Int. J. Aquat. Biol. (2017) 5(5): 321-327; DOI: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology Original Article The regeneration capacity of caudal fin in the common tooth-carp, Aphanius dispar (Rüppell, 1829) (Teleostei: Cyprinodontidae) Faezeh Zeinali, Mina Motamedi*1 Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, 76169-14111 Kerman, Iran. Article history: Received 26 August 2017 Accepted 22 October 2017 Available online 2 5 October 2017 Keywords: Regeneration Blastemal Caudal fin Cyprinodontids Aphanius Abstract: Regeneration ability is known for several Actinopterygii or ray-finned fishes. In order to assess the universality of regenerative potencies in this group of fishes, we have examined for the first time, caudal fin regeneration in tooth-carp, Aphanius dispar (Rüppell, 1829). The caudal fin is used because of its accessibility, simple structure and fast regeneration. The results revealed the regeneration ability in caudal fin of A. dispar. The initiation of the regenerative outgrowth was differing in three examined water temperatures. It is started approximately 5 days post amputation (5 dpa) at room temperature, and 2 dpa at 25°С and 28°С. Our finding indicates that water temperature more than 25°С promotes procedure in caudal fin regeneration of A. dispar. By considering the high regeneration ability in A. dispar, and also the relatively short life span of the members of the genus Aphanius, we concluded that these fishes could probably be used in regeneration researches. However, details of this mechanism in Aphanius need further examinations. Introduction The teleost fish together with some groups of amphibians among the vertebrates represent remark- able capability to regenerate their various organs (Pfefferli and Jazwinska, 2015). Due to the simplicity in manipulation and macroscopic examination, most studies on regeneration potential focused on external appendages such as fins and limbs in abovementioned animal groups. The first report on fish regeneration was documented for the gold fish (Broussonet, 1786). He discovered that in regeneration experiment of the fishes, caudal fin has advantage over other fin types, because it regenerates faster than pectoral, ventral and dorsal fin (Pfefferli and Jazwinska, 2015). Among the teleost fishes, zebrafish Danio rerio (Hamilton, 1822) has been used as powerful model for regeneration studies (e.g., Nechiporuk and Keating, 2002; Pfefferli and Jazwinska, 2015). Besides its simple architecture, its accessibility and its fast and robust regeneration, it is able to regenerate fins, scales, retina, spinal cord and heart among other internal organs (Iovine, 2007; Azevedo et al., 2011). In contrast to amphibians and fishes, other *Corresponding author: Mina Motamedi DOI: https://doi.org/10.22034/ijab.v5i5.375 E-mail address: m.motamedi@uk.ac.ir vertebrate such as reptiles, birds and mammals have a more limited capacity to regenerate complex body structures (Brockes and Kumar, 2008; Galis et al., 2003; Unguez, 2013). The limited capacity of humans to regenerate tissues and evolutionary loss of regenerative ability in animal groups attracts many biologists from medical perspective (Bely and Nyberg, 2010; Unguez, 2013), and therefore research- ers always attempt to elucidate the mechanisms responsible for the extensive regenerative capacities in specific groups of vertebrates. Caudal fin is composed of segmented bony rays, mesenchymal tissue, blood vessels and nerve axons. Each bony ray consists of two concave hemi-rays that define an inner space filled with intra-ray mesenchymal cells. The bifurcations are responsible for generating the characteristic shape of the caudal fin (Poss et al., 2003). Bony rays are produced and maintained by osteoblasts (also called scleroblasts), skeletogenic cells that secrete bone matrix (Hall, 2005). Based on the study of Poss et al. (2003), an amputation of caudal fin triggers a regenerative 322 Zeinali and Motamedi/ Regeneration in caudal fin of A. dispar program that occurs in three phases: (i) wound healing: the first phase after amputation started immediately after injury and continues within 12 hour-post-amputation (hpa). The