Iranian Journal of Sciences 15(4)1593-1608 2016

Otolith and scale morphologies of doctor (Garra rufa) inhabiting Kangal Balıklı Çermik thermal spring (Sivas, Turkey)

Yedier S.1; Kontaş S.1; Bostancı D. 1*; Polat N.2

Received: December 2015 Accepted: July 2016

Abstract Garra rufa is one of the most popular therapeutic and commercially important fish in ichthyotherapy. and scale morphology provide new and useful information for fish identification and classification. Left-right asteriscus and lapillus from juvenile and adult doctor fish, and the scales from 6 different regions of the juvenile and adult fish body have been examined in Kangal Balıklı Çermik thermal spring (Sivas, Turkey). The otolith and scale morphological characters such as type, size, shape, mesial surface, lateral surface, antirostrum and rostrum shapes, focus position, circuli appearance, radii type and posterior and anterior margin shapes were distinguishable features for the juvenile and adult G. rufa samples. Three different otolith shapes were observed such as squared and discoidal otolith shapes for asteriscus pairs and oval to elliptic otolith shapes for lapillus pairs in the juvenile and adult doctor fish. Seven different scale shapes were described from six different body regions of the G. rufa in the current study. The graphical illustration of wavelets was used for both asteriscus and lapillus pairs of the fish to discover otolith variabilities. This is one of the first otolith and scale morphology studies for the juvenile and adult doctor fish. All these otolith and scale characters and their morphologies could be used for best alternative tools to identification, classification, phylogenetic relationships among the different freshwater and marine fish species, genera, populations or stocks.

Keywords: Garra rufa, otolithology, Scale morphology, Shape indices

1-Department of Biology, Ordu University, Ordu, Turkey 2-Department of Biology, Ondokuz Mayis University, Samsun, Turkey *Corresponding author's Email: [email protected] 1594 Yedier et al., Otolith and scale morphologies of doctor fish (Garra rufa) inhabiting …

