Using community science data to assess the association between urbanization and the presence of invasive Aedes species in Hungary.

BACKGROUND: Urbanization can be a significant contributor to the spread of invasive mosquito vector species, and the diseases they carry, as urbanized habitats provide access to a great density of food resources (humans and domestic animals) and offer abundant breeding sites for these vectors. Although anthropogenic landscapes are often associated with the presence of invasive mosquito species, we still have little understanding about the relationships between some of these and the built environment. METHODS: This study explores the association between urbanization level and the occurrence of invasive Aedes species, specifically Aedes albopictus, Aedes japonicus, and Aedes koreicus, in Hungary, using data from a community (or citizen) science program undertaken between 2019 and 2022. RESULTS: The association between each of these species and urbanized landscapes within an extensive geographic area was found to differ. Using the same standardized approach, Ae. albopictus showed a statistically significant and positive relationship with urbanization, whereas Ae. japonicus and Ae. koreicus did not. CONCLUSIONS: The findings highlight the importance of community science to mosquito research, as the data gathered using this approach can be used to make qualitative comparisons between species to explore their ecological requirements.

Vector-borne protozoan and bacterial pathogen occurrence and diversity in ectoparasites of the Egyptian Rousette bat.

Bats are known reservoir hosts for a wide variety of parasites and pathogens, including bacteria and protozoans. Some of these pathogens are vector-borne, and although their role is poorly studied, ectoparasites may contribute significantly to their transmission. The aim of this study was to molecularly detect the presence of vector-borne microorganisms in bat-associated ectoparasites to explore their diversity and distribution in these insects. We tested the presence of Bartonella spp., Polychromophilus spp., and Trypanosoma spp. in bat flies and bat fleas collected from 56 Egyptian Rousette bats (Rousettus aegyptiacus), using conventional PCR. We found a high prevalence of 43.9% (47/107) of Bartonella spp. in bat flies, but a low prevalence of 6.6% (4/61) in bat fleas. Polychromophilus and Trypanosoma DNA were absent in both bat flies and bat fleas. Furthermore, we found novel gltA Bartonella sequences, as well as genotypes that are highly similar to recently described and potentially zoonotic ones. Our results show high diversity of Bartonella in bat flies, however, their role in pathogen transmission is still unknown and should be further explored.

Polyctenidae (Hemiptera: Cimicoidea) species in the Afrotropical region: Distribution, host specificity, and first insights to their molecular phylogeny.

Polyctenidae bugs are rarely studied, hematophagous, and highly specialized ectoparasites of bats. There are only 32 described species worldwide, including six species in the Afrotropical region. Knowledge on these parasites is limited, and most studies are restricted to the New World polyctenid species. Here we report additional records of Adroctenes horvathi from Kenya and South Africa, as well as Hypoctenes faini from Rwanda. We present an updated list of published polyctenid records in the Afrotropical region indicating their host specificity and their geographical distribution. We report global infection patterns and sex ratio of polyctenids based on previously published data, including Old and New World species. Lastly, we demonstrate the first molecular phylogeny of Polyctenidae, showing their phylogenetic relationship with the closely related family Cimicidae.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary.

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.

Latrocimicinae completes the phylogeny of Cimicidae: meeting old morphologic data rather than modern host phylogeny.

The family Cimicidae includes obligate hematophagous ectoparasites (bed bugs and their relatives) with high veterinary/medical importance. The evolutionary relationships of Cimicidae and their hosts have recently been reported in a phylogenetic context, but in the relevant study, one of the six subfamilies, the bat-specific Latrocimicinae, was not represented. In this study the only known species of Latrocimicinae, i.e., Latrocimex spectans, was analyzed with molecular and phylogenetic methods based on four (two nuclear and two mitochondrial) genetic markers. The completed subfamily-level phylogeny of Cimicidae showed that Latrocimicinae is most closely related to Haematosiphoninae (ectoparasites of birds and humans), with which it shares systematically important morphologic characters, but not hosts. Moreover, in the phylogenetic analyses, cimicid bugs that are known to infest phylogenetically distant bat hosts clustered together (e.g., Leptocimex and Stricticimex within Cacodminae), while cimicid subfamilies (Latrocimicinae, Primicimicinae) that are known to infest bat hosts from closely related superfamilies clustered distantly. In conclusion, adding Latrocimicinae significantly contributed to the resolution of the phylogeny of Cimicidae. The close phylogenetic relationship between Latrocimicinae and Haematosiphoninae is consistent with long-known morphologic data. At the same time, phylogenetic relationships of genera within subfamilies are inconsistent with the phylogeny of relevant hosts.

