Bat rabies, public health and European bat conservation.

Most records of European bat lyssaviruses (EBLVs) are confined to three species - the serotine bat for EBLV1 (900 records) and Daubenton's bat and the pond bat for EBLV2 (25 records). High levels of seroprevalence, which may vary from year to year, are also recorded. All bat vectors of EBLVs are synanthropic, some exclusively so. Despite this, there have been only five cases of human rabies resulting from EBLV infection in the 590 million people of greater Europe during the last 35 years. These have triggered major programmes of surveillance in many European countries. The emphasis on active versus passive surveillance and the intensity with which they have been carried out has varied from country to country. Both involve cooperation between bat researchers, virologists and public health officials and the latter, in particular, engages amateur bat workers and members of the public. Bat NGOs throughout Europe have worked to persuade the public not to handle bats or to do so only with gloved hands and, in the case of bat workers, to receive pre-exposure immunization. They have also countered negative media coverage of bat rabies. Householders with bat roosts in their dwellings have in general been persuaded to retain their bats. Attempts have been made to persuade all European countries to establish comparable EBLV surveillance programmes. In the last 25 years, virologists, public health officials, bat biologists and conservationists, both amateur and professional have worked closely and collaboratively for the protection of the public and the conservation of bats, with little polarization of views.

European bat lyssaviruses: Distribution, prevalence and implications for conservation.

Worldwide, there are more than 1100 species of the Order Chiroptera, 45 of which are present in Europe, and 16 in the UK. Bats are reservoirs of, or can be infected by, several viral diseases, including rabies virus strains (in the Lyssavirus genus). Within this genus are bat variants that have been recorded in Europe; European bat lyssavirus 1 (EBLV-1), European bat lyssavirus 2 (EBLV-2) and, four currently unclassified isolates. Since 1977, 783 cases of EBLVs (by isolation of viral RNA) have been recorded in Europe. EBLV-1 or EBLV-2 has been identified in 12 bat species, with over 95% of EBLV-1 infections identified in Eptesicus serotinus. EBLV-2 is associated with Myotis species (Myotis daubentonii and Myotis dasycneme). A programme of passive surveillance in the United Kingdom between 1987 and 2004 tested 4871 bats for lyssaviruses. Of these, four M. daubentonii (3.57% of submitted M. daubentonii) were positive for EBLV-2. Potential bias in the passive surveillance includes possible over-representation of synanthropic species and regional biases caused by varying bat submission numbers from different parts of the UK. In 2003, active surveillance in the UK began, and has detected an antibody prevalence level of 1-5% of EBLV-2 in M. daubentonii (n = 350), and one bat with antibodies to EBLV-1 in E. serotinus (n = 52). No cases of live lyssavirus infection or lyssavirus viral RNA have been detected through active surveillance. Further research and monitoring regarding prevalence, transmission, pathogenesis and immunity is required to ensure that integrated bat conservation continues throughout Europe, whilst enabling informed policy decision regarding both human and wildlife health issues.

Undernutrition of ewe lambs in utero and in early post-natal life does not affect hypothalamic-pituitary function in adulthood.

The effect of undernutrition in utero, during late gestation (from day 100), and early neonatal life on hypothalamic-pituitary function was investigated in female lambs born to ewes fed rations calculated to provide either 100% (high; H) or 70% (low; L) of the energy requirements to sustain a twin pregnancy. Following parturition in early spring, ewes and lambs were maintained on pasture with sward heights of 6 cm (H) or 4 cm (L) until week 8 of lactation and then sward heights of 5 cm (H) or 3 cm (L) until weaning at week 14. Mean lamb birth weights were 18% lower in L than H animals (P<0.05) and mean liveweights were 23% lower in the L animals (P<0.001) at weaning at 14 weeks of age. Liveweight differences were not significant at, or after, 26 weeks of age. There were no significant differences between pre-pubertal H and L animals, either before (26 weeks) or after ovariectomy (31 weeks), with respect to hypothalamic or pituitary activity, as measured by LH pulse frequency, pulse amplitude or mean plasma LH and FSH concentrations and the responses to GnRH injection as measured by LH peak amplitude, respectively. Similarly there were no differences in any of these variables in pubertal animals at 18 months of age. At 31 weeks of age, H animals had significantly lower pituitary GnRH receptor binding (P<0.01) and lower ERalpha mRNA content (P<0.05) than L lambs. There were no differences with treatment in the abundance of mRNA for LHbeta, FSHbeta or GnRH-receptor at 31 weeks of age or in pubertal animals aged 18 months, when there were no significant differences with treatment in GnRH receptor binding or ERalpha mRNA expression. It is concluded that effects on lifetime reproductive function of female sheep of undernutrition during late gestation and early neonatal life are unlikely to be expressed through permanent changes in hypothalamic-pituitary function and are therefore attributable to effects exerted directly on the ovary.

