Streblid flies parasitizing cave bats in Carajás, Amazonia, with a new record for Brazil

ABSTRACT Streblid flies (Diptera: Streblidae) are hematophagous and highly specialized parasitic insects, found only on bats (Chiroptera). Caves are important roosts for bats but, despite harboring high bat richness, relatively few studies exist on the ecology and biology of streblid flies in cave environments, especially in Amazonia, the largest domain in northern Brazil, with >140 bat species and thousands of caves. To fill some of the gaps in the geographical distribution and interspecific relationships for streblid flies in the region, we sampled bats in six caves in Carajás National Forest, a protected area in Pará state (Brazil). Thirteen of the 14 streblid species found are new records for Carajás, with three new records for Pará state and two for the northern region of Brazil. Nycterophilia fairchildi was recorded for the first time in Brazil. Most streblids had host-specific behavior, however, N. fairchildi, Trichobius caecus, and T. johnsonae were less host-specific, parasitizing different Pteronotus bat species. The gregarious behavior of Pteronotus species and the spatial distribution of their colonies within the caves may be important factors in the flies’ exchange among congener hosts and deserve special attention in future studies. Furthermore, studies on ecological interaction networks between bats and their ectoparasitic flies in caves will be useful for a broader understanding of how this relationship is structured over time and space, as well as its impact on both bats and flies.


INTRODUCTION
Although most insects are free-living, many have evolved a parasitic lifestyle and have numerous adaptations to live in or on their hosts (Poulin 2007).This is the case of streblids (Diptera: Streblidae), a morphologically diverse, highly specialized group of flies that exclusively parasitize bats (Wenzel et al. 1966).These flies spend their entire adult stage on the fur or wing membranes of bats, where both males and females feed on the host's blood (Wenzel et al. 1966).Many streblid flies are strikingly host-specific, being restricted to a single bat species (Barbier et al. 2019(Barbier et al. , 2021)).On the other hand, some species may show less host specificity, associating with different species of the same genus or even different genera of the same bat family (Dick and Dittmar 2014;Barbier and Bernard 2017).Like its host bats, streblid flies are worldwide in distribution, but much more diverse in the tropics (Dick and Patterson 2006;Dick and Dittmar 2014).
The Brazilian Amazon is, by far, the least studied domain of the distribution range of bat-streblid relationships (Lourenço et al. 2016;Barbier and Bernard 2017).Amazonia is one of the regions with the greatest bat species richness in the world (López-Baucells et al. 2016) and holds thousands of caves, which are important roosts for more than 70 bat species in Brazil (Oliveira et al. 2018).There is little research on bats and their parasites in northern Brazil, and even less in cave environments (Barbier and Benard 2017;Urbieta et al. 2022).However, recently there have been significant advances in the study of bat ectoparasitic flies in the region (Hrycyna et al. 2019;Palheta et al. 2020), revealing a rich diversity of streblid fauna and their potential host bat species.Here we fill a gap in the knowledge about the geographical distribution and host-parasite relationship of streblid flies on bats in caves in the Carajás National Forest, a protected area in eastern Brazilian Amazonia.

MATERIAL AND METHODS
We collected streblid flies on bats in June and July 2022 during bat monitoring surveys in six caves in the Carajás National Forest (eastern Amazonia), Pará state, Brazil (Table 1; Figure 1).In the region, the caves are located in the upper compartment of rocky ridges that stand out, supported by banded iron formations and canga (iron breccia) vegetation (Piló et al. 2015).In addition to the canga open vegetation, which is restricted to the iron ore rocky outcrops, the predominant vegetation is ombrophilous forest (Piló et al. 2015).The sampled caves are located at the foot of scarps in a ridge to the north (n = 5) and a ridge to the south (n = 1).The minimum and maximum distances between the caves are of approximately 110 m and 45 km, respectively (Figure 1).Fieldwork and sampling collections were authorized by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) through authorization SISBIO  Host bats were captured with hand nets and visually inspected for ectoparasites.Streblid flies were collected with featherweight forceps and preserved in labeled vials with 70% alcohol.Flies were identified at the species level under a stereomicroscope using diagnosis and identification keys proposed by Wenzel et al. (1966), Wenzel (1976), and Guerrero (1998).Voucher specimens were deposited in the entomological collection at Universidade Federal de Pernambuco (CE-UFPE) (Table 2).Bats were identified to the lowest possible taxonomic level following López-Baucells et al. (2016), Pavan (2019), andDíaz et al. (2021).Due to the similarity of external morphological features between Pteronotus alitonus and P. rubiginosus (Pavan 2019), we were not able to differentiate these two species in the field.In these cases, we kept the identification at the genus level (i.e.Pteronotus sp.).One bat of each species was collected, fixed, preserved in ethanol, and deposited as a voucher in the mammal collection at Universidade Federal de Pernambuco.
Based on a weighted matrix (i.e.frequency of observed interactions) containing the streblid fly species as columns and their host bats as rows, we used the 'visweb' function from the 'bipartite' package in R (Dormann et al. 2008;R Core Team 2021) to build a graph illustrating the observed hostparasite interactions.Photographs of fly species were taken at the Laboratory of Advanced Microscopy and Imagery at Universidade Federal de Pernambuco (LAMI-UFPE) using a stereomicroscope (Zeiss SteREO Discovery.V20).

