PHYTOPLANKTON COMPOSITION OF THE STOMACH CONTENTS OF THE MUSSEL MYTILUS EDULIS L.

FROM TWO POPULATIONS: COMPARISON WITH ITS FOOD SUPPLY

Author(s) :G. ROUILLON, J. GUERRA RIVAS, N. OCHOA, and E. NAVARRO Source: Journal of Shellfish Research, 24(1):5-14. 2005. Published By: National Shellfisheries Association DOI: 10.2983/0730-8000(2005)24[5:PCOTSC]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.2983/0730-8000%282005%2924%5B5%3APCOTSC%5D2.0.CO %3B2

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Journal of Shellfish Research, Vol. 24, No. 1, 514, 2005.

PHYTOPLANKTON COMPOSITION OF THE STOMACH CONTENTS OF THE MUSSEL MYTILUS EDULIS L. FROM TWO POPULATIONS: COMPARISON WITH ITS FOOD SUPPLY
G. ROUILLON,1* J. GUERRA RIVAS,1 N. OCHOA2 AND E. NAVARRO1 Departamento de Gentica, Antropologa Fsica y Fisiologa Animal, Facultad de Ciencia y Tecnologa. Universidad del Pas Vasco/Euskal Herriko Unibertsitatea. Apdo. 64448080, Bilbao, Espaa; 2Laboratorio de Ecologa Acutica, Facultad de Ciencias Biolgicas. Universidad Nacional Mayor de San Marcos, Ciudad Universitaria, Avda. Venezuela, cuadra 32 s/n Lima, Per

ABSTRACT Seasonal data on phytoplankton composition of seston and stomach contents of the mussel (Mytilus edulis Linnaeus, 1758) from two contrasting sites, an estuarine mud flat and a rocky open shore, were compared to ascertain: (a) the extent to which differential characteristics of both sites affect this composition and (b) the degree of similarity between stomach contents and microalgal composition of seston of these sites as an index reflecting the complex processes of selection taking place within the feeding-digestive system of mussels. Individuals and water samples were collected monthly from November 2001 to December 2002, when salinity, temperature, and total and organic particulate matter concentration were also recorded in the water column. Preserved samples of seston and stomach contents were analyzed by inverted microscopy according to the Utermhl method. Phytoplankton cells were counted and the different species grouped, taxonomically and, according to the habitat, into pelagic and tychopelagic. These data served to compute abundance (total cell count) and frequency index. Relative abundances of each group were compared for similarity between sampling sites and stomach and water samples in each site. Similarity analyses were performed using the index of Bray-Curtis, significant differences between samples being determined by the non parametric test of ANOSIM. Results of this test for the comparison between water and stomach contents resulted in significant differences: R 0.68 in the estuary and R 0.75 in the open shore area. Stomach contents presented a reduced average number of species (n 6 in mussels from both sites) and a greater proportion of tychopelagic forms for comparison with the water samples (n 20 and 24 in the estuary and open shore, respectively). Maximum phytoplankton density in water samples occurred in the May to October period, the group responsible for this increment being the diatoms. The stomach contents of marine mussels displayed two peaks of phytoplankton concentration in May (caused by the dinoflagellate Ensiculifera sp.) and in July (caused by the diatoms Pseudo-nitzschia pungens and Licmophora sp.). In the case of stomach contents of estuarine mussels, a single peak of abundance was recorded in the month of May and was mainly produced by Ensiculifera sp. To conclude, the main result coming from these comparisons is the increased abundance of dinoflagellates in the stomach contents relative to the corresponding seawater samples in the estuarine and open shore media. This result is discussed in the light of previous data concerning the differential utilization of species of phytoplankton by bivalve molluscs. KEY WORDS: phytoplankton, food supply, stomach content, mussels, Mytilus edulis INTRODUCTION

