Multiple bursts of pancreatic ribonuclease gene duplication in insect-eating bats

Pancreatic ribonuclease gene (RNASE1) was previously shown to have undergone duplication and adaptive evolution related to digestive efficiency in several mammalian groups that have evolved foregut fermentation, including ruminants and some primates. RNASE1 gene duplications thought to be linked to diet have also been recorded in some carnivores. Of all mammals, bats have evolved the most diverse dietary specializations, mainly including frugivory and insectivory. Here we cloned, sequenced and analyzed RNASE1 gene sequences from a range of bat species to determine whether their dietary adaptation is mirrored by molecular adaptation. We found that seven insect-eating members of the families Vespertilionidae and Molossidae possessed two or more duplicates, and we also detected three pseudogenes. Reconstructed RNASE1 gene trees based on both Bayesian and maximum likelihood methods supported independent duplication events in these two families. Selection tests revealed that RNASE1 gene duplicates have undergone episodes of positive selection indicative of functional modification, and lineage-specific tests revealed strong adaptive evolution in the Tadarida β clade. However, unlike the RNASE1 duplicates that function in digestion in some mammals, the bat RNASE1 sequences were found to be characterized by relatively high isoelectric points, a feature previously suggested to promote defense against viruses via the breakdown of double-stranded RNA. Taken together, our findings point to an adaptive diversification of RNASE1 in these two bat families, although we find no clear evidence that this was driven by diet. Future experimental assays are needed to resolve the functions of these enzymes in bats.     

Behavioural flexibility: the little brown bat, Myotis lucifugus, and the northern long-eared bat,M. septentrionalis , both glean and hawk prey

We present behavioural data demonstrating that the little brown bat, Myotis lucifugus, and the northern long-eared bat, M. septentrionalis, can glean prey from surfaces and take prey on the wing. Our data were collected in a large outdoor flight room mimicking a cluttered environment. We compared and analysed flight behaviours and echolocation calls used by each species of bat when aerial hawking and gleaning. Our results challenge the traditional labelling ofM. lucifugus as an obligate aerial-hawking species and show that M. septentrionalis, which is often cited as a gleaning species, can capture airborne prey. As has been shown in previous studies, prey-generated acoustic cues were necessary and sufficient for the detection and localization of perched prey. We argue that the broadband, high-frequency, downward-sweeping, frequency-modulated calls used by some bats when gleaning prey from complex surfaces resolve targets from background. First, because calls of lower frequency and narrower bandwidth are sufficient for assessing a surface before landing, and second, because there are few, if any, simple surfaces in nature from which substrate-gleaning behaviours in wild bats would be expected. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.     

Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.     

Tripartite parasitic and symbiotic interactions as a possible mechanism of horizontal gene transfer

Herbivory is a highly sophisticated feeding behavior that requires abilities of plant defense suppression, phytochemical detoxification, and plant macromolecule digestion. For plant‐sucking insects, salivary glands (SGs) play important roles in herbivory by secreting and injecting proteins into plant tissues to facilitate feeding. Little is known on how insects evolved secretory SG proteins for such specialized functions. Here, we investigated the composition and evolution of secretory SG proteins in the brown marmorated stink bug (Halyomorpha halys) and identified a group of secretory SG phospholipase C (PLC) genes with highest sequence similarity to the bacterial homologs. Further analyses demonstrated that they were most closely related to PLCs of Xenorhabdus, a genus of Gammaproteobacteria living in symbiosis with insect‐parasitizing nematodes. These suggested that H. halys might acquire these PLCs from Xenorhabdus through the mechanism of horizontal gene transfer (HGT), likely mediated by a nematode during its parasitizing an insect host. We also showed that the original HGT event was followed by gene duplication and expansion, leading to functional diversification of the bacterial‐origin PLC genes in H. halys. Thus, this study suggested that an herbivore might enhance adaptation through gaining genes from an endosymbiont of its parasite in the tripartite parasitic and symbiotic interactions.     

