Abstract
The copepod Pseudocaligus fugu (Siphonostomatoida: Caligidae) is a common parasite, collected from the body surface of the pufferfish Takifugu spp. in Japan. It was endowed with number of bacterial colonies, revealed through scanning electron microscopy (SEM) and by experiments. On the basis of bacterial colonies isolated, two types of isolates showed a high affinity for adhesion to the shrimp carapace. These two types were identified through 16S rRNA sequence analysis as Shewanella woodyi and Roseobacter sp. Representative isolates of these two adhesive bacteria were examined for tetrodotoxin (TTX) production by High Performance Liquid Chromatography (HPLC)–Fluorometric system, Gas chromatography–Mass Spectrometry (GC-MS) and Liquid Chromatography–Mass Spectrometry (LC-MS). From these results, it is evident that TTX and anhydroTTX are present in the isolate of Roseobacter sp. indicating the bacterial origin of TTX.
Introduction
In the marine fish aquaculture industry, parasitic copepods are causing serious consequences and important as pathogens particularly the “sea lice” belonging to the family Caligidae these copepod cause mortality or acting as disease agents, by creating a portal for entry of bacterial or other pathogens (Rosenberg, 2008). It is successful only through their feeding behavior on host mucus, tissues and blood. They could easily transmit disease -causing agents to other hosts (Cusack and Cone, 1986). Furthermore, it has been found that parasitic copepods are endowed with an abundance of bacteria on their exoskeleton (Venmathi Maran et al., 2011) which had been reported from the body surface of marine planktonic copepods also the attachment and abundance of bacteria can be host specific or site specific (Sochard et al., 1979). However, the significance of bacterial adhesion onto the surfaces of copepods or any living aquatic organisms has not been studied in detail.
Tetrodotoxin (TTX), known as pufferfish toxin, is one of the most potent non-protein neurotoxin because of frequent involvement in fatal food poisoning. It has a unique chemical structure and has specific action of blocking sodium channels of excitable membranes. The toxin derives its name from the pufferfish family Tetraodontidae, but past studies have revealed its wide distribution in both terrestrial and marine animals of vertebrate species which includes goby, newt and frog, and invertebrate species like octopus, gastropod mollusk, crab, starfish, nemertean and turbellarian (Noguchi et al., 2006). The origin of TTX and its biological significance in TTX-bearing animals have been investigated for a long time (Matsumura, 1995).
The puffers of the genus Takifugu (Actinopterygii: Tetraodontidae) is common in the Far East Asian countries, considered as delicacy and commercially important (Venmathi Maran et al., 2011). It has been proven that liver, ovary and other viscera of the fish are endowed with high level of TTX, and however varies depending on its species (Noguchi et al., 2006). Most of the puffers are infected with an ectoparasitic copepod P. fugu (Venmathi Maran et al., 2011), which has revealed accumulation of TTX (Ikeda et al., 2006). Interestingly, the abundance of rod-shaped bacteria that adhere to the body surface of P. fugu was noted. Although marine bacteria are likely to be a source of TTX, there has been no clear evidence to support the bacterial origin of TTX. On this basis, the adhesive bacteria on P. fugu were speculated that to be the producers of TTX. Thus the present study documents the characteristic feature of bacteria associated with the copepod, in producing TTX.
Observation of bacteria on copepod
The puffers Takifugu spp. (called as ‘fugu’ in Japanese) were collected from the central part of the Seto Inland Sea of Japan (Fig.1A). The infected parasitic copepod P. fugu was removed from the body surface of those pufferfish (Fig.1B). The copepods were desalinated by transferring into the distilled water for 1-3 hours and processed further to reveal the attachment of bacteria over the whole body of copepod through scanning electron microscope (SEM). Attached bacteria were observed throughout the body surface of the copepod P. fugu (Fig. 2A). These bacteria were rod-shaped, approximately 1.2–2.8 μm size in length with slimy materials and some bacteria were observed in dense masses on the cephalothorax (Fig. 2A).
Fig. 1A. Pufferfish infected with the parasitic copepod Pseudocaligus fugu; B. Ovigerous Pseudocaligus fugu, lu: lunule; ce: cephalothorax; gc: genital complex; ab: abdomen; es: egg sac.
Adhesion of bacteria
The bacteria present on copepods were cultured and isolated on Marine Agar 2216 at 25°C for 24h and subjected to the adhesion experiment using the shrimp carapace. There were several colony types attached on the carapace. Of those, only two types of bacteria showed a high degree of adhesion to the shrimp carapace. The level was evaluated based on the abundance and density of bacteria attached. The other types of bacteria showed less adhesion (attached sparsely). Control showed no bacterial attachment but slimy substances.
Fig. 2. Scanning electron micrographs of bacteria: A. Attached on the cephalothorax of the parasitic copepod; B. First type isolate; C. Second type isolate; D. The same at higher magnification.
Identification
The two bacterial isolate which showed high adhesion to the shrimp carapace were characterized by morphological and biochemical tests, and identified at generic level by 16S rRNA sequence analyses. The highly adhesive bacteria, were Gram-negative, oxidase-positive, short rods (1.2-2.8 μm in length). For the genus level identification, 16S rRNA sequence of both the isolates were amplified and sequencing was done. Through BLAST search, it was found that both of N- and C- terminus nucleotide sequences of the 16S rRNA gene of one of the bacterial isolate perfectly matched (100 % similarity) with only those of Shewanella woodyi (Fig. 2B). Other isolate, matched (99 % similarity) with Roseobacter sp. (Fig. 2C & D).
