First record of Eyndhovenia (Mesostigmata: Gamasina: Spinturnicidae) from Vietnam

Eyndhovenia is one of the twelve genera of Spinturnicidae which are highly specialised parasites of bats. Previously known hosts of this genus comprised 17 species of Old World bats: Eptesicus serotinus, Hipposideros larvatus, Miniopterus schreibersi, Myotis blythi, M. emarginatus, Pipistrellus pipistrellus, P. gaisleri, Rhinolophus axillaris, R. blasii, R. clivosus, R. cornutus, R. euryale, R. ferrumequinum, R. hipposideros, R. megachyllus, R. mehelyi, R. rouxi. Within Asia, Eyndhovenia was only recorded from two countries, China and Thailand. Between 2018 and 2020, we conducted a series of bats surveys and recorded of this genus from intermediate horseshoe bat, Rhinolophus affinis, in Vietnam. The present study exhibits the new record in both parasitological and geographical aspects.     

What Story Does Geographic Separation of Insular Bats Tell? A Case Study on Sardinian Rhinolophids

Competition may lead to changes in a species’ environmental niche in areas of sympatry and shifts in the niche of weaker competitors to occupy areas where stronger ones are rarer. Although mainland Mediterranean (Rhinolophus euryale) and Mehely’s (R. mehelyi) horseshoe bats mitigate competition by habitat partitioning, this may not be true on resource-limited systems such as islands. We hypothesize that Sardinian R. euryale (SAR) have a distinct ecological niche suited to persist in the south of Sardinia where R. mehelyi is rarer. Assuming that SAR originated from other Italian populations (PES) – mostly allopatric with R. mehelyi – once on Sardinia the former may have undergone niche displacement driven by R. mehelyi. Alternatively, its niche could have been inherited from a Maghrebian source population. We: a) generated Maxent Species Distribution Models (SDM) for Sardinian populations; b) calibrated a model with PES occurrences and projected it to Sardinia to see whether PES niche would increase R. euryale’s sympatry with R. mehelyi; and c) tested for niche similarity between R. mehelyi and PES, PES and SAR, and R. mehelyi and SAR. Finally we predicted R. euryale’s range in Northern Africa both in the present and during the Last Glacial Maximum (LGM) by calibrating SDMs respectively with SAR and PES occurrences and projecting them to the Maghreb. R. mehelyi and PES showed niche similarity potentially leading to competition. According to PES’ niche, R. euryale would show a larger sympatry with R. mehelyi on Sardinia than according to SAR niche. Such niches have null similarity. The current and LGM Maghrebian ranges of R. euryale were predicted to be wide according to SAR’s niche, negligible according to PES’ niche. SAR’s niche allows R. euryale to persist where R. mehelyi is rarer and competition probably mild. Possible explanations may be competition-driven niche displacement or Maghrebian origin.     

Divergent echolocation call frequencies in insular rhinolophids (Chiroptera): a case of character displacement?

Aims Because rhinolophids have been hypothesized to use echolocation call frequency to recognize conspecifics, sympatric species calling at similar frequencies should be subject to acoustic character displacement, i.e. a drift in frequency values to minimize the risk of misidentification of conspecifics. However, it has been proposed that insufficient geographical separation between populations in sympatry and allopatry may counter the establishment of frequency differences by character displacement. Here we tested the hypothesis that insular populations should exhibit acoustic divergence, and this should be revealed by comparing call frequencies with those observed in mainland, allopatric populations of conspecifics. We also tested whether the evolutionary pressure towards acoustic divergence should be especially strong at sites where rhinolophid species emitting similar call frequencies roost together in order to minimize interspecific frequency overlap. Location Sardinia and southern Italy (Campania, Lazio, Abruzzo). Methods Time‐expanded echolocation calls and body size were recorded from Sardinian populations of Rhinolophus mehelyi Matschie, Rhinolophus hipposideros (Bechstein) and Rhinolophus euryale Blasius. Both call frequencies and forearm length of insular R. hipposideros and R. euryale were compared with those of populations from mainland areas of Italy where R. mehelyi is absent, to explore the hypothesis that the presence of the latter species (which calls at frequency values intermediate between the other two) may determine acoustic divergence in the other species. For Sardinian R. mehelyi and R. euryale, we also carried out intraspecific comparisons of call frequencies between bats from monospecific colonies and those from mixed colonies. Results As hypothesized, Sardinian R. hipposideros and R. euryale called at frequencies higher and lower, respectively, than in the peninsula. In this way, overlap with R. mehelyi is avoided. Body size showed no difference between insular and peninsular populations, i.e. frequency differences are not a by‐product of difference in body size determined by insularity. Frequency values in Sardinian R. euryale from monospecific colonies did not differ from those of bats roosting together with R. mehelyi. However, R. mehelyi showed frequency values significantly higher when associated with R. euryale, possibly to minimize the risk of species misrecognition. Main conclusions At least under geographical isolation, character displacement may be a causal mechanism for shifts in call frequency of sympatric rhinolophids. Species recognition and facilitation of intraspecific communication (with possible implications for mate recognition) constitute the best candidate factors for the phenomenon we observed.     