Introduction psoriasis and eczema (Fackler and Otolith and scales are crucial and Augustin, 2001; De Groot and versatile research materials used in Conemans, 2004; Grassberger and ichthyological studies due to their Hoch, 2006). Nowadays, G. rufa is unique characters such as shape, becoming a much more popular fısh contour, size and shape indices species for therapeutic purposes and the (Esmaeili et al., 2014; Bostancı et al., doctor fish were listed as a locally and 2015). The otolith and scale commercially vulnerable fish species morphologies have recently increased for treatment and aquarium industry in in importance with the development of Turkey. image analysis systems and software Previous studies on G. rufa were programs using a Scanning Electron limited and some of them are focused Microscope (SEM) (Razak, 2014; on length-weight, length-length Bostancı et al., 2015). Previous studies relationship (Esmaeili and Ebrahimi, have indicated and otoliths 2006; Gerami et al., 2013; Hamidan were used successfully for and Britton, 2013; Hashemzadeh et al., identification and discrimination of fish 2015), karyotypes (Gorshkova et al., species and stocks (Jawad and Al- 2012), microbiological risks for human Jufaili, 2007; Esmaeili et al., 2014; (Heistinger et al., 2011), reproductive Bostancı et al., 2015), and they widely biology and age determination (Abedi used for age determination (Shimose et et al., 2011), and mortality (Majtán et al., 2015; Elzey et al., 2015), growth al., 2012). The uses of scales and (Romo-Curiel et al., 2015; Mayank et otoliths morphological and al., 2015), prey-predator (Esmaeili, morphometric characters have been 2001; Škeljo and Ferri, 2012) and shown as an important tool to genetic studies (Kumar et al., 2007; determinate and discriminate marine Annabi et al., 2013). and species (Esmaeili et The genus Garra is represented 73 al., 2014; Bostancı et al., 2015). There fish species in the family of . is little information related to the scale G. rufa is one of them and is small morphologies of G. rufa in . For that live in the Syria, Iran, instance, Keivany et al., (2015) Iraq and Turkey (Esmaeili et al., 2009; examined 28 morphometric characters Coad, 2014). G. rufa is also known as (standard length, body depth, body doctor fish because of the regular width, head length, etc.), 25 ratios (head treatment specialties. The doctor fish is length to standard length, body depth to one of the therapeutic fish which is standard length, pectoral fin length to used in ichthyotherapy for therapeutic standard length, etc.) and 10 meristic purposes (Özcelik et al., 2000), their characters ( scales, scale regular treatment specialties can greatly above the lateral line, scale below the improve skin treatment especially lateral line, predorsal scales, keel Iranian Journal of Fisheries Sciences 15(4) 2016 1595 scales, total vertebrae, etc.) of G. rufa and 90% ethanol for 30 minutes in different basins of Iran. However, respectively and they were dried on there are no studies about otolith filter paper to avoid curling (Lippitsch, morphology; therefore, the scales and 1990). Non-damaged otolith and scale otolith morphologies are largely samples were stored for further unknown for G. rufa in many areas. examination. The current study presents for the first While the scales were viewed under time the comprehensive variation a stereo binocular microscope with information on scales and left-right transmitted light and magnification asteriscus and lapillus otoliths in the strength of 10×, the asteriscus and juvenile and adult G. rufa inhabiting lapillus otolith pairs were photographed Kangal Balıklı Çermik thermal spring using Leica S8APO brand microscope (Sivas, Turkey). with Leica Application Suit (Ver. 3.7.0) software. The otoliths and scales Materials and methods morphometric parameters such as The doctor fish specimens were length, width, perimeter, and area were collected from Kangal Balıklı Çermik measured using Digimizer image thermal spring in Sivas, Turkey (Fig. analysis software (Ver. 4.5.1) program. 1). Total length (TL), fork length (FL), The left-right asteriscus and lapillus and standard length (SL) of each doctor otolith shape indices such as form fish was measured to the nearest 0.1 factor (FF), roundness (RD), aspect cm, and their weight (W) was recorded ratio (AR), circularity (C), to the nearest 0.1 g. Schematic shape of rectangularity (R), and ellipticity (E) the Garra rufa and the six body regions were calculated to describe the otolith for scale collection in the doctor fish shape for the juvenile and adult fish were shown in Fig. 2. The fish were (Tuset et al., 2003; Ponton, 2006). dissected and examined as a mature or Mesial and distal surfaces of the left- immature and then they were right otoliths were photographed for considered as a juvenile or adult. The each sample. Eleven otolith characters fish scales were gently removed from such as width, depth, shape, rostrum- six different body regions of the fish. antirostrum size and shape, anterior- Asteriscus and lapillus otoliths pairs of posterior regions, mesial-lateral surfaces each fish were removed using the were determined for each G. rufa. otolith removal procedure (Secor et al., SHAPE software (Ver. 1.3) program 1991). The scales and otoliths were was used to extract the contours of the gently cleaned using a fine brush and G. rufa otolith outline (Iwata and Ukai, then each otolith and scale were rinsed 2002) and AFORO database system by distilled water (Chugunova, 1963; (Lombarte et al., 2006) was used to Campana, 2001). The cleaned scales of determine contour wavelets of the the fish were dehydrated in 30, 50, 70 otolith for the juvenile and adult fish.

1596 Yedier et al., Otolith and scale morphologies of doctor fish (Garra rufa) inhaabbiting …

Figuure 1: Sampling location of the Garra rufa in Kangal Balıklı Çermik thermal spring (Sivas, Turkey).

Figuure 2: Schematic shape of the Garra rufaa; showing the six body regions for scale collection (modified from Coad, 2014).

