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Sheltering behavior of the abalone, Haliotis tuberculata L., in artificial and natural seawater: The role of calcium
Aquaculture 299 (2010) 67–72
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e
Sheltering behavior of the abalone, Haliotis tuberculata L., in artificial and natural seawater: The role of calcium Federica Cenni a, Giuliana Parisi b, Felicita Scapini a, Francesca Gherardi a,⁎ a b
Dipartimento di Biologia Evoluzionistica “Leo Pardi”, Università di Firenze, Via Romana 17, 50125 Firenze, Italy Dipartimento di Scienze Zootecniche, Università di Firenze, Via delle Cascine 5, 50144 Firenze, Italy
a r t i c l e
i n f o
Article history: Received 8 September 2009 Received in revised form 16 November 2009 Accepted 17 November 2009 Keywords: Artificial seawater Calcium Haliotis tuberculata L. Sheltering behavior
a b s t r a c t There is an increased interest worldwide in developing land-based closed systems to cultivate the abalone, Haliotis tuberculata L. Here, we analyzed whether artificial conditions, and particularly the use of artificial seawater, might affect sheltering, a critical behavior of this species. The study was composed of two experiments. In the first, we compared sheltering between two groups of 100 individuals each reared in either natural or artificial seawater. For 15 days, a score was assigned every day to the position occupied by each individual, i.e. “0” when it was out of the hide, “1” when it was on the bottom of the maintenance tank underneath the hide, and “2” when it occupied the inner spot of the hide. Our results show that artificial seawater significantly affects sheltering: only the individuals reared in natural seawater were able to reach the hide and maintain a position in it. Chemical analyses revealed that artificial seawater differed from natural seawater for a relatively low concentration of Ca2+. In the second experiment we compared H. tuberculata's sheltering behavior in three media, natural, artificial, and calcium-enriched artificial seawater. Sheltering was restored in enriched artificial seawater, which supports the hypothesis that calcium has an effect on this species' behavior. However, calcium concentration both in the artificial and “natural” water showed a progressive decrease with time likely due to the absorption by abalones, which suggests that in closed systems calcium supply is constantly required, even when the used medium is natural seawater. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Natural stocks of the abalone, Haliotis tuberculata L. (Mollusca: Haliotidae), are worldwide overexploited and in a state of serious decline (Guzmán del Proó, 1992; Parker et al., 1992; Tegner et al., 1992; Shpigel et al., 1999). Decreased fisheries, combined with the increasing demand for this species and its high market value, have resulted in accelerating the spread of abalone aquaculture (e.g. Oakes and Ponte, 1996) and the development of new projects that look into land-based closed or semi-closed systems (Basuyaux and Mathieu, 1999) to support traditional farming in natural environments. In order to maximize production, research has been mostly directed to understand the biology of the species (Mgaya and Mercer, 1994). Conversely, little is known about the environmental factors that are likely to influence the abalone's growth in closed systems (Basuyaux and Mathieu, 1999). Here, we analyzed whether the use of artificial seawater may alter the behavior of H. tuberculata. The rationale underlying this pivotal study is that, in farm settings, rearing techniques should be adjusted to it so as to maximize the health and growth performance of the animals
⁎ Corresponding author. Tel.: +39 055 2288216/215; fax: +39 055 222565. E-mail address: francesca.gherardi@unifi.it (F. Gherardi). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.11.016
(Huchette et al., 2003). This implies a good knowledge of the behavior of the reared species. Changes in behavior may in fact be used as early warning indicators of suboptimal rearing conditions. On the basis of the information about this species' biology in nature (e.g. Momma and Sato, 1969) and in the laboratory (Cenni et al., 2009), we used sheltering behavior as a proxy of the individuals' wellbeing. In the inhabited reefs, abalones take refuge during the day under rocks or in crevices, from which they emerge in the evening to hide again at dawn (Stephenson, 1924). As observed in several species of Haliotis, some spots of the reef are most often occupied as shelters possibly because of the better protection from predators they offer (Momma and Sato, 1969; Shepherd, 1973; Tarr, 1995). Laboratory studies on H. rubra (Leach) (Huchette et al., 2003) and H. tuberculata (Cenni et al., 2009) showed that abalones do not distribute evenly on the provided sheltering space in the tanks, but rather aggregate in particular spots, suggesting that this phenomenon, i.e. sheltering in the most suitable spots, also occurs in artificial environments. Based on these premises, firstly we compared sheltering behavior in H. tuberculata reared in either natural or artificial seawater; secondly, having found a significant difference between the two treatments, we analyzed the possible effect of calcium on this species' sheltering behavior, since natural and artificial seawater differed in calcium content.
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2. Materials and methods
df = 3, P N 0.05), with a similar sex ratio (after a G test after William's correction: G between 2.275 and 4.354, df = 3).
2.1. General set-up 2.3. Experiment 2 The experimental abalones (3–4 year old), originating from a French hatchery (“Les ormeaux du Cotentin”, Gouville sur Mer), had been reared before the study in an open system in Orbetello (Grosseto, Italy). The experiments were carried out at the University of Florence after at least two weeks of acclimation to the laboratory conditions. The sex of each animal was determined and its maximum shell length was measured with an electronic caliper to the nearest 0.1 mm. Abalones were individually marked using plastic tags (Hallprint, Adelaide, South Australia) attached to their shell with a superglue gel (Shepherd, 1988). Marking did not affect their behavior, as shown in preliminary tests. For our experiments we used Instant Ocean® artificial seawater (made with demineralized and distilled water, with the same salinity as natural seawater, i.e. 33.3 gmL− 1) [AS], the natural seawater used in the rearing system in Orbetello [NS], or artificial seawater that was enriched with calcium [ES]. The content in calcium was increased by adding 0.3 mL of the enriching Calcium Plus® Prodac per liter of seawater, to reach the same calcium concentration as natural seawater (approximately 1000 mgL− 1). AS, NS, and ES were analyzed for their chemical content in the Laboratory of Microanalysis at the University of Florence using the international official method for water minerals determination, i.e. E.P.A. method 3005 applied to ICP OES D.V. Perkin Elmer Optima 2000 instrumentation. Six hundred-L plastic tanks were used for Experiment 1. Experiment 2 was conducted in smaller containers, 50-L glass aquaria that allowed us to better manage calcium enrichment. Tanks and aquaria were cleaned daily using a hose; twice a week, 20% of their water was renewed. The room was maintained at a natural light:dark cycle (approximately 12:12 h) and at a constant air temperature of 18 °C. Abalones were fed ad libitum once every second day using artificial feed (ADAM & AMOS “7-mm Chip” Abalone Food, Australia). Each replicate of both experiments was run for two weeks. Using a VWR SympHony SP90M5 electronic tester, we measured twice a week in each tank/aquarium a number of water parameters, such as temperature, conductivity, salinity, and pH, together with the content of dissolved oxygen and ammonia/nitrite/nitrate to monitor the biological activity of the study animals. The two experiments were conducted between March 2007 and April 2009. The first experiment was aimed at analyzing the possible differences in the sheltering behavior of H. tuberculata reared in either natural or artificial seawater, whereas purpose of the second experiment was to analyze the possible effect of calcium on the abalones' behavior.
