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RESEARCH ARTICLE Measuring Body Size in Small Marine Fishes: A Comparison of Three Non-intrusive Methods Fisheries and

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RESEARCH ARTICLE Measuring Body Size in Small Marine Fishes: A Comparison of Three Non-intrusive Methods Fisheries and
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  RESEARCH ARTICLE Measuring Body Size in Small Marine Fishes: AComparison of Three Non-intrusive Methods Fisheries and  Aquaculture Journal,Vol. 2013: FAJ-71  Fisheries and Aquaculture Journal, Vol. 2013: FAJ-71http://astonjournals.com/faj1    A  s   t  o  n   J  o  u  r  n  a   l  s Measuring Body Size in Small Marine Fishes: A Comparisono Three Non-intrusive Methods Stanton G Belord 1,2 *, Nanette E Chadwick 3 , Maroo A Khala 4 1 Department o Curriculum and Teaching, 5040 Haley Center,Auburn University, Auburn, AL 36849-5212, USA. 2 Ofce o Diversity and Multicultural Aairs, 103 M. White Smith Hall,381 Mell Street, Auburn University, Auburn, AL 36849-5168, USA. 3 Department o Biological Sciences, 101 Rouse Lie Science Building,Auburn University, Auburn, AL 36849-5407, USA. 4 Department o Marine Biology, Faculty o Marine Sciences,The University o Jordan–Aqaba, P.O. Box 2595, Aqaba 77110, Jordan. *Correspondence: sgb0008@auburn.edu Accepted: Apr 2, 2013; Published: Apr 16, 2013 Abstract Studies o non-intrusive techniques are important in sheries biology, because research methods may inadvertently causedamage to the study organisms. In addition, current eects o human–environment interactions coupled with uture trends inglobal climate change likely will lead to increased monitoring o sh population dynamics. The aim o this study is to analyzethe eectiveness o three simple non-intrusive techniques to accurately obtain body length measurements o anemoneshand other small shes. Frequently used catch and re-capture methods are stressul to shes, and can alter their behaviorsupon release, thus negatively impacting eld ecological studies. Alternate methods to non-intrusive sizing o ree shesare needed, and these methods should be compared to determine the most eective and ecient means o collecting thetargeted data. Three non-intrusive techniques were employed to obtain accurate ork length (FL) measurements o the two-band anemonesh,  Amphiprion bicinctus. Comparison o these methods revealed that sh lengths rom visual estimatesby sel-contained under water breathing apparatus ( SCUBA) divers did not dier signicantly rom those estimatedusing both video-mirror and Tps-mirror techniques (ANOVA, F  (2,60) 5 1.572;  p   5 0.22). Under laboratory conditions, shsizes rom manual measurements also did not dier signicantly rom those obtained using either mirror method (ANOVA, F  (2,81) 5 0.489;  p   5 0.61), demonstrating that the mirror techniques accurately assess sh size under both laboratory andeld conditions. These methods were not eective in identiying or tracking individual sh among years in the eld, due tohigh rates o sh mobility and turnover. However, they were useul in determining short-term anemonesh migration amongsea anemone hosts. Keywords: Biodiversity; obligate symbiosis; population dynamics; ish body size; anemoneish; giant sea anemone. 1. Introduction Body length measurements are important or determining the growth rates and population size structure oshes. In sh populations that experience stable recruitment and mortality [1], body size requencies can also beapplied to the Beverton–Holt model to calculate productivity and population yield or the sustainable manage-ment o sheries [2]. This model was used to characterize not only the population dynamics o shes, but also omany other marine organisms, including some stony corals [3]. Data rom the Beverton–Holt shery model alsocan be tted to von Bertalany growth curves [4] to estimate age–size relationships in shes and other organisms.Common techniques used to acquire sh body size measurements, such as catch and release, hookand line, electro-shing and anesthetics can cause physical damage and physiological stress to the sh [5–7].Although these intrusive methods are oten used to collect sh length data, they