Hard-Part Counting for Identification of Marine Ornamental Fish Species

PROCEEDING 1st International Conference on Fisheries and Marine Research (ICoFMR 2020) – ISBN:978-602-72784-4-8

HARD-PART COUNTING FOR IDENTIFICATION OF MARINE ORNAMENTAL FISH SPECIES

Dewa Gede Raka Wiadnyaa*, Rifqi Al-Qodrib, Wahyu Endra Kusumaa, Anik Martinah Hariatib, Septiana Sri Astutic, Agung Pramana Warih Marhendrad

a*Ichthyo-Fauna Faculty of Fisheries and Marine Science Universitas Brawijaya, Malang, Indonesia bAquatic Biofloc Faculty of Fisheries and Marine Science Universitas Brawijaya, Malang, Indonesia cPhD Program, Faculty of Fisheries and Marine Science Universitas Brawijaya, Malang, Indonesia dDepartment of Biology Faculty of Mathematics and Science Universitas Brawijaya Malang, Indonesia

*Corresponding author: [email protected]

Abstract

Together with the Philippines, Indonesia is the biggest exporter of marine ornamental fishes for the aquarium trade. However, there was no data in place on the number and species involved in the trade. Species diversity and comparable characters among species within genera have made it difficult to identify up to species level. This study aimed to use internal hard part and fin spines-rays counting as complementary data for fish identification. In order not to impair specimens, staining and clearing procedures were implemented to the specimens. Specimens of reef-ornamental fishes for aquarium trade were collected from Bangsring Banyuwangi and Sumber Kima Singaraja Bali. Following photograph, coding and identification, all viscera and internal organs were taken out from each specimen. Bone staining and clearing was held based on alcian- blue acetic acid bone coloring, continued with trypsin and alizarin red soaking for 24-h and finally cleared with mixed solution of 0.5% KOH and glycerin for a period of 1-7 days. With the help of a standard photograph, vertebral column, fin-spines and rays were counted to confirm the species. The staining procedure was subject to trial and error but finally succeeded. Vertebral column, fin-spines, and rays from all 50 specimens were counted, confirming the species. This hard-part identification process resulted in 21 nominal (predicted) species. The technique can be applied to small-size and compressed forms of fish body- shape.

Keywords: aquarium trade, identification, ornamental-reef fishes, Indonesia

INTRODUCTION especially in morphological appearance [8]. FAO species identification sheet [9] applied 14 Before 1990, world trade on ornamental body elements to identify commercial fishery fishes was more than USD 7*109 year-1 [1]. species. But this is not enough for more than Indonesia is the leading country in exporting 30,000 fish species. Fish taxonomists the species [2] and contributed 85% of concluded that skeleton or other hard-part of specimens together with the Philippines. most actinopterygian could be used as an Located in the coral triangle [3], but with less indicator in studying the relationship among interest in taxonomic studies and limited fish group [10]. A fish can be described or research facilities, including financial support, identified based on meristic traits of these bone Indonesia is blessed with 4,786 fish species, and cartilage components and can be compared and 2,083 species (58% of total marine species) to its closest relatives. Alcian blue for cartilage are reef-associated [4]. High fishing pressure staining and alizarin red for bone are and habitat destruction due to unfriendly techniques commonly used to identify internal fishing practices [5] will cause reduced species hard-part of fishes [11] for many different diversity for this type of fishery. Both purposes [12][13]. situations may result in fish biodiversity losses Despite its large number of marine species before having a chance to be researched or [14], Indonesia recorded only 92 commercial even yet identified [6]. marine species in its annual fishery statistical Pisces is having the largest species within report and let marine ornamental species vertebrate’s superclass division [7]. Many fish remained unreported. Comparable to the species are superficially similar to one another, Philippines, Indonesia exported 997 marine

