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Hyperparasitism in caves: Bats, bat flies and ectoparasitic fungus interaction
Journal of Invertebrate Pathology 166 (2019) 107206
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Hyperparasitism in caves: Bats, bat flies and ectoparasitic fungus interaction a
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Katrine M. Jensen , Luísa Rodrigues , Thomas Pape , Anders Garm , Sergi Santamaria , ⁎ Ana Sofia P.S. Reboleiraa,
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Natural History Museum of Denmark, University of Copenhagen, 2100 København Ø, Denmark Instituto da Conservação da Natureza e das Florestas, IP, Divisão de Conservação da Biodiversidade, Avenida da República, 16 a 16B, 1050-191 Lisboa, Portugal Biological Institute, University of Copenhagen, 2100 København Ø, Denmark d Unitat de Botànica, Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain b c
ARTICLE INFO
ABSTRACT
Keywords: Subterranean ecosystems Parasitism Nycteribiidae Ectoparasites Hyperparasites Laboulbeniales
Bat flies (Diptera: Nycteribiinae) are highly specialized bloodsucking bat ectoparasites. Some of the ectoparasitic bat flies are themselves parasitized with an ectoparasitic fungus of the genus Arthrorhynchus (Laboulbeniales). Ascospores of the fungus attach to the cuticle of a bat fly and develop a haustorium that penetrates the host cuticle. This interaction defines the fungus as a hyperparasite. Both the fly and the fungus are obligate parasites and this peculiar case of hyperparasitism has remained largely unstudied. We studied the prevalence of Laboulbeniales, genus Arthrorhynchus, in natural populations of bat flies infesting the bat species Miniopterus schreibersii, Myotis bechsteinii, My. blythii, My. daubentonii, My. escalerai and My. myotis in Portuguese caves. Laboulbeniales were found infecting 10 of the 428 screened bat flies (2.3%) in natural populations, with fewer infections in winter. Images obtained with transmission electron microscopy show the fungal haustorium within the bat fly host tissue, from where it extracts nutrition.
1. Introduction
fungus.
Parasitism is defined as the relationship between two organisms where one organism, the parasite, benefits while the other organism, the host, is harmed by the relationship. Typically, the parasite extracts nourishment from the host and thereby reduces its fitness (Crofton, 1971). A parasite itself can be infected with another parasite, which defines the latter as a hyperparasite (Haelewaters et al., 2018a). Understanding hyperparasitism requires a complex multidisciplinary approach involving ecology, evolution and behaviour of the three participants in the interaction. The interest in hyperparasitic fungus Laboulbeniales began with the early work of Thaxter (1896, 1908, 1931), followed by Merola (1952, 1953), and Benjamin (1971), and more recent studies recognized the high potential of these models for developmental, ecological and evolutionary studies (Blackwell, 1980a, 1980b; Meola and Tavares, 1982; Haelewaters, 2018a, 2018b, 2019). In Europe, Laboulbeniales parasitize bat flies that rely on bats belonging to the suborder Microchiroptera as the primary hosts for nutrition and geographical dispersal. This study aims at quantifying prevalence and transmission of Laboulbeniales on their bat fly hosts from caves in Portugal, using for the first time a sampling design for natural populations, and documenting the haustorial structures of the parasitic
1.1. Bats and bat flies
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There are over 1,300 bat species (Chiroptera) globally, which places bats as the second-most species diverse order of mammals, only surpassed by rodents (Fenton and Simmons, 2014). Bats are parasitized by a variety of arthropods including mites, ticks, fleas, bat bugs, and bat flies (Lourenço and Palmeirim, 2007, 2008). Amongst these parasites, bat flies are the most distinctive (Dittmar et al., 2015; Haelewaters et al., 2017). Bat flies are specialized louse flies (family Hippoboscidae) and comprise the subfamily Nycteribiinae. The subfamily was previously split into streblids with around 230 species, and the completely wingless nycteribiids with around 275 species (Marshall, 2012; Haelewaters et al., 2017; Walker et al., 2018). However, growing evidence for streblid paraphyly (Dittmar et al., 2006; Petersen et al., 2007) required a new classification (Pape et al., 2011). Bat flies are bloodsucking obligate ectoparasitic dipterans that are highly specialized for living nearly their entire life cycle in the fur and on the wing membranes of bats (Marshall, 2012; Haelewaters et al., 2018a). Bat grooming is the biggest threat to adult bat flies (Dick and
Corresponding author. E-mail address: [email protected] (A.S.P.S. Reboleira).
https://doi.org/10.1016/j.jip.2019.107206 Received 3 May 2019; Received in revised form 25 May 2019; Accepted 28 May 2019 Available online 29 May 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 166 (2019) 107206
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Fig. 1. Life cycle of nycteribiid bat flies [Based on Dick and Patterson (2006)].
which is nourished internally by specialized glands until maturity, when it briefly leaves the bat to deposit the larva on the wall or ceiling of the roost (Fig. 1). This type of adenotrophic viviparity is called larviparity (Meier et al., 1999). The larva immediately forms a puparium. After 3–4 weeks the adult bat fly emerges from the puparium and searches for a host bat (Dick and Patterson, 2006). Twenty-five bat species occur in mainland Portugal (ICNB, 2010), with the most abundant cave-dwelling species being Miniopterus schreibersii (Rodrigues and Palmeirim, 2008). Miniopterus schreibersii is frequently parasitized by two bat fly species, Nycteribia schmidlii and Penicillidia conspicua (Lourenço and Palmeirim, 2008), both of which are known hosts for Laboulbeniales (Haelewaters et al., 2017, 2018a). 1.2. Laboulbeniales Laboulbeniales (Ascomycota) are ectobiont fungi living on the surface of arthropods, some are parasitic and develop specialized structures, the haustoria (Santamaría et al., 2018). Laboulbeniales do not form typical, filamentous hyphae. Instead, they form a compact, multicellular thallus consisting of a receptacle containing one or more perithecia and antheridia, and some appendages (Tavares, 1985; Weir and Beakes, 1995; Haelewaters et al., 2017). These fungi produce ascospores, which are formed in the perithecium and released by pressure, often elicited during contact between hosts (Blackwell, 1980a). The two-celled ascospores are encased in a sticky sheath that attaches to the cuticle of the new host (Fig. 2). In some species, parasitism occurs when cell I penetrates the host cuticle and forms the haustorium to
Fig. 2. Life cycle of Arthrorhynchus [Reconstructed based on slide mounts of A. nycteribiae combined with data from Tavares (1985)].
Patterson, 2006; Dittmar et al., 2015). To avoid this, they can move quickly in any direction. In addition, they have big tarsal claws and combs or ctenidia to secure them from dislodgement from the bat fur both during bat grooming and flight (Humphries, 1967; Dick and Pospischil, 2015). Upon mating, the female bat fly carries a single larva, 2
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Fig. 3. Map with sampling localities in Portugal.
2. Material and methods
draw nourishment from the host (Blackwell, 1980b; Weir and Beakes, 1995), and the cell II forms the foot and later the 3-celled receptacle (Walker et al., 2018). The receptacle bears the carpogenic cell, trichophoric cell and trichogyne, in early stages of development, and the antheridia (Tavares, 1985). Over 2,200 species of Laboulbeniales are known, mostly from insects, but also from arachnids and millipedes (Santamaría et al., 2016, 2017). Approximately 80% of the reported species live on Coleoptera whilst 10% are found on dipterans. The remaining 10% are divided between Acari, Opiliones, Diplopoda, Thysanoptera, Orthoptera, Dermaptera, Blattodea, Mallophaga, Heteroptera and Hymenoptera (Haelewaters et al., 2017; Reboleira et al., 2018; Santamaria et al., 2017; Walker et al., 2018; Weir and Hammond, 1997). Species of several genera of Laboulbeniales are known to be position-specific (Santamaria et al., 2014; Sundberg et al., 2018; Weir and Hammond, 1997); female hosts are infected on the dorsal surface of the abdomen while males are infected on the ventral surface. This pattern is related to the transmission effectuated by the mating behaviour of the hosts (Goldmann, 2015). However, the relative importance of direct and indirect fungal transmission is unknown for most of the Laboulbeniales species (De Kesel, 1995). Bat flies are the only hosts of Laboulbeniales of the genus Arthrorhynchus (Thaxter, 1908; Blackwell, 1980a). Infections by Arthrorhynchus are usually observed as clusters of thalli. The thalli are most frequently present on the membrane between tergites or sternites of the abdomen, or in the genital area (Blackwell, 1980a; Santamaria, 2006). Species of the genus Arthrorhynchus do not show particularly high position specificity, and the fungus may be present on the legs or thorax of host specimens of both sexes. Thus, other types of contacts would be necessary for the direct transmission between body parts outside the genital area (Blackwell, 1980a). There were no published records of bat fly-associated Laboulbeniales in Portugal until 2018 (Jensen et al., 2018; Haelewaters et al., 2018b; Szentiványi et al., 2018). Here, we studied the prevalence of Arthrorhynchus in bat flies from natural populations of cave bats in Portugal in different seasons of the year, and show evidence of the presence of the fungal haustorium within the bat fly host tissue. This is the first study to examine population dynamics of species of the Laboulbeniales genus Arthrorhynchus, and their relationship to fly and bat hosts.
