Acta Chiropterologica, 19(2): 239–254, 2017 PL ISSN 1508-1109 © Museum and Institute of Zoology PAS doi: 10.3161/15081109ACC2017.19.2.003
Diet determined by next generation sequencing reveals pest consumption and opportunistic foraging by bats in macadamia orchards in South Africa PETER JOHN TAYLOR1, 2, 8, EMMANUEL MATAMBA3, JACOBUS NICOLAAS (KOOS) STEYN4, TSHIFHIWA NANGAMMBI3, 5, M. LISANDRA ZEPEDA-MENDOZA6, and KRISTINE BOHMANN6, 7 1SARChI
Chair on Biodiversity Value and Change in the Vhembe Biosphere Reserve and Centre for Invasion Biology, School of Mathematical and Natural Sciences, University of Venda, P. Bag X5050, Thohoyandou 0950, Republic of South Africa 2 School of Life Sciences, University of KwaZulu-Natal, Durban 4001, Republic of South Africa 3 Department of Zoology, School of Natural and Mathematical Sciences, University of Venda, P. Bag X5050, Thohoyandou 0950, Republic of South Africa 4Department of Ecology and Resource Management, School of Environmental Sciences, University of Venda, P. Bag X5050, Thohoyandou 0950, Republic of South Africa 5 Department of Nature Conservation, Faculty of Science, Tshwane University of Technology, P. Bag X680, Pretoria 0001, Republic of South Africa 6 Section for Evolutionary Genomics, Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen K, Denmark. 7 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom 8 Corresponding author: E-mail:
[email protected] Recent studies have documented the economically significant impact of bats as predators of agricultural pest insects. We used Next Generation Sequencing (NGS) of the cytochrome oxidase I gene to elucidate the diet of six species of bats based on faecal pellets collected from individuals and roosts in macadamia orchards at Levubu, Limpopo Province, South Africa. For five of these species, we compared the molecular data with published results from microscopic analysis of faecal pellets, culled parts and stomach contents. We provide the first description of the molecular diet of the large African molossid bat, Mops midas. Expectations from skull morphology and a single limited study of stomach contents were that this species should be a beetle-specialist. However, NGS revealed that the diet of M. midas contained a much higher prevalence and diversity of lepidopteran (81 taxa from 17 families) compared to coleopteran (two taxa) prey. While this result is predicted by the allotonic frequency hypothesis for a bat species with low echolocation frequency, it could also be explained by unequal PCR amplification, a constraint of amplicon sequencing. Apart from the above-mentioned species where our sample was probably unbiased (24 pellets from multiple roosts and occasions), sample sizes of the other five species were very low and therefore potentially biased (1–6 pellets). Nevertheless, these samples revealed for each bat species surprisingly many prey taxa spanning several insect orders, indicating that individual bats were capable of consuming a wide diversity of prey during one or two nights of foraging. Contrary to expectations, bats of all foraging groups (clutter, clutteredge and open-air) fed opportunistically on mostly-flightless cockroaches (Order Blattodea). About one third of all faecal pellets tested from five species of bats of all foraging groups contained DNA from the significant macadamia pest species, Nezara viridula (Order Heteroptera), indicating the value of intact bat communities in the biological control of pest stink bugs in macadamia orchards. Contrary to the general expectations of the allotonic frequency hypothesis, all six bat species studied fed predominantly on tympanate versus non-tympanate species of moths (57–75% of lepidopteran prey taxa), even those ‘non-allotonic’ bat species having intermediate echolocation peak frequencies that encompass the frequency sensitivity of hearing (tympanate) moths. Key words: Chiroptera, macadamia, stink bugs, Limpopo Province, South Africa
INTRODUCTION Bats consume large numbers of nocturnal insects including many crop pests (Williams-Guillén, Perfecto and van der Meer, 2008; Jones et al., 2009; Bohmann et al., 2011; Kunz et al., 2011; McCracken et al., 2012). Bat predation of pest insects has
a significant economic benefit to farmers (Cleveland et al., 2006; Boyles et al., 2011; Maas et al., 2013, 2015; Lopez-Hoffman et al., 2014; Wanger et al., 2014; Maine and Boyles, 2015). There is an urgent need to further document the economic value of bats in agriculture to underline the ecological services they provide, thus promoting the need for their