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ornamental fish species to the US [15]. Also, new fresh one if it takes on a bluish Indonesia and the US contributed 53% of all color. The process was continued until ornamental fish species imported by European bone and cartilage were clearly visible countries [16]. In addition to morphological and all flesh retained transparent and pictorial guides applied in the 7) Transfer the specimen into a mixed identification of Indonesia’s ornamental fish solution containing 100 ml KOH 0.5% species [17], this study aimed to describe some and 10 mg alizarin red S until it turned morphometric traits of 50 marine ornamental into deep purple. Solution is replaced fish species using cartilage and bone staining. every three days until bones turned into red. METHOD 8) The specimen was finally soaked in 0.5% KOH-glycerin series 1:1, 1:2, 1:3 Collection of specimens and to pure glycerin A total of 50 specimens, consisting of 9) All specimens were stored in glass- predicted 21 species and eight different bottle with pure glycerin solution. families, were randomly selected from 653 Second photograph of each sample frozen (-20C) specimens of 169 species and was taken for comparison with the 30 different families of reef-ornamental fish original (before treatment) species. The specimens were collected from As the methods are depend on fish size local fishers who catch it from around Lesser category, some trials and errors were applied to Sunda of South-Eastern Indonesia. Each reach the optimal result. The procedure specimen was identified based on pictorial consisted of 26 steps at all, divided into 7 parts. guide [14, 17] and some morphological The first part, cleaning and preservation: explanation [18 – 20]. Following the specimen was cleaned with water and soak in photograph and coded for specimen formalin solution for 24 hours. depository, each specimen was eviscerated and There were nine external and internal fully descaled at one side of their body (right- hard-part structures identified from each lateral side when the fish is faced left). specimen, consisted of pleural spines (PU), neural-pleural spines (NPU), inter-neural spine Staining Procedure cartilage (CINPU), hypural plate (HYP), dorsal Cartilage and bone staining procedure fin spines (DFS), dorsal fin rays (DFR), caudal applied a modified traditional protocol [12, 21] fin rays (CFR), anal fin spines (AFS) and anal with some trials and errors. It completed in 26 fin rays (AFR) (Figure 1) steps, including: 1) Immersing specimen into 10% formalin for 2*24 hours and rinsed afterward several times with aquadest 2) Transferring into a mixed solution containing 10 mg alcian blue, 80 ml ethyl-alcohol 95% and 20 ml glacial acetic acid for 24 hours 3) Rinsed twice in ethyl-alcohol 95% for three hours period each change 4) Transferred through ethyl-alcohol 75%, 40% and 15% for three hours Figure 1. Internal and external hard-part each concentration structures of fish specimen for identification 5) Specimen was immersed into distilled water for maximum 3 hours until it sinks Landmark, digitation & shape analysis 6) Immersing in a mixed solution of Photographs from all specimens, before saturated (solution) sodium borrate + and after staining, were transferred into tps file 30 ml distilled water + 1 g trypsin. The (*.tps) based on tpsDig version 2.1 software solution is replaced (changed) with the [22]. Digitation and the coordinate number of

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anatomical landmark were done based on ten The results showed that we can calculate anatomical landmark points [23]. A total of 10 most of internal bones and cartilages of a fish landmark points were undertaken and digitized specimen (fin-rays). These parts included: to the sample (Table 1). The result of tpsDig Pleural spines (PU), neural-pleural spine digitation in the bilateral (left/right) sides (NPU), inter-neural spine cartilage (CINPU), transferred into Symmetry and Asymmetry hypural plate (HYP), and the different between Geometric Data Analysis (SAGE) software fin-spines and soft-rays. The species can be version 1.04 [24]. described using bone and cartilage for more clear description of the species during Table 1. Landmark point of fish specimen used identification process. for digitation and shape analysis POINT LANDMARK POINT NAME 1 Anterior most of mouth 2 Posterior end of nuchal spine 3 Anterior insertion of dorsal fin 4 Posterior insertion of dorsal fin 5 Dorsal insertion of caudal fin 6 Midpoint of caudal fin 7 Ventral insertion of caudal fin 8 Posterior insertion of anal fin 9 Anterior insertion of anal fin 10 Dorsal base of pelvic fin

RESULTS AND DISCUSSION

Total 21 morpho-species were identified Figure 2. Photo specimen of from all 50 specimens [Table 2]. The clearest Pseudocheilinus evanidus (SWC_02) prior hard-part structures explaining the species (above) and after staining (below) were the number of between fin-spines and fin- rays of dorsal and anal fins of the specimen. SAGE (symmetry-asymmetry geometric Fin-rays were clearly branching, forked and analysis) of showed no different in shape of segmented. Contrary, fin-spines were un- both prior and after staining and clearing of all branched and many times serrated. fish specimens (p > 0.05). Also, landmark Fish species are rarely identified based on points and truss were nestled closely (Figure its internal and hard structure. Rather than bone 3). Landmark point and shape analysis of hard- and cartilages, pictorial guide is more practical part is a good alternative method to analyze [17]. Staining procedure may be considered species differences based on its morphometric complicated and involved chemical materials characters. with in many cases hazardous. Hence, this technique may useful for species those were difficult di be identified based on morphological approaches. Comparison of digital picture for specimen prior to and after staining treatment revealed the appearance of cartilage and hard- part structure of the specimen (Figure 2). Except bones and other hard-part structures, all flash components were transparent. The head region mainly covered with many plates and bones and overlap one to another. This explained red color almost of the region. All Figure 3 MANONVA analysis indicating no the specimens were then stored in glass-bottle significant difference in the shape of the with glycerin solution (lower-left of Figure 2). specimen before and after staining