Due to the determination of were presented in Fig. 3. The aasteriscus graphical illustration of wavelets in the and lapillus otolith pairs morphological left-right asteriscus and lapillus otolith characters such as overall otoliith shape, contours, a total of 512 equidistant otolith with, otolith depth, rostrum size cartesian coordinates of the left and and shape, antirostrum size and shape, right otolith were extracted and they mesial and lateral surfaces, anterior and were analyzed using the wavelet posterior regions were examined for transformed. each G. rufa. Due to the small body of General otolith (A) and scale (B) the fish, six body regions were selected features of the doctor fish and the for scale morphology of G. rufa. terminology used in the current study Iranian Journal of Fisheries Sciences 15(4) 1597

Figure 3: Otolith (A) and scale (B) features of the Garra rufa, and their terminologgy used in the current study.

The scales morphological characters regions of the juvenile and adult doctor such as scale type, overall scale shape, fish are shown in Table 2 and also fork scale size, focus position, circuli length (FL), standard length (SL), W appearance, radii type, rostral and and TL are presented in Table 2. The caudal margins were determined in six left-right asteriscus and lapillus otoliths different body regions of the doctor morphological characters of the fish. juvenile and adult fish were separately determinated. In this study, 11 otolith Results morphological characters such as In this study, G. rufa samples weight overall otolith shape, otolith with, (W) and total length (TL) and the mean otolith depth, rostrum size and shape, values of their left-right asteriscus and antirostrum size and shape, mesial and lapillus otoliths measurements are lateral surfaces, anterior and posterior shown for the juvenile and adult fish regions were used, and the results are (Table 1). The mean values of otolith presented in Table 3. The overall otolith shape indices such as FF, RD, AR, C, surface views are shown for left and R, and E were calculated and the results right asteriscus (a;b) and lapillus (c;d) are shown for not only asteriscus and otoliths in the doctor fish (Fig. 4). lapillus otolith pairs but also the juvenile and adult fish (Table 1). The mean values of the scale measurements in six different body 1598 Yedier et al., Otolith and scale morphologies of doctor fish (Garra rufa) inhabiting …

Table 1: The mean values of the leftt - right asteriscus and lapillus measurements and thee otoliths shape indices of the juvenile and adult Garra ruufa.

Otolith Measurements Otolith Shape Indices

Side Form factorfactor Species Otolith Otolith Length Ellipticity Ellipticity Perimeter Roundness Circularity Aspect ratio Otolith Area Otolith Type Rectangularity Rectangularity Otolith Width

Left 0.157472 1.676127 0.506155 0.467697 0.7043675588 0.782612 1.082228 0.039491 17.84064 0.665205 e Asteriscus il Right 0.165401 1.656890 0.515218 0.465100 0.757112892 0.793353 1.107757 0.051124 16.59775 0.690241

uven Left 0.167274 1.620323 0.545638 0.420177 0.800635998 0.715368 1.298591 0.129902 15.69549 0.729611

J Lapillus Right 0.164867 1.617943 0.540573 0.406844 0.791438485 0.718348 1.328698 0.141151 15.87789 0.749638

Left 0.782242 3.846612 1.079345 0.989114 0.664345856 0.854930 1.091224 0.043622 18.91540 0.732714 Asteriscus Garra rufa Right 0.785028 3.767128 1.110066 1.041649 0.695143166 0.811143 1.065681 0.031796 18.07738 0.678914

Adult Left 0.659788 3.183537 1.019115 0.846896 0.8180775547 0.808850 1.203353 0.092293 15.36086 0.764454 Lapillus Right 0.643000 3.165159 1.009782 0.892159 0.806547174 0.802908 1.131841 0.061844 15.58045 0.713742

Table 2: The mean values of the scale measurements in six different body reegions of the juvenile and adult Garra rufa wiith fish weight (W), total lengtth (TL), fork length (FL), and standard length (SL).

