Trichosporon species isolated from guano samples obtained from bat-inhabited caves in Japan

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of approximately 700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.     

Trichosporon Species Isolated from Guano Samples Obtained from Bat-Inhabited Caves in Japan

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ~700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.     

Non-native pollen found in short-tailed bat (

We analysed pollen in short-tailed bat guano samples from Rangataua Forest and from guano and pollen found in bat holding bags used in the Kaimanawa Range, central North Island. Fifty seven percent of the pollen from Rangataua was from a previously unrecorded source and was tentatively identified as Trachycarpus fortunei (Chinese windmill palm). The significant remaining pollen was identified as Collospermum (15%) and Nothofagus (14%) from Rangataua, and Collospermum (90%) and Nothofagus (6%) from Kaimanawa. While the presence of Collospermum from both sites is consistent with previous work, pollen from T. fortunei, an exotic palm growing near Rangataua Forest, has not previously been found in association with short-tailed bats. Despite nocturnal surveillance with automated bat detectors and infra-red video cameras, we failed to confirm bat visitation to these palms. Nothofagus is wind-pollinated and pollen extracted from samples taken from both sites is probably wind-borne contamination. A collation of data from all available studies on the pollen found associated with short-tailed bats throughout New Zealand reveals that flowers from just four plant groups appear to be regularly used by bats: Collospermum spp., Knightia excelsa, Metrosideros spp. and, apparently, T. fortunei.     

Environmental information from guano palynology of insectivorous bats of the central part of the United States of America

Bat droppings accumulate in caves, and the resultant guano contains a stratigraphic record of the environment analogous to the record from lake sediment and peat. The bats forage at night for insects; they return to the cave during the day to sleep and care for their young. They attach themselves to suitable perches in the cave ceiling, and their excrement accumulates on the floor below. Flying requires a lot of energy, and bats of temperate regions consume large numbers of night-flying insects. In some situations the guano can reach a depth of meters in hundreds to thousands of years, and it has a valuable chronostratigraphy. The bat scats occur as small pellets that represent the non-digestible portion of the animal's diet in the preceding few hours; hence the diet provides information about the time of the year the feeding occurred. Bat guano contains, among other things, insect fragments, hair, pollen, and mineral matter. Night-flying insects do not normally visit flowers for the pollen; many species do not eat during the flying phase of their life cycle, and those that do generally are nectar feeders. Although the insects are not after the pollen, they do fly through a pollen-laden environment, and the pollen and dust adheres to their bodies. The insects essentially act as living traps for airborne debris. The bats also are furry pollen traps; during grooming they ingest pollen and dust enmeshed in their fur, and this also is excreted. The pollen in an individual scat contains a record of the atmospheric pollen during a single day in the past. This kind of detail is rarely available from lake sediment. Chemical analysis of individual bat scats in a time series can chart the changing environment caused by agriculture, industry, volcanic dust, and a host of other details that depend only on the cleverness of the researcher. Careful ¹⁴C analysis can isolate the times when bats did not use the cave, and that may be useful in interpreting past conditions. If the insect types in the guano change over time, that may provide evidence of changing climate. Pollen was analyzed from guano samples taken from Tumbling Creek Cave near Protem, Missouri, USA. The cave contains a maternal colony of the Grey Bat (Myotis grisescens) that occupies the cave for a short time each year. Scats collected from the base of a 70 cm thick cone of guano yielded an AMS ¹⁴C date of 2810 ± 40 yr BP. The fecal material has a crumbly structure below the surface; it was of mahogany color (7.5 YR 2/1 to 3/2) and had no noticeable odor. Guano can be processed like normal sediment, but simple washing in a weak detergent solution followed by acetolysis appears adequate.     

Is Histoplasma capsulatum a native inhabitant of Japan?

Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat‐inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma‐specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.     

