, when we observe our puzzle image, the period of highest abundance of Lu. longipalpis on the Argentinian side (the other piece of the puzzle) was early autumn, and the species was mainly distributed in the most urbanized areas [11]. In Latin America, several studies have reported that a higher prevalence of Lu. longipalpis is related to the rainy season, particularly in northeastern Brazil [47, 62–66]. In a VL focus in the city of Belo Horizonte, Minas Gerais (Central Brazil), this vector showed higher abundance from October to March, increasing progressively until February. Then, the population started to decrease in April until reaching the lowest levels from June to August [47, 49]. In the state of Mato Grosso do Sul, Central Brazil, a survey carried out by Oliveira et al. [67] for two consecutive years revealed the occurrence of several peaks of Lu. longipalpis: the first in February, and the second in April, with a higher frequency (72%) in the rainy season compared to the dry season (28%). In our survey, Ny. whitmani females in the intradomicile were mainly observed in May and June. In the State of Rio de Janeiro, Ny. whitmani was abundant in the coolest months (June, July, and August), although both occurred throughout the year [61]. In Argentina, Ny. whitmani mainly occupied less urbanized areas, showing abundance peaks in early spring and summer [12].
The aim here was to know which variables had significance for the presence of Lu. longipalpis in the intradomicile and peridomicile. The significant variables for the intradomicile were NDVI, NDWI, altitude, minimum temperature, and EW wind, as shown by Salomon [7]. In the peridomicile habitat, in addition to the previous variables, the minimum and maximum relative humidity were also significant. Other studies have supported that the distribution of Lu. longipalpis was associated with climatic conditions such as temperature, humidity, and wind [7, 67]. In addition, peridomicile characteristics such as the presence of chickens or dogs [68, 69] and the type and amount of vegetation cover, type of street paving, distance to water bodies, and landscape features were also related to the distribution of this species [10, 11, 13, 66].
In the present study, the intradomicile presence of Ny. whitmani was significantly related to the altitude, water distance, NDWI, EW wind, and maximum temperature. In the peridomicile, only the rainfall, EW wind, and wind speed were significant. The abundance of Nyssomyia whitmani was also correlated with weather conditions [61, 70], and this species was captured in large numbers in pig and chicken sheds [8]. The distribution of Ny. whitmani has also been associated with several landscape features [8, 11, 61, 71–73]
The infection rate by Leishmania was determined using the PCR approach followed by sequencing of the ITS1 target. Out of 792 traps (22 traps x 12 months x 3 nights), 36 (4.5%) contained infected females. Two sand fly species (Lu. longipalpis with 71.5% and Ny. whitmani with 19.9%) were found with L. infantum, with a mean infection rate of 14.1% (11.4 minimum – 17.1% maximum). In Argentina, Moya et al. [45] reported a 3.9% infection rate across the triple border. On the other hand, in the Alto Paraná Department (on the other side of the puzzle– Paraguay), the infection rate of Lu. longipalpis was 23.4% [46]. In the Americas, the studies conducted thus far showed that the infection rates can change according to the country/region, season, or vector species. For example, during two years of sampling in Colombia, the natural infection by L. infantum was 1.9% [74]. In Brazil, the estimated infection range was between 0.2 and 36.5%. Considering the different states that constitute the Brazilian territory, a 0.2% prevalence of infected females was observed in Bahia [75], 2.6% in Mato Grosso do Sul [76], 2.7 to 3.9% in Minas Gerais [49, 77], 3.7% in Maranhão [78], 36.5% in Ceará [32], and 4.8 to 7.2% in São Paulo [79]. The Old World is no different in that regard: Branco et al. [80] showed a 4% prevalence of infected females in Portugal, whereas Goméz-Saladín et al. [81] showed a 2.9% rate in italy. In Morocco, Mhaid et al. [82] used PCR with ITS as a target and obtained a prevalence rate of 7.3%. In Madrid, 58.5% of the Phlebotomus perniciosus specimens were positive for L. infantum infection using kDNA-PCR methods [83], whereas in Northern and Central Tunisia the prevalence of infection by L. infantum within P. perniciosus was 0.2% using nested ITS-PCR [84]. It would be certainly interesting to apply the same methodology in order to compare infection rates. In addition, reverse transcription should be used to differentiate infection only by the presence of DNA, using a robust methodology to obtain more reliable results. Control measures must be appropriate in order to reduce the risk of L. infantum infection in sand flies. Therefore, it is important to know the infection rates and the periods in which it occurs. Nyssomyia whitmani and Ny. neivai have been implicated in the transmission of L. braziliensis [85, 86] under sympatric conditions [13]. Thus, we show that the pathogenic complex (Leishmania/hosts) can be composed of several vectors and genotypes of the parasite and a main reservoir: the dog. Another aspect that has drawn attention is that these two elements (vector, and reservoir) are closely linked to humans. In this context, the theoretical (genesis of the focus) and practical (fight strategy) interests are fundamental to propose control measures.
At this stage, we have gone beyond the critical inventory of collected data, also revealing that: 1) The population of Lu. longipalpis increases in the peridomicile and intradomicile from the summer to autumn; 2) Population increases in the intradomicile could be a response to reductions in temperature and environmental humidity, leading females to enter the houses to seek shelter.
The ‘microfocus’ of L. infantum can be maintained and rapidly propagated below the 54S parallel in areas where conditions are now favorable and climate change has already been observed through temperature increases of about 2°C, as shown by De La Rocque et al. [87]. Therefore, innovative measures are necessary to control infections, especially in humans. As a result, it is imperative that the medical service be trained to recognize this disease in new areas where it was not endemic, thus preventing treatment delays. The increase in the canine population of all continents will help maintain the macrofocus of VL. The dog population tends to grow due to their proximity to humans, who are increasingly attached to pets as emotional support. There are many dogs (reservoirs) abandoned in city streets, which, due to cultural and social factors, are raised for guarding purposes. In many cities, these animals sleep outside residences, increasing their contact with the vectors in the peridomicile. Infected dogs have spread across cities and countries (especially in border areas) without being assessed for VL infection.
In this study, the periods with the highest rates of sand fly infection with L. infantum were the autumn and spring. However, there were infected females in ten out of the 12 months surveyed. Chemical control could be a way to control vector populations when the first population (August, September) appears. In the winter period, it could be possible to reduce the population by using chemical control inside human houses and animal shelters. These measures would be easily performed with knowledge about vector density peaks outside and inside residences. Moreover, continued surveillance can provide new insights on a global scale and particularly when it comes to this complex biological ecosystem of vector-parasite-host-environment-climate, especially in countries and regions that have recently become endemic [88, 89]. Vulnerable populations need assistance and education to understand the life cycle of Leishmania and sand flies and prevent the transmission and worsening of leishmaniasis.
Finally, it has been shown that the methodology employed here should be used by responsible administrators to implement vector population control and reduction policies; moreover, identifying hotspots will allow targeted control measures. New vector colonization is concentrated in urban and peri-urban hotspots, with some individuals being present in various parts of the city. However, few sites show a high abundance of vectors. This distribution suggests that the risk of actual contact between humans and parasitic vectors in urban areas during epidemic periods is associated with patches of peri-urban vegetation, with subsequent dispersion into urban areas; the period of higher transmission of L. infantum is from January to May. Therefore, if measures are taken to reduce access to infection sources, there will be fewer female sand fly infections, and, consequently, reduced human infection. If intervention measures are implemented for vector control from September to December, the population of sand flies will be smaller, and the risk of protozoan transmission to humans will be reduced.