Nghiên cứu Detritivory ở Cua Sesarmid Parasesarma erythodactyla - Luận án Tiến sĩ

Detritivory in the mangrove sesarmid crab Parasesarma erythodactyla Thi Hong Hanh Bui, B. NUS Griffith School of Environment Griffith Sciences Group Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy August 2014 ABSTRACT Sesarmid crabs have important ecological roles in the mangroves of the Indo-west-Pacific biogeographic region. By processing large amount of leaf litter, these crabs retain organic matter in the mangroves, increase the rate of mineralisation, and fuel additional trophic links with the processed material. However, the dietary composition of the mangrove sesarmids is still a controversy.

Gut content analyses suggest a dominant contribution of mangrove leaf litter in the crab diet but stable isotope data seem to support the assimilation of organic matter derived from the microphytobenthos but not mangrove litter. In addition, ability of the sesarmid crabs to obtain sufficient nutrition from mangrove litter, which is rich in structural carbon (cellulose) but poor in other nutrients such as nitrogen, has been questioned. The over- arching goal of this thesis was to fill these gaps of knowledge in detritivory of the mangrove sesarmids, using Parasesarma erythodactyla (Hess, 1865), the dominant shredder in sub- tropical Australian mangroves, as an animal model. The role of mangrove leaf litter in the diet of P.

erythodactyla was evaluated though a series of feeding experiments. Using natural abundance and dual-labelled (13C and 15N) mangrove leaf litter as feed, I showed that the crabs were well sustained on the leaf litter only diet for an extended period (9 weeks), and assimilated mangrove carbon and nitrogen at the efficiency of 36% and 56. Experimentally determined carbon and nitrogen trophic discrimination values (Δδ13C and Δδ15N) between P. erythodactyla and its three potential food sources, namely mangrove leaf litter, microphytobenthos and prawn muscle, were significantly different from the global mean values (~+1 ‰ for Δδ13C and +3 ‰ for Δδ15N) commonly used for interpretation of stable isotope data on mangrove food webs.

The close proximity between Δδ13C(crab-mangrove) determined by this study (+5.45‰ ) and the difference ii in δ13C values of the sesarmid-mangrove litter pairs reported in the liturature (mean = +5.8‰ ) suggests that the same Δδ13C(crab-mangrove) values may apply to other mangrove sesarmids feeding on mangrove litter. Dietary composition calculated by the IsoConc mixing model based on trophic discrimination values determined in this study supports a dominant contribution of mangrove leaf litter to the crab’s diet (89% in terms of biomass). Together, these results reinforce the notion that sesarmid crabs are the key initial processor of mangrove primary production, particularly in the Indo-west-Pacific, where mangrove sesarmids are most diverse and abundant. Unlike other detritivores, sesarmid crabs are capable of assimilating fresh mangrove leaf litter before significant microbial decomposition can occur, although aged litter is usually preferred.

Mechanisms by which these animals could exploit this low-quality food source, however, remains unclear. Using a molecular approach, I showed that mangrove detritivorous crabs are physiologically adapted to utilizing cellulose-rich mangrove detritus, the most abundant carbon source in this ecosystem, by possessing endogenous cellulose hydrolysis enzymes. Direct evidence of endogenous cellulase production not only in P. erythodactyla but also in other crabs of Sesarmidae, Varunidae, Macrophthalmidae, Heloeciidae, and Ocypodidae families is presented.

The cDNA sequences encoding endo-β-1,4-glucanase, one of the key enzymes required for complete digestion of cellulose, identified in these crabs are the first endogenous cellulase gene sequences reported for brachyuran crabs. I also demonstrated that the essential amino acid Threonine, Isoleucine, and Phenylalanine and the long-chain polyunsaturated fatty acid, docosahexaenoic acid (DHA), could be de novo synthesized in P. erythodactyla, possibly with assistance from its gut microflora. In addition to nutrients derived from animal and microphytobenthos food, these de novo synthesised iii nutrients represent a subsidy for the nitrogen and LC-PUFA deficient mangrove litter diet of the crab.