2.1. Sampling and studied material Bat flies were collected at 11 different roost sites (Fig. 3) from 2016 to 2018 during monitoring programs of cave dwelling bats coordinated by the Portuguese Institute for Nature Conservation and Forests (ICNF). SimpleMappr (www.simplemappr.net) was used to produce the distribution map. Sampling was done during bat maternity season (summer), in the autumn migration period and in hibernation season (winter). In the winter, bats were captured directly by hand from the wall and ceiling or with handheld nets (Fig. 4A), while in the summer the bats were captured with harp traps at the cave entrance. Harp traps were set at sunset and were checked for bats every 5–10 min. Bats were kept in cotton bags, 10 individuals per bag until they were examined. The species, sex and age class of each bat (when possible) was recorded. All bats were visually inspected for bat flies, which were removed with forceps and immediately deposited in vials with 96% ethanol (one vial per bat). The vials are deposited in the collection of the Natural History Museum of Denmark (NHMD). 2.2. Bat fly species identification, Laboulbeniales screening and documentation Collected bat flies were identified and screened for presence of Laboulbeniales using a stereomicroscope (Wild M8 Heerbrugg). Presence of Laboulbeniales was photo documented using a Visionary Digital system (http://www.visionarydigital.com), and Adobe Photoshop Lightroom 5.4 (Adobe Inc.; Seattle, WA, USA). Image stacks were combined with Zerene Stacker Version 1.04 (Zerene Systems LLC; Richland, WA, USA). Background of some images was cleaned with Adobe Photoshop CS6 (Adobe Inc.; Seattle, WA, USA). 2.3. Scanning electron microscopy (SEM) A heavily infected female bat fly was transferred to 100% ethanol, critical point-dried in a Tousimis Autosamdri 815 Series A critical point dryer, mounted on an aluminium stub, coated with platinum/palladium and observed under a JEOL JSM-6335F scanning electron microscope (SEM). 3
Journal of Invertebrate Pathology 166 (2019) 107206
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Fig. 4. Sampling bats and bat flies. (A) Miniopterus schreibersii captured with a handheld net (February 2018); (B) Myotis blythii; (C) M. schreibersii with Penicillidia conspicua.
2.4. Transmission electron microscopy (TEM)
Table 2 Number of individuals of each bat fly species.
A heavily infected female bat fly preserved in 96% alcohol was prepared for TEM by rinsing in 100% acetone 2 × 20 min. and placed in a 1:1 mix of 100% acetone and Epon 812 on a wave motion platform shaker overnight. The following morning the lid was removed for 2 h to allow the acetone to evaporate. The specimen was transferred to pure Epon 812 and placed on the wave motion platform shaker again for 3 h and subsequently transferred to a mold with Epon 812 and placed in an oven to polymerize for 2 days at 60 °C. Ultrathin sections (70 nm) were made on a Leica V ultratome, placed on single slot grids and contrasted with lead citrate and uranyl acetate. Sections were observed in a JEOL 1010 microscope equipped with a Gatan SC1000 camera.
Bat fly species
N° of individuals
Penicillidia dufourii Penicillidia conspicua Nycteribia schmidlii Nycteribia vexata Nycteribia latreillei Nycteribia kolenatii Total
227 138 34 24 4 1 428
My. escalerai and My. myotis. A total of 428 bat flies were collected, belonging to six species: Penicillidia dufourii, P. conspicua, Nycteribia schmidlii, N. vexata, N. latreillei and N. kolenatii (Tables 1 and 2). Miniopterus schreibersii was the most abundant bat (Fig. 4A, C), followed by My. blythii (Fig. 4B) and My. myotis (Table 1). Among the 428 bat flies screened, only 10 were infected with the hyperparasite A. nycteribiae (Figs. 5 and 6, Table 3) resulting in an overall infection rate of 2.3%. Nine of the 10 infected bat flies were P. conspicua and one was P. dufourii (Table 1). Eight bat flies were collected in autumn (September 2016), 240 collected in summer (July 2016) and 180 collected in winter (January–March 2018). In autumn no Laboulbeniales infected bat flies were found. In winter one infected bat fly was found and in summer nine infected bat flies were found, an infection rate in summer of 3.8% and in winter of 0.6%.
2.5. Differential interference contrast microscopy (DIC) The thalli of the fungus on an infected bat fly specimen were removed using an insect pin and mounted with lactophenol on a slide where it was observed and imaged in a DM2500 Leica microscope with DIC, following the methodology of Santamaría et al. (2018). 3. Results 3.1. Infection rate Bat flies were found on 239 bats belonging to six species: Miniopterus schreibersii, Myotis bechsteinii, My. blythii, My. daubentonii,
Table 1 An overview of number of bat species, number of bat fly species per bat species, and number of infected bat flies. Bat species
N° of individuals (bats)
Bat fly species
N° of individuals (bat flies)
N° of infected bat flies
Miniopterus schreibersii
127
Myotis bechsteinii Myotis blythii
1 59
Myotis daubentonii Myotis escalerai Myotis myotis
1 2 49
Nycteribia schmidlii Penicillidia conspicua Penicillidia dufourii Nycteribia vexata Nycteribia latreillei Nycteribia vexata Penicillidia conspicua Penicillidia dufourii Nycteribia kolenatii Penicillidia dufourii Nycteribia latreillei Nycteribia vexata Penicillidia conspicua Penicillidia dufourii
34 135 7 1 3 12 2 95 1 2 1 11 1 123
0 9 0 0 0 0 0 0 0 0 0 0 0 1
4
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Fig. 5. Arthrorhynchus nycteribiae on DIC. Arrows indicate the host cuticle. Below this point the haustorium continues into the bat fly. (A) Small thallus; (B) Fully developed thallus; (C) Detail of the foot; (D) Thallus with trichogyne. Scale bars: A = 100 μm; B-D = 50 μm.
3.2. Bat flies sex ratio
3.3. Dispersal of infected bat flies
The sex ratio of bat flies observed in the samples was 245 females:183 males (Table 3). Also, more females were infected with the fungus than males (Table 5). Of the 10 infected bat flies, seven were females and three were males, i.e., more females in absolute numbers but a lower percentage of these were infected.
Infected bat flies were found in three of 11 sampling sites: Tomar I, Odemira VII, and Alvaiázere I (Fig. 3, Table 4). All infected bat flies found in summer were from Tomar I and Odemira VII. The only infected bat fly found in winter was from Alvaiázere I.
Fig. 6. SEM images of Arthrorhynchus nycteribiae infecting a female Penicillidia conspicua. (A) Dorsal view; (B) Detail of the group of thalli; (C) Host cuticle with attached thalli; (D) Detail of the foot attached to an intersegmental membrane. Scale bars: A = 1 mm; B = 100 μm; C–D = 10 μm. 5
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flies of the species N. schmidlii were only found on bats of the species Mi. schreibersii (Table 1). The bats My. blythii and My. myotis were both heavily infested with P. dufourii and to a lesser degree with N. vexata. On My. blythii, 84.8% (n = 95) of bat flies were P. dufourii while 10.7% (n = 12) are N. vexata. Similarly, 90.4% (n = 123) of bat flies on My. myotis, were P. dufourii while 8.1% (n = 11) were N. vexata. The bats My. bechsteinii, My. daubentonii, and My. escalerai are not represented in Fig. 7, because they had very low bat fly infestation rates with only one fly on each My. bechsteinii and My. daubentonii, and two on My. escalerai. All fungal infected P. conspicua were found on the bat Mi. schreibersii (Fig. 4C, Table 1). The single specimen of infected P. dufourii was found on the bat My. myotis (Figs. 4B and 7).
Table 3 Numbers of females and males with dorsal, ventral and lateral position of the Laboulbeniales. Sex of bat fly
Position of Laboulbeniales
N° of individuals
Female
Dorsal Ventral Lateral Both ventral and dorsal Dorsal Ventral Lateral Both ventral and dorsal
4 2 1 0 1 1 0 1
Male
Table 4 The relation of sampling sites and infected bat flies.
3.5. Position of the fungus
Official name of roost site
N° of infected bat flies
Species of bat fly
Tomar I
4 1 4 1
P. P. P. P.
Odemira VII Alvaiázere I
The position of the fungus on the bat fly body was variable. Three females were infected dorsally on the left side of abdomen, two were infected ventrally on the left side of abdomen, one was infected dorsally on the right side of abdomen, and one was infected laterally on left mid coxa (Fig. 8A–C). One male was infected dorsally on the right side, one was infected ventrally on the left side, and one was infected on the left side both dorsally and ventrally (Fig. 8D–F, Table 6).
conspicua dufourii conspicua conspicua
Table 5 Number of males and females for each bat fly species. Bat fly species
N° of males
N° of females
P. dufourii P. conspicua N. schmidlii N. vexata N. latreillei N. kolenatii Total
103 60 11 5 3 1 183
124 78 23 19 1 0 245
3.6. Structure of the haustorium Tube-like structures were found in the bat fly tissue just under the fungal thallus (Fig. 9). The tubules were not made of insect cuticle and the structure resembled the thallus (compare Fig. 9 B2, C2 and D2). Furthermore, the presence of cytoplasm inside the structures (Fig. 9B–D) indicate that these structures are not part of the tracheal system of the fly. The haustorium of Arthrorhynchus nycteribiae was apparent inside the host tissue, recognizable because the fungal cell walls show no layering (Fig. 9A).
3.4. Host specificity of bat flies and of infected bat flies Miniopterus schreibersii was primarily infested with two bat fly species: P. conspicua 76.7% (n = 135), N. schmidlii 19.3% (n = 34). Bat
Fig. 7. Percentage of bat fly species for each bat species with multiple infections. 6
Journal of Invertebrate Pathology 166 (2019) 107206
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Fig. 8. Penicillidia conspicua, infected with Arthrorhynchus nycteribiae. (A) Female bat fly infected dorsally; (B) Female infected ventrally; (C) Female infected laterally on the left mid coxa; (D) Male infected dorsally; (E) Male infected laterally; (F) Male infected both dorsally and ventrally. Scale bars: 0.5 mm.
the genital area on the dorsal side of females and ventral sides of males but probably not for infections in other body parts (Blackwell, 1980a). However, it is possible that other body parts of the male and female are in contact during courtship, which could explain patterns found where the thorax of the male touches the female. Further behavioural studies will shed light on the transmission mechanisms. Infection elsewhere on the body of bat flies is most likely to happen randomly by direct contact between bat flies due to their limited microhabitat (the host bat surface).