Host conservation through their parasites: molecular surveillance of vector-borne microorganisms in bats using ectoparasitic bat flies.

Most vertebrates host a wide variety of haematophagous parasites, which may play an important role in the transmission of vector-borne microorganisms to hosts. Surveillance is usually performed by collecting blood and/or tissue samples from vertebrate hosts. There are multiple methods to obtain samples, which can be stored for decades if properly kept. However, blood sampling is considered an invasive method and may possibly be harmful to the sampled individual. In this study, we investigated the use of ectoparasites as a tool to acquire molecular information about the presence and diversity of infectious microorganism in host populations. We tested the presence of three distinct vector-borne microorganisms in both bat blood and bat flies: Bartonella bacteria, malaria-like Polychromophilus sp. (Apicomplexa), and Trypanosoma sp. (Kinetoplastea). We detected the presence of these microorganisms both in bats and in their bat flies, with the exception of Trypanosoma sp. in South African bat flies. Additionally, we found Bartonella sp. in bat flies from one population in Spain, suggesting its presence in the host population even if not detected in bats. Bartonella and Polychromophilus infection showed the highest prevalence in both bat and bat fly populations. Single, co- and triple infections were also frequently present in both. We highlight the use of haematophagous ectoparasites to study the presence of infectious microorganism in host blood and its use as an alternative, less invasive sampling method.

TITLE: Conservation des hôtes grâce à leurs parasites : surveillance moléculaire des microorganismes à transmission vectorielle chez les chauves-souris à l'aide de mouches ectoparasites. ABSTRACT: La plupart des vertébrés hébergent une grande variété de parasites hématophages, qui peuvent jouer un rôle important dans la transmission de microorganismes à transmission vectorielle à leurs hôtes. La surveillance est généralement effectuée en prélevant des échantillons de sang et/ou de tissus sur des hôtes vertébrés. Il existe plusieurs méthodes pour obtenir des échantillons, qui peuvent être conservés pendant des décennies dans des bonnes conditions. Cependant, le prélèvement sanguin est considéré comme une méthode invasive et peut éventuellement être nocif pour l'individu prélevé. Dans cette étude, nous avons étudié l'utilisation d'ectoparasites comme outil pour acquérir des informations moléculaires sur la présence et la diversité des microorganismes infectieux dans les populations hôtes. Nous avons testé la présence de trois microorganismes distincts, transmis par des vecteurs, dans le sang et les mouches des chauves-souris : les bactéries Bartonella, Polychromophilus sp. (Apicomplexa) et Trypanosoma sp. (Kinetoplastea). Nous avons détecté la présence de ces microorganismes à la fois chez les chauves-souris et chez leurs mouches des chauves-souris, à l'exception de Trypanosoma sp. chez les chauves-souris sud-africaines. De plus, nous avons trouvé Bartonella sp. chez les mouches des chauves-souris d'une population en Espagne, ce qui suggère sa présence dans la population hôte même si elle n'est pas détectée chez les chauves-souris elles-mêmes. Les infections à Bartonella et Polychromophilus ont montré la prévalence la plus élevée dans les populations de chauves-souris et de mouches des chauves-souris. Des infections simples, doubles et triples étaient également fréquemment présentes dans les deux cas. Nous mettons en évidence l'utilisation d'ectoparasites hématophages pour étudier la présence de microorganismes infectieux dans le sang de l'hôte et son utilisation comme méthode alternative et moins invasive d'échantillonnage.

On the Fly: Tritrophic Associations of Bats, Bat Flies, and Fungi.

Parasitism is one of the most diverse and abundant modes of life, and of great ecological and evolutionary importance. Notwithstanding, large groups of parasites remain relatively understudied. One particularly unique form of parasitism is hyperparasitism, where a parasite is parasitized itself. Bats (Chiroptera) may be parasitized by bat flies (Diptera: Hippoboscoidea), obligate blood-sucking parasites, which in turn may be parasitized by hyperparasitic fungi, Laboulbeniales (Ascomycota: Laboulbeniomycetes). In this study, we present the global tritrophic associations among species within these groups and analyze their host specificity patterns. Bats, bat flies, and Laboulbeniales fungi are shown to form complex networks, and sixteen new associations are revealed. Bat flies are highly host-specific compared to Laboulbeniales. We discuss possible future avenues of study with regard to the dispersal of the fungi, abiotic factors influencing the parasite prevalence, and ecomorphology of the bat fly parasites.

