Hectopsylla pulex (Haller, 1880) (Siphonaptera: Tungidae) infestation on Eptesicus furinalis (Chiroptera: Vespertilionidae) in the Central Andes of Colombia

Bat ectoparasites have a complex natural history narrowly tied to their hosts at ecological, behavioral, and evolutionary scales. As flying and social organisms, bats represent a potential mechanism of dispersal, a source of feeding, and a roost for ectoparasite reproduction. The chiggerflea Hectopsylla pulex (Siphonaptera: Tungidae) is widely distributed across the Neotropics. Females of this ectoparasite have been found in their neosomal form on bats of the family Molossidae, Noctilionidae, Phyllostomidae, and Vespertilionidae. Here we present the record of infestation of chiggerfleas on the Argentine bat, Eptesicus furinalis (Vespertilionidae) in Colombia, representing the first record of the flea on this species, and providing novel genetic information of this poorly known flea species.


INTRODUCTION
The coexistence within species is mainly determined by abiotic and biotic filters that act as stabilizing agents of fitness and niche differences (HilleRisLambers et al., 2012). In the world of an ectoparasite, a host represents a habitat filter with potential resources, complex movement patterns, varied social systems, roosting behaviors, and immunologic responses (Balashov, 2006;Dick & Ditmar, 2014). As a tradeoff, an ectoparasite requires a set of morphological, behavioral, and physiological adaptations in response to their host's traits, which drives coevolution (Balashov, 2006;Patterson et al., 2007).
Several host features could induce ectoparasite infections: first, hosts with high mobility enhance parasite colonization of new habitats (Presley & Willig, 2008). Second, selection for a host roosting site with suitable environmental conditions for development could induce an ectoparasite interaction (Patterson et al., 2007). Third, when social hosts are spatially aggregated, parasite infections are likely to increase between individuals of the same and different sex, and between species (ter Hofstede & Fenton, 2005;van Schaik et al., 2014). And, finally, stressed or immunologically suppressed hosts are more likely to be infected with ectoparasites than immunologically active or healthier hosts (Dick & Ditmar, 2014). Conversely, to suppress the predatory pressure of an ectoparasite, the host tends to decrease its resting time and invest metabolic energy into auto-or allogrooming behaviors and immunologic responses (Balashov, 2006;ter Hofstede & Fenton, 2005).
Approximately 48 species of vespertilionid bats live in South America (Díaz et al., 2016), of which few species have been recorded carrying chiggerfleas (Hastriter et al., 2014). The family Vespertilionidae comprises a cos-mopolitan and highly diverse lineage of bat species with relatively stable roosting habits, cohesive social organizations, and varied feeding behaviors (Lack & Van Den Bussche, 2010). Vespertilionids feed mainly on animal items, preying on insect populations or other invertebrate or vertebrate taxa (Kasso & Balakrishnan, 2013). A highly developed sense of echolocation allows them to hunt insects while releasing a diverse range of wide harmonic signals during flight (Jones & Teeling, 2006). Several vespertilionids are highly tolerant of anthropogenic landscape disturbance levels, which enables them to select for a wide range of permanent or temporary roosts (Jung & Threlfall, 2016). Roosting sites vary from natural cavities in rocks or trees to human domestic, commercial, and industrial structures (Kunz, 1982).
The lack of studies on living vespertilionid bats with temporal flea infections makes it difficult to infer host-parasite interaction patterns within the family (Marinkelle & Grose, 1981). Hence, documenting chiggerfleas-bat interactions is important to obtain a clearer idea of the relationships between both host and parasite. Here, we report the first record of H. pulex infestation on a bat of the genus Eptesicus in Colombia and update to the distribution of this ectoparasite on the family Vespertilionidae in South America.

