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A molecular perspective on mammalian evolution from the gene encoding interphotoreceptor retinoid binding protein, with convincing evidence for bat monophyly.

The evolutionary relationships of the various orders of placental mammals remain an issue of uncertainty and controversy. Molecular studies of mammalian phylogeny at the DNA level that include more than just a few orders are still relatively meager. Here we report results on mammalian phylogeny deduced from the coding sequence of the single-copy nuclear gene for the interphotoreceptor retinoid binding protein (IRBP). Analysis of 13 species representing eight eutherian orders and one marsupial yielded results that falsify the hypothesis that megachiropteran bats are "flying primates," only convergently resembling microchiropteran bats. Instead, in agreement with more traditional views, as well as those from other recent molecular studies, the results strongly support a monophyletic Chiroptera (micro- and megabats grouped together). The IRBP results also offer some rare molecular support for the Glires concept, in which rodents and lagomorphs form a superordinal grouping. Also in congruence with other recent molecular evidence, IRBP sequences do not support the view of a superorder Archonta that includes Chiroptera along with Dermoptera (flying lemur), Scandentia (tree shrew), and Primates. IRBP was not however, without its shortcomings as a molecular phylogenetic system: high levels of homoplasy, evident in the marsupial outgroup, did not allow us to properly root the tree, and several of the higher level eutherian clades were only weakly supported (e.g., a Carnivora/Chiroptera clade and an Artiodactyla/Carnivora/Chiroptera clade). We suggest that these shortcomings may be diminished as the phylogenetic density of the data set is increased.

Amino Acid Sequence↗

Azimuth-dependent recovery cycle affects directional selectivity of bat inferior collicular neurons determined with sound pulses within a pulse train.

In our previous study, we have shown that the recovery cycle of most neurons in the inferior colliculus (IC) of the big brown bat, Eptesicus fuscus, is typically longer at ipsilateral azimuth than at contralateral azimuth under free-field stimulation conditions. The present study is to test the hypothesis that this azimuth-dependent recovery cycle may contribute to the variation of directional selectivity of IC neurons with sequential presented sound pulses within a pulse train. A 300-ms pulse train containing nine sound pulses of 4-ms with an inter-pulse interval of 33.3 ms was delivered at several selected azimuthal angles between +/-80 degrees lateral in the frontal auditory space of a bat. A family of nine directional selectivity curves was plotted with a neuron's number of impulses in response to each individual pulse against the azimuthal angles. The type and sharpness of these directional selectivity curves were then compared in relation to pulse position within the pulse train. All 675 directional selectivity curves obtained from 75 IC neurons could be described as directionally selective (423, 63%), hemifield (220, 32%), or non-directional (32, 5%). The directional selectivity curves of 45 (60%) neurons did not vary with pulse position. However, those of the remaining neurons (30, 40%) changed from one type to another such that the number of neurons with directionally selective curves progressively increased and the number of neurons with hemifield and non-directional selectivity curves decreased with increasing pulse position within the pulse train. Among 68 IC neurons whose directional selectivity curves were compared quantitatively, directional selectivity determined with sequentially presented sound pulses significantly increased in 38 (56%) neurons; decreased in 18 (26%) neurons but did not change in 12 (18%) neurons. This change of directional selectivity was due to the variation in recovery cycle of these IC neurons with azimuthal angle as we hypothesized.

Acoustic Stimulation↗

Corticofugal modulation of directional sensitivity in the midbrain of the big brown bat, Eptesicus fuscus.