Each individual was randomly assigned to one of three aquaria (= treatments) containing artificial seawater [AS, Aquarium A], natural seawater [NS, Aquarium B], or artificial seawater enriched with calcium [ES, Aquarium C]. We ran three replicates per aquarium, with 20 animals per replicate (total: 180 abalones); records were taken once a day, reaching a total of 15 records per replicate. AS, NS and ES were analyzed for their calcium content three times, i.e. at the start of the experiment, after a week, and at the end. Here, the same materials and methods as in Experiment 1 were used except as follows. Instead of the baskets, we used single glass aquaria (50 × 35× 35 cm) containing 50 L of water filtered in continuum by two 60 L pumps. Each aquarium was provided with a semi-cylindrical concrete hide as above and hosted 20 individuals (density: ca. 350 m− 2; overall biomass: ca. 0.20 kg) of similar size (40–55 mm; F between 0.463 and 0.781, df= 8, P N 0.05) and sex ratio (after a G test after William's correction: G = 1.481, df = 8, P N 0.1). 2.4. Data recording Records were taken at 9:00 when each basket/aquarium was photographed with a digital camera. The position of each abalone with respect to the shelter was later determined from the photos; a score was given to the position occupied by each individual per record, i.e. 0 when it was out of the hide, 1 when it was on the bottom of the basket/aquarium underneath the hide, and 2 when it occupied the inner spot of the hide. To analyze differences among days, each basket/aquarium was associated with the total daily sum of individual scores, i.e. the sum of every daily score totalized by each of its inhabitants per day. To analyze overall differences between treatments, the total sum of scores, i.e. the sum of the scores totalized by all its inhabitants during the entire study period, was used. To analyze the preference of abalones for the three different positions with respect to the shelters, we measured the alpha selectivity coefficient, slightly modified after Chesson (1978), from the formula: αi = ð ri = pi Þ = ð∑ ri =pi Þ i
where pi is the ratio between the surface corresponding to each of the three scores (i = 0, 1, or 2) and the whole available area of the basket, and ri is the proportion of the abalones found in the i position. A low mortality of abalones (ca. 3%) was observed during the acclimation period possibly due to handling and transportation stress. No abalone died during the experiments.
2.2. Experiment 1 2.5. Statistical analyses Each individual was randomly assigned to one of two plastic tanks (= treatments) containing either artificial seawater (made as above) [AS, Tank A], or natural seawater [NS, Tank B]. Three replicates per treatment, with 100 animals per replicate (total: 600 abalones), were run, and records were taken once a day, reaching a total of 15 records per replicate. Each tank (160 × 100 × 60 cm) contained 600 L of seawater filtered in continuum by two 500 L pumps and four squared-section baskets (23 × 23 × 27 cm), each subject to a constant water flow and positioned at the same distance from the light sources and the walkway in order to maintain equal the possible effects on behavior of light intensity and vibrations. Each basket was provided with a semi-cylindrical concrete hide (length: 15 cm; diameter: 10 cm) and hosted 25 individuals (density: ca. 400 m− 2; overall biomass: ca. 0.25 kg) of similar size (35–65 mm; F between 0.134 and 1.933,
The data that met the assumptions for normality and homogeneity of variance after the Kolmogorov-Smirnov and Levene test were analyzed using parametric tests. Specifically, differences between the two treatments for the water parameters were analyzed using two tailed Student's t-tests (statistic: t); differences among baskets and aquaria for abalones' dimensions were analyzed with one-way ANOVAs (statistic: F); GLM Analyses For Repeated Measures (statistic: F) followed by multiple comparison tests were used to estimate the overall difference among AS, NS, and CE. For the other data, nonparametric techniques were used. Differences among days were analyzed using a Friedman test (statistic: Fr), whereas those among records, replicates, baskets and aquaria, along with the differences in Chesson's alpha value (Experiment 2), were analyzed using Kruskal Wallis tests (statistic: H). Mann–Whitney tests (statistic: U) were used
F. Cenni et al. / Aquaculture 299 (2010) 67–72 Table 1 Experiment 1: differences between natural (NS) and artificial (AS) seawater for the concentration of the most common seawater ions (mgL− 1) using Mann–Whitney tests (N = 3). Significant values are denoted in bold. Ion
Al3+ (mgL− 1) B3+ (mgL− 1) Ba2+ ( mgL− 1) Ca2+ (mgL− 1) Cd2+ (mgL− 1) Cr2+(mgL− 1) Cu2+(mgL− 1) Fe3+ (mgL− 1) Hg2+ (mgL− 1) K+ (mgL− 1) Li+ (mgL− 1) Mg2+ (mgL− 1) Mn3+ (mgL− 1) Ni3+ (mgL− 1) Pb2+ (mgL− 1) Se6+ (mgL− 1) Sr2+ (mgL− 1) Ti3+ (mgL− 1) Zn2+ (mgL− 1)
NS
AS
Statistics
Mean (SE)
Mean (SE)
U
P
1.8 (0.5) 0 0.06 (0.01) 803.1 (74.0) 0.005 (0.002) 0 0.28 (0.11) 0.07 (0.03) 0 407.8 (48.22) 0.47 (0.05) 370.86 (35.8) 0.23 (0.08) 0.17 (0.02) 0 0.25 (0.06) 17.57 (6.41) 0 0.32 (0.18)
0.03 (0.01) 5.09 (0.92) 0.02 (0.005) 269.48 (31.6) 0 0 0 0.06 (0.03) 0 385.39 (36.2) 0.16 (0.06) 366.92 (38.4) 0.01 (0.003) 0.03 (0.001) 0 0 6.65 (1.98) 0 0
3.9 1.5 2 0 1.99 40.5 1.6 4 40.5 3.5 2.1 4 1.7 3.8 40.5 1.6 2.2 40.5 1.8
0.827 0.114 0.261 3.52 × 10− 5 0.275 1 0.141 0.897 1 0.671 0.293 0.935 0.141 0.569 1 0.135 0.312 1 0.199
Table 3 Experiment 1: differences among replicates in natural (NS) and artificial (AS) seawater for the parameters measured (N = 3).
