The nuclei of the lateral lemniscus in the rufous horseshoe bat,Rhinolophus rouxi

In the rufous horseshoe bat, Rhinolophus rouxi, responses to pure tones and sinusoidally frequency modulated (SFM) signals were recorded from 289 single units and 241 multiunit clusters located in the nuclei of the lateral lemniscus (NLL). The distribution of best frequencies (BFs) of units in all three nuclei of the lateral lemniscus showed an overrepresentation in the range corresponding to the constant-frequency (CF) part of the echolocation signal ('filter frequency' range): in the ventral nucleus of the lateral lemniscus (VNLL) 'filter neurons' represented 43% of all units encountered, in the intermediate nucleus (INLL) 33%, and in dorsal nucleus (DNLL) 29% (Fig. 2a). Neurons with best frequencies in the filter frequency range had highest Q10dB-values (maxima up to 400, Fig. 2c) and only in low-frequency units were values comparable to those found in other mammals. On the average, filter neurons in ventral nucleus had higher Q10dB-values (about 220) than did those in intermediate and dorsal nucleus (both about 160, Fig 2d). Response patterns and tuning properties showed higher complexity in the dorsal and intermediate nucleus than in the ventral nucleus of the lateral lemniscus (Figs. 4 and 6). Multiple best frequencies were found in 12 neurons, nine of them with harmonically related excitation maxima (Fig. 5c, d). Best frequencies of six of these harmonically tuned units could not be correlated with any harmonic components of the echolocation signal. Half of all multiple tuned neurons were located in the caudal dorsal nucleus the other half in the caudal intermediate nucleus. Synchronization of responses to sinusoidally frequency modulated (SFM) signals occurred in VNLL-units in the average up to modulation frequencies of 515 Hz (maximum about 800 Hz) whereas in the intermediate and dorsal nucleus of the lateral lemniscus responses were synchronized in the average only up to modulation frequencies of about 300 Hz (maximum about 600 Hz) (Figs. 7 and 8). A tonotopic arrangement of units was found in the intermediate nucleus of the lateral lemniscus with units having high best frequencies located medially and those with low best frequencies laterally. In the dorsal nucleus the tonotopic distribution was found to be fairly similar to that in the intermediate nucleus but much less pronounced. In more rostral parts of the dorsal nucleus additionally higher best frequencies predominated whereas in caudal areas of that nucleus and also of the intermediate nucleus low BFs were found more regularly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogenesis of auditory fovea representation in the inferior colliculus of the Sri Lankan rufous horseshoe bat,Rhinolophus rouxi

Summary This report describes the ontogenesis of tonotopy in the inferior colliculus (IC) of the rufous horseshoe bat (Rhinolophus rouxi). Horseshoe bats are deaf at birth, but consistent tonotopy with a low-to-high frequency gradient from dorsolateral to ventromedial develops from the 2nd up to the 5th week. The representation of the auditory fovea is established in ventro-mediocaudal parts of the IC during the 3rd postnatal week (Fig. 3). Then, a narrow frequency band 5 kHz in width, comprising 16% of the bat's auditory range, captures 50–60 vol% of the IC (Fig. 3c). However, foveal tuning is 10–12 kHz (1/3 octave) lower than in adults; foveal tuning in females (65–68 kHz) is 2–3 kHz higher than in males (62–65 kHz). Thereafter, foveal tuning increases by 1–1.5 kHz per day up to the 5th postnatal week, when the adult hearing range is established (Figs. 4, 5). The increase of sensitivity and of tuning sharpness of single units also follows a low-to-high frequency gradient (Fig. 6).Throughout this development the foveal tuning matches the second harmonic of the echolocation pulses vocalised by these young bats. The results confirm the hypothesis of developmental shifts in the frequency-place code for the foveal high frequency representation in the IC.Abbreviations BF best frequency - CF constant frequency - FM frequency modulation - IC inferior colliculus - IHC inner hair cell; - OHC outer hair cell - RR Rhinolophus rouxi     

Coding of sinusoidally amplitude modulated acoustic stimuli in the inferior colliculus of the rufous horseshoe bat,Rhinolophus rouxi

