Vampire bats have three ‘leaf pits’ that surround the nose () and receive input from low-threshold heat-sensitive nerve fibers responding to stimuli >29°C (refs. 1
). Closely related fruit bats (Carollia brevicauda
) have a different nasal structure devoid of pit organs () and thus cannot detect infrared stimuli 3
. Sensory ganglia of fruit bats showed a typical size distribution of neurons resembling that seen in other mammals (). In contrast, TG from vampire bats showed marked skewing towards large diameter neurons, much like that observed in TG of pit bearing snakes 6
, suggesting that anatomical specialization of TG in vampire bats has similarly evolved to suit a predominant role in infrared sensation. Consistent with this, vampire bat DRG showed a normal size distribution resembling sensory ganglia of fruit bats or other mammals (). To our knowledge, vampire bats provide the only such example of TG specialization among mammalian species.
Anatomy of fruit bat and vampire bat sensory ganglia
Given the distinct anatomy of vampire bat TG versus DRG, we asked whether these ganglia show differential patterns of gene expression that might highlight molecules specifically involved in infrared sensation, as in the case of infrared sensing snakes 6
. Thus, we performed ‘deep sequencing’ of cDNAs from bat TG and DRG. Initially, transcriptomes from these ganglia appeared to be indistinguishable (SFig. 1
). However, examination of cDNAs encoding candidate thermosensors revealed a novel short isoform of the capsaicin receptor, TRPV1, an excitatory ion channel that is activated by noxious heat (>43°C) (refs. 13
). This novel isoform (TRPV1-S) lacks 62 amino acids from the C-terminus ( and SFig. 2
) and is uniquely expressed in TG from vampire bat, constituting between 35–46% of all TRPV1 transcripts in TG, but <3% in DRG, as determined from transcriptome data or direct cloning and sequencing of RT-PCR products amplified from sensory ganglia (). Moreover, TRPV1-S transcripts were barely detected (<6%) in fruit bat TG or DRG (), and represented ≤1.8% of TRPV1 transcripts from TG of three other fruit, nectar, or insect feeding bat species (U. bilobatum
, S. lilium
, and A. cultrata
). Taken together, these observations support the notion that TRPV1-S contributes to the specialized function of vampire bat TG, most notably infrared sensation.
Sequence and distribution of vampire bat TRPV1
Discrete cellular expression of TRPV1 in vampire bat sensory ganglia was confirmed by in situ
hybridization. Consistent with RNA-seq analysis, TRPV1 transcripts were found in similar percentages of TG and DRG neurons (31.1 ± 1.5% and 49.2 ± 1.5%, respectively) (). Specificity of this pattern was confirmed by examining the distribution of TRPA1 transcripts, which were present in a larger percentage of TG and DRG neurons (43.6 ± 1.7 and 75.4 ± 1.0, respectively) compared to TRPV1 ( and SFig. 3
If TRPV1-S contributes to infrared detection in vampire bats, then this isoform should exhibit appropriate temperature sensitivity. We expressed and characterized TRPV1-S and TRPV1-L in HEK293 cells or Xenopus
oocytes using calcium imaging or electrophysiological assays, respectively. Indeed, we observed a marked difference in temperature sensitivity such that the TRPV1-S isoform was activated at a substantially lower threshold compared to TRPV1-L (30.5 ± 0.7°C versus 39.6 ± 0.4°C in HEK293 cells and 31.2 ± 1.5°C versus 40.2 ± 0.7 in oocytes) ( and SFigs. 4, 5
). Similar thresholds were observed for fruit bat TRPV1 isoforms (SFigs. 4, 6
). This ~10°C threshold differential between TRPV1-S and TRPV1-L, together with unique expression of the short isoform in vampire bat TG, is consistent with a role for TRPV1-S in infrared detection. In contrast, heterologously expressed TRPA1 channels from either bat species were heat insensitive (SFig. 7
Functional analyses of vampire bat TRPV1 isoforms
Our histological analysis does not allow us to discriminate between TRPV1 isoforms. We therefore asked whether their co-expression would produce channels having lowered thermal thresholds compared to TRPV1-L alone. Injection of oocytes with equal amounts of TRPV1-S and -L cRNAs produced an intermediate temperature response curve with an activation threshold of 33.9 ± 1.2°C (SFig. 5
), rather than biphasic thresholds, suggesting that short and long isoforms can produce functional heterotetrameric complexes. Oocytes expressing mostly one isoform (10S:1L or 1S:10L) showed a thermal threshold defined by the predominant species (SFig. 5
). Thus, if individual TG neurons express equivalent levels of short and long isoforms, then they will have lower thermal thresholds compared to cells predominately expressing the long form.