Data
to check differences in ion concentrations, total sum of scores, and Chesson's alpha values between the two treatments. Spearman's correlations (rs) were performed between sums of scores and treatments and between behavior and size. Frequencies of sex ratio, positions, and transition among positions were analyzed with G tests after William's correction (statistic: G). The level of significance under which the null hypothesis was rejected is α = 0.05. 3. Results 3.1. Experiment 1 3.1.1. Water analyses We analyzed the concentration of the most common seawater ions (Al3+, Ba2+, Ca2+, Cd2+, Cr2+, Cu2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn3+, Ni3+, Pb2+, Se6+, Ti3+, and Zn2+) (Table 1); when compared with NS, AS was relatively poor in calcium , while the two types of water were similar for the other ions (U = 0, N = 9, P b 0.05; for the other ions: U between 40.5 and 1.5, N = 9). Temperature, conductivity, salinity, pH, dissolved oxygen, and ammonia/nitrite/nitrate did not significantly differ between treatments (t between 9.839 and 11.490, df = 6) (Table 2). 3.1.2. Behavioral data Since replicates did not differ for any variable in either AS (H ranging between 0.062 and 3.919, df= 2) or NS (H ranging between 0.008 and 3.033, df= 2) (Table 3), the daily sums of scores were compared among the baskets analyzed separately per treatment, which showed a significant difference among days (Fig. 1), both in AS (Fr = 36.426, df= 14,
69
NS
Statistics
AS
Statistics
Mean (SE)
H
Mean (SE)
H
P
Length (mm) 45.6 (4.2) 2.01 P N 0.1 Daily sum of scores 20.65 (6.24) 3.03 P N 0.1 Chesson's α 0.16 (0.01) 0.08 P N 0.1 (out of the hide) Chesson's α 0.37 (0.02) 0.21 P N 0.1 (underneath the hide) Chesson's α 0.47 (0.02) 1.02 P N 0.1 (inner spot of the hide)
P
45.4 (7.2) 2.09 P N 0.1 7.2 (1.23) 3.919 P N 0.1 0.48 (0.02) 0.128 P N 0.1 0.27(0.03) 0.062 P N 0.1 0.25 (0.01) 1.12
P N 0.1
P = 0.002) and in NS (Fr = 26.374, df= 14, P = 0.045). However, the daily sums of scores were significantly correlated between the two tanks (rs = 0.878, n = 15, P b 0.001), suggesting that this trend was the same in the two types of seawater, as a possible result of the abalones changing their activity in association with food provision (Fig. 1). Another hypothesis is that the abalones change their behavior (activity) in association with their increased familiarization with the experimental tank. However, as also shown by Fig. 1, this does not appear to be the case. The daily sums of scores did not differ among baskets within each tank (AS: H between 3.986 and 4.130, df = 3; NS: H between 6.872 and 7.292, df = 3), whereas the total sum of scores significantly differed between treatments (U = 0, N = 24, P b 0.001), with higher values in NS. This was confirmed by analyzing the behavior of individual abalones. In fact, as revealed by assigning to each individual the mode of the scores computed for the entire period, those inhabiting NS showed a larger use of the hide (scores 1 and 2) (G = 121.861 df = 2, P b 0.001), whereas those in AS most often scored 0 (G = 161.367, df = 2, P b 0.001), with a significant difference in the frequency distribution of scores between the two treatments (AS: mean = 106.4, SE = 16. 81; NS: mean = 311.66, SE = 26.14; G = 48.221, df = 2, P b 0.001). The recorded difference was not related to the animal size (AS: rs = − 0.02, N = 600, P = 0.986; NS: rs = 0.052, N = 600, P = 0.524). The transitions from one record to the subsequent in the positions taken were counted and their probability was analyzed (G between 266.144 and 7.123, df = 1) (Fig. 2). The comparison between AS and NS for the hiding behavior revealed that the abalones in NS were more mobile than those in AS, and that they more often managed to remain in the hide. The overall frequencies of transitions differed within (AS: G = 2908.992, df = 24, P b 0.001; NS: G = 701.856, df = 24, P b 0.001) and between the two treatments (G = 634.465, df = 24, P b 0.001), the abalones in AS being more often found out of the hide (Fig. 3). To support the above results, the three positions “out of the hide”, “underneath the hide”, and “inner spot of the hide” were compared between artificial seawater (AS) and natural seawater (NS).
Table 2 Experiment 1: differences between natural (NS) and artificial (AS) seawater for the water parameters measured after t-tests for independent data. NS
AS
Statistics
Data
Mean (SE)
Mean (SE)
t
Temperature (°C) Conductivity (mS/cm) Salinity (‰) pH Dissolved O2 (mg/L) Ammonia/Nitrite/Nitrate (mg/L)
16.35 (0.25) 503.28 (24.6) 34.07 (0.22) 8.12 (0.29) 77.67 (2.76) 4.06 (0.89)
16.21 (0.27) 0.388 2 507.3 (28.9) 0.127 2 34.22 (0.13) − 0.614 2 8.11 (0.09) 0.476 2 77.15 (2.49) 0.138 2 4.11(1.02) − 0.513 2
df P 0.705 0.911 0.551 0.643 0.892 0.583
Fig. 1. Experiment 1: comparison between artificial seawater (AS) and natural seawater (NS) for the positions taken by the abalones (daily sums of scores) in the 15 records (observations) of the study. Arrows denote when food was supplied.
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Fig. 2. Experiment 1: transitions between the three positions taken by the abalones compared between artificial seawater (AS) and natural seawater (NS). Arrows indicate the direction of the transitions between two positions within two subsequent records. If there was no transition the arrow is curve. The signs “b” and “N” indicate in which medium (AS or NS) the number of transitions was higher. G values are indicted over the arrows (⁎⁎ = P b 0.001;⁎ = P b 0.01).
Fig. 4. Experiment 1: differences between Chesson's alpha for the three positions (a: “out of the hide”, b: “underneath the hide”, and c: “inner spot of the hide”) compared between artificial seawater (AS) and natural seawater (NS).
The abalones in AS and NS also differed for the Chesson's alpha value (out of the hide: U = 0, N = 15, P b 0.001; underneath the hide: U = 445.0, N = 15, P = 0.005; inner spot of the hide: U = 7.0, N = 15, P b 0.001) (Fig. 4). 3.2. Experiment 2 3.2.1. Water analyses Within each treatment, replicates showed a similar calcium content (H ranging between 1.988 and 2.101, df = 2) (Table 4) so that the data were pooled to estimate the overall difference among AS, NS, and CE. A GLM analysis for repeated measures showed a significant effect of time (F = 18.681, df = 2, P b 0.001), with calcium decreasing its concentration in each treatment, but also a significant difference among AS, NS, and CE as expected (F = 64.465, df = 2, P b 0.001; NS = CE N AS) (Fig. 5). The interaction between time and treatment was also significant (F = 166.247, df = 2, P b 0.001), revealing that the decrease had a different speed in the two types of water, being slower in AS. Table 4 Experiment 2: differences among replicates in natural (NS), artificial (AS), and calciumenriched artificial (CE) seawater for initial calcium concentration (mgL− 1) (N = 3). Fig. 3. Experiment 1: transitions between two subsequent records (denoted by arrows) in the three positions taken by the abalones in artificial seawater (AS) and natural seawater (NS). The curved arrow indicates no transition. The percentage of individuals in the corresponding position is indicated over each arrow. The signs “−” and “+” denote if the corresponding frequency of transition is lower and higher, respectively, than the expected one.
Ca2+ (mgL− 1)
Statistics
Water
Mean (SE)
H
P
NS AS CE
1043.43 (64.31) 584.97 (41.12) 981.26 (56.17)
1.298 1.876 2.101
P N 0.1 P N 0.1 P N 0.1
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Fig. 6. Experiment 2: comparison among artificial seawater (AS), natural seawater (NS), and calcium-enriched artificial seawater (CE) for the position taken by the abalones (daily sums of scores) in the 15 records of the study. Arrows denote when food had been supplied.
Fig. 5. Experiment 2: comparison among artificial seawater (AS), natural seawater (NS), and calcium-enriched artificial seawater (CE) for the mean concentration of Ca2+ (± SE) analyzed at the beginning of the experiment, 10 days from the start, and at the end.