Summary Single neuron responses to sinusoidally amplitude modulated (SAM) signals were studied in the inferior colliculus of the horseshoe bat,Rhinolophus rouxi.57% of the neurons responded to SAM stimuli with periodical discharges synchronized to the modulation cycle. The proportion of cells driven by amplitude modulated signals was independent of the best frequency of the neurons. Best modulation frequencies were at or below 100 Hz in about 70% of the neurons. Synchronized activity could be elicited by modulation frequencies up to 400 Hz.Best SAM responses were observed at stimulus intensities 10 dB above threshold. Generally the BMF of a neuron did not change with intensity. The BMF decreased with decreasing modulation depth of the amplitude modulation.A trend for a topographical organization of neurons according to best modulation frequencies was detected. The results did not reveal any significant specialization of the bat's auditory system for coding of amplitude modulations as compared to other mammals.Abbreviations BF best frequency - BMF best modulation frequency - CF constant frequency - FM frequency modulation - IC inferior colliculus - SAM sinusoidal amplitude modulation - SFM sinusoidal frequency modulation     

Directional sensitivity of echolocation in the horseshoe bat,Rhinolophus ferrumequinum

Summary The directionality of sound emission by a horseshoe bat (Rhinolophus ferrumequinum) has been determined for the constant frequency component of its orientation sounds. The bat was fixed in the center of an acoustic perimeter and the SPL of the orientation sounds measured with a scanning microphone at different angles compared with the SPL measured by another microphone located in the direction perpendicular to the plane of the horseshoe-like structure of the nose-leaf. The maximum SPL was always found in this direction which also corresponds to the flight direction of a bat in horizontal flight. Above and lateral to this direction the SPL decreases steadily with -6 dB-points at 24 above and 23 lateral. Below the flight direction we found a prominent side lobe with a -6 dB-point at 64 .When the present data are combined with measurements of the behavioral directionality of hearing at the same frequency (Grinnell and Schnitzler, 1977), the directionality diagram of the entire echolocation system is very narrow and points in the flight direction. The prominent downward side lobe of emission does not conspicuously increase echolocation effectiveness in the direction of the ground, since hearing sensitivity is falling off so steeply in that direction. However, without this downward beam of emission, signals from below the bat would be that much less effective.Interference with the structure of the nose-leaf by covering the upper part with vaseline or plugging the left nostril destroyed the smoothness of the normal sound field and demonstrated that this complex organ is a highly functional structure optimized in the course of evolution.With differences in mood or attention, the emitted pulses varied by as much as 20 dB (80–100 dB). The emission directionality pattern also varied. In most cases, as orientation sounds increased in SPL, the acoustic beam became smaller.Supported by Deutsche Forschungsgemeinschaft, grant No. Schn 138/1-6, Stiftung Volkswagenwerk, grant No. 111 858, and the Alexander von Humboldt StiftungWe thank W. Hollerbach and C. Nitsche for technical assistance.     

Binaural influences on Doppler shift compensation of the horseshoe bat Rhinolophus rouxi

The flying horseshoe bat Rhinolophus rouxi compensates for Doppler shifts in echoes of their orientation pulses. By lowering the frequency of subsequent calls the echo's constant frequency is stabilized at the so-called reference frequency centered in a narrow and sensitive cochlear filter. This audio-vocal behaviour is known as Doppler shift compensation. To investigate whether the bats depend on binaural cues when compensating, three animals were tested for compensation on a swing before and after unilateral deafening. In each case compensation was severely impaired by unilateral deafening. Individual animals' compensation amplitude was reduced to 28–48% of the preoperational compensation of a +1.8 kHz shift. Doppler shift compensation performance did not recover to control levels during the observed period of 24 h after surgery. In contrast, unilateral middle ear removal which induces a unilateral auditory threshold increase of 9–14 dB does not impair compensation performance on the swing. To mimick Doppler shifts in a fixed setup, the frequencies of recorded echolocation calls were experimentally shifted between 0 and +2 kHz and played back via earphones to six animals. The bats completely compensated the experimental shifts only as long as the interaural intensity difference of the playback did not exceed 20 dB. No animal compensated with monaural playback. Accepted: 27 August 1999     

Determinants of echolocation call frequency variation in the Formosan lesser horseshoe bat (Rhinolophus monoceros)

The origin and maintenance of intraspecific variation in vocal signals is important for population divergence and speciation. Where vocalizations are transmitted by vertical cultural inheritance, similarity will reflect co-ancestry, and thus vocal divergence should reflect genetic structure. Horseshoe bats are characterized by echolocation calls dominated by a constant frequency component that is partly determined by maternal imprinting. Although previous studies showed that constant frequency calls are also influenced by some non-genetic factors, it is not known how frequency relates to genetic structure. To test this, we related constant frequency variation to genetic and non-genetic variables in the Formosan lesser horseshoe bat (Rhinolophus monoceros). Recordings of bats from across Taiwan revealed that females called at higher frequencies than males; however, we found no effect of environmental or morphological factors on call frequency. By comparison, variation showed clear population structure, with frequencies lower in the centre and east, and higher in the north and south. Within these regions, frequency divergence was directional and correlated with geographical distance, suggesting that call frequencies are subject to cultural drift. However, microsatellite clustering analysis showed that broad differences in constant frequency among populations corresponded to discontinuities in allele frequencies resulting from vicariant events. Our results provide evidence that the processes shaping genetic subdivision have concomitant consequences for divergence in echolocation call frequency.     