Table 6 Overview of sex of bat fly with indicated position and side of Laboulbeniales. Bat fly ID
Sex of bat fly
Position of Laboulbeniales
Side of Laboulbeniales
S91 S86 W39 S58 S66 S47 S92 S85 S51 S59
Female Female Female Female Female Female Female Male Male Male
Dorsal abdomen Dorsal abdomen Dorsal abdomen Ventral abdomen Ventral abdomen Dorsal abdomen Lateral thorax (coxa) Dorsal abdomen Ventral abdomen Ventral abdomen Dorsal abdomen
Left Left Left Left Left Right Left Right Left Left Left
4.2. Fungal distribution We record for the first time the presence of the fungus Arthrorhynchus nycteribiae in central Portugal. This species was recently found in the southernmost province of the country, the Algarve, an area clearly marked by a strong Mediterranean influence (Szentiványi et al., 2018). Arthrorhynchus nycteribiae was previously reported from Australia, Africa (Kenya, Zambia), Asia (Sri Lanka), and across Europe (Austria, Bulgaria, Croatia, Czech Republic, Denmark, France, Hungary, Italy, Netherlands, Poland, Romania, Russia, Serbia, Slovakia, Spain, Sweden, Switzerland) (Blackwell, 1980a; Haelewaters et al., 2017).
4. Discussion 4.1. Seasonal infection rates Bat flies infected with Laboulbeniales exhibit a generally low infection prevalence (Blackwell, 1980a; Haelewaters et al., 2017). In this study we found an overall prevalence of 2.3%, with fewer infections in winter. Bat flies collected in summer yielded an infection rate of 3.8%, contrasting to 0.6% in winter samples. Blackwell (1980a) screened 2,517 bat flies from 404 entomological collections for the presence of Laboulbeniales and found 56 infected individuals, leading to an infection rate of 2.2%. Data on season was not included. Haelewaters et al. (2017) found an infection rate of 3.0% in 1,494 bat flies collected in Hungary and Romania, which is again remarkably similar to what we report here. Again, data on season was not included. Szentiványi et al. (2018) found an infection rate of 9% (in 667 bat flies collected in Albania, Croatia, Hungary, Italy, Portugal, Slovakia, Spain and Switzerland). These bat flies were collected in the summer period (April–September), which could explain the higher infection rate. The lower infection rate in winter may be explained by the hibernation of bats, when the bat flies show a decreased level of activity and, therefore, decreased contact. Some tropical bat flies are known to mate year-round, but this might not be the case for bat flies on hibernating bats (Blackwell 1980a). It is known that the reproductive cycle of bat flies is synchronized with the reproductive cycle of their bat hosts for this geographic region (Lourenço and Palmeirim, 2008). Blackwell (1980a) states that transmission probably is not highly dependent on mating. Copulation of bat flies happens by the male mounting the female, which relates directly to fungal transmission in
4.3. Female-biased sex ratio Ectoparasitic insects typically display a sex ratio biased toward females (Dick and Patterson, 2008), confirmed in our study. This is especially observed among permanent parasites and groups of temporary ectoparasites; bat flies are permanent parasites. Females and males apparently are produced in equal numbers but, because of greater longevity in females, the sex ratio is skewed (Marshall, 1981; Dick and Patterson, 2008). In a study on the bat fly Basilia hispida Theodor, 1967, males were found to have an approximate 97-day life-span versus 156 days for females (Haelewaters et al., 2017). Another factor permitting a female-biased sex ratio could be sampling bias. The females of some bat fly species are larger than males, especially during pregnancy. In addition, females may be more sedentary while males may be more active (Marshall, 1981; Dick and Patterson, 2008), leading to a sampling bias towards excess of females. Climate may also affect this bias. A study by Hurka (1964) on N. latreillei showed that the female-biased sex ratio increased towards the north, possibly because females cope better with the colder climate (Marshall, 1981). Females also showed a higher Laboulbeniales infection rate, possibly because of their larger size, especially during pregnancy (larger surface area bringing a higher probability of contact with spores), or 7
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Fig. 9. TEM image of the bat fly Penicillidia conspicua tissue with haustorium of Arthrorhynchus nycteribiae. (A) White circles show sections of the fungal haustorium in the bat fly tissue. (B1, B2 and C1, C2) Details of the fungal haustorium. (D1, D2) Cell wall of fungal haustorium. Scale bars: A, B1, C1 = 5 μm; D1 = 2,5 μm; B2, C2, D2 = 200 μm.
which correlates with earlier studies where P. conspicua is the most common host for A. nycteribiae (Blackwell, 1980a; Haelewaters et al., 2017). Some Laboulbeniales species, including those in the genus Arthrorhynchus, can infect several host species (Haelewaters et al., 2017), and Blackwell (1980a) found two species A. nycteribiae and A. eucampsipodae on several genera of Nycteribiinae. The high host specificity for bat species is exhibited among nycteribiid bat flies and also observed in studies on the streblid bat flies (Dick, 2007; Dick and Patterson, 2007). High host specificity is a common trait in parasitic species with low dispersal capabilities, an evolutionary effect from low probability of encounters with other potential hosts, or due to acquired structural or immunological incompatibility (Dick, 2007). The hosts we studied are cave-dwelling bats that hibernate in winter. The roosts are often shared by several bat species, and colonies of different species can be adjacent or even intermixed (Rodrigues et al., 2003). The fact that the bat fly puparium is glued to the walls or ceiling of the roost (not on the host body), should make bat flies prone to dispersal to other host species using the same roosts. Strong position-specificity has not been observed in Arthrorhynchus. The most common positions were in the membrane between two sclerites or between tergites/sternites on the abdomen or in the genital region. Several specimens are found to be infected on legs or thorax (Blackwell, 1980a), indicating that transmission does not rely solely on mating behavior, but also on other types of host contacts, or even via the substrate (bat fur). Cave bats live in a high humidity environment, which might allow for the survival of ascospores released in the bat fur until another bat fly crawls by.
longevity that increases the exposure time for infection. Pregnant females store large fat reserves that are of high nutritional value for the fungus (Haelewaters et al., 2017), but this is not relevant because the fungus cannot actively select its host. It also possibly can be linked with sexual transmission and differences in male and female behaviour. Males mate with several females, so if the fungus is primarily transmitted during mating, each infected male produces multiple infected females. Males are more active than females (often the case in insects), which would make males more prone to being lost through host grooming and explaining the biased sex ratio. Also, high male activity may allow males to spread the fungus to several females even if not accepted as mates by all of them. Haelewaters et al. (2017)Haelewaters et al. (2017) hypothesized that the fungus develops slowly, in which case female bat flies may provide a more long-lived host for development of the fungus than males, and a long-lived female can infect multiple males. This, however, contrasts with a study by Blackwell (1980a), who hypothesized that Arthrorhynchus has a relatively short life cycle since thalli found in clusters had several stages of maturity. 4.4. Low dispersal Infected bat flies were found at three of 11 sampling sites. The dispersal of the fungus between different bat roosts is dependent on the dispersal of infected bat flies during bat migrations, known to occur in this area in spring and autumn (Rodrigues et al., 2010). 4.5. Host and position specificity In our study all bat flies collected were infected with A. nycteribiae, 8
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4.6. Structure of the haustorium