may alter subsequent sh  Research Articlehttp://astonjournals.com/faj2    A  s   t  o  n   J  o  u  r  n  a   l  s behavior during long-term eld studies. Reduction o sh stress thereore requires sizing methods that relyon observation rom a distance, but the non-intrusive methods employed to date had limited success. Brockinitially used visual census to assess sh body sizes on coral rees [8], but it was suggested that it was dicultto obtain accurate sh lengths by visual estimation underwater [9]. Furthermore, problems were reported withobservations at a distance using an underwater auto-ocus video camera mounted on a Remotely OperatedVehicle [10]. Consequently, laser-tagging was used to collect sh measurements, which proved to be a moreaccurate but much more expensive method.The use o video cameras in conjunction with mirrors may allow accurate determination o live shlengths, because many shes are attracted to their mirror images, and even display parallel swimming withtheir images, causing them to line up closely with length markings on the mirror surace [8]. This video-mirrormethod also provides a visual record o sh appearance, thus potentially allowing long-term identication oindividuals. This method has been applied ar only to assess measurement eciency, in terms o the numbero video clips required to obtain length measurements or each sh during a single sel-contained under waterbreathing apparatus (SCUBA) dive [8].Little is known about the long-term growth rates o anemoneshes in the eld, in part because thesesh are negatively impacted by standard catch and re-capture methods [11–13], hence there is a need todevelop a non-intrusive method to identiy them and measure their body sizes. The accuracy o the video-mirrortechnique can be tested easily in laboratory aquaria, where the sh are accustomed to handling and thus lessnegatively impacting by manual measurements o body size.In the Red Sea, endemic two-band anemonesh  Amphiprion bicinctus are obligate mutualists withthree species o giant sea anemone hosts: Entacmaea quadricolor  , Heteractis crispa and Heteractis magnifca  [12–14]. These sot-bodied sea anemones provide a unique habitat or anemoneshes, which are protectedrom piscivorous shes by the anemones’ nematocysts. Furthermore, host anemones benet rom the pres-ence o anemoneshes as they are aggressive against specialized anemone predators such as chaetodontids,and attack them more than they do non-predatory shes in close vicinity [15]. Recent research has revealedphysiological benets rom anemonesh to host anemones in the orm o transerred nutrients [16–18] andenhanced gas exchange [19].The abundance o  A. bicinctus is highest in Jordan at the northern tip o the Gul o Aqaba, Red Sea,in comparison with the central and southern coasts o the Red Sea [20]. The average abundance o  A. bicinctus per 100 m ree transect is 25.22 in Jordan, ollowed by 2.77 in Egypt, 3.91 in Saudi Arabia, 0.11 in Yemenand only 1.06 in southern Djibouti rees on the nearby Gul o Aden [20]. However, these high requencies oanemonesh on Jordanian rees are threatened by recent coastal development.Over the last 30 years, industrial growth in the Red Sea cities o Eilat and Aqaba has led to increase incommercial port, aquaculture and tourism activities, resulting in rising domestic and industrial efuents suchas oil, ertilizers and pesticides on coral rees along the coasts o Israel [21, 22] and Jordan [23]. These anthro-pogenic stressors likely impact patterns o sea anemone and anemonesh recruitment, growth and mortalitydue to alteration o the environmental conditions on nearshore coral rees and eld methods are needed toaccurately size the anemonesh and determine these demographic changes. The purpose o the present studywas to assess the three methods o measuring small marine shes including anemoneshes, using inexpensivetechniques in the laboratory and the eld. Specically, the useulness o the video-mirror method was examinedas a tool to accurately assess sh body size and identiy individuals. 