Development of a novel molecular tool to study molecular ecology of Ornithomya (Hippoboscidae) avian louse flies.

Louse flies (Diptera: Hippoboscidae) are obligatory hematophagous ectoparasites of birds and mammals. These widely distributed parasitic flies may have a significant impact on wild and farm animals by feeding on their blood and transmitting bloodborne pathogens. However, despite their ecological importance, louse flies are clearly underrepresented in host-parasite research and implementation of genetic approaches in this group is generally hampered by lacking molecular tools. In addition, louse flies that parasitize long-distance migrants can travel long distances with their avian hosts, facilitating the large-scale spread of pathogens across landscapes and geographic regions. Given the wide diversity of louse flies that parasitize a variety of avian hosts, their direct negative impact on host survival, and their high potential to transmit bloodborne pathogens even along avian migration routes, it is surprising that our knowledge of louse fly ecology is rather modest and incomplete. Here, we aimed to develop a novel molecular tool for polyxenous avian louse flies from the genus Ornithomya, which are among the most common and widely distributed representatives of Hippoboscidae family, to improve research of their genetic population structure and molecular ecology. Using the Illumina Mi-seq sequencing, we conducted a genome-wide scan in Ornithomya avicularia to identify putative microsatellite markers. A panel of 26 markers was selected to develop amplification protocols and assess polymorphism in the Central European population of O. avicularia, as well as to test for cross-amplification in a congeneric species (O. chloropus). A genome-scan in O. avicularia identified over 12 thousand putative microsatellite markers. Among 26 markers selected for a population-wide screening; one did not amplify successfully and three were monomorphic. 22 markers were polymorphic with at least two alleles detected. Two markers showed presence of null alleles. A cross-amplification of microsatellite markers in O. chloropus revealed allelic polymorphism at 14 loci, with the mean allelic richness of 3.78 alleles per locus (range: 2-8). Our genome-wide scan in O. avicularia provides a novel and powerful tool for molecular research in Ornithomya louse flies. Our panel of polymorphic microsatellite loci should allow genotyping of louse flies from geographically distinct populations and from a wide spectrum of avian hosts, enhancing population genetic and phylogeographic research in Ornithomya.

Influence of colony traits on ectoparasite infestation in birds breeding in mixed-species colonies.

Coloniality in birds is often associated with an increase in parasite burden, but whether the co-occurrence of several host species influences the prevalence and abundance of ectoparasites and their relationship with colony size or density remains poorly known. Here, we studied mixed-species breeding colonies formed after the provision of artificial breeding structures for restoring the lesser kestrel (Falco naumanni) population in Portugal, to investigate the influence of colony traits on ectoparasite infestation. We sampled four groups of ectoparasites (carnid flies, haematophagous mites, louse flies and chewing lice) in four hosts: lesser kestrels, European rollers (Coracias garrulus), feral pigeons (Columba livia) and spotless starlings (Sturnus unicolor). Each host species had a distinct infracommunity of ectoparasites, regardless of colony traits such as size, density or host richness. The abundance of the most common ectoparasite, Carnus hemapterus, was influenced by colony composition ­ number of nests of each host species ­ rather than by colony size or density, with its abundance being diluted with increasing numbers of less suitable hosts such as starlings. The increased contact between multiple species of hosts in breeding colonies may complexify host­parasite interactions and challenge our current knowledge on the ecological relationships between host sociality and parasitism.

Evolutionary adaptations in four hippoboscid fly species belonging to three different subfamilies.

Lipoptena cervi (Linnaeus, 1758), Lipoptena fortisetosa Maa, 1965, Hippobosca equina Linnaeus, 1758, and Pseudolynchia canariensis (Macquart, 1840) (Diptera: Hippoboscidae) are haematophagous ectoparasites that infest different mammal and bird species and occasionally attack humans. They are known for the health implications they have as vectors of pathogens to humans and animals, and for the injuries they inflict on their host's skin. This study focused on the morphological structures evolved by parasites in terms of their biology and the different environment types that they inhabit. To this aim, we examined four hippoboscid species, as well as their hosts' fur (ungulate and horse), and feather (pigeon) through light and Scanning Electron Microscopy (SEM) observations in order to highlight the main morphological features that evolved differently in these flies and to explain the effect of hosts' fur/feather microhabitats on the morphological specializations observed in the investigated ectoparasites. The studied species showed main convergent characters in mouthparts while remarkable differences have been detected on the antennal sensillar pattern as well as on the leg acropod that displayed divergent characters evolved in relation to the host.

