Ecological aspects of Phlebotomines (Diptera: Psychodidae) and the transmission of American cutaneous leishmaniasis agents in an Amazonian/ Guianan bordering area

Background An entomological study was conducted in the municipality of Oiapoque (lower Oyapock River Basin) in the Brazilian side bordering French Guiana to gain information on the transmission pattern of American cutaneous leishmaniasis (ACL) in that region, presumed to reflect the classical Amazonian/Guianan enzootic scenario. Methods Three ecologically isolated forested areas near urban environments were surveyed during the rainy and dry seasons of 2015 and 2016, using a multi-trapping approach comprising ground-level and canopy light traps, black and white colored cloth Shannon traps and manual aspiration on tree bases. Female phlebotomines were dissected to find infections and isolate flagellates from Leishmania spp. The strains were characterized by restriction fragment length polymorphism analysis and compared with those of local ACL cases and World Health Organization reference strains. Results Nyssomyia umbratilis, Trichopygomyia trichopyga and Evandromyia infraspinosa were the most frequently found species. Findings on relative abundance, spatiotemporal vector/ACL congruence, natural infections and anthropophilic insights strengthened the Guianan classical transmission of Leishmania (Viannia) guyanensis by Ny. umbratilis and suggested further investigations for Ev. infraspinosa. Nyssomyia umbratilis showed an eclectic feeding habit, including bird blood. Ecological data and literature reports also included Psychodopygus squamiventris maripaensis and Bichromomyia flaviscutellata on the list of suspected vectors. Conclusions These findings contributed to understanding ACL ecoepidemiology in the Amazonian/Guianan scenario. Local studies are required to better comprehend the Leishmania spp. enzootic mosaic in specific ecotopes.

The present study assessed potential ACL transmission cycles in the lower Oyapock River Basin to promote knowledge on phlebotomine ecology, mainly focusing on species composition, multi-trapping stratification, blood-source investigation and natural Leishmania spp. infections. The isolates obtained were also compared with human isolates.

Study area
The municipality of Oiapoque (03°49'29"N, 51°49'05"W) is in the Oyapock River Basin, a border region between Brazil and the Ultramarine Department of French Guiana. It is the northernmost municipality of the Brazilian State of Amapá (AP) and is limited by the AP municipalities of Calçoene, Serra do Navio and Pedra Branca do Amapari to the south, by Laranjal do Jari to the west, by the Atlantic Ocean to the east, and by the French-Guianan communes of Camopi and Saint Georges de l'Oyapock to the north. Oiapoque has a population of approximately 24,263 distributed over 22,625 km 2 [17]. During 2008-2017, a total of 1299 new ACL cases were registered by the health services in Oiapoque (average of 118 cases/year), with 560 shown to be autochthonous for that municipality (average of 50 cases/year). Because of Oiapoque's border characteristics, ACL epidemiology is a mosaic of "binational" infections, as half of ACL-notified cases were autochthonous from Brazil and half were likely imported from French Guiana [18].

Sampling sites
Located in the lower Oyapock River Basin, the urban area of Oiapoque is surrounded by different forested environments with slightly distinct ecological characteristics. Thus, three "terra-firme" (dry-land) forested sites, approximately 7 km apart, were selected for sampling as follows ( Fig. 1 an impacted area in southern urban Oiapoque with evidence of human activities, such as wood extraction. (iii)Clevelândia do Norte Road (3°49'4.14"N, 51°5 1'6.35"W): an old colonized area on the western side of urban Oiapoque, where the original vegetation was partially suppressed and replaced by secondary forest. It is currently an environmentally protected area by the Brazilian Army.

