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Molecular scatology: how toimprove prey DNA detectionsuccess in avian faeces?
 JOHANNES OEHM, ANITA JUEN, KARIN NAGILLER, SIGRID NEUHAUSER* and MICHAELTRAUGOTT
 Mountain Agriculture Research Unit, Institute of Ecology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria
Abstract
The analysis of prey DNA in faeces is a non-invasive approach to examine the diet of birds. However, it is poorly knownhow gut transition time, environmental factors and laboratory treatments such as storage conditions or DNA extraction pro-cedures affect the detection success of prey DNA. Here, we examined several of these factors using faeces from carrioncrows fed with insect larvae. Faeces produced between 30 min and 4 h post-feeding tested positive for insect DNA, repre-senting the gut transition time. Prey detection was not only possible in fresh but also in 5-day-old faeces. The type of sur-face the faeces were placed on for these 5 days, however, affected prey DNA detection success: samples placed on soilprovided the lowest rate of positives compared to faeces left on leaves, on branches and within plastic tubes. Exposing fae-ces to sunlight and rain significantly lowered prey DNA detection rates (17% and 68% positives in exposed and protectedsamples, respectively). Storing faeces in ethanol or in the freezer did not affect molecular prey detection. Extracting DNAdirectly from larger pieces of faecal pellets resulted in significantly higher prey detection rates than when using smallamounts of homogenized faeces. A cetyltrimethyl ammonium bromide-based DNA extraction protocol yielded signifi-cantly higher DNA detection rates (60%) than three commercial kits, however, for small amounts of homogenized faecesonly. Our results suggest that collecting faeces from smooth, clean and non-absorbing surfaces, protected from sunlightand rain, improves DNA detection success in avian faeces.
Keywords
:
 Corvus corone corone,
 diet analysis, feces,
 Melolontha melolontha,
 prey detection,
 Tenebrio molitor,
 transition time,trophic interactions
Received 12 November 2010; revision received 20 January 2011; accepted 26 January 2011
Introduction
Examining the dietary choices of birds is needed tounderstand their biology and the role of birds in foodwebs as well as to protect and manage them. To investi-gate the diet of birds under natural conditions, the stom-ach content has been analysed after killing and dissectingthem (Miller & McEwen 1995; Scribner & Bowman 1998).Gut content analysis where the consumer has to be killed,however, is limited because of ethical reasons. Hence,non-lethal approaches have been used as well, includingthe use of neck-collars (Exnerova
 et al.
 2003), flushing thecrop or stomach (Hull 1999; Moorman
 et al.
 2007), forcedregurgitation (Valera
 et al.
 1997) or collecting preyremains from boluses and faeces to retrieve dietaryremnants for morphological identification (Xavier
 et al.
2005; Gamez-Virues
 et al.
 2007). Although the latterapproach is non-invasive, identification of the preyremains is often impossible as soft-bodied prey, such asinsect larvae, rarely provides morphologically discern-able remains. Moreover, although stomach flushing may be practicable for large and ratite birds (e.g. penguins),catching and treating passerine birds this way is muchmore difficult and might be harmful to them. Molecularapproaches of prey detection (King
 et al.
 2008) are aversatile tool to examine birds’ feeding ecology, forexample, Jarman
 et al.
 (2002) identified krill species from both stomach contents and faeces of Adelie penguins(
Pygoscelis adeliae
), while Deagle
 et al.
 (2007, 2010) investi-gated the diet of two other penguin species (
Eudypteschrysolophus
 and
 Eudyptula minor
) by either cloning andsequencing of PCR products or pyrosequencing 16SmtDNA of prey in faeces. Similarly, Sutherland (2000)identified invertebrate prey DNA from faeces of five
Correspondence: Dr Michael Traugott, Fax: ++43 512 507 2817;E-mail: Michael.Traugott@uibk.ac.at*Present address: Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria.
 2011 Blackwell Publishing LtdMolecularEcologyResources(2011)
11
,620628 doi:10.1111/j.1755-0998.2011.03001.x
 
