Development of the hyolaryngeal architecture in horseshoe bats: insights into the evolution of the pulse generation for laryngeal echolocation.

BACKGROUND: The hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage. RESULTS: We found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency. CONCLUSIONS: The unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

Description of three new bat-associated species of hard ticks (Acari, Ixodidae) from Japan.

In Eurasia, the geographically most widespread ixodid tick species of the bat families Rhinolophidae Gray, Vespertilionidae Gray, and Miniopteridae Dobson were considered to belong to four species, Ixodesvespertilionis Koch, I.collaris Hornok, I.ariadnae Hornok, and I.simplex Neumann. Previous data attest that bat-associated tick species from Eastern Asia show remarkable genetic difference from the above four tick species, but in the absence of detailed morphological comparison these were regarded as conspecific. In this study we compensate for this lack of data on three bat-associated tick species, reporting their morphological comparison, as well as molecular and phylogenetic relationships. According to the results we describe the females of three tick species new to science, i.e., I.nipponrhinolophi Hornok & Takano, sp. nov., I.fuliginosus Hornok & Takano, sp. nov., and I.fujitai Hornok & Takano, sp. nov. In case of all three new tick species the cytochrome c oxidase subunit (coxI) gene showed remarkably high sequence differences from the species that they previously were thought to belong to, well exceeding the average limit delineating ixodid tick species. This, as well as observed morphological differences fully justify their taxonomical status as new species.

Anatomy and homology of the caudal auricular muscles in greater short-nosed fruit bat (Cynopterus sphinx).

Bats can be phylogenetically classified into three major groups: pteropodids, rhinolophoids, and yangochiropterans. While rhinolophoids and yangochiropterans are capable of laryngeal echolocation, pteropodids lack this ability. Delicate ear movements are essential for echolocation behavior in bats with laryngeal echolocation. Caudal auricular muscles, especially the cervicoauricularis group, play a critical role in such ear movements. Previously, caudal auricular muscles were studied in three species of bats with laryngeal echolocation, but to our knowledge, there have been no studies on non-laryngeal echolocators, the pteropodids. Here, we describe the gross anatomy of the cervicoauricularis muscles and their innervation in Cynopterus sphinx by using diffusible iodine-based contrast-enhanced computed tomography and 3D reconstructions of immunohistochemically stained serial sections. A previous study on bats with laryngeal echolocation reported that rhinolophoids have four cervicoauricularis muscles and yangochiropterans have three. We observed three cervicoauricularis muscles in the pteropodid C. sphinx. The number of cervicoauricularis muscles and their innervation pattern were comparable to those of non-bat boreoeutherian mammals and yangochiropterans, suggesting that pteropodids, and yangochiropterans maintain the general condition of boreoeutherian mammals and that rhinolophoids have a derived condition. The unique nomenclature had been previously applied to the cervicoauricularis muscles of bats with laryngeal echolocation, but given the commonality between non-bat laurasiatherians and bats, with the exception of rhinolophoids, maintaining the conventional nomenclature (i.e., M. cervicoauricularis superficialis, M. cervicoauricularis medius, and M. cervicoauricularis profundus) is proposed for bats.

Caudal auricular muscle variations and the evolution of echolocation behavior in pteropodid bats.

Among bats, rhinolophoids and yangochiropterans, but not pteropodids, exhibit laryngeal echolocation. Although Rousettus has been regarded as the only pteropodid capable of echolocation using tongue clicks, recent evidence suggests that other species of pteropodids are also capable of echolocation using wing clicks. Studies on laryngeal echolocators suggest that delicate ear movements are essential for the echolocation behavior of bats and that the cervicoauricularis muscles play a critical role in such ear movements. In this study, we observed the gross anatomy of cervicoauricularis muscles in three species of pteropodids (Cynopterus sphinx, Eonycteris spelaea, and Rousettus leschenaultii) to examine whether ear muscle anatomy varies among pteropodids with different echolocation types and between pteropodids and laryngeal echolocating bats. We found that M. cervicoauricularis profundus originates from the nuchal crest in tongue-click echolocators (R. leschenaultii) and from the midline in wing-click echolocators (C. sphinx and E. spelaea). In general, tongue-click echolocation using high click rates is considered to be more sophisticated in terms of sonar performance than wing-click echolocation. M. cervicoauricularis profundus originating from the nuchal crest (CPNC) is not common in non-bat laurasiatherian mammals, but can be found in laryngeal echolocating bats. As it pulls the ear pinna caudally in the horizontal plane and increases the access to sound, CPNC found in R. leschenaultii and laryngeal echolocating bats may be a key characteristic of the sophisticated active echolocation behavior of bats.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents.

