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INTRODUCTION

Corythoichthys intestinalis, known commonly as the Scribbled pipefish, is a species of

marine fish in the family Syngnathidae, same family as seahorses. Most of these species has

been utilized as an ancient medication in the traditional Chinese medicine, particularly in

Southeast Asia (e.g. Japanese and Korean traditional medicine) according to Vincent (1996).

Pipefishes are usually captured for Chinese medicinal trade in the wild and considered as a

medicinally potent as seahorses, they are ground and mixed with various herbs and are used

for "whole-body" treatment, while the seahorses are generally used to target specific

ailments (Burhans, 2012). Due to the increasing demand as part of the Traditional Chinese

Medicine (TCM), pipefishes and seahorses population have become vulnerable and

depleting (Lam et al., 2016).

The Syngnathidae family (e.g. pipefishes and seahorses) has a role to play in increasing

and balancing vital energy flows within the body, as well as a curative role for ailments,

such as impotence and infertility, asthma, high cholesterol, goiter, kidney disorders, and skin

conditions (Moreau et al., 2000). Thus, both possess the same morphological features, such

as head and snout. Their mouths are small, narrow and toothless and create a vacuum to

suck tiny crustaceans (Van Wassenbergh et al., 2008). Pipefishes have slender, straight

bodies capable of good camouflage with grasses and weeds of the sea to protect themselves

from predators (Dawson 1985; Kuiter, 2003). Syngnathids do not have scales like most other

fish (LePage, 2012). Instead their long, rigid bodies seem to have armor plating that protects

them from predators and they are known to possess a plethora of microorganisms that

adhere to the cells of the flame cone covered by mucus caps found in their skin (LePage

2012; Bremer 1972).

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In line with this, hypothesis assumes that the therapeutic claims of C. intestinalis could be

attributed to the bacteria isolated from its epithelial surface and not the species itself.

Meanwhile, microorganisms are ubiquitous and they can be found everywhere. In

oceans, microbial life is accountable for 98 percent of the primary production and mediation

of all the biogeochemical cycles (Sogin et al., 2006). Bacteria are widespread in nature as

they can adapt to any extreme environmental conditions and perform various physiological

activities (Dash et al., 2012). Thus, it is apparent that wild marine organisms are

continuously exposed to the microorganisms present in water, sediment and in the

environment around the fish were there are greater contaminations coming from various

sewage and therefore external surfaces of the fish exposed and intimately in contact with

microorganisms. The organisms coming into contact with fish surfaces may be inhibited by

the resident microflora or by natural inhibitory compounds present on or in the fish (Austin,

2006). Hence, pipefishes are tend to live in various habitat in every parts of the ocean and

most prefer in marine or brackish water (Kuiter, 2003). Given this information there is a

high possibility that C. intestinalis were exposed intimately with various bacteria that adhere

and colonize their epithelial surface. Thus, the microorganisms that are harbored by the

pipefishes may support the source of TCM and not to the species itself since bacteria species

also have medicinal properties.

In related studies, Bacteria species having medicinal properties are taking popularity in

the field of research. Study shows that they have antiviral effects, kill pathogens and can

enhance the immune response of an organism (Balcazar et.al, 2006). One of these is the

Family Bacillaceae that have various medicinal properties and is used widely in

pharmaceuticals. This family offers higher acid tolerance and better stability during heat
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processing and low temperature storage (Bader, et al., 2012). They have also been shown to

possess pathogen exclusion, anti-oxidant, antimicrobial, immuno-modulatory (Lefevre et

al., 2015; Shobharani et al., 2015; Ripert et al., 2016) and food fermentation abilities

(Terlabie et al., 2006). Different species of Bacillus have also been used for the production

of additional nutraceuticals including vitamins (e.g., riboflavin, cobalamin, and inositol) and

carotenoids for the synthesis of several health supplements for human consumption

(Mohammed et al., 2014; Tanaka et al., 2014; Takano, 2016). Bacteria of clinical

significance that have been described to affect syngnathids are mostly limited to

Mycobacterium spp. and Vibrio spp. (Alcaide et al., 2001; Bombardini et al., 2006; Balcázar

et al., 2010; Martins et al., 2010; Balcázar et al., 2011). Mycobacteriosis in syngnathids is

usually caused by atypical or non-tuberculous species; Mycobacterium chelonae, M.

fortuitum and M. marinum (Koldewey 2005; LePage, 2012). Gross lesions are mainly

restricted to skin ulcerations, which can be mild or very severe with ulcers extending to

bone. Histologically, mycobacterial lesions are observed as granulomatous inflammation

within the central nervous system, kidneys and other organs leading to neurological deficits

and associated organ failure (Bombardini et al., 2006: LePage, 2012). Vibriosis can be

observed clinically as hemorrhagic to necrotic skin lesions in seahorses and pipefish

(Alcaide et al., 2001; Tendencia 2004; Bombardini et al., 2006; Balcázar et al., 2010;

Martins et al., 2010).

Moreover, in spite of the clinical significance of these bacteria, there is still a great

dispute of identifying it accurately since most researchers solely uses presumptive

identifications such as morphological characterization and biochemical tests in identifying

the isolated bacteria just up to its family level resulting to have an obscure and unreliable
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data. In this study, a tool is needed to identify and establish the credibility of the bacteria

present (Yan et al., 2013). By DNA barcoding of the DNA samples of bacteria, enable the

researchers to generate a phylogenetic hypothesis and a taxonomic identification up to its

species level (Barberán, 2010; J.Wang, 2012; Lebonah, 2014). Through this tool it helps the

researchers to distinguish precisely the bacteria present in the epithelial surface of C.

Intestinalis. After the bacterial properties are associated, bacterial groups present may prove

to have the therapeutic claims of the TCM, ruling out that the bacteria are the one that has

medicinal properties and not the seahorses itself, thus, saving the population of pipefishes.