Effect of maternal overnutrition during pregnancy on pituitary gonadotrophin gene expression and gonadal morphology in female and male foetal sheep at day 103 of gestation.

The aim was to determine whether nutritionally mediated restriction of placental growth alters foetal body growth, pituitary gonadotrophin gene expression and gonadal development at Day 103 of gestation. Embryos recovered from adult ewes inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After transfer, adolescent ewes were offered a high (H, n=16) or moderate (M, n=12) level of a complete diet. Ewes were slaughtered at 103+/-0.2 days of gestation and foetal blood, brain, pituitary and gonads were collected. Mean placental weight was lower (P< 0.01) in H than in M groups but foetal weight and reproductive organ weights were similar. Maternal nutrition did not influence LHbeta or FSHbeta mRNA expression in either sex but FSHbeta mRNA expression was higher (P< 0.001) in female (n=11) than in male (n=17) foetal pituitaries. Mean foetal plasma gonadotrophin concentrations were not influenced by dietary intake in either sex. Plasma progesterone concentrations were lower (P=0.001) in foetuses derived from H compared with M intake dams. Compared with M foetuses (n=5), ovaries from H foetuses (n=6) had fewer primordial follicles (P< 0.05) and fewer follicles in total (P< 0.005). In contrast, maternal nutritional status did not influence either seminiferous cord or Sertoli cell numbers in male foetuses (H, n=10; M, n=7). It is concluded that high maternal nutrient intakes restricted placental growth and altered foetal ovarian follicular development prior to the end of the second third of gestation. The latter effect was independent of gonadotrophin secretion.Crown

Impact of maternal nutrition during pregnancy on pituitary gonadotrophin gene expression and ovarian development in growth-restricted and normally grown late gestation sheep fetuses.

The influence of maternal nutrition during pregnancy on anterior pituitary gonadotrophin gene expression and ovarian development in sheep fetuses during late gestation was investigated. Embryos recovered from superovulated adult ewes that had been inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After embryo transfer, adolescent ewes were offered a high or moderate amount of a complete diet. Pregnancies were terminated at day 131 +/- 0.6 of gestation and the fetal brain, anterior pituitary gland and gonads were collected. Gonadotrophin gene expression (LHbeta and FSHbeta subunits) in the fetal pituitary gland was examined using in situ hybridization. Ovarian follicular development was quantified in haematoxylin- and eosin-stained ovarian sections embedded in paraffin wax. Six dams that were offered a high nutrient intake carried normal-sized fetuses (weight within +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet) and 13 dams carried growth-restricted fetuses (weight

Effect of acoustic clutter on prey detection by bats.

Bats that capture animal prey from substrates often emit characteristic echolocation calls that are short-duration, frequency-modulated (FM) and broadband. Such calls seem to be suited to locating prey in uncluttered habitats, including flying prey, but may be less effective for finding prey among cluttered backgrounds because echoes reflecting from the substrate mask the acoustic signature of prey. Perhaps these call designs serve primarily for spatial orientation. Furthermore, it has been unclear whether the acoustic image conveyed by FM echoes enables fine texture discrimination, or whether gleaning bats that forage in echo-cluttering environments must locate prey by using other cues, such as prey-generated sounds. Here we show that two species of insectivorous gleaning bats perform badly when compelled to detect silent and immobile prey in clutter, but are very efficient at capturing noisy prey items among highly cluttered backgrounds, and both dead or live prey in uncluttered habitats. These findings suggest that the short, broadband FM echolocation calls associated with gleaning bats are not adapted to detecting prey in clutter.

Influence of placentally mediated fetal growth restriction on the onset of puberty in male and female lambs.