Marine coastal areas are characterized by great space-temporal fluctuations in both phytoplankton abundance and specific composition. In the temperate area, dominant species of phytoplankton exhibit seasonal variations and a natural succession between phytoplankton groups that accompany the occurrence of blooms (Berg & Newell 1986, Varela 1996). Superimposed on these long-term cycles, short-term pulses of microphytobenthos resuspension caused by wind- and tide-driven currents are also relevant regarding the chlorophyll concentration and microalgal composition of the seston (Roman & Tenore 1978, Baillie & Welsh 1980, Muschenheim 1987, Grant et al. 1990, de Jonge & van Beusekom 1992, Newell & Shumway 1993). This stands particularly true for soft-bottomed shallow tidal flats common to estuaries and sheltered areas of the coast, where tidal variability of chlorophyll concentration can exceed the seasonal range (Grant et al. 1993, Smaal & Haas 1997). Hummel (1985) studying the diatom composition of the water column above a tidal mud-flat in the Wadden Sea reported a greater presence of benthic species at the most landward sampling points for comparison with deeper points. Consequently, microalgal composition at these sites would appear, to a great extent, affected by short-term tidal changes in contrast with the open shores where seasonal variability would be dominant.

*Corresponding author. E-mail: ggbrozeg@lg.ehu.es

This fact constitutes one of the sources of spatial variation along the coast. Mussels Mytilus edulis Linnaeus, 1758 are conspicuous inhabitants of both open-shore and estuarine habitats where they form important populations. The interaction of mussel populations with phytoplankton as food source has long been documented. For example, Blanton et al. (1987), van der Veer (1989) and Hickman et al. (1991) reported that phytoplankton blooms were accompanied by increased growth and production and improved condition index of various mussel species in culture plots. All these observations support the idea that mussels generally rely on phytoplankton as their main food supply. However, different species of phytoplankton seems to behave in a different way as regards its food value for bivalves under culture conditions (Epifanio 1979, Enright et al. 1986, Laing & Millican 1986). When available, field observations appear to confirm the above extremes: Beukema and Cadee (1991) compared the patterns of growth of Macoma balthica Linnaeus, 1758 with the patterns of abundance of different phytoplankton components over 15 y, to conclude faster growth and better condition associated with higher abundance of diatoms for comparison with years when flagellates were dominant. Given that, the ability exhibited by several bivalves (mussels included) for the preferential utilization of species of phytoplankton (Cucci et al. 1985, Shumway et al. 1985, Bougrier et al. 1997, Rouillon & Navarro 2003) should be considered a useful mechanism ensuring a complete exploitation of this resource under the conditions of variability prevailing in the marine coastal habitat. 5

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which spatial differences in phytoplankton composition reflect the particular conditions of both sites, specially in which concerns to the balance between pelagic and tychopelagic microalgae and (b) the degree of similarity between stomach contents of mussels from the two sites and the microalgal composition of the water column at these sites as an index reflecting the possible occurrence of differential retention, ingestion, and selective digestion of species of phytoplankton within the feeding apparatus of mussels.
MATERIALS AND METHODS

Figure 1. Map of the study area. Sampling sites are indicated

In dealing with this phenomenon of differential utilization, a useful approach under field conditions would be the comparison of gut contents, and the water column or superficial sediments, in terms of phytoplankton species composition. Using these procedures, Kamermans (1994) reported that species composition in gut contents of various epifaunal and infaunal bivalves living in mud flats resembled more the composition of the water column than that in superficial sediments, but no evidence of selective feeding can be concluded from her data. Shumway et al. (1987) and Muetn-Gmez et al. (2001) also reported similar patterns of seasonal variation of species composition in seawater and stomachs of Placopecten magellanicus Gmelin, 1791 and Spondylus leucacanthus Broderip 1833, respectively. However, Ciocco and Gayoso (2002) observed that stomach content composition of ribbed mussels (Aulacomya atra Molina, 1782) did not reflect the occurrence of diatom blooms recorded in the area of study. The present study compares seasonal data on phytoplankton composition of seston and stomach contents of mussels (M. edulis) sampled along a year. The study was conducted at two sites presenting different characteristics, a muddy flat in an estuary and a rocky shore from an exposed beach, to ascertain (a) the extent to