Mutations in the VNTR of the carboxyl-ester lipase gene (CEL) are a rare cause of monogenic diabetes

We have previously shown that heterozygous single-base deletions in the carboxyl-ester lipase (CEL) gene cause exocrine and endocrine pancreatic dysfunction in two multigenerational families. These deletions were found in the first and fourth repeats of a variable number of tandem repeats (VNTR), which has proven challenging to sequence due to high GC-content and considerable length variation. We have therefore developed a screening method consisting of a multiplex PCR followed by fragment analysis. The method detected putative disease-causing insertions and deletions in the proximal repeats of the VNTR, and determined the VNTR-length of each allele. When blindly testing 56 members of the two families with known single-base deletions in the CEL VNTR, the method correctly assessed the mutation carriers. Screening of 241 probands from suspected maturity-onset diabetes of the young (MODY) families negative for mutations in known MODY genes (95 individuals from Denmark and 146 individuals from UK) revealed no deletions in the proximal repeats of the CEL VNTR. However, we found one Danish patient with a short, novel CEL allele containing only three VNTR repeats (normal range 7–23 in healthy controls). This allele co-segregated with diabetes or impaired glucose tolerance in the patient’s family as six of seven mutation carriers were affected. We also identified individuals who had three copies of a complete CEL VNTR. In conclusion, the CEL gene is highly polymorphic, but mutations in CEL are likely to be a rare cause of monogenic diabetes.     

Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme

Background

Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism.

Principal Findings

Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition.

Conclusions

Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism.     

Improved Analysis of Long-Term Monitoring Data Demonstrates Marked Regional Declines of Bat Populations in the Eastern United States

Bats are diverse and ecologically important, but are also subject to a suite of severe threats. Evidence for localized bat mortality from these threats is well-documented in some cases, but long-term changes in regional populations of bats remain poorly understood. Bat hibernation surveys provide an opportunity to improve understanding, but analysis is complicated by bats'' cryptic nature, non-conformity of count data to assumptions of traditional statistical methods, and observation heterogeneities such as variation in survey timing. We used generalized additive mixed models (GAMMs) to account for these complicating factors and to evaluate long-term, regional population trajectories of bats. We focused on four hibernating bat species – little brown myotis (Myotis lucifugus), tri-colored bat (Perimyotis subflavus), Indiana myotis (M. sodalis), and northern myotis (M. septentrionalis) – in a four-state region of the eastern United States during 1999–2011.Our results, from counts of nearly 1.2 million bats, suggest that cumulative declines in regional relative abundance by 2011 from peak levels were 71% (with 95% confidence interval of ±11%) in M. lucifugus, 34% (±38%) in P. subflavus, 30% (±26%) in M. sodalis, and 31% (±18%) in M. septentrionalis. The M. lucifugus population fluctuated until 2004 before persistently declining, and the populations of the other three species declined persistently throughout the study period. Population trajectories suggest declines likely resulted from the combined effect of multiple threats, and indicate a need for enhanced conservation efforts. They provide strong support for a change in the IUCN Red List conservation status in M. lucifugus from Least Concern to Endangered within the study area, and are suggestive of a need to change the conservation status of the other species. Our modeling approach provided estimates of uncertainty, accommodated non-linearities, and controlled for observation heterogeneities, and thus has wide applicability for evaluating population trajectories in other wildlife species.     

Histochemical and biochemical properties of flight muscle fibers in the little brown bat,Myotis lucifugus

Summary The purpose of this investigation was (1) to determine the fiber composition of pectoralis muscle of the little brown bat,Myotis lucifugus; (2) to compare the fiber composition of this muscle with two of the animal's accessory flight muscles; and (3) to study the effect of hibernation on pectoralis muscle fiber composition. Bat skeletal muscle fibers were also compared with those of white laboratory rats (Rattus norvegicus). Bat pectoralis muscles possessed exceptionally high oxidative capacities as indicated by their succinate dehydrogenase activities, but relatively low glycolytic potentials (phosphofructokinase activities). Muscle histochemistry demonstrated that fiber composition of bat pectorlis muscle was homogeneous; all fibers possessed high aerobic and low glycolytic potentials, and high myofibrillar ATPase activities indicating fast contractile properties. In contrast, accessory flight muscles possessed three distinguishable fiber types. During hibernation there was a significant decline in oxidative potential, no change in glycolytic potential, and no alteration in basic fiber composition of bat pectoralis muscle. The findings of this study suggest that pectoralis muscles ofM. lucifugus may approach the ultimate adaptation of a mammalian locomotory muscle for aerobic generation of muscular power.Abbreviations FG fast-twich glycolytic - FOG fast-twitch-oxydative-glycolytic - -GPDH -glycerophosphate dehydrogenase - LDH lactate dehydrogenase - NADH-D reduced nicotinamide adenine dinucleotide diaphorase - PFK phosphofructokinase - SDH succinate dehydrogenase - SO slowtwich-oxidative     

