A minimally invasive method of field sampling for genetic analyses of the Florida manatee (Trichechus manatus latirostris)

A minimally invasive method of field sampling for genetic analyses of the Florida manatee (Trichechus manatus latirostris)
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  See discussions, stats, and author profiles for this publication at: Minimally invasive method of fieldsampling for genetic analyses of theFlorida manatee (Trichechusmanatus latirostris)  Article   in  Marine Mammal Science · September 2007 DOI: 10.1111/j.1748-7692.2007.00155.x CITATIONS 3 READS 65 7 authors , including: Some of the authors of this publication are also working on these related projects: Using genetic tracking and acoustic telemetry to estimate spawningpopulation size   View projectSusan Lynne CarneyHood College 58   PUBLICATIONS   383   CITATIONS   SEE PROFILE Michael D. TringaliFlorida Fish and Wildlife Conserv… 98   PUBLICATIONS   656   CITATIONS   SEE PROFILE All content following this page was uploaded by Susan Lynne Carney on 08 August 2014. The user has requested enhancement of the downloaded file.  MARINE MAMMAL SCIENCE, 23(4): 967–975 (October 2007) C   2007 by the Society for Marine MammalogyDOI: 10.1111/j.1748-7692.2007.00155.x A MINIMALLY INVASIVE METHOD OF FIELD SAMPLING FORGENETIC ANALYSES OF THE FLORIDA MANATEE( TRICHECHUS MANATUS LATIROSTRIS ) S USAN  L. C ARNEY  E LLEN  E. B OLEN 1 S HERI  L. B ARTON K ERRI  M. S COLARDI C AROLYN  C. E NGLUND Mote Marine Laboratory,1600 Ken Thompson Parkway,Sarasota, Florida 34236, U.S.A.E-mail: M ICHAEL  D. T RINGALI Florida Fish and Wildlife Conservation Commission,Fish and Wildlife Research Institute,100 Eighth Avenue SE,St. Petersburg, Florida 33701, U.S.A.  J OHN  E. R EYNOLDS  III Mote Marine Laboratory,1600 Ken Thompson Parkway,Sarasota, Florida 34236, U.S.A. Samplingfromlivinganimalshasanumberofadvantagesoverusingsamplesfromonlydeceasedanimalswhengeneticstudiesareconducted.Oncegeneticmarkerswiththe appropriate level of variability are developed, they can be used to generate DNAgenotypes to distinguish individuals, and comparisons of genotypes can permit theelucidation of familial relationships (siblings, parents, and offspring). Better under-standingofsocialrelationshipscanbegarneredfromthisknowledgewhencombinedwith information from photo-identification records. In addition, multilocus geno-types of individuals can serve as a type of “tag” for use in mark-recapture, movement,and population size estimation studies with samples from live animals (Wilson andRannala 2003, Bellemain  et al  . 2005, Peakall  et al  . 2006).Most of the genetic samples that exist for the Florida manatee,  Trichechus manatuslatirostris ,havebeenobtainedfromtissuesandboneofdeceasedanimalsacquiredfromthe Florida Fish and Wildlife Conservation Commission’s (FWC) Marine MammalPathobiology Laboratory, or from captured animals using dermatome (“cookie”)biopsy punches or, occasionally, blood samples (McClenaghan and O’Shea 1988;Bradley  et al.  1993; Garcia-Rodriguez  et al.  1998, 2000; Cantanhede  et al.  2005; 1 Currentaddress:NicholasSchooloftheEnvironment,DukeUniversity,Box90328,Durham,NorthCarolina 27708, U.S.A. 967  968  MARINE MAMMAL SCIENCE, VOL. 23, NO. 4, 2007 Vianna  et al.  2005). Most of the current methods of sampling living animals areexpensive, require the coordinated effort of many researchers, or are invasive andquite disruptive to the animal, as they require hands-on contact either in or out of the water and removal of a substantial sample from the fluke (up to 20 g, althoughthe sample may be used for other research in addition to genetics 2 ). Studies fromother marine mammals have described how it is possible to obtain DNA from fe-cal material (Tikel  et al.  1996, Parsons 2001). Although this might be feasible insome situations for Florida manatees, it is often impossible to discern from whichanimal a fecal sample came. Biopsy darting, commonly done with cetaceans (Lam-bertsen 1987; Whitehead 1990; Brown  et al.  1991; Weinrich  et al.  1991, 1992;Barrett-Lennard  et al.  1996; Weller  et al.  1997; Hooker  et al.  2001), is a less