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

Timing of organogenesis underscores the evolution of neonatal life histories and powered flight in bats.

Bats have undergone one of the most drastic limb innovations in vertebrate history, associated with the evolution of powered flight. Knowledge of the genetic basis of limb organogenesis in bats has increased but little has been documented regarding the differences between limb organogenesis in bats and that of other vertebrates. We conducted embryological comparisons of the timelines of limb organogenesis in 24 bat species and 72 non-bat amniotes. In bats, the time invested for forelimb organogenesis has been considerably extended and the appearance timing of the forelimb ridge has been significantly accelerated, whereas the timing of the finger and first appearance of the claw development has been delayed, facilitating the enlargement of the manus. Furthermore, we discovered that bats initiate the development of their hindlimbs earlier than their forelimbs compared with other placentals. Bat neonates are known to be able to cling continuously with their well-developed foot to the maternal bodies or habitat substrates soon after birth. We suggest that this unique life history of neonates, which possibly coevolved with powered flight, has driven the accelerated development of the hindlimb and precocious foot.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

ChiroVox: a public library of bat calls.

Recordings of bat echolocation and social calls are used for many research purposes from ecological studies to taxonomy. Effective use of these relies on identification of species from the recordings, but comparative recordings or detailed call descriptions to support identification are often lacking for areas with high biodiversity. The ChiroVox website (https://www.chirovox.org) was created to facilitate the sharing of bat sound recordings together with their metadata, including biodiversity data and recording circumstances. To date, more than 30 researchers have contributed over 3,900 recordings of nearly 200 species, making ChiroVox the largest open-access bat call library currently available. Each recording has a unique identifier that can be cited in publications; hence the acoustic analyses are repeatable. Most of the recordings available through the website are from bats whose species identities are confirmed, so they can be used to determine species in recordings where the bats were not captured or could not be identified. We hope that with the help of the bat researcher community, the website will grow rapidly and will serve as a solid source for bat acoustic research and monitoring.

On the sequence heterochrony of cranial ossification of bats in light of Haeckel's recapitulation theory.

Haeckel's recapitulation theory has been a controversial topic in evolutionary biology. However, we have seen some recent cases applying Haeckel's view to interpret the interspecific variation of prenatal ontogeny. To revisit the validity of Haeckel's recapitulation theory, we take bats that have undergone drastic morphological changes and possess a characteristic ecology as a case study. All members of Rhinolophoidea and Yangochiroptera can generate an ultrasonic pulse from the larynx to interpret surrounding objects (laryngeal echolocation) whereas Pteropodidae lacks such ability. It is known that the petrosal bone is particularly derived in shape and expanded in laryngeal echolocators. If Haeckel's recapitulation theory holds, the formation of this derived trait should occur later than those of other bones. Therefore, we compared the prenatal ossification timing of the petrosal in 15 bat species and five outgroup species. We found that the ossification of the petrosal is accelerated in laryngeal echolocators while it is the last bone to ossify in non-laryngeal echolocating bats and non-volant mammals, which runs counter to the prediction generated by Haeckel's recapitulation theory. We point out the evolutionarily labile nature of trait developmental timing and emphasize that Haeckel's recapitulation theory does not hold in many cases. We caution that generating predictions on ancestral conditions and evolutionary history leading from Haeckel's recapitulation theory is not well supported.

A novel SARS-CoV-2 related coronavirus in bats from Cambodia.