Aerial insectivorous bat activity in relation to moonlight intensity

It is commonly assumed that aerial insectivorous bats in the tropics respond to moonlight intensity by decreasing their foraging activity during bright nights due either to an increase in predation risk, or to a reduction in insect availability.The effect of moonlight on bat activity can be measured both between nights and within a single night. However, few studies have simultaneously used both approaches, and most authors generally compare bat activity with lunar phases. Our main aim was to evaluate how moonlight influences aerial insectivorous bat activity at different time scales: between nights and within the same night. Activity of five bat species was measured using autonomous ultrasound recording stations and moonlight intensity percentages retrieved from the Moontool program nightly throughout a 53-day sampling period. Only one species (Myotis riparius) responded negatively to moonlight, while two species (Pteronotus parnellii and Saccopteryx leptura) increased their foraging activity in moonlight. For Cormura brevirostris and S. bilineata, moonlight intensity did not affect activity level. Bat activity was greater for all species at the beginning of the night, independent of the presence of the moon, indicating that foraging just after the sunset is adaptive. Thus, bat response to the effect of moonlight intensity is more apparent between nights than within a single night and may depend on species-specific traits, such as flight speed, flexibility in habitat use and body size.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

Seasonal reliance on nectar by an insectivorous bat revealed by stable isotopes

Many animals have seasonally plastic diets to take advantage of seasonally abundant plant resources, such as fruit or nectar. Switches from insectivorous diets that are protein rich to fruits or nectar that are carbohydrate rich present physiological challenges, but are routinely done by insectivorous songbirds during migration. In contrast, insectivorous bat species are not known to switch diets to consume fruit or nectar. Here, we use carbon stable isotope ratios to establish the first known case of a temperate bat species consuming substantial quantities of nectar during spring. We show that pallid bats (Antrozous pallidus) switch from a diet indistinguishable from that of sympatric insectivorous bat species in winter (when no cactus nectar is present) to a diet intermediate between those of insectivorous bats and nectarivorous bats during the spring bloom of a bat-adapted cactus species. Combined with previous results that established that pallid bats are effective pollinators of the cardon cactus (Pachycereus pringlei), our results suggest that the interaction between pallid bats and cardon cacti represents the first-known plant-pollinator mutualism between a plant and a temperate bat. Diet plasticity in pallid bats raises questions about the degree of physiological adaptations of insectivorous bats for incorporation of carbohydrate-rich foods, such as nectar or fruit, into the diet.     

Adaptive Evolution of the Hox Gene Family for Development in Bats and Dolphins

Bats and cetaceans (i.e., whales, dolphins, porpoises) are two kinds of mammals with unique locomotive styles and occupy novel niches. Bats are the only mammals capable of sustained flight in the sky, while cetaceans have returned to the aquatic environment and are specialized for swimming. Associated with these novel adaptations to their environment, various development changes have occurred to their body plans and associated structures. Given the importance of Hox genes in many aspects of embryonic development, we conducted an analysis of the coding regions of all Hox gene family members from bats (represented by Pteropus vampyrus, Pteropus alecto, Myotis lucifugus and Myotis davidii) and cetaceans (represented by Tursiops truncatus) for adaptive evolution using the available draft genome sequences. Differences in the selective pressures acting on many Hox genes in bats and cetaceans were found compared to other mammals. Positive selection, however, was not found to act on any of the Hox genes in the common ancestor of bats and only upon Hoxb9 in cetaceans. PCR amplification data from additional bat and cetacean species, and application of the branch-site test 2, showed that the Hoxb2 gene within bats had significant evidence of positive selection. Thus, our study, with genomic and newly sequenced Hox genes, identifies two candidate Hox genes that may be closely linked with developmental changes in bats and cetaceans, such as those associated with the pancreatic, neuronal, thymus shape and forelimb. In addition, the difference in our results from the genome-wide scan and newly sequenced data reveals that great care must be taken in interpreting results from draft genome data from a limited number of species, and deep genetic sampling of a particular clade is a powerful tool for generating complementary data to address this limitation.     

