The settlement of abalone (Haliotis discus hannai ino) larvae on culture layers of different diatoms.

ABSTRACT An innovative method was developed to quantitatively measure the attractiveness of cultured diatoms for early stages of abalone larvae settlement. Larvae of the abalone Haliotis discus hannai were offered, in a petri dish, a choice of several toed patches. each made of a diatom monoculture layer. The distribution of the larval settlement on the patches of 17 diatom species was determined. Nitzschia laevis induced the most abalone larvae settlement, followed by Navicula cf lenzii and Amphora luciae. The attractiveness of these diatoms for abalone larvae settlement was not directly related to algal biochemical composition or algal color. It is suggested that compounds excreted by algal cells determine their inductive properties for larval settlement. The larval chemosensory response depends on the concentration of these inductive substances. Sonicated diatom cell suspensions strongly induced larvae attachment, whereas dilute suspensions did not. Boiling settlement-inductive sonicated diatom cell suspensions did not affect their induction ability, implying that native proteins are net essential for the response. Aspartic acid was the only free amino acid excreted by two tested diatoms. It was also the only pure amino acid found inductive for larval settlement. It is therefore suggested that aspartic acid is a component of the settlement induction complex.

KEY WORDS: abalone, Haliotis discus hannai, larvae settlement, larval feeding, abalone, diatoms, mariculture 

INTRODUCTION

The process of abalone larval settlement consists of several characteristic behaviors. Descent and exploration of the settlement substrata by the swimming larva is followed by attachment, which includes inspection and orientation at a favorable spot, and is concluded finally by metamorphosis (Seki & Taniguchi 1996, Seki 1997). The induction of larval settlement is a most critical stage in abalone recruitment in the wild and in seed production in culture. Abalone larvae require highly specific cues for attachment and metamorphosis stimulation (Morse & Morse 1984, Morse 1985, Roberts 2001). The absence of cues lead to low settlement rates and low survival in early post larval growth stages (Searcy-Bernal et al. 1992, Slattery 1992, Roberts et al. 1999, Daume et al, 1999, Takami et al. 2002). A better understanding of the settlement processes and the identification of a reliable approach for efficient recruitment and early growth in abalone larvae, are therefore of paramount importance for furthering both abalone recruitment research and abalone commercial reproduction.

Substances that have been reported to induce settlement in abalone larvae are crustose and coralline algae (Morse & Morse 1984, McShane 1992, Shepherd & Turner 1985), diatom layers (Slattery 1992, Takami et al. 1997, Daume et al. 1999), chemicals like GABA [gamma-amino butyric acid] (Morse 1985, Trapido-Rosental & Morse 1986, Morse & Morse 1984, Morse & Morse 1988), abalone mucus trails (Slattery 1992, Searcy Bernal et al. 1992, Seki & Taniguchi 1996, Seki 1997. Takami et al. 1997, Bryan & Qian 1998), and bacterial films (Bryan & Qian 1998, Roberts 2001).

The induction of the settlement process in larval abalone Haliotis florescence on crustose red macroalgae (such as Lithothamnium californicum) is based on chemosensory recognition of morphogenic and regulatory molecules (such as amino acids), occur ring on the macroalgae outer surfaces and the surrounding water (Morse & Morse 1984, Trapido-Rosenthal & Morse 1986, Morse 1992).

Naturally recruited diatom films have long been used to induce larval settlement in abalone hatcheries around the world, but with a limited awareness or management of suitable settlement-inductive species and the properties that make them attractive to abalone larvae (Roberts 2001). Understanding the factors that regulate effective settlement, post larval growth, and the selection of cultivable diatoms that promote it, are therefore essential (Matthews & Cook 1995, Roberts 2001).

Diatoms release into their surrounding water substances that can induce settlement (Helebust 1974, Admiraal et al. 1986, Searcy-Bernal et al. 1992). It is hypothesized that the selective settlement of abalone larvae on a film of a certain species of algae is associated with larval attracting chemicals, which "attractive" algae have on their cellular exterior or secrete into the water around them (Kuehn 1997). Such chemicals can create gradients detectable by small aquatic animals even in turbulent water (Finelli et al. 1999), and abalone larvae are known to have the physiologic ability to sense them (Morse & Morse 1984, Morse & Morse 1993, Morse 1992, Baxter & Morse 1992). This article describes a method developed to quantitatively measure the preference of swimming larvae for attachment onto a range of cultured diatoms (the term "settlement" is used henceforth, even though metamorphosis was not studied). "Attractive" and "not attractive" diatoms were selected by this method and then used to probe the chemical basis for the chemosensory settlement response of abalone larvae.