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conservation. As part of integrated pest management (IPM), increasing local bat populations (e.g., by bat houses) may prove to be an effective and less environmentally destructive means of controlling insect pests than simply increasing chemical pesticide use. Also, pesticide use may ultimately prove counterproductive if populations of beneficial top insect predators, such as insectivorous spiders, bats and birds decline through poisoning (Wickramasinge et al., 2003; Pocock and Jennings, 2008). Very few studies on Africa have examined the economic impact of bats as biocontrol agents of agricultural pest insects. In sugar cane plantations in Swaziland, Noer et al. (2012) showed that bats selectively forage over sugar cane fields, while Bohmann et al. (2011) used Next Generation Sequencing (NGS) of mitochondrial DNA minibarcode markers to show that two molossid bat species (Chaerephon pumilus and Mops condylurus) feed on insect pest species such as stinkbugs (Order Heteroptera, Family Pentatomidae). Stinkbugs are the major pests of macadamias worldwide, including South Africa (Jones and Caprio, 1994; Vincent et al., 2001; Schoeman, 2013, 2015). A variety of local bat species forage in macadamia orchards in Limpopo Province, South Africa, and their seasonal patterns of foraging activity at these sites coincide generally with annual outbreaks of pest stinkbugs (Heteroptera: Pentatomidae: Bathycoelia distincta and Nezara viridula) in late austral summer to autumn (Taylor et al., 2013c). Furthermore, using both microscopic and NGS analyses, Taylor et al. (2011, 2012, 2013a) reported generally higher proportions of bugs (Heteroptera) in the diet of bats foraging in macadamia orchards compared with bats of the same species foraging in natural environments, as well as the presence of at least one major crop pest species (the Southern green vegetable bug, N. viridula) in the diet of multiple insectivorous bat species. While Taylor et al. (2013a) reported briefly on the occurrence of pest stink bugs in the diet of five species of bats occurring in macadamia orchards, the present article extends this study to a more detailed analysis of the diet of six species of bats foraging in macadamia orchards based on prey items (molecular operational taxonomic units or MOTU’s) identified from NGS data. We examined the diet of six bat species having different foraging strategies, including clutterfeeders (the slit-faced bat, Nycteris thebaica and Sundevall’s leaf-nosed bat, Hipposideros caffer), clutter-edge foragers (the African pipistrelle,
Pipistrellus hesperidus and the yellow house bat, Scotophilus dinganii) and open-air foragers (Mops free-tailed bat, Mops midas and the little free-tailed bat, Chaerephon pumilus) (see Monadjem et al., 2010 for a summary of bat foraging strategies). We collected faecal pellets from known roosts of these bats in the study area as well as from captured individuals flying in and presumably foraging in or near macadamia orchards. We used NGS techniques (Bohmann et al. 2011; Pompanon et al., 2012) for comparison with previous studies (Taylor et al., 2011, 2013a) using microscopic methods (Whitaker and Kunz, 1988; Whitaker et al., 2009). Whilst the latter technique usually allows identification only to order or family, the former provides greater taxonomic resolution but biases may be present due to unequal PCR amplification and other technical obstacles, rendering accurate quantitative estimates of dietary composition using NGS to be generally suspect (Pompanon et al., 2012). For this reason, we report only on % occurrence of dietary items (number of pellets) when reporting on our NGS data. Based on axioms in the literature, it would be predicted that clutter and clutter-edge bats would be more likely than open-air feeding bats to prey on predominantly slow-flying stinkbugs, which usually fly close to vegetation and are immobile at temperatures below 18°C (Schoeman, 2009). However, Bohmann et al. (2011) recorded DNA of stinkbugs (Family Pentatomidae) in eight out of 89 pellets (14%) analysed from aerial-feeding free-tailed bats (M. condylurus and C. pumilus) in a sugarcanedominated landscape in Swaziland. We thus predicted that most of the bat species sampled in our study would prey on locally abundant pest