Journal of Chemical Ecology, Vol. 15, No. 4, 1989
CHEMICAL COMPOSITION OF DEGRADING MANGROVE LEAF LITTER AND CHANGES PRODUCED AFTER CONSUMPTION BY MANGROVE CRAB Neosarmatium smithi (Crustacea: Decapoda: Sesarmidae)
J. N E I L S O N l a n d G E O F F R E Y
N. RICHARDS 2
Department of Chemistry and Biochemistry James Cook University of North Queensland Townsville, Queensland 4811, Australia (Received April 20, 1988; accepted June 13, 1988)
Abstract--The leaves of the mangrove Ceriops tagal contained 3.2-4.1% (all percentages relate to dry weight) of D-l-O-methyl-muco-inositol previously unreported in mangroves. They consisted of 37% aqueous acetonewater-soluble material, 18% water-insoluble polysaccharides, and ca. 50% polyphenols, which include soluble and insoluble tannins and lignin. The polysaccharide component sugars were glucose, arabinose, uronic acids, mannose, xylose, galactose, and rhamnose in the proportions 28:26:22:10:7:5:2, respectively. The leaves were pectate rich, and the low level of glucan was presumed to consist mainly of cellulose. After four weeks of biodegradation, ca. 60 % of the acetone-water-soluble material was lost from the leaves. Degradation processes greatly altered the polysaccharide components in the leaves. Pectates were rapidly degraded, while other polysaccharides, although reduced proportionately, resisted degradation at about the same level, and all component sugars were found in the 8-week-old leaves. "Apparent lignin" contents increased from 15 to >30% during biodegradation up to eight weeks. The yields of the major fractions in corresponding fecal material from Neosarmatium smithi showed a similar trend to the diets. An enrichment of the insoluble residue was noticeable due to the digestion of dialyzable material. The fecal carbohydrate content was greatly reduced (7-11070) and the "apparent lignin" increased (27-39070)due to its resistance to degradation. All dietary polysaccharide component sugars were found in the Present address: Group Research Unit, Associated Pulp and Paper Mills, Burnie, Tasmania, 7320, Australia. 2Present address: Wood Chemistry Laboratory, University of Montana, Missoula, Montana, 59812. 1267 0098-0331/89/0400-1267 $06.130/0 9 1989 Plenum Publishing Corporation
NEILSON AND RICHARDS
fecal residues, including some uronic acids. The leaves also contained a readily water-soluble fraction (15%) which consisted of pectates strongly complexed with proanthocyanidins. Key Words--Mangrove, detritus, tannins, carbohydrates, polysaccharides, D-l-O-methyl-muco-inositol, Neosarmatium smithi.
The degradation of mangrove leaf litter is a dominant factor in the ecology of mangrove swamps, and the detritus is an essential nutrient source for the ecosystem food chain (Odum and Heald, 1975). For example, litter may be decomposed by microorganisms (Bunt et al., 1979; Cundell et al., 1979; Benner and Hodson, 1985) and crustacea (Malley, 1978; Leh and Sasekumar, 1985; Giddins et al., 1986; Poovachiranon et al., 1986; Robertson, 1986). Detailed chemical studies have been made on leaf composition with respect to its elements (Spain and Holt, 1980; Kotmire and Bhosale, 1980), organicslipids, sterols, hydrocarbons (Hogg and Gillan, 1984; Ghosh et al., 1985), and low-molecular-weight carbohydrates (Popp, 1984). Other studies (Jamale and Joshi, 1976; Bhosle et al., 1976; Sumitra-Vijayaraghavan et al., 1980) have used proximate analyses only to obtain information about leaf composition and decomposition. However, there have been few specific studies (Benner et al., 1984) on the detailed chemistry of mangrove leaf cell walls, in particular the polysaccharides and their primary degradation processes. Understanding the factors that control the degradation of the walls is of great ecological significance. Plant cell walls inherently resist digestion in animals, due largely to their water-insoluble constituents. For example, only half the wall polysaccharides are digested during passage through a ruminant (Dekker et al., 1972). This study looks at specific chemical changes occurring in leaf litter degrading at the mud surface (Giddins et al., 1986) and also provides information on the detailed composition of undigested leaf material after passage through the mangrove crab N. smithi (Crustacea: Decapoda: Sesarmidae). Interest also focused on the low-molecular-weight carbohydrates (or free sugars), since they arguably play a significant role in osmotic adjustment in halophytes such as mangroves (Flowers et al., 1977; Popp, 1984).
