Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae)

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi–Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis–parasite combinations, as well with Lu. longipalpis–uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.


Introduction
Phlebotomines (Diptera: Psychodidae) are medically important insects implicated in the transmission of several pathogens, mainly Leishmania protozoa (Kinetoplastea: Trypanosomatidae) [1], which are the agents of leishmaniases, a group of neglected tropical diseases affecting millions of people worldwide [2].
A fundamental aspect in determining whether phlebotomines are incriminated by the transmission of Leishmania is the differentiation of infectious metacyclic forms [14].In this same sense, the parasitosis and placement of late-stage infection constitute important parameters for evaluating the vector competence of a particular phlebotomine species for the developmental success of a given Leishmania sp.[15].
In the Brazilian Amazon, a particular tegumentary leishmaniasis (TL) transmission scenario occurs mainly because of the etiology of L. (Leishmania) amazonensis, L. (Viannia) lainsoni and L. (V.) lindenbergi in the forest fragments of Belém city [16].In these TL foci, with respect to L. (L.) amazonensis, Bichromomyia flaviscutellata has well-established vector evidence [17]; for L. (V.) lainsoni, species of Trichophoromyia, particularly Th.Ubiquitalis and Th.brachipyga, have been shown to be involved in transmission [18]; ultimately, for L. (V.) lindenbergi, Nyssomyia antunesi has received attention due to its abundance, dominance, spatiotemporal convergence with human disease, blood feeding on human and potential reservoirs of Leishmania [19,20].Despite the lack of evidence of true and species-specific identifiable Leishmania infection, the vector role of Ny. antunesi remains undefined, not advancing on suspect status.
However, studies on the interactions between Leishmania and its vectors are required to advance the understanding of the processes involved in parasite development and transmission [6].Some parasite-vector combinations have been studied under laboratory conditions; however, the majority of binomials inferred by field evidence still require laboratory investigation.Therefore, the present study aimed to fill the gap in vector knowledge on the development of L. (V.) lainsoni and L. (V.) lindenbergi in Ny. antunesi and Lu.longipalpis.A L. (L.) chagasi-Lu.longipalpis combination was used as a 'positive control'.

Phlebotomines
Wild-caught Ny. antunesi were obtained from the Bosque Rodrigues Alves-Jardim Botânico da Amazônia (1 • 25 ′ 48 ′′ S; 48 • 27 ′ 25 ′′ W), an urban park of Belém city in which the phlebotomine fauna has already been surveyed [21].Captures were performed with CDC light traps set 1.5 m above ground level (n = 4) and 20 m above ground level (n = 2), operating from 6:00 p.m. to 6:00 a.m., from May to August 2023.The phlebotomines were visually screened, aspirated from the primary cage in the field, and transported to the laboratory under 80 ± 10% relative humidity and 10% glucose solution offered ad libitum [22,23].The phlebotomines were immediately transferred to a secondary nylon cage.Congested, gravid or semigravid females were excluded from the experiments [24].

Parasite-Vector Systems
Experimental infections were carried out according to the artificial blood feeding protocol proposed by Sánchez Uzcátegui et al. [24].Briefly, the groups of both wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were artificially fed in 30 cm 3 nylon cages for 3 h through a sausage membrane installed in a circulator device containing previously heat-inactivated serum (56 • C for 1 h) human blood and 5 × 10 6 /mL promastigotes [5] from log-phase cultures [29].For Lu. longipalpis, females that had fed on uninfected blood were also assessed.The engorged females were confined to 200 mL flasks, and the recipients were lined with moistened filter paper and given a 10% sucrose diet until dissection [5].Females from each species/experiment were divided into two groups for dissection: one group was dissected before the females defecated (early stage of infection) on day 2 post-blood meal (pbm), and the other group was dissected after defecation (late stage of infection) on day 8 pbm [5,25].