Two sampling sites were chosen to represent open shore and estuarine habitats: a rocky area at Atxabiribil beach (4323 29 LN; 0259 28 LW) and a muddy-flat in the Ra de Plencia (4324 39 LN; 0256 52 LW), respectively (Fig. 1). Water temperature and salinity were recorded each time with a LF330/SET Standard Conductivity Cell (TetraCon 325, Germany). Sampling was performed during the ebb tide from a point situated approximately 3 m. above the chart datum. Mussels recently emerged (<30 min.) and superficial water samples were collected monthly at these two sites, from October 2001 to December 2002, in the case of water samples and from November 2001 to October 2002 in the case of mussels. Water samples (5 L.) were divided into two aliquots, for particulate seston analysis and phytoplankton composition, respectively. Total particulate matter and organic content of seston were determined by standard procedures: Known volumes of water sampled were filtered on ashed and preweighed glass-fiber filters that were then rinsed with seawater-isotonic ammonium formate dried at 100C for 48 h, weighed, ashed at 450C for 6 h and weighed again. The increment of dry weight and the subsequent weight loss on ignition experienced by these filters, both divided by the volume filtered, represented the total particulate matter (TPM: mg L1) and particulate organic matter (POM: mg L1), respectively; organic content (f) was the ratio of POM to TPM.
Phytoplankton Composition of Water Samples

Water samples were immediately fixed by the addition of glutaraldehyde to the sample to give a final concentration of 1%. Quantitative analysis was made following a modification of the method of Utermhl (Hasle 1978): 50 mL of water were allowed to settle for 3648 h in a combined chamber and cylinder. Identification and counting followed under an inverted microscope (Nikon, phase contrast DIAPHOT). The aliquot counted represented a 5% of total chamber volume. Identification of phytoplankton species was performed to the closest taxon possible according to the following references: Cupp (1943), Balech (1988), Ricard (1987), Tomas (1993; 1996), Van den Hoek et al. (1995). Microalgal species were grouped, according to the habitat, in two categories: pelagic and tychopelagic, the latter including both intermediate and benthic non-adhering forms (Gailhard et al. 2002).

TABLE 1. Range of environmental variables in the two sampling sites, measured over a 14 month period. Salinity S 18.030.0 32.635.2 TPM mg 11 4.03643.44 1.78517.755 Organic Content f 0.1150.216 0.1090.521 Cells Density (N ml1) 15.4662.8 12.53746.18

Site Name Plencia estuary Atxabiribil beach

Habitat Estuarine Marine (open-shore)

Temperature C 10.222.5 11.021.5

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Figure 2. Seasonal variation of phytoplankton abundance of the main taxonomic groups. Note that the scale is divided by 105 in the case of stomach content of mussels. Stomach Content Analysis

Mussels collected in the field were immediately preserved in a fixative mixture made up of formaldehyde and glutardialdehyde in the proportion 3:2, diluted with filtered seawater to a final concentration of 5%. This mixture is assumed to simultaneously ensure optimal preservation of animal tissues (formaldehyde) and features of phytoplankton cells (glutardialdehyde). Gapping of valves observed after 1015 min exposure of mussels to the fixative allowed access to the mussels soft tissues, stomach contents included. Mussels that did not gape were discarded; this circumstance only occurred 1 mo (June). In the laboratory, valves were separated apart to allow stomach contents collection by means of a hypodermic syringe. After recording the volume collected, the stomach content was added to 100 mL of glutardialdehyde 1% and 50 mL of it were allowed to settle for 3648 h in a combined chamber and cylinder. A five percent of chamber volume was analyzed under the inverted microscope following Utermhl method. Cell density or abundance was obtained by dividing the number of cells counted in an aliquot by the aliquot volume. Relative density of a given species or group of species was defined as the fraction this species or group represent of the total cell number frequency index of a species or group is defined as the number of samples presenting this species or group divided by the total number of samples.
Statistical Analysis

differences in cell density between water and stomach samples, statistical comparisons were based on relative density values (percentage of cell number) to obviate the concentration factor that affects the stomach contents. According to Clarke and Warwick (2001), these data were normalized by means of square root transformation before performing similarity analysis using the BrayCurtis index, significant differences between samples being determined by the non parametric test of similarity (ANOSIM) using PRIMER v5 software (Clarke & Gorley 2001).
RESULTS

Density of phytoplankton cells in the water column fluctuated between broad limits along the year, but the range was very similar in both the sites (15.4662.8 cell.mL1 in the estuary and 12.53 746.18 cells.mL1 in the open shore). This contrasts with signifiTABLE 2. Summary of ANOSIM results. Samples Marine vs. estuarine water Marine water versus stomach contents Estuarine water versus stomach contents * Significant differences Factors Month habitat Month Compartment Month Compartment R 0.956 0.931 0.796 0.77 0.683 0.653 P 0.001* 0.001* 0.001* 0.001* 0.001* 0.001*

We used 3 replicates for water samples and 3 individuals to analyze of stomach contents per site and per month. Given the

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Figure 3. Dendrogram for hierarchical clustering of water samples.