The onset of puberty in prenatally growth-restricted versus normally grown lambs of both sexes, born in April and housed under natural photoperiod, was examined. Singleton pregnancies were established and adolescent ewes were offered a high or moderate nutrient intake throughout gestation. Placental mass was reduced (P < 0.001) in high compared with moderate intake dams and resulted in the birth of growth-restricted and normal birth weight offspring, respectively. At birth, female lambs weighed 3.43 kg versus 5.03 kg (P < 0.001; n = 14 per group) and male lambs weighed 2.75 kg versus 5.18 kg (P < 0.001; n = 7 per group) in growth-restricted and normal birth weight groups, respectively. Lambs suckled for 12 weeks and thereafter were fed ad libitum until week 43 of age. Growth-restricted lambs had lower preweaning live weight gains and this difference was more pronounced in male (P < 0.05) than in female lambs (P = 0.07). Thereafter, live weight remained lower (P < 0.05) in growth-restricted than in normally grown lambs of both sexes until week 25 of age. In females, the time of onset of puberty was similar in the two groups. All females ovulated and there were no differences in the number of ovarian cycles recorded or in the incidence of aberrant ovarian function. In males, testosterone concentrations and testicular volume were lower in growth-restricted compared with normally developed lambs from birth until weeks 28 and 35 of age, respectively (P < 0.05). The seasonal increase in plasma testosterone concentrations occurred later in growth-restricted than in normally developed lambs (P < 0.01) but the timing of maximum peak concentrations was similar. Peak testosterone concentrations were lower (P < 0.05) in growth-restricted than in normal male lambs.

Mating patterns, relatedness and the basis of natal philopatry in the brown long-eared bat, Plecotus auritus.

The brown long-eared bat, Plecotus auritus, is unusual among temperate zone bats in that summer maternity colonies are composed of adult males and females, with both sexes displaying natal philopatry and long-term association with a colony. Here, we describe the use of microsatellite analysis to investigate colony relatedness and mating patterns, with the aim of identifying the evolutionary determinants of social organization in P. auritus. Mean colony relatedness was found to be low (R=0.033 +/- 0.002), with pairwise estimates of R within colonies ranging from -0.4 to 0.9. The proportion of young fathered by males in their own colony was investigated using a Bayesian approach, incorporating parameters detailing the number of untyped individuals. This analysis revealed that most offspring were fathered by males originating from a different colony to their own. In addition, we determined that the number of paternal half-sibs among cohorts of young was low, inferring little or no skew in male reproductive success. The results of this study suggest that kin selection cannot account for colony stability and natal philopatry in P. auritus, which may instead be explained by advantages accrued through the use of familiar and successful roost sites, and through long-term associations with conspecifics. Moreover, because the underlying causes of male natal dispersal in mammals, such as risk of inbreeding or competition for mates, appear to be avoided via extra-colony copulation and low male reproductive skew, both P. auritus males and females are able to benefit from long-term association with the natal colony.

Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus).

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.

Matrilineal genetic structure and female-mediated gene flow in red grouse (Lagopus lagopus scoticus): an analysis using mitochondrial DNA.

DNA sequence variation at the hypervariable 5' end of the mitochondrial control region was examined in 247 individuals to detect genetic divergence among 14 populations of red grouse (Lagopus lagopus scoticus) in northeastern Scotland. Ten haplotypes were resolved, several of which were shared among populations. Analysis of molecular variance, Nei's gamma ST, and a cladistic estimate of the amount of gene flow indicated a lack of overall population differentiation. Patterns of overall panmixia are in stark contrast to previous reports of localized subdivision among the same set of populations detected using hypervariable microsatellite markers. Because grouse cocks are territorial and show extreme natal philopatry and females are the dispersing sex, such discordance could be explained by sex-biased dispersal, with extensive female-mediated gene flow preventing mitochondrial DNA divergence. However, it is difficult to reconcile how effective dispersal of females would not homogenize both mitochondrial and nuclear structure simultaneously. We use a model that examines the spatial and temporal dynamics of diparentally and uniparentally inherited genes to show that, under realistic ecological scenarios and with specific differences in the dispersal of males and females, the local effective size of the nuclear genome can be less than that of the mitochondrial and the patterns of structuring we observe are meaningful.

Influence of torpor on milk protein composition and secretion in lactating bats.

In the pipistrelle bat (Pipistrellus pipistrellus), the metabolic load of lactation is not met to any significant extent by increased food intake or mobilization of body reserves, and aerial foraging accounts for most of the animal's energy expenditure even during lactation. Energy conservation must, therefore, play a critical role in maintaining lactation. The principal mechanism for energy conservation appears to be the bat's ability to enter torpor, but this may itself interrupt milk synthesis and secretion unless the pipistrelle mammary gland is adapted to counteract its effect. The effect of torpor on mammary tissue function was studied in mammary tissue explant cultures prepared in weeks 1-3 of lactation, when milk water yield was 0.20, 0.88, and 0.30 mL/d respectively. Protein synthesis measured by incorporation of radiolabeled amino acids was 44% lower (P < 0.001) in bat tissue explants cultured at ambient temperature (22 degrees C) compared with 37 degrees C. The reduction was similar to that observed in mouse mammary tissue (57%) and was unaffected by stage of lactation. Analysis of explant protein after [35S]methionine labelling showed the majority of proteins synthesised in culture to be milk proteins; it also demonstrated that the decrease in protein synthesis at ambient temperature was a general phenomenon: synthesis of both secretory and intracellular mammary proteins was reduced at the lower culture temperature. The results suggest that bat mammary tissue has no mechanism to counteract the effect of reduced body temperature and that periods of lactational torpor are likely to cause a pronounced diurnal variation in the rate of milk secretion.