Analyses of TTX
Along with authentic TTX standards, both the extracts were examined for TTX and related substances by high performance liquid chromatography (HPLC). A small amount of these Fr. I and II mentioned above were subjected to gas chromatography –mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry. As authentic toxins, TTX standard containing several percent of 4epi-TTX and anhydrotetrodotoxin (anhyTTX) was prepared from the ribbon worm Cephalothrix sp. (Asakawa et al., 2000). HPLC analysis of Roseobacter sp., two peaks, with retention times (Rt) of 14.1 and 16.3 min, corresponded well to the retention times of TTX and anhyTTX, respectively. TTX and anhyTTX, were detected from the culture supernatant of Roseobacter sp., by HPLC and GC-MS. Mass spectral analysis showed a protonated molecule ion of (M+H)+ at m/z 320 and the other ion of (M+H-H2O)+ at m/z 302.
Discussion
Naturally-occurring rod-shaped bacteria are seen on both dorsal and lateral parts of the cephalothorax of P. fugu. It was reported that Vibrio spp. predominantly attached on the body surface of the planktonic copepods and in gut (Sochard et al., 1979) in contrast to our study on parasitic copepods. Multiplication of Vibrio takes place on the body surface, thereby enhancing the possibility of disease. After colonization by bacteria, multiplication takes place on the copepod surface rather than in the water samples. After multiplication, the cells are joined by additionally attaching cells and leads to the formation of microcolonies (Nagasawa, 1986).
The adhesion of bacteria to the parasitic copepod has considerable ecological significance. These bacteria, with their high adhesive ability, could colonize and degrade the chitinous material comprising the cuticle of the copepod. Usually, nutrient less location are selected by bacteria for their attachment. In the adhesion experiment, only two types of bacteria were virulent with high adhesive affinity to the carapace. The present method, experimental infection of bacteria-free shrimp carapace, is useful to determine whether the isolates have a high adhesive ability or not (Venmathi Maran et al., 2007).
The γ-proteobacteria S. woodyi, a Gram-negative, facultatively anaerobic, motile, short rod was first isolated from the squid ink and sea water samples from depths of 200-300m in the Alboran Sea. During the last decade, the genus Shewanella has received significant attention due to its important role in co-metabolic bioremediation of halogenated organic pollutants. The α-proteobacteria Roseobacter is a Gram-negative, aerobic, motile rod (Holt et al., 1994). Roseobacter species are ecologically significant because they play a major role in the production of toxin in dinoflagellates. When the non-toxic dinoflagellate Alexandrium tamaranse mixed with Roseobacter sp., it can produce the toxin.
The TTX-bearing animals are considered not to synthesize the toxin by themselves but to accumulate TTX through food chain starting from the marine bacteria that produce TTX. It is strongly suggested that bacteria like Vibrio, Pseudomonas, Shewanella, Alteromonas and others have been shown to produce TTX, although the produced amount of TTX was very small. In addition to TTX, bacterial production of anhyTTX was also reported. Although TTX-productivity of the bacteria isolated from the positive strain is much less, due to the culture conditions. Research on the mechanism of TTX synthesis and also on optimization of culture conditions in laboratory could be helpful. However, it was reported that Vibrio alginolyticus produced 213 MU of TTX in the medium containing 1% NaCl and 1% Phytone peptone in 72 hr culture. These facts suggested that bacteria are closely related to the toxification of pufferfish. More research is needed on aspects such as, whether puffer fish accumulate the TTX-producing bacteria through the food or come from the environment (Noguchi et al., 2006).
The bacteria Shewanella alga and Alteromonas tetraodonis isolated from a red alga Jania sp. produce TTX. Shewanella putrefaciens from the pufferfish Takifugu niphobles and many other marine bacteria isolated from TTX-bearing organisms have also been reported to produce TTX. In relation to that, the two high adhesive types of bacteria have been isolated from P. fugu for TTX. Though Shewanella is known as a TTX producer (Simidu et al., 1987), TTX and its derivatives are not detected in S. woodyi. On the other hand, Roseobacter sp. exhibited productivity of TTX Though some Roseobacter strains are known to be toxic, for the first time it is reported that the genus Roseobacter as a tetrodotoxin producer (Venmathi Maran et al., 2007).
In general, TTX produced in the host puffers mainly due to the bacteria and also by the ingestion of toxic diets. In this study, our earlier hypothesis was that the isolated bacteria would be a pathogen for the fishes and could act as a vector however, the results shown that they are not pathogenic. On the other hand, they are involved in the production of TTX. At present, Roseobacter strain is considered as a TTX producer and its analog, anhydro-TTX for the first time. Even though, other bacteria Shewanella was not positive in HPLC analysis, still presume that they could be involved in the production of TTX, since previous studies suggested Shewanella as the toxin producer. Although, we investigated only in limited strains for TTX, the results indicate that TTX-producing bacteria are quite widespread among various attached bacterial groups. The exact mechanism of the synthesis of TTX by bacteria and the role of TTX in the bacteria themselves are still unknown. It seems reasonable to postulate that TTX is synthesized solely by bacteria which could be transferred from the host puffer to the parasite. More research is needed to elucidate the mechanism of TTX synthesis and the role of TTX in bacteria (Venmathi Maran et al., 2011).
Acknowledgments
I am thankful to Professors S. Ohtsuka, T. Nakai and M. Asakawa, Hiroshima University, for their support during this study. I also thank Korea Research Council of Fundamental Science and Technology (KRCF) and Korea Institute of Ocean Science and Technology (KIOST) projects (PK08080) for financial support to prepare the article.
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