Molecular Evolution And Functional Studies On Short Wavelength-Sensitive Type 1 Pigment In Bats

Bats belong to the order Chiroptera,which is the second largest group within mammals.Bats are nocturnal animals.The microbats have the ability of echolocation to detect preys and orientate.On the other hand,some other bats cannot use echolocation and mainly rely on vision and olfaction.There are some studies suggest that bats can detect ultraviolet?UV?light.However,the studies suggesting the UV vision of bats are only limited to electrophysiological experiments and sequence analyses,and the bat SWS1 pigments have not been verified by in vitro functional assays.Moreover,researchers found that the SWS1 genes lost in some cave roosting fruit-eating bats because of the dark environments.Interestingly,bats using a sophisticated type of echolocation also lost the SWS1 gene,which maybe due to the sensory trade-offs between hearing and vision.Therefore,we determined the function of SWS1 pigments in representative bats in vitro here and also studied the evolution of SWS1 gene in Pteronotus parnellii,species using high duty cycle echolocation.The first part of this study is the molecular evolution and functional verification of UV vision in bats.Here,we studied 11 bat species and then amplified and sequenced their SWS1 genes.Interestingly,we found that the SWS1 genes of common vampire bat?Desmodus rotundus?and white-winged vampire bat?Diaemus youngi?are pseudogenes.In order to study the molecular evolution of bat SWS1 genes,we also downloaded 81 mammalian SWS1 sequences for selective pressure analysis.The results showed that bats SWS1 genes were highly conservative during their evolution.In addition,we determined the function of the SWS1 visual pigments from eight extant bats and also their common ancestor in vitro.The results show that all of them are sensitive to UV light with the maximum absorption wavelength(?_max)ranging from 357 nm to 359 nm.The results showed that UV vision is very important for many bats,but not the case in some other species.In the second part of this study,the function of SWS1 visual pigment in P.parnellii was verified.Firstly,we amplified the Cytb gene of two P.parnellii individuals and confirmed that the two individuals belongs to either P.mexicanus or P.sp1.Then,we amplified the SWS1 gene sequences from the two individuals,and found that the coding region of their SWS1 genes were complete.We analyzed the selection pressures on the SWS1 genes in mustache bats.The results showed that compared with other mammals and other mustache bats with low duty cycle echolocation,the SWS1 genes from P.mexicanus,P.sp1 and the ancestor of P.parnellii complex were close to neutral evolution.The in vitro assay results showed that both SWS1 pigments from the ancestor of P.parnellii complex and P.quadridens are sensitive to ultraviolet light,with ?max values at 357 nm.However,the SWS1 visual pigments of P.mexicanus and P.sp1,both of which have high duty cycle echolocation,had no spectral sensitivity,indicating independently functional lost during evolution.Subsequently,we validated the expression of P.mexicanus SWS1 opsin.We detected the SWS1 opsin expression by both Western Blot and liquid chromatography-mass spectrometry.The results showed that the purified solution contains SWS1 pigment,but not functional.Finally,in order to reveal the molecular mechanism of the functional loss,we confirmed that there are total three key amino acid substitutions related,G6 R,G83A and R314 W.The function of P.mexicanus SWS1 pigments could be destroyed if anyone of the three mutated.Reverse mutations with all three sites,6,83 and 314 can restore its function.In summary,we amplified and analyzed representative bat SWS1 genes,and verified the function of the SWS1 pigments.The experimental results show that UV vision plays a very important role in many bats,but not the others.In addition,we found that SWS1 gene is intact in P.mexicanus,but it's not functional.Subsequent studies have found that three key amino acid substitution can leads to the functional loss of SWS1 pigment.Therefore,the results show that the existence of SWS1 gene does not necessarily means the bat has ultraviolet vision.