Iron age to medieval entomogamous vegetation and Rhinolophus hipposideros roost in South-Eastern Wales (UK)

Karst cave systems are well developed in Wales (UK) and, in some instances, constitute important bat roosts. Ogof Draenen, near Blaenavon in south-east Wales, is the most recent major cave discovery (1994) with already > 70 km of passages explored spanning a vertical range of 148 m.With the exception of one small chamber (Siambre Ddu) located directly above the main Ogof Draenen system, very few bats have been noticed inside. Extensive accumulations of guano, attributable to Rhinolophus hipposideros, are however found in parts of the Ogof Draenen system. In places covering many square metres and sometimes building heaps > 0.5 m thick, these represent volumes not yet found in any other cave system in the British Isles.Although the date of the abandonment of the main Ogof Draenen system as a bat roost remains unknown, six radiocarbon dates on guano from Ogof Draenen place the occupation in the Iron Age to Medieval period at least. Palynological analysis was undertaken on ten samples distributed through the cave. Comparisons were made with a moss polster and a lake mud sample from the area to provide a first approximation of the regional modern pollen rain and with two modern guano samples, one from Siambre Ddu and one from Agen Allwedd cave (5 km to the north-west) to provide a temporal comparison with the fossil guano. Agen Allwedd cave currently is one of the largest active roosts for Lesser Horseshoe bats in Britain and lies close to the present northern limit of this endangered species in Europe.The main results are that (1) the cave appears to have been used both as a summer and a winter roost; (2) most of the Ogof Draenen guano is formed within c. 1600 ¹⁴C years and, if the largest heap is continuous, it has accumulated within 750 ¹⁴C years, i. e. 0.16 mm yr^− 1; 3) the fossil guano samples reflect a relatively closed oak forest with more abundant ivy (Hedera) and holly (Ilex) than at present; (4) insect-pollinated plants such as Ilex, Acer, Hedera and Impatiens glandulifera are over-represented in the guano samples; (5) in addition to the usual causes of bat roost decline (pesticides, pollution), in the case of Ogof Draenen, we may add entrance blocked by rock collapse and decline of the local forest cover as well as change in its composition.     

Detection of Environmental Sources of Histoplasma capsulatum in Chiang Mai, Thailand, by Nested PCR

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.     

Pollen assemblages of tauber traps and surface soil samples in steppe areas of China and their relationships with vegetation and climate

We use 86 pollen trap and surface soil pollen samples in steppe areas of China to explore the relationships between modern pollen, vegetation, and climate. The modern pollen spectra from both sources have comparable compositions with regard to the major pollen taxa. However, the number of taxa in the traps was higher than in the surface soil samples. Both pollen accumulation rates and pollen concentrations are higher in the typical steppe areas than in the desert steppe areas. Discriminant analysis indicates that pollen spectra from trap and surface soil samples roughly reflect the vegetation zones of desert steppe and typical steppe, especially in the case of the trap samples. Detrended canonical correspondence analysis suggests that pollen assemblages have a significant relationship with the temperature of the coldest month and the mean annual precipitation.     

Diverse archaeal community of a bat guano pile in Domica Cave (Slovak Karst,Slovakia)

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5–3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.     

Guano exposed: Impact of aerobic conditions on bat fecal microbiota

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large‐scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost‐effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12 hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environment—much faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non‐exposed samples could still be recovered after 12 hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.     

Occurrence of Dermatophytes in Fresh Bat Guano

Evidence is presented in support of the hypothesis that fresh bat guano serves as a means of pathogenic fungi dissemination in caves. A total of 371 guano samples were collected from caves in southeastern New Mexico. Each sample was agitated in sterile saline and sand. The supernatant fluid was treated with an antibiotic and streaked on differential media. Cultures were incubated at 25 and 37 C and examined at intervals over a 4-week period. For animal inoculation, highly concentrated inoculum was injected intraperitoneally into white Swiss mice. Animals were sacrificed 4 weeks later, and portions of their lung, liver, and spleen were cultured on selective media, incubated at 25 C, and examined at intervals over a 4-week period. Microsporum gypseum was isolated at all 10 collecting stations with an incidence of 22.4%, Trichophyton mentagrophytes at 7 stations with an incidence of 5%, T. rubrum at 3 stations with an incidence of 3%, and T. terrestre at 1 station with an incidence of 0.5%. From a total of 60 pools of liver-spleen-lung suspensions, 6 pools yielded positive cultures of Histoplasma capsulatum and 1 pool yielded T. mentagrophytes. No significant difference was found among the different selective media with respect to recovery of dermatophytes. Among the human pathogenic fungi isolated were Candida sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans, H. capsulatum, M. gypseum, T. mentagrophytes, T. rubrum, T. terrestre, and Sporotrichum sp.     