stinkbug and other pest species, especially during the peak stinkbug outbreak season (Taylor et al., 2013c). We present for the first time a detailed analysis of the diet of a large African molossid bat, M. midas. Based on its strong jaw structure, and a single study of stomach contents, it has been predicted that this species may be a beetle-specialist (Monadjem et al., 2010). On the other hand, based on its low echolocation peak frequency (14 kHz — Monadjem et al., 2010; Taylor et al., 2013b), we expect from the allotonic frequency hypothesis a high occurrence of moths in the diet of this species, as predicted for specialist moth-feeding bat species having frequencies either above or below the typical frequency range of hearing (approximately 20–60 kHz) of tympanate (hearing) moths (Jacobs, 2000; Schoeman and Jacobs, 2003, 2011). A very high frequency of moths has been reported in the diet of Otomops
Bat diet in macadamia orchards
mariensseni, another large African molossid with a low frequency (12 kHz) echolocation frequency (Rydell and Yalden, 1997; Fenton et al., 2004). Since NGS methods were able to resolve moths from 17 distinct moth families in the bat diet, and, hence, whether or not these moths possess tympani (Sierra and Arlettaz, 1997; Zha et al., 2009), we can further predict that the diet of M. midas should include both hearing and non-hearing moths. Similarly, in the case H. caffer and N. thebaica, since they are also predicted to be allotonic, avoiding moth hearing by having high peak frequencies (142 and 90 kHz, respectively — Monadjem et al., 2010), we predict that like M. midas, they should also have a diet including both hearing and nonhearing moths. We further predict that the ‘non-allotonic’ bat species in our study having intermediate peak frequencies within the range of moth hearing (P. hesperidus, S. dinganii, C. pumilus) should feed on a significantly lower proportion of hearing versus non hearing moths, compared with the three allotonic species in our study. MATERIALS AND METHODS Study Area The study area is located in the southern foothills of the Soutpansberg Range, 20 km east of the town of Louis Trichardt, encompassing the Levuvhu Valley subtropical fruit-growing area in the south, which is dominated by extensive monocultures of macadamias, pecan nuts, avocados, bananas, pine and gum plantations (Fig. 1). It is bordered to the north by mountains covered predominantly by dense thickets classified by Mucina and Rutherford (2006) as ‘Soutpansberg Mountain Bushveld’, as well as by extensive commercial plantations of gums and pines. Annual rainfall in the study area was around 930 mm in 2010 and 960 mm in 2011 and fell mostly between November and April. Daily maximum temperatures frequently exceed 35°C in summer (October to March) but rarely exceed 40°C and minimum daily summer temperature seldom fell below 15°C, whereas winters are much colder, with minimum daily temperatures dropping to just over 0°C on a few days in June and July and maximum daily temperatures reaching 25°C.
Collection of Faecal Pellets All the faecal pellets obtained from day or night roosts (for N. thebaica and M. midas — Table 1) were collected onto 1 × 2 m boards covered in ‘Gladwrap’ placed under the position of roosting bats approximately one week prior to collection. Pellets of the remaining four species were obtained from captured individual bats that were held individually in cloth bags until they yielded sufficient numbers of pellets (Table 1). In the case of P. hesperidus (n = 3) and H. caffer (n = 1), pellets were obtained directly from harp-trapped individuals, whereas in S. dinganii (n = 3) and C. pumilus (n = 1), pellets were taken from individuals roosting in an attic, which flew accidentally
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into the house and were captured and retained in cloth bags until they produced pellets. Collection sites included farm subdivisions (‘portions’) 9 (day-roost of M. condylurus), 10 (day roost of both M. midas and M. condylurus), 28 (day roost of S. dinganii and C. pumilus), and 36 (harp trap site for capture of individual P. hesperidus and H. caffer) of the Welgevonden farm, with one site located approximately 10 km to the northwest (Vlakfontein farm, a day-roost of M. midas) and another 30 km to the east (Laatsgevonden farm, a night-roost of N. thebaica) (Fig. 1). Pellets were kept in zip lock bags in a freezer or placed in 90% ethanol.