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PROCEEDING 1st International Conference on Fisheries and Marine Research (ICoFMR 2020) – ISBN:978-602-72784-4-8

The staining and clearing processes, that quick result, X-Ray procedure may far better resulted in good final specimen, spent more and need shorter time to deal with this internal than one month. It involved some chemical hard-part of fish. compounds those not always available within laboratory. So, comparing with simple and

Table 2 Hard-part counting of all specimens of ornamental fishes NO SCIENTIFIC NAME PU NPU CINPU DORSAL_F ANAL_F 1 Dascyllus reticulatus 24 23 25 DXII;13 AII;13 2 Pomacentrus amboinensis 24 24 15 DXIII;11 AII;11 3 Dascyllus melanurus 24 22 12 DXI;13 AIII;10 4 Valenciennea sexguttata 26 24 25 DVII;18 AII;17 5 Chrysiptera talboti 25 23 23 DXIII;11 AII;12 6 Canthigaster valentini 24 22 9 DIX;11 AII;11 7 Dascyllus trimaculatus 24 22 12 DXI;14 AII;13 8 Halichoeres prosopeion 24 22 9 DIX;12 AII;11 9 Coris gaimard 24 22 16 DIX;11 AIII;12 10 Halichoeres marginatus 24 22 9 DIX;11 AII;11 11 Halichoeres chrysus 23 23 9 DIX;11 AII;9 12 Cetoscarus bicolor 24 23 13 DX;10 AII;11 13 Neosynchiropus ocellatus NA NA NA D?;8 A?;7 14 Amphiprion polymnus 26 24 24 DX;16 AII;13 15 Ptereleotris heteroptera 25 24 12 DVI;23 AI;30 16 Cirrhilabrus solorensis 26 24 19 DXI;9 AIII;10 17 Pseudocheilinus evanidus 26 25 12 DXII;10 AIII;13 18 Macropharyngodon negrosensis NA NA NA D_;9 A_;9 19 Cirrhilabrus exquisitus 23 21 18 DX;9 AIII;9 20 Cirrhilabrus flavidorsalis 26 26 17 DXI;10 AIII;10 21 Pseudanthias huchtii 26 24 15 DX;17 AIII;8 Note: PU = pleural spines; NPU = neural-pleural spines; CINPU = inter-neural spine cartilage; DORSAL_F = dorsal fin spines and rays; ANAL_F = number of anal fin rays

ACKNOWLEDGEMENT CONCLUSION This research was fully funded by Hibah In short, it can be concluded from the Doktor of Faculty of Fisheries and Marine findings that: Science Universitas Brawijaya. Authors 1) cartilage and bone staining can help to appreciate wish to thank for all the support. complete morphological identification of Also, we thank Conference committee of fish species, the first technique prior to ICoFMR-2020, especially Kadhafi who DNA analysis; helped in the arrangement of the manuscript 2) this technique is essential for species group with sexual di- and poly-morphism REFERENCES characteristics 3) body-shape of the specimen (photograph [1] C. Andrews, “The ornamental fish comparison) was still the same after trade and fish conservation,” J. treatment of staining and clearing; Fish Biol., vol. 37, pp. 53–59, 1990. 4) staining procedures are species, and size- [2] K. E. Lunn and M.-A. Moreau, dependent resulted in trials and errors “Unmonitored trade in marine before a fair, and standard product can be ornamental fishes: the case of achieved Indonesia’s Banggai cardinalfish