Body size as an important factor determining trophic niche partitioning in three syntopic rhinolophid bat species

We investigated a community of syntopically occurring horseshoe bats (Rhinolophus hipposideros, R. euryale, R. ferrumequinum) in southern Slovakia. The faecal pellets of these bat species were collected in the field and later analysed under a dissecting microscope. The three species studied are known to be very similar as far as their ecology, echolocation and preferred habitats are concerned, but they diverge significantly in their body sizes. In this study, all three species fed predominantly on moths [59–80 percentage frequency (%f); 87–95 percentage volume (%vol)], but their diet compositions differed in the size of individuals consumed. The smallest bat species (R. hipposideros) fed only on the smallest moths (%f = 59; %vol = 87), the medium-sized species (R. euryale) mainly on medium-sized moths (%f = 60; %vol = 74) and the largest one (R. ferrumequinum) especially on the largest moths (%f = 54; %vol = 89). Despite similar preferred habitat and the main prey category, the rates of trophic niche overlap were surprisingly low. The trophic niche percentage overlap was 7–31% (computed from %f data) and 1–20% (computed from %vol data), respectively and suggests an extraordinary importance of mere divergences of bats in their body sizes for trophic niche partitioning and stable species coexistence.     

Does the survival of greater horseshoe bats and Geoffroy’s bats in Western Europe depend on traditional cultural landscapes?

Greater horseshoe bats (Rhinolophus ferrumequinum) and Geoffroy’s bats (Myotis emarginatus) are two sympatric species that have undergone a serious population decline in Central Europe. In both species, population decline is likely to have been caused by habitat deterioration and habitat loss resulting in a decrease of carrying capacity. We examined the spatial ecology, habitat use and key landscape features in vital populations in R. ferrumequinum and M. emarginatus in the northern part of their current European distribution in Luxembourg. In total, 3,559 fixes from 26 radiotracked individuals were calculated. The tracked individuals of both species showed commuting flights along hedgerows, streams and small-forested patches. Maximum flight distances, home range sizes and habitat diversity did not differ between species, but R. ferrumequinum had significantly larger foraging ranges than adult M. emarginatus. Contrary to other studies, both bat species preferred semi-open, but richly structured traditional farmland habitats such as orchards, pastures and parkland habitats instead of available large broad-leaved deciduous forests. M. emarginatus frequently preyed on abundant arthropod resources in cowsheds, sometimes even exclusively throughout the night. The landscape configuration around the buffered radio fixes of adult R. ferrumequinum and M. emarginatus differed significantly from that around analysed random buffers. Radio fixes were concentrated in a landscape characterised by higher habitat diversity than in the surrounding region. Our results support the concept of a ‘biocultural link’ between traditional land use and biodiversity, and we therefore strongly recommend the conservation of extensive traditional land use practices, which will help to preserve both endangered bat species along with the local biodiversity.     