Krasnov, B., Poulin, R. (Eds.), Micromammals and Macroparasites: From Evolutionary Ecology to Management. Springer, Tokyo, pp. 179–194. https://doi. org/10.1007/978-4-431-36025-4_11. Dick, C.W., Patterson, B.D., 2007. Against all odds: Explaining high host specificity in dispersal-prone parasites. Int. J. Parasitol. 37, 871–876. https://doi.org/10.1016/j. ijpara.2007.02.004. Dick, C.W., Patterson, B.D., 2008. An excess of males: skewed sex ratios in bat flies (Diptera: Streblidae). Evol. Ecol. 22 (6), 757–769. https://doi.org/10.1007/s10682007-9201-9. Dick, C.W., Pospischil, R., 2015. Nycteribiidae (bat flies). In: Mehlhorn, H. (Ed.), Encyclopedia of Parasitology. Springer-Verlag, Berlin. https://doi.org/10.1007/9783-642-27769-6_3462-1. Dittmar, K., Porter, M.L., Murray, S., Whiting, M.F., 2006. Molecular phylogenetic analysis of nycteribiid and streblid bat flies (Diptera: Brachycera, Calyptratae): Implications for host associations and phylogeographic origins. Mol. Phylogenet. Evol. 38, 155–170. Dittmar, K., Morse, S., Dick, C.W., Patterson, B.D., 2015. Bat fly evolution from the eocene to the present (Hippoboscoidea, Streblidae and Nycteribiidae). In: Morand, S., Krasnov, B., Littlewood, T. (Eds.), Parasite Diversity and Diversification: Evolutionary Ecology Meets Phylogenetics. Cambridge University Press, Cambridge, pp. 246–264. https://doi.org/10.1017/CBO9781139794749.015. Fenton, M.B., Simmons, N.B., 2014. Bats: A World of Science and Mystery. University of Chicago Press, Chicago. Goldmann, L.M., 2015. Investigations into position specificity and the molecular systematics of the Laboulbeniomycetes. PhD-thesis from State University of New York, Syracuse, New York. Published by ProQuest LLC. Haelewaters, D., Pfliegler, W.P., Szentiványi, T., Földvári, M., Sándor, A.D., Barti, L., Camacho, J.J., Gort, G., Estók, P., Hiller, T., Dick, C.W., Pfister, D.H., 2017. Parasites of parasites of bats: Laboulbeniales (Fungi: Ascomycota) on bat flies (Diptera: Nycteribiidae) in central Europe. Parasit. Vectors. 10 (1), 96. https://doi.org/10. 1186/s13071-017-2022-y. Haelewaters, D., Hiller, T., Dick, Carl, W., 2018a. Bats, bat flies, and fungi: A case of hyperparasitism. Trends Parasitol. 34 (9), 784–799. https://doi.org/10.1016/j.pt. 2018.06.006. Haelewaters, D., Page, R., Pfliegler, W.P., 2018b. Laboulbeniales hyperparasites (Fungi, Ascomycota) of bat flies: Independent origins and host associations. Ecol. Evol. https://doi.org/10.1002/ece3.4359. Haelewaters, D., Boer, P., Báthori, F., Rádai, Z., Reboleira, A.S.P.S., Tartally, A., De Kesel, A., Pfiegler, W.P., Nedvéd, O., 2019. Studies of Laboulbeniales on Myrmica ants (IV): host-related diversity and thallus distribution patterns of Rickia wasmannii. Parasite 26. https://doi.org/10.1051/parasite/2019028. Humphries, D.A., 1967. Function of combs in ectoparasites. Nature 215 (5098), 319. Hurka, K., 1964. Distribution, bionomy and ecology of the European bat flies with special regard to the Czechoslvak fauna (Dip., Nycteribiidae). ACTA Univ. Carol. Biologica 167–234. Jensen, K., Rodrigues, L., Pape, T., Garm, A., Santamaria, S., Reboleira, A.S.P.S., 2018. Hyperparasitism in caves: bats, bat flies and ectoparasitic fungus. e29967. ARPHA Conference Abstracts 1. https://doi.org/10.3897/aca.1.e29967. ICNB, 2010. Implementation Report of EUROBATS in Portugal (March 2006 to May 2010). Report presented to the 6th Session of the Meeting of the Parties. Lourenço, S., Palmeirim, J.M., 2007. Can mite parasitism affect the condition of bat hosts? Implications for the social structure of colonial bats. J. Zool. 273 (2), 161–168. https://doi:10.1111/j.1469-7998.2007.00322.x. Lourenço, S., Palmeirim, J.M., 2008. Which factors regulate the reproduction of ectoparasites of temperate-zone cave-dwelling bats? Parasitol. Res. 104, 127–134. https://doi.org/10.1007/s00436-008-1170-6. Marshall, A.G., 1981. The sex ratio in ectoparasitic insects. Ecol. Entomol. 6, 155–174. https://doi.org/10.1111/j.1365-2311.1981.tb00602.x. Marshall, S.A., 2012. Flies: The Natural History and Diversity of Diptera. Firefly Books, Ontario. Meier, R., Kotrba, M., Ferrar, P., 1999. Ovoviviparity and viviparity in the Diptera. Biol. Rev. 74 (3), 199–258. https://doi.org/10.1111/j.1469-185X.1999.tb00186.x. Meola, S., Tavares, I.I., 1982. Ultrastructure of the haustorium of Trenomyces histophthorus and adjacent host cells. J. Invertebr. Pathol. 40, 205–215. Merola, A., 1952. Interessante ritrovamento di labulbeniologia cavernicola: Arthrorhynchus acrandros n. sp. (con considerazioni sul gen. Arthrorhynchus). Boll. Soc. Nat. Napoli 60, 1–30. Merola, A., 1953. Unisessualità di una Labulbeniacea omotallica e suoi rapporti con il dioicismo delle Labulbeniacee eterotalliche. Bul. Orto Bot. Univ. Napoli, n. s 60, 62–92. Pape, T., Blagoderov, V., Mostovski, M.B., 2011. Order DIPTERA Linnaeus, 1758. In: Zhang, Z.-Q. (Ed.), Animal Biodiversity: An Outline of Higher-level Classification and Survey of Taxonomic Richness. Zootaxa, pp. 222–229. Petersen, F.T., Meier, R., Kutty, S.N., Wiegmann, B.M., 2007. The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers. Mol. Phylogenet. Evol. 45 (1), 111–122. https://doi.org/10. 1016/j.ympev.2007.04.023. Reboleira, A.S.P.S., Enghoff, H., Santamaria, S., 2018. Novelty upon novelty visualized by rotational scanning electron micrographs (rSEM): Laboulbeniales on the millipede order Chordeumatida. PLoS ONE 13 (11), e0206900. https://doi.org/10.1371/ journal.pone.0206900. Rodrigues, L., Zahn, A., Rainho, A., Palmeirim, J., 2003. Contrasting the roosting behaviour and phenology of an insectivorous bat (Myotis myotis) in its southern and northern distribution ranges. Mammalia 67 (3), 321–336. Rodrigues, L., Palmeirim, J., 2008. Migratory behaviour of the Schreiber's bat: When, where and why do cave bats migrate in a Mediterranean region? J. Zool. 274 (2),
Despite ethanol fixation of flies, it was possible to observe parts of the haustorium inside the fly with TEM. This is the first record of haustorial sections inside the body cavity of a bat fly. Furthermore, between the fungal cell wall layer and the cuticle of the host there is a dark region, also observed by Blackwell (1980b) and described as a carbonaceous patch. Blackwell (1980b) proposed that the dark region may be due to growth of the fungus and breakdown of the surrounding cuticle of the bat fly), but it is likely to be a defence response to infection of the bat fly because melanization is an ancestral response to wounding in arthropods, even conserved in albino cave species (Bilandžija et al., 2017). 5. Future perspectives Knowledge of the biology of Laboulbeniales is still very immature (Haelewaters et al., 2017, 2018a). More research on the potential for seasonal differences in infection rates is needed to elucidate the effect of bat hibernation on both bat flies and fungus (Blackwell, 1980a). Furthermore, understanding of behavioural transmission of the fungus between bat flies is still incomplete because transmission may be associated with behaviours other than mating of the bat flies (Blackwell, 1980a). In addition, the structure of the haustorium of Arthrorhynchus, the response to infection and effect of infection on the bat flies should be further examined (Blackwell, 1980a). Lastly, many Laboulbeniales species are cryptic and difficult to distinguish based on morphological characters. For this, a molecular phylogenetic approach should be applied to clarify the taxonomic status (Haelewaters et al., 2017) and to understand the genetic structure of populations. Declaration of Competing Interest There are no conflicts of interest to be declared. Acknowledgements We are grateful to Jonas Winding Christensen (Erhvervsakademi Sjælland) for illustrations of life cycles, to the Portuguese Institute for Nature Conservation and Forests (ICNF), to Paulo Barros (University of Trás-os-Montes e Alto Douro, Portugal) and to Margarida Augusto (BioInsight, Portugal) for support in collecting nycteribiids under the National Bats Monitoring Program in Portugal, and to Lis Munk Frederiksen (Biological Institute of Copenhagen University) for help with TEM preparation. All the specimens were collected under permits of the ICNF. ASR is supported by a research grant (15471) from the VILLUM FONDEN. References Bilandžija, H., Laslo, M., Porter, M.L., Fong, D.W., 2017. Melanization in response to wounding is ancestral in arthropods and conserved in albino cave species. Sci. Rep. 7 (1), 17148. https://doi.org/10.1038/s41598-017-17471-2. Blackwell, M., 1980a. Incidence, host specificity, distribution and morphological variation in Arthrorhynchus nyctyeribiae and Arthrorhynchus eucampsipodae (Laboulbeniomycetes). Mycologia 72 (1), 143–158. https://doi.org/10.2307/ 3759427. Blackwell, M., 1980b. Developmental Morphology and Taxonomic Characters of Arthrorhynchus Nycteribiae and Arthrorhynchus eucampsipodae (Laboulbeniomycetes). Mycologia 72 (1), 159–168. https://doi.org/10.2307/3759428. Benjamin, R.K., 1971. Introduction and supplement to Roland Thaxter’s contribution toward a monograph of the Laboulbeniaceae. Biblioth. Mycol. 30, 1–155. Crofton, H., 1971. A model of host–parasite relationships. Parasitol. 63 (3), 343–364. https://doi.org/10.1017/S0031182000079890. De Kesel, A., 1995. Relative importance of direct and indirect infection in the transmission of Laboulbenia slackensis (Ascomycetes, Laboulbeniales). Belgian J. Bot. 128 (2), 124–130. Dick, C.W., 2007. High host specificity of obligate ectoparasites. Ecol. Entomol. 32, 446–450. https://doi.org/10.1111/j.1365-2311.2007.00836.x. Dick, C.W., Patterson, B.D., 2006. Bat flies: obligate ectoparasites of bats. In: Morand, S.,