METHODS AND MATERIALS
General. The conditions for drying samples were: 1 mm Hg, P 2 0 5 , 90% recovered as glucose when analyzed by the method.
RESULTS AND DISCUSSION
Green (66 % moisture) and senescent (68 % moisture) leaves were first fractionated using a classical approach for isolation of plant polysaccharides (Blake and Richards, 1970). Plant material was isolated and separated sequentially into
NEILSON AND RICHARDS TABLE 1. PRECISION OF RELATIVE RESPONSE FACTORS IN ALDITOL ACETATE PROCEDURE RELATIVE TO METHYL t~-D-GLucOPYRANOSIDE (INTERNAL STANDARD) AS UNITY
Rharnnose Fucose Arabinose Xylose Me-a-glucose Mannose Galactose Glucose
2.210 1.714 0.983 1.068 1.000 1.182 1.113 1.250
2.113 1.627 0.884 0.987 1.000 1.180 1.165 1.296
2.270 1.747 0.994 1.106 1.000 1.095 1.047 1.184
Average +_ standard deviation 2.198 1.696 0.954 1.054 1.000 1.152 1.108 1.243
+ + + + _ + + +
0.079 0.062 0.061 0.061 0.000 0.041 0.059 0.056
operationally defined fractions, namely, hot water (HW), cold water (CW), ammonium oxalate (pectic substances), chlorite (removes lignin, which produces a cellulose and hemicelluloses fraction) and sodium hydroxide (hemicelluloses) (Table 2). Apparent lignin was also measured using 72 % sulfuric acid (Adams, 1965). Similar fraction yields were obtained for the two types of leaves except for CW extracts (9.1 vs. 16.9%) and lignin (17.9 vs. 12.1%). Since both CW extracts contained similar proportions of carbohydrate (6.2 vs. 5.4% TABLE 2. DISTRIBUTION OF COMPONENTS IN MATURE GREEN AND SENESCENT LEAVES OF Ceriops tagal OBTAINED BY TRADITIONAL FRACTIONATION PROTOCOL (BLAKE AND RICHARDS, 1970)
Leaf dry wt (%) Fraction
80% ethanol solublea Chloroform soluble Cold water soluble Hot water soluble Pectic substances Lignin Hemicelluloses A Hemicelluloses B a-Cellulose
38.8 0.5 9.1 4.6 3.8 17.9 0.2 4.6 10.6
36.7 0.2 16.9 5.3 6.3 12.1 0.6 4.3 8.7
Includes low-molecular-weight carbohydrates (free sugars), salt(s), and water-insoluble material.
D E G R A D I N G M A N G R O V E L E A F LITTER
of leaf dry weight), this major difference could only be attributed to a change in the noncarbohydrate, predominantly proanthocyanidin component (see later). The major fraction was obtained as the 80% ethanol extract (ca. 37 %), and this material was analyzed for free sugars. Bonded-phase HPLC analysis of the water-soluble extract from 80% ethanol-soluble material showed two well-resolved peaks, I and II (area ratio 4: 1), which coeluted with fructose and sucrose standards, respectively. TLC using detection systems 1 and 2, confirmed the presence of sucrose but not fructose or glucose. Both systems detect fructose, but, although system 1 showed apparent fructose in the sample, system 2 did not. GLC analysis of the TMS derivatives of the water-soluble components showed two components: sucrose and a compound almost coeluting with authentic fructose, retention time relative to inositol (Rti), 0.79 vs. 0.78. Following isolation by preparative HPLC, [1H]NMR spectroscopy confirmed that peak I was not fructose. The presence of a singlet resonance at 63.40 indicated a methoxyl group, and the remaining partially resolved resonances at 64.03-3.54 suggested that peak I may be a monomethyl cyclitol (Angyal and Odier, 1983) and in particular 1-O-methyl-muco-inositol (Angyal and Kondo, 1980). Integration of the spectrum supported this identification. A [1H]NMR spectral comparison showed that peak I was not pinitol, which has a methoxyl group shift of 63.59. Furthermore, there was little similarity in the respective ring proton patterns. [~3C]NMR spectroscopy of peak I with complete decoupling showed that the molecule had a plane of symmetry. Chemical shifts relative to acetone at 630.6 were observed at: 82.1 (_CC-OCH3),72.6 (2 • C), 70.4, 70.2, 68.5, and 58.1 (OCH3). Peak I was observed to be optically active [ot]~~ = - 2 5 . 2 ~ (c 0.28, water), and these combined data suggested that the unknown compound was (-)-l-O-methyl-muco-inositol (MMI) (Angyal et al., 1967). Comparative [~3C]- and [1H]NMR spectroscopy with authentic MMI gave unequivocal identification. Quantitative HPLC analysis with relative responses determined from authentic compounds gave only MMI and sucrose as 3.2% and 0.8% of the total dry weight of fresh green C. tagal leaves. Senescent leaves were shown to contain MMI (4.1%) and sucrose (0.8 %); a minor peak with retention time relative to MMI (0.80) was also observed but not identified. MMI has been found in most gymnosperms and some angiosperms (Dittrich et al., 1971). Its occurrence has not been reported in mangrove species, although a significant amount of pinitol has been reported in various mangrove species including C. tagal (Popp, 1984). However, GC alone was used for the identification of the "pinitol." Since it was not possible to resolve pinitol from MMI using the various chromatographic conditions described in our work, the now positive identification of MMI, by NMR spectrometry, intimates that Popp was mistaken in designating the O-methyl derivative as pinitol.