Parasite Detection and Development
Phlebotomines were monitored daily to account for dead females, and survival curves were constructed.Dead females were dissected, and only Leishmania-positive females were counted [30].The proportion survived (lx) was calculated according to Rabinovich [31].Survivorship curves were obtained for different parasite-vector combinations and were compared by the log-rank test using BioEstat 5.3 software [32].On days 2 and 8 pbm, the females were removed from the oviposition glasses using a Castro aspirator, and placed at 4 • C for thermal immobilization by cooling.The females were washed once with a 0.9% NaCl solution plus 5% neutral detergent, and twice with 0.9% NaCl for subsequent dissection.Phlebotomines were placed in a drop of phosphate-buffered saline (PBS) on a microscope slide, and the head was separated from the thorax before the intestine was extracted through the apex of the abdomen.The intestines were individually observed under an optical microscope and examined to determine the development of flagellates in the guts [33] following the taxonomic statement of Lainson and Shaw [34], and classified with the semiquantitative parasitosis scale described by Myskova et al. [15], whereby parasite loads were graded as absent (0 parasites per gut), weak (less than 100 parasites per gut), moderate (100-1000 parasites per gut) or heavy (more than 1000 parasites per gut).Promastigotes from the gut were Giemsa-stained and microscopically assessed to infer distinguishable evolutive forms based on morphologic/morphometric criteria modified by Ticha et al. [5], focusing on identifying metacyclic-like forms on day 8 pbm.All experiments were repeated at least three times for each parasite-vector combination.Parasitoses were compared using the G test with BioEstat 5.3 software [32].In all statistical analysis, p ≤ 0.05 was considered to indicate a 95% confidence interval.

Ethical Approval
The capture and processing of invertebrate fauna (phlebotomines) were authorized by the 'Sistema de Autorização e Informação em Biodiversidade' under protocol no.70142-2.Animals used for the blood feeding of phlebotomine colonies were maintained and handled at the Instituto Evandro Chagas animal facility, in accordance with institutional guidelines and Brazilian legislation (Federal Law no.11.794, 8 October 2008).In vivo blood feeding standard operational procedures were approved by the Ethics Committee on Animal Use (CEUA/IEC), under certificate no.30/2021.
Regarding the placement of parasites in the gut, as promastigotes were limited to the endoperitrophic space on day 2 pbm, this parameter was only considered on day 8 pbm, when 83% of the Ny.antunesi-L.(V.) lainsoni combinations did not sustain infection; 8.3% presented peripylarian development with colonization in the hindgut (HG) and abdominal midgut (AMG); and 8.3% presented suprapylarian development, with colonization in the AMG, thoracic midgut (TMG) and cardia (CA) (Figure 2A).In Ny. antunesi-L.(V.) lindenbergi combinations, 63.6% of the strains did not sustain infection, and 36% presented suprapylarian development, 18.2% colonization in the AMG and 18.2% suprapylarian development with colonization in the AMG, TMG and CA (Figure 2B).Parasitoses were classified into four categories: weak (less than 100 parasites per gut), moderate (100-1000 parasites per gut) and heavy (more than 1000 parasites per gut).The number of females evaluated can be found above the columns.
Regarding the placement of parasites in the gut, as promastigotes were limited to the endoperitrophic space on day 2 pbm, this parameter was only considered on day 8 pbm, when 83% of the Ny.antunesi-L.(V.) lainsoni combinations did not sustain infection; 8.3% presented peripylarian development with colonization in the hindgut (HG) and abdominal midgut (AMG); and 8.3% presented suprapylarian development, with colonization in the AMG, thoracic midgut (TMG) and cardia (CA) (Figure 2A).In Ny. antunesi-L.(V.) lindenbergi combinations, 63.6% of the strains did not sustain infection, and 36% presented suprapylarian development, 18.2% colonization in the AMG and 18.2% suprapylarian development with colonization in the AMG, TMG and CA (Figure 2B).
On the other hand, the results of the evaluation of parasites among the Leishmania species were significant on day 8 pbm (Table 1).
On the other hand, the results of the evaluation of parasites among the Leishmania species were significant on day 8 pbm (Table 1).

