Contrasting laboratory and field outcomes of bat–moth interactions

Predator–prey interactions are important but difficult to study in the field. Therefore, laboratory studies are often used to examine the outcomes of predator–prey interactions. Previous laboratory studies have shown that moth hearing and ultrasound production can help prey avoid being eaten by bats. We report here that laboratory behavioral outcomes may not accurately reflect the outcomes of field bat–moth interactions. We tested the success rates of two bat species capturing moths with distinct anti-bat tactics using behavioral experiments. We compared the results with the dietary composition of field bats using next‐generation DNA sequencing. Rhinolophus episcopus and Rhinolophus osgoodi had a lower rate of capture success when hunting for moths that produce anti-bat clicks than for silent eared moths and earless moths. Unexpectedly, the success rates of the bats capturing silent eared moths and earless moths did not differ significantly from each other. However, the field bats had a higher proportion of silent eared moths than that of earless moths and that of clicking moths in their diets. The difference between the proportions of silent eared moths and earless moths in the bat diets can be explained by the difference between their abundance in bat foraging habitats. These findings suggest that moth defensive tactics, bat countertactics, and moth availability collectively shape the diets of insectivorous bats. This study illustrates the importance of using a combination of behavioral experiments and molecular genetic techniques to reveal the complex interactions between predators and prey in nature.Excel and WINRARFunding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number: 31872681Funding provided by: Human Frontier Science ProgramCrossref Funder Registry ID: http://dx.doi.org/10.13039/100004412Award Number: CDA00009/2019-CFunding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number: 32071492Funding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number: 32271558We investigated the success rate of a bat capturing a tethered moth in flight in a recording room. The acoustic signals of the bat and the moth were recorded using an ultrasonic microphone (UltraSoundGate CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) connected to an ultrasound recording interface (UltraSoundGate 116Hm), and their behavior was recorded using two digital video cameras (Sony FDR-AX60, Sony, Tokyo, Japan). The success rate of a bat capturing a moth was calculated as the reciprocal of the number of capture attempts multiplied by 100%. When a bat failed to catch a moth during a five-minute foraging period, the capture success of the bat was defined as zero. We analyzed the diet composition of fecal samples of the bat species using next-generation DNA sequencing. We extracted DNA from 100 mg feces per sample using the E.Z.N.A. DNA Extraction Kit (Omega Bio-tek) following the manufacturer's protocol. DNA extracts were amplified in three replicates using two primer pairs. A 225 bp fragment of the cytochrome c oxidase subunit I (COI) was amplified using the primers LCO‐1490 (5′‐GGTCAACAAATCATAAAGATATT GG‐3′) and ZBJ‐ArtR2c (5′‐WACTAATCAATTWCCAAATCCTCC‐3′). A 105 bp fragment of the mitochondrial 16S ribosomal DNA was amplified using the primers Coleop_16Sc (TGCAAAGGTAGCATAATMATTAG) and Coleop_16Sd (TCCATAGGGTCTTCTCGTC). We quantified the diet composition of each bat species using the weighted percentage of occurrence (wPOO) and the relative read abundance (RRA) of prey orders, families, and species. We used moth traps (6W 12V portable heath moth trap, Natural History Book Service, Totnes, UK) to attract moths and investigate moth availability in the bat foraging habitats. We estimated the abundance of the earless moths, silent eared moths, and moths capable of producing anti-bat clicks by summing the number of individuals of each category collected on three nights at three locations. We referred to terHofstede & Ratcliffe (2016, Journal of Experimental Biology, 219, 1589–1602) to define whether a moth family had ultrasound-sensitive ears. In particular, Sphingidae except for Acherontiini and Choerocampina were listed as earless moths. We examined the acoustic responses of captured moths to tactile stimulation and attacking bats. First, we lightly compressed and touched the moth's head, thorax, abdomen, or tail to simulate handling by predators. Second, we tethered a moth in flight after dark and recorded its acoustic response to an attacking bat. We considered a moth species to be responsive if we recorded ultrasound production in at least one individual in response to tactile stimuli and/or the bat. We examined the acoustic responses of captured moths to tactile stimulation and attacking bats. First, we lightly compressed and touched the moth's head, thorax, abdomen, or tail to simulate handling by predators. The acoustic responses of moths were recorded using an UltraSoundGate microphone and interface. The microphone was placed 10 cm from the tested moth. Second, we tethered a moth in flight after dark and recorded its acoustic response to an attacking bat. The experimental setup and equipment were the same as those for the experiment on the capture success rates of foraging bats