Scale I- II- V- VI- Species W TL FL SL III-Region IV-Region (g) (cm) (cm) (cm) Measurements Region Region Region Region

Area 2.419 5.709 4.582 4.442 5.691 4.593 Perimeter 6.615 9.531 8.780 8.648 10.014 8.814 0.7 4.6 4.2 3.7 Length 1.898 3.009 3.052 2.935 2.973 2.976

Juvenile Juvenile Width 1.797 2.678 1.942 2.004 2.799 1.981 Area 4.021 7.380 7.090 5.460 8.231 5.534

Garra rufa Garra rufa Perimeter 8.063 11.141 12.120 10.341 12.156 9.986 4.9 8.0 7.2 6.5 Length 2.444 3.493 3.637 3.229 3.664 3.358 Adult Width 2.235 3.129 2.834 2.229 3.339 2.182

Figuure 4: Surface views of the asteriscus (a;b) and lapilluus (c;d) otolith pairs in the Garra rufa ((A)- TL: 4.6 cm; (B)-TL: 8.0 cm).

Iranian Journal of Fisheries Sciences 15(4) 2016 1599

The asteriscus and lapillus otolith scale type, overall scale shape, scale shapes are characterized as squared and size, focus position, circuli appearance, oval to elliptic for asteriscus and radii type, rostral and caudal margins lapillus otolith in juvenile doctor fish, were used during the study and the and discoidal and oval to elliptic for determined scales morphological adult doctor fish asteriscus and lapillus characters are shown in Table 4. otolith shape, respectively (Table 3). Different types of cycloid scale shape The rostrum and antirostrum are present were found in the six different body for asteriscus otolith for the G. rufa, but regions of the doctor fish and generally they are absent for lapillus otoliths of juvenile and adult fish morphological the juvenile and adult fish (Fig. 4). scales characters are varied in six body Moreover the lapillus width is regions especially overall shape, rostral determined as thick, asteriscus width is and caudal margins (Table 4; Fig. 6). thin in the G. rufa, while anterior While the scale focus is distinct for II- margin of the otoliths is characterized Region on both the juvenile and adult by double-peaked for asteriscus and fish bodies, the focus is indistinct for rounded for lapillus in the juvenile and III-Region on body (Table adult fish (Fig. 4). Whereas the 4; Fig. 6). Moreover, the scale size is posterior margin of the otoliths is varied in six different body regions of determined as an oblique for lapillus, it the fish (Table 2; Fig. 6). The circuli is rounded for asteriscus (Table 3; Fig. appearance on the scales are district for 4). Nine wavelet functions were the six different body regions of the determined for asteriscus and lapillus fish. In addition, the radii are otoliths using the AFORO in the determined as primary and secondary juvenile and adult fish. The wavelet 5-6 for each region scale from the juvenile gave high variations of the otolith and adult doctor fish (Fig. 6). contour and the wavelet 6 was selected as an intermediate function in the current study. The graphical illustration of wavelet number 6 showed specific variations associated with prominent features of the left-right lapillus and asteriscus otolith contours (Fig. 5). Six different body regions were selected based on the removed scales areas in the G. rufa (Fig. 2). General scale shapes from six different body regions were presented for the juvenile and adult fish, separately (Fig. 6). Eight scales morphological characters such as

1600 Yedier et al., Otolith and scale morphologies of doctor fish (Garra rufa) inhabiting …

Table 3: The morphological characters of the asteriscus and lapillus otolith pairs for the juvenile and adult Garra rufa. Juvenile Adult Otolith Asteriscus Lapillus Asteriscus Lapillus Characters Left Right Left Right Left Right Left Right Oval to Oval to Oval to Oval to Otolith Shape Squared Squared Discoidal Discoidal elliptic elliptic elliptic elliptic Moderately Moderately Moderately Moderately Otolith Width Thin Thin Thick Thick thick thick thin thin Otolith Depth Shallow Shallow Shallow Shallow Shallow Shallow Shallow Shallow Rostrum Size Short Short Absent Absent Short Short Absent Absent Moderately Moderately Rostrum Shape Round Round Absent Absent Absent Absent Pointed Pointed Well Well Well Antirostrum Size Well Absent Absent Absent Absent development development development development Moderately Antirostrum Shape Pointed Absent Absent Pointed Pointed Absent Absent Pointed Mesial Surface Concave Concave Concave Concave Concave Concave Concave Concave Lateral Surface Convex Convex Concave Concave Convex Convex Concave Concave Double Double Double Double Anterior Region Rounded Rounded Rounded Rounded Peaked Peaked Peaked Peaked Posterior Region Rounded Rounded Oblique Oblique Rounded Rounded Oblique Oblique