Numerical and Functional Responses of Forest Bats to a Major Insect Pest in Pine Plantations

Global change is expected to modify the frequency and magnitude of defoliating insect outbreaks in forest ecosystems. Bats are increasingly acknowledged as effective biocontrol agents for pest insect populations. However, a better understanding is required of whether and how bat communities contribute to the resilience of forests to man- and climate-driven biotic disturbances. We studied the responses of forest insectivorous bats to a major pine defoliator, the pine processionary moth pityocampa, which is currently expanding its range in response to global warming. We used pheromone traps and ultrasound bat recorders to estimate the abundance and activity of moths and predatory bats along the edge of infested pine stands. We used synthetic pheromone to evaluate the effects of experimentally increased moth availability on bat foraging activity. We also evaluated the top-down regulation of moth population by estimating T. pityocampa larval colonies abundance on the same edges the following winter. We observed a close spatio-temporal matching between emergent moths and foraging bats, with bat activity significantly increasing with moth abundance. The foraging activity of some bat species was significantly higher near pheromone lures, i.e. in areas of expected increased prey availability. Furthermore moth reproductive success significantly decreased with increasing bat activity during the flight period of adult moths. These findings suggest that bats, at least in condition of low prey density, exhibit numerical and functional responses to a specific and abundant prey, which may ultimately result in an effective top-down regulation of the population of the prey. These observations are consistent with bats being useful agents for the biocontrol of insect pest populations in plantation forests.     

Plasma proteomic profiles differ between European and North American myotid bats colonized by Pseudogymnoascus destructans

Emerging fungal diseases have become challenges for wildlife health and conservation. North American hibernating bat species are threatened by the psychrophilic fungus Pseudogymnoascus destructans (Pd) causing the disease called white‐nose syndrome (WNS) with unprecedented mortality rates. The fungus is widespread in North America and Europe, however, disease is not manifested in European bats. Differences in epidemiology and pathology indicate an evolution of resistance or tolerance mechanisms towards Pd in European bats. We compared the proteomic profile of blood plasma in healthy and Pd‐colonized European Myotis myotis and North American Myotis lucifugus in order to identify pathophysiological changes associated with Pd colonization, which might also explain the differences in bat survival. Expression analyses of plasma proteins revealed differences in healthy and Pd‐colonized M. lucifugus, but not in M. myotis. We identified differentially expressed proteins for acute phase response, constitutive and adaptive immunity, oxidative stress defence, metabolism and structural proteins of exosomes and desmosomes, suggesting a systemic response against Pd in North American M. lucifugus but not European M. myotis. The differences in plasma proteomic profiles between European and North American bat species colonized by Pd suggest European bats have evolved tolerance mechanisms towards Pd infection.     

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.     

Isolation and characterization of microsatellite marker loci in the Wagner’s mustached bat <Emphasis Type="BoldItalic">Pteronotus psilotis</Emphasis> (Chiroptera: Mormoopidae) and cross-amplification in other related species

Pteronotus psilotis, a mormoopid bat, is an insectivorous, gregarious and strict cave-dwelling species that is found areas between the sea level and an elevation of about 1000 masl. This species is present in diverse habitats ranging from rain forest to dry deciduous forest. Nine microsatellite loci were developed for Wagner’s mustached bat, Pteronotus psilotis using the next-generation sequencing approach, and their utility for population genetics studies was assessed. All loci were polymorphic (7–15 alleles) and characterized in 30 individuals from three P. psilotis populations, with the levels of observed and expected heterozygosity ranging from 0.280 to 0.867 and 0.584 to 0.842, respectively. One locus showed significant departures from Hardy–Weinberg expectations after Bonferroni correction. Cross-amplification in 11 other bat species was tested, for which eight microsatellites were successfully amplified, and of these seven were polymorphic. The development of these new microsatellite loci will contribute to investigations of genetic population structure, genetic diversity and gene flow in P. psilotis populations, as well as in other closely related bat species.