de-sirable method for manatees owing to their tough skin and their proximity tothe public, who might view the darting process as unacceptable. To date, a min-imally invasive method of directly sampling manatees in the field has not beendescribed.We present here a method of skin sampling that can be done by a researcher froma boat, dock, or seawall while manatees are in their natural habitat. We demonstratethat our method yields high-molecular-weight genomic DNA that is suitable for usein mitochondrial and nuclear genotyping studies. Our sampling is less disruptive tothe animals than other current methods of obtaining tissue, and its simple, low-costdesign should render it useful in settings where researchers have the appropriateaccessibility to the animals.Our sampling device (Fig. 1) consists of a 7.5-cm by 6-cm piece of aluminumthat has been perforated using a nail and bent around a 6-cm-long piece of 1.9-cm-diameter nylon rod (Modern Plastics, Bridgeport, CT). We have experimented witha wooden rod and do not recommend using wood because components of the woodand wood treatment leach into the digestion buffer; no problems have been notedwith nylon rods. A hole drilled into the center of the rod allows it to be attached,using a screw, to the end of an approximately 2-m-long pole made out of 3.8-cm-diameterPVCpipe.Sampleacquisitionoccursbyfirmlyrubbingthesampleragainsta manatee’s skin, ideally at an area of its back or side with little to no barnacle oralgal coverage, while it is within reach of a boat or a point on land. The nylon rodbehind the perforated aluminum aids with the retention of skin that is scraped off.Thealuminum/nylonsamplerisremovedfromthepoleaftereachsampleistakenandplaced into a 50-mL tube of 75% ethanol buffered with 1 × TE (pH 8) for transportback to the laboratory inside a cooler. In our case, DNA extractions were generallyperformed within days of field sampling; however, successful extractions have beendone on tissues stored at  − 20 ◦ C for up to 6 mo. For samples that will be storedlong-term before extractions are done, it would be wise to remove tissue from thesampler as soon as possible after sampling for storage in an alternate medium, suchas a DMSO–salt solution, in order to increase the high-quality DNA yield from thestored tissue (Kilpatrick 2002). 2 Personal communication from Robert K. Bonde, Biologist, Sirenia Project, U.S. Geological Survey,FloridaIntegratedScienceCenter,2201NW40thTerrace,Gainesville,Florida32605-3574,April2007.  NOTES  969 Figure 1.  Photograph of manatee tissue sampler. A. The perforated aluminum is wrappedaround a cylindrical nylon rod, which is attached to the end of an approximately 2-m-long“pole” made out of PVC pipe. This assembly can be easily removed between sampling events.B. The aluminum and rod have holes drilled through them (arrow) and are firmly held ontothe PVC cap/rod with a screw. C. A close-up view of the aluminum/nylon sampler. WetestedaprototypeofoursampleroncapturedanimalsinDecember2005usingsamplers with a range of perforations, from dull to very sharp. Perforations werecreated by hammering different varieties of nails and screws to different depths intothe aluminum to create a range of hole depths and diameters. We confirmed that thesamplerobtainedsufficientandsuitabletissueforDNAextraction,andwealsonotedthattherewasnodifferenceintheamountorqualityofDNAobtainedfromscrapingsthatelicitedminorbleedingversusthosethatdidnot.Forthisreason,weoptedtousemoderatelysharpperforations( ∼ 1mmdeep,2mmwide,spacedabout10mmapart,made by hammering a 1 #12 oval-head screw) for future samplers in order to collectsufficientskinwithoutcausinganybleeding.WenexttriedthecurrentsamplerdesignonMoteMarineLaboratory’scaptivemanatees(followingapprovalbyanInstitutionalAnimal Care and Use Committee) to ensure that sampling would be possible in thewater and that it would not elicit a strong behavioral response (or bleeding). Afterthiswassuccessful( i.e. ,neitheranimalshowedamarkedreactionorinjury),webeganto sample animals in the field. Thus far we have acquired skin tissue from seventeenwild manatees (confirmed by photo-identification) and one