Harmonic and frequency structure used for echolocation sound pattern recognition and distance information processing in the rufous horseshoe bat

Summary The rufous horseshoe bat, Rhinolophus rouxi, was trained to discriminate differences in target distance. During the discrimination trials, the bats emitted complex FM/CF/FM pulses containing first harmonic and dominant second harmonic components.Loud free running artificial pulses, simulating the CF/FM part of the natural echolocation components, interfered with the ability of the bat to discriminate target distance. Changes in the frequency or frequency pattern of the artificial pulses resulted in systematic changes in the degree of interference. Interference occurred when artificial CF/FM pulses were presented at frequencies near those of the bat's own first or second harmonic components.These findings suggest that Rhinolophus rouxi uses both the first and second harmonic components of its complex multiharmonic echolocation sound for distance discrimination. For interference to occur, the sound pattern of each harmonic component must contain a CF signal followed by an FM sweep beginning near the frequency of the CF.Abbreviations CF constant frequency - FM frequency modulated     

Development and characterization of novel microsatellite markers from the Chinese rufous horseshoe bat (Rhinolophus sinicus) with cross-species amplification in closely related taxa

We isolated nine polymorphic microsatellite markers from the Chinese rufous horseshoe bat (Rhinolophus sinicus) using an enriched library method. We assessed genetic polymorphism at these loci in 42 individuals from a single population. We recorded high genetic diversity with four to 17 alleles per locus, and estimated expected and observed heterozygosity values ranging from 0.492 to 0.910 and from 0.462 to 0.881, respectively. No locus departed from Hardy-Weinberg equilibrium following Bonferroni correction, and no linkage disequilibrium was detected. Most loci successfully cross-amplified congeneric species. These loci will be used to characterize phylogeographical history of Rhinolophus sinicus in China.     

Parentage, reproductive success and breeding behaviour in the greater horseshoe bat (Rhinolophus ferrumequinum)

Female greater horseshoe bats form maternity colonies each summer in order to give birth and raise young. During the mating period, females visit males occupying territorial sites, copulation takes place and sperm are stored until ovulation occurs, normally in April. Using microsatellite markers and a likelihood method of parentage analysis, we studied breeding behaviour and male reproductive success over a five-year period in a population of bats in south-west Britain. Paternity was assigned with 80% confidence to 44% of young born in five successive cohorts. While a small annual skew in male reproductive success was detected, the variance increased over five years due to the repeated success of a few individuals. Mating was polygynous, although some females gave birth to offspring sired by the same male in separate years. Such repeated partnerships probably result from fidelity for either mating sites or individuals or from sperm competition. Females mated with males born both within and outside their own natal colony; however, relatedness between parents was no less than the average recorded for male female pairs. Gene flow between colonies is likely to be primarily mediated by both female and male dispersal during the mating period rather than more permanent movements.     

Echolocation sounds and hearing of the greater Japanese horseshoe bat (Rhinolophus ferrumequinum nippon)

Summary The relationship between the orientation sounds and hearing sensitivity in the greater Japanese horseshoe bat,Rhinolophus ferrumequinum nippon was studied.An orientation pulse consisted of a constant frequency (CF) component followed by a short downward frequency-modulated (FM) component. Sometimes, an initial upward FM component preceded the CF component. Duration of pulses was about 30 ms and the CF of resting pulses (RF) averaged 65.5 kHz. The best frequency (BF) at the lowest threshold in audiograms as measured by the pinna reflex averaged 66.1 kHz. Audiograms showed remarkable sharp cut-offs on both sides near the BF. The frequency difference between the BF and the RF was about 0.6 kHz, and the RF was always below the BF. The values of RF and BF were characteristically different from those of the European subspecies,Rhinolophus ferrumequinum ferrumequinum.Abbreviations BF best frequency - CF constant frequency - FM frequency modulated - RF resting frequency     

A gating mechanism for sound pattern recognition is correlated with the temporal structure of echolocation sounds in the rufous horseshoe bat