Perch-hunting in insectivorous Rhinolophus bats is related to the high energy costs of manoeuvring in flight

Foraging behaviour of bats is supposedly largely influenced by the high costs of flapping flight. Yet our understanding of flight energetics focuses mostly on continuous horizontal forward flight at intermediate speeds. Many bats, however, perform manoeuvring flights at suboptimal speeds when foraging. For example, members of the genus Rhinolophus hunt insects during short sallying flights from a perch. Such flights include many descents and ascents below minimum power speed and are therefore considered energetically more expensive than flying at intermediate speed. To test this idea, we quantified the energy costs of short manoeuvring flights (<2 min) using the Na-bicarbonate technique in two Rhinolophus species that differ in body mass but have similar wing shapes. First, we hypothesized that, similar to birds, energy costs of short flights should be higher than predicted by an equation derived for bats at intermediate speeds. Second, we predicted that R. mehelyi encounters higher flight costs than R. euryale, because of its higher wing loading. Although wing loading of R. mehelyi was only 20% larger than that of R. euryale, its flight costs (2.61 ± 0.75 W; mean ± 1 SD) exceeded that of R. euryale (1.71 ± 0.37 W) by 50%. Measured flight costs were higher than predicted for R. mehelyi, but not for R. euryale. We conclude that R. mehelyi face elevated energy costs during short manoeuvring flights due to high wing loading and thus may optimize foraging efficiency by energy-conserving perch-hunting.     

Winter diet analysis in Rhinolophus euryale (Chiroptera)

We investigated the winter food of Mediterranean horseshoe bats (Rhinolophus euryale) in four winter cave roosts in southern Slovakia and northern Hungary and investigated the relationship between food and ambient temperature. The bats were active during the whole winter period and they produced excrement throughout the entire hibernation period, even when outside temperatures dropped below zero. The guano was in two forms, containing (1) prey items and (2) non-prey items. The identifiable items belonged to lepidopteran species, but only one was identified, on the basis of the genital fragments, the moth Colotois pennaria, which was the main prey species in autumn and early winter. Our results shed light on the extraordinarily high level of activity in this bat species during winter hibernation, which in temperate regions is a strategy that enables bats to survive when prey is reduced or absent. In R. euryale, the torpor in the course of hibernation is not continuous and our results help to explain how energy losses caused by bat movements are covered.     

Brevity is prevalent in bat short-range communication

Animal communication follows many coding schemes. Less is known about the coding strategy for signal length and rates of use in animal vocal communication. A generalized brevity (negative relation between signal length and frequency of use) is innovatively explored but remains controversial in animal vocal communication. We tested brevity for short-range social and distress sounds from four echolocating bats: adult black-bearded tomb bat Taphozous melanopogon, Mexican free-tailed bat Tadarida brasiliensis, adult greater horseshoe bat Rhinolophus ferrumequinum, and adult least horseshoe bat Rhinolophus pusillus. There was a negative association between duration and number of social but not distress calls emitted. The most frequently emitted social calls were brief, while most distress calls were long. Brevity or lengthiness was consistently selected in vocal communications for each species. Echolocating bats seem to have convergent coding strategy for communication calls. The results provide the evidence of efficient coding in bat social vocalizations, and lay the basis of future researches on the convergence for neural control on bats’ communication calls.     

Characteristics of the northernmost population ofRhinolophus ferrumequinum in the Carpathian Basin

Roost utilisation byRhinolophus ferrumequinum (Schreber, 1774) was investigated between 1984 and 1998 in north-eastern Hungary. Exploration of summer and winter roosts, monitoring and bat-banding were implemented to find movements between the colonies. Data on roost utilisation by this species in south-eastern Slovakia, collected in a similar way, were included for comparison. Twenty-two marked bats were recaptured. The studied bats created nursery colonies in Hungarian churches and moved to Slovakian mines and caves to hibernate in winter. The population used two main hibernacula, two large nursery roosts and one temporary roost but several other roosts were also visited. The area occupied by the population was 5180 km².R. ferrumequinum living in SE Slovakia and NE Hungary formed probably a separate population on the northern edge of the species range. This population is a part of the metapopulation of the species.     

Assessing road effects on bats: the role of landscape, road features, and bat activity on road-kills