Molecular and morphological analysis revealed a new Lipoptena species (Diptera: Hippoboscidae) in southern Spain harbouring Coxiella burnetii and bacterial endosymbionts.

Hippoboscid flies (Diptera: Hippoboscidae) are obligate bloodsucking ectoparasites of animals. In Europe, limited research has been conducted on this family until the recent introduction of the deer ked Lipoptena fortisetosa Maa, 1965. A new species of the genus Lipoptena, Lipoptena andaluciensis sp. nov., was found in southern Spain after extensive sampling with carbon-dioxide baited suction traps. A total of 52 females and 32 males were collected at 29 out of 476 sites examined over eight months in 2023. Lipoptena andaluciensis sp. nov. was characterized morphologically and molecularly. The new Lipoptena species can be differentiated from the closely related L. fortisetosa by size, chaetotaxy of the dorsal and ventral thorax, abdominal plates, and genitalia. Based on DNA-barcoding, our specimens showed the highest similarity with Melophagus ovinus (Linnaeus, 1758) (88.4 %) and with L. fortisetosa (86-88 %). Individual screening of Lipoptena specimens (n = 76) for seven important zoonotic pathogens such as bacteria (Anaplasmataceae family: Bartonella spp., Borrelia spp., Coxiella burnetii and Rickettsia spp.) and protozoans (Babesia spp. and Theileria spp.) by conventional PCR and RT-PCR was performed. DNA of C. burnetii was detected in one specimen, while two other specimens harboured Anaplasmataceae (Wolbachia spp., 100 % homology and another endosymbiont probably related to Arsenophonus sp., 95.3 % homology, respectively), all representing the first records of these bacteria in the Lipoptena spp. from Europe. Carbon dioxide traps probed its effectiveness as a reliable passive method for keds surveillance. Our study highlights the existence of a new Lipoptena species, presumably widely distributed in southern Spain. The role of this species in the transmission cycle of pathogens of medical-veterinary relevance needs to be considered in the area.

Detection of Pathogens and Ticks on Sedentary and Migratory Birds in Two Corsican Wetlands (France, Mediterranean Area).

Birds are one of the most species-diverse vertebrate groups and are susceptible to numerous hematophagous ectoparasites. Migratory birds likely contribute to the circulation of these ectoparasites and their associated pathogens. One of the many migration paths crosses the Mediterranean islands including Corsica and its wetlands, which are migration stopovers. In our study, we collected blood samples and hematophagous ectoparasites in migratory and sedentary bird populations in two coastal lagoons: Biguglia and Gradugine. A total of 1377 birds were captured from which 762 blood samples, 37 louse flies, and 44 ticks were collected. All the louse flies were identified as Ornithomya biloba and all the ticks were from the Ixodes genus: Ixodes sp. (8.5%), I. accuminatus/ventalloi (2.9%), I. arboricola/lividus (14.3%), I. frontalis (5.7%) and I. ricinus (68.6%). Five pathogens were detected: Anaplasma phagocytophilum, Erhlichia chaffeensis, and Rickettsia helvetica in ticks, and Trypanosoma sp. in louse flies. Ehrlichia chaffeensis and the West Nile virus were both detected in bird blood samples in Corsica. This is the first report of these tick, louse fly and pathogen species isolated on the bird population in Corsica. Our finding highlights the importance of bird populations in the presence of arthropod-borne pathogens in Corsican wetlands.

Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria.

BACKGROUND: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria. METHODS: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses. RESULTS: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida. CONCLUSIONS: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.

Louse flies in Azorean and mainland populations of four Passeriformes species: A new perspective to parasite Island syndromes.