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses.

Inferring the ecological niche of bat viruses closely related to SARS-CoV-2 using phylogeographic analyses of Rhinolophus species.

The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the coronavirus disease 2019 (COVID-19) pandemic. To date, viruses closely related to SARS-CoV-2 have been reported in four bat species: Rhinolophus acuminatus, Rhinolophus affinis, Rhinolophus malayanus, and Rhinolophus shameli. Here, we analysed 343 sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (CO1) from georeferenced bats of the four Rhinolophus species identified as reservoirs of viruses closely related to SARS-CoV-2. Haplotype networks were constructed in order to investigate patterns of genetic diversity among bat populations of Southeast Asia and China. No strong geographic structure was found for the four Rhinolophus species, suggesting high dispersal capacity. The ecological niche of bat viruses closely related to SARS-CoV-2 was predicted using the four localities in which bat viruses were recently discovered and the localities where bats showed the same CO1 haplotypes than virus-positive bats. The ecological niche of bat viruses related to SARS-CoV was deduced from the localities where bat viruses were previously detected. The results show that the ecological niche of bat viruses related to SARS-CoV2 includes several regions of mainland Southeast Asia whereas the ecological niche of bat viruses related to SARS-CoV is mainly restricted to China. In agreement with these results, human populations in Laos, Vietnam, Cambodia, and Thailand appear to be much less affected by the COVID-19 pandemic than other countries of Southeast Asia. In the climatic transitional zone between the two ecological niches (southern Yunnan, northern Laos, northern Vietnam), genomic recombination between highly divergent viruses is more likely to occur. Considering the limited data and the risk of recombinant bat-CoVs emergence as the source of new pandemics in humans, the bat populations in these regions should be under surveillance.

Prenatal Developmental Trajectories of Fluctuating Asymmetry in Bat Humeri.

Fluctuating asymmetry (random fluctuations between the left and right sides of the body) has been interpreted as an index to quantify both the developmental instabilities and homeostatic capabilities of organisms, linking the phenotypic and genotypic aspects of morphogenesis. However, studying the ontogenesis of fluctuating asymmetry has been limited to mostly model organisms in postnatal stages, missing prenatal trajectories of asymmetry that could better elucidate decoupled developmental pathways controlling symmetric bone elongation and thickening. In this study, we quantified the presence and magnitude of asymmetry during the prenatal development of bats, focusing on the humerus, a highly specialized bone adapted in bats to perform under multiple functional demands. We deconstructed levels of asymmetry by measuring the longitudinal and cross-sectional asymmetry of the humerus using a combination of linear measurements and geometric morphometrics. We tested the presence of different types of asymmetry and calculated the magnitude of size-controlled fluctuating asymmetry to assess developmental instability. Statistical support for the presence of fluctuating asymmetry was found for both longitudinal and cross-sectional asymmetry, explaining on average 16% of asymmetric variation. Significant directional asymmetry accounted for less than 6.6% of asymmetric variation. Both measures of fluctuating asymmetry remained relatively stable throughout ontogeny, but cross-sectional asymmetry was significantly different across developmental stages. Finally, we did not find a correspondence between developmental patterns of longitudinal and cross-sectional asymmetry, indicating that processes promoting symmetrical bone elongation and thickening work independently. We suggest various functional pressures linked to newborn bats' ecology associated with longitudinal (altricial flight capabilities) and cross-sectional (precocial clinging ability) developmental asymmetry differentially. We hypothesize that stable magnitudes of fluctuating asymmetry across development could indicate the presence of developmental mechanisms buffering developmental instability.

On the Embryonic Development of the Nasal Turbinals and Their Homology in Bats.