passerine bird species (
Sturnus vulgaris, Emberiza citro-nella, Parus major, Parus caeruleus
 and
 Hirundo rustica
)using restriction digest, cloning and sequencing. Hence,DNA-based analysis of faecal samples provides a clearadvantage to study the diets of birds: (i) it is non-lethaland non-invasive and (ii) it allows identifying the dietaryremains on a species-specific level.However, currently, little information is availableexamining DNA-based faecal analysis on a more experi-mental basis. Here, we present experiments, conductedwith carrion crows and insect prey. First, we examinedthe minimum and maximum time after prey ingestionduring which insect prey DNA can be amplified fromfaecal material. Then, we investigated how environmen-tal conditions, such as sunlight and rain or different sur-faces the faeces were placed on, affected prey DNAdetection rates. Finally, sample storage and DNA extrac-tion protocols were compared with regard to prey DNAdetection success.
Materials and methods
Gut transition time of insect prey
To determine how early and how long after feeding theDNA of the prey is detectable in faecal pellets of birds,two carrion crows (
Corvus corone corone
) were offered lar-vae of the cockchafer,
 Melolontha melolontha
 (Coleoptera:Scarabaeidae). In addition, standard food (approximately20–60% of the diet) was provided that consisted of cattleheart, mealworms (
Tenebrio
 spp.), as well as a mixture of insects, meat and curd. The experiments were carried out between 20th June and 1st July 2004 in an aviary at theAlpenzoo Innsbruck. Each feeding experiment lasted between 4 and 6.5 h and started with a feeding period of 0.5–2.25 h where each bird was fed with 7–17 second-instar cockchafer larvae. The birds’ droppings were col-lected continuously throughout the experimental time;all droppings produced from a bird within 1 h werepooled and stored in a 20-mL collection tube. Within twoof four feeding experiments, additional faecal pelletswere obtained 24 h after the birds had been feeding onthe cockchafer larvae: all faecal pellets produced within30 min by the individual birds were collected. The faecalsamples were cooled immediately upon collection andsubsequently stored at
 )
28
 
C. Additionally, samplesfrom the standard food sources and saliva samples fromeach bird were collected and the DNA extracted. ForDNA extraction, three subsamples of approximately0.1 g were taken from each faecal sample. The subsam-ples were extracted following a cetyltrimethyl ammo-nium bromide (CTAB) protocol and purified using asilica-based purification kit as described in Juen &Traugott (2005). Eight faecal samples were extracted asecond time, as the first extracts tested negative in a PCRwith general metazoan primers.
The influence of the surface underlying the faeces on preyDNA detection
For this and all following experiments, four carrioncrows were fed with larvae of 
 Tenebrio molitor
 (Coleop-tera: Tenebrionidae). Feeding experiments were con-ducted between 8th and 15th August 2007 in aviaries of the Institute of Ecology at the University of Innsbruck.Each bird was starved overnight and fed with 30 meal-worms in the morning. One and 2 h after this initial feed-ing event, each bird received another 30 mealworms (i.e.90 mealworms per bird in 3 h). One hour after each feed-ing event, all droppings produced within 1 h were col-lected with plastic spoons and transferred individuallyinto collection tubes (one dropping per tube). After thethree mealworm feeding events, grains (
 Avena sativa
,
 Zeamays
), fruits (
 Malus
 sp.,
 Vitis vinifera
), cheese (from
 Bostaurus
) and cat food (including
 Bos taurus
 and
 Gallus gal-lus
) were offered to the crows as standard food. To facili-tate faecal collection and to ensure that only droppingsproduced within the feeding experiments were taken, the basement of each aviary was covered with a thin plasticfoil every morning before the feeding experimentsstarted.One hundred freshly collected droppings were trans-ferred onto four different surfaces: open plastic tubes,maple leaves, willow branches and potting soil. Ten pro-tected (under cover of roof) and 15 unprotected drop-pings per surface were exposed to direct sunlight andrain. The droppings were left on the surfaces for 5 days(17 August 2007–21 August 2007), and a data loggerrecorded air temperature and humidity. Thereafter, allremaining pellets were scratched from the surfaces withplastic spoons. DNA was extracted following the CTABprotocol (Juen & Traugott 2005).
The influence of storage conditions on prey DNAdetection
Upon collection, 20 faecal samples were stored in ethanol(70%). After 14 months, these droppings were homoge-nized using a Vortex (Scientific Industries), and aliquotsof 900
 l
L were incubated overnight at
 )
28
 
C. To testwhether the addition of salt improves DNA precipitation,additional aliquots of 900
 l
L of each sample were incu- bated overnight after adding 600
 l
L NH
4
Ac (5
 M
). Afterspinning the samples at 4
 
C and 21250
 g
 for 30 min, thesupernatant was removed and the remaining pellet wassubjected to the CTAB protocol to extract the DNA (Juen& Traugott 2005). The success of prey DNA detectionwas compared between the samples kept in ethanol
 2011 Blackwell Publishing Ltd
IMPROVING PREY DNA DETECTION IN AVIAN FAECES
 621
 