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K.M. Jensen, et al. 116–125. https://doi.org/10.1111/j.1469-7998.2007.00361.x. Rodrigues, L., Ramos Pereira, M., Rainho, A., Palmeirim, J., 2010. Behavioural determinants of gene flow in the bat Miniopterus schreibersii. Behav. Ecol. Sociobiol. 64 (5), 835–843. https://doi.org/10.1007/s00265-009-0900-9. Santamaria, S., 2006. New or interesting Laboulbeniales (Fungi, Ascomycota) from Spain, V. Nova Hedwigia 82 (3–4), 349–363. Santamaria, S., Enghoff, H., Reboleira, A.S.P.S., 2014. Laboulbeniales on millipedes: the genera Diplopodomyces and Troglomyces. Mycologia 106 (5), 1027–1038. Santamaria, S., Enghoff, H., Reboleira, A.S.P.S., 2016. Hidden biodiversity revealed by collections-based research—Laboulbeniales in millipedes: genus Rickia. Phytotaxa 243 (2), 101–127. Santamaria, S., Enghoff, H., Gruber, J., Reboleira, A.S.P.S., 2017. First Laboulbeniales from harvestmen: the new genus Opilionomyces. Phytotaxa 305 (4), 285–292. Santamaria, S., Enghoff, H., Reboleira, A.S.P.S., 2018. New species of Troglomyces and Diplopodomyces (Laboulbeniales, Ascomycota) from millipedes (Diplopoda). Eur. J. Taxon. 429, 1–20. https://doi.org/10.5852/ejt.2018.429. Sundberg, H., Kruys, Å., Bergsten, J., Ekman, S., 2018. Position specificity in the genus Coreomyces (Laboulbeniomycetes, Ascomycota). Fungal Systemat. Evol. 1 (1), 217–228. Szentiványi, T., Haelewaters, D., Pfliegler, W.P., Clément, L., Christe, P., Glaizot, O., 2018. Laboulbeniales (Fungi: Ascomycota) infection of bat flies (Diptera:
Nycteribiidae) from Miniopterus schreibersii across Europe. Parasit. Vectors. 11 (1), 395. https://doi.org/10.1186/s13071-018-2921-6. Tavares, I.I., 1985. Laboulbeniales (Fungi, Ascomycetes), Mycologia Memoir no. 9, J. Cramer, Braunschweig. Thaxter, R., 1896. Contribution towards a monograph of the Laboulbeniaceae. Mem. Am. Acad. Arts Sci. 12, 187–429. Thaxter, R., 1908. Contribution toward a Monograph of the Laboulbeniaceæ: Part II. Mem. Am. Acad. Arts Sci., New Series 13 (6), 219–469. https://doi.org/10.2307/ 25058090. Thaxter, R.R., 1931. Contribution towards a monograph of the Laboulbeniaceae. Part V. Mem. Am. Acad. Arts Sci., New Series 16, 1–435. Walker, M.J., Dorrestein, A., Camacho, J.J., Meckler, L.A., Silas, K.A., Hiller, T., Haelewaters, D., 2018. A tripartite survey of hyperparasitic fungi associated with ectoparasitic flies on bats (Mammalia: Chiroptera) in a neotropical cloud forest in Panama. Parasit. 25, 19. https://doi.org/10.1051/parasite/2018017. Weir, A., Beakes, G., 1995. An introduction to the Laboulbeniales: A fascinating group of entomogenous fungi. Top. Catal. 9 (1), 6–10. https://doi.org/10.1016/S0269-915X (09)80238-3. Weir, A., Hammond, P.M., 1997. Laboulbeniales on beetles: host utilization patterns and species richness of the parasites. Biodivers. Conserv. 6, 701–719. https://doi.org/10. 1023/A:1018318320019.
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Hyperparasitism in caves: Bats, bat flies and ectoparasitic fungus interaction a
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a
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d
Katrine M. Jensen , Luísa Rodrigues , Thomas Pape , Anders Garm , Sergi Santamaria , ⁎ Ana Sofia P.S. Reboleiraa,
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Natural History Museum of Denmark, University of Copenhagen, 2100 København Ø, Denmark Instituto da Conservação da Natureza e das Florestas, IP, Divisão de Conservação da Biodiversidade, Avenida da República, 16 a 16B, 1050-191 Lisboa, Portugal Biological Institute, University of Copenhagen, 2100 København Ø, Denmark d Unitat de Botànica, Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain b c
ARTICLE INFO
ABSTRACT
Keywords: Subterranean ecosystems Parasitism Nycteribiidae Ectoparasites Hyperparasites Laboulbeniales
Bat flies (Diptera: Nycteribiinae) are highly specialized bloodsucking bat ectoparasites. Some of the ectoparasitic bat flies are themselves parasitized with an ectoparasitic fungus of the genus Arthrorhynchus (Laboulbeniales). Ascospores of the fungus attach to the cuticle of a bat fly and develop a haustorium that penetrates the host cuticle. This interaction defines the fungus as a hyperparasite. Both the fly and the fungus are obligate parasites and this peculiar case of hyperparasitism has remained largely unstudied. We studied the prevalence of Laboulbeniales, genus Arthrorhynchus, in natural populations of bat flies infesting the bat species Miniopterus schreibersii, Myotis bechsteinii, My. blythii, My. daubentonii, My. escalerai and My. myotis in Portuguese caves. Laboulbeniales were found infecting 10 of the 428 screened bat flies (2.3%) in natural populations, with fewer infections in winter. Images obtained with transmission electron microscopy show the fungal haustorium within the bat fly host tissue, from where it extracts nutrition.
1. Introduction
fungus.
Parasitism is defined as the relationship between two organisms where one organism, the parasite, benefits while the other organism, the host, is harmed by the relationship. Typically, the parasite extracts nourishment from the host and thereby reduces its fitness (Crofton, 1971). A parasite itself can be infected with another parasite, which defines the latter as a hyperparasite (Haelewaters et al., 2018a). Understanding hyperparasitism requires a complex multidisciplinary approach involving ecology, evolution and behaviour of the three participants in the interaction. The interest in hyperparasitic fungus Laboulbeniales began with the early work of Thaxter (1896, 1908, 1931), followed by Merola (1952, 1953), and Benjamin (1971), and more recent studies recognized the high potential of these models for developmental, ecological and evolutionary studies (Blackwell, 1980a, 1980b; Meola and Tavares, 1982; Haelewaters, 2018a, 2018b, 2019). In Europe, Laboulbeniales parasitize bat flies that rely on bats belonging to the suborder Microchiroptera as the primary hosts for nutrition and geographical dispersal. This study aims at quantifying prevalence and transmission of Laboulbeniales on their bat fly hosts from caves in Portugal, using for the first time a sampling design for natural populations, and documenting the haustorial structures of the parasitic
1.1. Bats and bat flies
⁎
There are over 1,300 bat species (Chiroptera) globally, which places bats as the second-most species diverse order of mammals, only surpassed by rodents (Fenton and Simmons, 2014). Bats are parasitized by a variety of arthropods including mites, ticks, fleas, bat bugs, and bat flies (Lourenço and Palmeirim, 2007, 2008). Amongst these parasites, bat flies are the most distinctive (Dittmar et al., 2015; Haelewaters et al., 2017). Bat flies are specialized louse flies (family Hippoboscidae) and comprise the subfamily Nycteribiinae. The subfamily was previously split into streblids with around 230 species, and the completely wingless nycteribiids with around 275 species (Marshall, 2012; Haelewaters et al., 2017; Walker et al., 2018). However, growing evidence for streblid paraphyly (Dittmar et al., 2006; Petersen et al., 2007) required a new classification (Pape et al., 2011). Bat flies are bloodsucking obligate ectoparasitic dipterans that are highly specialized for living nearly their entire life cycle in the fur and on the wing membranes of bats (Marshall, 2012; Haelewaters et al., 2018a). Bat grooming is the biggest threat to adult bat flies (Dick and
Corresponding author. E-mail address: [email protected] (A.S.P.S. Reboleira).
https://doi.org/10.1016/j.jip.2019.107206 Received 3 May 2019; Received in revised form 25 May 2019; Accepted 28 May 2019 Available online 29 May 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. Life cycle of nycteribiid bat flies [Based on Dick and Patterson (2006)].
which is nourished internally by specialized glands until maturity, when it briefly leaves the bat to deposit the larva on the wall or ceiling of the roost (Fig. 1). This type of adenotrophic viviparity is called larviparity (Meier et al., 1999). The larva immediately forms a puparium. After 3–4 weeks the adult bat fly emerges from the puparium and searches for a host bat (Dick and Patterson, 2006). Twenty-five bat species occur in mainland Portugal (ICNB, 2010), with the most abundant cave-dwelling species being Miniopterus schreibersii (Rodrigues and Palmeirim, 2008). Miniopterus schreibersii is frequently parasitized by two bat fly species, Nycteribia schmidlii and Penicillidia conspicua (Lourenço and Palmeirim, 2008), both of which are known hosts for Laboulbeniales (Haelewaters et al., 2017, 2018a). 1.2. Laboulbeniales Laboulbeniales (Ascomycota) are ectobiont fungi living on the surface of arthropods, some are parasitic and develop specialized structures, the haustoria (Santamaría et al., 2018). Laboulbeniales do not form typical, filamentous hyphae. Instead, they form a compact, multicellular thallus consisting of a receptacle containing one or more perithecia and antheridia, and some appendages (Tavares, 1985; Weir and Beakes, 1995; Haelewaters et al., 2017). These fungi produce ascospores, which are formed in the perithecium and released by pressure, often elicited during contact between hosts (Blackwell, 1980a). The two-celled ascospores are encased in a sticky sheath that attaches to the cuticle of the new host (Fig. 2). In some species, parasitism occurs when cell I penetrates the host cuticle and forms the haustorium to
Fig. 2. Life cycle of Arthrorhynchus [Reconstructed based on slide mounts of A. nycteribiae combined with data from Tavares (1985)].
Patterson, 2006; Dittmar et al., 2015). To avoid this, they can move quickly in any direction. In addition, they have big tarsal claws and combs or ctenidia to secure them from dislodgement from the bat fur both during bat grooming and flight (Humphries, 1967; Dick and Pospischil, 2015). Upon mating, the female bat fly carries a single larva, 2
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Fig. 3. Map with sampling localities in Portugal.