Captures
Systematic field expeditions were initially performed during 2015-2016 to provide information on the predominantly rainy (February-May) and dry (September-November) Guianan/Amazonian seasons of the three sampling sites. Phlebotomines were obtained by surveying a horizontal transect from the edge inside each sampling site's forested area, using a multi-trapping approach described elsewhere [8]. The approach comprised captures using CDC light traps (John W. Hock Company, Gainesville, USA) set up from 6:00 h to 18:00 h at 1.5 m (ground level; n = 8 traps/night) and at 20 m (canopy level; n = 2 traps/night), captures from 6:00 h to 20:00 h with modified Shannon black and white colored cloth [19], and manual aspiration on tree bases from 6:00 h to 20:00 h.

Processing and identification of phlebotomines
Phlebotomines were immediately processed in the field laboratory. Females were dissected under sterile conditions [20]. Flagellate infection was semi-quantified (in a cross '+' scale) according to Freitas et al. [21], and parasite development was classified by Lainson & Shaw [22]. The guts of infected females were triturated and inoculated into Difco B45 culture media (Becton, Dickinson and Company, Franklin Lakes, USA) to isolate the parasites. Phlebotomine species were identified under fresh conditions using morphological characteristics. Unidentified specimens were processed for mounting on glass slides using Canada balsam. Morphology and taxonomic criteria, terminology and generic abbreviations were adopted following Galati [23] and Galati et al. [24].

Investigation of phlebotomine blood sources
Intestines dissected from engorged females were macerated in PBS (pH 7.2, 0.001 M) and stored at -20°C until processing by Enzyme-Linked Immunosorbent Assay according to Afonso et al. [25]. Based on local observation and the antisera available for testing, the panel chosen comprised bird, armadillo, opossum, dog, rodent and human antisera, obtained from the Immunodiagnostics Laboratory, Department of Biological Science, Escola Nacional de Saúde Pública Sérgio Arouca, FIOCRUZ, Brazil.

Investigation of ACL cases
Patients residing the study area who required diagnosis in the field laboratory (Oiapoque) or in the Ralph Lainson Leishmaniasis Laboratory, Instituto Evandro Chagas (Ananindeua, Brazil), were investigated according to the standard procedures of the Programa de Vigilância e Controle da Leishmaniose Tegumentar Americana (ACL Surveillance and Control Programme, Brazil). When clinically epidemiologically suggestive, patients were diagnosed by parasitological demonstration (Giemsa-stained smears of exudates from ACL lesions), the Montenegro skin test (inactivated promastigotes of L. (V.) braziliensis -MHOM/BR/M17323 -1 × 10 7 parasites/ml) and parasite isolation (inoculating exudates from ACL lesions in Difco B45 media) [2,26].

Leishmania spp. characterization
In both cases (phlebotomines and ACL patients), Leishmania DNA was obtained from successfully isolated strains. If no growth or contamination occurred, parasites were recovered from the remaining dissection slide contents (phlebotomines) or Giemsa-stained slides (ACL patients) using the DNeasy Tissue and Blood Kit (Qiagen, Hilden, Germany). Species were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) of a 615 bp region of the RNA polymerase II gene amplified using the primers RPOF2 (5'-AGA ACA TGG GCG GCC-3') and RPOR2 (5'-CGA GGG TCA CGT TCT TG-3') and digested with TpsRI and HgaI endonucleases following previously established and validated methodology [27]. Digestion profiles were compared with those of the WHO Leishmania reference strains occurring in the Guiana Shield.