Findings of this study re-affirm the important role of the sesarmid crabs in mangrove organic matter dynamics. Through the sesarmid link, the unpalatable and hard-to-digest mangrove leaf litter is converted to either crab secondary production or faecal materials of improved nutritive values, supporting the nutrition of fauna in the mangrove and connected habitats. iv STATEMENT OF ORIGINALITY This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

Thi Hong Hanh Bui v TABLE OF CONTENTS ABSTRACT .ii STATEMENT OF ORIGINALITY. v LIST OF FIGURES. viii LIST OF TABLES. ix LIST OF ABBREVIATIONS .2 Role of sesarmid crabs in tropical mangrove ecosystems .3 Feeding ecology of grapsoid crabs .4 Parasesarma erythodactyla – the dominant sesarmid crab in the eastern Australian subtropical mangroves.5 Rationale of this thesis .5 Aims and objectives the thesis .6 Structure of the thesis.

DOES ‘YOU ARE WHAT YOU EAT’ APPLY TO MANGROVE GRAPSID CRABS? .3 Materials and Methods. ENDOGENOUS CELLULASE PRODUCTION IN THE LEAF LITTER FORAGING MANGROVE CRAB PARASESARMA ERYTHODACTYLA .2 Materials and Methods .4 Results and Discussion. POTENTIAL CONTRIBUTIONS OF GUT MICROFLORA TO THE NUTRITION OF THE DETRITIVOROUS SESARMID CRAB PARASESARMA ERYTHODACTYLA .3 Materials and Methods .2 Implications for the trophic role of detritivorous crabs in tropical mangrove ecosystems .3 Implications for the dynamics of mangrove primary production in estuarine food webs .4 Future research directions. 140 vii LIST OF FIGURES Figure 2.

Influence of available organic matter on the fitness of P. Temporal changes in δ13C and δ15N values of P. erythodactyla fed enriched mangrove leaves. δ13C and δ15N profiles of crabs on the mangrove leaf, MPB, and prawn diet.

Estimation of the diet composition of P. erythodactyla by the IsoConc mixing model. Effect of (A) pH (n = 8) and (B) temperature (n = 10) on total cellulase activity in the stomach juice of P. Full-length cDNA and the deduced amino acid sequence of PeEG.

Phylogenetic analysis of PeEG and GHF9 EGs from other animals. Expression of endo-β-1,4-glucanase genes in the hepatopancreas of the mangrove crabs. Effect of antibiotic treatment on number of gut microflora in crabs on the 13C- enriched glucose (GNE & GAE) and palmitic acid (PNE and PAE) supplemented diets. δ13C values of non-essential and essential (in boldface) amino acids of crabs fed 13 C-enriched glucose (GNE, GAE); non-enriched glucose diet (GNN); and the field crabs (F).

Relative enrichment in 13C of amino acids in muscle tissues of crabs fed on 13C- enriched glucose with (GAE) and without (GNE) antibiotic treatment. δ13C values of crabs fed glucose (A - C) and palmitic acid (D - F) supplements. Effect of antibiotic treatment on 13C compositions of FAs synthesised by crabs fed 13C-enriched glucose (A) and palmitic acid (B). Trophic transfer and transformation of mangrove primary production through the coastal food webs via the grapsoid link.

135 viii LIST OF TABLES Table 1. Reported rates of litter removal by sesarmid crabs recorded from mangroves throughout the world. C/N ratios of fresh mangrove leaf litter. Data used in the estimation of relative contributions of mangrove leaf, MPB and animal tissue in the diet of P.

erythodactyla using the IsoConc mixing model. Difference in δ13C (‰) values of the mangrove grapsid and their associated mangrove leaf litter from the literature. Oligonucleotide primers used for molecular work. Mangrove crabs used for the endo-β-1,4-glucanase gene expression study and the GenBank accession numbers of the 204 bp endo-β-1,4-endoglucanase cDNA fragments amplified from them.