Survival Curves
The survival of both phlebotomine species decreased up to day 8 pbm for all combinations (Figure 6).The survival of Lu. longipalpis was affected differently in some Lu.longipalpis combinations, and the survival was higher when the phlebotomine species were infected with L. (V.) lindenbergi than when they were infected with L. (V.) lainsoni (p < 0.0001) or L. (L.) chagasi (p < 0.0001).Moreover, the survival was higher when the phlebotomine species were infected with L. (V.) lainsoni compared with L. (L.) chagasi (p < 0.0001); and when they were infected with uninfected blood compared with those infected with L. (V.) lindenbergi (p = 0.0246), or L. (L.) chagasi (p < 0.0001) (Table 2).

Survival Curves
The survival of both phlebotomine species decreased up to day 8 pbm for all combinations (Figure 6).The survival of Lu. longipalpis was affected differently in some Lu.longipalpis combinations, and the survival was higher when the phlebotomine species were infected with L. (V.) lindenbergi than when they were infected with L. (V.) lainsoni (p < 0.0001) or L. (L.) chagasi (p < 0.0001).Moreover, the survival was higher when the phlebotomine species were infected with L. (V.) lainsoni compared with L. (L.) chagasi (p < 0.0001); and when they were infected with uninfected blood compared with those infected with L. (V.) lindenbergi (p = 0.0246), or L. (L.) chagasi (p < 0.0001) (Table 2).

Discussion
Several parasite-vector combinations were studied under laboratory conditions to evaluate interaction patterns, including the ability of vectors to support the late-stage development of parasites, suggesting the well-recognized classification of restrictive and permissive vectors.In the former category, phlebotomines present a remarkable specificity for a single (or some closely related) Leishmania species; while in the latter, phlebotomines allow for the development of a broad range of apart-related Leishmania species [35,36].In this sense, the present study assessed Ny. antunesi and Lu.longipalpis in the development of medically important parasites in the Amazon biome, L. (V.) lainsoni and L. (V.) lindenbergi, until day 8 pbm, when late-stage promastigote forms were supposed to colonize the foregut, thus providing advanced inferences on their susceptibility.
After exhaustive attempts, the unsuccessful colonization of Ny. antunesi led the researchers to challenge wild-caught specimens.Although the unknown life status of these specimens and an apparently low number of assessments may compromise experimental reproducibility, it is believed that the field background brought about by nature adds pivotal elements for genuine parasite-vector interactions.Nyssomyia antunesi has been recognized as a suspected vector of L. (V.) lindenbergi based on some eco-epidemiological evidence [19][20][21], although no natural infection has been ascribed to this parasite.The present findings demonstrate that Ny. antunesi can develop at least a small population of late-stage promastigotes of both L. (V.) lainsoni and L. (V.) lindenbergi (a taxonomically distinct species) with no difference in parasitosis on day 8 pbm, suggesting that this species could be further investigated as a possible permissive vector, although it has never been found to be naturally infected by L. (V.) lainsoni in wild-caught specimens that have been examined.Interestingly, the present findings support early microscopic and current molecular-based evidence, which suggests that Ny. antunesi can harbor Trypanosoma sp.[37,38], Porcisia sp.[39], and a wide range of Leishmania spp.[40][41][42][43][44][45][46][47][48][49].Most of these detections do not provide evidence of late-stage promastigote forms, which are insufficient to characterize Ny. antunesi as a true vector.Other supporting information for the present results is related to O-glycosylated proteins with N-acetylgalactosamine (GalNAc) epitopes, which are likely reported exclusively for permissive species [35], as has been suggested to be present in the midgut epithelial cells of Ny. antunesi [50].
On day 8 pbm, L. (V.) lainsoni and L. (V.) lindenbergi were observed up to the cardia of Ny. antunesi, probably because these Leishmania species need more time to advance to the stomodeal valve under laboratory conditions, as has been suggested for the binomial Phlebotomus arabicus-L.(L.) infantum [15].On several occasions, it was noted that Ny. antunesi only partially fed under experimental conditions (Sánchez-Uzcátegui, personal observation), which could possibly be important information from an epidemiological point of view.In this sense, multiple bloodmeals during a single gonadotrophic cycle have been reported for Lu.longipalpis [51], which has a potential impact on survival and Leishmania transmission, as suggested by Killick-Kendrick [52], for Ph.papatasi and L. major.Moreover, this characteristic would improve vector competence since the development of a successful infection in wild phlebotomines is a gradual process that depends on the parasite's action, which is amplified and enhanced by the ingestion of multiple blood meals [53].Thus, all these facts add weight to the hypothesis that Ny. antunesi is an important vector from a medical point of view, but other criteria still need to be evaluated.
When evaluating the experimental infection of Lu. longipalpis with L. (V.) lainsoni and L. (V.) lindenbergi, it has been demonstrated that both parasite species can develop up to day 8 pbm with no difference in parasitosis between these combinations.Preliminary experimental infections have already been performed and the descriptions of these experiments with the two Leishmania species have focused on determining the developmental pattern for taxonomic purpose [19,38], thus not extending to the observation of late-stage promastigote forms.Lutzomyia longipalpis is well known as the major natural vector of L. (L.) chagasi [54], which is laboratory-supported as a permissive vector and competent for experimentally transmitting L. (V.) braziliensis [55], L. (L.) chagasi [56], L. (L.) mexicana [57], L. (L.) major [58,59] and L. (L.) amazonensis [60].
As expected, the predominant gut development reported for the studied Leishmania species (i.e., peripylarian for L. (V.) lainsoni and L. (V.) lindenbergi, and suprapylarian for L. (L.) chagasi) is in agreement with the taxonomic positions of these species originally described by Lainson and Shaw [34].Few specimens with hindgut development were recorded for all combinations, which was exclusively attributed to heavy parasite loads throughout the phlebotomine gut.On that gut site, only adhered promastigotes were considered, avoiding artifactual observation due to back-wash [70].In contrast, in the midgut, free-living promastigotes were considered.Killick-Kendrick [71] has shown striking features separating metacyclic forms from others in the phlebotomine gut, including a lack of attachment to the epithelium, high motility, a small body size and the presence of a long free flagellum.
The survival curves significantly decreased for all combinations, but were differentially affected in Lu. longipalpis-parasite combinations and uninfected blood.The reduced longevity of experimentally Leishmania-infected phlebotomines was documented [30,72,73], without evidence of a strain-specific impact [30].Moreover, the results reported herein support the classical hypothesis that successful transmission in nature also depends on equilibrate parasitosis, which is sufficient for the inoculum but within the limits of vector tolerance, preserving its longevity [74].

