Fatty acid composition of the Caprellidea ( Crustacea : Amphipoda ) from the Strait of Gibraltar *

The total fatty acid composition of nine species of caprellidean amphipods collected from the Strait of Gibraltar was investigated. All caprellids were characterised by high levels of polyunsaturated fatty acids, mainly eicosapentaenoic acid, 20:5(n-3), and docosahexaenoic acid, 22:6(n-3); other major fatty acids were the saturate palmitic acid, 16:0, and the monounsaturate oleic acid, 18:1(n-9). In spite of this general uniformity, univariate and multivariate analysis showed that Caprella acanthifera and C. grandimana differed from the remaining species (C. danilevskii, C. equilibra, C. liparotensis, C. penantis, C. santosrosai, Phtisica marina and Pseudoprotella phasma). These two species (C. acanthifera and C. grandimana) showed higher concentrations of 18:1(n-7) and 20:4(n-6) and lower percentages of 22:6(n-3). These results, together with higher values of the biomarker ratio 20:5(n-3)/22:6(n-3) and a lower ratio 18:1(n-9)/18:1(n-7), suggested a greater contribution of diatoms and macroalgae in the diet of C. acanthifera and C. grandimana and a lesser contribution of flagellates than in the remaining species. The cluster classification of the caprellid specimens from the Strait of Gibraltar based on the fatty acid composition was in agreement with the Cluster output from data of feeding behaviour. Sex-related changes in the fatty acid composition were also explored in C. acanthifera, C. danilevskii and C. penantis; the percentage of 18:0, 20:4(n-6), and 20:5(n-3) was significantly higher in males, whereas 16:1(n-7) was higher in females. The intraspecific differences of fatty acids in different environmental conditions seem to indicate that the consumption of diatoms in contrast to flagellates increases with the degree of eutrophication.


INTRODUCTION
Caprellidean amphipods are small crustaceans which inhabit many littoral zones.They are common and diverse on erect hydrozoans and bryozoans, and on plant substrata such as macroalgae and seagrasses (McCain, 1968).They feed on suspended materials, prey on other organisms or graze on epibiotic fauna and flora (Caine, 1974).Locally, caprellids are important preys for many coastal fish species (Caine, 1989(Caine, , 1991) ) and can be useful as bioindicators of water quality (Guerra-García and García-Gómez, 2001).
The Strait of Gibraltar is an interesting zoogeographical area, especially from the point of view of the interchange of species between the Atlantic and the Mediterranean, and the European and African faunas.Recently, the Marine Biology Laboratory of the University of Seville has been conducting research programmes focusing on taxonomy, ecology and behaviour of the caprellid communities of the Strait of Gibraltar (Guerra-García, 2001a;Guerra-García and García-Gómez, 2001;Guerra-García and Takeuchi, 2002;Guerra-García et al., 2002).
The fatty acid composition of marine amphipods is known to be related to water temperature and feeding modes (Kawashima et al., 1999;Graeve et al., 2001) and a fatty acid approach can be useful to explore habitat preferences and feeding strategies.However, there is a lack of studies dealing with the fatty acid composition in the Caprellidea.Data of fatty acids have been recorded only in five caprellid species before this study, all of them collected from Japanese waters (Kawashima et al., 1999): an unidentified caprellid species and four other species (C. acanthogaster, C. danilevskii, Caprella mutica and C. penantis) inhabiting Otsuchi and Mutsu Bays in northern Japan.
The objective of this paper is to analyse the fatty acid composition of various caprellid species from the Strait of Gibraltar and to explore the sex-related changes and the intraspecific variations in fatty acids depending on the environmental conditions.These aspects had not been studied in Caprellidea before this study.

Study area
The Strait of Gibraltar is situated on the border of the Mediterranean Sea and the Atlantic Ocean.Three areas of different environmental characteristics were selected for sampling the caprellids: El Portil and Tarifa island in southern Spain, and Ceuta in north Africa (Fig. 1).El Portil is located in the estuary area of the river Piedras.The place is subject to important fluctuations in salinity and temperature and receives a considerable organic enrichment due to the river influence.Average water temperatures in summer are close to 25ºC (Guerra-García et al., 2000a).In contrast, Tarifa island, as a part of a Natural Park, is a very well protected area characterised by clean and cooler waters (mean values of about 17ºC in summer) and highly biodiverse marine benthic communities (Guerra-García and García-Gómez, 2000;Guerra-García et al., 2000b).Ceuta is located on the north African side of the Strait of Gibraltar and the water temperature in summer is 502 J.M. GUERRA-GARCÍA et al. about 19-20ºC (Guerra-García, 2001b).The degree of human influence in the littoral area of Ceuta is intermediate between the Tarifa island (no anthropogenic influence) and El Portil (a high level of anthropogenic influence).Therefore, in selecting these three areas, a range of conditions is encompassed to check intraspecific variability in the fatty acid composition.