In our recent study of corticofugal modulation of collicular amplitude sensitivity of the big brown bat, Eptesicus fuscus, we suggested that the corticofugal modulation is based upon the best frequency (BF) differences and the relative amplitude sensitivity difference between collicular (IC) and cortical (AC) neurons but not the absolute amplitude sensitivity of IC and AC neurons. To show that corticofugal modulation is systematic and multiparametric, we studied corticofugal modulation of directional sensitivity in 89 corticofugally inhibited IC neurons in the same bat species under free field stimulation conditions. A neuron's directional sensitivity was expressed with the azimuthal range (AR) at 50% below the maximum of each directional sensitivity curve and the best azimuth (BAZ) at which the neuron discharged maximally. Cortical electrical stimulation did not affect the directional sensitivity of 40 (45%) neurons with BF(IC-AC) differences of 7.3+/-4.4kHz but sharpened the directional sensitivity of other 49 (55%) neurons with BF(IC-AC) differences of 2.3+/-1.8kHz. Corticofugal modulation sharpened directional sensitivity curves of IC neurons by decreasing the AR and shifting collicular BAZ toward cortical BAZ. The decrease in AR and the shift in BAZ increased significantly with AR(IC-AC) and BAZ(IC-AC) differences but not with absolute AR and BAZ of IC and AC neurons or BF(IC-AC) differences. Corticofual modulation also shifted collicular BF toward cortical BF. The shift in BF increased significantly with BF(IC-AC) differences but not with the BF of IC and AC neurons or BAZ shift. Consonant with our previous study, these data indicate that corticofugal modulation of collicular directional sensitivity is based on topographic projections between the IC and the AC and the difference in directional sensitivity but not the absolute directional sensitivity of IC or AC neurons.

Acoustic Stimulation↗

Threshold minima and maxima in the behavioral audiograms of the bats Artibeus jamaicensis and Eptesicus fuscus are not produced by cochlear mechanics.

Behavioral audiograms of Artibeus jamaicensis and Eptesicus fuscus are characterized by two threshold minima separated by a threshold maximum at 40 kHz, for A. jamaicensis, and 45 kHz, for E. fuscus [Koay, G., Heffner, H.E., Heffner R.S., 1997. Audiogram of the big brown bat (Eptesicus fuscus). Hear. Res. 105, 202-210; Heffner, R.S., Koay, G., Heffner H.E., 2003. Hearing in American leaf-nosed bats. III: Artibeus jamaicensis. Hear. Res. 184, 113-122.]. To investigate whether these characteristics are due to cochlear properties, we recorded distortion product otoacoustic emissions (DPOAEs) and calculated DPOAE threshold curves. We found that in both species cochlear sensitivity, assessed by DPOAE recordings, does not show local threshold maxima. The DPOAE threshold curve calculated for A. jamaicensis reveals a broadly tuned minimum for frequencies between 20 and 50 kHz and the threshold curve of E. fuscus shows a broad sensitive area for frequencies between 15 and 60 kHz. In none of the two species any pronounced threshold irregularities were found. The characteristic pattern of a threshold maximum followed by a minimum observed in behavioral studies seems to be shaped by transfer characteristics of the outer ear and/or neuronal processing in the ascending auditory pathway rather than by cochlear mechanics.

Animals↗

Detection and strain differentiation of European bat lyssaviruses using in situ hybridisation.

A protocol suitable for the detection of rabies virus and the related European bat lyssaviruses type 1 and 2 is described. In situ hybridisation, employing digoxigenin labelled riboprobes was used for the detection of lyssavirus RNA in mouse-infected brain tissue. The principal advantage of this technique, compared to routine methods used for histopathology, is that this method is robust, highly sensitive, and specific for assessing the presence of RNA in different tissues. An additional advantage is that there is no longer any requirement for high laboratory bio-containment, once the tissue under investigation has been safely fixed. Using this method, both genomic and messenger RNA were detected. The ability to detect messenger RNA is indicative of the presence of replicating virus and therefore, this technique is a powerful diagnostic tool for the routine detection of strains of rabies virus including the European bat lyssaviruses.

Animals↗

Development and biological evaluation of 99mTc-BAT-tropane esters.

