First Record and Morphological Identification of the Seahorse Hippocampus kelloggi Great Seahorse Jordan and Snyder (1902) in Coastal Waters of Pakistan

The first record of seahorse Hippocampus kelloggi commonly known as the great Seahorse from the coastal waters of Pakistan based on morphological and morphometric characteristics. The Seahorses belong to family Syngnathidae and about 50 species have been reported across the world. They have been found to inhabit coral reefs, seagrass beds and also coastal mangroves. All seahorse species ( Hippocampus sp .) are vulnerable and according to the global conservation status of Hippocampus kelloggi is also among them. H. kelloggi is listed as data deficient since 2003 and then classified as IUCN Red List (http://www.iucnredlist.org) Category & Criteria: A2cd version 3.1 (2017). The Hippocampus kelloggi previously recorded from the Indo Pacific regions, including China; India; Indonesia; Japan; Malaysia; Philippines; Thailand; United Republic of Tanzania; Viet Nam. The current study confirmed the presence of H. kelloggi from the coastal waters of Pakistan.


Introduction
This is the first record of seahorse Hippocampus kelloggi commonly known as the great seahorse from the coastal waters of Pakistan as provide morphological identification and range extension of the species. The seahorses are representative of family Syngnathidae that includes, pipe fishes, pipe horses and sea dragons. There are about 50 species reported across the world and have been found to inhabit various home ranges, i.e., Sea grass beds, coastal mangroves, and coral reefs, although characterized as sedentary fish species with patchy distribution [1][2][3]. H. kelloggi previously described for the low mobility, fecundity, prolonged parental care, mate fidelity and reproduction information [4][5][6]. In Philippines, H. kelloggi reported at maximum depth of 152 m and associated morphological revisions of seahorse have been also carried out [7]. According [5].
Dried seahorse used in the traditional Chinese medicine market. The trade of seahorse observed throughout its distribution range bycatch during shrimp trawling [10][11][12][13]. Whereas currently legal and illegal trade also enduring (UNEP-WCMC 2012 a, b) that why the stock was substantially declines between 1995 and 2000 [6], in addition Singapore, United Arab Emirates and Hong Kong import 4.34 tons of sea horse from India during the year 2001-200 [5,[14][15][16].
According to 70% decline in Seahorse population included H. kelloggi [12,15,17] due to the over exploitation at the Palk Bay and Gulf of Mannar in the South-east coast of India, and to overcome this issue, India Government prohibited the trade and fishing of representatives of family Syngnathidae as a protected species under the Schedule I (Part 2A) of the Indian Wildlife (Protection) Act, 1972 through a Notification No. 1-4/95 WL1 dated 11 July, 2001, [12,15], UNEP-WCMC 2012b. Molecular identification of dried seahorse and pipe fish was done, that was sold on the Chinese market by using two mitochondrial COI and 16S rRNA genes and revealed the exploitation of representatives of family Syngnathids by illegal and not regulated trade. Previously, there is no record of sea horse species from the coastal water of Pakistan, the current study provides the evidence for the presence and range extension of H. kelloggi from the coastal water of Pakistan [18,19].

Hippocampus kelloggi Jordan and Snyder 1902
Common names Kellogg

Colour/pattern
Greenish brown in color.

Morphological Identification and Morphometric Measurements
The description of the seahorse was made based on the observation of a single specimen which was caught in a rock pool at Mubarak village, rocky cum sandy shore found in the rocky pool at a depth of four feet, from the coastal waters of Pakistan ( Figure  2), on dated 18 th April 2019. Morphological identification based on the taxonomic characters and all Morpho-measurements were made according to the standard protocol. Length of specimen is given as standard length (from the tip of snout to end of the tail) up to the nearest 0.1 mm. The comparison of morphometric and meristic character was made based on the earlier literature [7]. Later the Morphometric characters were examined and compared with earlier observations ( Table 2). As per earlier description, this species shares the characters similar to the Hippocampus spinosissimus with the presence of distinct high coronet and with five short spines.

Diagnostic characteristics (Figure 3 & 4).
Seahorse species have elongated body encased in thick trunk rings, back-pointing, small pectoral fins and a single dorsal fin, thick snout with no teeth, maximum recorded height is 28 cm, with tiny white spots running in vertical lines. Body narrows with thick snout (tube like), deep head. The species characterized by the distinct high coronet, with five short spines with high plate in front of coronet and a prominent rounded eye spine. They have long, slightly backward pointing, rounded cheek spine. Spines low and rounded; slightly better developed in younger specimens, but still blunt tipped. Other distinctive characteristics: Long, slightly backwards-pointing, rounded cheek spine; deep head; narrow body; thick body rings; prominent, rounded eye spine; thick snout.

Taxonomic Remarks
According to literature [5], Pakistan included in the distribution range of H. kelloggi however, there is no previous record of H. kelloggi from the coastal waters of Pakistan, the current study is the first detailed study and describe the range extension of H. kelloggi.

SDS and Native PAGE (Polyacrylamide gel electrophoresis) of General protein (GP) EC (nonspecific)
Seahorses are economical, commercially important and traded internationally, used in traditional Chinese medicine and its derivatives for the treatment includes: the respiratory disorders (asthma), sexual dysfunctions (Erectile dysfunction), and general lethargy due to the insufficient sleep, overexertion, overburdening, stress, lack of exercise, improper nutrition, boredom, and pains [10]. During the present study, an estimation was made for the various size of proteins by using the electrophoresis technique. Two types of electrophoresis were performed, SDS for general protein and Native PAGE for the specific protein (specific protein by using the specific subtract of enzyme). SDS and NativePAGE (Polyacrylamide gel electrophoresis) of General protein (GP) EC (non-specific) banding pattern was estimated in muscle tissue of K. kelloggi. Approximely 250-300 mg was removed and homogenized in 1 ml of Tris Citrate II extraction buffer, and the sample was centrifuged at 15,000 rpm for 15 minues, supernatant was used as protein source.
Electrophoresis was performed [20] accordingly, whereas the gel was stained (Coomassie Brilliant Blue) for General protein (GP) EC (non-specific), Bovine (BSA serum albumen) used as standard protein. Electrophoresis was performed in vertical native polyacrylamide gels (Native-PAGE) as described by [20]. A total of four protein bands were observed in SDS PAGE electrophoreis ( Figure 5), the Rf values ranges from 0.08 to 0.451, the standard band (5 KDa standard of BSA) was observed in between gel that showed that the higher and lower molecular weight protein was observed in H. kelloggi sample whereas a total of five protein bands were observed in Native PAGE electrophoresis ( Figure 5), the Rf values ranges from 0.098 to 0.633. The further detailed biochemical study and characterization of protien can reveal some intresting results from these species.