MATERIALS AND METHODS

Larval Culture

Larvae of Haliotis discus hannai were obtained from an in-house abalone hatchery in Eilat, Gulf of Aqaba, Red Sea. Adults were induced to spawn using ultraviolet light (Kikuchi & Uki 1974). Fertilized eggs were collected and transferred into 20-L culture aquaria at a concentration of 12 eggs per mL. Rafamycin antibiotic was added at a concentration of 1.5 mg/L. Hatched larvae were kept at 22[degrees]C, with a 12 L: 12 D light cycle (60-70 [micro]mol photons [m.sup.-2][s.sup.-1]), for 4-5 days, until they were competent for settlement. Larval competency was assessed based on swimming behavior, as described in Seki and Taniguchi 1996. The competent larvae were then used for the settlement preference experiments.

Diatom Cultures

Cultures of benthic diatoms were isolated from local waters and elsewhere (Table 1). Diatom cultures were made axenic by an antibiotic mixture of Penicillin G (800 [micro]g/mL), Ampicillin and Streptomycin (400 [micro]g/mL) and Chloramphenicol (160 [micro]g/mL) for 24 hours (Ucko 1996). The cultures were then transferred into Gentamycin sulfate (200 [micro]g/mL) for 24 hours. The axenity of the treated cultures was confirmed by plating them on a rich nutrient agar (DIFCO) petri dishes, which can clearly show any bacterial contamination. The axenic algal cultures were cultured at 22[degrees]C to 24[degrees]C in a slightly modified f/2 medium (Guillard & Ryther 1962). The cultures were maintained in test tubes under continuous light (~110 [micro]mol photons [m.sup.-2][s.sup.-1]).

For the settlement preference experiments, algae were axenically grown on a Bacto-agar (DIFCO) solid medium, which was prepared with modified f/2 medium as mentioned earlier, or with artificial sea water (ASW) (Jones et al. 1963) enriched with silica. Petri agar dishes were inoculated by spreading the diatoms uniformly on the agar surface and left to grow for about 2 weeks, when a uniform diatom layer was formed.

The Experimental System, Developed to Quantify Larval Settlement Preference

Preparation of the Experimental Petri Dishes

The larval settlement preference experimental system was a modification of the "cafeteria" method used by Lee et al. (1977). The top of an alcohol-sterilized 16 mm test tube was used to remove diatom-covered agar plugs from the diatom culture dishes and to insert them into the same size holes in Agarose agar (Sigma) layers in the experiment dishes. These were prepared by pouring 25 mL of 1% Agarose agar into 90 mm petri dishes, in thin layers. Preliminary experiments showed that other types of agar negatively affected the settlement and growth of the larvae. After cooling and hardening of the agar, four plugs were cut out and removed from the Agarose agar layer in each dish with an alcohol-sterilized 16 mm test tube. Three of the round empty spaces that remained were filled, as described earlier, with diatom covered Bacto-agar plugs of the same size, whereas the fourth empty space was filled with a control Bacto agar plug, without any algal layer (Fig. 1). Usually 10 replicates were prepared of each experimental dish, with the same three diatom species and a control plug. Special care was taken to reduce microbial contamination during the experiments once the axenity was compromised with the addition of the larvae. The experiments took place in a clean culture room and manipulations took place in a sterile laminar flow hood.

[FIGURE 1 OMITTED]

Assessment of the Settlement Preference of Larval Japanese Abalone onto Layers of Different Diatoms

Forty mL of 32 ppt filtered sea water was carefully pipetted onto the surface of the Agarose agar layer in the experimental dishes, to make aqueous layers (0.6 cm in depth) for the swimming larvae. Competent larvae (200-250) were gently introduced to the center of each dish. The larval density was selected after a set of preliminary tests that suggested this density as optimal for counting and statistics, with no harm to the settlement process. The dishes were left covered, at 22[degree]C and under continuous light (~60 [micro]mol photons [m.sup.-2][s.sup.-1]). These conditions involved minimal technical difficulties in the laboratory, and fell within the range of conditions reported in the literature. No preliminary tests were made to optimize temperature and light conditions.