Cellulase activities in the digestive juice of P. Summary of experimental set-up. 103 ix LIST OF ABBREVIATIONS AA Arachidonic acid CBM Carbohydrate binding module CMC Carboxymethyl cellulose CSIA Compound-specific isotope analysis DHA Docosahexaenoic acid EAA Essential amino acid EG Endo-β-1,4-glucanase EPA Eicosapentaenoic acid FA Fatty acid FAME Fatty acid methyl ester GC-IRMS Gas chromatography - isotope ratio mass spectrometry GHF9 Glycosyl hydrolase family 9 HSI Hepatosomatic index HUFA Highly unsaturated fatty acid IWP Indo-west Pacific LA Linoleic acid LC-PUFA Long-chain polyunsaturated fatty acid LNA Linolenic acid MPB Microphytobenthos MUFA Monounsaturated fatty acid ORF Open reading frame PBS Phosphate buffer saline PKS Polyketide synthase PUFA Polyunsaturated fatty acid RT-PCR Reverse transcription polymerase chain reaction SAFA Saturated fatty acid UTR Untranslated region x ACKNOWLEDGEMENTS First and formost, I would like to express my deep gratitude to my supervisor, Prof. Shing Yip (Joe) Lee, for his encouragement, continuous support, and great guidance.

From this enthusiastic, thoughtful and dedicated scientist, I have learned a great deal about doing research. I especially appreciate how he always encourages me to think independently and be confident in what I am doing. I sincerely thank the financial support from the Endeavour Postgraduate Scholarship scheme of the Australian Government, without which I would not have had the opportunity to conduct this work. I would also like to offer my special thanks to Prof.

Brian Fry (Griffith University), Dr Wenqing Wang (Xiamen University, China), and Ben Matthews (Smart Water Research Centre) for constructive feedback and useful advice on the stable istope and molecular work. Technical assistance in analysis of stable isotope and fatty acid samples provided by Daniel Tonzing, Rene Diocares, and Radoslaw Bak is greatly appreciated. I also wish to acknowledge Alan Richards, Dr Tish Pankhurst, Assoc. Dave Welsh, Dr Helen Stratton, Prof.

Jane Hughes, Shimony Mohanty, and Dr Marxim Sheludchenko for sharing resources for my experimental work. Also, thanks to Hee Lee for helpful assistance in administrative matters throughout my candidature. Many thanks to my friends, Anh, Patricia, Augustina, Yibing, Jason, Maizah, Liana, James, Ariella, Jean, Xiaolu, Dona, Shafagh, and Robert for general discussion on the project, providing good feedback for the pratice talks, and making life fun. Finally, I would like to thanks my family, especially my husband and daughter, and my best friend, Loan Le.

Their unconditional love, patience, and support meant so much to me.1 General overview Grapsoid crabs are some of the most abundant brachyurans in mangroves (Jones, 1984). The majority of mangrove grapsoids belongs to family Sesarmidae (previously Sesarminae (Lee, 1998) but see (Ng et al. The highest species diversity of grapsoids is recorded in the mangroves of the Indo-Pacific region (Lee, 1998), with up to 51 species (44 sesarmid species) described in Singapore-Malaysia (Tan and Ng, 1994) and 61 species (48 sesarmid species) in Australia (Davie, 2002). Being the initial processors of mangrove leaf litter, the grapsoids have an important role in cycling of organic matter in the mangroves.

By ingesting high percentages of leaf litter, these crabs retain a significant amount of autochthonous primary production in the mangroves (Cannicci et al. Crab litter processing also generates faecal material, which, upon microbial enrichment, has improved nutritive values and smaller fragment sizes than mangrove litter, potentially valuable to the nearshore consumers (Lee, 1997; Werry and Lee, 2005). In addition, mangrove grapsoids themselves are source of food for higher trophic levels. Remnants of grapsoids are prominent items in the diet of fishes in the mangroves of Malaysia (Leh et al., 2012; Sasekumar et al.

Despite the reported significant trophic role of the sesarmid crabs, their actual diet remains controversial. The high rates of litter removal (Table 1.1) and the high abundance of leaf litter relative to other food items found in the stomach of these crabs (Malley, 1978; Poovachiranon and Tantichodok, 1991) suggest a dominant contribution of mangrove leaf litter to their diet. Large deviations (~5‰) of carbon isotopic values (δ13C) of the grapsoid crabs from those of mangrove leaf litter, however, question a leading dietary role of this food source (Bouillon et al., 2002; Guest and Connolly, 1 2004; Mazumder and Saintilan, 2010). It must be noted, however, that past interpretations of mangrove crab stable isotope data were based on the means of the highly variable (among pairs of consumer and food source) trophic discrimination values reported in the literature rather than the true trophic discrimination values between the grapsoids and their potential food sources.