RESULTS
We collected 107 streblid flies (62 males and 45 females) belonging to 14 species, parasitizing 28 bats belonging to nine species and four families (Table 2).Except for Strebla proxima, the other 13 streblid species are the first records for the Carajás National Forest.Furthermore, Trichobius dugesii is a new record for Pará state, T. anducei and T. pallidus are new records for the northern region of Brazil, while Nycterophilia fairchildi is a new record for Brazil.Of the 14 streblid species, 11 (79%) occurred on a single bat species (Figure 2).Trichobius caecus and N. fairchildi were the most generalist streblid species, found parasitizing Pteronotus gymnonotus, P. personatus, and Pteronotus sp.Nycterophilia fairchildi was more abundant on Pteronotus sp., while T. caecus was more abundant on P. gymnonotus (Table 2; Figure 2).
Throughout its known geographic distribution, this fly species seems to occur primarily in association with bats of the genus Pteronotus.Nycterophilia fairchildi resembles N. parnelli, but is easily distinguishable by the density of setae present on the thorax (sparser in N. parnelli) and by the females having four macrosetae at the distal end of the abdomen (instead of two in N. parnelli) (see Wenzel et al. 1966) (Figure 3).Remarks: This fly species is relatively common, having C. perspicillata as its primary host.It occurs throughout the entire distribution range of this bat, from southern Mexico to southern Brazil (Wenzel et al. 1966;Barquez et al. 2015;Barbier and Bernard 2017).Strebla guajiro has been recorded for Brazilian Amazonia, including Pará and neighboring states (Santos et al. 2009;Hrycyna et al. 2019;Palheta et al. 2020).
Strebla proxima Wenzel, 1976 (Figure 4b) Remarks: Second record for Brazil.In 2018, this fly species was recorded for the first time in the country, also in the Carajás National Forest and in association with P. kappleri (Barbier et al. 2018).The only other known records for this fly species are from Venezuela (Wenzel 1976).Remarks: This fly species is a specific parasite for the common vampire bat, D. rotundus, and frequently co-occurs with Trichobius parasiticus (Wenzel et al. 1966;Wenzel 1976), however, in some areas as the Caatinga dry forest region in Brazil, it seems to be less abundant than the latter (Barbier et al. 2019(Barbier et al. , 2021)).
The distance of approximately 1,700 km between the two most far apart records indicates a wide distribution range for T. anducei in the country, which agrees with the fact that its host, C. perspicillata, has one of the widest distributions in the Neotropics (Barquez et al. 2015).

Remarks:
Although there is a wide list of bats as transient/ non-primary hosts for T. caecus, this fly species is considered a primary parasite of Mormoopidae, especially of the genus Pteronotus (Guerrero 1994).The species has been collected previously in northern Brazil, also in Pará state, on an unidentified Pteronotus species by Palheta et al. (2020).

Remarks:
Trichobius dugesii is a typical parasite of the widely distributed G. soricina (Guerrero 1995).The species was already known from the border of the Amazon biome in the northeastern Brazilian state of Maranhão (e.g.Santos et al. 2009), and further north from the Brazilian Amazonian state of Amapá (Hrycyna et al. 2019).Our study provides the first record of the species in the state of Pará.
Remarks: Although T. johnsonae has a wide distribution, parasitizing Pteronotus species in Mexico, Panama, Colombia, and Venezuela, in Brazil it was recorded for the first time only in 2015 (Guerrero 1994;Guerrero and Morales-Malacara 1996;Figueiredo et al. 2015).The scarcity of records in Brazil is likely related to the few records of their hosts in traditional bat surveys, since most Pteronotus bats are captured inside or when emerging from caves.Palheta et al. (2020) recorded this fly species in Pará state, but, interestingly, on Carollia perspicillata and Lophostoma carrikeri (Allen, 1910), which are likely non-primary hosts.
Remarks: A specific parasite of F. horrens, T. pallidus can be considered a rare species (Graciolli and Azevedo 2011).
Here, it is recorded for the first time in the northern region of Brazil, and, consequently, for Pará state.Previous records in the country included the northeast and southeast regions (Graciolli and Azevedo 2011;Graciolli and Carvalho 2012;Barbier et al. 2016).Furipterus horrens is a nationally threatened bat species in Brazil (MMA 2022).

