Species on the Menu of a Generalist
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Molecular Ecology (2009) 18, 2532–2542 doi: 10.1111/j.1365-294X.2009.04184.x SpeciesBlackwell Publishing Ltd on the menu of a generalist predator, the eastern red bat (Lasiurus borealis): using a molecular approach to detect arthropod prey ELIZABETH L. CLARE,* ERIN E. FRASER,† HEATHER E. BRAID*, M. BROCK FENTON† and PAUL D. N. HEBERT* *Department of Integrative Biology, University of Guelph, Guelph, ON, Canada N1G2W1, †Department of Biology, University of Western Ontario, London, ON, Canada N6A 5B7 Abstract One of the most difficult interactions to observe in nature is the relationship between a predator and its prey. When direct observations are impossible, we rely on morphological classification of prey remains, although this is particularly challenging among generalist predators whose faeces contain mixed and degraded prey fragments. In this investigation, we used a poly- merase chain reaction and sequence-based technique to identify prey fragments in the guano of the generalist insectivore, the eastern red bat (Lasiurus borealis), and evaluate several hypotheses about prey selection and prey defences. The interaction between bats and insects is of significant evolutionary interest because of the adaptive nature of insect hearing against echolocation. However, measuring the successes of predator tactics or particular prey defences is limited because we cannot normally identify these digested prey fragments beyond order or family. Using a molecular approach, we recovered sequences from 89% of the fragments tested, and through comparison to a reference database of sequences, we were able to identify 127 different species of prey. Our results indicate that despite the robust jaws of L. borealis, most prey taxa were softer-bodied Lepidoptera. Surprisingly, more than 60% of the prey species were tympanate, with ears thought to afford protection against these echolocating bats. Moths of the family Arctiidae, which employ multiple defensive strategies, were not detected as a significant dietary component. Our results provide an unprecedented level of detail for the study of predator–prey relationships in bats and demonstrate the advantages which molecular tools can provide in investigations of complex ecological systems and food- web relationships. Keywords: bats, mammals, predator–prey interactions, molecular scatology, insects, species interactions Received 15 December 2008; revised 15 February 2009; accepted 18 February 2009 arctiid moths produce ultrasonic clicks in response to bat Introduction echolocation calls and thus deter some bat attacks (Ratcliffe Although species’ interactions underlie many evolutionary & Fullard 2005; Ratcliffe et al. 2008) or advertise their unpala- and ecological principles, observing and describing these tability (Dunning 1968; Surlykke & Miller 1985) while other relationships can be challenging. This is particularly true arctiids mimic these aposematic signals (Ratcliffe et al. 2008). for predator–prey interactions (Agusti et al. 1999; Sheppard The ‘allotonic frequency’ hypothesis (AFH) (Fullard 1987) et al. 2004). The relationship between insectivorous bats suggests that bats can counter these adaptations by produc- and moths is a classic example of predator–prey adaptation. ing echolocation calls outside the hearing range of moths, Bats use echolocation to detect and track insect prey and potentially leading to a co-evolutionary arms race between bat insects use hearing-based defences to detect and evade echo- echolocation and moth hearing. The AFH also predicts that locating bats (Roeder 1967; Fenton & Fullard 1979). Some bats calling within that threshold should have a diet devoid of tympanate (eared) species (Schoeman & Jacobs 2003). Correspondence: Elizabeth L. Clare, Fax: 519-767-1656; E-mail: When direct observations of predation are impossible, we [email protected] use morphological classification of digested prey remains. © 2009 Blackwell Publishing Ltd MOLECULAR DETECTION OF BAT–INSECT INTERACTION 2533 Since insectivores rapidly and thoroughly chew their food, attempted to use a single marker analytical technique for the species-level identification of prey is extremely difficult. As dietary analysis of insectivorous bats that can be employed a result, published data about the diet of insectivorous bats in natural settings to survey prey diversity across the full rarely provide identifications beyond order (see Table S1, range of feeding behaviours — from specialist to generalist Supporting information). A number of generalizations about — and that can identify both unexpected and expected prey. bat hunting styles, predator preferences and prey defences Comparing sequences recovered from prey items with a have been proposed