Two (99m)Tc-BAT-tropane conjugates, i.e., technetium(V)-oxo-3-[N-(2-mercaptoethyl), N-(N'-(2-mercaptoethyl)-2-aminoethyl)]-aminopropyl 3beta-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1]octane-2beta-carboxylate and the corresponding norchloro derivative, were prepared and evaluated as potential imaging agents for the central nervous dopamine transporter (DAT) system. In these compounds, a tropane and a (99m)Tc-BAT moiety were linked through an ester bond. Both compounds were formed as a mixture of two diastereomers which could be purified and isolated using reversed-phase high-performance liquid chromatography (HPLC). Radio-LC-MS analysis supported the hypothesised structure of the synthesised technetium complexes. After intravenous injection in mice and rats, the compounds were stable in vivo, and no important metabolites were found in plasma or urine. In vitro testing suggested specific competitive binding to the DAT system, but in vivo experiments in rats showed no significant brain uptake for the diastereomers of both compounds; neither was there any specific uptake in the striatum. The results suggest that replacement of a methylene linker by an ester does not seriously affect the binding properties of the tropane conjugates to the dopamine transporter but results in a drastic reduction of passage over the blood-brain barrier (BBB).

Animals↗

Rabies human diploid cell vaccine elicits cross-neutralising and cross-protecting immune responses against European and Australian bat lyssaviruses.

The ability of antibodies elicited against the rabies human diploid cell vaccine (HDCV) to neutralise European bat Lyssaviruses (EBLV types-1 and -2), Australian bat Lyssavirus and classical rabies virus (RABV) has been evaluated using modified fluorescent antibody virus neutralisation (mFAVN) assays. Ninety-six percent (48 of 50) of the human post-vaccinated sera tested cross-neutralised these viruses (>or=0.5 IU/ml). Cross-protection experiments using inbred mice (RIII, k/k haplotype) were also assessed. Mice were given HDCV (twice by the intra-peritoneal route) and challenged (intra-cranially or peripherally) with a lethal dose (25 MLD(50)) of the individual viruses. The vaccine conferred statistically significant protection in 80--100% of animals challenged via the peripheral route. Levels of protection were lower following intra-cranial (i.c.) challenge. Our data provides strong evidence for broad spectrum cross-neutralisation and cross-protection of phylogroup I lyssaviruses using rabies HDCV.

Animals↗

Genetic characterization of Yokose virus, a flavivirus isolated from the bat in Japan.

Yokose virus (strain Oita-36) was isolated from the bat in Japan in 1971. In the present study, we determined complete nucleotide sequences of Yokose virus using RT-PCR and RACE techniques. Yokose virus genome consists of 10,857 nucleotides in length (accession no. AB114858), containing a single open reading frame (3425 amino acids) encoding 11 viral proteins. We deduced the boundaries of each protein in the polyprotein sequence according to the protein cleavage sites of other flaviviruses. The nucleotide sequences of the 5' and 3' nontranslated region (NTR) and amino acid sequences of individual proteins of the virus were compared with those of six other flaviviruses including Japanese encephalitis virus, dengue-2 virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, and Rio Bravo virus or Modoc virus. Yokose virus demonstrated the highest similarity to yellow fever virus. Yokose virus also has CS1 motif, which are well-conserved specifically in mosquito-born flaviviruses, in its 3' NTR. When a part of the NS5 amino acid sequence (345 amino acids) was compared with those of other four flaviviruses, Entebbe bat virus, Sokuluk virus, Sepik virus, and yellow fever virus, the three former viruses are more closely related to Yokose virus than yellow fever virus. Human sera from dengue-virus-infected case and yellow fever vaccine reacted with the viral proteins. Moreover, human serum from a yellow fever vaccine weakly neutralized Yokose virus. Our results suggest that there are cross-reactive antigenicities among Yokose virus and other flaviviruses.

3' Untranslated Regions↗

Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition.

The nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) genes of Irkut and West Caucasian bat viruses (WCBV) were sequenced and compared with those of other lyssaviruses. N gene nucleotide identities provided unequivocal separation of all lyssavirus genotypes with an identity threshold of 82%. On this basis, Irkut virus should be considered as a new genotype with particular relatedness to genotypes 4 and 5 (78.0-78.6% identity for N gene nucleotides and 90.4-92.6% for amino acids). Furthermore, genotypes 4-6, together with Aravan, Khujand and Irkut viruses, present a solid phylogroup of Old World bat lyssaviruses. This relationship is apparent using all three viral genes, and causes overlap between intragenotype and intergenotype identities for the P gene (Aravan, Khujand viruses and genotype 6) and for the G gene (Aravan, Khujand, genotypes 5 and 6). WCBV is the most divergent of known lyssaviruses with only limited relatedness to genotypes 2 and 3.