Figure 4. Cell density of 10 more abundant species. Black bars correspond to pelagic species, white bars correspond to tychopelagic species. d, indicates dinoflagellates species.

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cantly higher values of total particulate matter and lower organic contents found in the estuary for comparison with the open shore. Average temperatures were slightly higher in the estuary. Salinity mean values were 25.4 in the estuary and 33.8 in the open shore (Table 1).
Seasonal Variation of Phytoplankton Abundance and Composition in the Water Column

Temporal variation of abundance of the different phytoplankton groups is compared between estuarine and open shore water samples in Figure 2a and b. Data for the estuarine area suggests the existence of 2 blooms, in spring (April to June) and autumn (late September). Two centric diatoms (Chaetoceros costatus and Leptocylindrus danicus), and C. costatus and the pennate diatom Asterionellopsis glacialis appeared as the main species responsible for the spring and autumn peaks, respectively (Fig. 2a). In the open shore (Fig. 2b), the onset of the period of high abundance was delayed to May and the two spring and autumn peaks tended to merge in this area giving rise to a period of high abundance that extended from May to early October. This period was characterized by the following succession of species: Skeletonema costatum (centric diatom) and L. danicus, that formed a first peak, followed by C. costatus forming a second peak, and Pseudo-nitzschia pungens (pennate diatom) a third peak of abundance. Results of a two-layout analysis of similarity based on comparing the abundance of different phytoplankton species (ANOSIM) between sampling dates and sites (estuarine vs. open-shore) are summarized in Table 2. According to these results, phytoplank-

ton composition was dissimilar in both the seasonal comparison (R 0.956) and when comparing estuarine and marine waters (R 0.931). Results from ANOSIM performed on water samples were expressed as dendrograms of similarity (Fig. 3). Season appears as the main component allowing differentiation of two groups sharing a similarity <30% and constituted, approximately, by the samples from springsummer on one side and the rest of seasons on the other. Analogously, two secondary groups constituted by autumn and winter samples shared <45% of similarity. Site origin of water samples was a less discriminating component, although similarity between estuarine and open shore samples was never >55%. The highest values corresponded to comparisons of samples taken in the same month, and particularly in the summer season due to the simultaneous occurrence in both media of blooms dominated by the pelagic centric diatoms: C. costatus, L. danicus, S. costatum, and Chaetoceros curvisetus. Figure 4a and b compares the 10 most abundant species (highest cell density) between estuarine and open shore samples. All these species were diatoms and about 40% of more abundant diatoms were centric species, which contributed more (15% to 20%) than any other group to total abundance. As to the habitat, pelagic group constituted 60% to 80% of most abundant species, compared with 10% to 30% of tychopelagic. Species more abundant do not necessarily represent the commonest species. Consideration of the frequency index computed for the 10 commonest species found in water samples (Fig. 5a,b) was aimed to provide complementary information, that can be

Figure 5. Frequency index of the 10 commonest species. Legends as in Fig. 4.

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summarized as follows: (a) The totality of the 10 most frequent species were also diatoms, but in this case 90% of species were pennates (b) 60% of species recorded were common to both areas, these species also presenting a high frequency index (0.745 0.938). Two centric chain-forming diatoms, Stephanopysis turris (estuarine) and Guinardia delicatula (open shore) and 6 pennate diatoms: Gyrosigma fasciola, Navicula elegans, Synedropsis sp. (estuarine) and Climacosphenia sp., Licmophora sp, and Pseudonitzschia pungens (open shore) accounted for the main differences between both sites.

