DOI: 10.5061/dryad.4mw6m90fr
title: Contrasting laboratory and field outcomes of bat–moth interactions

description: 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.
description: We 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. 
description: Excel and WINRAR
description: # Contrasting laboratory and field outcomes of bat–moth interactions ##
 Description of the data and file structure We obtained the success rates
 of two bat species, *Rhinolophus episcopus* and *Rhinolophus osgoodi,*
 capturing earless moths, silent eared moths, and clicking moths in the
 laboratory. We obtained the proportions of insect orders/families in the
 diets of *R. episcopus* and *R. osgoodi*. Moreover, We provided the
 behavioral and ecological information of the moth species captured in the
 bat foraging habitats. We provided sound files of the acoustic response of
 moths to tactile stimulation and attacking bats. We 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
 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, 15891602) 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 moths
 head, thorax, abdomen, or terminal segment of the abdomen 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 moths 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.
 There are three data files and two sound files, "Table S1 The success
 rates of Rhinolophus episcopus and R. osgoodi capturing earless moths,
 silent eared moths, and clicking moths in the laboratory.csv",
 "Table S3 Behavioral and ecological information of the moth species
 captured in the bat foraging habitats.csv", "Table S4 Details of
 the proportions of insect orders/families in the diets of Rhinolophus
 episcopus and Rhinolophus osgoodi.csv", acoustic response of moths to
 attacking bats, and acoustic response of moths to tactile stimulation.
 Table S1 includes the following information: Bat species, Bat ID, Number
 of capture attempts, succeeded or failed to catch the moth, Capture
 success rate, Prey, Prey type, and Produced clicks or not in response to
 attacking bats. Table S3 includes the following information: Moth species,
 Family and Subfamily of the moth species, eared or earless, moth response
 to tactile stimuli and an attacking bat, Palatability, Wing length,
 Abundance in habitats (number of individuals), and proportion of moth
 species in the diets of *Rhinolophus episcopus* and *Rhinolophus osgoodi*.
 Table S4 includes the following information: Bat species, Primers, order
 and family information of prey classified in bat diets, eared or earless,
 proportion of prey in the diets, and methods used for diet quantification
 (wPOO/RRA). The file named “acoustic response of moths to attacking bats”
 includes sound recordings of 10 moth species that produce ultrasonic
 clicks in response to attacking *R. episcopus* or *R. osgoodi*. The file
 named "acoustic response of moths to tactile stimulation"
 includes sound recordings of 19 moth species that produce ultrasonic
 clicks in response to tactile stimulation. For more information, please
 see the manuscript titled "Contrasting laboratory and field outcomes
 of bat-moth interactions" by Lin et al. Contact:
 linaq376@nenu.edu.cn.