Counts of settled larvae on top of the algal patches were carried out using a dissecting microscope several times during 24 hours. Attractiveness of a certain algal species to the larvae was determined by the number of larvae that settled (even if metamorphosis was not completed) on its patch relative to the number of larvae settled on the other diatom patches and on the control patch up to a certain time (usually 10 h) after initiation of the experiment. Seventeen species of algae were tested for larval settlement preference in 50 experiments, each consisting of 10 replicate petri dishes, each with the same group of three diatoms.

Elucidation of Possible Factors Influencing Settlement Preference by the Larvae of the Japanese Abalone

Settlement Preference of the Larvae onto Sonicated and Boiled Diatom Cells

This experiment used the settlement preference measurement system described above to evaluate a possible linkage between algal internal biochemical composition and larval preference to settle on it. Concentrated algal cells were broken with an ultrasonic cell disruptor (Microson) for 5 minutes (microscopy check assured cell disruption) and then mixed with cooled (40-45[degrees]C), still liquefied Bacto agar. Larval settlement preference was tested towards Nitzschia laevis (2.05 x [10.sup.6] cells/plug), which in its native state had been found very attractive to the larvae, and Amphora cf tenerrima (2) (3.9 x [10.sup.5] cells/plug), which in its native state had been found to be unattractive or even repulsive to the larvae.

A second experiment was conducted as earlier stated, but the sonicated cells were boiled for 15 minutes before mixing with agar, to denature the proteins. The aim of this experiment was to determine the role of native proteins in attractiveness to the larvae.

Settlement Preference of the Larvae onto Agar Plugs Soaked with Algal Growing Medium and Amino Acids

Settlement preference of abalone larvae was tested towards liquid culture media in which algae had been axenically grown. Algal cultures were centrifuged (3000 rpm for 5 min in a clinical centrifuge) and the clear media were then filtered through a Nucleopore filter (0.2 [mu]) to remove any cell debris, and used with 1% agar to prepare an agar layer from which plugs could be cut for the assays.

Settlement preference of the larvae was also tested towards agar plugs soaked with solutions (final concentration in agar 10 mM) of the following L-amino acids: methionine, threonine, serine, phenylalanine, tryptophan, glycine, DL-alanine, proline, betaine, leucine, taurine, valine, cysteine, histidine, ornithine, arginine, lysine and aspartic acid (All Sigma reagent grade).

Statistical Analyses

The data were compared by ANOVA (one way) with Duncan's multiple range test. Results in percentages were arcsine square root-transformed prior to ANOVA to homogenise variances (Sokal & Rohlf 1969).

The Effect of Light on the Settlement of Abalone Larvae

Settlement preference of abalone larvae was tested towards agar plugs with three different sonicated algal species (made as earlier stated). Two series of 10 replicate algal dishes were made. One set was put in the dark, and the other in light.

RESULTS

Assessment of the Settlement Preference of Larval Japanese Abalone (Haliotis discus hannai) onto Layers of Different Diatoms

In most cases larvae settled during 10 hours from the experiment initiation (Fig. 2). Strong settlement stimulants, like sonicated cell suspensions, speeded up the settlement to 1.5 hours to 5 hours.

[FIGURE 2 OMITTED]

The larval abalone preferred, by a large margin, to settle on patches of Nitzschia laevis in each of the experiments in which this diatom was offered (Table 2). On average, almost half the larvae settled on N. laevis in each relevant individual experiment, in comparison to much smaller fractions on the other diatoms offered and on the control (Table 3). Significant (1-w ANOVA, P < 0.05) preference of the abalone larvae for the diatoms Navicula cf lenzii and Amphora luciae was obtained in 82% and 75% of relevant experiments, respectively (see Table 2). On the average, about a quarter to a third of the larvae preferred these two diatoms in each relevant individual experiment (Table 3).

Nitzschia laevis (the symbiont clone) and Amphora coffeiformis were also found attractive to the larvae in individual experiments (see Table 3). However, over half of the diatoms tested were not found to be attractive to larval settlement in most of the experiments (Tables 2 and 3).