3.2.2. Behavioral data Since replicates were similar for all the analyzed variables in AS (H ranging between 0.041 and 2.936, df = 2), NS (H ranging between 0.012 and 4.103, df = 2), and CE (H ranging between 0.021 and 3.188, df = 2) (Table 5), the daily sums of scores of each replicate were pooled to make an overall comparisons among treatments. A significant difference was found among days (Fig. 6), in both AS (Fr = 41.628, df = 14, P = 0.001) and NS (Fr = 36.374, df = 14, P = 0.005). However, the daily sums of scores were significantly correlated among treatments (rs = 0.746, N = 15, P b 0.001), suggesting that this trend was the same between types of seawater, as a possible result of the abalones changing their activity in association with food provision, as also seen in Experiment 1 (Figs. 1 and 6). The daily sums of scores did not differ among aquaria within each tank (AS: H between 1.764 and 1.908, df = 2; NS: H between 4.650 and 5.070, df = 2; CE: between 2.866 and 3.162, df = 2), whereas the total sum of scores significantly differed between treatments (U = 0, N = 9, P b 0.001), with lower values in AS and similarly higher values in NS and CE. This was confirmed by analyzing the behavior of individual abalones. The individuals inhabiting NS and CE showed a larger use of the hide (scores 1 and 2) (NS: G = 121.861, df = 2, P b 0.001; CE: G = 116.672, df = 2, P b 0.001), whereas those in AS most often scored 0 (G = 161.367, df = 2, P b 0.001), with a significant difference in the frequency distribution of scores between the two types of seawater (AS: mean = 106.4, SE = 16. 81; NS: mean = 311.66, SE = 26.14; CE: mean = 295.87, SE = 31.24; G = 51.226, df = 2, P b 0.001). The recorded difference was not related to the animal size (AS: rs = − 0.018, N = 60, P = 0.961; NS: rs = 0.048, N = 60, P = 0.522; CE: rs = 0.041, N = 60, P = 0.532).
The comparison among the treatments for the transitions from one record to the subsequent in the positions taken revealed that the abalones in NS were more mobile than those in AS, and that they more often managed to remain in the hide. Overall frequencies of transitions differed within (AS: G = 2506.225, df = 8, P b 0.001; NS: G = 509.765, df = 8, P b 0.001; CE: G = 488.567, df = 8, P b 0.001) and among the three treatments (G = 592.967, df = 8, P b 0.001), the abalones in AS being more often found out of the hide . The abalones in AS, NS, and CE also differed for the Chesson's alpha (out of the hide: H = 0.021, df = 2, P b 0.001; underneath the hide: H = 0.038, df = 2, P b 0.001; inner spot of the hide: H = 0.04921, df = 2, P b 0.001), with significant differences between AS and NS (out of the hide: U = 0, N = 15, P b 0.001; underneath the hide: U = 338.0, N = 15, P = 0.004; inner spot of the hide: U = 8.0, N = 15, P b 0.001), AS and CE (out of the hide: U = 1.0, N = 15, P b 0.001; underneath the hide: U = 546.0, N = 15, P = 0.006; inner spot of the hide: U = 6.0, N = 15, P b 0.001), but not between NS and CE (out of the hide: U = 108.0, N = 15, P = 0.850; underneath the hide: U = 92.0, N = 15, P = 0.390; inner spot of the hide: U = 102.5, N = 15, P = 0.605). 4. Discussion The results of Experiment 1 clearly show that sheltering behavior in H. tuberculata is independent of the individuals' body size but it is highly affected by their rearing in artificial seawater. Although all individuals displayed a tendency to improve their position with respect to the shelter, only the abalones reared in natural seawater were able to reach and maintain a position in the hide. A recent study investigating the use of space by H. tuberculata in an artificial environment (Cenni et al., 2009) showed that each individual, independently of its sex, adopts one of two locomotion strategies, behaving as either a “wanderer” or a “sedentary”, with wanderers covering longer distances and occupying the hides, particularly their inner spots, more often than the sedentary individuals. Although the experimental animals were randomly extracted from a seemingly homogeneous sample, equilibrium was soon reached between the
Table 5 Experiment 2: differences among replicates in natural (NS), artificial (AS), and calcium-enriched artificial (CE) seawater for the parameters measured. (N = 3). NS
Statistics
AS
Statistics
CE
Statistics
Data
Mean (SE)
H
P
Mean (SE)
H
P
Mean (SE)
H
P
Length (mm) Daily sum of scores Chesson's α (out of the hide) Chesson's α (underneath the hide) Chesson's α (inner spot of the hide)
44.9 22.78 0.14 0.38 0.49
3.02 4.103 0.012 0.145 0.603
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
45.1 6.8 0.51 0.28 0.21
2.701 2.936 0.198 0.041 1.01
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
44.8 22.72 0.15 0.37 0.48
2.911 3.188 0.234 0.799 0.021
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
(4.9) (7.07) (0.02) (0.03) (0.02)
(5.3) (1.98) (0.04) (0.01) (0.03)
(3.1) (5.11) (0.04) (0.05) (0.03)
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two behavioral strategies. Our results here showed that living in artificial seawater increased the number of sedentary animals (i.e. animals that did not change their position), suggesting some properties of the medium that might shift the equilibrium between the two behavioral strategies. The chemical analyses showed that the artificial seawater used had a lower content in calcium than natural seawater. Experiment 2 indicated that low calcium concentration may affect the behavior of H. tuberculata. In fact, abalones reared in calcium-enriched artificial seawater restored the behavior exhibited in natural seawater. There are several explanations to this phenomenon. Indeed, the lack of calcium may impede a number of functions in this taxon, including shell building, blood clotting, muscle function, sperm motility and metabolism, and nerve transmission (Azuma, 1976; Tash and Means, 1983; Lovell, 1989; Coote et al., 1996; Tan et al., 2001; Moulis, 2006). In turn, an affected neural transmission due to a lack of calcium may decrease locomotion rate and the ability to home in abalones (Nakamura and Soh, 1997). How calcium can be obtained by abalone is debated (Coote et al., 1996). Similarly to fish (e.g. Ogino and Takeda, 1978; Shim and Ho, 1989; but see Robinson et al., 1986, 1987), shrimp (Deshimaru et al., 1978) and other mollusks (Thomas and Lough, 1974; Coote et al., 1996), abalones are likely to obtain adequate calcium directly from the surrounding seawater, although the mechanisms of calcium absorption has not been yet established (Coote et al., 1996; Tan et al., 2001). These results may be supported by our second experiment, in which a progressive decrease in calcium was recorded. This result also suggests that in closed systems (as currently used in aquaculture), a calcium supply, together with periodical water changes, is constantly required, even when the used medium is natural seawater. At this point of the study, our suggestion is that, to farm this species in closed systems, natural seawater should be preferred over artificial seawater, and that this should be enriched with calcium to keep its concentration constant through time. Obviously, further experiments are needed to determine the optimal concentration of calcium for the on-farm management of H. tuberculata and of other abalone species. Acknowledgements We thank Laura Aquiloni, Leonardo Barsalini, Ilaria Galigani, Gianluca Giorgi, Riccardo Innocenti, Silvano Lancini, and Susanna Pucci for their help in the experiments and analyses. ARSIA (“Agenzia Regionale per lo Sviluppo e l'Innovazione nel Settore Agricoloforestale”) is acknowledged for having funded the supply of the study animals. References Azuma, N., 1976. Calcium sensitivity of abalone, Haliotis discus, myosin. Journal of Biochemistry 80, 187–189. Basuyaux, O., Mathieu, M., 1999. Inorganic nitrogen and its effect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck. Aquaculture 174, 95–107. Cenni, F., Parisi, G., Gherardi, F., 2009. Use of space and costs/benefits of locomotion strategies in the abalone, Haliotis tuberculata. Ethology Ecology & Evolution 21, 15–26.