Hippoboscid flies, also known as louse flies, are obligate blood-feeders ectoparasites of birds and mammals. By studying louse fly parasites of four Passeriformes species, Eurasian blackbird (Turdus merula), Eurasian blackcap (Sylvia atricapilla), common chaffinch (Fringilla coelebs) and European robin (Erithacus rubecula), with dissimilar time of colonization of Azores islands, we tested whether: (i) island host populations have lower parasite richness than the mainland one; (ii) island host populations undergo higher parasite prevalence, mean intensities and mean abundance than the mainland one; (iii) island parasite diversity are composed exclusively by specific parasites and (iv) parasite richness is positively correlated with the island area and proximity to the continent. For these purposes, 775 birds were sampled for presence of louse flies, by modified fumigation chamber method, from Azores Islands (São Miguel, Terceira and Flores) and Portugal mainland. Insular and mainland parasite assemblages were statistically compared. We record for the first time to Azores, Ornithomya fringillina and Icosta minor from Portugal mainland. Louse flies had highest prevalence and abundance from Azores Islands compared to those observed in mainland birds, especially blackbirds. The insular parasite diversity of Azores blackbirds, blackcaps and chaffinches was richer than the one observed in mainland population. None of the hippoboscid flies observed on the islands and mainland were host-specific. Thus, our findings provide an upgrade of parasite island syndromes knowledge, in the context of the ectoparasites, namely to the hippoboscid flies case.

Molecular characterization of Lipoptena cervi from environmental samples collected in Poland.

The activity of Lipoptena cervi has intensified in Poland in recent years. The population genetics of this ectoparasite in Poland has never been described in the literature. The objectives of this study were to investigate the population genetics of L. cervi in selected regions of Poland, to evaluate molecular differences between L. cervi populations, and to determine phylogenetic relationships with other L. cervi sequences obtained in previous studies. In 2019, louse flies were sampled in natural mixed forests in five Polish voivodeships. Seven samples of L. cervi were collected from each voivodeship, and a total of 35 insects were analyzed molecularly. In the first step, Lipoptena spp. were identified to species level under a stereoscopic microscope. A fragment of the rRNA 16S gene was used as a marker to identify L. cervi by the PCR assay. The sequences were assigned accession numbers MT337409 to MT337416. A total of eight haplotypes were identified, two of which were dominant. In the obtained sequences, intraspecific pairwise genetic distances varied between 0.000 and 0.0496 (m = 0.0135; SD = 0.0149; SE = 0.0006; V = 110.11). Mean interpopulation diversity was d = 0.0135 (SE = 0.0027). The acquired nucleotide sequences were highly similar to the sequences from the Czech Republic (MF495940, AF322437), Lithuania (MN889542-MN889544) and Poland (MF541726-MF541729). The similarity with GenBank sequences ranged from 97.24% to 100%. This study revealed two dominant haplotypes of L. cervi in Poland, MT337410 and MT337413. Fragments of the analyzed sequences were detected in only one voivodeship. These findings suggest that the two dominant sequences are the oldest sequences that gave rise to the locally identified haplotypes. The lack of significant correlations with the sequences obtained in regions situated west of the research sites suggests the presence of other genetic populations in Europe.

Molecular Detection of Selected Pathogens with Zoonotic Potential in Deer Keds (Lipoptena fortisetosa).

Deer keds are obligatory hematophagous ectoparasites of birds and mammals. Cervids serve as specific hosts for these insects. However, ked infestations have been observed in non-specific hosts, including humans, companion animals, and livestock. Lipoptena fortisetosa is a weakly studied ectoparasite, but there is evidence to indicate that it continues to spread across Europe. The existing knowledge on deer keds' impact on wildlife is superficial, and their veterinary importance is enigmatic. Lipoptena fortisetosa is a species with vectorial capacity, but potential pathogen transmission has not been assessed. The objective of this study was to evaluate the prevalence of selected pathogens in L. fortisetosa collected from cervids and host-seeking individuals in the environment. Out of 500 acquired samples, 307 (61.4%) had genetic material from at least one tested pathogen. Our research suggests that L. fortisetosa may be a potential vector of several pathogens, including A. phagocytophilum, Babesia spp., Bartonella spp., Borellia spp., Coxiella-like endosymbionts, Francisiella tularensis, Mycoplasma spp., Rickettsia spp., and Theileria spp.; however, further, more extensive investigations are required to confirm this. The results of the study indicate that keds can be used as biological markers for investigating the prevalence of vector-borne diseases in the population of free-ranging cervids.

Temporal-Microclimatic Factors Affect the Phenology of Lipoptena fortisetosa in Central European Forests.

