Fatty acids in an estuarine mangrove ecosystem

Fatty acids have been successfully used to trace the transfer of organic matter in coastal and estuarine food webs. To delineate these web connections, fatty acid profiles were analyzed in species of microbes (Azotobacter vinelandii, and Lactobacillus xylosus), prawns (Metapenaeus monoceros and Macrobrachium rosenbergii) and finfish (Mugil cephalus), that are associated with decomposing leaves of two mangrove species, Rhizophora apiculata and Avicennia marina. The fatty acids, except long chain fatty acids, exhibit changes during decomposition of mangrove leaves with a reduction of saturated fatty acids and an increase of monounsaturated fatty acids. The branched fatty acids are absent in undecomposed mangrove leaves, but present significantly in the decomposed leaves and in prawns and finfish, representing an important source for them. This revealed that the microbes are dominant producers that contribute significantly to the fishes and prawns in the mangrove ecosystem. This work has proved the fatty acid biomarkers as an effective tool for identifying the trophic interactions among dominant producers and consumers in this mangrove. Rev. Biol. Trop. 58 (2): 577-587. Epub 2010 June 02.

Mangroves are one among the most productive ecosystems and their productivity is attributed to litter degradation and efficient recycling of nutrients, which are supplied by both autochthonous and allochthonous inputs from natural and anthropogenic sources (Heald 1971, Odum & Heald 1975, Lee 1990, Kathiresan & Bingham 2001).Microbial processing of litter is an important mechanism for preserving nutrients and energy in the mangrove ecosystem (Fell et al. 1975, Fell & Master 1981, Raghukumar et al. 1994, Kathiresan & Bingham 2001, Rajendran & Kathiresan 2007).Mangrove litter of low nutritive value is decomposed and converted into nutrient-rich which serves as food for fishes (Odum 1971, Lee 1990, Kathiresan & Bingham 2001, Rajendran & Kathiresan 1998, 1999a, 2000, 2004& 2007, Ashton et al. 1999).The litter decomposition occurs typically in three, often-simultaneous phases: (i) leaching of soluble components; (ii) microbial oxidation of refractory components such as cellulose and lignin; and (iii) physical and biological fragmentation with microbial enrichment (Valiela et al. 1985).In these processes, the flow of carbon and nitrogen from primary producers to consumers takes place, but the flow is not clearly understood for the mangrove ecosystem.
Food web in mangroves is often under debate and contribution of the mangroves as primary producers is much clear even when advanced techniques like stable isotopes have been used (Canuel et al. 1995, Bouillon et al. 2002).Fatty acid trophic marker (FATM) concept is based on the observation that marine primary producers lay down certain fatty acid patterns that may be transferred conservatively to primary consumers (Dalsgaard et al. 2003).Fatty acids can be specific to each species and are often used as food web markers (Kiyashko et al. 1998, Kharlamenko et al. 2001, Dalsgaard et al. 2003).This FATM concept has been successfully used to trace the transfer of organic matter in coastal and estuarine food webs (Kharlamenko et al.1995, Napolitano et al.1997, Meziane & Tsuchiya 2000, Bachock et al. 2003, Hall et al. 2006).However, such studies are not available for estuarine mangrove ecosystems and associated flora and fauna of India.To fill this lacuna, the present study was made to trace the potential of fatty acids as biomarkers in identifying the trophic interactions, in such a way to delineate the food web in mangrove ecosystem.