but, in the absence of prey identifica- reference database is an advantage in the study of predator– tions, many are difficult to confirm. For instance, specific prey relationships because it requires no a priori knowledge evolutionary aspects of predator–prey relationships such as of prey identity. If the reference database is comprehensive, prey size selection by predators or the effectiveness of arctiid both expected and unexpected prey will be diagnosed with colouration and sound emission are difficult to assess if we similar efficiency. Since DNA fragments rapidly degrade cannot identify specific prey species in guano or stomach during digestion (Zaidi et al. 1999), studies need to target contents. small, fast-evolving multicopy gene regions (Symondson Molecular approaches provide new opportunities to chara- 2002). Despite occasional pseudogenes (Bensasson et al. 2001), cterize predator–prey relationships in complex food webs mitochondrial DNA has emerged as a likely target since it from laboratory and field studies. These techniques prima- has high copy number and sufficient variation to allow rily target trace materials (e.g. Zaidi et al. 1999) and have species-level diagnosis (Hebert et al. 2003). Until recently, now been applied to many target predator groups, including the lack of a comprehensive reference sequence database wasps (Kasper et al. 2004), mosquitoes (Coulson et al. 1990; represented the primary barrier to such analysis (Hadrys Gokool et al. 1993), carnivore communities (Farrell et al. 2000), et al. 1992). However, databases of mitochondrial sequence marine vertebrates (Jarmin et al. 2002; Jarmin & Wilson 2004), information from vouchered specimens are rapidly expand- marine invertebrates (Blankenship & Yayanos 2005) and ing for standardized gene regions (Ratnasingham & Hebert captive species such as sea lions (Deagle et al. 2005). Even 2007) and can be used to retrieve identifications for unknown ancient DNA contained in coprolites has been recovered sequences. Vouchered databases are particularly advant- (Poinar et al. 1998; Hofreiter et al. 2000). Similar techniques ageous for these analyses because they provide an extra level can also track secondary predation (Sheppard et al. 2005). of taxonomic precision to the identification of unknowns. While trophic connections have been made using a variety In this investigation, we employ molecular techniques to of molecular approaches (Symondson 2002; King et al. 2008), describe the diet of the eastern red bat, Lasiurus borealis, and substantial analytical limitations remain. In particular, many to test several existing hypotheses about the prey of these techniques are taxon-specific (e.g. Bacher et al. 1999), requir- bats. While PCR and sequence-based approaches for dietary ing a priori knowledge of prey species. For instance, mono- analyses are not novel (Symondson 2002; King et al. 2008), clonal antibodies (Symondson 2002) are best used to confirm they have not previously been applied to bats. Eastern red the predators of a target prey species rather than to reveal bats range across most of eastern North America where the dietary complexity of generalist predators (e.g. Chen et al. they frequently forage in concentrations of insects around 2000). Most existing antibody techniques are particularly streetlights (Acharya & Fenton 1999). The diet of these bats is difficult to apply (and cost-prohibitive) for species that con- particularly interesting because of both morphological and sume multiple prey taxa (Sheppard et al. 2004; King et al. behavioural peculiarities in their hunting style. First, alth- 2008). ough they have extremely robust jaws (Freeman 1981) similar New approaches based on polymerase chain reaction to Coleoptera specialists, previous morphological dietary (PCR) are promising (Höss et al. 1992; Kohn & Wayne 1997; data indicate that these bats eat a range of prey (Ross 1961; Zaidi et al. 1999), but their application is complicated by Whitaker 1972) and may specialize on moths (Acharya 1995; several factors. Multiple genetic markers have been employed Hickey et al. 1996; Acharya & Fenton 1999) which are softer- depending on the target group (reviewed by King et al. 2008); bodied. Furthermore, eastern red bats emit echolocation calls however, this can increase the analytical complexity and between 30 and 65 kHz (Obrist 1995), making them audible cost. For example, Scribner & Bowman (1998) required two to tympanate insects, including moths on which they are microsatellite loci to identify a limited number of bird spe- thought to specialize. cies as prey of glaucous gulls, and could not identify further We used a PCR-based approach to obtain COI sequences taxonomic groups without additional markers. For this from prey items recovered from the guano of eastern red bats reason,