2. Methods Preliminary trials o video-mirror laboratory experiments were conducted in aquaria [17] at Auburn Universityin January 2009. Anemonesh that were srcinally transported to Auburn in 2006 rom a culture acility atoceans, rees and aquariums (ORA, Florida, USA) were observed in laboratory aquaria to which mirrors hadbeen added. These preliminary trials aided in selection o the mirror size to use or later laboratory and eldsh measurements, also determined the period o time needed or anemonesh to adjust their aggressive  3Fisheries and Aquaculture Journal, Vol. 2013: FAJ-71http://astonjournals.com/faj    A  s   t  o  n   J  o  u  r  n  a   l  s behavior and to begin parallel swimming adjacent to the mirror. In September 2010, a total o 28 anemonesh(16 adults, 12 juveniles) were measured in the laboratory using (a) hand-held calipers (i.e., manually), (b) thevideo-mirror technique and (c) the Tps-mirror technique. These methods are discussed in detail in the laboratorysection (below).In June 2009, 21 anemonesh on the coral ree adjacent to the Marine Science Station at Aqaba,Jordan (N 29 31’, E 35 0’) were selected. Divers using SCUBA recorded these anemonesh ork lengths (FL)using visual estimates, and the video-mirror and Tps-mirror techniques. These measurements were used tocompare these three non-intrusive techniques in the eld. 2.1. Laboratory measurements Fish body size measurements were made under laboratory conditions on 16 adult (FL  60.1 mm, [24]) two-bandanemonesh  A. bicinctus (FL 5 113.7  12.0 mm, mean  sd) and 12 juveniles (FL  60 mm, 59.0  13.7 mm,or details o culture conditions see [17]). To obtain video-mirror measurements o sh body size, a 20  20 cmglass mirror bordered by alternating 1 cm orange marks (or scale bars) was placed inside the home aquarium oeach sh. Based on preliminary observations, each individual was allowed to acclimate 1 min to the presence othe mirror, and then videotaped or 30–60 s using a digital camera (Samsung Digimax A503). Images rom eachvideo sequence later were viewed on a computer screen, and analyzed to obtain sh lengths [8].In the video sequences, individuals o  A. bicinctus were observed to display parallel swimming backand orth adjacent to the mirror surace. The video playback speed was slowed during these sequences, andimages viewed until one was obtained o the sh positioned parallel and close to the mirror surace. The videowas paused at this image, and the video rame number recorded. Hand-held calipers were used to obtain anon-screen FL measurement, ollowed by a FL measurement using the scale bar markings on the mirror. A cor-rection coecient was calculated rom the ratio o these measurements (scale bar markings = on-screen shlength, ater [8]). The actual video-mirror sh length was calculated by multiplying the correction coecient bythe on-screen sh length measurement.A morphometric computer program TpsDig 2 (http://lie.bio.sunysb.edu/morph/) was applied to assessthe accuracy o the video-mirror technique, and this modied technique was termed the Tps-mirror method[25]. This sotware was designed to digitize landmarks and outlines or morphometric analyses, and eachselected video rame was stored as an extension le or a top speed (Tps) Database, which is a type o lethat saves data entries, one entry at a time. This sotware was used to analyze the above video rames, as anadditional sh body size analysis to compare with the video-mirror method. Each recorded video was openedin the TpsDig 2 sotware, as the one described above or the video-mirror method was captured, saved andre-opened in the Tps-utility program, where a digital scale allowed or more accurate calculation o sh lengthmeasurements.Ater each sh was videotaped under laboratory conditions, it was removed rom its home aquariumusing a ne mesh net, transerred to a paper towel, briefy blotted to remove excess water, and its FL measuredmanually using calipers (tip o snout to posterior end o middle caudal rays, www.shbase.org). Each sh wasout o water or  30 sec during this manual measurement o body size, and all sh appeared to swim normallywithin a ew minutes ater return to their home aquaria. These manual FL measurements provided the exactbody length o each sh, and were compared to the other two methods above. 