Settlement of Japanese Abalone Larvae on Sonicated Diatom Cell Suspensions

The larvae preferred by a large margin (P < 0.001) to settle onto Nitzschia laevis layers rather than onto Amphora cf tenerrima (2) layers or the control (no algae) (Fig. 3). Whole cells of A. cf tenerrima (2) were found unattractive to larvae in this experiment and others (see Table 2), On the contrary, the abalone larvae showed a strong preference (70% of the settled larvae: P < 0.001) for settlement onto sonicated cells of the same A. cf tenerrima (2). Surprisingly, the settlement on sonicated N. laevis was much lower than that on A. cf tenerrima (2) and in one experiment it did not differ from the control (no algae) (see Fig. 3).

[FIGURE 3 OMITTED]

The temporal dynamics of the settlement process were also different with the sonicated diatoms. When comparing larval settlement onto whole cells and onto sonicated cells in the same petri dish, larvae explored and attached first (after 1.3 h) onto the sonicated cells, and gradually over time moved to settle onto the whole cells (Fig. 4). After 22 hours, larval settlement onto patches of intact cell layers was significantly (1-w ANOVA, P < 0.001) higher than onto patches of the sonicated cell suspensions or the control (no algae).

[FIGURE 4 OMITTED]

Settlement of Abalone Larvae on Boiled Sonicated Diatom Cells

Larval preference for settlement onto sonicated cells of three of the tour diatom species tested was not significantly affected (1-w ANOVA, P > 0.05) by boiling them (Fig. 5). Only with sonicated Amphora luciae cells, did the larval preference significantly drop (1-w ANOVA, P < 0.001) after boiling, even though it still remained significantly above the preference (1-w ANOVA, P = 0.001) for the control (no algae) (Fig. 5).

[FIGURE 5 OMITTED]

Settlement of Japanese Abalone larvae on Agar Plugs Soaked with Algal Growing Medium and Amino Acid Solutions

The abalone larvae settlement on agar plugs soaked with algal growing medium, in which attractive diatoms had been grown, did

not produce significant results (1-w ANOVA, P > 0.05) (Table 4). Settlement on agar plugs soaked with 10 mM of aspartic acid was consistently much (13 times) higher than on the control (no algae) plug (P < 0.001 Table 5). However, settlement on 16 other amino acids tested, singly and in mixtures, could not be distinguished (1-w ANOVA, P > 0.05) from the control (see Table 5).

The Effect of Light on the Settlement of Abalone Larvae

There was no significant effect of light (1-w ANOVA. P > 0.05) on the settlement of larvae (Fig. 6).

[FIGURE 6 OMITTED]

DISCUSSION

Post larvae of abalone are able to distinguish between diatom species, feeding on some and avoiding others (Norman-Boudreau et al 1986, Matthews & Cook 1995, Siqueiros-Beltrones & Voltolina 2000). This behavior is similar to that which has been observed in many meiofauna that were attracted to and retained by patches of micro-algae that they selected (Lee et al. 1977). In this context it was not surprising that the larvae of Haliotis discus hannai in our experiment preferred to selectively attach and settle on some of the diatom patches offered to them but not on others. The chemosensory nature of the process, by which swimming abalone larvae select where to attach and where to settle, has been confirmed by the success of our novel "cafeteria" method by which "good" diatoms were selected. This method was based on the ability of abalone to sense and select the site of settlement and source of food according to several proposed environmental cues (Morse 1985, Morse 1992, Baxter & Morse 1992, Searcy-Bernal et al. 1992, Fleming 1995, Bryan & Qian 1998, Roberts 2001).

Not many diatoms are known to be consistently suitable for abalone larval recruitment (Roberts 2001). Most of the diatoms we tested were indeed unsuitable (as has also been reported in Daume et al. 1999), even though they formed flat colonies, which had been reported as suitable for abalone larval settlement (Kawamura 1996). Nevertheless we have found three highly attractive diatoms: Nitzschia laevis, which was by far the best, followed by Navicula lenzii and Amphora luciae.

In accordance with the results of the study by Morse and Morse (1984) on Haliotis rufescens larvae, the cue that allows the Japanese abalone larvae to select one diatom over another for settlement was not visual or physical, but probably chemical. Apparently, the larvae swim towards settlement-inducing substances that are released by the diatoms and descend around them (Searcy-Bernal et al. 1992). If the diatoms are nutritious, the larvae attach and metamorphose: otherwise they ascend and swim on.