MATERIALS AND METHODS
Experimental work: The present study was conducted from January to March 2007, post monsoon season of the study area.Senescent leaves of Rhizophora apiculata Blume and Avicennia marina (Forsk).Vier were collected from a mangrove forest, growing along the Vellar estuary (11º29'11'' N-9º46'17'' E) located in southeast coast of India.The leaves were shade-dried and packed at 50g in each of six nylon bags (15x10cm) and mesh size of 2mm.Pits were constructed linearly in the inter-tidal mangrove area with an equi-distance of 1m and a dimension of 1x1x1m.The temperature during the study period varied between 29°C and 34°C.The bags containing the leaves of three different species were submerged separately in each pit and one pit was kept as control (without any mangrove leaves).Three replicates for all the groups of pits were also maintained.Each bag was submerged in water by placing a stone weighing 0.25kg inside the bag.The inner side of each pit was covered with nylon net of 2mm mesh size to trap the juvenile fishes.This experiment was conducted for 70 days.Fishes attracted towards the litter bags were collected daily during the time of low tide by lifting the inner net.
Collection and processing of microbes: For microbial analysis, the decomposing leaf samples were taken randomly from each nylon bag during 0,20,30,40,50,60 and 70 days of experiment.They were cut in to small pieces and were washed thoroughly in sterilized seawater in order to remove debris on the leaves.For enumeration of total colony forming units, the Zobell's marine agar medium was used for total heterotrophic bacteria (THB) and the Winogradsky's medium for azotobacters.All the media components were purchased from Hi-media Chemicals, Mumbai, India.The microbes were enumerated by adopting spread plate method.The plates were incubated in an inverted position at 28±2°C.All the determinations were carried out in triplicates.After the incubation period of 2 to 3 days for THB and 7 to 10 days for azotobacters, the colonies were counted and calculated for colony forming units per gram leaf tissue.The dominant microbial colonies were identified based on morphological and biochemical characters (Buchanan & Gibbons 1974).
Collection and processing of prawns, finfish and mangrove leaves: The collected samples of prawns, finfish and mangrove leaves were brought to laboratory and washed in the tap water.The prawns and finfish were identified using standard guidelines (Fischer & Bianchi 1984, Paulpandian & Ramasamy 1991) and were used for further studies.
Analysis of fatty acids: Fatty acid profile was analyzed for the mangrove leaves of R. apiculata and A. marina at different days of decomposition, and also for the dominant species of microbes and prawns and finfish (muscles) associated with the decomposing mangrove leaves.Fatty acids were extracted by standard methods (Bligh & Dyer 1959, Meziane & Tsuchiya 2000).The extracts were saponified at 80°C for 90min with a mixture of sodium hydroxide and methanol in a ratio of 1:2.The fatty acid methyl ester (FAME) was prepared and analyzed by using a gas chromatograph (Agilent-GC 6890N) equipped with flame ionization detector.Capillary column HP Ultra 2 with 2m long and 0.2mm inner diameter, coated with 5% phenyl methyl silaxane and 0.33µm thicknesses was used.The rate of hydrogen carrier gas flow was maintained at 30ml/min.Most of the FAME were identified by using a calibration standard software of MIDI.Some of FAME peaks were identified by comparing their retention times with those of authentic standards (Supelco Inc.).
Statistical analysis: To evaluate significance in fatty acid composition, one way ANOVA test followed by Post hoc test was used with the help of SPSS, Version 13.0 (SPSS, Illinois).