Testis-like steroidogenesis in the ovotestis of the European mole, Talpa europaea.

The female European mole (Talpa europaea) presents a vivid paradox in relation to our contemporary understanding of mammalian sexual differentiation. These animals are exceptional among female mammals in that they possess bilateral ovotestes. The ovotestis contains a morphologically normal ovarian component that develops during the spring breeding season and a histologically defined testicular region, the interstitial gland, which enlarges during autumn when the ovarian component decreases in size. In correlation with this unusual gonadal situation, the female mole displays a penile clitoris traversed by a urethral canal. Although the histology of the ovotestis is well documented and has recently been extended to an additional three species of the genus Talpa, there have been no clear indications of the physiological function, particularly androgen production, of the ovotestis in these female moles. This paper presents the first clear evidence of seasonal variation in plasma testosterone concentrations, which parallel the growth and regression of the "testicular" interstitial gland, in T. europaea. Plasma androstenedione did not show significant seasonal variation, but plasma testosterone (1.06 +/- 0.2 ng/ml) and gonadal testosterone concentration (1.57 +/- 0.65 microgram/mg protein) in females in autumn were significantly higher (p < 0.02) than plasma (0.4 +/- 0.2 ng/ml) and gonadal (0.24 +/- 0.21 microgram/mg) concentrations in pregnant or immediately postpartum females in spring. Our data also reveal selective metabolic production of testosterone from radiolabeled steroid precursors (progesterone and androstenedione) by these ovarian interstitial tissues and male testes; estradiol is produced by ovarian tissue but not interstitial gland or testis.

The effect of prenatal photoperiodic history on the postnatal endocrine status of female lambs.

Postnatal photoperiodic experience plays a pivotal role in determining the timing of ovarian activity in female lambs. This study examines whether a photoperiodic history gained while in utero is able to influence this timing. Pregnant Soay ewes were maintained in either long days (n = 7, 18 h light: 6 h dark; group PLD) or short days (n = 12, 6 h light: 18 h dark; group PSD) from 25 days of gestation. At birth, female lambs (n = 8 per group) were transferred to long days for 10 weeks, and then placed under short days until the end of the experiment at 38 weeks of age. Blood samples were collected from lambs on the day of birth and three times weekly for the duration of the study and the resulting plasma assayed for progesterone and prolactin. Although both gestational photoperiods produced, at best, abbreviated periods of ovarian activity, lambs born to ewes which experienced long days during gestation (group PLD) exhibited elevated plasma progesterone concentrations significantly earlier (P < 0.05) than lambs born to ewes exposed to short days during gestation (group PSD) (mean +/- SEM, 193 +/- 17 versus 244 +/- 14 days for PLD and PSD groups, respectively. Plasma prolactin concentrations in newborn lambs born between late December and early April were not affected by the ambient photoperiod, but reflected the artificial daylength experienced by their mothers during gestation. Lambs born to ewes maintained under long days during gestation (group PLD) had significantly higher prolactin concentrations on the day of birth than lambs born to ewes maintained under short days during gestation (group PSD) (45 +/- 5.4 ng/ml versus 7 +/- 3.7 ng/ml respectively, P < 0.001). The mean birth weight, rate of live weight gain and live body weight of lambs at the end of the experiment did not vary significantly between treatment groups. These results suggest that the ovine foetus is sensitive to photoperiodic information prior to birth, and develops a photoperiodic history which, under the present experimental conditions, modulates the subsequent endocrine status of the neonatal lamb.

Effect of undernutrition of ewes from the time of mating on fetal ovarian development in mid gestation.