MATERIAL AND METHODS
We report an individual of the Argentine brown bat, Eptesicus furinalis D'Orbigny & Gervais, 1847, infested with Hectopsylla pulex on a village located at 1,590 m in the western slopes of the Central Cordillera, Northern Andes of Colombia. The village is in a peri-urban area on the northeastern portion of the Dosquebradas municipality (04°51′34.96″N, 75°39′52.53″W), Department of Risaralda (Fig. 1). The landscape is a mosaic of human On 19 th June 2020, at 18:20 h, an adult, non-lactating female of E. furinalis was found resting while attached to the wall of a house. The bat specimen was captured by hand, sexed, and identified following taxonomic keys and discrete characters including external and cranial sizes (Gardner, 2008;Díaz et al., 2016). We identified the bat as Argentine brown bat, E. furinalis based on the combination of intermediate size (forearm length, 41.98 mm), short dorsal hair (5.5 mm), condylo-incisive length (14.92 mm), and length of the maxillary toothrow (5.88 mm). We collected the specimen and deposited the skin, skull, and ectoparasites in the Museo de Historia Natural, Universidad de Caldas (MHN-UCa), under the catalog number MHN-UCa-M 3299. Hectopsylla pulex individuals were distinguished based on the narrowly pointed palpus-bearing lobe of the maxilla; a rounded anterior margin of the head; an S-shaped spermathecal with a conical projection of this orifice; short and inclusive terga and sterna 2-7, lateral portions without reaching contact each other in females; a highly expanded abdomen coupled with an autoseverence of appendages (Fig. 2).
The individuals of H. pulex were counted and collected from the bat specimen by examining the fur using fine point forceps and photographed with a Nikon Coolpix B500 semiautomatic camera with a millimeter scale to estimate total body length for each flea specimen. Fleas were preserved and stored in 70% alcohol, at room temperature. We externally identified the chiggerfleas as H. pulex individuals using the taxonomic keys of Hastriter & Méndez (2000).
After the morphological identification, two H. pulex specimens were individually processed for molecular analyses. Genomic DNA was extracted with the Wizard® Genomic DNA Purification kit (Promega Corporation) following the standard protocol indicated by the manufacturer. DNA quality and quantity were measured using a NanoVueTM Plus spectrophotometer. PCR amplification was performed using primers flanking two target mtDNA genes. The 12S rRNA gene fragment (≈340 pb) was amplified using the primer pair T1B 5′-AAACTAGGATTAGATACCCT-3′ and T2A 5′-AATGAGAGCGACGGGCGATGT-3′ reported by Beati & Keirans (2001). Primers LCO1490 (F) 5′-GGTCAACAAATCATAAAGATATTGG-3′ y HCO2198 (R) 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ were used to amplify a fragment of the cytochrome oxidase I (COI; ≈700 pb) gene (Folmer et al., 1994). The amplifications were performed on a Techne TCPLUS thermocycler, according to the following conditions: initial denaturation at 94℃ for 2 min, followed by 7 cycles at 94℃ for 30 s, 45℃ for 30 s, and 72℃ for 45 s, followed by 28 cycles at 94℃ for 30 s, 48℃ for 30 s, and 72℃ for 45 s, completing the reaction with a final extension cycle at 72℃ for 7 min, for the 12S rRNA gene. Initial denaturation at 95℃ for 5 min, followed by 5 cycles at 94℃ for 5 min, 46℃ for 1 min 30 s, and 72℃ for 1 min 30 s, followed by 35 cycles at 94℃ for 1 min, 53℃ for 1 min, and 72℃ for 1 min, completing the reaction with a final extension cycle at 72℃ for 5 min, for the COI gene. All PCR products were visualized by horizontal electrophoresis on 1% agarose gels with 1X TBE pH 8.0 running buffer at 110 v/50 mA. Gels were stained with ethidium bromide and visualized on a GelDocIt® 2 310 Imager (UVP). PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega®), estimated the amount of With the obtained sequences for both genes, we performed independent and concatenated phylogenetic analyses together with sequences of related taxa available in GenBank database (Sayers et al., 2020; NIH genetic sequence database of the National Center for Biotechnology Information) ( Table 1). The final dataset was represented by a total of 408 positions for the 12S and 789 for the COI genes. The analysis involved seven nucleotide sequences for 12S and eight for COI. The sequences were aligned using ClustalW (Thompson et al., 1997), and the best substitution model for each gene was selected using MEGA X (Kumar et al., 2018). The best evolutionary models were T92 (Tamura 3-parameter; Tamura, 1992) for 12S rRNA, and GTR + I for COI. Based on both the alignment (single and concatenated sets of mitochondrial genes) and substitution model, intraspecific and interspecific genetic distances were calculated. The phylogenetic tree was constructed using Maximum Likelihood (ML), 1000 bootstrap replications, using MEGA X (Nei & Kumar, 2000). The rate variation model allowed for some sites to be evolutionarily invariable (+ I, 40.24% sites) for COI. For the concatenated analysis, we selected the best-fitting models of sequence evolution (GTR + I), using the Akaike Information Criterion (AIC) calculated with ModelFinder in PhyloSuite (Zhang et al., 2020). The Maximum Likelihood (ML) analysis was conducted with IQ-TREE (Nguyen et al., 2015), 5,000 ultrafast bootstraps (Minh et al., 2013); as well as Shimodaira-Hasegawa-like approximate likelihood-ratio test (SH-like aLRT) for branches with 1,000 replicates (Guindon et al., 2010), all included in PhyloSuite platform (Zhang et al., 2020). Finally, we used the graphical viewer of phylogenetic trees FigTree v.1.4.3 (Rambaut, 2007). The species Dorcadia ioffi was used as outgroup, since it was a close genus for which we found COI and 12S sequences in GenBank.

