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EXOSYMBIONTS OF POLYCHEATS FROM KERALA COAST –POTENTIAL ANTAGONISTIC RESOURCE ?

Mathan MageshDepartment of Aquatic Biology & Fisheries, University of Kerala,Thiruvananthapuram, 695581, India.Email: maheshmathan2008@gmail.com

Abstract: Biological diversity of every ecosystem currently receives attention for conservation and sustainableutilization due to the increasing threats on the ecosystems from anthropogenic interventions. Proper samplingand identif ication of organisms are necessary to monitor biodiversity at any level. This paper documents Polychaete-associated Streptomycetes and anaerobic photosynthetic Purple Sulphur Bacteria (PSB) in the estuarine ecosystemsof Kerala coast, India. The microbial population from the gut of Namalycastis abiuma species complex, Namalycastisjaya and their inhabiting soil samples were studied. The occurrences of these exo-symbionts were positively correlatedwith seasons. Among the polychaetes, Namalycastis jaya harbored the maximum PSB (56 ± 4.7×04 ml-1) andStreptomyces density (18 ± 1.67) was recorded in the months of April and May. Even though soil showed considerablyhigher microbial density, only the extracts of the polychaete-associated microbes showed more potential than soilcounterparts against aquatic fungal f ish pathogen, Saprolegnia parasitica. Compared to PSB, Sreptomyces sppexhibited higher activity. Most potential strains were identif ied by using standard morphological and physiologicaltechniques. Mass production of these microbial compounds and its usage as other pathogens remains to be studied.

Key words: Namalycastis jaya, Streptomycetes, Photosynthetic bacteria, Gut association, Antifungal.

Received on: 10 October 2013, accepted on: 12 December 2013

INTRODUCTION

The interaction of microorganisms with aquaticbiota is unique and diverse. Microbes presentin the gut of polychaetes indirectly indicatetheir feeding habit, food preference and thetype of nutrients required for their growth.This in turn, will enable one to infer theirimportance in nutrition. The association ofmicrobes with tissues of the gastrointestinaltract of animals during evolution has resultedin a balanced relationship between residentmicrobes and the host. Numerous biochemical,physiological and immunological features, areconsidered to be the intrinsic characteristicsof animal species, are actually the responses bythe animal to the physical presence andmetabolic activities of the normal indigenousmicro biota. This microbial challenge hasmodif ied the course of evolution in animalsresulting in the selection of animal microberelationships. The gastrointestinal microbialcommunity is considered by its highpopulation density, wide diversity andcomplexity of interactions. Gut associatedbacteria are widespread within nearly allinvertebrate animals.

Phototrophic sulphur bacteria (PSB) are aspecialized group of microorganisms thatcarryout the oxidation of sulphide underanaerobic conditions (Pfennig, 1977; Pfennigand Truper, 1981; Sucharita et al., 2010). It isshown that phototrophic bacteria play animportant role in the preservation of themangrove environment (Kabayashi et al., 1978).The Genus Streptomyces was proposed byWaksman and Henric (1943) for aerobic andspore forming Actinomycetes. Streptomycetesare the largest antibiotic producing groups inthe microbial world discovered so far. A generalrevision of the Genus Streptomyces was madewith the ISP (International StreptomycesProject) Shirling and Gottlieb, 1966) as a resultof which in the approved list Streptomyces spp.remains just 459 of the total initially described1000. Studied on the occurrence anddistribution of antagonistic Streptomycetes havebeen carried out in different region in aroundthe world (Waker and Colwell, 1975; Okazakiand Okami, 1976; Dharmaraj and Alagarsamy,2009; Magesh and Dhevendaran, 2012). Themarine Polychaeta, Namalycastis jaya, Magesh

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2012 and Namalycastis abiuma species complex(species group) were also collected from sameregion already reported from the southern coastof Kerala, India. The association betweenpolychaetes N. jaya of with microorganisms arecarryout in this f irst time and the results showedvoluble sources of antibiotics against lifethreatening diseases.

MATERIAL AND METHODS

Location and Sample collection

The study was carried out from January 2008to December 2008 (12 months) in retting zonelocated at Murukumpuzha of Trivandrum coast.A number of polycheates were collected andtransported to laboratory in sterile and livecondition with in sterilized polythene bag andbottles.

Morphological Observation of Polychaetes

The polychaetes associated with retting coconuthusk were collected by breaking the coconuthusk with hammer and chisel. Collection andidentif ications were made by previouscontributions (Day, 1967 (1 & 2); Glasby 1999;Magesh et al., 2012).