Sonication that causes release of cellular material into the surrounding water enhanced Amphora cf tenerrima (2) attractiveness for larval settlement, whereas intact cells were not found attractive. Morse and Morse (1984) obtained similar results in their study with Haliotis rufescens by surface abrasion of the seaweed Porphyra sp. Evidently, sonication or abrasion of "non-attractive" algae releases or speeds up the release of attractive substances that are tightly contained naturally in their intact cells.

The high preference of the abalone larvae to descend onto broken (sonicated) cells, in contrast to the lack of descent onto plugs soaked with algal growth medium, implies that attachment and settlement are concentration dependent and may explain the link between density and age of an algal layer and its suitability for settlement, as reported in previous studies (Kawamura & Kikuchi 1992, Daume et al. 1999).

The process of larval swimming, descent on several sites and attachment to a certain diatom patch as seen in our work and noted earlier by others (Seki 1997, Bryan & Qian 1998) is a serial cue process. Our data and observations confirm that in the process of substratum exploration, the decent of larvae to a certain spot usually depends on a cue, different in kind or in quantity from the cue for attachment and metamorphosis. The attachment is most probably induced by the mutual and perhaps synergistic action of several cues (Trapido-Rosental & Morse 1986). If larvae are given a choice, attachment and metamorphosis will most probably depend on specific attractive and repellent compounds (see below), in addition to the obvious cue of the nutritive value of the patch. Repellant or harmful substances can reduce descent or elicit ascent of the larvae (Seki 1997, Bryan & Qian 1998, Roberts 2001). This can explain why larvae that quickly descended in large numbers on a sonicated diatom suspension patch, eventually moved on and settled on patches of intact algae, apparently nutritive even though comparatively less attractive for settlement. Seki and Taniguchi (1996) and Takami et al. (1997) found similar results with mucus induction; abalone larvae settled preferentially on trails of apparently more nutritive mucus, secreted by juvenile or adult abalone during grazing of diatoms, than on mucus secreted by adults during crawling without feeding. Aspartic acid strongly induced descent and attachment of swimming larval abalone. It was also the only free amino acid traced in the growth media of two diatoms, the attractive Nitzschia laevis and the unattractive Amphora cf tenerrima (2). However, the concentrations of aspartic acid in these media were two orders of magnitude lower than inside algal cells, and three orders of magnitude lower than the level needed to induce larval attachment in our experiments. Aspartic acid might therefore be only one of several factors concurrently involved in larval settlement induction. The rate of the chemical release to the water by the intact cells and the sharpness of the resulting chemical concentration gradients around them might also be important aspects, as suggested by the inability of the other diatom (A. cf tenerrima (2)) to attract larval descent with this amino acid in its surroundings. Chemical gradients that are sufficient for chemosensory attraction of larvae can be maintained even in turbulent water, let alone still water as in these experiments (Turner et al. 1994).

Complex native proteins are unlikely to be responsible for the attractiveness of a diatom to larval settlement, because heat denaturation of proteins in sonicated diatom suspensions hardly affected the results of the settlement experiments. Still, small peptides or free amino acids, not affected by heat-denaturation and therefore not excluded by the boiling procedure, have been proposed as possible as chemical cues for abalone larval settlement (Morse & Morse 1984, Morse & Morse 1988, Morse 1985).

Layers made of certain diatoms, such as the three species selected in the research reported here, but not many others, apparently provide the swimming larvae with the whole battery of cues they need for descent, attachment, metamorphosis, and nutritious feeding. Layers of diatoms such as those selected here improve attachment and settlement and supply proper nutrition for early post larval stages. These qualities greatly improve survival and growth of the abalone early post larvae (Stator 1992, Searcy-Bernal et al. 1992, Matthews & Cook 1995, Daume et al. 1999, Roberts 2001). This understanding helps explain the interrelationship between diatom biodiversity and the recruitment of animals, such as abalone, both in natural settings and in aquaculture.

TABLE 1.
The diatoms evaluated in this study. Numbers (in parenthesis) after
species names indicate isolates numbers; see Taxonomic Appendix
for taxonomic details.