RESULTS
The Argentine brown bat, E. furinalis was infested with seven females of H. pulex, five of them distributed on the dorsal surface at the base of the right ear and the remaining two on the dorsal surface at the base of the left ear (Fig. 2).
The Maximum Likelihood tree with the highest log likelihood (-844.31) was obtained for the 12S gene (Fig. 3), and log likelihood (-1739.63) obtained for the COI gene (Fig. 4). The species H. pulex was recovered as monophyletic, composed of species that parasitized the two hosts, E. furinalis and M. molossus from Colombia ( Fig. 3  and Fig. 4). The ML tree with the highest log likelihood (-2677.397) was obtained for the concatenated analyses, with proportion of invariable sites of 0.445 and base frequencies of A: 0.327 C: 0.128 G: 0.129 T: 0.417 (Fig. 5).
Based on the intraspecific genetic distance, H. pulex showed average distances between 0,0% for the 12S, 0,0%-1,6% for the COI fragments, and distances between 0.0%-1.0% for the concatenated analyses; while genetic divergence values between H. pulex and H. cypha varied between 4%, 16.1-17,4% and 10,9-17,4% for 12S, COI and the two genes concatenated, respectively. The genetic divergence values between H. pulex and D. ioffi were 56-61%, 17.8-18.91% and 16.1-30% for 12S, COI and the two genes concatenated, respectively.  Xiang et al. (2017) Regarding the H. pulex occurrence in Colombia, we found seven previously confirmed localities (Table 2) for its occurrence associated with six bat species and three bat families (Noctilionidae, Molossidae, and Phyllostomidae). Our record is the first documented for the family Vespertilionidae in Colombia. We also gathered five confirmed localities and records of infestation of H. pulex in bat species of the family Vespertilionidae in South America, in the countries of Argentina, Brazil, Peru, and Venezuela (Fig. 1).