2. Material and methods
draw nourishment from the host (Blackwell, 1980b; Weir and Beakes, 1995), and the cell II forms the foot and later the 3-celled receptacle (Walker et al., 2018). The receptacle bears the carpogenic cell, trichophoric cell and trichogyne, in early stages of development, and the antheridia (Tavares, 1985). Over 2,200 species of Laboulbeniales are known, mostly from insects, but also from arachnids and millipedes (Santamaría et al., 2016, 2017). Approximately 80% of the reported species live on Coleoptera whilst 10% are found on dipterans. The remaining 10% are divided between Acari, Opiliones, Diplopoda, Thysanoptera, Orthoptera, Dermaptera, Blattodea, Mallophaga, Heteroptera and Hymenoptera (Haelewaters et al., 2017; Reboleira et al., 2018; Santamaria et al., 2017; Walker et al., 2018; Weir and Hammond, 1997). Species of several genera of Laboulbeniales are known to be position-specific (Santamaria et al., 2014; Sundberg et al., 2018; Weir and Hammond, 1997); female hosts are infected on the dorsal surface of the abdomen while males are infected on the ventral surface. This pattern is related to the transmission effectuated by the mating behaviour of the hosts (Goldmann, 2015). However, the relative importance of direct and indirect fungal transmission is unknown for most of the Laboulbeniales species (De Kesel, 1995). Bat flies are the only hosts of Laboulbeniales of the genus Arthrorhynchus (Thaxter, 1908; Blackwell, 1980a). Infections by Arthrorhynchus are usually observed as clusters of thalli. The thalli are most frequently present on the membrane between tergites or sternites of the abdomen, or in the genital area (Blackwell, 1980a; Santamaria, 2006). Species of the genus Arthrorhynchus do not show particularly high position specificity, and the fungus may be present on the legs or thorax of host specimens of both sexes. Thus, other types of contacts would be necessary for the direct transmission between body parts outside the genital area (Blackwell, 1980a). There were no published records of bat fly-associated Laboulbeniales in Portugal until 2018 (Jensen et al., 2018; Haelewaters et al., 2018b; Szentiványi et al., 2018). Here, we studied the prevalence of Arthrorhynchus in bat flies from natural populations of cave bats in Portugal in different seasons of the year, and show evidence of the presence of the fungal haustorium within the bat fly host tissue. This is the first study to examine population dynamics of species of the Laboulbeniales genus Arthrorhynchus, and their relationship to fly and bat hosts.
2.1. Sampling and studied material Bat flies were collected at 11 different roost sites (Fig. 3) from 2016 to 2018 during monitoring programs of cave dwelling bats coordinated by the Portuguese Institute for Nature Conservation and Forests (ICNF). SimpleMappr (www.simplemappr.net) was used to produce the distribution map. Sampling was done during bat maternity season (summer), in the autumn migration period and in hibernation season (winter). In the winter, bats were captured directly by hand from the wall and ceiling or with handheld nets (Fig. 4A), while in the summer the bats were captured with harp traps at the cave entrance. Harp traps were set at sunset and were checked for bats every 5–10 min. Bats were kept in cotton bags, 10 individuals per bag until they were examined. The species, sex and age class of each bat (when possible) was recorded. All bats were visually inspected for bat flies, which were removed with forceps and immediately deposited in vials with 96% ethanol (one vial per bat). The vials are deposited in the collection of the Natural History Museum of Denmark (NHMD). 2.2. Bat fly species identification, Laboulbeniales screening and documentation Collected bat flies were identified and screened for presence of Laboulbeniales using a stereomicroscope (Wild M8 Heerbrugg). Presence of Laboulbeniales was photo documented using a Visionary Digital system (http://www.visionarydigital.com), and Adobe Photoshop Lightroom 5.4 (Adobe Inc.; Seattle, WA, USA). Image stacks were combined with Zerene Stacker Version 1.04 (Zerene Systems LLC; Richland, WA, USA). Background of some images was cleaned with Adobe Photoshop CS6 (Adobe Inc.; Seattle, WA, USA). 2.3. Scanning electron microscopy (SEM) A heavily infected female bat fly was transferred to 100% ethanol, critical point-dried in a Tousimis Autosamdri 815 Series A critical point dryer, mounted on an aluminium stub, coated with platinum/palladium and observed under a JEOL JSM-6335F scanning electron microscope (SEM). 3
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Fig. 4. Sampling bats and bat flies. (A) Miniopterus schreibersii captured with a handheld net (February 2018); (B) Myotis blythii; (C) M. schreibersii with Penicillidia conspicua.
2.4. Transmission electron microscopy (TEM)
Table 2 Number of individuals of each bat fly species.
A heavily infected female bat fly preserved in 96% alcohol was prepared for TEM by rinsing in 100% acetone 2 × 20 min. and placed in a 1:1 mix of 100% acetone and Epon 812 on a wave motion platform shaker overnight. The following morning the lid was removed for 2 h to allow the acetone to evaporate. The specimen was transferred to pure Epon 812 and placed on the wave motion platform shaker again for 3 h and subsequently transferred to a mold with Epon 812 and placed in an oven to polymerize for 2 days at 60 °C. Ultrathin sections (70 nm) were made on a Leica V ultratome, placed on single slot grids and contrasted with lead citrate and uranyl acetate. Sections were observed in a JEOL 1010 microscope equipped with a Gatan SC1000 camera.
Bat fly species
N° of individuals
Penicillidia dufourii Penicillidia conspicua Nycteribia schmidlii Nycteribia vexata Nycteribia latreillei Nycteribia kolenatii Total
227 138 34 24 4 1 428
My. escalerai and My. myotis. A total of 428 bat flies were collected, belonging to six species: Penicillidia dufourii, P. conspicua, Nycteribia schmidlii, N. vexata, N. latreillei and N. kolenatii (Tables 1 and 2). Miniopterus schreibersii was the most abundant bat (Fig. 4A, C), followed by My. blythii (Fig. 4B) and My. myotis (Table 1). Among the 428 bat flies screened, only 10 were infected with the hyperparasite A. nycteribiae (Figs. 5 and 6, Table 3) resulting in an overall infection rate of 2.3%. Nine of the 10 infected bat flies were P. conspicua and one was P. dufourii (Table 1). Eight bat flies were collected in autumn (September 2016), 240 collected in summer (July 2016) and 180 collected in winter (January–March 2018). In autumn no Laboulbeniales infected bat flies were found. In winter one infected bat fly was found and in summer nine infected bat flies were found, an infection rate in summer of 3.8% and in winter of 0.6%.
2.5. Differential interference contrast microscopy (DIC) The thalli of the fungus on an infected bat fly specimen were removed using an insect pin and mounted with lactophenol on a slide where it was observed and imaged in a DM2500 Leica microscope with DIC, following the methodology of Santamaría et al. (2018). 3. Results 3.1. Infection rate Bat flies were found on 239 bats belonging to six species: Miniopterus schreibersii, Myotis bechsteinii, My. blythii, My. daubentonii,
Table 1 An overview of number of bat species, number of bat fly species per bat species, and number of infected bat flies. Bat species
N° of individuals (bats)
Bat fly species
N° of individuals (bat flies)
N° of infected bat flies
Miniopterus schreibersii
127
Myotis bechsteinii Myotis blythii
1 59
Myotis daubentonii Myotis escalerai Myotis myotis
1 2 49
Nycteribia schmidlii Penicillidia conspicua Penicillidia dufourii Nycteribia vexata Nycteribia latreillei Nycteribia vexata Penicillidia conspicua Penicillidia dufourii Nycteribia kolenatii Penicillidia dufourii Nycteribia latreillei Nycteribia vexata Penicillidia conspicua Penicillidia dufourii
34 135 7 1 3 12 2 95 1 2 1 11 1 123
0 9 0 0 0 0 0 0 0 0 0 0 0 1
4
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Fig. 5. Arthrorhynchus nycteribiae on DIC. Arrows indicate the host cuticle. Below this point the haustorium continues into the bat fly. (A) Small thallus; (B) Fully developed thallus; (C) Detail of the foot; (D) Thallus with trichogyne. Scale bars: A = 100 μm; B-D = 50 μm.
3.2. Bat flies sex ratio
3.3. Dispersal of infected bat flies
The sex ratio of bat flies observed in the samples was 245 females:183 males (Table 3). Also, more females were infected with the fungus than males (Table 5). Of the 10 infected bat flies, seven were females and three were males, i.e., more females in absolute numbers but a lower percentage of these were infected.
Infected bat flies were found in three of 11 sampling sites: Tomar I, Odemira VII, and Alvaiázere I (Fig. 3, Table 4). All infected bat flies found in summer were from Tomar I and Odemira VII. The only infected bat fly found in winter was from Alvaiázere I.
Fig. 6. SEM images of Arthrorhynchus nycteribiae infecting a female Penicillidia conspicua. (A) Dorsal view; (B) Detail of the group of thalli; (C) Host cuticle with attached thalli; (D) Detail of the foot attached to an intersegmental membrane. Scale bars: A = 1 mm; B = 100 μm; C–D = 10 μm. 5
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flies of the species N. schmidlii were only found on bats of the species Mi. schreibersii (Table 1). The bats My. blythii and My. myotis were both heavily infested with P. dufourii and to a lesser degree with N. vexata. On My. blythii, 84.8% (n = 95) of bat flies were P. dufourii while 10.7% (n = 12) are N. vexata. Similarly, 90.4% (n = 123) of bat flies on My. myotis, were P. dufourii while 8.1% (n = 11) were N. vexata. The bats My. bechsteinii, My. daubentonii, and My. escalerai are not represented in Fig. 7, because they had very low bat fly infestation rates with only one fly on each My. bechsteinii and My. daubentonii, and two on My. escalerai. All fungal infected P. conspicua were found on the bat Mi. schreibersii (Fig. 4C, Table 1). The single specimen of infected P. dufourii was found on the bat My. myotis (Figs. 4B and 7).