Chesson, J., 1978. Measuring preference in selective predation. Ecology 59, 211–215. Coote, T.A., Hone, P.W., Kenyon, R., Maguire, G.B., 1996. The effect of different combinations of dietary calcium and phosphorus on the growth of juvenile Haliotis laevigata. Aquaculture 145, 267–279. Deshimaru, O., Kuroki, K., Sakamoto, S., Yone, Y., 1978. Absorption of labelled calcium Ca45 by prawn from sea water. Bulletin of the Japanese Society of Scientific Fisheries 44, 975–977. Guzmán del Proó, S.A., 1992. A review of the biology of abalone and its fishery in Mexico. In: Shepherd, S.A., Tegner, M.J., Guzmán del Proó, S.A. (Eds.), Abalone of the World. Blackwell Scientific Publications, Cambridge, USA, pp. 370–383. Huchette, S.M.H., Koh, C.S., Day, R.W., 2003. The effects of density on the behaviour and growth of juvenile blacklip abalone (Haliotis rubra). Aquaculture International 11, 411–428. Lovell, R.T., 1989. Nutrition and Feeding of Fish. Van Nostrand Reinhold, New York. 260 pp. Mgaya, Y.D., Mercer, J.P., 1994. A review of the biology, ecology, fisheries and mariculture of the European abalone Haliotis tuberculata Linnaeus 1758 (Gastropoda: Haliotidae). Biology and Environment Proceedings of the Royal Irish Academy 94 B, 285–304. Momma, H., Sato, R., 1969. The locomotion behaviour of the disc abalone, Haliotis discus hannai, Ino, and the Siebold's abalone, Haliotis sieboldi, Reeve, in the fishing grounds. Journal of Agricultural Research 20, 150–157. Moulis, A., 2006. The action of RFamide neuropeptides on molluscs, with special reference to the gastropods Buccinum undatum and Busycon canaliculatum. Peptides 27, 1153–1165. Nakamura, K., Soh, T., 1997. Mechanical memory hypothesized in the homing abalone Haliotis diversicolor supertexta under experimental conditions. Fisheries Sciences 63, 854–861. Oakes, F.R., Ponte, R.D., 1996. The abalone market: opportunities for cultured abalone. Aquaculture 140, 187–195. Ogino, C., Takeda, H., 1978. Requirements of rainbow trout for dietary calcium and phosphorus. Bulletin of the Japanese Society of Scientific Fisheries 44, 1019–1022. Parker, D.O., Haaker, P.L., Togstadt, H.A., 1992. Case histories for three species of California abalone: Haliotis corrugatae, H. fulgens and H. crucherodii. In: Shepherd, S.A., Tegner, M.J., Guzmán del Proó, S.A. (Eds.), Abalone of the World. Blackwell Scientific Publications, Cambridge, USA, pp. 370–383. Robinson, E.H., Rawles, S.D., Brown, P.B., Yette, H.E., Greene, L.W., 1986. Dietary calcium requirement of channel catfish Ictalurus punctatus, reared in calcium-free water. Aquaculture 53, 263–270. Robinson, E.H., LaBomascus, D., Brown, P.B., Linton, T.L., 1987. Dietary calcium and phosphorus requirements of Oreochromis aureus reared in calcium-free water. Aquaculture 64, 267–276. Shepherd, S.A., 1973. Studies on Southern Australian abalone (genus Haliotis). 1. Ecology of five sympatric species. Australian Journal of Marine and Freshwater Research 24, 217–257. Shepherd, S.A., 1988. Studies on Southern Australian abalone (genus Haliotis). 8. Growth of juvenile H. laevigata. Australian Journal of Marine and Freshwater Research 39, 177–183. Shim, K.F., Ho, C.S., 1989. Calcium and phosphorus requirements of guppy Poecilia reticulata. Bulletin of the Japanese Society of Scientific Fisheries 55, 1947–1953. Shpigel, M., Ragg, N.L., Lupatsch, I., Neori, M., 1999. Protein content determines the nutritional value of the seeweed Ulva lactuca L. for the abalone Haliotis tuberculata L. and H. discus hannai Ino. Journal of Shellfish Research 18, 227–232. Stephenson, T.A., 1924. Notes on Haliotis tuberculata. Marine Biological Association of the United Kingdoom 13, 480–495. Tan, B., Mai, K., Liufu, Z., 2001. Response of juvenile abalone, Haliotis discus hannai, to dietary calcium, phosphorus and calcium/phosphorus ratio. Aquaculture 198, 141–158. Tarr, R.J.Q., 1995. Growth and movement of the South African abalone Haliotis midae: a reassessment. Marine and Freshwater Research 46, 583–590. Tash, J.S., Means, A.R., 1983. Cyclic adenosine 3′, 5′-monophosphate, calcium and protein phosphorylation in flagellar motility. Biology of Reproduction 28, 75–104. Tegner, M.J., DeMartini, J.D., Karpov, K.A., 1992. The California red abalone fishery: a case study in complexity. In: Shepherd, S.A., Tegner, M.J., Guzmán del Proó, S.A. (Eds.), Abalone of the World. Blackwell Scientific Publications, Cambridge, USA, pp. 370–383. Thomas, J.D., Lough, A., 1974. The effects of external calcium concentration on the rate of uptake of this ion by Biomphalaria glabratus. Journal of Animal Ecology 43, 861–871.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e
Sheltering behavior of the abalone, Haliotis tuberculata L., in artificial and natural seawater: The role of calcium Federica Cenni a, Giuliana Parisi b, Felicita Scapini a, Francesca Gherardi a,⁎ a b
Dipartimento di Biologia Evoluzionistica “Leo Pardi”, Università di Firenze, Via Romana 17, 50125 Firenze, Italy Dipartimento di Scienze Zootecniche, Università di Firenze, Via delle Cascine 5, 50144 Firenze, Italy
a r t i c l e
i n f o
Article history: Received 8 September 2009 Received in revised form 16 November 2009 Accepted 17 November 2009 Keywords: Artificial seawater Calcium Haliotis tuberculata L. Sheltering behavior
a b s t r a c t There is an increased interest worldwide in developing land-based closed systems to cultivate the abalone, Haliotis tuberculata L. Here, we analyzed whether artificial conditions, and particularly the use of artificial seawater, might affect sheltering, a critical behavior of this species. The study was composed of two experiments. In the first, we compared sheltering between two groups of 100 individuals each reared in either natural or artificial seawater. For 15 days, a score was assigned every day to the position occupied by each individual, i.e. “0” when it was out of the hide, “1” when it was on the bottom of the maintenance tank underneath the hide, and “2” when it occupied the inner spot of the hide. Our results show that artificial seawater significantly affects sheltering: only the individuals reared in natural seawater were able to reach the hide and maintain a position in it. Chemical analyses revealed that artificial seawater differed from natural seawater for a relatively low concentration of Ca2+. In the second experiment we compared H. tuberculata's sheltering behavior in three media, natural, artificial, and calcium-enriched artificial seawater. Sheltering was restored in enriched artificial seawater, which supports the hypothesis that calcium has an effect on this species' behavior. However, calcium concentration both in the artificial and “natural” water showed a progressive decrease with time likely due to the absorption by abalones, which suggests that in closed systems calcium supply is constantly required, even when the used medium is natural seawater. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Natural stocks of the abalone, Haliotis tuberculata L. (Mollusca: Haliotidae), are worldwide overexploited and in a state of serious decline (Guzmán del Proó, 1992; Parker et al., 1992; Tegner et al., 1992; Shpigel et al., 1999). Decreased fisheries, combined with the increasing demand for this species and its high market value, have resulted in accelerating the spread of abalone aquaculture (e.g. Oakes and Ponte, 1996) and the development of new projects that look into land-based closed or semi-closed systems (Basuyaux and Mathieu, 1999) to support traditional farming in natural environments. In order to maximize production, research has been mostly directed to understand the biology of the species (Mgaya and Mercer, 1994). Conversely, little is known about the environmental factors that are likely to influence the abalone's growth in closed systems (Basuyaux and Mathieu, 1999). Here, we analyzed whether the use of artificial seawater may alter the behavior of H. tuberculata. The rationale underlying this pivotal study is that, in farm settings, rearing techniques should be adjusted to it so as to maximize the health and growth performance of the animals
⁎ Corresponding author. Tel.: +39 055 2288216/215; fax: +39 055 222565. E-mail address: francesca.gherardi@unifi.it (F. Gherardi). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.11.016
(Huchette et al., 2003). This implies a good knowledge of the behavior of the reared species. Changes in behavior may in fact be used as early warning indicators of suboptimal rearing conditions. On the basis of the information about this species' biology in nature (e.g. Momma and Sato, 1969) and in the laboratory (Cenni et al., 2009), we used sheltering behavior as a proxy of the individuals' wellbeing. In the inhabited reefs, abalones take refuge during the day under rocks or in crevices, from which they emerge in the evening to hide again at dawn (Stephenson, 1924). As observed in several species of Haliotis, some spots of the reef are most often occupied as shelters possibly because of the better protection from predators they offer (Momma and Sato, 1969; Shepherd, 1973; Tarr, 1995). Laboratory studies on H. rubra (Leach) (Huchette et al., 2003) and H. tuberculata (Cenni et al., 2009) showed that abalones do not distribute evenly on the provided sheltering space in the tanks, but rather aggregate in particular spots, suggesting that this phenomenon, i.e. sheltering in the most suitable spots, also occurs in artificial environments. Based on these premises, firstly we compared sheltering behavior in H. tuberculata reared in either natural or artificial seawater; secondly, having found a significant difference between the two treatments, we analyzed the possible effect of calcium on this species' sheltering behavior, since natural and artificial seawater differed in calcium content.