Sample collection
Nine species of caprellid amphipods were sampled for the study (Table 1).The specimens were collected by hand in intertidal areas and using snorkelling and scuba diving in infralittoral zones.Immediately after sampling, the individuals were sorted into species and stored at -80 ºC for later analysis.

Fatty acid analysis
Samples of the different species of caprellids (25-30 mg) were homogenised with 0.9% NaCl.Fatty acid methyl esters were prepared by a direct transesterification reaction according to Lepage and Roy (1987): 2 ml of methanol-benzene 4:1 (v/v) was added to 0.2 ml of homogenate and then, while stirring, 0.2 ml of acetyl chloride was slowly added.Tubes were tightly closed and maintained at 100ºC for 1 h.After cooling, 5 ml of 6% K 2 CO 3 solution was added.The tubes were then shaken and centrifuged and an aliquot of the benzene upper phase was injected into the chromatograph for fatty acid analysis.
Analysis of fatty acid methyl esters was performed by gas-liquid chromatography using a Hewlett Packard 5890 gas chromatograph equipped with a 30 m long x 0.25 mm internal diameter TR-WAX fused silica capillary column (Teknokroma, Barcelona, Spain).Peaks were identified by comparison with known standards (Supelco, Bellefonte, PA, USA), and the results were reported as area percentages.

Statistical analysis
The possible differences in the fatty acid composition between species, together with the intraspecific differences between sexes and sample sites, were tested.The data was verify for normality using the Kolmogorov-Smirnov test and Levene's test was used to check the homogeneity of variances.Provided that there was not normality and/or homogeneity of variances, the non-parametric Kruskal-Wallis test was used.The affinities between species based on the fatty acid composition were established through Cluster analysis using the UPGMA method (unweighted Pair Group Method using arithmetic Averages) (Sneath and Sokal, 1973).An MDS (nonmetric Multidimensional Scaling) analysis was also conducted and the Kruskal stress coefficient was calculated to test the ordination (Kruskal and Wish, 1978).The Primer computer package v.5 (Clarke and Gorley, 2001) and the program PC-ORD (PC-Ordination) v. 3.05 (McCune and Mefford, 1997) were used for multivariate analyses.For univariate analyses the BMDP (BioMedical Data Programs) was used (Dixon, 1983).

RESULTS
A detailed description of mean values of fatty acid percentages together with the biomarker ratios is given in Table 2 and Figures 2 and 3.At least 29 fatty acids with a number of carbon atoms from 14 to 22 were present in the studied caprellid species.Unsaturated acids were the major fatty acids, accounting for 69-75% of total fatty acids.Polyunsaturated fatty acids (PUFA) were the dominant unsaturated fatty acids, making up 50% of the total fatty acids.Eicosapentaenoic acid (20:5(n-3)) and docosahexaenoic acid (22:6(n-3)) were the two major PUFA; other major fatty acids found in all caprellid species were palmitic acid (16:0) and oleic acid (18:1(n-9)).
In general, the fatty acid composition was similar in all species (Table 2).However, the percentage of 504 J.M. GUERRA-GARCÍA et al.  the fatty acids 18:1(n-7) and 20:4(n-6) were significantly higher in C. acanthifera and C. grandimana than in the remaining species (Kruskal-Wallis test, p<0.05).Significant differences were also obtained for the percentage of 22:6(n-3), which was lower in C. acanthifera and C. grandimana (Fig. 2).
The biomarker ratio 18:1(n-9)/18:1(n-7) was significantly lower in C. acanthifera and C. grandi-mana than in other species, whereas the ratio 20:5(n-3)/22:6(n-3) was higher (Kruskal-Wallis test, p<0.05) (Fig. 3) When the fatty-acid matrix was used in the multivariate analyses to explore the similarity between species, the pattern obtained with the univariate analysis was confirmed (Fig. 4 and 5).rated from the remaining species in both the MDS ordination output (Fig. 4) and the dendrogram (Fig. 5).The Cluster analysis based on the Euclidean distance showed that two species groups can be differentiated (Fig. 5).The first includes C. acanthifera and C. grandimana, characterised by higher contents of 18:1(n-7) and 20:4(n-6) and lower contents of 22:6(n-3).The second group can be split into two subgroups: the species C. penantis and C. danilevskii in one subgroup, and C. equilibra, C. santosrosai, C. liparotensis, Phtisica marina and Pseudoprotella phasma in the other subgroup; the first subgroup was characterised by slightly higher concentrations of 16:0 and 18:2(n-6) than the second subgroup.

Intraspecific variation of fatty acids
Only the species Caprella penantis was collected in the three study areas (Tarifa, Ceuta, El Portil).
Caprella acanthifera was found in Tarifa and Ceuta but not in El Portil, and Pseudoprotella phasma was collected from Ceuta and El Portil but specimens from Tarifa were not available for this study.There was a common pattern of variation in two of the major fatty acids, 16:1(n-7) and 22:6(n-3), for the three caprellid species (Fig. 7).If we consider a growing gradient of eutrophication from Tarifa to El Portil, an increase in the percentage of 16:1(n-7) and a decrease in 22:6(n-3) could be observed.