RESULTS
Fish and microbial counts: The trends in fishes and microbial counts with decomposing mangrove leaves are depicted in the Fig. 1 and 2. In general, fish groups increased towards decomposing leaves up to 40 days, and then declined (Fig. 1).A similar trend was also recorded with counts of total heterotrophic bacteria and azotobacters (Fig. 2).The prawns, finfish and microbes behaved similarly with increasing days of leaf decomposition up to 40 days in both species of mangroves, R. apiculata and A. marina.
Dominant species attracted towards the decomposing leaves were Metapenaeus monoceros, Macrobrchium rosenbergii, and Mugil cephalus that belong respectively to penaeid prawns, non-penaeid prawns and finfish.Two predominant species of microbes isolated from the decomposing mangrove leaves were Azotobacter vinelandii and Lactobacillus xylosus.
Changes of fatty acid groups during decomposition of mangrove leaves: Levels of fatty acid groups in the mangrove leaves of R. apiculata and A. marina at 0, 40 and 70 days of decomposition are shown in table 1. Fatty acid groups varied significantly (p<0.05) with mangrove species.Among the fatty acid groups, saturated fatty acids were the most abundant one, which decreased significantly (p<0.05) with increasing days of decomposition.Long chain fatty acids did not show any significant change during leaf decomposition.Monounsaturated fatty acids were significantly (p<0.05)higher in decomposed leaves than fresh leaves.Branched fatty acids exhibited a different trend: they were significantly higher (p<0.05) at 40 days of decomposition than at 70 days and/or fresh leaves in both mangrove species.Polyunsaturated fatty acids decreased significantly (p<0.05) with leaf decomposition only in the case of A. marina, but, there was no such trend in R. apiculata.Levels of fatty acid groups in fishes and microbes: Levels of fatty acid groups in prawns, finfish and microbes associated with decomposing leaves of two mangrove species is shown in table 1.The fishes and microbes contained high levels of branched chain fatty acids followed by saturated fatty acids.However, these species were devoid of detectable quantities of long chain fatty acids.Polyunsaturated fatty acid groups were higher in fishes than microbes; in contrast, saturated fatty acid group was higher in microbes than fishes.The monounsaturated fatty acid group did not show any significant variation between microbes and fishes.
In the present study we observed an abundance of Azotobacter vinelandii and Lactobacillus xylosus in the decomposing mangrove leaves, besides Metapenaeus monoceros, Macrobrchium rosenbergii and Mugil cephalus.These fishes are well-known as a detritivorous organisms commonly occurring in the mangrove waters (Rajendran & Kathiresan 1998, 1999a, 1999b).Azotobacters are capable of fixing nitrogen and building protein in the form of microbial biomass in the decomposing leaves of mangroves, thereby enhancing palatability of detritus food to the fishes (Ravikumar 1995, Kathiresan & Masilamani 2005).The genus Lactobacillus is a beneficial microbial flora, present in guts of the fishes (Fuller 1989).
Composition of fatty acids changed significantly in the decomposing leaves of mangroves, from saturated straight chain fatty acids to monounsaturated fatty acids and more Branched Fatty Acids (BrFAs) (Table 1).These BrFAs were absent in fresh mangrove leaves, but detected during leaf decomposition and this was due to the growth of microbes that were rich in BrFAs.However, there was no change in the long chain fatty acids present in the mangrove leaves on decomposition (Table 1).
The branched chain fatty acids (15:0 and 17:0 ISO and ANTEISO) and the monounsaturated fatty acids (18:1 n-7) are known to be synthesized predominantly by bacterial communities (Jeffries 1972, Volkman et al. 1980) and consequently, they are useful as bacterial biomarkers and indicators of bacterial biomass (Parkes 1987).Levels of the bacterial fatty acids were higher in 40 days decomposed leaves than fresh leaves and/or 70 days  Values are mean ± standard deviation of 3 replicates in each group Values not sharing a common superscript differ significantly at p<0.05 (DMRT) decomposed ones.This could be attributed to the bacterial abundance in those leaves (Fig. 2).The fish assemblage was also higher with 40 days decomposed leaves.This link could be attributed to enrichment of microbial biomass coupled with enhancement of nutrients of the decomposing leaves, required for diet of the detritivorous fish (Rajendran & Kathiresan 2000, 2007).
Long chain fatty acids (LCFAs) with more than 24 carbon atoms are synthesized only by vascular plants (Hogg & Gillan 1984), and consequently the LCFAs can be used as biomarkers of vascular plant origin in animal tissues.The LCFAs are present mainly in epicuticular wax of the vascular plants including mangroves (Meziane et al.2002, Mfilinge et al. 2003).In the present study, LCFAs were found as common constituent in the leaves of the two mangrove species at all the days of decomposition (Table 1).There was no significant change in LCFAs during mangrove leaf decomposition as they are resistant to microbial transformation (Wannigama et al. 1981, Mfilinge et al. 2003).The LCFAs could not be detected in the tissues of prawns and fish.Thus the LCFAs do not contribute to the consumer fishes, and this fact makes them weak biomarkers.In support of this, Hall et al. (2006) have observed that crabs do not assimilate the LCFAs due to lack of necessary enzymes for break down of LCFAs and hence release them through feces.
As microorganisms usually do not accumulate large lipid reserves, fatty acid trophic marker (FATM) may be of less relevance to trace trophic link in ecosystems.However, despite their lack of storage lipids, heterotrophic bacteria, which contribute significantly to these systems, are still recognizable by specific fatty acids.Considering the fast turnover rates of microorganisms, Dalsgaard et al. (2003) therefore hypothesize that FATM may help resolve trophic interactions in microbial loop food webs and they support the strengthening of fatty acid research in this area, also by Strom (2000), recognizing the importance of these systems in the global carbon budget.The present study has also reiterated that the bacterial fatty acids are important component which contribute significantly to the food web of mangrove ecosystem.

ACKNOWLEDGMENTS
The authors are thankful to authorities of Annamalai University for providing facilities and to Ministry of Environment & Forests, Govt. of India, New Delhi for providing financial assistance.

Fig. 1 .
Fig. 1.Abundance of penaeids, non-penaeids and finfish at different days of leaf decomposition in two species of mangroves (A) Rhizophora apiculata; and (B) Avicennia marina.

Fig. 2 .
Fig. 2. Total heterotrophic bacteria (THB) and azotobacters at different days of leaf decomposition in two species of mangroves (A) Rhizophora apiculata; and (B) Avicennia marina, expressed in colony forming units per gram leaf tissue.

TABLE 2
Concentration of fatty acid biomarkers (% of total) in microbes, prawns, and fish predominantly associated with 40-days decomposed leaves of two mangrove species (Rhizophora apiculata and Avicennia marina)