Modern and ancient olive stands near Sagalassos (south-west Turkey) and reconstruction of the ancient agricultural landscape in two valleys

Aim To study the present‐day olive stands and their ecology in the eastern part of the territory of the ancient city of Sagalassos, to study the variation of olive pollen production and dispersal near the olive stands, to establish a modern pollen reference model, and to compare Hellenistic–Roman pollen data from two wetlands with this modern reference model. Location Eastern part of the territory of the ancient city of Sagalassos, western Taurus mountain range in south‐west Turkey. Methods The study is based on field survey, pollen analysis of surface samples, multivariate statistics of modern pollen data and the use of ‘modern analogues’ in comparison with Hellenistic–Roman pollen samples. Results A field survey revealed the presence of 35 olive stands in the study area. These are mainly small‐scale stands. The olive pollen representation in the surface samples is highly variable. Two groups of modern ‘olive’ pollen spectra could be distinguished: (1) a group representing mainly olive stands from lush and moist mixed orchards; and (2) a group representing mainly olive stands from open small‐scale olive stands in combination with annual crop agriculture. Although no ‘perfect’ modern analogue was found for the Hellenistic–Roman pollen data, the fossil pollen data show similarities with modern spectra from the second group, due to the presence of relatively high pollen values for secondary anthropogenic indicators. Main conclusion A well‐organized and diverse, but time‐ and energy‐consuming, agricultural system was maintained nearby the wetlands of Çanakl? soils, presumably to maximize the yields in both valleys.     

Feeding by the short‐tailed bat (Mystacina tuberculata) on fruit and possibly nectar

New Zealand's short‐tailed bat (Mystacina tuberculata Gray, 1843) feeds on fruit, insects, and possibly nectar in North Island kauri (Agathis australis) forest. Fruits eaten by members of a colony of 500 bats in May included those of Freycinetia baueriana (Pandanaceae), Collospermum hastatum, and C. microspermum (Liliaceae). Pollen analyses of bat guano, and of the stomach contents of 4 short‐tailed bats from Omahuta Forest (Lat. 35°10'S) and 3 from Stewart Island and adjacent islands (Lat. 47°15'S), showed that most of the pollen was from flowers of Metrosideros and Leptospermum (Myrtaceae), Knightia excelsa (Proteaceae), and Collospermum, and that spores of the tree fern Cyathea (Cyatheaceae) were present also. Both Metrosideros and Knightia have abundant nectar. The partially extensile tongue of Mystacina is tipped with a brush of fine papillae, possibly to extract nectar and pollen; but the pollen and spores in the bat stomachs and guano could have come from insects eaten by the bats. Transverse ridges on the tongue may assist removal of juice from ripe fruits. These bats may disperse the small seeds of Freycinetia baueriana. The anatomical modifications of Mystacina for terrestrial and arboreal locomotion may have evolved primarily in response to its frugivorous and suspected nectarivorous habits.     

Larval dispersal and cannibalism in a natural population of Zophobas atratus (Coleoptera: Tenebrionidae)

A natural population, of the tenebrionid beetle Zophobas atratus was studied in an attic in El Zamorano, Honduras. Larvae were living in bat guano and were, densest in the deepest part of the guano. No pupae or pharate pupae were found in the guano: they were always located a small distance away from the guano pile. Adults occurred on the guano surface and the beams and tiles of the roof. When larvae of three different ages and weights were marked and released in the guano, only the older, larger larvae were recaptured away from the guano, indicating a correlation between larval dispersal and increased weight and/or age. When 50 pharate pupae were exposed to a natural population of active larvae, only 4 (8%) survived after 5 days. This intense cannibalism of pharate pupae is consistent with the hypothesis that inhibition of pupation by crowding and the associated pre-pupal dispersal evolved, at least partly, in response to cannibalism.     

Leptogenys khammouanensis sp. nov. (Hymenoptera: Formicidae). A possible troglobitic species of Laos,with a discussion on cave ants

The new species Leptogenys khammouanensis sp. nov. is described from two caves of the Khammouan karst (Laos). It is characterized by a set of striking morphological characters (reduced eyes, light pigmentation, slender body and very elongated legs and antennae), which recall the troglobiomorphic traits of cave arthropods. Relations between caves and ants are discussed at this occasion, in the light of the recent biological explorations of caves in Southeast Asia. The classical view that ants are rare and unimportant in caves is challenged. Ants are actually major and regular components of guano assemblages in many caves of the region, but none of these guano species exhibit cave-related adaptation in its external morphology. Conversely, ants are very rare in low-resources habitats, where only accidental occurrence of outside species are reported in Southeast Asia. Leptogenys khammouanensis has been found only in such an oligotrophic environment, very deep in the cave and far from any guano deposits. Its presence there, together with its troglobiomorphic traits, support the idea that Leptogenys khammouanensis might be the first truly troglobitic ant.     