and 48 frozen ones; the latter were homogenizedand extracted with the CTAB protocol (Juen & Traugott2005).
The influence of DNA extraction methods on prey DNAdetection
DNA from 96 faecal samples was extracted using fourdifferent protocols to evaluate extraction-specific differ-ences in prey DNA detection success: a CTAB-basedmethod (Juen & Traugott 2005) and three silica-basedkits, UltraClean
Fecal DNA Kit (MoBio Laboratories,Carlsbad CA, USA), ExtractMaster
Fecal DNA Extrac-tion Kit (Epicentre Biotechnologies, Madison WI, USA)and QIAamp DNA Stool Mini Kit (Qiagen, Hilden,Germany). The mass of each dropping was determinedto the nearest 0.01 mg in 48 samples. Thereafter, drop-pings were dissolved in PBS buffer in a ratio of 1:1.8(w:v) and homogenized, and 20-
l
L aliquots weretaken. Within each DNA extraction method, 48 aliquotswere extracted (‘small amount of homogenized sam-ples’). The remaining 48 faecal samples were dividedinto four equal parts, and each part was extracted byone of the four DNA extraction protocols (‘largerpieces of faeces’).
Diagnostic PCR
Detection of cockchafer DNA was achieved using the
 Melolontha
-specific primers M-387f and M-387r, targetingthe mitochondrial cytochrome subunit one gene andamplifying a fragment of 387 bp (Juen & Traugott 2005).Each PCR was carried out in a 20-
l
L assay, containing6
 l
L of extracted DNA, 0.2 m
M
 dNTPs (GeneCraft), 1
 l
M
of each primer, 3 m
M
 MgCl
2
, 1
·
 PCR buffer and 0.95 UHotStarTaq DNA Polymerase (Qiagen). PCR cycling con-ditions consisted of 15 min at 95
 
C, followed by 40cycles of 30 s at 94
 
C, 120 s at 64
 
C, 100 s at 72
 
C andfinally 10 min at 72
 
C. To check for false-negativeresults, all samples that did not produce a prey ampliconwere tested in a separate PCR using the general meta-zoan primers CO1-S38 (5
¢
-ttt tca act aac cat aaa gat att ggaac-3
¢
) and CO1-A36 (5
¢
-taa act tct ggg tga cca aaa aatca-3
¢
). The PCR mix and the cycling conditions werethe same as described earlier, except for the annealingtemperature that was lowered to 52
 
C.Detection of mealworm DNA was achieved using
 Ten-ebrio
-specific primers. Part of the cytochrome subunit onegene was sequenced from three mealworms (GenBankaccession numbers HQ891143 HQ891145) using theprimers of Folmer
 et al.
 (1994). These sequences wereused to design the primer pair Ten-mol-S210 (5
¢
-tac cgttat tcg tat gag cag tag-3
¢
) and Ten-mol-A212 (5
¢
-cgc tgggtc aaa gaa gga t-3
¢
) which amplified a 128-bp product.PCRs were carried out in 10-
l
L assays, containing 1.5
 l
Lof extracted DNA, 0.2 m
M
 dNTPs (GeneCraft), 1
 l
M
 of each
 Tenebrio
 primer, 3 m
M
 MgCl
2
, 1
·
 PCR buffer, 1
 l
g bovine serum albumin (BSA; Applichem) and 0.75 U TaqDNA polymerase (GeneCraft). PCR cycling conditionsconsisted of 2 min at 94
 
C, followed by 40 cycles of 20 sat 94
 
C, 30 s at 66
 
C, 45 s at 72
 
C and 3 min at 72
 
C.All samples that tested negatively for prey DNA weretested with the universal metazoan primers of Folmer
et al.
 (1994) and the minibarcode-primer (Meusnier
 et al.
2008) to check for false-negative results. PCR mix andcycling conditions were the same as described for detec-tion of mealworm DNA, except for the 48
 
C annealingtemperature. DNA extracts from faecal samples left ondifferent surfaces where no DNA could be amplified, nei-ther by prey-specific nor by universal primers, werespiked with DNA from
 Agriotes
 spp. (Coleoptera: Elateri-dae) to test for potential inhibition of the PCR. The 10-
l
Lreaction mix contained 1.5
 l
L of extracted faecal DNA,1
 l
L of 
 Agriotes
 DNA, 0.2 m
M
 dNTPs (GeneCraft), 1
 l
M
of each of the universal primers (Folmer
 et al.
 1994),3 m
M
 MgCl
2
, 1
·
 PCR buffer, 1
 l
g BSA (Applichem) and0.75 U Taq DNA polymerase (GeneCraft). Cycling condi-tions were the same as described earlier for PCR withuniversal primers.Within all PCRs, positive (diluted DNA of the targetprey species) and several negative (molecular gradewater instead of DNA) controls were included to checkfor amplification success and DNA contamination,respectively. The PCR products were separated electro-phoretically on ethidium bromide-stained agarose gelsand visualized using a UV-transilluminator. The specific-ity of the
 Melolontha
 and
 Tenebrio
 assays was testedagainst DNA samples from the birds and from standardfood sources.
Data analysis
We determined the minimum and the maximum timeinterval between consumption and defecation duringwhich detection of the ingested
 Melolontha
 in the drop-pings was possible. Below, these two time intervals arereferred to as the minimum and maximum gut transitiontime. As the duration of feeding differed within the firstset of experiments, we calculated the time intervals afterconsumption of the first and the last
 Melolontha
 larva foreach pooled faecal sample set. For these calculations, weused the medium time of collection for the pooled drop-pings. The resulting two values for each pooled faecalsample set were plotted against each other and used toestimate minimum and maximum transition time of detectable
 Melolontha
 DNA.Mean air temperature was compared betweenprotected and unprotected sample locations using
 2011 Blackwell Publishing Ltd
622
 J. OEHM
 ET AL.

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