Amino Acid Sequence↗

Mitochondrial phylogeography of the long-eared bats (Plecotus) in the Mediterranean Palaearctic and Atlantic Islands.

Long-eared bats of the genus Plecotus are widespread and common over most of the western Palaearctic. Based on recent molecular evidence, they proved to represent a complex of several cryptic species, with three new species being described from Europe in 2002. Evolutionary relationships among the different lineages are still fragmentary because of the limited geographic coverage of previous studies. Here we analyze Plecotus mitochondrial DNA sequences from the entire Mediterranean region and Atlantic Islands. Phylogenetic reconstructions group these western Palaearctic Plecotus into two major clades which split at least 5 Myr ago and that are each subdivided into further subgroups. An 'auritus group' includes the traditional P. auritus species and its sister taxon P. macrobullaris (=P. alpinus) plus related specimens from the Middle East. P. auritus and P. macrobullaris have broadly overlapping distributions in Europe, although the latter is apparently more restricted to mountain ranges. The other major clade, the 'austriacus group,' includes the European species P. austriacus and at least two other related taxa from North Africa (including P. teneriffae from the Canary Islands), the Balkans and Anatolia (P. kolombatovici). The sister species of this 'austriacus group' is P. balensis, an Ethiopian endemic. Phylogenetic reconstructions further suggest that P. austriacus reached Madeira during its relatively recent westward expansion through Europe, while the Canary Islands were colonized by a North African ancestor. Although colonization of the two groups of Atlantic Islands by Plecotus bats followed very distinct routes, neither involved lineages from the 'auritus group.' Furthermore, the Strait of Gibraltar perfectly segregates the distinct lineages, which confirms its key role as a geographic barrier. This study also stresses the biogeographical importance of the Mediterranean region, and particularly of North Africa, in understanding the evolution of the western Palaearctic biotas.

Animals↗

Corticofugal inhibition compresses all types of rate-intensity functions of inferior collicular neurons in the big brown bat.

Recent studies have shown that the auditory corticofugal system modulates and improves signal processing in the frequency, time and spatial domains. In this study, we examine corticofugal modulation of rate-intensity functions of inferior collicular (IC) neurons of the big brown bat, Eptesicus fuscus, by electrical stimulation in the primary auditory cortex (AC). Cortical electrical stimulation compressed all types of rate-intensity functions so as to increase the slope but decrease the dynamic range of IC neurons. Cortical electrical stimulation also shifts the responsive intensity of IC neurons to higher levels. These data indicate that corticofugal modulation also improves subcortical signal processing in intensity domain. The implication of these findings to bat echolocation is discussed.

Acoustic Stimulation↗

Vasopressin location and relative quantification within the vampire bat (Desmodus rotundas) brain.

Vasopressin was localized in the supraoptic nucleus (SON), paraventricular nucleus, amygdala, habenula, and the posterior pituitary. Microdensitometry analysis revealed an inversely proportionate relationship between the relative vasopressin levels in the SON and the posterior pituitary when exposed to varying periods of dehydration. Short term dehydrated bats displayed increased vasopressin in the SON and decreased levels in the posterior pituitary. Chronically dehydrated bats displayed decreased vasopressin in the SON with increased levels in the posterior pituitary.

Animals↗

Cytomegalovirus in the principal submandibular gland of the little brown bat, Myotis lucifugus.

In the course of a large scale study of salivary gland ultrastructure in chiropterans, enlarged cells infected with numerous virus particles were encountered in some acinar cells in the principal submandibular glands of two of 34 little brown bats (Myotis lucifugus). The characteristic morphology of the viruses, together with the cytomegaly that they induced, led to their identification as cytomegalovirus (CMV). In a reversal of the situation in other animal species, bat virus particles within cytoplasmic vacuoles lacked capsomeres, whereas the latter were prominent in particles free in the cytosol. The generally accepted schemes for CMV production cannot explain this seemingly aberrant morphology. This report extends to four the number of mammalian orders in which CMV has been documented by means of electron microscopy as occurring in salivary glands.

