Materials and methods Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol246.Issue2.Apr2000:

S . de Lestang et al. J. Exp. Mar. Biol. Ecol. 246 2000 241 –257 243 are restricted to one or the other of those two systems cf. Loneragan et al., 1986; Rose, ¨ 1994; Potter et al., 1997; Durr and Semeniuk, 2000; Semeniuk, 2000; Semeniuk and Wurm, 2000. We have thus compared the dietary compositions of P . pelagicus in these two estuaries to ascertain whether any of the latter differences in potential prey are reflected in a significant difference between the compositions of the food ingested by crabs in those two systems. Comparisons between the dietary compositions of crabs of different size and shell state and in the two estuaries were facilitated by the use of non-metric ordination, which is particularly well suited for analysing dietary data Platell et al., 1998.

2. Materials and methods

2.1. Sampling regime in the Peel–Harvey and Leschenault estuaries Portunus pelagicus was collected from numerous sites throughout the basins of the o Peel–Harvey 32 40 9S and 1158409E and Leschenault estuaries 338129S and 115 8409E. In both estuaries, the water depth is usually , 2.5 m and the substratum is relatively homogeneous, comprising mainly sand and silt, with aquatic macrophytes sometimes being present. Although most crabs were caught by seine netting, some were collected by otter trawling. The size compositions of crabs caught by these two methods were similar. The seine nets used in the Peel–Harvey and Leschenault estuaries, which were 10.5 and 21.5 m long, respectively, and consisted of 3 mm mesh in the bunt, fished upwards to a height of 1.5 m. The otter trawl net, which contained 25 mm mesh in the bunt, was towed by boat at a speed of ca. 3–4 km h for ca. 250 m. Portunus pelagicus were removed from samples collected in each of the two estuaries at four or six-weekly intervals between November and April of 1995 1996 and 1996 1997, during which months of the year this portunid is most abundant in these systems Potter et al., 1983; Potter and de Lestang, 2000. Up to 20 crabs, that covered a wide size range, were collected for dietary analysis on each sampling occasion in each estuary. Every attempt was made to ensure that each shell state for definitions, see below was well represented in the total sample of crabs collected from the Peel–Harvey Estuary. Since the catches of particularly the small P . pelagicus were far lower in the Leschenault Estuary, the crabs used for examining the dietary compositions of this species in this estuary were restricted to the intermoult stage, which was the most abundant shell state. The crabs were frozen immediately after capture. Prior to examining the contents of its stomach, each crab was sexed and its carapace width CW measured to the nearest 1 mm and a record kept of its shell state. The criteria of Warner 1977 and Williams 1982 were used to identify the following shell states: premoult old shell splitting, brittle sutures, newly moulted soft shell with minimal calcification, recently moulted flexible shell with some calcification and intermoult hard shell with calcification essentially complete. The foregut of each crab was removed and the fullness of its cardiac stomach sensu Warner, 1977 recorded using a scale of 0 empty to 10 fully distended with food. N.B. The contents of the cardiac stomach had not yet undergone extensive grinding by 244 S . de Lestang et al. J. Exp. Mar. Biol. Ecol. 246 2000 241 –257 the gastric mill and were thus usually readily identifiable. Food was never found in the stomachs of any newly-moulted crab. The stomach contents of crabs of each of the other three shell states in the Peel–Harvey Estuary and of intermoult crabs in the Leschenault Estuary were stored in 70 ethanol. They were subsequently examined under a binocular microscope and each dietary item identified to the lowest possible taxon. For dietary analyses, each of the various dietary items was allocated to one of a number of broader dietary categories. The number of times that each dietary category was found in the stomach contents of premoult, recently-moulted and intermoult crabs was used to calculate its percentage frequency of occurrence F in the diets of crabs in each of those three shell states. The percentage volumetric contributions of each dietary category to the total volume of the diet V of each crab in these shell states was expressed using the points method, which takes into account stomach fullness Hynes, 1950; Hyslop, 1980. The volumetric data for dietary categories were calculated separately for sequential size classes, i.e. , 30, 30–59, 60–89 mm CW etc., of recently-moulted and intermoult crabs in the Peel–Harvey Estuary and of intermoult crabs in the Leschenault Estuary. N.B. Since premoult crabs had not apparently been recently feeding see Results, they were not subjected to the same detailed dietary analyses. Sediment and unidentifiable material contributed 12.0 and 2.8, respectively, to the volume of the stomachs of recently-moulted crabs in the Peel–Harvey Estuary and 12.1 and 4.6, respectively, to those of intermoult crabs in the Peel–Harvey and Leschenault estuaries collectively. These two dietary categories were not included in the dietary analyses. 2.2. Data analyses One-way analysis of variance ANOVA was used to test whether the stomach fullness of crabs in the Peel–Harvey Estuary varied significantly amongst shell states, i.e. premoult, recently moulted and intermoult. Because Cochran’s C test showed that these data were heteroscedastic, they were log transformed, which resulted in homoscedas- ticity and therefore satisfied the assumptions of ANOVA. In those cases where ANOVA ´ detected significant differences, Scheffe’s a posteriori test was used to determine which means were significantly different at P , 0.05. Since the stomachs of all newly-moulted crabs were empty and thus yielded zero values for fullness, the crabs of this shell state were not included in the ANOVA. The mean percentage volumetric contributions of each dietary category to the diets of recently-moulted and intermoult crabs of sequential size classes in the Peel–Harvey Estuary was root-transformed and a similarity matrix constructed using the Bray–Curtis similarity coefficient. A Bray–Curtis similarity matrix was also constructed in the same manner, using a combination of data for the diets of intermoult crabs of sequential size classes in both the Peel–Harvey and Leschenault estuaries. Classification, involving hierarchical agglomerative cluster analysis with group-average linking, and ordination, employing non-metric multi-dimensional scaling MDS, were performed on each of the two similarity matrices using the PRIMER package Clarke and Warwick, 1994. One-way analyses of similarities ANOSIM were carried out to determine whether the dietary compositions of recently-moulted and intermoult crabs in the Peel–Harvey Estuary and of intermoult crabs in the Leschenault Estuary changed significantly with S . de Lestang et al. J. Exp. Mar. Biol. Ecol. 246 2000 241 –257 245 body size Clarke, 1993. One-way ANOSIM and two-way crossed ANOSIM were used to compare the dietary data for the corresponding size classes of intermoult crabs in the Peel–Harvey and Leschenault estuaries, in order to determine whether the dietary compositions of intermoult crabs in these two estuaries were significantly different. N.B. Although Clarke and Warwick 1994 recommend using replicates for tests employing ANOSIM, the contents of each crab stomach replicate often comprise only three or four of the 14 dietary categories recorded overall in stomachs. Thus, to reduce the number of zero values and thereby construct a more appropriate similarity matrix for testing with ANOSIM, the dietary data for up to six groups of three randomly-selected stomachs in each size class of each shell state in each estuary were pooled. It should be noted that, when different trios of randomly-selected stomachs were subjected to ANOSIM, they always yielded the same results. In those cases where significant differences were detected with ANOSIM, similarity percentages SIMPER were employed to identify those dietary categories which made the greatest contribution to those differences Clarke, 1993. Multivariate dispersion MVDISP was used to determine the degree of dispersion of the dietary samples of crabs representing different size classes and shell states and occupying different estuaries Somerfield and Clarke, 1997.

3. Results

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