NEILSON AND RICHARDS
Anomalies in anticipated carbohydrate compositions were observed in the major operationally defined fractions obtained using traditional methods, e.g., o~-cellulose contained arabinose and glucose in equal proportions, and contained 7% uronic acids instead of mostly glucose. High levels of uronic acids and arabinose also were found in most of the other fractions isolated in this way. So, although the yield of pectic substances from conventional oxalate extraction was low (4-6%), it was apparent that in total the leaves consisted of unusually high proportions of pectic substances. To circumvent the difficulties in interpreting these compositional data and consistent with a desire to preserve the chemical integrity of the leaf constituents, a simpler approach was chosen that produced extractives, EDTA (or water) -soluble and -insoluble components (Table 3). These fractions were then analyzed in detail (Table 4 and 7). Corresponding N. smithi fecal material was also studied using this approach (Tables 5 and 6). As mangrove leaves contain a high content of (poly)phenolic material, an effort was made to estimate the contribution of flavonoids to the total phenolics in absolute methanol extracts of the major components of the leaves, namely the aqueous acetone extracts and EDTA-insoluble fractions (Table 3). The FolinCiocalteu reagent (Price and Butler, 1977) for total phenolics and the vanillin reaction (Bums, 1971; Ribereau-Gayon, 1972b), specific for phloroglucinol ring functionality in condensed tannins were evaluated. Quantification was precluded by the presence of a colloidal haze in the Folin-Ciocalteu assay solutions of all samples, except intact senescent leaves (SO), which could not be removed by centrifugation (3000g, 10 min). Nevertheless, in qualitative terms, the total phenolics decreased from very high to very low in the extracts from SO through to $8. The disappearance of these TABLE 3. FRACTION DISTRIBUTION IN
Ceriops tagal LEAF SAMPLES
Fraction (% leaf dry wt) Leaf sample Green c Senescent c $2 d $4 $6 $8
22.9 22. I 12.3 9.6 9.0 7.4
15.2 14.7 12.6 5.6 8.8 2.9
62.9 59.3 67.5 60.2 69.0 61.7
0.0 3.9 7.6 24.6 13.2 28.0
aIncludes pigments, free sugars, flavonoids, and salt(s). bCalculated by difference and represents water-soluble material < 14,000 mol wt. CGreen and senescent leaves contain 7.4 and 7.2 % sodium chloride, respectively. dSenescent leaves subjected to biodegradation for two weeks, etc . . . . (Giddins et al., 1986).
