The Diversity And Function Of Social Vocalizations Of Big-Footed Myotis (Myotis Macrodactylus) During Foraging

As an information carrier of acoustic communication,animal vocalizations can encode a multitude of information,including internal motivation,emotion of the sender,and information on the environment.Animal acoustic signals are not dependent on light and confer advantages in regard to long–range transmission,detection,and location compared with visual and olfactory signals.Animal acoustic signals play a key role in their foraging,courtship,fighting,and anti-predator defense.In many animals,foraging behaviors is one of the most important events in the life history that are linked to resource exploitation and energy supply,and ultimately determines individual survival and reproduction.Food-associated calls could signal detailed information about food availability,which largely improve foraging efficiency of foraging companions and therefore mediate resource competition and sharing between conspecifics.To date,however,the function of food-associated calls remains poorly investigated in most animal taxa.The relationships among food-associated vocalizations,food resource diversity,and conspecific activities in animals remain uncertain.Echolocating bats fly fast and seek foraging at night sky,with a comparatively poor performance on the vision,tactility and olfaction system.They rely heavily on acoustic signals to search for food and communicate information,providing an ideal model for bioacoustic research.Here,we aim to elucidate the diversity and function of social vocalizations of big-footed myotis(Myotis macrodactylus)during foraging based upon field monitoring,DNA metabarcoding,and playback experiment.We determined foraging habitats utilized by big-footed myotis via the line transect method based on acoustic sampling.Using acoustic recording,spectro-temporal feature analysis and statistical classification,we characterize social vocalizations of big-footed myotis during foraging.Our analyses indicate that social calls of big-footed myotis in foraging context consisted of 6 simple syllables and 2 composites,namely bent downward frequency modulation(bDFM),steep downward frequency modulation(sDFM),flattened downward frequency modulation(fDFM),wrinkled downward frequency modulation(wDFM),sinusoidal frequency modulation(SFM),rectangular broadband noise burst(rBNB),chevron frequency modulation-downward frequency modulation(CFM-DFM)and downward paraboloid frequency modulation-downward frequency modulation(dPFM-DFM).Discriminant function analyses associated with a subset-validation procedure revealed that 76.4%of syllable types were correctly classified,which was significantly higher than a random classification score of 14.3%.In addition,multidimensional scaling of multiple acoustic parameters further confirmed notable difference among syllables in a three-dimensional space.Euclidean distance analysis also showed that SFM calls overlaps spectrally temporally with echolocation pulses particularly with feeding buzzes emitted by conspecific.The majority of social calls in big-footed myotis are characterized by a relatively long duration and low frequency compared with echolocation pulses.We non-invasively collected the feces from 72 bat individuals(?:24,?:and 48).We employed next-generation sequencing of fecal DNA to perform sequencing and sequence alignment analysis.To assess bats'dietary composition,we computed the weighted percentage of occurrence data(wPOO)and the relatively read abundance data(RRA)of prey.To estimate the availability of insect in the core foraging areas of bats,we sampled night-flying insects using a light trapping.We found that big-footed myotis consumed a diverse range of insects belonging to 59 families in 10 orders.The dietary composition of the big-footed myotis was dominated by Diptera,Lepidoptera,and Trichoptera reaching up to 90%of prey identified in the feces.Permutational Multivariate Analysis of Variance using Soensen dissimilarity matrix of presence or absence of insect types,or Bray-Curtis dissimilarity matrix of abundance of insects confirmed that the abundance and types of insect taxa from light traps were remarkably similar to those from fecal samples.These results suggested that populations of big-footed myotis adopt a generalist feeding strategy consuming various kinds of insect species.In addition,we found that the preference of big-footed myotis for Ephemeroptera was the weakest through euclidean distance analysis(d=6.192,P<0.05).To disentangle the relationships between bat social vocalizations and associated predictive factors,we monitored the availability of insect,conspecific activities,and climatic factors in the core foraging patches of big-footed myotis.A multiple linear regression model(MLS)identified a tight link between insect abundance and social vocalizations'rate(MLS:Estimate=1.2E-3±4.4E-4,P=0.010).The rate of social vocalizations were negatively associated with Pielou's evenness index of insects(MLS:Estimate=-0.60±0.27,P=0.038),but were positively associated with the level of conspecific activities(MLS:Estimate=1.3E-3±4.7E-4,P=0.011).This suggested that a decrease in insect evenness would cause an intensification of food resource competition,leading an increase in the output of social calls.Further analysis of our data showed that insect Pielou's evenness index was remarkably predicted by the relative abundance of Lepidoptera.There was also a negative association between the relative abundance of Lepidoptera and the rate of syllables(R~2=0.32,P=0.001).Hierarchical partitioning algorithm revealed that the differences in rate of social vocalizations were largely explained by the level of conspecific activities in foraging areas(IE=22.55%).The relationships among the rate of syllables,ambient temperature,and humidity were not statistically significant.Field playbacks consisting of experimental stimuli,echolocation pulses,and silence were presented to experimental bats.The foraging efforts of conspecifics were reduced 1.29-1.90 fold during playback of social calls compared to silent control.Food consumption was reduced 1.40-1.60 fold in the presence of social calls compared to silent control.By contrast,playback of echolocation pulses leaded to an increase in feeding activity among experimental bats.Together,these findings indicate that:(1)Group-living big-footed myotis possess a broad social call repertoire in the context of social foraging.(2)These bats consumes a diverse range of insects dominated by Diptera,Lepidoptera,and Trichoptera.Furthermore,the available insect resources significantly affect the emission of social calls in big-footed myotis during foraging.(3)The social calling phenomena were significantly shaped by the emergence of foraging conspecifics.Foraging bats employ social calls to engage in intraspecific food competition and claim the ownership of limited food.(4)In addition to use of echolocation pulses for spatial orientation and foraging,bats also employ echolocation signals to provides reliable information about food availability.This study firstly described and analyzed the spectro-temporal parameters of the social vocalizations of bats in their natural foraging habitat.Our works further demonstrated that bats'vocalizations during agonistic foraging interactions function in resource defense.Our findings will provide supporting evidences for further work on the neural pathways underlying vocal control in mammals and the ecological principle of intraspecific competition for food resources and animal conservation,and will be helpful to understanding the evolutionary mechanisms of acoustic communication in bats,and will be beneficial to uncover the auditory adaptation and maintenance of social stability in nocturnal mammals.