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2. Materials and methods
df = 3, P N 0.05), with a similar sex ratio (after a G test after William's correction: G between 2.275 and 4.354, df = 3).
2.1. General set-up 2.3. Experiment 2 The experimental abalones (3–4 year old), originating from a French hatchery (“Les ormeaux du Cotentin”, Gouville sur Mer), had been reared before the study in an open system in Orbetello (Grosseto, Italy). The experiments were carried out at the University of Florence after at least two weeks of acclimation to the laboratory conditions. The sex of each animal was determined and its maximum shell length was measured with an electronic caliper to the nearest 0.1 mm. Abalones were individually marked using plastic tags (Hallprint, Adelaide, South Australia) attached to their shell with a superglue gel (Shepherd, 1988). Marking did not affect their behavior, as shown in preliminary tests. For our experiments we used Instant Ocean® artificial seawater (made with demineralized and distilled water, with the same salinity as natural seawater, i.e. 33.3 gmL− 1) [AS], the natural seawater used in the rearing system in Orbetello [NS], or artificial seawater that was enriched with calcium [ES]. The content in calcium was increased by adding 0.3 mL of the enriching Calcium Plus® Prodac per liter of seawater, to reach the same calcium concentration as natural seawater (approximately 1000 mgL− 1). AS, NS, and ES were analyzed for their chemical content in the Laboratory of Microanalysis at the University of Florence using the international official method for water minerals determination, i.e. E.P.A. method 3005 applied to ICP OES D.V. Perkin Elmer Optima 2000 instrumentation. Six hundred-L plastic tanks were used for Experiment 1. Experiment 2 was conducted in smaller containers, 50-L glass aquaria that allowed us to better manage calcium enrichment. Tanks and aquaria were cleaned daily using a hose; twice a week, 20% of their water was renewed. The room was maintained at a natural light:dark cycle (approximately 12:12 h) and at a constant air temperature of 18 °C. Abalones were fed ad libitum once every second day using artificial feed (ADAM & AMOS “7-mm Chip” Abalone Food, Australia). Each replicate of both experiments was run for two weeks. Using a VWR SympHony SP90M5 electronic tester, we measured twice a week in each tank/aquarium a number of water parameters, such as temperature, conductivity, salinity, and pH, together with the content of dissolved oxygen and ammonia/nitrite/nitrate to monitor the biological activity of the study animals. The two experiments were conducted between March 2007 and April 2009. The first experiment was aimed at analyzing the possible differences in the sheltering behavior of H. tuberculata reared in either natural or artificial seawater, whereas purpose of the second experiment was to analyze the possible effect of calcium on the abalones' behavior.
Each individual was randomly assigned to one of three aquaria (= treatments) containing artificial seawater [AS, Aquarium A], natural seawater [NS, Aquarium B], or artificial seawater enriched with calcium [ES, Aquarium C]. We ran three replicates per aquarium, with 20 animals per replicate (total: 180 abalones); records were taken once a day, reaching a total of 15 records per replicate. AS, NS and ES were analyzed for their calcium content three times, i.e. at the start of the experiment, after a week, and at the end. Here, the same materials and methods as in Experiment 1 were used except as follows. Instead of the baskets, we used single glass aquaria (50 × 35× 35 cm) containing 50 L of water filtered in continuum by two 60 L pumps. Each aquarium was provided with a semi-cylindrical concrete hide as above and hosted 20 individuals (density: ca. 350 m− 2; overall biomass: ca. 0.20 kg) of similar size (40–55 mm; F between 0.463 and 0.781, df= 8, P N 0.05) and sex ratio (after a G test after William's correction: G = 1.481, df = 8, P N 0.1). 2.4. Data recording Records were taken at 9:00 when each basket/aquarium was photographed with a digital camera. The position of each abalone with respect to the shelter was later determined from the photos; a score was given to the position occupied by each individual per record, i.e. 0 when it was out of the hide, 1 when it was on the bottom of the basket/aquarium underneath the hide, and 2 when it occupied the inner spot of the hide. To analyze differences among days, each basket/aquarium was associated with the total daily sum of individual scores, i.e. the sum of every daily score totalized by each of its inhabitants per day. To analyze overall differences between treatments, the total sum of scores, i.e. the sum of the scores totalized by all its inhabitants during the entire study period, was used. To analyze the preference of abalones for the three different positions with respect to the shelters, we measured the alpha selectivity coefficient, slightly modified after Chesson (1978), from the formula: αi = ð ri = pi Þ = ð∑ ri =pi Þ i
where pi is the ratio between the surface corresponding to each of the three scores (i = 0, 1, or 2) and the whole available area of the basket, and ri is the proportion of the abalones found in the i position. A low mortality of abalones (ca. 3%) was observed during the acclimation period possibly due to handling and transportation stress. No abalone died during the experiments.