injury is healed through migration of epidermal cells that cover and close the wound. (ii) blastema formation: the wound epithelium thickens forming an apical epidermal cap (AEC). Formation of blastema, is accomplished by the dedifferentiation and proliferation of cells in the disorganized mesenchymal tissue under AEC. (iii) Regenerative outgrowth: the onset of regenerative outgrowth starts after establishment of the blastema, at 48 hpa. The third phase is characterized by the extensive proliferation of cells. Up to now, no information is available on the regeneration phenomenon in tooth-carp, genus Aphanius Nardo, 1827. To determine whether Aphanius can regenerate its body parts, we used Aphanius dispar from southern Iran as model, and removed its caudal fin from the basal part. Materials and Methods Study model: The common tooth-carp, A. dispar inhabits brackish and freshwater environments almost in the desert regions (Wildekamp, 1993) (Fig. 1). More especially, its principal habitats are coastal lagoons, but owing to its high tolerance to ecological changes, it can live in inland waters and hot sulphuric springs (Wildekamp, 1993; Teimori et al., 2012a, b). In this study, A. dispar specimens were captured from Bandar Abbas city in southern Iran and transferred to the laboratory aquariums equipped with heater, thermometer and constantly aerated water. The animals were fed every day during experiment period with freeze-dried live food and also dry food that contain macronutrients, trace elements and vitamins necessary to keep captive fish in good health. The individuals were kept in three separate aquariums in pH 6.8-7, and each settled with specific temperature (Room Temperature (RT)=23°С, 25°С and 28°С). Throughout the examination, temperature was recorded twice a day. Fin amputation: In total, 12 fish individuals were examined for this experiment. They placed in 180 ppm of aqueous clove (Eugenia caryophyllata) solution, and the anesthesia was induced after approximately 3 minutes. Once fish asleep and immobile, it was moved to the dry lid of the Petri dish, oriented so the head was positioned to the left, the caudal fin was fanned out with a clean razor blade. The fish was then transferred back to a tank of fresh water, observed for gill movement, and allowed to awaken. The caudal fins of four fish individuals (two males and two females) in each temperature were imaged 2, 5, and 7 days post amputation (dpa) (Fig. 2). Each captured image was used for measurement in Figure 1. A fresh male common tooth-carp, Aphanius dispar from southern Iran, studied as model in this study (Scale bar is about 1 cm; fish photo provided by H.R. Esmaeili). Figure 2. Stages of the caudal fin including amputation: Uncut (before cutting), Amputation (immediately after cutting) and 2, 5, and 7 days after amputation (dpa). The dashed line indicates position of the cutting and the regeneration processes. The white arrows show the measurements from point of amputation. 323 Int. J. Aquat. Biol. (2017) 5(5): 321-327 Image-J software (Schneider et al., 2012). The length of caudal fin regeneration from point of amputation was measured in three times (in millimeter, mm) to obtain an average values of the growth rate. Eventually the obtained data was analyzed using IBM SPSS Statistics 24. The T-test (P<0.05) was used to show if significant differences exist for the regener- ation rates among different days at different water temperatures. Results The results clearly indicate for the first time the regeneration ability in tooth-carp, A. dispar. The results reveal that A. dispar can obviously repair its caudal fin a short time (approximately 2 days) after traumatic injury as described below. There was little bleeding after caudal fin amputation. Few hours after amputation, the wound is covered by epidermal layer which is called wound healing phase. Next step in A. dispar fin regeneration is blastema formation which is occurred in 12-48 hours after amputation (2 dpa in Fig. 3). During blastema formation, epidermis accumulates additional layers by cell migration. The hall mark of blastema is a proliferative mass of mesenchymal cells which sits atop each fin ray and gives rise to the new structures of the caudal fin. The last step in fin regeneration is switch from blastema formation to regenerative outgrowth. This transition occurs at approximately 5 dpa at RT, and 2 dpa at 25°С and 28°С (Fig. 3). Therefore, for quantitative analysis, we only compared regeneration rates for 2 and 7 days post amputation at 25°С and 28°С water temperature in males and females separately. Descriptive analysis of regeneration rate in different days and water Figure 3. The regeneration processes in caudal fin of Aphanius dispar in different days at three water temperatures. 