Conclusions
In summary, wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis have been suggested to be experimentally susceptible to L. (V.) lainsoni and L. (V.) lindenbergi, which results in the development of at least a small population of late-stage parasites up to day 8 pbm in the cardia or stomodeal valve.The putative permissiveness of Ny. antunesi has not been discarded, however, still requiring further assessment.The unsuccessful establishment of life cycles with these parasite-vector combinations in nature may result from non-negligible ecological field-driven elements.Indeed, the putative susceptibility of phlebotomines suggested herein is worthy of important epidemiological consequences because it enables a successful adaptation of Leishmania.A successful colonization of Ny. antunesi could provide a considerable number of specimens that allow for an in vivo and in vitro assessment of Leishmania-phlebotomine interactions, and thus definitively determine its permissiveness and vector competence status.Data Availability Statement: All data supporting the conclusions are included within the article.The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Figure 6 .
Figure 6.Survival curves of Nyssomyia antunesi and Lutzomyia longipalpis blood-fed on a Leishmania spp.suspension (or not) up to day 8 post-blood meal.

Table 2 .Figure 6 .
Figure 6.Survival curves of Nyssomyia antunesi and Lutzomyia longipalpis blood-fed on a Leishmania spp.suspension (or not) up to day 8 post-blood meal.

Table 2 .
Log-rank test significance on the comparison of survival curves obtained for the different parasite-vector combinations up to day 8 post-blood meal.Significant differences are highlighted in bold.