Table 3 Numbers of females and males with dorsal, ventral and lateral position of the Laboulbeniales. Sex of bat fly
Position of Laboulbeniales
N° of individuals
Female
Dorsal Ventral Lateral Both ventral and dorsal Dorsal Ventral Lateral Both ventral and dorsal
4 2 1 0 1 1 0 1
Male
Table 4 The relation of sampling sites and infected bat flies.
3.5. Position of the fungus
Official name of roost site
N° of infected bat flies
Species of bat fly
Tomar I
4 1 4 1
P. P. P. P.
Odemira VII Alvaiázere I
The position of the fungus on the bat fly body was variable. Three females were infected dorsally on the left side of abdomen, two were infected ventrally on the left side of abdomen, one was infected dorsally on the right side of abdomen, and one was infected laterally on left mid coxa (Fig. 8A–C). One male was infected dorsally on the right side, one was infected ventrally on the left side, and one was infected on the left side both dorsally and ventrally (Fig. 8D–F, Table 6).
conspicua dufourii conspicua conspicua
Table 5 Number of males and females for each bat fly species. Bat fly species
N° of males
N° of females
P. dufourii P. conspicua N. schmidlii N. vexata N. latreillei N. kolenatii Total
103 60 11 5 3 1 183
124 78 23 19 1 0 245
3.6. Structure of the haustorium Tube-like structures were found in the bat fly tissue just under the fungal thallus (Fig. 9). The tubules were not made of insect cuticle and the structure resembled the thallus (compare Fig. 9 B2, C2 and D2). Furthermore, the presence of cytoplasm inside the structures (Fig. 9B–D) indicate that these structures are not part of the tracheal system of the fly. The haustorium of Arthrorhynchus nycteribiae was apparent inside the host tissue, recognizable because the fungal cell walls show no layering (Fig. 9A).
3.4. Host specificity of bat flies and of infected bat flies Miniopterus schreibersii was primarily infested with two bat fly species: P. conspicua 76.7% (n = 135), N. schmidlii 19.3% (n = 34). Bat
Fig. 7. Percentage of bat fly species for each bat species with multiple infections. 6
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Fig. 8. Penicillidia conspicua, infected with Arthrorhynchus nycteribiae. (A) Female bat fly infected dorsally; (B) Female infected ventrally; (C) Female infected laterally on the left mid coxa; (D) Male infected dorsally; (E) Male infected laterally; (F) Male infected both dorsally and ventrally. Scale bars: 0.5 mm.
the genital area on the dorsal side of females and ventral sides of males but probably not for infections in other body parts (Blackwell, 1980a). However, it is possible that other body parts of the male and female are in contact during courtship, which could explain patterns found where the thorax of the male touches the female. Further behavioural studies will shed light on the transmission mechanisms. Infection elsewhere on the body of bat flies is most likely to happen randomly by direct contact between bat flies due to their limited microhabitat (the host bat surface).
Table 6 Overview of sex of bat fly with indicated position and side of Laboulbeniales. Bat fly ID
Sex of bat fly
Position of Laboulbeniales
Side of Laboulbeniales
S91 S86 W39 S58 S66 S47 S92 S85 S51 S59
Female Female Female Female Female Female Female Male Male Male
Dorsal abdomen Dorsal abdomen Dorsal abdomen Ventral abdomen Ventral abdomen Dorsal abdomen Lateral thorax (coxa) Dorsal abdomen Ventral abdomen Ventral abdomen Dorsal abdomen
Left Left Left Left Left Right Left Right Left Left Left
4.2. Fungal distribution We record for the first time the presence of the fungus Arthrorhynchus nycteribiae in central Portugal. This species was recently found in the southernmost province of the country, the Algarve, an area clearly marked by a strong Mediterranean influence (Szentiványi et al., 2018). Arthrorhynchus nycteribiae was previously reported from Australia, Africa (Kenya, Zambia), Asia (Sri Lanka), and across Europe (Austria, Bulgaria, Croatia, Czech Republic, Denmark, France, Hungary, Italy, Netherlands, Poland, Romania, Russia, Serbia, Slovakia, Spain, Sweden, Switzerland) (Blackwell, 1980a; Haelewaters et al., 2017).
4. Discussion 4.1. Seasonal infection rates Bat flies infected with Laboulbeniales exhibit a generally low infection prevalence (Blackwell, 1980a; Haelewaters et al., 2017). In this study we found an overall prevalence of 2.3%, with fewer infections in winter. Bat flies collected in summer yielded an infection rate of 3.8%, contrasting to 0.6% in winter samples. Blackwell (1980a) screened 2,517 bat flies from 404 entomological collections for the presence of Laboulbeniales and found 56 infected individuals, leading to an infection rate of 2.2%. Data on season was not included. Haelewaters et al. (2017) found an infection rate of 3.0% in 1,494 bat flies collected in Hungary and Romania, which is again remarkably similar to what we report here. Again, data on season was not included. Szentiványi et al. (2018) found an infection rate of 9% (in 667 bat flies collected in Albania, Croatia, Hungary, Italy, Portugal, Slovakia, Spain and Switzerland). These bat flies were collected in the summer period (April–September), which could explain the higher infection rate. The lower infection rate in winter may be explained by the hibernation of bats, when the bat flies show a decreased level of activity and, therefore, decreased contact. Some tropical bat flies are known to mate year-round, but this might not be the case for bat flies on hibernating bats (Blackwell 1980a). It is known that the reproductive cycle of bat flies is synchronized with the reproductive cycle of their bat hosts for this geographic region (Lourenço and Palmeirim, 2008). Blackwell (1980a) states that transmission probably is not highly dependent on mating. Copulation of bat flies happens by the male mounting the female, which relates directly to fungal transmission in
4.3. Female-biased sex ratio Ectoparasitic insects typically display a sex ratio biased toward females (Dick and Patterson, 2008), confirmed in our study. This is especially observed among permanent parasites and groups of temporary ectoparasites; bat flies are permanent parasites. Females and males apparently are produced in equal numbers but, because of greater longevity in females, the sex ratio is skewed (Marshall, 1981; Dick and Patterson, 2008). In a study on the bat fly Basilia hispida Theodor, 1967, males were found to have an approximate 97-day life-span versus 156 days for females (Haelewaters et al., 2017). Another factor permitting a female-biased sex ratio could be sampling bias. The females of some bat fly species are larger than males, especially during pregnancy. In addition, females may be more sedentary while males may be more active (Marshall, 1981; Dick and Patterson, 2008), leading to a sampling bias towards excess of females. Climate may also affect this bias. A study by Hurka (1964) on N. latreillei showed that the female-biased sex ratio increased towards the north, possibly because females cope better with the colder climate (Marshall, 1981). Females also showed a higher Laboulbeniales infection rate, possibly because of their larger size, especially during pregnancy (larger surface area bringing a higher probability of contact with spores), or 7
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Fig. 9. TEM image of the bat fly Penicillidia conspicua tissue with haustorium of Arthrorhynchus nycteribiae. (A) White circles show sections of the fungal haustorium in the bat fly tissue. (B1, B2 and C1, C2) Details of the fungal haustorium. (D1, D2) Cell wall of fungal haustorium. Scale bars: A, B1, C1 = 5 μm; D1 = 2,5 μm; B2, C2, D2 = 200 μm.
which correlates with earlier studies where P. conspicua is the most common host for A. nycteribiae (Blackwell, 1980a; Haelewaters et al., 2017). Some Laboulbeniales species, including those in the genus Arthrorhynchus, can infect several host species (Haelewaters et al., 2017), and Blackwell (1980a) found two species A. nycteribiae and A. eucampsipodae on several genera of Nycteribiinae. The high host specificity for bat species is exhibited among nycteribiid bat flies and also observed in studies on the streblid bat flies (Dick, 2007; Dick and Patterson, 2007). High host specificity is a common trait in parasitic species with low dispersal capabilities, an evolutionary effect from low probability of encounters with other potential hosts, or due to acquired structural or immunological incompatibility (Dick, 2007). The hosts we studied are cave-dwelling bats that hibernate in winter. The roosts are often shared by several bat species, and colonies of different species can be adjacent or even intermixed (Rodrigues et al., 2003). The fact that the bat fly puparium is glued to the walls or ceiling of the roost (not on the host body), should make bat flies prone to dispersal to other host species using the same roosts. Strong position-specificity has not been observed in Arthrorhynchus. The most common positions were in the membrane between two sclerites or between tergites/sternites on the abdomen or in the genital region. Several specimens are found to be infected on legs or thorax (Blackwell, 1980a), indicating that transmission does not rely solely on mating behavior, but also on other types of host contacts, or even via the substrate (bat fur). Cave bats live in a high humidity environment, which might allow for the survival of ascospores released in the bat fur until another bat fly crawls by.