Multiple corrugated cartilaginous structures are formed within the mammalian nasal capsule, eventually developing into turbinals. Due to its complex and derived morphology, the homologies of the bat nasal turbinals have been highly disputed and uncertain. Tracing prenatal development has been proven to provide a means to resolve homological problems. To elucidate bat turbinate homology, we conducted the most comprehensive study to date on prenatal development of the nasal capsule. Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we studied in detail the 3D prenatal development of various bat species and non-bat laurasiatherians. We found that the structure previously identified as "maxilloturbinal" is not the true maxilloturbinal and is only part of the ethmoturbinal I pars anterior. Our results also allowed us to trace the evolutionary history of the nasal turbinals in bats. The turbinate structures are overall comparable between laurasiatherians and pteropodids, suggesting that pteropodids retain the ancestral laurasiatherian condition. The absence of the ethmoturbinal I pars posterior in yangochiropterans and rhinolophoids has possibly occurred independently by convergent evolution.

Embryonic evidence uncovers convergent origins of laryngeal echolocation in bats.

Bats are the second-most speciose group of mammals, comprising 20% of species diversity today. Their global explosion, representing one of the greatest adaptive radiations in mammalian history, is largely attributed to their ability of laryngeal echolocation and powered flight, which enabled them to conquer the night sky, a vast and hitherto unoccupied ecological niche. While there is consensus that powered flight evolved only once in the lineage, whether laryngeal echolocation has a single origin in bats or evolved multiple times independently remains disputed. Here, we present developmental evidence in support of laryngeal echolocation having multiple origins in bats. This is consistent with a non-echolocating bat ancestor and independent gain of echolocation in Yinpterochiroptera and Yangochiroptera, as well as the gain of primitive echolocation in the bat ancestor, followed by convergent evolution of laryngeal echolocation in Yinpterochiroptera and Yangochiroptera, with loss of primitive echolocation in pteropodids. Our comparative embryological investigations found that there is no developmental difference in the hearing apparatus between non-laryngeal echolocating bats (pteropodids) and terrestrial non-bat mammals. In contrast, the echolocation system is developed heterotopically and heterochronically in the two phylogenetically distant laryngeal echolocating bats (rhinolophoids and yangochiropterans), providing the first embryological evidence that the echolocation system evolved independently in these bats.

Variation in cross-sectional shape and biomechanical properties of the bat humerus under Wolff's law.

Bats use their forelimbs in different ways, but flight is the most notable example of morphological adaptation. Foraging and roosting specializations beyond flight have also been described in several bat lineages. Understanding postcranial evolution during the locomotory and foraging diversification of bats is fundamental to understanding bat evolution. We investigated whether different foraging and roosting behaviors influenced humeral cross-sectional shape and biomechanical variation, following Wolff's law of bone remodeling. The effect of body size and phylogenetic relatedness was also tested, in order to evaluate multiple sources of variation. Our results suggest strong ecological signal and no phylogenetic structuring in shape and biomechanical variation in humeral phenotypes. Decoupled modes of scaling of shape and biomechanical variation were consistently indicated across foraging and roosting behaviors, suggesting divergent allometric trajectories. Terrestrial locomoting and upstand roosting species showed unique patterns of shape and biomechanical variation across all our analyses, suggesting that these rare behaviors among bats place unique functional demands on the humerus, canalizing phenotypes. Our results suggest that complex and multiple adaptive pathways interplay in the postcranium, leading to the decoupling of different features and regions of skeletal elements optimized for different functional demands. Moreover, our results shed further light on the phenotypical diversification of the wing in bats and how adaptations besides flight could have shaped the evolution of the bat postcranium.

Phylogeny and foraging behaviour shape modular morphological variation in bat humeri.