Molecular Diet Analysis Identification of Prey-DNA in faecal pellets DNA was extracted separately from each of 37 faecal pellets using a QIAamp DNA Stool Mini Kit (Handbook 06/2012) (Qiagen, Valencia, CA) according to the manufacturer’s instructions with modifications following Zeale et al. (2011). Extraction blanks were included. Amplifications of ca. 157 bp prey insect cytochrome oxidase I (COI) fragments were performed using insect-generic COI primers (MZArtF and R [Zeale et al., 2011] which were 5’ nucleotide barcoded (Binladen et al., 2007), i.e. 20 forward and 20 reverse primers, all varying by 8 bp of the 5’ barcode. By using different forward and reverse primer combinations for amplifying all extracts, all amplicons (227 bp in total, 157 bp fragment + 70 bp primers) from each extract were uniquely labelled. To discriminate PCR and/or sequencing artefacts from true biological sequences, each extract was amplified twice, each with a unique primer combination. PCR reactions were performed on a 2720 Thermal Cycler (Applied Biosystems) in 25 μl reactions using the Amplitaq Gold enzyme system (Roche, Basel, Switzerland). PCR blanks were included. Each reaction contained 1 μl DNA template, 1× PCR Gold buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1 unit AmpliTaq Gold, and 0.4 μM of each primer. Conditions were 95°C for 5 mins, then 40 cycles of 95°C for 15 seconds, 52°C for 30 seconds and 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes. 5 μl of each of the PCR products were visualised with GelRed Nucleic Acid Stain (Biotium) on 2% agarose gels. Positive amplicons were pooled at approximately equimolar ratios according to gel band strength. The amplicon pool was purified using the MinElute PCR Purification Kit (Qiagen, Valencia, CA). The concentration of the purified PCR pool was measured using a Qubit Flourometer (Invitrogen). The pool was subsequently converted into an Illumina sequencing library, using the NEBNext ‘DNA Library Prep Master Mix Set for 454’ (#E6070L) although using blunt end Illumina adapters (Meyer and Kircher, 2010) in place of Roche/454 FLX adaptors. A control library blank was also constructed principally following Schnell et al., 2015. The resulting indexed libraries were purified on QIAquick columns and eluted in 30 μl Buffer EB. The library, the library blank and the PCR blank were visualized on a 2100 Bioanalyzer (Agilent Technologies) and sequenced 150 bp paired-end on an Illumina MiSeq platform at the Danish National High-Throughput DNA Sequencing Centre.
Sequencing of pest insects Detached legs from collected specimens were taken for each of the 11 insect (Heteroptera) species collected from local macadamia plantations and identified by Dr. M. Stiller of the Agricultural Research Council, South Africa. This list included three
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A
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Sand R. SOUTPANSBERG Levuvhu R.
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Study area Polokwane
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Nzhelele R.
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FIG. 1. Map of study area showing location of study area in South Africa and Limpopo Province (A) and detailed location of faecal pellet collection sites within the study area (B). Grey shading indicates the extent of vegetation types associated with the Soutpansberg Mts. Numbers represent localities as follows: 1) Farm Vlakfontein, 2–5) Farm Welgevonden, 6) Farm Laatsgevonden
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TABLE 1. Summary of methods used to collect faecal samples from bats and their roosts from Levubu, South Africa Species N. thebaica S. dinganii P. hesperidus C. pumilus H. caffer M. midas Total
Sample months
No. pellets analysed
Aug 2010, May and Aug 2012 Feb 2011, Feb 2012 Aug 2011, Jan 2012 Apr 2012 Aug 2012 Aug and Nov 2012
6 2 3 1 1 24 37
known macadamia pest species and an additional eight species which are not known to be pests. The pest species included Nezara viridula (Pentatomidae; green vegetable stink bug), Pseudotheraptus wayi (Coreidae; coconut bug) and Bathycoelia rodhaini (Pentatomidae; yellow-spotted stink bug). The nonpest species included Dalsira costalis (Pentatomidae; stink bug), Caura rufiventris (Pentatomidae; stink bug), Coridius nubilis (Pentatomidae; stink bug), Anoplecnemis curvipes (Coreidae; giant coreid bug), Leptoglossus australis (Coreidae; leaffooted plant bug), Veterna sp. (Pentatomidae; grass stink bugs) and Dysdercus nigrofasciata (Pyrrhocoridae; cotton stainer, a major pest of cotton). DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) Handbook 07/ 2006, Protocol: Purification of Total DNA from Animal Tissues (Spin-Column Protocol). Extracts were amplified with the above mentioned conditions using the primers LCO1490 and HCO2198 (Folmer et al., 1994) amplifying the ca. 648 bp of the COI barcode region. Purification and Sanger bi-directional sequencing of the products was undertaken by the commercial facility offered by Macrogen (Seoul, South Korea).
Sequence analyses We first assessed the quality of the Illumina paired-end reads with FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/) before remaining adapter sequences were removed and low quality bases at the end of the reads were trimmed (q