DISCUSSION
In the present study the total fatty acids of the caprellid specimens were analysed.Some previous studies (e.g.Kawashima et al. 1999) differentiated the fatty acids of phospholipids from the fatty acids of triacylglycerols.However, Graeve et al. (2001) showed that the differences between the fatty acid composition of phospholipids as structural components of membranes and triacylglycerols as storage lipids are small.These authors proposed that future studies of benthic amphipods should be done with analysis of the total lipid fatty acid composition.
The fatty acid composition of caprellids has only been previously reported in a study carried out by Kawashima et al. (1999) in northern Japan.Caprellids from the Strait of Gibraltar showed a fatty acid composition similar to that described for the Japanese caprellids, except for 18:1(n-9) and 20:5(n-3), which showed lower values in the species collected in the Strait of Gibraltar.These differences may be related to differences in water temperature, as it has been reported that the amount of 20:5(n-3) increases when water temperature decreases (Kawashima et al., 1999).In the present study the percentage of 20:5(n-3) measured in the caprellid species from Tarifa (characterised by colder waters, 17 ºC in summer) ranged from 18 to 22%, whereas in the species from El Portil (characterised by warmer waters, 25 ºC in summer) ranged from 11 to 17%.These ranges seem to support the influence of the temperature on fatty acid composition, although the number of specimens from El Portil is small, so the data must be taken cautiously.In addition to the temperature, the type of diet could also be involved in these variations, as discussed later.
The dendrogram obtained in the present study based on the fatty acid composition (Fig. 5) is in agreement with the dendrogram represented by Guerra-García et al. (2002) based on the clinging behaviour associated with feeding strategies.The species C.danilevskii and C. penantis spend most of their time in a "parallel" posture on the substrate, feeding mainly by scraping, and both species are grouped together in the feeding cluster (Guerra-García et al., 2002).Similarly, Phtisica marina, Pseudoprotella phasma, C. equilibra and C. santosrosai are in the same group because they have a similar feeding behaviour, filtering and predating in an "upright" position.In the fatty-acid cluster output, these species are also very close based on similar percentages of fatty acids.Therefore, it seems that fatty acid composition is significantly related to feeding modes in caprellids.
Although the available data for differentiating the fatty acid composition in males and females are scarce, several clear patterns were obtained for three of the nine studied species.The percentage of 18:0, 20:4(n-6), and 20:5(n-3) was significantly higher in males of the three compared species (C.acanthifera, C. danilevskii and C. penantis), whereas 16:1(n-7) was higher in females.Similar patterns (except for 18:0) have been reported recently for the gammaridean amphipod Gammarus lacusta (Correia et al., 2003).These differences in fatty acid composition could reflect slight differences in feeding strategies, such as the greater consumption of diatoms by the females, maybe due to reproductive purposes.Correia et al. (2003) also found age-related changes in the fatty acid composition in Gammarus lacusta.The percentage of 16:1(n-7) decreased with age, while 18:1(n-9), 20:1(n-9) and 20:1(n-11) increased in adult specimens, although the age-related changes seem to be related to the peroxidation status of the animals in gammarids (Correia et al., 2003).Further research in future is encouraged to explain the implications of sex-and age-related changes of fatty acids in caprellidean amphipods.
Similar to the sex-related data, the data of intraspecific variation of the fatty-acid composition in the three studied areas were also limited because only three species could be collected in more than one area; Caprella penantis was found in the three studied areas, C. acanthifera in Tarifa and Ceuta and Pseudoprotella phasma in Ceuta and El Portil.However, an increasing trend of 16:1(n-7) and a decrease in 22:6(n-3) was observed when the level of eutrophication increased from Tarifa to El Portil.This could indicate that the consumption of diatoms in contrast to flagellates increases when the level of eutrophication becomes higher.However the effect of the water temperature could also be involved in the changes in the percentages of these two fatty acids, and it is difficult to separate the influence of the two factors.Future experimental studies should be addressed to further investigating the environmental implications of this intraspecific variation.
FIG. 1. -Map of the Strait of Gibraltar showing the sampling localities: El Portil, Tarifa and Ceuta.
FIG. 5. -Dendrogram of similarity between the caprellid species, produced from percentage data of fatty acids.
FIG. 7. -Comparison of the percentage of 16:1(n-7) and 22:6(n-3) along an increasing gradient of eutrophication from Tarifa (unpolluted area) and El Portil (eutrophic area) in the caprellids Caprella acanthifera, C. penantis and Pseudoprotella phasma.Arrows indicate that the species was not collected from that area.The values are expressed as mean and standard deviation.

Table 1 .
Caprellids from the Strait of Gibraltar used for the study.Details of locality, data, depth and substrata are given.[n=number of samples; each sample contains a number of specimens enough for chemical analysis (25-30 mg)]

TABLE 2 .
-Percentage of fatty acid composition in the Caprellidea from the Strait of Gibraltar.