             Algal Species                       Source

 1 Nitzschia laevis Hustedt 1939        Sediment pond, IOLR,
                                          Eilat, Israel
 2 Navicula. cf lenzii Hustedt 1939             -//-
 3 Navicula cf viminoides Giffen 1975           -//-
 4 Navicula salinicola Hustedt 1939             -//-
 5 Amphora cf tenerrima (1) Aleem               -//-
     and Hustedt 1951
 6 Amphora cf tenerrima (2) Aleem               -//-
     and Hustedt 1951
 7 Amphora sp. (2)                              -//-
 8 Amphora luciae Cholnoky emend        Martha's Vineyard
     Archibald 1983                       Island, lake Tashmoo,
                                          Massachusetts
 9 Nitzschia laevis Hustedt 1939        Gulf of Aqaba, Eilat,
     (Symbiont clone)                     Israel
10 Amphora sp. (1)                              -//-
11 Amphora sp. (4)                              -//-
12 Amphora sp. (6)                              -//-
13 Amphora coffeiformis Agardh          Ecuador
14 Navicula sp. (1)                     Lizard Island, Great
                                          Barrier Reef, Australia
15 Navicula sp. (2)                             -//-
16 Navicula sp. (3)                     Carlsbad farm C/A.
                                          California
17 Amphora sp. (3)                              -//-

TABLE 2.
The attraction rank (column A) of each of the examined diatoms
(column B). The diatoms were ranked according to their
attractiveness to larvae, based on the number of experiments each
diatom was offered to abalone larvae (column C), and the number
of experiments (with % of experiments from column C in
parenthesis) in which larvae significantly (1-w ANOVA, P < 0.05)
preferred to attach onto that diatom species (column D).

                                                          D
                                                        No. of
    A                                     C          Experiments
Attraction             B              Number of    with Significant
  Raking        Diatom Species       Experiments      Attraction

    1        Nitzschia laevis            31           31 (100%)
    2        Navicula cf lenzii          28           23 (82%)
    3        Amphora luciae              12            9 (75%)
    4        Nitzschia laevis            11            7 (64%)
               (symbiont clone)
    5        Amphora coffeiformis         6            4 (66%)
    6        Amphora sp. (1)              6            4 (67%)
    7        Navicula cf vimnoides        8            4 (50%)
    8        Navicula sp. (1)             4            2 (50%)
    9        Navicula sp. (2)             2            1 (50%)
   10        Navicula salinicula          9            3 (33%)
   11        Amphora sp. (2)              7            2 (28%)
   12        Amphora sp. (3)              7            2 (28%)
   13        Amphora cf tenerrima         9            1 (11%)
               (1)
   14        Amphora sp. (4)              5            1 (20%)
   15        Navicula sp. (3)            11            1 (9%)
   16        Amphora cf tenerrima         5            0
               (2)
   17        Amphora sp. (6)              5            0

TABLE 3.
The settlement preference of Japanese abalone larvae for different
diatoms in 8 typical positive comparisons, calculated for species that
had been found significantly (1-w ANOVA, P < 0.05) attractive in
50% or more of the experiments in which they had been offered. Means
include all experiments with the specific combination. Each
combination tested attraction toward a control of Bacto-agar plus
without algae, and a reference plug with a highly attractive alga
(Nitzschia laevis or Navicula lenzii).

Combination                Diatom                     % Attraction
    No.                    Species                   [+ or -] SE (*)