longevity that increases the exposure time for infection. Pregnant females store large fat reserves that are of high nutritional value for the fungus (Haelewaters et al., 2017), but this is not relevant because the fungus cannot actively select its host. It also possibly can be linked with sexual transmission and differences in male and female behaviour. Males mate with several females, so if the fungus is primarily transmitted during mating, each infected male produces multiple infected females. Males are more active than females (often the case in insects), which would make males more prone to being lost through host grooming and explaining the biased sex ratio. Also, high male activity may allow males to spread the fungus to several females even if not accepted as mates by all of them. Haelewaters et al. (2017)Haelewaters et al. (2017) hypothesized that the fungus develops slowly, in which case female bat flies may provide a more long-lived host for development of the fungus than males, and a long-lived female can infect multiple males. This, however, contrasts with a study by Blackwell (1980a), who hypothesized that Arthrorhynchus has a relatively short life cycle since thalli found in clusters had several stages of maturity. 4.4. Low dispersal Infected bat flies were found at three of 11 sampling sites. The dispersal of the fungus between different bat roosts is dependent on the dispersal of infected bat flies during bat migrations, known to occur in this area in spring and autumn (Rodrigues et al., 2010). 4.5. Host and position specificity In our study all bat flies collected were infected with A. nycteribiae, 8
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4.6. Structure of the haustorium
Krasnov, B., Poulin, R. (Eds.), Micromammals and Macroparasites: From Evolutionary Ecology to Management. Springer, Tokyo, pp. 179–194. https://doi. org/10.1007/978-4-431-36025-4_11. Dick, C.W., Patterson, B.D., 2007. Against all odds: Explaining high host specificity in dispersal-prone parasites. Int. J. Parasitol. 37, 871–876. https://doi.org/10.1016/j. ijpara.2007.02.004. Dick, C.W., Patterson, B.D., 2008. An excess of males: skewed sex ratios in bat flies (Diptera: Streblidae). Evol. Ecol. 22 (6), 757–769. https://doi.org/10.1007/s10682007-9201-9. Dick, C.W., Pospischil, R., 2015. Nycteribiidae (bat flies). In: Mehlhorn, H. (Ed.), Encyclopedia of Parasitology. Springer-Verlag, Berlin. https://doi.org/10.1007/9783-642-27769-6_3462-1. Dittmar, K., Porter, M.L., Murray, S., Whiting, M.F., 2006. Molecular phylogenetic analysis of nycteribiid and streblid bat flies (Diptera: Brachycera, Calyptratae): Implications for host associations and phylogeographic origins. Mol. Phylogenet. Evol. 38, 155–170. Dittmar, K., Morse, S., Dick, C.W., Patterson, B.D., 2015. Bat fly evolution from the eocene to the present (Hippoboscoidea, Streblidae and Nycteribiidae). In: Morand, S., Krasnov, B., Littlewood, T. (Eds.), Parasite Diversity and Diversification: Evolutionary Ecology Meets Phylogenetics. Cambridge University Press, Cambridge, pp. 246–264. https://doi.org/10.1017/CBO9781139794749.015. Fenton, M.B., Simmons, N.B., 2014. Bats: A World of Science and Mystery. University of Chicago Press, Chicago. Goldmann, L.M., 2015. Investigations into position specificity and the molecular systematics of the Laboulbeniomycetes. PhD-thesis from State University of New York, Syracuse, New York. Published by ProQuest LLC. Haelewaters, D., Pfliegler, W.P., Szentiványi, T., Földvári, M., Sándor, A.D., Barti, L., Camacho, J.J., Gort, G., Estók, P., Hiller, T., Dick, C.W., Pfister, D.H., 2017. 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Order DIPTERA Linnaeus, 1758. In: Zhang, Z.-Q. (Ed.), Animal Biodiversity: An Outline of Higher-level Classification and Survey of Taxonomic Richness. Zootaxa, pp. 222–229. Petersen, F.T., Meier, R., Kutty, S.N., Wiegmann, B.M., 2007. The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers. Mol. Phylogenet. Evol. 45 (1), 111–122. https://doi.org/10. 1016/j.ympev.2007.04.023. Reboleira, A.S.P.S., Enghoff, H., Santamaria, S., 2018. Novelty upon novelty visualized by rotational scanning electron micrographs (rSEM): Laboulbeniales on the millipede order Chordeumatida. PLoS ONE 13 (11), e0206900. https://doi.org/10.1371/ journal.pone.0206900. Rodrigues, L., Zahn, A., Rainho, A., Palmeirim, J., 2003. Contrasting the roosting behaviour and phenology of an insectivorous bat (Myotis myotis) in its southern and northern distribution ranges. Mammalia 67 (3), 321–336. Rodrigues, L., Palmeirim, J., 2008. 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Despite ethanol fixation of flies, it was possible to observe parts of the haustorium inside the fly with TEM. This is the first record of haustorial sections inside the body cavity of a bat fly. Furthermore, between the fungal cell wall layer and the cuticle of the host there is a dark region, also observed by Blackwell (1980b) and described as a carbonaceous patch. Blackwell (1980b) proposed that the dark region may be due to growth of the fungus and breakdown of the surrounding cuticle of the bat fly), but it is likely to be a defence response to infection of the bat fly because melanization is an ancestral response to wounding in arthropods, even conserved in albino cave species (Bilandžija et al., 2017). 5. Future perspectives Knowledge of the biology of Laboulbeniales is still very immature (Haelewaters et al., 2017, 2018a). More research on the potential for seasonal differences in infection rates is needed to elucidate the effect of bat hibernation on both bat flies and fungus (Blackwell, 1980a). Furthermore, understanding of behavioural transmission of the fungus between bat flies is still incomplete because transmission may be associated with behaviours other than mating of the bat flies (Blackwell, 1980a). In addition, the structure of the haustorium of Arthrorhynchus, the response to infection and effect of infection on the bat flies should be further examined (Blackwell, 1980a). Lastly, many Laboulbeniales species are cryptic and difficult to distinguish based on morphological characters. For this, a molecular phylogenetic approach should be applied to clarify the taxonomic status (Haelewaters et al., 2017) and to understand the genetic structure of populations. Declaration of Competing Interest There are no conflicts of interest to be declared. Acknowledgements We are grateful to Jonas Winding Christensen (Erhvervsakademi Sjælland) for illustrations of life cycles, to the Portuguese Institute for Nature Conservation and Forests (ICNF), to Paulo Barros (University of Trás-os-Montes e Alto Douro, Portugal) and to Margarida Augusto (BioInsight, Portugal) for support in collecting nycteribiids under the National Bats Monitoring Program in Portugal, and to Lis Munk Frederiksen (Biological Institute of Copenhagen University) for help with TEM preparation. All the specimens were collected under permits of the ICNF. ASR is supported by a research grant (15471) from the VILLUM FONDEN. References Bilandžija, H., Laslo, M., Porter, M.L., Fong, D.W., 2017. Melanization in response to wounding is ancestral in arthropods and conserved in albino cave species. Sci. Rep. 7 (1), 17148. https://doi.org/10.1038/s41598-017-17471-2. Blackwell, M., 1980a. Incidence, host specificity, distribution and morphological variation in Arthrorhynchus nyctyeribiae and Arthrorhynchus eucampsipodae (Laboulbeniomycetes). Mycologia 72 (1), 143–158. https://doi.org/10.2307/ 3759427. Blackwell, M., 1980b. Developmental Morphology and Taxonomic Characters of Arthrorhynchus Nycteribiae and Arthrorhynchus eucampsipodae (Laboulbeniomycetes). Mycologia 72 (1), 159–168. https://doi.org/10.2307/3759428. Benjamin, R.K., 1971. Introduction and supplement to Roland Thaxter’s contribution toward a monograph of the Laboulbeniaceae. Biblioth. Mycol. 30, 1–155. Crofton, H., 1971. A model of host–parasite relationships. Parasitol. 63 (3), 343–364. https://doi.org/10.1017/S0031182000079890. De Kesel, A., 1995. Relative importance of direct and indirect infection in the transmission of Laboulbenia slackensis (Ascomycetes, Laboulbeniales). Belgian J. Bot. 128 (2), 124–130. Dick, C.W., 2007. High host specificity of obligate ectoparasites. Ecol. Entomol. 32, 446–450. https://doi.org/10.1111/j.1365-2311.2007.00836.x. Dick, C.W., Patterson, B.D., 2006. Bat flies: obligate ectoparasites of bats. In: Morand, S.,
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Nycteribiidae) from Miniopterus schreibersii across Europe. Parasit. Vectors. 11 (1), 395. https://doi.org/10.1186/s13071-018-2921-6. Tavares, I.I., 1985. Laboulbeniales (Fungi, Ascomycetes), Mycologia Memoir no. 9, J. Cramer, Braunschweig. Thaxter, R., 1896. Contribution towards a monograph of the Laboulbeniaceae. Mem. Am. Acad. Arts Sci. 12, 187–429. Thaxter, R., 1908. Contribution toward a Monograph of the Laboulbeniaceæ: Part II. Mem. Am. Acad. Arts Sci., New Series 13 (6), 219–469. https://doi.org/10.2307/ 25058090. Thaxter, R.R., 1931. Contribution towards a monograph of the Laboulbeniaceae. Part V. Mem. Am. Acad. Arts Sci., New Series 16, 1–435. Walker, M.J., Dorrestein, A., Camacho, J.J., Meckler, L.A., Silas, K.A., Hiller, T., Haelewaters, D., 2018. A tripartite survey of hyperparasitic fungi associated with ectoparasitic flies on bats (Mammalia: Chiroptera) in a neotropical cloud forest in Panama. Parasit. 25, 19. https://doi.org/10.1051/parasite/2018017. Weir, A., Beakes, G., 1995. An introduction to the Laboulbeniales: A fascinating group of entomogenous fungi. Top. Catal. 9 (1), 6–10. https://doi.org/10.1016/S0269-915X (09)80238-3. Weir, A., Hammond, P.M., 1997. Laboulbeniales on beetles: host utilization patterns and species richness of the parasites. Biodivers. Conserv. 6, 701–719. https://doi.org/10. 1023/A:1018318320019.
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