ACTA AMAZONICA
Remarks: Like Strebla wiedemanni, T. parasiticus is a specific parasite of the common vampire bat, D. rotundus (Wenzel 1976), and, like its host, it has a wide distribution in South America.The occurrence of T. parasiticus on host species other than D. rotundus is considered a non-primary association (Barbier et al. 2021).
Remarks: Although T. uniformis is known to occur in Brazil since the 1930s (Guimarães 1937), its presence in Pará state was only recently confirmed (Palheta et al. 2020).Trichobius uniformis, along with T. dugesii, are the streblid species most often found parasitizing G. soricina (Barbier and Bernard 2017).

DISCUSSION
We recorded 14 streblid fly species, and provided three new species records for the state of Pará and two for the northern region of Brazil, in addition to the first record of Nycterophilia fairchildi for Brazil.Also, except for a single previous record of Strebla proxima parasitizing Peropteryx kappleri (Barbier et al. 2018), there was no information on bat ectoparasitic flies in the Carajás National Forest.
The majority of the streblid flies recorded here had a species-specific association, corroborating the findings of previous studies (e.g.Dick and Dittmar 2014; Barbier et al. 2019).On the other hand, three species (Nycterophilia fairchildi, Trichobius caecus, and T. johnsonae) showed lower host specificity, being collected on two to three bat species of the genus Pteronotus.It is worth noting that, in the surveyed caves, different Pteronotus species often used the same chambers as roost, keeping colonies very close to each other and even forming mixed colonies (E.Barbier, personal observation).This roosting behavior has already been recorded in other caves in the Neotropical region (see Pavan 2019).So, the frequent proximity between congener hosts over time may have facilitated the exchange of their streblid fly species.Furthermore, sheltering closely in perennial roosts such as caves has been pointed out as a possible important driver of host specificity in streblid flies (e.g.ter Hofstede and Fenton 2005).
The distribution range in Brazil of at least six streblid species reported in this study (N.fairchildi, N. parnelli, S. proxima, T. caecus, T. johnsonae, and T. pallidus) is littleknown and these species can be considered rare.As an example, T. pallidus is a specific parasite for the nationally threatened cave bat F. horrens (the only known species of Furipteridae in Brazil).Some studies have indicated high host specificity as the factor most likely to directly influence the possibility of coextinction when hosts are threatened (e.g.Moir et al. 2010;Poulin et al. 2011;Colwell et al. 2012).Due to the unique association of T. pallidus with F. horrens, the conservation status of this streblid species is also of concern due to the threat level to its host.Therefore, assessing host specificity can be pointed out as a crucial step in assessing the risk of coextinction (Colwell et al. 2012).
Considering the importance of caves for dozens of bat species, and that all endangered bat species in Brazil are cavedwelling (MMA 2022), it is urgent to improve the knowledge on these species, including basic parameters of host-parasite relationships such as parasite prevalence, abundance, and population structure.

CONCLUSIONS
In this study, we presented new records of streblid flies in a biodiversity-rich but undersampled region in northern Brazil, providing information on the geographic distribution and interspecific interaction of these ectoparasites and their host cave bats.Further studies that evaluate the interactions among cave bats and their ectoparasites in the context of ecological interaction networks will also be of great importance to shed light on questions about how such interactions are modulated in these unique environments, and what impact this has on both bats and parasites.

Figure 1 .
Figure 1.Location of the study area in eastern Brazilian Amazonia and location of the six caves sampled for bats and streblid flies in the Carajás National Forest in June and July 2022.This figure is in color in the electronic version.

Figure 2 .
Figure 2. Interactions among cave bats (rows) and streblid flies (columns) in six caves sampled in the Carajás National Forest (eastern Amazonia), Brazil.The darker the cells, the more frequent the observed interaction.

Figure 3 .
Figure 3. Nycterophilia species collected on cave bats in the Carajás National Forest, eastern Brazilian Amazonia.A -female of Nycterophilia fairchildi, lateral view; B -female of Nycterophilia parnelli, lateral view.Arrows highlight the density of setae present on the thorax and the number of macrosetae at the distal end of the abdomen.Scale bars = 0.5 mm.This figure is in color in the electronic version.

Figure 4 .
Figure 4. Dorsal view of streblid fly species collected on cave bats in the Carajás National Forest, in eastern Brazilian Amazonia.A -female of Strebla guajiro; B -female of Strebla proxima; C -female of Strebla wiedemanni; D -female of Trichobius anducei; E -female of Trichobius caecus; F -male of Trichobius dugesii; G -female of Trichobius dugesioides; H -male of Trichobius joblingi; I -female of Trichobius johnsonae; J -female of Trichobius pallidus; K -male of Trichobius parasiticus; L -male of Trichobius uniformis.Scale bars = 0.5 mm.This figure is in color in the electronic version.

Table 1 .
Geographic coordinates of the six bat caves sampled in the Carajás National Forest, Pará state, Brazil, in June-July 2022.