Environmental assessment and data analysis
Forest cover degree was estimated using digital hemispherical images captured 1.6 m above ground, pointing directly upward in the CDC-ground trap sites (8 images per sampling site). Canopy coverage in each image was determined using CAN-EYE v.6.314 hemispherical image analysis software. Microclimate parameters (temperature and humidity) were measured using data loggers placed in the CDC traps. Species composition at the three sampling sites was analyzed using the Shannon-Wiener diversity index (H') using PAST version 2.12 software [28]. All comparisons were performed using Student's t-test to determine significance level (P ≤ 0.05). Sampling effort, species infection rate (SIR) and number of phlebotomines per hour were calculated according to Souza et al. [8].
Based on 4608 h of CDC ground, 1152 h of CDC canopy trap, 48 h of white Shannon, 48 h of black Shannon, and 24 h of aspiration on tree bases (total sampling effort: 5880 h), white Shannon traps rendered 5.6 phlebotomines per hour. The greatest number of phlebotomines found per hour for these capture methods included Ny. umbratilis in the CDC canopy trap (0.96 ♀♀), white Shannon trap (0.93 ♀♀), and on aspiration on tree bases (1.16 ♀♀; 1.62 ♂♂). Evandromyia infraspinosa was also found on the black Shannon trap (0.97 ♀♀), and Ps. s. maripaensis was found on the white Shannon trap (2.29 ♀♀). These three species attempted to bite the professionals during capture ( Table 2).
Twelve flagellate strains were successfully isolated (Table 3), all without visible blood meals, and with infections varying from ++ to ++++. Isolates occurred in 10 Ny. umbratilis at sites I (n = 3), II (n = 6) and II (n = 1) and in one Sc. fluviatilis (site I) and one Vi. furcata (site I).
Twenty-eight infections were not isolated. The PCR-RFLP for the remaining DNA fixed on the glass dissection slides allowed characterizing L. (V.) guyanensis from one Ny. umbratilis and two Ev. infraspinosa specimens (Fig 4).
Species with more than one blood source in the same specimen included Ny. umbratilis (sample 13: dog and opossum; sample 16: bird, dog and armadillo) and Ps. claustrei (sample 3: dog, opossum and rodent). One Ny. umbratilis naturally infected with flagellates morphologically compatible with Leishmania spp. was positive for bird antiserum.

American cutaneous leishmaniasis cases
During 2015-2016, ten patients belonging to the Guiana Shield asked for ACL diagnostics in our laboratory, and eight believed that they had been infected on the Brazilian-French Guianan/Oyapock border (six from gold mining and two from agricultural settlements) ( Table 5). All Leishmania strains were isolated and characterized as L. (V.) guyanensis. A Brazilian agricultural settlement with L. (V.) guyanensis isolated from an ACL patient (MHOM/BR/2017/M32218) is located in site I, near the forested area subjected to the captures. Figure 3 shows a PCR-RFLP analysis of Leishmania isolates from infected phlebotomines and a human ACL case compared against the WHO L. (V.) guyanensis reference strain.