Enumeration of gut content from selectedworm

Body surface of the live polychaetes werewashed with sterile water and swabbed with 60-70% ethanol for surface sterilization. Then thepolychaetes were homogenized with steriledistilled water. Worms were dissected and thegut behind the gizzard was equally divided intothe three parts. The gut contents of the middleregion were released by squeezing the intactworms. Gut contents of the same species werepooled to obtain samples (1 gm) taken for theisolation of Streptomycetes and anaerobic PSB.

Enumeration of Photosynthetic bacteriaand Streptomycetes from polychaete

1 ml of 10-4 serially diluted polychaete sampleswere transferred into sterile petridishescontaining selective medium, Glycerolasparagines agar (Pridham and Lyons 1961) andincubated at 28°C for 7-10 days. Remaining partof homogenized materials were transferred intosterile screw capped test tubes containingselective, PSB enrichment media (Pfenning

Lippert, 1966; Arunasri et al., 2005) andincubated with continues illumination of 200watt bulb at room temperature for 10-15 days.

Identification of PSB

After 10 days of incubation, the positive tubes(green, red or purple) taken for furtheridentif ication using the standard methods(Pfenning Lippert, 1966; Arunasri et al., 2005).

Identification of Streptomycetes

After 7 days of incubation the total numbersof Streptomycetes colonies in each petridisheswere counted using standard plate counttechnique. The identif ications are made bystandard procedures (Shirling and Gottieb,1966; Locci, 1989).

RESULTS AND DISCUSSION

Polychaete, Namalycastis spp.

The positive association of Streptomycetes andPSB population in the gut of polychaete fromretting soil of Murukkumpuzha, Kerala wasnoted. However, studies on the ecology ofpolychaete gut associated microbes especially,Streptomyces spp. and Marichromatium spp(PSB) are scarce. In the present investigation,selected polychaetes were identif ied asNamalycastis abiuma species complex andNamalycastis jaya by using standard proceduresand taxonomic key (Magesh et al., 2012). Allpolychaete samples shown positive associationwith Streptomyces spp. and Marichromatiumspp. Among these isolates, selected 40Streptomyces spp. and 10 PSB showed antifungalactivity against aquatic f ish pathogen,Saprolegnia parasitica. The antagonistic strainswere identif ied in genus level.

Polychaete associated Streptomycetes

In this present study, the Colony Forming Unitof Actinomycetes population in polychaete,Namalycastis abiuma was ranged from 2 ± 0.12to 17 ± 1.26. The minimum CFU of 2 ± 0.12 wasrecorded in the month of July and maximumColony forming unit was recorded in the monthof March. In the polychaete, Namalycastis jayathe minimum population density (2 ± 0.10) wasrecorded in July and the maximum populationdensity (18 ± 1.67) was recorded in the monthof April. In the sediments, the minimum density

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(2 ± 0.15) was recorded in August and themaximum density (21 ± 2.10) was observed inMarch (Fig. 1).

The correlation was analysed betweenActinomycetes population from Namalycastisabiuma species group and Namalycastis jayawith soil. The signif icant was at 5% level. The

correlation between Actinomycetes populationof Namalycastis abiuma species group andsediment samples (Fig. 2) were moderatelypositively correlated (r- 0.6208). The correlationbetween Actinomycetes population ofNamalycastis jaya and soil samples revealed thatthey are signif icantly and positively correlated(Fig.3) with lower level (r - 0.3113). Generally,Actinomycetes populations varied broadly intheir density in different body parts ofinvertebrates and most marine invertebratesharbour microorganisms within their tissueswhere they reside in the extra and intracellularspaces (Dhevendaran, 1984).

Polychaete associated PSB

The density of PSB population isolated from thepolychaete worms namely, Namalycastisabiuma species group and Namalycastis jayawas shown in the Fig. 4. In Namalycastis jaya,the maximum population (56 ± 4.7 × 04 ml-1) andminimum population (6 ± 0.78 × 104ml-1) wasrecorded in the month of April and Novemberrespectively. During sample collectionpolychaete were not recorded in the month ofJuly, August, October and December. InNamalycastis abiuma species complex, theminimum density (6±0.52×104 ml-1) was recordedin December and the maximum density (49±4.67×104 ml-1) was observed in March. In sediments,the maximum population (61±5.20×104 g -1) wasrecorded in the month of May and the minimum(11 ± 0.96×104g-1) in August. In June, no bacterialpopulation was recorded in all samples. On theother hand, it has been reported thatMarichromatium purpuratum was able to