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(Pterapogon kauderni),” Coral [10] K. Fujita, “Nomenclature of Reefs, vol. 23, no. 3, pp. 344–351, cartilaginous elements in the caudal 2004. skeleton of teleostean fishes,” [3] A. L. Green and P. J. Mous, Japanese J. Ichthyol., vol. 36, no. 1, “Delineating the Coral Triangle, its pp. 22–29, 1989. ecoregions and functional [11] P. J. Gavaia, C. Sarasquete, and M. seascapes,” in Report on an expert L. Cancela, “Detection of workshop, held at the Southeast mineralized structures in early Asia Center for Marine Protected stages of development of marine Areas, Bali, Indonesia (April 30– Teleostei using a modified alcian May 2, 2003). Version, 2008, vol. 1. blue-alizarin red double staining [4] R. Froese and D. Pauly, “Fishbase: technique for bone and cartilage,” World wide web electronic Biotech. Histochem., vol. 75, no. 2, publication,” 2019. pp. 79–84, 2000. [5] G. R. Allen and T. B. Werner, [12] G. Dingerkus and L. D. Uhler, “Coral reef fish assessment in the “Enzyme clearing of alcian blue ‘coral triangle of southeastern stained whole small vertebrates for Asia,” Environ. Biol. Fishes, vol. demonstration of cartilage,” Stain 65, no. 2, pp. 209–214, 2002. Technol., vol. 52, no. 4, pp. 229– [6] J. Pini, S. Planes, E. Rochel, D. 232, 1977. Lecchini, and C. Fauvelot, [13] A. E. Gosztonyi, “The use of “Genetic diversity loss associated enzyme-based laundry ‘presoaks’ to high mortality and for clearing small vertebrates for environmental stress during the alizarin red staining of bony recruitment stage of a coral reef tissues,” Stain Technol., vol. 59, no. fish,” Coral Reefs, vol. 30, no. 2, 5, pp. 305–306, 1984. pp. 399–404, 2011. [14] R. Froese and D. Pauly, [7] S. Magadan, O. J. Sunyer, and P. “FishBase.” Fisheries Centre, Boudinot, “Unique features of fish University of British Columbia, immune repertoires: particularities 2010. of adaptive immunity within the [15] A. L. Rhyne, M. F. Tlusty, P. J. largest group of vertebrates,” in Schofield, L. E. S. Kaufman, J. A. Pathogen-Host Interactions: Morris Jr, and A. W. Bruckner, Antigenic Variation v. Somatic “Revealing the appetite of the Adaptations, Springer, 2015, pp. marine aquarium fish trade: the 235–264. volume and biodiversity of fish [8] W. A. Gosline, “The osteology and imported into the United States,” relationships of certain gobioid PLoS One, vol. 7, no. 5, p. e35808, fishes, with particular reference to 2012. the genera Kraemeria and [16] M. C. Leal et al., “Marine Microdesmus,” 1955. ornamental fish imports in the [9] W. Fischer and P. J. P. Whitehead, European Union: an economic FAO species identification sheets perspective,” Fish Fish., vol. 17, for fishery purposes: Eastern no. 2, pp. 459–468, 2016. Indian Ocean (Fishing Area 57) [17] H. R. Kuiter and T. Tonozuka, and Western Central Pacific “Pictorial guide to: Indonesian reef (Fishing Area 71) vol. 1. Food and fishes Part 1, 2, 3,” PT. Dive Dive’s. Agriculture Organization of the Denpasar, Bali, 2003. United Nations, 1974. [18] K. E. Carpenter and V. H. Niem,

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The living marine resources of the [22] F. J. Rohlf, “TPS dig v 1.4. Western Central Pacific. Volume 3. Department of Ecology and Batoid fishes, chimaeras and bony Evolution, State University of New fishes part 1 (Elopidae to York, Stony Brook, NY.” 2004. Linophrynidae), vol. 3. 1999. [23] K. F. Lagler, J. E. Bardach, R. R. [19] K. E. Carpenter and V. H. Niem, Miller, and D. R. M. Passino, The living marine resources of the “Ichthyology. John Willey and Western Central Pacific, Volume 4: Sons,” Inc., New York, vol. 396, Bony fishes part 2 (Mugilidae to 1962. Carangidae). Rome, Italy: FAO, [24] M. Eladio, “Sage: Symmetry and 1999. Asymmetry in Geometric Data [20] K. E. Carpenter and V. H. Niem, Version 1.05 (compiled09/17/08).” The living marine resources of the 2007. Western Central Pacific, Volume 5: Bony fishes part 3 (Menidae to Pomacentridae). Rome, Italy: FAO, 2001. [21] H. Sakata-Haga et al., “A rapid and non-destructive protocol for whole-

mount bone staining of small fish

and Xenopus,” Sci. Rep., vol. 8, no. 1, pp. 1–7, 2018.