Animals↗

In vivo blood oxygen binding in hypoxic lesser spear-nosed bats: relationship to control of breathing.

The hypoxic ventilatory threshold of many mammals correlates with their hemoglobin-oxygen affinity (P50). Yet, in some small mammals ventilation actually declines, rather than increases, with exposure to decreasing PaO2; their air convection requirement (V(E)/V(O2)), however, is elevated in hypoxia. We propose that the threshold of the hypoxic V(E)/V(O2) of small mammals coincides with the inflection ('knee') of their in vivo O2 equilibrium curve (O2EC). In vivo blood gas and pH data were obtained from normoxic and hypoxic lesser-spear nosed bats, Phyllostomus discolor; in vitro blood O2EC were also generated for normoxic bats at 32 and 37 degrees C and at three P(CO2)'s. The hypoxic V(E)/V(O2) threshold of P. discolor occurs at PaO2 = 39 Torr; the corresponding in vivo O2 saturation is 0.70, approximating the inflection of the O2EC. This animal has a high blood O2 affinity (P50 = 27.5 Torr at pH 7.40 and 37 degrees C; P50 = 30.8 Torr at in vivo pH of 7.31 and TB of 37.4 degrees C). As PaO2 is reduced, a pronounced hypoxia-induced respiratory alkalosis and hypothermia help maintain SaO2 near the O2EC inflection (0.64-0.70 S(O2)).

Acid-Base Equilibrium↗

Encoding of sound motion by binaural brainstem units in a Horseshoe bat.

In order to study how and if single brainstem units respond to moving compared with stationary sounds, radially moving sound sources were presented to the bat, Rhinolophus ferrumequinum. This time-variant binaural stimulation was simulated dichotically through earphones (closed-acoustic-field for the virtual azimuth range of +/-40 degrees from the midline). Neurophysiologically recorded responses primarily showed a function of interaural intensity difference (IID) which is considered a direct correlate of the sound source's azimuth angle. However, this is only true for the stationary case. Unit's response did not remain unaffected by the dynamic stimulus cues of sound source movement (velocity and direction). Maximal discharge rate became a function of motion velocity as well as the slopes of the response profiles. Hence, coding of IID became ambiguous as, depending on the unit, the response profiles and therefore a unit's receptive field, became spatially shifted with respect to one another when the direction of the sound source movement was reversed. Shifts within the movement direction (hysteresis) as well as against it (termed here 'advance') were observed: hysteresis is typical for units with non-monotonic, stationary rate/intensity functions, whereas those units with monotonic functions predominantly show advances. Further dynamic response features in form of transient peaks and troughs, superimposed on the response profiles, were registered. It appears that the ongoing firing rate no longer represents azimuth position alone, but vigorously reproduces the dynamic cues (velocity and movement direction), too. With respect to the neural mechanisms leading to dynamic response features, it is proposed that, as long excitation and inhibition act with similar short time constants, neural activity can rapidly and faithfully follow changing IIDs. Different time constants for excitation, inhibition, facilitation, and depression may be responsible for the dynamic 'features' such as transient responses and hysteresis/advance. They may provide biologically relevant information for nocturnally hunting bats to efficiently guide their flight maneuvers.

Animals↗

Neuronal sensitivity to interaural time differences in the sound envelope in the auditory cortex of the pallid bat.