Discussion
Few studies have been conducted on ACL ecology in the lower Oyapock basin. The first available information on that region (the French Guianan side) came from entomological studies conducted in the 1940s and 1950s, which provided much information on phlebotomine taxonomy and ecology [29]. The available commented checklist including that region can be found elsewhere [30] as a metabarcoding-based local inventory [31]. Evidence on ACL etiology was only provided recently [15].  With reasonable sampling efforts (5880 h) for the multi-trapping approach, the present findings showed high species diversity (48 spp.). We preferred to use different capture methods because, although light-baited suction traps are one of the most widely used tools for vector surveillance, they have biases and limitations in terms of their effect on collection efficiency, population data, and pathogen detection [32]. Multi-trapping approaches with large samplings may offer a broader picture on the surveyed fauna, as shown by Souza et al. [33] in the Lower Amazon Basin (68 spp.) and by Freitas et al. [21] (46 spp.) and Souza et al. [8] (63 spp.) in Amapá. However, the use of CDC light traps in long term surveys and/or with strategical placement (i.e. with spatial stratification biasing to find feeding/resting sites) may, in part, supply some of these limitations. The present results are also reasonably compatible with 'CDC trap-based' surveys recently conducted in the  [46,47].
Differences in the degree of canopy cover between the three sampling sites were congruent with their respective Shannon indices (H), suggesting forest cover as an eligible variable for maintaining species diversity, although deforestation associated with human settlements can also produce environmental conditions suitable for maintaining  the life-cycles of several sand fly species that are adaptable to these environments [48,49]. Conversely, high SIR was found at sites with high degrees of canopy cover. Dense and humid substrate provided by a well-covered canopy may contribute to vector/host availability. In addition, low light penetration in the denser forest can provide suitable conditions for vector/host movements, as observed with the inverse correlation of phlebotomine density on light traps versus moonlight [50]. However, a minimum light is important for these insects to fly [51]; thus, vector-host interactions may result from equilibrium between these factors. Numerous nematodes were found in the body cavity of five phlebotomine species. Although these flies were captured at different sampling sites and different vertical strata (ground/canopy level), some entomopathogenic nematode species infect phlebotomines on the ground, during the larval stage [52], suggesting that these infected flies may share the same breeding site.
Gregarines found in Bi. flaviscutellata (three specimens) were morphologically similar to Psychodiella sp. oocysts, although only molecular sequencing could confirm the species. Insect-host specificity between Bi. flaviscutellata and the gregarine species supports the hypothesis of a long, strong coevolutionary association between them [53].
Only 14.4% of blood-fed phlebotomines tested were positive by ELISA. This result could be attributed to the low blood content in the specimens as well as the blood recuperation procedure for the dissected slides, which may have contributed to the loss of material. Another possibility is the presence of animal blood; this was not accounted for by the available test panel since anteaters and sloths, for example, are presumably present in the Guianan ecosystem, acting as potential reservoir hosts of Undoubtedly, Ny. umbratilis is closely associated with L. (V.) guyanensis and has been consequently implicated as the main ACL vector in Oyapock based on data consistent with its well-recognized regional importance in the Guiana Shield [14] and in the wide Amazonian region [7]. Infection rates for this fly vary greatly in the literature, with some being consistent with the present findings [8,56,57]. Higher rates are usually biased by captures performed in the dry season [58] or supporting the dissection of fed and gravid females [21]. Infected specimens were captured in both levels of CDC traps and tree bases; however, they may have been infected at other sites. A natural vertical migration of these flies is well documented [13] and may explain dissociation movements between infection/resting sites as supposed for L. (V.) naiffi in the Lower Amazonian basin, where a canopy of Ps. davisi was found infected by that parasite, whose only recognized   [32]. Blood contents from Ny. umbratilis reacted mainly with bird antisera (9/12). The role of birds in the population dynamics of phlebotomine species has been discussed [59][60][61][62]. Nyssomyia umbratilis being found with bird blood and concomitantly with a flagellate (likely leishmanine parasites) infection could be an occasional finding or suggests that this blood source provided suitable conditions for L. (V.) guyanensis development, as has been demonstrated in experiments between Gallus gallus blood and L. (L.) mexicana [63]. The findings on the eclectic food habits of Ny. umbratilis are consistent with those of other studies [64,65]. Rodents, for example, appear to be alternative blood sources in disturbed environments [66], contributing to a possible groundward vertical migration of this traditionally canopy-loving phlebotomine species, as presumed to occur in a hydroelectric system-affected area of Jari River Basin [36].
