Molecular Dietary Analysis of the Endangered Ozark Big-Eared Bat (<i>Corynorhinus townsendii ingens</i>)

Bussche, Ronald A. Van Den; Lee, Dana N.; Judkins, Megan E.; Dyer, Jenny E.; Thompson, Denise M.; Stark, Richard; Puckette, William L.; Fuller, Brian Charles

doi:10.3161/15081109acc2016.18.1.008

Cited by 8 publications

(4 citation statements)

References 43 publications

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“…High‐throughput amplicon sequencing (HTS) has become the preferred method for rapid molecular identification of members of mixed ecological communities. HTS is now also increasingly used to identify the arthropod dietary components of a wide taxonomic range of animals including mammals (Bussche et al, ; Clare et al, ; Clare, Symondson, & Fenton, ; Mallott, Malhi, & Garber, ; Rydell et al, ; Vesterinen et al, ), birds (Crisol‐Martínez, Moreno‐Moyano, Wormington, Brown, & Stanley, ; Jedlicka, Vo, & Almeida, ; Trevelline, Latta, Marshall, Nuttle, & Porter, ), reptiles (Kartzinel & Pringle, ), fish (Harms‐Tuohy, Schizas, & Appeldoorn, ) and arthropods (Krehenwinkel, Kennedy, Pekár, & Gillespie, ). Identification of the DNA of dietary components is accomplished by “metabarcoding,” which involves extracting DNA from faecal samples, amplifying one or more barcoding loci, preparing DNA libraries and finally sequencing, bioinformatics and data analysis.…”

Section: Introductionmentioning

confidence: 99%

“…The ZBJ primers were designed to target a short fragment to amplify the presumably degraded DNA present in guano, and coincidentally, the length of the amplicon generated is compatible with many HTS platforms. Subsequently, numerous researchers have employed the ZBJ primers in HTS studies that analyse diets of insectivorous animals, including bats (Bussche et al, ; Clare et al, ; Rydell et al, ; Vesterinen et al, ) and birds (Crisol‐Martínez et al, ; Jedlicka et al, ; Trevelline et al, ). Although the ZBJ primers have been widely utilized, there are indications that they have a narrow taxonomic range (Brandon‐Mong et al, ; Clarke et al, ; Mallott et al, ).…”

Section: Introductionmentioning

confidence: 99%

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An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

Jusino

Banik

Palmer

et al. 2019

Molecular Ecology Resources

142

117

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DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre-and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24-40 of 59 taxa (41%-68%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization. K E Y W O R D SAMPtk, arthropod mock community, bat guano, dietary analysis, insectivore, next-generation sequencing *Indicates shared first authorship based on equal contributions.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

Jusino

Banik

Palmer

et al. 2019

Molecular Ecology Resources

142

117

View full text Add to dashboard Cite

show abstract

“…High-throughput amplicon sequencing (HTS) has become the preferred method for rapid molecular identification of members of mixed ecological communities. HTS is now also increasingly used to identify the arthropod dietary components of a wide taxonomic range of animals including mammals (Bussche et al 2016;Clare et al 2014a;Clare et al 2014b;Mallott et al 2015;Rydell et al 2016;Vesterinen et al 2016), birds (Crisol-Martínez et al 2016;Jedlicka et al 2016;Trevelline et al 2016), reptiles (Kartzinel & Pringle 2015), fish (Harms-Tuohy et al 2016), and arthropods (Krehenwinkel et al 2016). Identification of the DNA of dietary components is accomplished by "metabarcoding", which involves extracting DNA from fecal samples, amplifying one or more barcoding loci, preparing DNA libraries, and finally sequencing, bioinformatics, and data analysis.…”

Section: Introductionmentioning

confidence: 99%

“…The ZBJ primers were designed to target a short fragment in order to amplify the presumably degraded DNA present in guano and coincidentally the length of the amplicon generated is compatible with many HTS platforms. Subsequently, numerous researchers have employed the ZBJ primers in HTS studies that analyze diets of insectivorous animals, including bats (Bussche et al 2016;Clare et al 2014a;Clare et al 2014b;Rydell et al 2016;Vesterinen et al 2016) and birds (Crisol-Martínez et al 2016;Jedlicka et al 2016;Trevelline et al 2016). Although the ZBJ primers have been widely utilized, there are indications that they have a narrow taxonomic range (Brandon-Mong et al 2015;Clarke et al 2014;Mallott et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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Supplemental Information 1: Appendix S1 – DNA Extraction Details for the CLS Extraction for Arthropods

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DNA analysis of predator feces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (CO1). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML". To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) CO1 sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre-and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%) whereas ZBJ amplified 24 of 59 taxa (41%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly four-fold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.

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In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

Tournayre

Leuchtmann

Filippi‐Codaccioni

et al. 2020

Ecology and Evolution

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During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.

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Molecular Dietary Analysis of the Endangered Ozark Big-Eared Bat (Corynorhinus townsendii ingens)

Cited by 8 publications

References 43 publications

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

Untitled

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

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