2.2. Experiment 1 2.5. Statistical analyses Each individual was randomly assigned to one of two plastic tanks (= treatments) containing either artificial seawater (made as above) [AS, Tank A], or natural seawater [NS, Tank B]. Three replicates per treatment, with 100 animals per replicate (total: 600 abalones), were run, and records were taken once a day, reaching a total of 15 records per replicate. Each tank (160 × 100 × 60 cm) contained 600 L of seawater filtered in continuum by two 500 L pumps and four squared-section baskets (23 × 23 × 27 cm), each subject to a constant water flow and positioned at the same distance from the light sources and the walkway in order to maintain equal the possible effects on behavior of light intensity and vibrations. Each basket was provided with a semi-cylindrical concrete hide (length: 15 cm; diameter: 10 cm) and hosted 25 individuals (density: ca. 400 m− 2; overall biomass: ca. 0.25 kg) of similar size (35–65 mm; F between 0.134 and 1.933,
The data that met the assumptions for normality and homogeneity of variance after the Kolmogorov-Smirnov and Levene test were analyzed using parametric tests. Specifically, differences between the two treatments for the water parameters were analyzed using two tailed Student's t-tests (statistic: t); differences among baskets and aquaria for abalones' dimensions were analyzed with one-way ANOVAs (statistic: F); GLM Analyses For Repeated Measures (statistic: F) followed by multiple comparison tests were used to estimate the overall difference among AS, NS, and CE. For the other data, nonparametric techniques were used. Differences among days were analyzed using a Friedman test (statistic: Fr), whereas those among records, replicates, baskets and aquaria, along with the differences in Chesson's alpha value (Experiment 2), were analyzed using Kruskal Wallis tests (statistic: H). Mann–Whitney tests (statistic: U) were used
F. Cenni et al. / Aquaculture 299 (2010) 67–72 Table 1 Experiment 1: differences between natural (NS) and artificial (AS) seawater for the concentration of the most common seawater ions (mgL− 1) using Mann–Whitney tests (N = 3). Significant values are denoted in bold. Ion
Al3+ (mgL− 1) B3+ (mgL− 1) Ba2+ ( mgL− 1) Ca2+ (mgL− 1) Cd2+ (mgL− 1) Cr2+(mgL− 1) Cu2+(mgL− 1) Fe3+ (mgL− 1) Hg2+ (mgL− 1) K+ (mgL− 1) Li+ (mgL− 1) Mg2+ (mgL− 1) Mn3+ (mgL− 1) Ni3+ (mgL− 1) Pb2+ (mgL− 1) Se6+ (mgL− 1) Sr2+ (mgL− 1) Ti3+ (mgL− 1) Zn2+ (mgL− 1)
NS
AS
Statistics
Mean (SE)
Mean (SE)
U
P
1.8 (0.5) 0 0.06 (0.01) 803.1 (74.0) 0.005 (0.002) 0 0.28 (0.11) 0.07 (0.03) 0 407.8 (48.22) 0.47 (0.05) 370.86 (35.8) 0.23 (0.08) 0.17 (0.02) 0 0.25 (0.06) 17.57 (6.41) 0 0.32 (0.18)
0.03 (0.01) 5.09 (0.92) 0.02 (0.005) 269.48 (31.6) 0 0 0 0.06 (0.03) 0 385.39 (36.2) 0.16 (0.06) 366.92 (38.4) 0.01 (0.003) 0.03 (0.001) 0 0 6.65 (1.98) 0 0
3.9 1.5 2 0 1.99 40.5 1.6 4 40.5 3.5 2.1 4 1.7 3.8 40.5 1.6 2.2 40.5 1.8
0.827 0.114 0.261 3.52 × 10− 5 0.275 1 0.141 0.897 1 0.671 0.293 0.935 0.141 0.569 1 0.135 0.312 1 0.199
Table 3 Experiment 1: differences among replicates in natural (NS) and artificial (AS) seawater for the parameters measured (N = 3).
Data
to check differences in ion concentrations, total sum of scores, and Chesson's alpha values between the two treatments. Spearman's correlations (rs) were performed between sums of scores and treatments and between behavior and size. Frequencies of sex ratio, positions, and transition among positions were analyzed with G tests after William's correction (statistic: G). The level of significance under which the null hypothesis was rejected is α = 0.05. 3. Results 3.1. Experiment 1 3.1.1. Water analyses We analyzed the concentration of the most common seawater ions (Al3+, Ba2+, Ca2+, Cd2+, Cr2+, Cu2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn3+, Ni3+, Pb2+, Se6+, Ti3+, and Zn2+) (Table 1); when compared with NS, AS was relatively poor in calcium , while the two types of water were similar for the other ions (U = 0, N = 9, P b 0.05; for the other ions: U between 40.5 and 1.5, N = 9). Temperature, conductivity, salinity, pH, dissolved oxygen, and ammonia/nitrite/nitrate did not significantly differ between treatments (t between 9.839 and 11.490, df = 6) (Table 2). 3.1.2. Behavioral data Since replicates did not differ for any variable in either AS (H ranging between 0.062 and 3.919, df= 2) or NS (H ranging between 0.008 and 3.033, df= 2) (Table 3), the daily sums of scores were compared among the baskets analyzed separately per treatment, which showed a significant difference among days (Fig. 1), both in AS (Fr = 36.426, df= 14,
69
NS
Statistics
AS
Statistics
Mean (SE)
H
Mean (SE)
H
P
Length (mm) 45.6 (4.2) 2.01 P N 0.1 Daily sum of scores 20.65 (6.24) 3.03 P N 0.1 Chesson's α 0.16 (0.01) 0.08 P N 0.1 (out of the hide) Chesson's α 0.37 (0.02) 0.21 P N 0.1 (underneath the hide) Chesson's α 0.47 (0.02) 1.02 P N 0.1 (inner spot of the hide)
P
45.4 (7.2) 2.09 P N 0.1 7.2 (1.23) 3.919 P N 0.1 0.48 (0.02) 0.128 P N 0.1 0.27(0.03) 0.062 P N 0.1 0.25 (0.01) 1.12
P N 0.1
P = 0.002) and in NS (Fr = 26.374, df= 14, P = 0.045). However, the daily sums of scores were significantly correlated between the two tanks (rs = 0.878, n = 15, P b 0.001), suggesting that this trend was the same in the two types of seawater, as a possible result of the abalones changing their activity in association with food provision (Fig. 1). Another hypothesis is that the abalones change their behavior (activity) in association with their increased familiarization with the experimental tank. However, as also shown by Fig. 1, this does not appear to be the case. The daily sums of scores did not differ among baskets within each tank (AS: H between 3.986 and 4.130, df = 3; NS: H between 6.872 and 7.292, df = 3), whereas the total sum of scores significantly differed between treatments (U = 0, N = 24, P b 0.001), with higher values in NS. This was confirmed by analyzing the behavior of individual abalones. In fact, as revealed by assigning to each individual the mode of the scores computed for the entire period, those inhabiting NS showed a larger use of the hide (scores 1 and 2) (G = 121.861 df = 2, P b 0.001), whereas those in AS most often scored 0 (G = 161.367, df = 2, P b 0.001), with a significant difference in the frequency distribution of scores between the two treatments (AS: mean = 106.4, SE = 16. 81; NS: mean = 311.66, SE = 26.14; G = 48.221, df = 2, P b 0.001). The recorded difference was not related to the animal size (AS: rs = − 0.02, N = 600, P = 0.986; NS: rs = 0.052, N = 600, P = 0.524). The transitions from one record to the subsequent in the positions taken were counted and their probability was analyzed (G between 266.144 and 7.123, df = 1) (Fig. 2). The comparison between AS and NS for the hiding behavior revealed that the abalones in NS were more mobile than those in AS, and that they more often managed to remain in the hide. The overall frequencies of transitions differed within (AS: G = 2908.992, df = 24, P b 0.001; NS: G = 701.856, df = 24, P b 0.001) and between the two treatments (G = 634.465, df = 24, P b 0.001), the abalones in AS being more often found out of the hide (Fig. 3). To support the above results, the three positions “out of the hide”, “underneath the hide”, and “inner spot of the hide” were compared between artificial seawater (AS) and natural seawater (NS).