Presence and diversity of Chlamydiae bacteria in Spinturnix myoti, an ectoparasite of bats.

Chlamydia spp. and Chlamydia-like organisms are able to infect vertebrates such as mammals, reptiles and birds, but also arthropods and protozoans. Since they have been detected in bats and bat feces, we expected Chlamydiae bacteria to also be present in the mite Spinturnix myoti, an ectoparasite of mouse-eared bats (Myotis spp.). The prevalence of Chlamydiales in 88 S. myoti was 57.95% and significantly depended on bat host species. In addition, the prevalence was significantly different between bat species living in sympatry or in allopatry. While there was uninterpretable sequencing for 16 samples, eight showed best BLAST hit identities lower than 92.5% and thus corresponded to new family-level lineages according to the established taxonomy cut-off. The four remaining sequences exhibited best BLAST hit identities ranging from 94.2 to 97.4% and were taxonomically assigned to three different family-level lineages, with two of them belonging to the Parachlamydiaceae, one to the Simkaniaceae, and one to the Chlamydiaceae. These results highlighted for the first time the presence of Chlamydia-like organisms and the possible zoonotic origin of Chlamydia sp. in S. myoti ectoparasites of bats, and therefore suggest that these ectoparasites may play a role in maintaining and/or transmitting members of the Chlamydiae phylum within Myotis spp. bat populations. Our results further highlight that the wide diversity of bacteria belonging to the Chlamydiae phylum is largely underestimated.

TITLE: Présence et diversité des bactéries Chlamydiae chez Spinturnix myoti, un acarien ectoparasite de chauve-souris. ABSTRACT: Les Chlamydia spp. et les organismes apparentés aux Chlamydia sont capables d'infecter des vertébrés tels que les mammifères, les reptiles et les oiseaux mais aussi des arthropodes et des protozoaires. Puisqu'elles ont été détectées dans des chauves-souris et des excréments de chauves-souris, nous nous attendions à ce que les bactéries du phylum Chlamydiae soient également présentes dans des Spinturnix myoti, des acariens ectoparasites de chauves-souris du groupe des murins (Myotis spp.). La prévalence des Chlamydiales dans 88 S. myoti était de 57,95 % et dépendait de manière significative des espèces hôtes de chauves-souris. De plus, la prévalence était significativement différente entre les chauves-souris vivant en sympatrie ou en allopatrie. Alors qu'il y avait un séquençage ininterprétable pour 16 échantillons, huit présentaient des résultats d'analyse de type de type BLAST avec une similarité inférieure à 92.5% et à 92,5 % et correspondaient donc à de nouvelles familles selon les seuils utilisés en taxonomie par les chlamydiologistes. Les quatre séquences restantes présentaient des résultats BLAST allant de 94,2 à 97,4 % et ont été taxonomiquement attribuées à trois familles ; deux d'entre elles appartenant aux Parachlamydiaceae, une aux Simkaniaceae et enfin une aux Chlamydiaceae. Ces résultats ont mis en évidence pour la première fois la présence d'organismes de type Chlamydia mais aussi d'organisme pouvant amener à des zoonoses tel que Chlamydia sp. chez Spinturnix myoti, un ectoparasite de chauves-souris. Ces résultats suggèrent donc que ces ectoparasites pourraient jouer un rôle dans le maintien et/ou la transmission des membres de l'embranchement des Chlamydiae au sein des populations de chauves-souris du genre Myotis. Nos résultats soulignent en outre que la grande diversité des bactéries appartenant à l'embranchement des Chlamydiae est largement sous-estimée.

Effects of fungal infection on the survival of parasitic bat flies.

BACKGROUND: Parasites are able to alter numerous aspects of their hosts' life history, behaviour and distribution. One central question in parasitology is to determine the degree of impact that parasites have on their hosts. Laboulbeniales (Fungi: Ascomycota) are ectoparasitic fungi of arthropods. Even though these fungi are widely distributed, little is known about their ecology and their possible physiological effects on their hosts. We used a highly specific bat fly-fungi association to assess the effect of these fungal parasites on their dipteran hosts. METHODS: We collected bat flies (Diptera: Nycteribiidae) belonging to two species, Nycteribia schmidlii and Penicillidia conspicua from their bat host Miniopterus schreibersii (Chiroptera: Miniopteridae). We experimentally tested the effect of infection on the lifespan of bat flies. RESULTS: The prevalence of Laboulbeniales fungi was 17.9% in N. schmidlii and 64.8% in P. conspicua. Two fungi species were identified, Arthrorhynchus eucampsipodae and A. nycteribiae, both showing strict host specificity with N. schmidlii and P. conspicua, respectively. We found that fungal infection reduced by half the survival rate of P. conspicua regardless of sex, whereas N. schmidlii was not affected by the infection. Moreover, the intensity of infection showed negative correlation with the lifespan of P. conspicua. CONCLUSIONS: To our knowledge, this is the first indication that fungal infection can alter bat fly survival and thus may play a significant role in the population dynamics of these bat ectoparasites.