The objective of this study was to determine the correlations between the abundance of Lipoptena fortisetosa on new potential hosts and selected temporal-microclimatic conditions in a forest at the beginning of the host-seeking period. Louse flies were collected between 6 May and 15 July of 2019 and 2020 in a natural mixed forest in Poland. Keds were collected by three investigators walking along the same forest route during each sampling session. The number of captured keds and the date (time), temperature (°C), relative humidity (%), air pressure (hPa) and wind speed (km/h) were recorded. A total of five measurements were performed during each sampling session. The influence of temporal-microclimatic conditions on the number of collected ectoparasites was evaluated with the use of a Generalized Additive Model (GAM). A total of 1995 individuals were obtained during field surveys. The results of the GAM revealed a correlation between the number of host seeking L. fortisetosa vs. time, temperature, relative humidity, and wind speed. An increase in temperature was most highly correlated with the abundance of louse flies in the environment.

A revised annotated checklist of louse flies (Diptera, Hippoboscidae) from Slovakia.

The list of all known locality and host records from the literature on louse flies from Slovakia are summarized, with the addition of new collection data. New locality data are provided for five species. Three species are added to the Slovakian list: Icostaminor (Bigot in Thomson, 1858), which was erroneously cited for Moravia instead of Slovakia in the previous checklist, and Ornithophilametallica (Schiner, 1864) and Ornithomyachloropus (Bergroth, 1901), which were overlooked from the last checklist. As a result, the louse fly fauna of Slovakia increases to 19 species: 12 autochtonous species and seven rare, non-native species only occasionally imported to Slovakia or migrating to the country with their hosts. This is by far the largest regional fauna of Hippoboscidae in Central Europe, and matches the richest southern European faunas. In total, 78 host-parasite associations concerning 46 bird-host species from eight orders and nine species of mammals, including humans, have been found from a literature review in Slovakia. Two host-parasite associations are reported from Slovakia for the first time: Ornithomyaavicularia (Linnaeus, 1758) on Prunellamodularis (Linnaeus, 1758) (Aves: Prunellidae) and Lipoptenafortisetosa Maa, 1965 on Homosapiens Linnaeus, 1758 (Mammalia: Hominidae).

Arsenophonus and Sodalis replacements shape evolution of symbiosis in louse flies.

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis, Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis, suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies.

Occurrence of the louse fly Ornithoctona erythrocephala Leach (1817) (Diptera: Hippoboscidae) on a free-living red-legged seriema (Cariama cristata) / Ocorrência da mosca Ornithoctona erythrocephala Leach, 1817 (Diptera: Hippoboscidae) em seriema (Cariama cristata) de vida livre

Abstract The aim of this study was to report, for the first time, the occurrence of the hippoboscid fly Ornithoctona erythrocephala on a red-legged seriema (Cariama cristata). A Diptera specimen was found among the feathers of a free-living red-legged seriema, which was referred to necropsy at the Wild Animal Pathology Service, UNESP Jaboticabal. The fly was collected, stored in absolute alcohol, and shipped to the Institute of Biosciences of the Federal University of Mato Grosso do Sul for proper identification. Based on morphological characters, the specimen was identified as a female of Ornithoctona erythrocephala. This study provides a report on a new host for O. erythrocephala.

Resumo O objetivo deste estudo foi descrever pela primeira vez a ocorrência da mosca Hippoboscidae Ornithoctona erythrocephala em uma seriema (Cariama cristata). Um espécime de díptero foi encontrado entre as penas de dessa ave, que havia sido encaminhada para necropsia ao Serviço de Patologia de Animais Selvagens, UNESP, Jaboticabal. A mosca foi armazenada em álcool absoluto e enviada ao Instituto de Biociências da Universidade Federal do Mato Grosso do Sul para identificação por um especialista. Baseado em características morfológicas, o espécime foi identificado como fêmea de Ornithoctona erythrocephala. Este relato oferece a descrição de um novo hospedeiro para O. erythrocephala.