Recent studies suggest that roads can significantly impact bat populations. Though bats are one of the most threatened groups of European vertebrates, studies aiming to quantify bat mortality and determine the main factors driving it remain scarce. Between March 16 and October 31 of 2009, we surveyed road-killed bats daily along a 51-km-long transect that incorporates different types of roads in southern Portugal. We found 154 road-killed bats of 11 species. The two most common species in the study area, Pipistrellus kuhlii and P. pygmaeus, were also the most commonly identified road-kill, representing 72 % of the total specimens collected. About two-thirds of the total mortality occurred between mid July and late September, peaking in the second half of August. We also recorded casualties of threatened and rare species, including Miniopterus schreibersii, Rhinolophus ferrumequinum, R. hipposideros, Barbastella barbastellus, and Nyctalus leisleri. These species were found mostly in early autumn, corresponding to the mating and swarming periods. Landscape features were the most important variable subset for explaining bat casualties. Road stretches crossing or in the vicinity of high-quality habitats for bats—including dense Mediterranean woodland (“montado”) areas, water courses with riparian gallery, and water reservoirs—yielded a significantly higher number of casualties. Additionally, more road-killed bats were recorded on high-traffic road stretches with viaducts, in areas of higher bat activity and near known roosts.     

Low genetic variability of <Emphasis Type="Italic">Rhinolophus mehelyi</Emphasis> (Mehely's horseshoe bat) in Romania

Rhinolophus mehelyi (Mehely's horseshoe bat) is a vulnerable species with an increasingly fragmented distribution. In Romania, populations of R. mehelyi have experienced a dramatic decline over the past 50 years, and the current population size is estimated at only 100 adult individuals inhabiting almost exclusively the Limanu cave. In the present study, we investigated the genetic consequences of population decline for the viability of the remaining population of R. mehelyi in Romania. We sequenced and analyzed a 359-bp fragment of the mitochondrial control region from the only known Romanian population and compared it with two geographically close colonies from Bulgaria. A single haplotype was found in the Romanian population compared to 10 in the Bulgarian population, suggesting genetic isolation.     

Impact of predation by a cave-dwelling bat, Rhinolophus ferrumequinum, on the diapausing population of a troglophilic moth, Goniocraspidum preyeri

The predation pressure of the greater horseshoe bat, Rhinolophus ferrumequinum, on the diapausing population of the noctuid moth, Goniocraspidum preyeri, was examined at an abandoned mine in central Japan. These bats did not prey on the moths in summer. The number of moths preyed on was largest in March. More than 60% of the diapausing moths were eaten by only three or four bats, which accounted for over 90% of all the moth deaths. This moth was an important source of food at the end of the hibernating period when the bat had already used most of its stored subcutaneous fat. The predation on G. preyeri may have raised the overwintering success rate of R. ferrumequinum.     

Antioxidant Defenses in the Brains of Bats during Hibernation

Hibernation is a strategy used by some mammals to survive a cold winter. Small hibernating mammals, such as squirrels and hamsters, use species- and tissue-specific antioxidant defenses to cope with oxidative insults during hibernation. Little is known about antioxidant responses and their regulatory mechanisms in hibernating bats. We found that the total level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the brain of each of the two distantly related hibernating bats M. ricketti and R. ferrumequinum at arousal was lower than that at torpid or active state. We also found that the levels of malondialdehyde (product of lipid peroxidation) of the two hibernating species of bats were significantly lower than those of non-hibernating bats R. leschenaultia and C. sphinx. This observation suggests that bats maintain a basal level of ROS/RNS that does no harm to the brain during hibernation. Results of Western blotting showed that hibernating bats expressed higher amounts of antioxidant proteins than non-hibernating bats and that M. ricketti bats upregulated the expression of some enzymes to overcome oxidative stresses, such as superoxide dismutase, glutathione reductase, and catalase. In contrast, R. ferrumequinum bats maintained a relatively high level of superoxide dismutase 2, glutathione reductase, and thioredoxin-2 throughout the three different states of hibernation cycles. The levels of glutathione (GSH) were higher in M. ricketti bats than in R. ferrumequinum bats and were significantly elevated in R. ferrumequinum bats after torpor. These data suggest that M. ricketti bats use mainly antioxidant enzymes and R. ferrumequinum bats rely on both enzymes and low molecular weight antioxidants (e.g., glutathione) to avoid oxidative stresses during arousal. Furthermore, Nrf2 and FOXOs play major roles in the regulation of antioxidant defenses in the brains of bats during hibernation. Our study revealed strategies used by bats against oxidative insults during hibernation.     