Table 4: The scale morphological characters in six different body regions of the juvenile and adult Garra rufa. Scale I-Region II-Region III-Region IV-Region V-Region VI-Region Characters Scale Juvenile Cycloid Cycloid Cycloid Cycloid Cycloid Cycloid Type Adult Cycloid Cycloid Cycloid Cycloid Cycloid Cycloid Moderately Moderately Moderately Moderately Juvenile Small Medium Scale Large Medium Large Medium Size Adult Medium Large Large Medium Large Medium Elongate Juvenile Rectangular Square Oblong Square Oblong Scale Pentagonal Shape Elongate Adult Rounded Pentagonal Pentagonal Pentagonal Hexagonal Pentagonal Focus Juvenile Centric Non-centric Indistinct Indistinct Non-centric Non-centric Position Adult Non-centric Non-centric Non-centric Non-centric Non-centric Non-centric Circuli Juvenile Distinct Distinct Distinct Distinct Distinct Distinct Appearance Adult Distinct Distinct Distinct Distinct Distinct Distinct Primary Primary Primary Primary Primary Primary Juvenile Radii Secondary Secondary Secondary Secondary Secondary Secondary Type Primary Primary Primary Primary Primary Primary Adult Secondary Secondary Secondary Secondary Secondary Secondary Relatively Relatively Relatively Juvenile Undulate Undulate Lobed Smooth Wavy Smooth Rostral Margin Relatively Relatively Adult Lobed Wavy Wavy Undulate Wavy Wavy Juvenile Roundly Roundly Triangular Roundly Roundly Roundly Caudal Margin Adult Roundly Roundly Triangular Triangular Roundly Triangular Iranian Journal of Fisheries Sciences 15(4) 2016 1601

Figure 5: Signals of wavelet 6 from the asteriscus and lapillus otolith pairs in the juuvenile (A, C) and adult (B, D) Garra rufa.