Bats show a remarkable ecological diversity that is reflected both in dietary and foraging guilds (FGs). Cranial ecomorphological adaptations linked to diet have been widely studied in bats, using a variety of anatomical, computational and mathematical approaches. However, foraging-related ecomorphological adaptations and the concordance between cranial and postcranial morphological adaptations remain unexamined in bats and limited to the interpretation of traditional aerodynamic properties of the wing (e.g. wing loading [WL] and aspect ratio [AR]). For this reason, the postcranial ecomorphological diversity in bats and its drivers remain understudied. Using 3D virtual modelling and geometric morphometrics (GMM), we explored the phylogenetic, ecological and biological drivers of humeral morphology in bats, evaluating the presence and magnitude of modularity and integration. To explore decoupled patterns of variation across the bone, we analysed whole-bone shape, diaphyseal and epiphyseal shape. We also tested whether traditional aerodynamic wing traits correlate with humeral shape. By studying 37 species from 20 families (covering all FGs and 85% of dietary guilds), we found similar patterns of variation in whole-bone and diaphyseal shape and unique variation patterns in epiphyseal shape. Phylogeny, diet and FG significantly correlated with shape variation at all levels, whereas size only had a significant effect on epiphyseal morphology. We found a significant phylogenetic signal in all levels of humeral shape. Epiphyseal shape significantly correlated with wing AR. Statistical support for a diaphyseal-epiphyseal modular partition of the humerus suggests a functional partition of shape variability. Our study is the first to show within-structure modular morphological variation in the appendicular skeleton of any living tetrapod. Our results suggest that diaphyseal shape correlates more with phylogeny, whereas epiphyseal shape correlates with diet and FG.

A new genus of vespertilionid bat: the end of a long journey for Joffre's Pipistrelle (Chiroptera: Vespertilionidae).

Knowledge as to the taxonomic status of enigmatic bat species often is hindered by limited availability of specimens. This is particularly true for aerial-hawking bats that are difficult to catch. One such species, "Hypsugo" joffrei, was originally described in Nyctalus due to its long and slender wings, but subsequently transferred to Pipistrellus, and most recently to Hypsugo, on the basis of morphology. Analysis of newly available material, which more than doubles the known specimens of this taxon, demonstrates that it is morphologically and genetically distinct from all other bat genera. We accordingly describe it as belonging to a new, monotypic genus. We provide a detailed description of its external and craniodental traits, measurements, and assessment of genetic relationships, including barcode sequences to facilitate its rapid identification in future. The new genus belongs to a group that includes the recently described Cassistrellus, as well as Tylonycteris, and its closest relative, Philetor. We also describe the echolocation calls emitted by members of the taxon in different situations, which may facilitate finding them in previously unsampled locations. Based on the new data, the species occurs from Nepal to North Vietnam and China, which suggests that it could be more widespread than previously thought.

Description of the male and the larva of Ixodes collaris Hornok, 2016 with drawings of all stages.

BACKGROUND: Ixodes collaris Hornok, 2016 is a recently discovered tick species associated with bats in Asia. This study provides the description of the male and the larva, as well as high quality drawings of all stages. METHODS: Ticks were collected from cave walls and bats in Phia Oac (Vietnam). DNA was extracted from one individual of each stage/sex, while another was morphometrically analysed. Based on two genetic markers, all ticks were identified as I. collaris. RESULTS: The male of I. collaris has long legs (i.e. the length of Haller's organ exceeds the maximum diameter of tarsus I), unlike the male of I. simplex Neumann, 1906, but similarly to males of I. vespertilionis Koch, 1844 and I. ariadnae Hornok, 2014. The lateral and medial edges of the palpi of male I. collaris are both convexly curved, unlike in I. ariadnae and I. simplex, but similarly to I. vespertilionis. The male of I. collaris has long palpal setae (up to 210 µm), unlike the males of I. ariadnae (30-100 µm) and I. simplex (20-80 µm), but similarly to I. vespertilionis (100-200 µm). Males of I. collaris have sparse distribution of long palpal setae (vs dense in I. vespertilionis) and posteriorly diverging, sclerotized trapezoid ridge dorsally on the basis capituli (posteriorly convergent, U-shaped and less evident in I. vespertilionis). The larva of I. collaris has long legs (unlike the larva of I. simplex, but similarly to I. vespertilionis and I. ariadnae), elongated club-shaped palpi (240 × 70 vs 200 × 90 µm in I. ariadnae, 200 × 70 µm in I. vespertilionis; and 140 × 60 µm in I. simplex:), pentagonal scutum, which is longer than broad (different from I. ariadnae and I. simplex, but similar to that of I. vespertilionis). The larva of I. collaris has strongly concave caudolateral margin of ventral basis with perpendicular angle (vs slightly concave, with obtuse angle in I. vespertilionis) and a prominent, dark sclerotized edge, "collar" (absent in I. vespertilionis). CONCLUSION: Several features allow to distinguish the male and the larva of I. collaris morphologically from those of other bat-associated ixodid tick species.