Summary The rufous horseshoe bat, Rhinolophus rouxi, was trained to discriminate differences in target distance. Loud free running artificial pulses, simulating the bat's natural long-CF/FM echolocation sounds, interfered with the ability of the bat to discriminate target distance. Interference occurred when the duration of the CF component of the CF/FM artificial pulse was between 2 and 70 ms. A brief (2.0 ms) CF signal 2–68 ms before an isolated FM signal was as effective as a continuous CF component of the same duration. When coupled with the bat's own emissions, a 2 ms FM sweep alone was effective in interfering when it came 42 to 69 ms after the onset of the bat's pulse. The coupled FM artificial pulses did not interfere when they began during the bat's own emissions.It appears that the onset of the CF component activates a gating mechanism that establishes a time window during which FM component signals must occur for proper neural processing. A comparison with a similar gating mechanism in Noctillo albiventris, which emits short-CF/FM echolocation sounds, reveals that the temporal parameters of the time window of the gating mechanism are species specific and specified by the temporal structure of the echolocation sound pattern of each species.Abbreviations FM frequency modulated - CF constant frequency     

Spectral and temporal gating mechanisms enhance the clutter rejection in the echolocating bat,Rhinolophus rouxi

Summary Doppler shift compensation behaviour in horseshoe bats, Rhinolophus rouxi, was used to test the interference of pure tones and narrow band noise with compensation performance. The distortions in Doppler shift compensation to sinusoidally frequency shifted echoes (modulation frequency: 0.1 Hz, maximum frequency shift: 3 kHz) consisted of a reduced compensation amplitude and/or a shift of the emitted frequency to lower frequencies (Fig. 1).Pure tones at frequencies between 200 and 900 Hz above the bat's resting frequency (RF) disturbed the Doppler shift compensation, with a maximum of intererence between 400 and 550 Hz (Fig. 2). Minimum duration of pure tones for interference was 20 ms and durations above 40 ms were most effective (Fig. 3). Interfering pure tones arriving later than about 10 ms after the onset of the echolocation call showed markedly reduced interference (Fig. 4). Doppler shift compensation was affected by pure tones at the optimum interfering frequency with sound pressure levels down to –48 dB rel the intensity level of the emitted call (Figs. 5, 6).Narrow bandwidth noise (bandwidth from ± 100 Hz to ± 800 Hz) disturbed Doppler shift compensation at carrier frequencies between –250 Hz below and 800 Hz above RF with a maximum of interference between 250 and 500 Hz above resting frequency (Fig. 7). The duration and delay of the noise had similar influences on interference with Doppler shift compensation as did pure tones (Figs. 8, 9). Intensity dependence for noise interference was more variable than for pure tones (-32 dB to -45 dB rel emitted sound pressure level, Fig. 10).The temporal and spectral gating in Doppler shift compensation behaviour is discussed as an effective mechanism for clutter rejection by improving the processing of frequency and amplitude transients in the echoes of horseshoe bats.Abbreviations CF constant frequency - FM frequency modulation - RF resting frequency - SPL sound pressure level     

Control of doppler shift compensation in the greater horseshoe bat,Rhinolophus ferrumequinum

Summary FlyingRhinolophus ferrumequinum lower the frequency of the constant frequency part (f_A) of the emitted sounds in order to compensate for Doppler shifts caused by the flight speed. The echo frequency (f_E) is kept constant within a frequency band of about 200 Hz, the center frequency of which is about 150 Hz above the average or resting frequency (f_R) emitted by roosting bats shortly before take off. For the compensation they use a feedback control system in which the emission frequency is changed to hold the echo frequency at a criterion value. This feedback system was demonstrated by experiments with bats flying in an experimental wind tunnel and in a He-O₂-micture. In the wind tunnelRhinolophus lowers the emission frequency in order to compensate for Doppler shifts which are caused by the ground speed flown by the bat. In the He-O₂-mixtureRhinolophus compensates for Doppler shifts which correspond to the different sound speeds in the gas mixture.I would like to thank D. R. Griffin for his generous support and stimulating criticism. I express my appreciation to the New York Zoological Society for the use of its facilities and to R. Brown for technical assistance. The work was supported by grant number GB 7155 from the National Science Foundation to the New York Zoological Society. I also thank J. D. Pye for his suggestions.     

Characterization of 14 polymorphic microsatellite loci for the lesser horseshoe bat,Rhinolophus hipposideros (Rhinolophidae,Chiroptera)

Fourteen polymorphic microsatellites isolated in the lesser horseshoe bat, Rhinolophus hipposideros, are described. A subset of eight loci successfully coamplified in a multiplex polymerase chain reaction (PCR), and allowed to confirm that expected heterozygosity was relatively high for the eight loci (0.56–0.83). This set of eight microsatellites indeed permits to build genetic tags that distinguish all individuals in colonies consisting of up to more than 1000 individuals.     