First record of Microlynchia pusilla (Diptera: Hippoboscidae) in northeastern Brazil / Primeiro registro de Microlynchia pusilla (Diptera: Hippoboscidae) no nordeste do Brasil

The present paper reports the occurrence of Microlynchia pusilla in the state of Rio Grande do Norte (RN) on Leptotila verreauxi approximans from a deciduous forest fragment located in the municipality of Macaíba. A specimen of L. v. approximans was collected in June 2012, wrapped in paper towels and kept under refrigeration in a plastic bag for later analysis of parasite fauna, taxidermy, and eventual storage in the Ornithological Collection of the Federal University of Rio Grande do Norte (UFRN). During the search for ectoparasites, a specimen of M. pusilla was found among feathers of the ventral region; it was collected and stored in the Entomological Collection 'Adalberto Antônio Varela-Freire' of the same university. This report extends the knowledge about geographical distribution and confirms the association of M. pusilla with hosts Columbiformes, contributing to the knowledge of the family Hippoboscidae in the country.

Este trabalho registra a ocorrência de Microlynchia pusilla no estado do Rio Grande do Norte em Leptotila verreauxi approximans oriunda de um fragmento de floresta estacional decidual localizado no município de Macaíba. Um espécime de L. v. approximans foi coletado em junho de 2012, envolvido em papel toalha e conservado sob refrigeração em saco plástico para posterior análise da fauna parasitária, taxidermia e depósito na Coleção Ornitológica da Universidade Federal do Rio Grande do Norte (UFRN). Durante a busca por ectoparasitos foi encontrado um espécime de M. pusilla nas penas da região ventral, o qual foi coletado e depositado na Coleção Entomológica Adalberto Antônio Varela-Freire da mesma universidade. Este relato amplia o conhecimento sobre a distribuição geográfica de M. pusilla e confirma sua associação com hospedeiros Columbiformes, contribuindo para o conhecimento da família Hippoboscidae no país.

Pseudolynchia canariensis (Diptera: Hippoboscidae) em Buteogallus aequinoctialis (Ciconiiformes: Accipitridae) no estado do Rio de Janeiro, Brasil / Pseudolynchia canariensis (Diptera:Hippoboscidae) on Buteogallus aequinoctialis (Ciconiiformes: Accipitridae) in the State of Rio de Janeiro, Brasil

Registro de Pseudolynchia canariensis em dois gaviões caranguejeiros de vida livre atendidos no Hospital Veterinário da Fundação RioZoo. Os dezenoves exemplares coletados foram identificados no Laboratório de Diptera da Fundação Instituto Oswaldo Cruz. O encontro de P. canariensis fora do hospedeiro natural (Columba livia), representa uma contribuição aos estudos da família Hippoboscidae, visto que não há registros sobre aves nativas do continente americano parasitadas por P. canariensis.

The record of Pseudolynchia canariensis on two Rufous Crab-Hawk in situ taken care of the Hospital Veterinarian of the RioZoo Foundation. The nineteen collected specimens had been identified in the Laboratory of Diptera, Fundação Instituto Oswaldo Cruz. The findings of P canariensis out natural hosty (Columba livia) it represents a contribution to the studies of the family Hippoboscidae considering that it does not have records about native birds of the american continent parasitized by P canariensis.

Associação forética dos ácaros Myialges anchora Sergent & Trouessart (Acaridida, Epidermoptidae) e Ornithocheyletia hallae Smiley (Actinedida, Cheyletiellidae) com Pseudolynchia canariensis (Macquart) (Diptera, Hippoboscidae) / Phoretic association of the mites Myialges anchora Sergent & Trouessart (Acaridida, Epidermoptidae) and Ornithocheyletia hallae Smiley (Actinedida, Cheyletiellidae) with Pseudolynchia canariensis (Macquart) (Diptera, Hippoboscidae)

A partir de 45 espécimes de Pseudolynchia canariensis examinados, foram encontrados 23 deles hiperparasitados por ácaros. É registrada a ocorrência de Myialges anchora e Ornithocheyletia hallae no estado do Rio de Janeiro, Brasil. Dados sobre o grau de infestação desses hipoboscídeos, bem como a distribuição topográfica dos ácaros sobre o corpo desses insetos são brevemente discutidos.

Twenty three louse-flies, from a total of 45 specimens of Pseudolynchia canariensis examined, were found parasitised by mites of the species Myialges anchora and Ornithocheyletia hallae. These mites are recorded for the first time for the State of Rio de Janeiro, in Brazil. Data on levels of infestation, as well as on the topographical distribution of the mites on the body of the louse-flies are briefly discussed.