Phytoplankton in Stomach Contents: Comparison With the Food Supply

The patterns of monthly variation of phytoplankton abundance in the stomach contents of estuarine and marine mussels was char-

acterized by a peak in May contributed by the dinoflagellate Ensiculifera sp. A secondary peak in July, composed of Pseudonitzschia pungens (pelagic pennate) and Licmophora sp. (tychopelagic, pennate) was only apparent in the stomach of marine specimens (Fig. 2c,d). Results of two-layout ANOSIM tests (Table 2) comparing phytoplankton composition in stomach contents and water indicated significant differences for both estuarine and open shore habitats (R > 0.5). Dendrograms of similarity (Fig. 6) showed that water and stomach content samples were grouped apart, particularly in open shore samples where similarity in composition of both groups was <20%. In general, similarity between water samples and their corresponding stomach content was always <20%, with the exceptions of April in the estuary (>60%) and November to December in the open shore (>40%). Main contribution to phytoplankton abundance in stomach contents corresponded with the dinoflagellate Ensiculifera sp., which

Figure 6. Dendrogram for hierarchical clustering of water and stomach samples from estuarine and open-shore sites.

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was accounted for by 56% and 30% of total cell density in estuarine and open shore mussels, respectively. None of the remaining microalgal species, belonging to a diversity of groups (diatoms, dinoflagellates, naked flagellates, chlorophyta, and coccoliphorids), contributed more than 10% (Fig. 4c,d). The proportion of pelagic among the 10 most abundant species of phytoplankton decreased from 60% or 80% in water samples form the estuary and open shore sites, respectively, to 40% in stomach contents. Diatoms and dinoflagellates were the most frequent microalgal groups in the stomach contents of mussels, irrespective of the sampling area. Presence of centric diatoms increased from 10% in either samples to 20% or 50% in stomachs of open shore and estuarine mussels (Fig. 5c,d).
DISCUSSION

Fernndez (1990) and Varela (1996), described phytoplankton patterns in the Cantabrian Sea as typical from temperate seas, (with characteristically low density or biomass in winter) spring blooms, and summer stratification inducing a high primary productivity during these periods. Sporadic small blooms during autumn have also been described. In this work, we registered high microalgal abundance from May to October and a near-monthly succession of few species blooms. Present results thus appear to corroborate the earlier mentioned pattern, except that the onset of high-density periods was delayed to late spring and extended to the autumn, without a clear decline of phytoplankton abundance during the summer as that reported by Elosegui et al. (1987) in an area close to our study sites. These differential features were particularly marked in the estuary. Labry et al. (2001) recorded a winter phy-

toplankton bloom in water samples from the Gironde plume in the north of the Bay of Biscay in contrast with our lowest record of abundance during this season. Total phytoplankton abundance presented similar seasonal trends in the two places of study; however frequency and relative density of microalgal species differed between estuarine and open shore sites. These differences concern mainly to the habitat of the species, particularly diatoms. Benthic microalgae are abundant in shallow sediments and non-adhering species may be easily resuspended (Baillie & Welsh 1980, de Jonge 1985, Asmus & Asmus 1993), so that a greater presence of benthic and intermediate species of phytoplankton (here collectively designed as tychopelagic) could be expected in samples taken from the estuary. Indeed, 42% of diatoms density corresponded to tychopelagic species in the estuary, compared with 35% in the open shore (Fig. 7a,b). The species composition recorded in gut contents differed from that from the water surrounding the mussels in 2 respects: (a) An increase of cell density corresponding to the tychopelagic group of diatoms in stomach contents for comparison with water samples can be observed, irrespective of the sampling site (Fig. 7). In view of results reported by various authors working with different species of bivalves (Davis & Marshall, 1961, Shumway et al. 1987, Newell et al. 1989, Kamermans 1994) this could be considered a rather general rule and would be attributed to the possibility that diet available in the near bottom included a greater proportion of resuspended microphytobenthos compared with the water column above. Muschenheim & Newell (1992) analyzed data of chlorophyll and carbon concentration and direct cell count in the water at 35 cm above a mussel bed, to conclude that mussels fed prefer-

Figure 7. Relative abundance (annual average values) of diatoms from different habitats