2.2. Field measurements During June 2009, the body sizes o two-band anemonesh  A. bicinctus on a coral ree adjacent to the MarineScience Station at Aqaba, Jordan (N 29 31’, E 35 0’) was measured. SCUBA divers visually estimated the FL oeach anemonesh at the study site (N 5 112), using scale bars marked in cm on their underwater data slates.Divers careully extended their slates as close to each sh as possible, then visually estimated FL, rounding to thenearest 0.5–1.0 cm. During these visual estimations, each dive slate with a scale bar was held  10 cm rom eachmeasured sh, because even though the sh did not desert their host sea anemones during measurements,they actively avoided the dive slates.  Research Articlehttp://astonjournals.com/faj4    A  s   t  o  n   J  o  u  r  n  a   l  s O the 112 sh measured by visual estimation, hal (56) were selected randomly or video-mirror assess-ment, due to limited time underwater or videotaping. Preliminary observations underwater urther reducedthis number to 21 sh that were logistically the easiest to record on videotape, due to the orientations o theirsea anemones on the coral ree, lack o obstructing nearby ree structures, and sh behavior in relation to themirror surace. A marked mirror (Figure 1A and 1B) was placed adjacent to the sea anemone host o eachselected sh, then the diver (in all cases S.G. Belord) moved to a distance o 0.75–1.0 m rom the sea anemone.Fish were allowed to acclimate to the mirror or 30 s, then videoed or 60 s using a Sea & Sea DX-860G digitalcamera and underwater housing. In most cases, images o each sh swimming parallel and close to the mirrorwere observed during this initial 60 s video period; i not, an additional 60 s was recorded. Fish ork lengths romvideo sequences obtained under eld conditions then were analyzed and compared to those obtained usingthe other methods described above (eld visual estimation and the three laboratory measurement methods). Figure 1: Video-mirror images or the analysis o body size (FL) in the anemonefsh  A. bicinctus, shown herewith the giant sea anemone E. quadricolor  on a coral ree at Aqaba, Jordan during June 2009. (A) Two fshoriented obliquely to the mirror during the 30 s acclimation period. (B) One fsh beginning to parallel-swimadjacent to the surace o the mirror, near the start o 60 s o video recording. Note that the 1 cm scale markssurrounding the edges o the mirror are clearly visible in the video images. 3. Results3.1. Laboratory measurements Anemonesh FL did not dier signicantly among the three laboratory measurement methods (manual,video-mirror and Tps-mirror (ANOVA, F  (2,81) 5 0.489;  p   5 0.61)). Manual measurements using calipers wereslightly but not signicantly smaller (113.7  12 mm or adults; 59.1  13.7 mm or juveniles) than thoseusing both the video-mirror (123.2  15.4 mm or adults; 64.4  13.4 mm or juveniles) and Tps-mirrormethods (116.2  12.2 mm or adults; 57.8  14.4 mm or juveniles). Manual lengths correlated tightly withthose obtained rom both video-mirror ( r    5 0.980) and Tps-mirror methods ( r    5 0.993, Figure 2A and 2B).O the two non-intrusive sh sizing methods, the Tps-mirror method was the most ecient as required muchless time than the video-mirror method, which required both on-screen and reerence measurements, and thencalculation o a correction coecient. Additionally, the Tps-mirror method did not require sh removal romaquaria, nor did it cause sh to increase their respiratory activity, which usually results rom stressul situations. 3.2. Field measurements Anemonesh FL did not dier signicantly among the three eld measurement methods tested (visual estima-tion, video-mirror and Tps-mirror (ANOVA, F  (2,60) 5 1.572;  p   5 0.22)). Fish body lengths estimated visuallyby SCUBA divers were shorter than those obtained by both video-mirror and Tps-mirror methods, which didnot dier signicantly rom each other in the sh lengths obtained. The sh lengths estimated visually under-water correlated with those obtained by both video-mirror ( r    5 0.865) and Tps-mirror methods ( r    5 0.827) inthe eld, but these correlations were much looser than those between sh measurements obtained manuallyversus with mirrors under laboratory conditions (Figure 2C and 2D).
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