     1        Nitzschia laevis                    38.4 [+ or -] 3.2 (a)
              Navicula cf lenzii                  27.5 [+ or -] 2.3 (b)
              Control                             12.6 [+ or -] 1.6 (c)
     2        Navicula of lenzii                  32.8 [+ or -] 2.9 (a)
              Amphora luciae                      26.6 [+ or -] 2.5 (a)
              Control                              9.9 [+ or -] 1.1 (b)
     3        Navicula cf lenzii                  28.2 [+ or -] 3.5 (a)
              Nitzschia laevis (symbiont clone)   24.2 [+ or -] 4.2 (a)
              Control                             10.4 [+ or -] 1.8 (b)
     4        Navicula cf lenzii                  37.0 [+ or -] 2.3 (a)
              Amphora coeffeiformis               22.4 [+ or -] 2.4 (b)
              Control                             11.6 [+ or -] 1.2 (c)
     5        N. laevis                           41.8 [+ or -] 5.0 (a)
              Amphora sp. (1)                     18.6 [+ or -] 2.2 (b)
              Control                             13.6 [+ or -] 1.9 (b)
     6        N. laevis                           61.4 [+ or -] 6.0 (a)
              Navicula cf vimnoides               12.4 [+ or -] 1.1 (b)
              Control                             13.5 [+ or -] 3.4 (b)
     7        Navicula cf lenzii                  35.9 [+ or -] 3.4 (a)
              Navicula sp. (1)                    22.5 [+ or -] 2.7 (b)
              Control                             14.8 [+ or -] 2.6 (b)
     8        N. laevis                           45.7 [+ or -] 2.0 (a)
              Amphora cf tenerrima (2)            15.4 [+ or -] 6.4 (b)
              Control                             19.2 [+ or -] 4.6 (b)

* Data with the same letter indicate treatments that are not
significantly different from each other (ANOVA, Duncan's
multiple range test. P > 0.05).

TABLE 4.
Larval settlement, tested toward agar layers made with liquid media
in which diatoms had been grown, in comparison to whole cell
diatom layers on agar. Mean number of settled larvae [+ or -] SE;
n = 10; data with the same letter indicate treatments that are not
significantly different (ANOVA, Duncan's multiple range test,
P > 0.05).

                             No. of Larvae       No. of Larvae
                            Settled on Agar       Settled on
                              Plugs with          Agar Plugs
     Diatom Species           Whole Cells         with Media

Nitzchia laevis            44 [+ or -] 3.6 a   38 [+ or -] 2.9 a
Amphora cf tenerrima (2)   28 [+ or -] 3.2 b   29 [+ or -] 3.4 a
Control                    28 [+ or -] 3 b     33 [+ or -] 2.8 a

TABLE 5.
Larval settlement tested towards solutions (10 mM final
concentration) of four amino acids soaked into agar plugs.

Amino Acid      Attracted larvae (%)

Alanine             4 [+ or -] 1
Proline             3 [+ or -] 1
Leucine             7 [+ or -] 2
Aspartic acid      81 [+ or -] 4
Control             6 [+ or -] 3

Mean [+ or -] S.E.; n = 10.

ACKNOWLEDGMENTS

The authors thank H. Krogliak for providing technical help, M. Ben-Shaprut for the editorial assistance, and all the reviewers for their constructive comments.

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TAXONOMIC APPENDIX

The isolates used in this study have been identified to the closest described taxa. We recognize that species concept in diatoms has been interpreted in many ways. Some have very rigid "morpho-species" concepts that allow very little deviation from the type specimen (e,g., Clavero et al. 2000) whereas others (e.g., Lee et al. 2000) have looked at a wide variance in overlapping populations obtained from different field collections. We acknowledge the problem, as it relates to our isolates, but do not wish to resolve this conundrum here.

Some of the diatoms were easier to identify than were others. Four species were problematic. We chose to identify the organism #3 in Table 1 as Navicula cf viminoides Giffen. The clone culture we used fit the original description and illustrations of the species.

Navicula cf lenzii Hustedt 1945 was described as a freshwater species. In his 1961 monograph (see Literature Cited), Hustedt illustrated and described the variations he considered as belonging to this taxon. Although we would be skeptical to find a freshwater species in a eutrophic marine environment, the species with which we worked is more closely related to this taxon than to any other.

Organisms numbered 5 and 6 in Table I were referred to Amphora cf tenerrima Aleem and Hustedt. They were morphologically distinct from each other in SEM micrographs, but resembled the original light microscopic description of the species by Aleem and Hustedt.

NURIT GORDON, (1) MUKI SHPIGEL, (1) SHEENAN HARPAZ, (2) JOHN J. LEE (3) AND AMIR NEORI (1) *

(1) Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel; (2) Department of Aquaculture, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel; and (3) Department of Biology City College of City University of New York, Convent Ave at 138 St, New York, New York 10031

This work was supported by the Israeli Ministry for National Infrastructures (N.G., M.S. and A.N.), by grants from the European Commission (M.S. and A.N.) and NIH/NIGMS 08168-22 (J.J.L.).

* Corresponding author. E-mail: [e-mail]