Three infections found in Ev. infraspinosa were not isolated, but two were successfully characterized as L. (V.) guyanensis from the DNA content on the dissection slides. Considerable infections (++) and absence of blood observed with these peripylaric parasites suggest the necessity to continuously investigate this fly's possible involvement in the L. (V.) guyanensis enzootics. In addition, this species was frequent in Shannon captures (mainly in the black cloth, 0.97 females/h), with some specimens attempting to bite the professionals. It was impossible to determine whether Ev. infraspinosa could feed on potential L. (V.) guyanensis reservoirs. The present results indicate that this phlebotomine can feed on armadillos. The rodent Dasyprocta leporina is the only known blood source for this species [65] although anuran trypanosomatid isolated from this species from the western Brazilian Amazon suggests it feeds on coldblooded rather than warm-blooded vertebrates [67].
In addition to Ny. umbratilis and Ev. infraspinosa, flagellate infections have been found in Mi. migonei, Sc. fluviatilis, Sc. sordellii and Vi. furcata. Negative PCR-RFLP for the other infected specimens suggested low DNA for Leishmania-typing or that they were other trypanosomatids. The apparent high infection rates of some of these species may have been biased by the low number of dissected females and thus cannot yield conclusive findings.
Other phlebotomine species were found with flagellates. The trypanosomatid isolated from Sc. fluviatilis will be further characterized. Parasites found in the blood of Pa. dendrophyla should be cautiously interpreted; this species shares the same ecotope as Ny. umbratilis, and some specimens likely receive occasional parasite ingestions, as observed by Freitas et al. [21]. These considerations can be extended to Vi. furcata and Pi. damascenoi [13].
Although no Leishmania infections were found for the following two fly species, they should still be discussed as putative vectors in the lower Oyapock River Basin based on the current Guianan/Amazonian ACL epidemiological background: (i) Psychodopygus s. maripaensis has been included on the long list of possible vectors of L. (V.) naiffi based on infection findings in Régina, French Guiana [35] and Serra do Navio (AP) [8], extending its epidemiological relevance in other Brazilian/Guianan regions [36]. In addition, the Ps. s. maripaensis specimens tested for blood sources reacted positively to antiserum from an armadillo, the recognized potential reservoir of L. (V.) naiffi. Interestingly, DNA from L. (V.) braziliensis was detected in a pooled sample of Ps. s. maripaensis [referred to as P. squamiventris (s.l.)] in Sabajo Heuvels, Suriname, suggesting an additional putative vector role [68]. However, L. (V.) braziliensis transmission in Oyapock remains unclear. The most females found per hour in our Shannon captures (2.29; white colored cloth), with some attempting to bite the professionals, demonstrates the aggressive behavior of this species.
(ii) Bichromomyia flaviscutellata is the vector of L. (L.) amazonensis [7]. The presence of this sand fly in the surveyed sites, mainly site I, is noteworthy because of the pathological spectrum of its associated parasite [9] despite only the cutaneous form being documented in French Guiana [14,15]. The synanthropic behavior of the Guianan population of Bi. flaviscutellata, which appears to adapt to environments under ecological pressures and human-made modifications [36,69], has been documented. One Bi. flaviscutellata from a CDC canopy trap was positive for bird antiserum, raising the hypothesis that this species could migrate vertically to search for alternative blood sources. Domestic birds, such as chickens, may be attractive for this phlebotomine species, triggering it to adapt to modified environments. Our preliminary results from a peridomiciliary-forest stratification study have shown that some Bi. flaviscutellata specimens are captured outdoors, where animal shelters can stimulate phlebotomines to cross a 200 m gradient between the forest border and households (Vasconcelos dos Santos, unpublished data).
Most ACL isolates (6/8) were from patients infected while gold mining, showing that L. (V.) guyanensis ACL hotspots may be concentrated in these environments (upper Oyapock River Basin) [14,15,18]. Conversely, the present entomological results showed considerable infection rates of enzootics near urban cities, in which less economically attractive periurban forests (absence of gold mining) may reflect less human exposure to the disease (and consequently few ACL cases) in that area.

Conclusions
Our findings show that ACL transmission in the Oyapock River Basin reflects the Guianan/Amazonian classical ecosystem, where Ny. umbratilis remains the main vector. A putative alternative transmission by Ev. infraspinosa is possible, but circumstantial parasite ingestion is also likely, as seen with other biologically compatible phlebotomine species cohabiting the same potential L. (V.) guyanensis reservoir ecotopes. Conversely, epidemiological relevance of these putative alternative transmission cycles cannot be estimated with certainty. Literature-based evidence indicates that others fly species, such as Ps. s. maripaensis and Bi. flaviscutellata are also epidemiologically relevant, and we included them on the priority list for vector surveillance in the lower Oyapock basin. Local studies on ACL enzootics should be encouraged, since each an ecological mosaic is unique. The ACL etiology shows that the transmission pattern in the upper Oyapock may differ slightly from the lower basin, but only further surveys of the former environment can confirm this hypothesis.