The identification of C-terminal TRPV1 splice variants in vampire bats is somewhat surprising since, to our knowledge, such isoforms have not been previously reported for other species. Alignment between vampire bat TRPV1-S and -L cDNAs highlighted a 23-base pair insertion in the former (SFig. 8a
) containing a stop codon that accounts for production of the short isoform. We used sequences flanking this insert to amplify and characterize the organization of the vampire bat TRPV1 locus in this vicinity. The resulting 5.5 kb fragment contained a tiny 23-bp exon (e14a) flanked by two introns (2.1 and 3.1 kb), each marked by canonical –GT/AG- donor-acceptor sites required for U2-dependent splicing, as well as obligate polypyrimidine tracts preceding potential splicing sites 16,17
, allowing for formation of two splice variants that incorporate or bypass e14a to produce TRPV1-S or TRPV1-L, respectively (SFig. 9
To determine whether exon 14a-based splicing is a hallmark of other Laurasiatheria orders, we carried out transcriptome and/or genomic analysis from Cetartiodactyla (cow, pig), Carnivora (dog), and Lipotyphla (Coast mole), as well as several additional bat species representing both Chiroptera suborders, including microbats and megabats 18–20
. Two types of TRPV1 splicing events can occur (): in microbats, megabats, and moles, e14a contains a premature stop codon accounting for production of a low threshold TRPV1-S isoform (SFigs. 4–6 and 10
). In cows, pigs, and dogs e14a produces an in-frame insertion generating an extra-large isoform (TRPV1-XL). Thus, while members of the Laurasiatheria superorder exploit the same intronic region for TRPV1 modification, different suborders exhibit distinct e14a sequences and positions (relative to exons 14 and 15), indicative of independent evolutionary events leading to modification of this intronic region. Interestingly, the architecture of splice sites in microbats differs from that of other Laurasiatheria members in that the pyrimidine-rich tract is followed by a strong tandem splice site (CAGCAG), and a weak exon 15 acceptor site (TAG in microbats versus CAG in most other species) (). Consistent with our failure to observe TRPV1-S-like isoforms in mouse sensory ganglia (not shown), neither rodent nor human TRPV1 gene contains an e14a equivalent, limiting splicing to a single isoform.
Genomic organization of mammalian TRPV1 locus
Although cows and moles have the potential to produce TRPV1 splice variants, we found that <6% of TRPV1 transcripts corresponded to TRPV1-XL or TRPV1-S in TG of cow or mole, respectively, suggesting that this form of post-transcriptional regulation is not physiologically relevant in these animals. In cows, this is further underscored by the fact that TRPV1-L and TRPV1-XL isoforms are indistinguishable in regard to thermal response profiles (thresholds of 43.0 ± 0.8 and 42.7 ± 0.4°C, respectively) (SFigs. 4 and 11
). Our analysis of TRPV1 gene organization in different orders (including Chiroptera, Cetartiodactyla, Carnivora, Lipotyphla, Afrotheria, and Rodentia) ( and SFigs. 9 and 12
) is consistent with an independent molecular phylogeny of mammals 21,22
and supports the conclusion that bats are more closely related to cows, moles, and dogs 19,20
, rather than to rodents as initially posited based on anatomical and morphological criteria23
TRPV1 genomic sequences for vampire and other microbats in the vicinity of exon 14-14a-15 splice junctions are highly conserved (>90% identity). We therefore asked whether differential splicing was observed when mini-genes containing these genomic regions were introduced into HEK293 cells. Direct splicing of exon 14 to 15, with exclusion of exon 14a, was observed in all cases (). In mini-gene constructs where direct splicing between exon 14 and 15 was not possible, splicing to or from exon 14a was readily observed (), indicating that exon 14a is competent to engage in splicing, but disfavored by exon 14-15 competition. These results show that vampire and fruit bat genes exhibit the same default pattern of exon 14 to 15 splicing in this non-neural context, irrespective of small differences in gene structure. Indeed, this ‘constitutive’ splicing pattern predominates in vampire bat DRG, fruit bat TG and DRG, as well as cow and mole TG, suggesting that efficient splicing to exon 14a in vampire bat TG requires a specialized environment. Furthermore, we have used this assay to identify a putative exon 14a from megabat species, such as Pteropus vampyrus
, where transcriptome data are not available ( and SFig. 13
Our findings suggest that variation within the TRPV1 C-terminus represents a genetic mechanism for tuning the thermal response profile of the channel in a species- or tissue-specific manner. We therefore asked whether more primitive and evolutionarily distant species use a similar strategy for thermosensory adaptation. Indeed, we found that zebrafish TRPV1 is activated with a threshold of 32.9 ± 1.2°C (SFig. 14a–d
), consistent with physiological adaptation of the animal to its environment of 25–33°C (ref. 24
). When compared to rat TRPV1, the zebrafish channel has a gap of 12 amino acids within the C-terminus corresponding precisely to the location of the splice junction in the vampire bat channel (SFig. 14e
). This gap results from a polymorphism within exon 15 of the zebrafish TRPV1 gene, and thus zebrafish and vampire bats use different mechanisms to generate TRPV1 C-terminal variants.
RNA splicing extends the coding potential of the genome and enhances functionality of the proteome 25
. The vampire bat uses this strategy to produce physiologically distinct channels, thereby generating a hypersensitive detector (TRPV1-S) within a specific thermosensory organ, without sacrificing somatic thermo-sensation and/or –nociception. Moreover, our results demonstrate that sequence variation within a specific region of the cytoplasmic C-terminus accounts for differential thermal response profiles of vampire bat TRPV1 isoforms. Indeed, we previously proposed that this region of the channel interacts with membrane phospholipids to modulate sensitivity to thermal and chemical stimuli 26
In addition to illuminating mechanisms of thermosensation and sensory adaptation, the analysis of TRP channel gene structure provides a physiologically relevant marker for assessing phylogenetic relationships. Our findings support recent molecular classification in which bats are grouped together with horses, dogs, cows, moles, and dolphins (Laurasiatheria superorder), rather than with humans, monkeys, flying lemur, mouse, rat, and rabbits (Euarchontoglires superorder) as originally proposed on the basis of anatomical criteria.