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entially a high concentration of resuspended benthic diatoms. (b) The distribution of taxonomic groups of microalgae differed greatly as a consequence of the increased presence of dinoflagellate and chlorophyta in stomach contents compared with water samples (Fig. 8). Particularly dinoflagellates, that constitute a minor component in water samples, may reach, on average, 25% or 30% of microalgal abundance in stomachs of estuarine and open shore mussels, respectively. It could be argued that the stomach contents reflected the phytoplankton composition of the water column above the mussel beds 23 h before the specimens were sampled. However, the possibility this temporal shift might account for the differences recorded is remote, because preliminary reports on tidal variation of phytoplankton composition in the area of study revealed minor differences (own unpublished results). Moreover, the enrichment of stomach fluids in dinoflagellates and chlorophyta, in detriment of the major group of diatoms, has been broadly reported in different species of bivalves (Loret et al. 2000, Muetn-Gmez et al. 2001, Ciocco & Gayoso 2002, Rouillon et al. 2002). The process seems to be a selective one because Rouillon (1998) reported, in the scallop Argopecten purpuratus Lamarck 1719 that the abundance of dinoflagellates increased in the stomach contents, but the species diversity of this group decreased

in the stomach compared with that in the immediate habitat. In this work the abundance of dinoflagellates inside the mussels is mainly based on the noticeable accumulation of Ensiculifera sp. cells in the May to June interval, when seawater did not record a special presence of these species. This selectivity could be on the basis of the extraordinary capacity of accumulation exhibited by different bivalves in coincidence with short-lasting dinoflagellate blooms (Shumway et al. 1987, Shumway et al. 1994, Sidari et al. 1998), and acquires special relevance with the occurrence of episodic disease outbreaks associated with the blooming of toxic species. Concerning the mechanisms underlying the increased presence of dinoflagellates and chloropyta within the stomach contents of mussels from both populations, two hypothesis are at least possible: (1) these microalgae could be ingested in preference to diatoms through the differential retention in the ctenidia and/or selection during the pseudofeces forming process; (2) they would be far more resistant to extracellular digestion remaining longer within the gut and being preferentially voided with the feces. The fact that mussels remained emerged for 30 min before collection might have reinforced this effect. If not fully conclusive, available evidence on the subject based mainly on flow cytometry seems to support the second possibility:

Figure 8. Relative abundance (annual average values) of the main taxonomic groups of phytoplankton in water and stomach samples.

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In a series of experiments with seven species of bivalves fed a mixture of microalgae (a dinoflagellate, a diatom, and a cryptomonad flagellate), Shumway et al. (1985) and Cucci et al. (1985) found that most species accumulated dinoflagellate cells in the feces, although diatoms were also abundant in two cases. Ciocco and Gayoso (2002) reported the presence of two dinoflagellate species in the stomachs of A. atra mostly as intact cells, which suggest they are not assimilated by the ribbed mussel. Comparing the relative abundance of two microalgae (a diatom and a naked flagellate) in the diet, stomach contents, and feces, Rouillon and Navarro (2003) have demonstrated the preferential digestion of the diatom by the mussel M. edulis. This interpretation would be consistent with the reported long-term correlation between natural abundance of diatoms and growth of bivalves (Beukema & Cadee 1991, see Introduction). However, the preferential rejection of diatoms in the pseudofeces have also been documented (Shumway et al. 1985, Bougrier et al. 1997), pointing to an alternative mechanism for increasing the

relative abundance of other phytoplankton groups in the ingested diet under the conditions of high seston concentration that can be assumed during the occurrence of blooms. Precise knowledge of characteristics of natural diets available to bivalve populations, particularly the phytoplankton composition, is of prime importance as regards the understanding of growth and dynamics of these populations (Beukema & Cadee 1991). Comparison of stomach content and seston compositions in terms of frequency and abundance of phytoplankton species constitutes a realistic approach to this subject. However, results are very often difficult to interpret because of the various levels of selection interacting across the feeding processes of bivalves. Controlled feeding experiments with natural diets are currently underway at our laboratory, in order for these issues to be readily undertaken.
ACKNOWLEDGMENTS

This work was supported through the Research Group Project UPV 154.320-G07/99. G. R. was founded by an AECI grant.

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