D E G R A D I N G M A N G R O V E LEAF LITTER
components is due to leaching and to microbial degradation (Cundell et al., 1979; Giddins et al., 1986; Benner et al., 1986). Negligible amounts of the phenolics were found in the insoluble walls of the leaves, but further extraction of the walls with absolute acetone removed material that was soluble in absolute methanol and gave a strong Folin-Ciocalteu reaction with no haze. Although this approach therefore was not applicable to these extracts, the above observations did reflect the complex nature of the phenolics in the leaves and exemplifies the inherent difficulty in estimating the contribution of condensed tannins to total phenolics in ecological studies (Mole and Waterman, 1987). The leaching and degradative processes are further confirmed in Table 3 where there was a large reduction in the amount of acetone-soluble (pigments, free sugars, flavonoids, etc.) and polymeric water-soluble material contained in the aging leaf detritus. Without allowing for dry matter disappearance data, the relative proportion of the insoluble fraction which includes the cell walls, remained constant with time. The increased amount of dialyzable material ( < 14,000 mol wt) in the older leaves is also indicative of the depolymerization processes occurring during decomposition. Two capillary columns (BP20 and BP225) were evaluated for neutral sugar analysis. On the BP20 column, several artifact peaks eluted at R t (retention time relative to internal standard) < 0.488 (vs. rhamnose 0.615), while mannose coeluted with inositol (Rt = 1.537), and glucose eluted before galactose ( R t s = 1.636 and 1.676, respectively). The BP225 column, on the other hand, eluted the artifact peaks at R t < 0.344 (vs. rhamnose 0.485) and gave good separation of mannose, galactose, glucose, and inositol (Rts = 1.122, 1.212, 1.292, and 1.335, respectively). The carbohydrate data for the insoluble fraction (Table 4) show that green and senescent leaves have about the same amount of neutral sugars, while the senescent leaves have more uronic acids (predominantly galacturonic acid). The leaves have similar quantities of arabinose and glucose, which is unusual for the higher plants, and are rich in uronic acids. It is also worth noting that low yields of glucose indicate an unusually low concentration of cellulose in these mangrove leaves, especially since some of the glucose must be present as starch. This observation was supported by the low yield (ca. 9-11%) of c~-cellulose (Table 2). The analyses confirmed that mangrove leaves have a large amount of pectic material. The composition changed dramatically during degradation. After eight weeks, carbohydrates were reduced by 70 %, while apparent lignin content doubled, presumably because of the relative resistance of lignin to biodegradation. The apparent lignin material also includes insoluble polymers from the thick waxy cuticle that is characteristic of mangrove leaves. All of the carbohydrate constituents were reduced by biodegradation, but generally there was preferential digestion of pectates, reflected by the large reduction in arabinose
0.5 0.6 0.5 0.3 0.5 0.4
Green Senescent $2 $4 $6 $8
7.0 7.8 6.1 4.2 2.5 1.7
Arabinose 2.0 2.6 3.1 1.9 1.6 1.4
Xylose 2.6 1.4 3.2 0.9 0.7 1.0
Determined by colorimetric assay against D-galacturonic acid standard.
Leaf sample 1.4 1.7 2.2 1.3 1.0 0.9
TABLE 4. COMPOSITION (% DRY MATTER) OF INSOLUBLE FRACTION OF
7.6 6.2 7.6 4.1 3.7 3.4
Glucose 21.1 20.2 22.7 12.6 9.9 8.7
Total neutral sugars
6.0 10.4 6.2 1.2 0.8 0.6
Uronic acids a
Ceriops tagal LEAF SAMPLES FROM TABLE 3
33.2 25.2 47.9 50.8 51.9 49.3
z > 2:
DEGRADING MANGROVE LEAF LITTER
and uronic acids. These data indicate that biodegradation of mangrove leaves introduces 182 g/kg dry matter of total carbohydrates into the environment for utilization as an energy source by microorganisms and higher animals. The unaccounted-for material from the analyses was 39-44 % for green and senescent leaves and 23-41% for the degraded leaf samples. Since there is only ca. 3 % protein in both types of leaves (Giddins et al., 1986), the discrepancy is most probably explained by the presence of acid-labile polyphenolic material, which has not been investigated further. The problem is associated with the large amount of tannins in mangrove leaves. Residual insoluble tannins have been observed in the neutral detergent fiber residues of other plant species that contain high levels of phenolics (Reed, 1986; McArthur, 1987). Analysis of the N. smithi fecal material from crabs fed on leaf litter (Tables 5 and 6) shows that the crab does not utilize all of the available energy (carbohydrate) but releases into the environment energy in the form of undigested carbohydrates. All dietary constituent sugars were detected in the feces. The insoluble fraction contained a greatly reduced amount (7-11%) of carbohydrate and an enriched amount (27-39 %) of apparent lignin. However, unlike the diet material, the fecal matter has reached a constant level of residual carbohydrate and higher lignin contents, indicating that a digestion-resistant fraction containing ca. 13.5% carbohydrate, which includes uronic acids, survives passage through the gut of N. smithi. The relative proportions of carbohydrate show that all polysaccharides are degraded to some extent by N. smithi's digestive system and in particular there appears to be some cellulase activity, since the arabinoseto-glucose ratio increases from 0.68 to 0.90. The composition of the water-soluble fraction in mangrove leaf detritus is given in Table 7. The complex trends in glycose contents reflect the complex
TABLE 5. FRACTION DISTRIBUTION IN FECAL MATTER PRODUCED BY Neosarmatium
smithi FED Ceriops tagal LEAF DETRITUS(SEETABLE3)
Fraction (% of feces dry wt)
F2c F4 F6 F8
34.8 12.3 9.4 9.8
EDTA-soluble 23.6 2.3 1.3 2.7
EDTA-insoluble 50.6 71.5 79.6 80.9
Dialysate b 0.0 13.9 9.7 6.6
Unidentified. bCalculated by differenceand represents water-solublematerial < 14000 mol wt. CFecal matter produced when N. smithi was fed on litter types $2-$8 (Giddins et al., 1986).
