Litomosoides brasiliensis (Nematoda: Onchocercidae) infecting chiropterans in the Legal Amazon region, Brazil

Abstract Chiropterans play an important role in the maintenance of the environmental balance, since they are pollinators, seed dispersers and predators. They contribute to transmission and spreading of microorganisms such as helminths, fungi, protozoa, bacteria and virus. The aim of the present study was to investigate natural filariid infection among bats in the Legal Amazon region, Brazil, by means of parasitological and molecular analyses. Blood samples were collected from 82 bats for blood smears and for DNA extraction via the polymerase chain reaction (PCR) assay. Microfilariae were observed in blood smears from Carollia perspicillata (2), Artibeus lituratus (1), Artibeus fimbriatus (2), Dermanura gnoma (2) and Glossophaga soricina (1). Five positive samples were detected through the PCR assay and four of these were also positive in blood smears. From genome sequencing and comparative analysis with sequences deposited in GenBank, one sample showed 99.31% similarity to the species Litomosoides brasiliensis. The present study expands the geographical distribution of L. brasiliensis, to include the state of Maranhão as an area of occurrence of this species and includes D. gnoma and A. fimbriatus as hosts in Brazil.


Introduction
Brazil has a diverse chiropteran fauna that includes 9 families, 68 genera and 179 species (Feijó et al., 2015;Nogueira et al., 2014;Paglia et al., 2012). These form an important component of the mammalian fauna of Neotropical ecosystems, with regard to their richness and species diversity (Kalko, 1998;Marinho-Filho & Gastal, 2001). Moreover, bats play an important role in maintaining the environmental balance, since they are pollinators, seed dispersers and predators (Bianconi et al., 2004;Kalko, 1998;Menezes et al., 2015;Pedrozo et al., 2016). They are also considered to be important reservoirs of emerging and re-emerging zoonotic pathogens. Therefore, continuous surveillance and monitoring of these animals is essential in order to prevent pandemics (Brook & Dobson, 2015;Kadam et al., 2021;León et al., 2021).
Bats' characteristics of high flight mobility, great diversity of shelters, interaction among species in the same environment and remarkable social behavior make them interesting subjects for scientific studies. They contribute to transmission and spreading of microorganisms such as helminths, fungi, protozoa, bacteria and virus (Cabral et al., 2013;Frick et al., 2016;Roque & Jansen, 2014;Savani et al., 2009;Zetun et al., 2009).
Studies on parasite biodiversity are important for species management and conservation, since parasitism plays an essential role in ecosystems, through regulating host density, stabilizing food chains and structuring communities of animals (Poulin & Morand, 2000). Because of the high relevance of parasite diversity to ecosystems, any report on parasite infection provides understanding about ecology and environmental impacts (Rendón-Franco et al., 2019).
In Brazil, 59 species of helminths have been reported as infecting bats: 28 species of nematodes, 23 of trematodes, 6 of cestodes and 2 of acanthocephalans (Cardia et al., 2015;Santos & Gibson, 2015). The state of Maranhão is located in the Legal Amazon region, with a strategic position at the confluence of the Cerrado, Caatinga and Brazilian Amazon biomes, and comprises a mosaic of landscapes that are rich in fauna and flora. In this Neotropical region, studies have been conducted to provide data on chiropteran species (Bernard et al., 2011;Cruz et al., 2007;Olímpio et al., 2016). However, despite the high species diversity, studies on internal parasites in these biomes are scarce.
The aim of the present study was to investigate filariid species among chiropterans in the Legal Amazon region, in order to contribute to knowledge about the biodiversity of filariids among bats in Brazil.

Study area
This study was carried out in the municipalities of Turiaçu (  Bats were collected between 18:00 and 00:00, over 5-day periods, with the aid of mist nets. They were then anesthetized with ketamine, at a dose according to their weight, photographed and identified. Specimen identification was performed based on specialized classification (Dos Reis et al., 2007;Miranda et al., 2011;Uieda, 2008). Approximately 2 mL of blood were collected from each specimen by means of cardiac puncture, for blood smears and molecular assays.

Parasitological detection of filariids
Blood smears were made in triplicate, fixed with methanol, stained with Giemsa and analyzed under an optical microscope at 40x and 100x magnification. The blood smears were scanned to search for microfilariae.

Molecular assay
Genomic DNA was extracted from blood, in accordance with the protocol established for use in the Genejet Genomic DNA purification kit (Thermo Scientific, UAB, Lithuania), with previous incubation of 0.5 g of tissue, 20 μL of proteinase enzyme K (20 mg/ml) and 700 μL of lysis solution. Fragments of the gene 12S rDNA were amplified by means of the polymerase chain reaction (PCR), using the primer pairs Fila12SF (5'-CGGGAGTAAAGTTTTGTTTAAACCG-3') and Fila12SR (5'-CATTGACGGATGGTTTGTACCAC-3'), as described by Otranto et al. (2011), which amplify fragments of 330 bp from preserved filariids.
The amplified products were subjected to horizontal electrophoresis at 50 V/100 mA in Tris-acetate-EDTA (TAE) buffer, on 1.5% agarose gel stained with SYBR Safe (Invitrogen). The bands were viewed and photographed using a UV light transilluminator. The amplified products were purified using the ExoSAP-IT commercial product (USB Corporation), consisting of Exonuclease I (Exo I) to digest excess primers and shrimp alkaline phosphatase (SAP) to degrade excess nucleotides from the PCR. After purification, sequencing was performed using the Big Dye Terminator kit (Perkin Elmer), in accordance with the manufacturer's specifications, in an ABI PRISM 3500 automated sequencer (Life Technologies). The sequences obtained were edited in the SeqMan software (Lasergene, DNAstar, Madison, Wisconsin, United States) and were subjected to similarity analysis using the Basic Local Alignment Search Tool (BLAST two-sequence analysis) to verify their homology with corresponding sequences available in GenBank (Altschul et al., 1990).