Interaural time differences in the envelope of a sound (envelope ITDs) can potentially provide spatial information at high frequencies where interaural phase differences (IPDs) are not available. Interaural intensity differences (IIDs) also provide important spatial information at high frequencies. Both IIDs and envelope ITDs can influence spatial perception at high frequencies, but behavioral and physiological studies suggest that IIDs dominate perception. This study examines envelope ITD sensitivity in the auditory cortex of the pallid bat, a species that uses passive sound localization at the low end of its audible range to find prey. Its auditory system is entirely 'high-frequency' in that phase-locking does not occur at the low end of its audible range. If the bat uses ITDs, they must be derived from the envelope of the signal. A previous study of envelope ITD sensitivity in its inferior colliculus (IC) reported that neurons are sensitive to the small +/-70 micros range of available ITDs. This study extends these findings to the cortical level to assess the transformation of ITD sensitivity and the binaural response properties that underlie this sensitivity. Two measures of sensitivity were used. The dynamic ITD range measures the range of ITDs over which the maximum response of a neuron decreases by 80%. When presented with square-wave amplitude-modulated tones statically delayed in arrival time, the average dynamic ITD range in the IC is 304 micros, but dropped to 175 micros in auditory cortex. IC neurons average a 38% change in maximum response over the relevant ITD range, while cortical neurons average a 67% change. Also measured were time-intensity trading ratios, which index the extent to which a change in IID can cause a shift the dynamic ITD range. Average trading ratios are approximately the same in the IC and auditory cortex (17.9 micros/dB vs. 16.7 micros/dB, respectively). Binaural interactions changed from the IC to auditory cortex. In IC, ITD sensitivity is an inhibitory, subtractive process in which ITDs reduce the response evoked by contralateral monaural stimulation. In the auditory cortex, both binaural inhibition and facilitation occur. In the majority of cortical neurons, IID and ITD functions were remarkably similar in shape, having stepped, step-peaked or peaked functions. The binaural interactions (inhibition and/or facilitation) evoked by ITDs and IIDs were also typically similar. These results suggest that IIDs and envelope ITDs are having similar effects on output of the same binaural comparator system.

Acoustic Stimulation↗

Topographical distribution of delay-tuned responses in the mustached bat inferior colliculus.

In the mustached bat, delay-tuned neurons respond best to specific delays between the first harmonic frequency modulated (FM) component (FM1; 24-29 kHz) of the emitted biosonar pulse and a higher harmonic FM component in returning echoes (e.g. FM3, 72-89 kHz). These delay-tuned, combinatorial responses predominate in the inferior colliculus (IC) of the mustached bat. This study examined the topographical distribution of delay-tuned neurons in the 72-89 kHz frequency representation of the IC. We recorded and histologically localized 163 single units. Ninety units were facilitated and 41 were inhibited by the combination of two frequencies in the 24-29 kHz and 72-89 kHz ranges. The facilitatory responses were selective for delays up to 20 ms between the two signals. To determine if delay-tuned neurons were topographically organized, we plotted the dorsomedio-ventrolateral and caudo-rostral positions of each unit versus its best delay. Best delay was not correlated with either location. Response latency to best frequency tones was topographically organized, but was not correlated with best delay. This indicates that the latency axis in the IC is unrelated to the delay tuning of these combinatorial neurons. Because delay-tuned neurons are not topographically organized in the IC but are in the auditory cortex, our findings suggest that the creation and organization of delay-tuned neurons occur at different stages in the ascending auditory system.

Acoustic Stimulation↗

Hearing in American leaf-nosed bats. I: Phyllostomus hastatus.

We determined the audiogram of Phyllostomus hastatus (the greater spear-nosed bat), a large, omnivorous American leaf-nosed bat native to Central and South America. A conditioned suppression/avoidance procedure with a fruit juice reward was used for testing. At an intensity of 60 dB sound pressure level (SPL re 20 microN/m(2)), the hearing range of P. hastatus extends from 1.8 to 105 kHz, with a best sensitivity of 1 dB SPL at 20 kHz. Both its high-frequency and low-frequency hearing are not unusual for a small mammal. Despite its use of low-intensity echolocation calls there was no evidence for unusual sensitivity to either the frequencies used for echolocation or to the main frequencies of its communication calls, suggesting no selective "tuning" of the audiogram. Its behavioral pure-tone thresholds are lower than the multi-unit thresholds in the inferior colliculus.

Acoustic Stimulation↗