captive manatee. Fieldsampling took place in and around southwestern Florida from Terra Ceia Bay toEstero Bay between March and November 2006 and included individual animals,individualswithcalves,andinonecase,individualsinamatingherd.Forthefourteenanimals for which gender was known or was determined using genetics, an equalnumber of males and females were sampled. Samples were taken only from animalsthat were first clearly photographed and had identifiable scars in order to beginthe integration of genetic information into the existing Manatee Individual Photo-Identification System, a computerized database containing the sighting records, life  970  MARINE MAMMAL SCIENCE, VOL. 23, NO. 4, 2007 histories, and photographic images of distinctive individual manatees that have beendocumented throughout the state of Florida (Beck and Reid 1995). We recordedobservations of the sampled animals to note any behavioral reactions to sampling,generally for a period of at least 15 min before sampling and at least 15 min aftersampling (often longer for both cases). Responses were recorded as positive if theanimal pursued further contact with the sampling device or researcher. Negativeresponsesweregroupedintothreelevels:mild,wheretheanimalflinchedslightlybutdid not move away; moderate, where the animal slowly but deliberately moved away(horizontallyorvertically);andstrong,wheretheanimalexhibitedrapid,exaggeratedmovementawayfromthesampler,oftenwithaquickdivedown,forcefulswimming,and/or a tail thrash. In all of the animals from which we obtained tissue and DNA,thereweresevenanimalsthatshowednoresponse,twoanimalsthatshowedapositiveresponse, and nine animals that showed a negative response (1 mild, 5 moderate, and3 strong).Individual animals may vary in their behavioral responses to any stimulus owingto differences in gender, age, season, presence in a group or alone, or to additional,or a combination of, factors that may not be possible to determine. For the samplesreportedhere,withtheexceptionofoneanimal,themanateesgenerallydemonstratedminimalbehavioralresponsetosamplecollection.Samplingofoneadultfemale,whowastravelinginagroupofthreeanimals,resultedinamarkedtailsplash.Thisanimaland the cohort increased their speed in the same direction they had been previouslyheaded;werankedthisasastrongresponse.Subsequentsamplingoffouranimalsfromwinter field sites (warm-water refuges where large number of individuals are withinclose proximity to each other, primarily resting) resulted in the sampled animalssplashing their tails, submerging, and/or swimming away, and nearby animals alsotemporarilyswamawayorsubmerged.Mostoftheanimalsthatreactedresumedtheirpresampling behavior within 5 min and none left the refuge. We also ranked this asa strong response. Although we have arbitrarily called certain behavioral responses“strong” reactions because they are noticeable, they clearly would be considered asthe least problematic type of harassment, namely Level B ( i.e. , having the potentialto disturb, but not to injure; Marine Mammal Commission 2001).The intensity of the animals’ reactions that are reported here seemed to dependupon what the animal’s behavior was prior to sampling. For example, manatees thathad been involved in mating were less reactive to sampling than manatees that hadbeen resting. In terms of specific responses, one animal appeared startled when itwas sampled and splashed its tail. In 14 of the 18 cases from which we successfullyobtainedtissueandDNA,therewaseithernoresponseoramildtomoderatenegativereaction. For the mating herd, the most marked reaction from any of the animals wasabriefmovementbyoneanimalawayfrom,butthenareturnto,theherd.Nolastingbehavioral effects were noted for as long as we remained at sampling sites. Generallyspeaking, a sampled animal initially submerged or moved away from the samplerbut then returned almost immediately to its srcinal location and activity. It is notpossible at this point to discern whether the animals’ reactions were a response tobeingapproachedandtouchedortobeingsampled,orwhethertheyrespondedowingto some unrelated stimulus.
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