324 Zeinali and Motamedi/ Regeneration in caudal fin of A. dispar temperatures is shown in Table 1. In the 5 dpa, caudal fin regeneration was 0.77 mm at 25°С and 0.73 mm at 28°С in the males vs. 0.59 mm at 25°С and 0.83 mm at 28°С in the females. In the 7 dpa, at both 25°С and 28°С, the regeneration rate in the males was lower than the females (1.86 mm at 25°С and 2.05 mm at 28°С in the males vs 2.0 mm at 25°С and 2.90 mm at 28°С in the females) (Table 1). In the males, there were not significant differences for the regeneration rates at different water temperatures in 2- and 5 dpa, while the regeneration rates significantly differ between RT vs 25°C and RT vs. 28°C in 7 dpa (T-test, P<0.05) (Table 2 and Fig. 4). In the females, the regeneration rates differ significantly only between RT vs 28°C in both 5- and 7 dpa (T-test, P<0.05) (Table 2 and Fig. 4). Fin regeneration measurement revealed an overall increase in growth with increasing of water temperature. Regeneration outgrowth was seen after 5 dpa in 25°С and 28°С. However, in Rome Temperature (RT) it is detected after 7 dpa (Figs. 3-4). The increasing rate of regeneration was significant in Sex T°C Day 2 Day 5 Day 7 Mean S.D. Mean S.D. Mean S.D. Male RT 0.20 0.085 0.61 0.064 0.66 0.106 25 28 0.23 0.22 0.059 0.006 0.83 0.78 0.114 0.178 1.86 2.05 0.328 0.375 Female RT 0.32 0.162 0.44 0.080 0.45 0.160 25 0.31 0.014 0.59 0.050 1.98 0.070 28 0.32 0.028 0.73 0.028 2.89 1.163 The rate of regeneration is shown for different days at three water temperatures. RT=Room Temperature and S.D. = standard deviation. Table 1. Descriptive analysis of the regeneration rates between males and females of the Aphanius dispar. Sex Day T-test Mean differences Sig. Female Day 2 RT vs 25 0.0066 0.98 RT vs 28 0.0030 0.99 28 vs 25 0.0100 0.11 Day 5 RT vs 25 0.1575 0.91 RT vs 28 0.2925 0.03 28 vs 25 0.1350 0.61 Day 7 RT vs 25 1.5250 0.56 RT vs 28 2.4375 0.03 28 vs 25 0.9125 0.27 Male Day 2 RT vs 25 RT vs 28 0.0216 0.0183 0.89 0.92 28 vs 25 0.0033 0.99 Day 5 RT vs 25 0.2200 0.97 RT vs 28 0.4625 0.24 28 vs 25 0.0575 0.81 Day 7 RT vs 25 1.2009 0.00 RT vs 28 1.3895 0.00 28 vs 25 0.1885 0.48 Table 2. T-test analysis to show significant differences (P<0.05) of the regeneration rates for different days at three water temperatures in the examined Aphanius dispar. The analysis was carried out for the males and females separately. RT=Room Temperature and S.D.= standard deviation. Figure 4. Comparison of the rate of caudal fin regeneration in the male (above), and the female (below) specimens housed at three water temperatures over 7 days. 325 Int. J. Aquat. Biol. (2017) 5(5): 321-327 5 and 7 days post amputation in comparison to the 2 dpa (T-test, P<0.05). Also, our results indicate that no clear growths happened in the 2 dpa comparing the time of amputation (Figs. 2-4). Discussion Regenerative capacity in different organs and organisms is interesting for biologists, and clinicians. There are many studies which attempt to discover various aspect of regeneration (e.g. Poss et al., 2003; Azevedo et al., 2011; Rink, 2013; Hoppe et al., 2015; Kim et al., 2016). Therefore, it is in the center of attention to reveal regenerative capacity in organisms from evolutionary point of view, the role of stem cells, signals which initiate regeneration and its controlling mechanisms. Among teleost fishes, various degrees of regeneration abilities have been reported. Good examples are cyprinid fish, D. rerio which can regenerate lost parts repeatedly (Azevedo et al., 2011), and short-lived African turquoise killi- fish, Nothobranchius furzeri (e.g. Hoppe et al., 2015; Kim et al., 2016). However, some teleost fishes exist with total absence of regeneration such as some members in the family Blennidae (Wagner and Misof, 1992). In the present study, the possible occurrence of the regeneration ability in caudal fin of another relatively short-lived killifish, A. dispar is examined. The outcome of all these studied could help researchers to assess the universality of regenerative potencies in Actinopterygii fishes. The studied taxon is a small tooth-carp with a maximum length up to 8 cm (usually 45-55 mm in total length). It is widely distributed throughout the coastal environments of the Mediterranean Sea, Red Sea and Persian Gulf regions (Wildekamp et al., 1999; Hrbek and Meyer, 2003; Reichenbacher et al., 2009; Ferrito et al., 2013; Teimori et al., 2016; Esmaeili et al., 2017). Most of the Aphanius species tolerate euryhaline and eurythermal habitats as well as relatively low levels of oxygen and wide range of pollution. In addition, they are good candidates as biological control of malaria because they feed on mosquito larvae of Anopheles (Homski et al., 1994). Based on the finding of the present study, the regeneration phenomenon is happening in A. dispar, and probably could be occurred also in the other members of the genus Aphanius. The killifishes with special biological properties such as short life span and having regeneration ability are very interesting models for biomedical researchers. Therefore, besides the already known killifish, Nothobranchius furzeri (Hoppe et al., 2015; Kim et al., 2016), here we present genus Aphanius as a new candidate model organism to be used to study the mechanisms of regeneration. According to the previous study, the regeneration procedure contains the wound healing, blastema formation and growth (Pfefferli and Jazwinska, 2015). Blastema is a heterogeneous group of dedifferentiated/ proliferative cells, able to regrow and pattern a new complex organ (Pfefferli and Jazwinska, 2015). In this study, after the caudal fin amputation, the wound is healed in few hours. Thereafter, blastema formation is occurred in 12-48 hours. It is documented that B-catenin is one of the earliest molecular markers for fin regeneration in wound epidermal cells, induced in the first few hours after amputation and maintained through regeneration (Poss et al., 2000). Initiation of regenerative outgrowth is differing in three examined water temperatures of this study. It is started approximately 5 dpa at RT, and 2 dpa at 25°С and 28°С. Our finding indicates that temperature more than 25°С promotes procedure in caudal fin regenera- tion of A. dispar. As results, members of the genus Aphanius can probably be a suitable candidate to study regeneration phenomenon. 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(2017) 5(4): 321-327 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology چکیده فارسی : عالی استخوانی ماهیان) Aphanius dispar (Rüppell, 1829) معمولی گورخری ماهی در دمی باله ترمیم توانایی (داردندان کپورماهیان *معتمدی مینا زینلی، فائزه .ایران کرمان، کرمان، باهنر شهید دانشگاه علوم، دانشکده شناسی،زیست بخش گروه چکیده: نخستین برای ماهیان، از گروه این در ترمیم هایتوانایی پذیریقابلیت ارزیابی برای. است شده شناخته باله شعاع ماهیان از تعدادی در ترمیم توانایی راحت، دسترسی دلیلهب دمی باله مطالعه، این در. گرفت قرار بررسی مورد Aphanius disparمعمولی گورخری ماهی در دمی باله ترمیم بار، رشد ازآغ. است برخوردار دمی باله ترمیم توانایی از معمولی گورخری ماهی که داد نشان نتایج. گرفت قرار استفاده مورد سریع ترمیم و ساده ساختار 28 و 25 دمای در و( dpa5) برش اولین از پس روز 5 حدوداً ترمیم اتاق، دمای در. باشدمی متفاوت مطالعه، مورد آب حرارت درجه سه در ترمیمی موجب گرادسانتی درجه 25 از بیش آب دمای که دهدمی نشان مطالعه این هاییافته. شودمی شروع ترمیم برش از پس روز 2 گرادسانتی درجه اًنسبت عمر همچنین و معمولی گورخری ماهی در ترمیم باالی توانایی گرفتن نظر در با. گرددمی معمولی گورخری ماهی در ترمیم سرعت افزایش این با .گیرند قرار استفاده مورد ترمیم به مربوط مطالعات در توانندمی احتماال ماهیان این که نمود گیرینتیجه توانمی ماهیان، این اعضای کوتاه .باشدمی بیشتر مطالعه به نیاز ماهیان این در ترمیم مکانیسم جزئیات شناخت برای حال، .آفانیوس دار،دندان کپورماهیان دمی، باله بالستما، ترمیم، :کلمات کلیدی