Figure 6: Scales from six different body regions of the juvenile and adult Garra rufa. (Juvenile- TL: 4.6 cm; Adult-TL: 8.0 cm). Discussion features for left and right asteriscus and The current study describes the otolith lapillus otoliths were examined in and scale morphologies of juvenile and juvenile and adult fish. Additionally, 4 adult doctor fish, G. rufa from Turkey. morphometric and 8 morphological In addiition, this is the first time that 10 features were determined for scales of morphometric and 11 morphological the juvenile and adult G. rufa. The 1602 Yedier et al., Otolith and scale morphologies of doctor fish (Garra rufa) inhabiting … general architectural pattern of a present study suggest that differences in cycloid scale of doctor fish having asteriscus - lapillus otoliths and scales focus, circuli and radii. Esmaeili et al., measurements and their morphologies (2012) reported that focus of the scale are detectable in G. rufa. in Garra rossica is distinct and located Environmental and ecological factors in the anterior field and the focus is the might also influence fish otolith and first part of the scale to be formed scale morphologies defining fish during ontogenesis. Similarly, the focus species and populations for inhabiting of the scale in G. rufa is also distinct for natural waters (Volpedo and body regions I, III, and IV. Scale shape Echeverria, 2003; Vignon et al., 2008; in different fish species may vary such Bendoy et al., 2011). However, the as circular, triangular, rectangular and doctor fish is inhabiting thermal spring; oval (Helfman et al., 2009). In the therefore, the environmental and current study, six different scale shapes ecological factors are more stable with were determined such as rectangular, 7.8 pH, dissolved oxygen 2.9 ppm, and rounded, square, pentagonal, 35°C (Timur et al., 1983) and they are hexagonal, elongate and oblong in not directly influence the fish otoliths juvenile and adult fish. and scales. In addition, their scale The morphological otolith characters properties are varied in that habitat and can be used to distinguish fish species they may be a useful tool to identify the in different habitats, the same doctor fish in different habitat. measurements may also be used to Comparing the scales from six different predict unknown fish species (Zischke body regions in the same G. rufa, the et al., 2016). Bostancı et al., (2015) best body region is II-Region for the demonstrated that otolith characters fish scale with weak deformation and were varied among four valid species of distinct scale characters such as focus, the genus in different habitats circuli, and radii. Otolith morphology is of Turkey. In addition, the present study the most crucial criteria in revealed that the otolith characters are ichthyological studies, whereby a also varied in juvenile and adult doctor number of identification guides and fish. Morphometric and meristic atlases are published on scale and characters of hard parts such as otolith otolith morphologies (Patterson et al., and scale can be used as a tool for 2002; Tuset et al., 2008; Lin and identification of fish species in marine Chang, 2012). Nowadays, otolith and freshwater ecosystems. As the morphology (Jemaa et al., 2015a,b; otolith and scale being important parts Tuset et al., 2015) and scale of fish species, the study reveals that morphology (Ganzon et al., 2012; these bony structures can be used Chapman et al., 2015; Keszka et al., successfully to determine fish species at 2015) are much more popular and juvenile and adult stages. The results of Iranian Journal of Fisheries Sciences 15(4) 2016 1603 several studies have been published for their morphometric and morphological a variety of fish species. features were used to determination of The graphical illustration of one of the therapeutic fish in the wavelets was used for asteriscus and thermal spring (Turkey). The features lapillus pairs in the doctor fish to might be considered importantly for discover variabilities that could indicate ichthyologist, biologists, and scientists different type of otolith shape. The and they might be used for current study demonstrates that wavelet identification or discrimination of other analysis is a convenient method for freshwater and marine fish species in determination and discrimination of the further studies. juvenile and adult doctor fish. Although, the method is reliable Acknowledgement approach for species discriminations in We would like to thank Furkan Coşkun ichthyological studies, it was rarely for having kindly supplied the G. rufa used in the previous studies (Parisi- specimens in this study. Baradad et al., 2010, Sadighzadeh et al., 2012). For instance, the analysis References was successfully used to discriminate Abedi, M., Shiva, A.H, Mohammadi, for ten species of snappers Lutjanus H. and Malekpour, R., 2011. spp. (Sadighzadeh et al., 2014). In the Reproductive biology and age present study, wavelet analysis was determination of Garra rufa Heckel, detected as an alternative method to 1843 (: Cyprinidae) in identify juvenile and adult G. ruffa. central Iran. Turkish Journal of The previous ichthyological studies Zoology, 35(3), 317-323. indicated that molecular and genetic Annabi, A., Said, K. and methods are also important for Reichenbacher, B., 2013. Inter- determination and discrimination of fish population differences in otolith species and populations (Perrier et al., morphology are genetically encoded 2011; Shafee et al., 2013; Dorafshan et in the killifish Aphanius fasciatus al., 2014). However, using the (Cyprinodontiformes). Scientia morphometry and morphology of Marina, 77(2), 269-279. versatile research materials are cheaper Bendoy, C.P., Torres, M.A.J. and and more time-efficient methods than Demayo, C.G., 2011. Image genetic and molecular methods (Tracey analysis of fish intraspecific scale et al., 2006). variations. The First International In conclusion, this is the first time Conference on Interdisciplinary that the left-right asteriscus-lapillus Research and Development otoliths and scales of the juvenile and Bangkok, Thailand. adult doctor fish are examined. In Bostancı, D., Polat, N., Kurucu, G., addition, the otolith, scale shapes, and Yedier, S., Kontas, S. and Darcin,

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