Ewes were fed either 150% (High, H) or 50% (Low, L) of their energy requirements for maintenance of liveweight during early gestation. Effects of maternal nutrition on fetal ovarian size, histological structure and steroidogenic capacity were studied at Day 47 and on ovarian size and structure at Day 62 of gestation. At Day 47 of gestation, there were significantly higher concentrations of oogonia in the ovaries of L fetuses than H fetuses (105.9 v. 76.9 germ cells mm(-2); s.e. 4.94; P < 0.001). The capacity of the ovaries to secrete oestradiol (pg/ovary/24 h) at Day 47 was not affected by treatment when they were incubated either with (H, 773; L, 740; s.e. 179; not significant, n.s.) or without (H, 260; L, 290; s.e. 92.7; n.s.) ovine luteinizing hormone (oLH). At Day 62 of gestation, the process of germ cell degeneration was less advanced in L than H fetal ovaries, as indicated by higher oocyte concentrations in the former (68.4 v. 48.6 germ cells mm(-2); s.e. 3.85; P < 0 01). There was a greater percentage of meiotic cells in L ovaries (76.5 v. 18.6; s.e. 5.82; P < 0.001). It is concluded that undernutrition of the ewe from the time of mating significantly retards ovarian development in fetal ovaries.

Effects of naloxone, metaclopramide and domperidone on plasma levels of prolactin and LH following suckling in the female rabbit.

Saline, naloxone, domperidone or metaclopramide was injected into lactating rabbits immediately before suckling. Blood samples were taken prior to injection (0 minutes) and then at 15, 30, 45 and 60 minutes after the start of suckling, after which the samples were assayed for plasma prolactin and LH concentrations. In all the does there was a significant increase in prolactin concentration, which was highest 15 minutes after the start of suckling, and which declined exponentially thereafter to levels significantly higher than before suckling. The increase in prolactin concentration was similar in does given saline and naloxone, but it was significantly enhanced in does given metaclopramide; with domperidone the increase was intermediate and not significantly different from that following treatment with saline. In does given saline, domperidone, and metaclopramide plasma LH concentrations declined slowly during the hour after suckling but the concentration was increased significantly in does given naloxone. The inverse correlations between prolactin and LH were low weak and were not significant.

Effect of stage of lactation and milk accumulation on mammary cell differentiation in lactating bats.

Mammary cell differentiation was measured in lactating pipistrelle bats (Pipistrellus pipistrellus) by assay of key enzyme activities, and by determination of protein and lactose synthesis rates in short-term tissue cultures. By these criteria, mammary cell differentiation did not change significantly with stage of lactation, but depended on the extent to which the gland was filled with milk. Key enzyme activities and in vitro synthesis rates were significantly higher in glands suckled immediately before tissue collection, compared with contralateral glands that were engorged with milk. This indicates that mammary cell differentiation in the lactating bat is regulated locally within each gland by a mechanism sensitive to milk accumulation, to the extent that, unlike other species, this obscures any underlying effect of stage of lactation.

Oxygen consumption and evaporation during parturition in a vespertilionid bat (Pipistrellus pipistrellus).

Oxygen consumption and evaporation were measured in a single pregnant pipistrelle bat during labour and parturition of twins, using an open-flow respirometry system. During 233 min of measurements, three distinct phases were noted, which we suggest represent prelabour, labour and grooming or suckling the young. On the basis of this hypothesis, during labour and parturition, oxygen consumption was a maximum of 8.9% of daily energy expenditure, evaporation was 2.7% of daily water turnover, and total water loss was 5.5% of daily water turnover in free-living bats in early lactation. We estimated that, for the mother and young combined, oxygen consumption associated with grooming and suckling would be equivalent to 37.5% of daily energy expenditure of the mother, if carried out continuously. Similarly, evaporation due to grooming and suckling would be equivalent to 16.4% of daily water turnover. In terms of daily energy expenditure and daily water turnover, labour and parturition are therefore cheap, but grooming and suckling (even ignoring costs in terms of losses in the milk) are expensive.

The functional significance of ventilation frequency, and its relationship to oxygen demand in the resting brown long-eared bat, Plecotus auritus.

Mean oxygen consumption and simultaneous ventilation frequency of nine non-reproductive brown long-eared bats (body mass 8.53-13.33 g) were measured on 159 occasions. Ambient (chamber) temperature at which the measurements were made ranged from 10.8 to 41.1 degrees C. Apneic ventilation occurred in 22 of the 59 measurements made when mean oxygen consumption was less than 0.5 ml.min-1. No records of apneic ventilation were obtained when it was over 0.5 ml.min-1. The relationship between ventilation frequency and mean oxygen consumption depended on whether ventilation was apneic or non-apneic. When ventilation was non-apneic the relationship was positive and log-linear. When ventilation was apneic the relationship was log-log. Within the thermoneutral zone ventilation frequency was not significantly different from that predicted from allometric equations for a terrestrial mammal of equivalent body mass, but was significantly greater than that predicted for a bird. A reduction in the amount of oxygen consumed per breath occurred at ambient temperatures above the upper critical temperature (39 degrees C).