Integrating population genetics to define conservation units from the core to the edge of Rhinolophus ferrumequinum western range

The greater horseshoe bat (Rhinolophus ferrumequinum) is among the most widespread bat species in Europe but it has experienced severe declines, especially in Northern Europe. This species is listed Near Threatened in the European IUCN Red List of Threatened Animals, and it is considered to be highly sensitive to human activities and particularly to habitat fragmentation. Therefore, understanding the population boundaries and demographic history of populations of this species is of primary importance to assess relevant conservation strategies. In this study, we used 17 microsatellite markers to assess the genetic diversity, the genetic structure, and the demographic history of R. ferrumequinum colonies in the western part of its distribution. We identified one large population showing high levels of genetic diversity and large population size. Lower estimates were found in England and northern France. Analyses of clustering and isolation by distance suggested that the Channel and the Mediterranean seas could impede R. ferrumequinum gene flow. These results provide important information to improve the delineation of R. ferrumequinum management units. We suggest that a large management unit corresponding to the population ranging from Spanish Basque Country to northern France must be considered. Particular attention should be given to mating territories as they seem to play a key role in maintaining high levels of genetic mixing between colonies. Smaller management units corresponding to English and northern France colonies must also be implemented. These insular or peripheral colonies could be at higher risk of extinction in the near future.     

Classification of insects by echolocating greater horseshoe bats

Summary Echolocating greater horseshoe bats (Rhinolophus ferrumequinum) detect insects by concentrating on the characteristic amplitude- and frequency modulation pattern fluttering insects impose on the returning echoes. This study shows that horseshoe bats can also further analyse insect echoes and thus recognize and categorize the kind of insect they are echolocating.Four greater horseshoe bats were trained in a twoalternative forced-choice procedure to choose the echo of one particular insect species turning its side towards the bat (Fig. 1). The bats were able to discriminate with over 90% correct choices between the reward-positive echo and the echoes of other insect species all fluttering with exactly the same wingbeat rate (Fig. 4).When the angular orientation of the reward-positive insect was changed (Fig. 2), the bats still preferred these unknown echoes over echoes from other insect species (Fig. 5) without any further training. Because the untrained bats did not show any prey preference, this indicates that the bats were able to perform an aspect-anglein-dependent classification of insects.Finally we tested what parameters in the echo were responsible for species recognition. It turned out that the bats especially used the small echo-modulations in between glints as a source of information (Fig. 7). Neither the amplitudenor the frequencymodulation of the echoes alone was sufficient for recognition of the insect species (Fig. 8). Bats performed a pattern recognition task based on complex computations of several acoustic parameters, an ability which might be termed cognitive.Abbreviations AM amplitude modulation - CF constant frequency - FM frequency modulation - S+ positive stimulus - S- negative stimulus     

Comparison of Brain Transcriptome of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) in Active and Torpid Episodes

Hibernation is an energy-saving strategy which is widely adopted by heterothermic mammals to survive in the harsh environment. The greater horseshoe bat (Rhinolophus ferrumequinum) can hibernate for a long period in the hibernation season. However, the global gene expression changes between hibernation and non-hibernation season in the greater horseshoe bat remain largely unknown. We herein reported a comprehensive survey of differential gene expression in the brain between winter hibernating and summer active greater horseshoe bats using next-generation sequencing technology. A total of 90,314,174 reads were generated and we identified 1,573 differentially expressed genes between active and torpid states. Interestingly, we found that differentially expressed genes are over-represented in some GO categories (such as metabolic suppression, cellular stress responses and oxidative stress), which suggests neuroprotective strategies might play an important role in hibernation control mechanisms. Our results determined to what extent the brain tissue of the greater horseshoe bats differ in gene expression between summer active and winter hibernating states and provided comprehensive insights into the adaptive mechanisms of bat hibernation.     

Microsatellite loci for the greater horseshoe bat,Rhinolophus ferrumequinum (Rhinolophidae,Chiroptera) and their cross‐utility in 17 other bat species

We have isolated 14 polymorphic greater horseshoe bat, Rhinolophus ferrumequinum microsatellite loci. The number of alleles varied from two to 12 in 58 individuals. These loci will be used to assign paternity in order to characterize patterns of breeding and reproductive success in a wild R. ferrumequinum population. Loci were also tested in 17 other bat species. Twelve loci cross‐amplified in other species and three loci were polymorphic in all eight Rhinolophus species tested.     

Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum,with special reference to nose leaf formation

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a “nose leaf,” a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15–16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.     

Molecular identification of Eimeria species in Spanish bats

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.