Microfilariae (
From the PCR assays, five positive samples were detected; four of these were also found to be positive through blood smears. From genome sequencing and comparative analysis with sequences deposited in GenBank, one was found to show 99.31% similarity to the species Litomosoides brasiliensis (accession number: MW471081.1).

Discussion
The family Phyllostomidae was the most representative (68%) among the bats sampled, followed by Molossidae (30.66%). The high frequency of catching specimens of Phyllostomidae was due to its diversity in Brazil: this family accounts for approximately 57% of all bat species in this country.
According to Anderson & Bain (2009), the family Onchocercidae is composed of seven subfamilies, in which five genera have already been identified in bats: Litomosoides, Migonella, Chiropterofilaria, Josefilaria and Litomosa . The genus Litomosoides, which includes at least 31 species , and the genus Migonella are found in bats in South America (Santos & Gibson, 2015).
In studies conducted in Brazil, parasitism by species of Litomosoides in bats of the genus Artibeus and Carollia has also been reported (Albuquerque et al., 2015;Cardia 2012;Costa et al., 2021;Mello, 2017;Vicente et al., 1997).  However, these studies consisted of reports on adult parasites and not microfilaria in blood samples. In the present study, the diagnosis was based on findings of microfilaria in blood smears and on PCR-positive samples and genome sequencing.
Presence of microfilaria in peripheral blood allows transmission of the parasite within the bat population, both though hematophagous arthropods and transplacentally (Gazarini et al., 2012;Williams, 1948). Transmission is facilitated through the bats' habits, since they live in groups and use caverns and trees as shelters (Melaun et al., 2014;Sekiama et al., 2013).
Although the vectors of bat filariids are not well known, certain species of Litomosoides are transmitted by hematophagous mites (Williams, 1948), such as those in the families Dermanyssidae (Anderson, 2000) and Macronyssidae Guerrero et al., 2002). The latter family is also considered to act as vectors for filariids in rodents and marsupials .
Litomosoides spp. have also been detected through PCR on blood samples from Artibeus jamaicensis and through PCR on genetic material from Trichobius intermedius (Diptera: Streblidae) and Periglischrus iheringi (Acari: Spinturnicidae). However, it was not possible to incriminate these as vectors (Reeves et al., 2016).
In the present study, microfilariae of Litomosoides sp. were found in blood smears from C. perspicillata, A. lituratus, A. fimbriatus, D. gnoma and G. soricine. Reports of Litomosoides sp. in association with the genera Artibeus and Carollia are not uncommon; in fact, among chiropterans, this nematode seems to prefer host of the genus Artibeus Jiménez et al., 2017). These parasites are limited to only a few host species and are order specific. Thus, species that occur in bats do not occur in rodents and marsupials, and vice versa (Brant & Gardner, 2000;Guerrero et al., 2002;Vogeler et al., 2018).
It is important to highlight that among the bat species that presented microfilariae, four were fruit bats and one was a nectar-feeding bat. Both of these types of bat are fundamental for the biosystem and ecosystem, since they fly long distances searching for fruits and they disperse seeds along the way and act as pollinators. Hence, they are essential for forest regeneration and maintenance of plant diversity and ecosystem equilibrium (Estrada & Coates-Estrada, 2001;Medellin & Gaona, 1999). However, when infected by Litomosoides, they can present signs of weakness, tachypnea and hemorrhage, which leads to decreased efficiency of seed dispersion and pollination (Rendón-Franco et al., 2019).
The molecular analysis on the blood samples showed there was 99.31% similarity to L. brasiliensis. This species has been reported parasitizing the families Phyllostomidae and Vespertilionidae, which are widely distributed in South and Central (Albuquerque et al., 2015;Brant & Gardner, 2000;Costa et al., 2021;Guerrero et al., 2002;Jiménez et al., 2021;Mourão et al., 2002;Vicente et al., 1997;Vogeler et al., 2018). In Brazil, presence of L. brasiliensis has been reported in the states of Amapá, Minas Gerais, Mato Grosso, Mato Grosso do Sul, Pará, Piauí, Paraná, Rio de Janeiro and São Paulo (Albuquerque et al., 2015;Costa et al., 2021;Mourão et al., 2002). Now, we register it in the state of Maranhão. This study provides the first report of L. brasiliensis parasitizing D. gnoma and A. fimbriatus in Brazil.
Phylogenetic analyses have been used to reconstruct the evolution of Litomosoides and infer its history. Two decades ago, Brant & Gardner (2000) suggested that this genus was not a monophyletic group, although they identified 22 morphological characteristics with high levels of homoplasy. However, a recent study on a database of DNA sequences demonstrated that Litomosoides showed up as a monophyletic group that originated in Neotropical phyllostomid bats, with strong evidence of at least two events of host changing: one of them involving cricetid rodents and the other, mormoopids. The latter event included simultaneous geographical expansion of the parasite's range (Jiménez et al., 2021). Thus, studies on molecular detection of Litomosoides are relevant, to provide data for reconstructing the phylogeny of supposed changes to hosts and monophyly.
The present study expands the geographical distribution of L. brasiliensis, to include the state of Maranhão as an area of occurrence of this specie. This study also includes D. gnoma and A. fimbriatus as hosts in Brazil.