<Emphasis Type="Italic">Chrysosporium speluncarum</Emphasis>, a new species resembling <Emphasis Type="Italic">Ajellomyces capsulatus,</Emphasis> obtained from bat guano in caves of temperate Europe

A new species, Chrysosporium speluncarum, obtained from bat guano in caves in the Czech Republic, Slovakia, and Romania, is described, based on phenotype and rDNA sequences. It possesses tuberculate conidia similar to the Histoplasma anamorph of Ajellomyces capsulatus, but it is not dimorphic, does not grow at 37°C, lacks microconidia, and is nonpathogenic and nonkeratinolytic. All studied characteristics suggest a close relationship with Renispora flavissima. The morphologically similar C. chiropterorum, another species known from the bat environment, is a relative of C. lobatum and Polytolypa hystricis, but is distinct from A. capsulatus and C. speluncarum. The affinity for bats and the presence of conidia with prominent tuberculations has originated convergently in several groups of onygenalean fungi. In addition, the occurrence of fungi mimicking A. capsulatus in caves of temperate Europe is reviewed.     

Tracking dietary habits of cave arthropods associated with deposits of hematophagous bat guano: A study from a neotropical savanna

Carbon and nitrogen stable isotopes were used to track major dietary variations in arthropods found in a cave located in a neotropical savanna in Central Brazil. We determined the δ¹³C and δ¹⁵N for cave crickets, cockroaches, spiders, guano of a hematophagous bat, and leaf litter found on the ground near the cave entrance. The δ¹³C and δ¹⁵N for the cricket and cockroaches showed that bat guano was not the only food item for these arthropods. They had intermediate δ¹³C and δ¹⁵N between bat guano and leaf litter, which means that they consumed additional food resources other than guano in their diet, independent of distribution of guano deposits in the cave. The spiders, predators with great mobility, seemed to not have a preferential diet item since their isotope signals indicated they hunt both crickets and cockroaches. The δ¹³C of the bat guano (?15.4‰) indicated that the diet of these hematophagous bats relied on animals fed with C₄ plants. The conversion of native savanna vegetation of which the debris is mostly from C₃ plants, to pasture, based on C₄ African grass, a remarkably common land use transformation in the region, is indirectly influencing the diet of hematophagous bats, which basically relied on exotic fauna. The C and N stable isotope ratios showed that the arthropods inside the cave do not rely solely on bat guano for their diet, but interact directly with the external environment, through litter debris, indicating a significant exchange of energy and matter between the cave environment and the surrounding area.     

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.     

A taphonomic study of modern pollen assemblages from dung and surface sediments in arid environments of Spain

Coastal plant communities in arid southeastern Spain are characterized by insect-pollinated scrub species, which fail to occur in Quaternary pollen sequences from valleys, marshlands and marine cores. We investigate pollen–vegetation relationships in samples from soil surfaces, animal dung, and sediments in depressions or basins that, in theory, should have pollen spectra that are comparable to those from sedimentary basins elsewhere. Pollen spectra from basins or depressions are very susceptible to long-distance wind and water transport. This can mask representation of pollen from the surrounding insect-pollinated vegetation, as can over-representation of basin-margin halophilous and hydrophilous pollen. Pollen spectra from biogenic materials of animal origin are the best analogues of local and regional vegetation, and show the best analytical potential in terms of pollen concentration and taxon diversity. Pollination properties of the species studied indicate they will rarely be found in most conventional pollen records. It cannot be stressed too strongly that insight into Quaternary vegetation of arid regions demands complementary pollen analysis of coprolites, urine-cemented deposits, and cave sediments with preserved biotic remains, in addition to water-lain sediments.     

Isolation of Exophiala jeanselmei from bat guano

Exophiala jeanselmei was isolated from two out of 75 samples of bat guano. The isolates hydrolysed urea and tyrosine, assimilated sodium nitrate and produced a zone of haemolysis on blood agar, They were pathogenic for laboratory mice when injected subcutaneously, intraperitoneally or intravenously.