Fig. 1. Actinomycetes population in polychaetes,N. abiuma species complex, N. jaya & Soilduring 2008

Fig. 2. Correlation between Actinomycetespopulation in N.abiuma species complex & Soilduring 2008

Fig. 3. Correlation between Actinomycetespopulation in N. jaya and Soil during 2008

Fig. 4. PSB population in polychaetes, N. abiumaspecies comples and N. jaya. & in Soil during 2008

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produce antimicrobial compounds with a broadspectrum activity against f ilamentous fungi, yeastas well as gram-negative and gram-positivebacteria (Burgess et al., 1991).

The correlation was analysed between PSBpopulations from Namalycastis abiuma speciesgroup and Namalycastis jaya with soil and itis signif icant at 5% level. Fig. 5 shows thecorrelation between the PSB populationsisolated f rom Namalycastis jaya and soilsamples and it showed moderately positivelycorrelated (0.4549). Fig. 6 shown thecorrelation between PSB populations ofNamalycastis abiuma species complex and soilsamples and it also showed moderatelypositively correlated (r - 0.4581). Relatively,similar f indings were also recorded in thesediments of Cochin (west coast of India) andVellar estuary of East coast of India byChandrika et al. (1990) and Dhevendaran,(1984) respectively.

Antagonistic activity

In the present study, primarily 177Actinomycetes and 39 PSB strains (polychaete,gut associated) were randomly selected andtested for antifungal activity against the fungi,Saprolegnia parasitica (MTCC 718). Totally 50strains (40 Streptomyces spp. and 10 PSB)showed antifungal activity. Similarly suchoccurrence (Fish associated Actinomycetes) ofActinomycetes could be benef icial to the f isheseither for production of enzymes and vitaminswhich are useful for f ishes (Sivakumar et al.,2007). There are possibilities for some metabolitef rom marine animals which might haveoriginated from symbiotic bacteria within thehost rather than the host animals. This has beendiscussed intensely for many years (Proksch etal., 2002; Piel, 2004). Gut contents harbouredmore population density when compared toother parts of the f ishes and other invertebrateslike polychaetes. The reason for this could bethat the gut is exposed to variety of microbialpopulations along with diverse food particlesand more numbers of microbes can be retainedin the guts. Streptomycetes isolates showedGrey, green, cream and white colour of aerialmycelium and cream, pale yellow, yellow, pinkand black on the substrate mycelium. All strainsare Gram positive, non-motile and rectiflexiblespore chain and only 7 isolates were exhibitedpositive results of melanin production. Basedon the morphological obsevation, the selectedPSB isolates were (All strains are Purple redcoloured, rod shaped, gram negative and motilebacteria) identif ied as Marichromatium spp. Thedetails of identif ication of microbes weredescribed in materials and method of this paper.

The mode of association was not well knownbut both the host and microbes are benef ittedbecause it has been believed that theStreptomyces and Marichromatium (PSB)were known to control pathogens (Burgess etal., 1991; Magesh and Dhevendaran, 2012;Selvakumar and Dhevendaran, 2011) and itwas assumed that polychaete gut might serveas an excellent habitat for the microbes.Marine invertebrates have developed highlyspecif ic relationship with numerousmicroorganisms and these associations are ofrecognized ecological and biologicalimportance (Strahl et al., 2002).

Fig. 5. Correlation between PSB population inN.abiuma species complex & Soil during 2008

Fig. 6. Correlation between PSB population inN. jaya & Soil during 2008

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CONCLUSION

The symbiotic relationship of microbes with theaquatic organisms preferably the Polychaeteswould be of much importance for thebiotechnological applications especially Antibioticproduction. Streptomyces spp. and PSB wereidentif ied by genus level. Exosymbionts, bothStreptomyces spp. and Mrichromaium spp. ofpolycheats from Thiruvananthapuram, Keralacoast undoubtedly proved a promised source ofpotential antagonistic strains. Further researchon mass production of these Antagonisticmicrobial compounds and its usage as otherpathogens remains to be studied. However, thesemicrobial sources are potent enough to utilizethem on the commercial scale in the days tocome.

ACKNOWLEDGEMENTS

Thanks to Christopher J. Glasby, Museum andArt Gallery of the Northern Territory, Australiaand Dr. A. Bijukumar, Department Head,Aquatic biology and f isheries, University ofKerala, India for their constant support.

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