Molecular detection of vector-borne bacteria in bat ticks (Acari: Ixodidae, Argasidae) from eight countries of the Old and New Worlds.

BACKGROUND: Despite the increasingly recognized eco-epidemiological significance of bats, data from molecular analyses of vector-borne bacteria in bat ectoparasites are lacking from several regions of the Old and New Worlds. METHODS: During this study, six species of ticks (630 specimens) were collected from bats in Hungary, Romania, Italy, Kenya, South Africa, China, Vietnam and Mexico. DNA was extracted from these ticks and analyzed for vector-borne bacteria with real-time PCRs (screening), as well as conventional PCRs and sequencing (for pathogen identification), based on the amplification of various genetic markers. RESULTS: In the screening assays, Rickettsia DNA was only detected in bat soft ticks, whereas Anaplasma phagocytophilum and haemoplasma DNA were present exclusively in hard ticks. Bartonella DNA was significantly more frequently amplified from hard ticks than from soft ticks of bats. In addition to Rickettsia helvetica detected by a species-specific PCR, sequencing identified four Rickettsia species in soft ticks, including a Rickettsia africae-like genotype (in association with a bat species, which is not known to migrate to Africa), three haemotropic Mycoplasma genotypes in Ixodes simplex, and Bartonella genotypes in I. ariadnae and I. vespertilionis. CONCLUSIONS: Rickettsiae (from both the spotted fever and the R. felis groups) appear to be associated with soft rather than hard ticks of bats, as opposed to bartonellae. Two tick-borne zoonotic pathogens (R. helvetica and A. phagocytophilum) have been detected for the first time in bat ticks. The present findings add Asia (China) to the geographical range of R. lusitaniae, as well as indicate the occurrence of R. hoogstraalii in South Africa. This is also the first molecular evidence for the autochthonous occurrence of a R. africae-like genotype in Europe. Bat haemoplasmas, which are closely related to haemoplasmas previously identified in bats in Spain and to "Candidatus Mycoplasma haemohominis", are reported here for the first time from Central Europe and from any bat tick.

Prenatal cranial bone development of Thomas's horseshoe bat (Rhinolophus thomasi): with special reference to petrosal morphology.

Cochlear morphology has been regarded as one of the key traits to understand the origin and evolution of echolocation in bats, given its functionality and performance for receiving echolocation sonar. While numerous researchers have compared adult-stage morphology, few have studied the prenatal development of the cochlea. Here, we provide the first detailed three-dimensional description of the prenatal cranial development in bats, using Rhinolophus thomasi as a model, with particular interest to the petrosal which houses the cochlea. Results revealed that among all cranial bones the onset of the ossification of the petrosal is earlier in R. thomasi when compared to other reported mammals. Generally, the cochlea reaches adult size and shape before or around birth in placental mammals including bats, but we found that its shape and size growths continue until maturity in Rhinolophus species. The relationship of cochlear size and skull size is maintained constant throughout the postnatal ontogeny to adulthood in Rhinolophus, a pattern previously reported neither in any other bats nor other mammals. The peculiar developmental pattern in Rhinolophus possibly allows them to form their characteristically large cochlea and facilitate their distinctive echolocation behavior. A recent study reported that non-echolocating Pteropodidae shares a similar prenatal cochlear size to laryngeal echolocating bats. The apparent resemblance of fetal cochlear size was proposed to be a vestigial signal of large cochlear size in the last common ancestor of bats and thus as supporting evidence for the single origin of laryngeal echolocation. However, results from the present observations suggest that limited aspects of the cochlear development were captured in this previous investigation and that the resulting interpretations may be questionable. We point out that diversity and patterns of cochlear development among bats are still not resolved, and the controversy on the origins of laryngeal echolocation is still open to discussion.