DISCUSSION
Our record represents the first confirmed case of an Argentine bat infested with H. pulex, and for a member of the family Vespertilionidae in Colombia (Table 2), confirmed by both morphology and 12S rRNA, COI gene fragments and the concatenation of two genes (Fig. 3-Fig. 5). Previous records of the presence of H. pulex in Colombia were obtained mainly in bats of the family Molossidae (Tamsitt, 1970;Ramírez-Chaves et al., 2020), and in one species of Noctilionidae, and two of Phyllostomidae (Table 2). Similar situation occurs in the Neotropics where H. pulex has been recorded predominantly on bats of the families Molossidae and, to a lesser extent, in Noctilionidae, Phyllostomidae, and Vespertilionidae (Méndez, 1977;Hastriter et al., 2014). There are records of chiggerflea on vespertiliond bats from South America for only three genera: Eptesicus, Histiotus, and Myotis (Hastriter & Méndez, 2000). In Argentina, records of H. pulex on Myotis sp. has been reported from Buenos Aires (Tipton & Machado-Allison, 1972;Autino et al., 1999;Autino & Claps, 2000). In Brazil, the chiggerflea has been reported from one specimen of Eptesicus sp. from São Paulo state (Linardi, 2011), and one of Histiotus velatus from Pará state (Tipton & Machado-Allison, 1972;Hastriter & Méndez, 2000). In Peru, H. pulex was recorded from Histiotus sp. found in Huaura (Macchiavello, 1948). Finally, in Venezuela, specimens of E. fuscus and M. nigricans from Caracas (Hastriter & Méndez, 2000) has been reported carrying H. pulex females (Tipton & Machado-Allison, 1972).   (Table 2). In contrast, vespertilionid bats are well represented in other type of ectoparasite reports for South America, where approximately 77% of species have been found infested predominantly by ectoparasites of the orders Diptera and Siphonaptera (Frank et al., 2014). Within the siphonapterans, the families Ischnopsyllidae and Tungidae have parasitic interactions with several species in the genera Myotis, Histiotus, Lasiurus, and Eptesicus (Linardi, 2011;Hastriter et al., 2014;Autino et al., 2016).
Our observation is congruent with the female-bias host selection found for other species of Siphonaptera (Presley, 2012;Ramírez-Chaves et al., 2020) but, since we have a limited sample size, we cannot make further generalizations. Furthermore, the prevalence and intensity in this host-parasite system has been only eval-uated for two species of Molossus (Molossidae) in Brazil and Colombia (Esbérard, 2001;Luz et al., 2009;Ramírez-Chaves et al., 2020) limiting additional comparisons with other vespertilionids. Current evidence suggests that H. pulex infestations are localized on the bat ears and tragus (Esbérard, 2001;Luz et al., 2009;Hastriter et al., 2014;Ramírez-Chaves et al., 2020). These flea attachment areas could interfere with echolocation, and thus compromise the fitness of infected individuals (Hastriter et al., 2014). Previous studies on H. pulex suggest it is a vector of tungiasis, enhancing gram-negative bacterial infections on the host's damaged skin (Linardi & de Avelar, 2014), and this topic is also in need of additional research.
Finally, our results have implications for the historical association of H. pulex with bats of different families. Previous phylogenetic analyses for Siphonaptera using genetic data (Zhu et al., 2015) did not include the host Figure 5. Phylogenetic tree inferred from the concatenation of 12S and COI genes. The tree was inferred through Maximum Likelihood (ML) method with the GTR + I evolution model. The sequences obtained in this study (bold) and the GenBank accessions numbers are provided within brackets (12S; COI respectively). Numbers at nodes are above-selected branch support analysis from left to right: ultrafast bootstraps values, and Shimodaira-Hasegawa-like approximate likelihood-ratio test (SH-like aLRT). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.  (Ramírez-Chaves et al., 2020). Considering that Hectopsylla are strictly bat ectoparasites that do not exhibit the morphological features of other arthropod bat parasites (Whiting et al., 2018), the genetic distances observed between samples of H. pulex from different bat hosts are key to understand its generalist host selection behavior. However, additional H. pulex samples from different bat host are needed to draw definitive conclusions. An additional future challenge for the study of bats/H. pulex interactions is the drivers, times, and places where the interactions took place.

ACKNOWLEDGMENTS
We thank Valentina Cepeda and Daniel López from Primera Toma for their technical assistance in photography. Special thanks to Jim Patton and Julio Chacón-Pacheco and two anonymous reviewers for their helpful comments that improved the manuscript. We also thank the Vicerrectoría de Investigaciones y Posgrados of the Universidad de Caldas, "Convocatoria de Apoyo con Recursos Económicos a Grupos de Investigación de la Universidad de Caldas Año 2020", and for financing the seedbed project "Barreras de control peri-urbanas: el papel de los mamíferos silvestres en la circulación de hemoparásitos en la red Ecoparques de Manizales, Caldas" (2019). PAOL thanks Minciencias "Convocatoria del Fondo de Ciencia, Tecnología e Innovación del Sistema General de Regalías para la conformación de una lista de proyectos elegibles para ser viabilizados, priorizados y aprobados por el OCAD dentro del Programa de Becas de Excelencia Doctoral Bicentenario -2019".

AUTHORS' CONTRIBUTIONS
J.C.C.D. field data collection, specimen preparation, and wrote the paper. L.F.R.C. allocated H. pulex records in vespertilionids from South America. H.E.R.C., A.C.G., P.A.O.L., F.A.R.P. prepared maps, obtained genetic data, confirmed the bat and flea identification, analyzed the data, and wrote the paper.