Bat Flies and Their Microparasites: Current Knowledge and Distribution.

Bats are the second most diverse mammalian group, playing keystone roles in ecosystems but also act as reservoir hosts for numerous pathogens. Due to their colonial habits which implies close contacts between individuals, bats are often parasitized by multiple species of micro- and macroparasites. The particular ecology, behavior, and environment of bat species may shape patterns of intra- and interspecific pathogen transmission, as well as the presence of specific vectorial organisms. This review synthetizes information on a multi-level parasitic system: bats, bat flies and their microparasites. Bat flies (Diptera: Nycteribiidae and Streblidae) are obligate, hematophagous ectoparasites of bats consisting of ~500 described species. Diverse parasitic organisms have been detected in bat flies including bacteria, blood parasites, fungi, and viruses, which suggest their vectorial potential. We discuss the ecological epidemiology of microparasites, their potential physiological effects on both bats and bat flies, and potential research perspectives in the domain of bat pathogens. For simplicity, we use the term microparasite throughout this review, yet it remains unclear whether some bacteria are parasites or symbionts of their bat fly hosts.

Laboulbeniales (Fungi: Ascomycota) infection of bat flies (Diptera: Nycteribiidae) from Miniopterus schreibersii across Europe.

BACKGROUND: Bat flies (Diptera: Nycteribiidae and Streblidae) are obligate, blood-sucking ectoparasites of bats with specialized morphology, life-cycle and ecology. Bat flies are occasionally infected by different species of Laboulbeniales (Fungi: Ascomycota), microscopic fungal ectoparasites belonging to three genera: Arthrorynchus spp. are restricted to the Eastern Hemisphere, while species of Gloeandromyces and Nycteromyces occur on Neotropical bat flies. Little is known about the distribution and host specificity of Arthrorynchus spp. on bat flies. In this study, we focused on sampling bat flies from the cave-dwelling bat species Miniopterus schreibersii. Bat and ectoparasite collection took place in Albania, Croatia, Hungary, Italy, Portugal, Slovakia, Spain and Switzerland. Flies were inspected for Laboulbeniales infections. RESULTS: Six hundred sixty seven bat flies of five species were collected: Nycteribia latreillii, N. pedicularia, N. schmidlii, Penicillidia conspicua, and P. dufourii. Laboulbeniales infection was observed on 60 specimens (prevalence = 9%). Two Laboulbeniales species, Arthrorhynchus eucampsipodae and A. nycteribiae, were present on three bat fly species. All observations of A. eucampsipodae were on N. schmidlii, and A. nycteribiae was present on P. conspicua and P dufourii. Arthrorhynchus eucampsipodae is, for the first time, reported from Slovakia and Spain. Arthrorhynchus nycteribiae represents a new country record for Portugal and Slovakia. There were no significant differences among infection rates in different countries. Females of N. schmidlii showed a higher infection rate than males with an observable trend (P = 0.0502). No sex differences in infection rate for P. conspicua and P. dufourii were detected. Finally, thallus density was significantly lower in N. schmidlii compared to P. conspicua and P. dufourii. CONCLUSIONS: With this study, we contribute to the knowledge of the geographical distribution and host specificity of Laboulbeniales fungi associated with ectoparasitic bat flies within Europe. We discuss parasite prevalence and host specificity in the light of our findings and the available literature. Penicillidia conspicua is unambiguously the main host species for A. nycteribiae based on our and previous findings. Differences in parasite intensity and sex-biased infections of the fungi are possible depending on the species.

Density-dependent sex ratio and sex-specific preference for host traits in parasitic bat flies.