0.3 0.3 0.2 0,3
2.3 1.8 2.3 2.5
Arabinose 1.5 1.2 1.1 1.3
Xylose 0.8 0.9 0.7 0.5
~Determined by colorimetric assay against D-galacturonic acid standard.
Fecal sample 0.9 0.9 0,8 1.1
Galactose 3.4 3.3 3.0 2.8
Ceriops tagal LEAF DETRITUS (SEE TABLE 5)
9.1 8.2 8.1 8.3
Total neutral sugars
4.4 3.9 5.7 5.1
53.9 49.5 47.7 47.7
TABLE6. COMPOSITION (% DRY MATTER) OF INSOLUBLE FRACTION FROM FECAL MATTER PRODUCED BY Neosarmatium smithi FED
tra 1.1 1.0 2.0 2.4 tr
2.8 3.8 3.3 5.1 5.0 tr
Green Senescent $2 $4 $6 $8
19.9 22.2 15.7 5.2 8.3 4.0
Arabinose 2.8 2.8 1.9 2.4 2.7 tr
Xylose 3.9 2.8 tr 3.9 2.5 tr
Carbohydrates (mole %)
15.0 18.2 11.4 12.4 8.6 3.4
TABLE 7. COMPOSITION OF POLYMERIC EDTA-SOLUBLE FRACTION OF
15.3 14.7 6,4 18.1 10.0 3,4
Glucose 40.5 34.5 60,3 52.0 60,6 89,1
11 16 10 23 17 5
9.3 10.9 12.4 36.6 17.2 13.4
Dry matter (%)
Ceriops tagal LEAF SAMPLES FROM TABLE 3
NEILSON AND RICHARDS
chemical nature of this material and of the degradative processes. The large quantity of acid-insoluble residue (phlobaphenes) and the formation of pink anthocyanidins in the acid hydrolysates is indicative of the high proportion of proanthocyanidin material in this fraction (Ribereau-Gayon, 1972c). The amount of anthocyanidins was highest in the extracts from green leaves and declined rapidly to be just detectable in $4 leaves. The neutral monosaccharide composition of the water-soluble fraction after hydrolysis was similar to that of the insoluble fraction, with arabinose, galactose, and glucose being the major sugars, while rhamnose, fucose, xylose, and mannose were the minor ones. The polysaccharide component in these fractions contained 35-89% uronic acids. These results confirm that pectates are complexed with or bonded to the proanthocyanidin. Poor recoveries were obtained from the analysis of these watersoluble fractions, and once again this loss could only be explained by the presence of a large amount of polyphenolic material that is acid-labile. In this case, agreement was obtained between GC and corrected phenol-sulfuric acid values for the carbohydrate measurements. However, it should be noted that due to interference by flavonoids, significant errors may result when the carbohydrate contents in flavonoid-rich materials are determined by phenol-sulfuric acid analysis (Rahman and Richards, 1987). A corresponding soluble fraction from Rhizophora stylosa was designated flavologlycan (Neilson et al., 1986a) during the preliminary investigation of the diagenesis of mangrove leaves, implying covalent linkages between flavonoid and polysaccharide components. One of us (G.N.R.) has subsequently found evidence that condensed tannin-polysaccharide complexes involve strong noncovalent interpolymer bonding (Rahman and Richards, 1988), and further studies are continuing on these complexes. The leachate from mangrove leaves introduces a large amount of organic material into the mangrove environment. If the tannin concentrations in the soluble fraction are high, then the leachate is inhibitory to the uptake by organisms (Benner et al., 1986; Alongi, 1987), and this inhibition will extend to any carbohydrate material that may be bound to the tannins. Furthermore, leachable tannins have been demonstrated to significantly reduce consumption rates of mangrove leaves by N. smithi (Neilson et al., 1986b) prior to leaching. Nevertheless, Benner et al. (1986) have shown that 42 % of some leachates can be utilized by microorganisms and are therefore available to animals in the estuarine food web.
Acknowledgments--AuthenticD-l-O-methyl-muco-inositolwas kindly provided by Professor S.J. Angyal. This study was financially supported by grants from the Australian Marine Science and Technology Grants Scheme.
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