Development of 19 polymorphic microsatellite loci for the intermediate horseshoe bat, Rhinolophus affinis (Rhinolophidae, Chiroptera)

The intermediate horseshoe bat (Rhinolophus affinis) is a widespread species in Southeast Asia. We developed 19 novel microsatellite loci from an enriched genomic library of the bat, and tested their polymorphism using a single population from Guangdong province, southern China. The number of alleles ranged from 3 to 15 per locus with the expected and observed heterozygosity ranging from 0.397 to 0.920 and 0.280–0.926, respectively. Three markers significantly deviated from the Hardy-Weinberg expectations after Bonferroni correction and no linkage disequilibrium was detected in any of loci. These microsatellite loci will be useful in studying the phylogeography of this species.     

Population genetic structure and demographic history of the endemic Formosan lesser horseshoe bat (Rhinolophus monoceros)

Intraspecific phylogenies can provide useful insights into how populations have been shaped by historical and contemporary processes. Taiwan formed around 5 million years ago from tectonic uplift, and has been connected to mainland Asia several times since its emergence. A central mountain range runs north to south, bisecting the island, and potentially impedes gene flow along an east-west axis. The Formosan lesser horseshoe bat (Rhinolophus monoceros) is endemic to Taiwan, where it is found mainly at low altitude. To determine the population structure and the demographic and colonization history of this species, we examined variation in the mitochondrial DNA control region in 203 bats sampled at 26 sites. We found very high haplotype and nucleotide diversity, which decreased from the centre to the south and north. Population differentiation followed a pattern of isolation by distance, though most regional genetic variance was attributable to differences between the relatively isolated southern population and those from other regions. A haplotype network was consistent with these findings and also suggested a southward colonization, followed by subsequent secondary contact between the south and other regions. Mismatch distributions were used to infer a past population expansion predating the last glacial maximum, and a neighbour-joining tree showed that R. monoceros formed a monophyletic grouping with respect to its sister taxa. Taken together, our results suggest that this taxon arose from a single period of colonization, and that demographic growth followed in the late Pleistocene. Current genetic structure reflects limited gene flow, probably coupled with stepwise colonization in the past. We consider explanations for the persistence of the species through multiple glacial maxima.     

Geographical variation in echolocation call and body size of the Okinawan least horseshoe bat, Rhinolophus pumilus (Mammalia: Rhinolophidae), on Okinawa-jima Island, Ryukyu Archipelago, Japan

The Okinawan least horseshoe bat, Rhinolophus pumilus, is a cave-dwelling species endemic to the central and southern Ryukyus, Japan. We analyzed variation in the constant frequency (CF) of the echolocation call and in forearm length (FAL) of this species on Okinawa-jima Island on the basis of data for 479 individuals from 11 caves scattered over the island. CF values in samples from six caves, all located in the southwestern half of Okinawa-jima, were significantly higher than those in samples from five caves in the northeastern half of the island. Also, FAL was significantly greater in the latter group than in the former group, although the ranges of variation in this character substantially overlapped between the two groups. These results suggest substantial differentiation between R. pumilus populations on Okinawa-jima. The implications of our findings for the conservation of this endangered bat species are briefly discussed.     

The photoreceptor populations in the retina of the greater horseshoe bat Rhinolophus ferrumequinum

Recently, we reported the existence of AII "rod" amacrine cells in the retina of the greater horseshoe bat Rhinolophus ferrumequinum (Jeon et al., 2007). In order to enhance our understanding of bat vision, in the present study, we report on a quantitative analysis of cone and rod photoreceptors. The average cone density was 9,535 cells/mm2, giving a total number of cones of 33,538 cells/retina. The average rod density was 368,891 cells/mm2, giving a total number of rods of 1,303,517 cells. On average, the total populations of rods were 97.49%, and cones were 2.51% of all the photoreceptors. Rod: cone ratios ranged from 33.85:1 centrally to 42.26:1 peripherally, with a mean ratio of 38.96:1. The average regularity index of the cone mosaic in bat retina was 3.04. The present results confirm the greater horseshoe bat retina to be strongly rod-dominated. The rod-dominated retina, with the existence of AII cells discovered in our previous study, strongly suggests that the greater horseshoe bat retina has a functional scotopic property of vision. However, the existence of cone cells also suggests that the bat retina has a functional photopic property of vision.