BACKGROUND: Deviation of sex ratios from unity in wild animal populations has recently been demonstrated to be far more prevalent than previously thought. Ectoparasites are prominent examples of this bias, given that their sex ratios vary from strongly female- to strongly male-biased both among hosts and at the metapopulation level. To date our knowledge is very limited on how and why these biased sex ratios develop. It was suggested that sex ratio and sex-specific aggregation of ectoparasites might be shaped by the ecology, behaviour and physiology of both hosts and their parasites. Here we investigate a highly specialised, hematophagous bat fly species with strong potential to move between hosts, arguably limited inbreeding effects, off-host developmental stages and extended parental care. RESULTS: We collected a total of 796 Nycteribia kolenatii bat flies from 147 individual bats using fumigation and subsequently determined their sex. We report a balanced sex ratio at the metapopulation level and a highly variable sex ratio among infrapopulations ranging from 100% male to 100% female. We show that infrapopulation sex ratio is not random and is highly correlated with infrapopulation size. Sex ratio is highly male biased in small and highly female biased in large infrapopulations. We show that this pattern is most probably the result of sex-specific preference in bat flies for host traits, most likely combined with a higher mobility of males. We demonstrate that female bat flies exert a strong preference for high host body condition and female hosts, while the distribution of males is more even. CONCLUSIONS: Our results suggest that locally biased sex ratios can develop due to sex-specific habitat preference of parasites. Moreover, it is apparent that the sex of both hosts and parasites need to be accounted for when a better understanding of host-parasite systems is targeted.

Parasites of parasites of bats: Laboulbeniales (Fungi: Ascomycota) on bat flies (Diptera: Nycteribiidae) in central Europe.

BACKGROUND: Bat flies (Streblidae and Nycteribiidae) are among the most specialized families of the order Diptera. Members of these two related families have an obligate ectoparasitic lifestyle on bats, and they are known disease vectors for their hosts. However, bat flies have their own ectoparasites: fungi of the order Laboulbeniales. In Europe, members of the Nycteribiidae are parasitized by four species belonging to the genus Arthrorhynchus. We carried out a systematic survey of the distribution and fungus-bat fly associations of the genus in central Europe (Hungary, Romania). RESULTS: We encountered the bat fly Nycteribia pedicularia and the fungus Arthrorhynchus eucampsipodae as new country records for Hungary. The following bat-bat fly associations are for the first time reported: Nycteribia kolenatii on Miniopterus schreibersii, Myotis blythii, Myotis capaccinii and Rhinolophus ferrumequinum; Penicillidia conspicua on Myotis daubentonii; and Phthiridium biarticulatum on Myotis capaccinii. Laboulbeniales infections were found on 45 of 1,494 screened bat flies (3.0%). We report two fungal species: Arthrorhynchus eucampsipodae on Nycteribia schmidlii, and A. nycteribiae on N. schmidlii, Penicillidia conspicua, and P. dufourii. Penicillidia conspicua was infected with Laboulbeniales most frequently (25%, n = 152), followed by N. schmidlii (3.1%, n = 159) and P. dufourii (2.0%, n = 102). Laboulbeniales seem to prefer female bat fly hosts to males. We think this might be due to a combination of factors: female bat flies have a longer life span, while during pregnancy female bat flies are significantly larger than males and accumulate an excess of fat reserves. Finally, ribosomal DNA sequences for A. nycteribiae are presented. CONCLUSIONS: We screened ectoparasitic bat flies from Hungary and Romania for the presence of ectoparasitic Laboulbeniales fungi. Arthrorhynchus eucampsipodae and A. nycteribiae were found on three species of bat flies. This study extends geographical and host ranges of both bat flies and Laboulbeniales fungi. The sequence data generated in this work contribute to molecular phylogenetic studies of the order Laboulbeniales. Our survey shows a complex network of bats, bat flies and Laboulbeniales fungi, of which the hyperparasitic fungi are rare and species-poor. Their host insects, on the other hand, are relatively abundant and diverse.

Checklist of host associations of European bat flies (Diptera: Nycteribiidae, Streblidae).

Bat flies are obligate blood-sucking ectoparasites of bats. They are divided into two families: Nycteribiidae and Streblidae. Europe has 17 species of bat flies and 45 species of bats (Mammalia: Chiroptera). This checklist is based on both published records and our own field data and provides updated information on all associations between bat flies and their hosts in Europe. The host-parasite association between Basilia italica Theodor and Plecotus auritus (Linnaeus) is reported for the first time. Moreover, our records of B. italica on Myotis alcathoe Helversen & Heller, B. nana Theodor & Moscona on Plecotus auritus, Nycteribia kolenatii Theodor & Moscona on M. bechsteinii (Kuhl) and Penicillidia dufourii (Westwood) on M. daubentonii (Kuhl) represent new host associations for Hungary.