Table 2 Experiment 1: differences between natural (NS) and artificial (AS) seawater for the water parameters measured after t-tests for independent data. NS
AS
Statistics
Data
Mean (SE)
Mean (SE)
t
Temperature (°C) Conductivity (mS/cm) Salinity (‰) pH Dissolved O2 (mg/L) Ammonia/Nitrite/Nitrate (mg/L)
16.35 (0.25) 503.28 (24.6) 34.07 (0.22) 8.12 (0.29) 77.67 (2.76) 4.06 (0.89)
16.21 (0.27) 0.388 2 507.3 (28.9) 0.127 2 34.22 (0.13) − 0.614 2 8.11 (0.09) 0.476 2 77.15 (2.49) 0.138 2 4.11(1.02) − 0.513 2
df P 0.705 0.911 0.551 0.643 0.892 0.583
Fig. 1. Experiment 1: comparison between artificial seawater (AS) and natural seawater (NS) for the positions taken by the abalones (daily sums of scores) in the 15 records (observations) of the study. Arrows denote when food was supplied.
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Fig. 2. Experiment 1: transitions between the three positions taken by the abalones compared between artificial seawater (AS) and natural seawater (NS). Arrows indicate the direction of the transitions between two positions within two subsequent records. If there was no transition the arrow is curve. The signs “b” and “N” indicate in which medium (AS or NS) the number of transitions was higher. G values are indicted over the arrows (⁎⁎ = P b 0.001;⁎ = P b 0.01).
Fig. 4. Experiment 1: differences between Chesson's alpha for the three positions (a: “out of the hide”, b: “underneath the hide”, and c: “inner spot of the hide”) compared between artificial seawater (AS) and natural seawater (NS).
The abalones in AS and NS also differed for the Chesson's alpha value (out of the hide: U = 0, N = 15, P b 0.001; underneath the hide: U = 445.0, N = 15, P = 0.005; inner spot of the hide: U = 7.0, N = 15, P b 0.001) (Fig. 4). 3.2. Experiment 2 3.2.1. Water analyses Within each treatment, replicates showed a similar calcium content (H ranging between 1.988 and 2.101, df = 2) (Table 4) so that the data were pooled to estimate the overall difference among AS, NS, and CE. A GLM analysis for repeated measures showed a significant effect of time (F = 18.681, df = 2, P b 0.001), with calcium decreasing its concentration in each treatment, but also a significant difference among AS, NS, and CE as expected (F = 64.465, df = 2, P b 0.001; NS = CE N AS) (Fig. 5). The interaction between time and treatment was also significant (F = 166.247, df = 2, P b 0.001), revealing that the decrease had a different speed in the two types of water, being slower in AS. Table 4 Experiment 2: differences among replicates in natural (NS), artificial (AS), and calciumenriched artificial (CE) seawater for initial calcium concentration (mgL− 1) (N = 3). Fig. 3. Experiment 1: transitions between two subsequent records (denoted by arrows) in the three positions taken by the abalones in artificial seawater (AS) and natural seawater (NS). The curved arrow indicates no transition. The percentage of individuals in the corresponding position is indicated over each arrow. The signs “−” and “+” denote if the corresponding frequency of transition is lower and higher, respectively, than the expected one.
Ca2+ (mgL− 1)
Statistics
Water
Mean (SE)
H
P
NS AS CE
1043.43 (64.31) 584.97 (41.12) 981.26 (56.17)
1.298 1.876 2.101
P N 0.1 P N 0.1 P N 0.1
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Fig. 6. Experiment 2: comparison among artificial seawater (AS), natural seawater (NS), and calcium-enriched artificial seawater (CE) for the position taken by the abalones (daily sums of scores) in the 15 records of the study. Arrows denote when food had been supplied.
Fig. 5. Experiment 2: comparison among artificial seawater (AS), natural seawater (NS), and calcium-enriched artificial seawater (CE) for the mean concentration of Ca2+ (± SE) analyzed at the beginning of the experiment, 10 days from the start, and at the end.
3.2.2. Behavioral data Since replicates were similar for all the analyzed variables in AS (H ranging between 0.041 and 2.936, df = 2), NS (H ranging between 0.012 and 4.103, df = 2), and CE (H ranging between 0.021 and 3.188, df = 2) (Table 5), the daily sums of scores of each replicate were pooled to make an overall comparisons among treatments. A significant difference was found among days (Fig. 6), in both AS (Fr = 41.628, df = 14, P = 0.001) and NS (Fr = 36.374, df = 14, P = 0.005). However, the daily sums of scores were significantly correlated among treatments (rs = 0.746, N = 15, P b 0.001), suggesting that this trend was the same between types of seawater, as a possible result of the abalones changing their activity in association with food provision, as also seen in Experiment 1 (Figs. 1 and 6). The daily sums of scores did not differ among aquaria within each tank (AS: H between 1.764 and 1.908, df = 2; NS: H between 4.650 and 5.070, df = 2; CE: between 2.866 and 3.162, df = 2), whereas the total sum of scores significantly differed between treatments (U = 0, N = 9, P b 0.001), with lower values in AS and similarly higher values in NS and CE. This was confirmed by analyzing the behavior of individual abalones. The individuals inhabiting NS and CE showed a larger use of the hide (scores 1 and 2) (NS: G = 121.861, df = 2, P b 0.001; CE: G = 116.672, df = 2, P b 0.001), whereas those in AS most often scored 0 (G = 161.367, df = 2, P b 0.001), with a significant difference in the frequency distribution of scores between the two types of seawater (AS: mean = 106.4, SE = 16. 81; NS: mean = 311.66, SE = 26.14; CE: mean = 295.87, SE = 31.24; G = 51.226, df = 2, P b 0.001). The recorded difference was not related to the animal size (AS: rs = − 0.018, N = 60, P = 0.961; NS: rs = 0.048, N = 60, P = 0.522; CE: rs = 0.041, N = 60, P = 0.532).
The comparison among the treatments for the transitions from one record to the subsequent in the positions taken revealed that the abalones in NS were more mobile than those in AS, and that they more often managed to remain in the hide. Overall frequencies of transitions differed within (AS: G = 2506.225, df = 8, P b 0.001; NS: G = 509.765, df = 8, P b 0.001; CE: G = 488.567, df = 8, P b 0.001) and among the three treatments (G = 592.967, df = 8, P b 0.001), the abalones in AS being more often found out of the hide . The abalones in AS, NS, and CE also differed for the Chesson's alpha (out of the hide: H = 0.021, df = 2, P b 0.001; underneath the hide: H = 0.038, df = 2, P b 0.001; inner spot of the hide: H = 0.04921, df = 2, P b 0.001), with significant differences between AS and NS (out of the hide: U = 0, N = 15, P b 0.001; underneath the hide: U = 338.0, N = 15, P = 0.004; inner spot of the hide: U = 8.0, N = 15, P b 0.001), AS and CE (out of the hide: U = 1.0, N = 15, P b 0.001; underneath the hide: U = 546.0, N = 15, P = 0.006; inner spot of the hide: U = 6.0, N = 15, P b 0.001), but not between NS and CE (out of the hide: U = 108.0, N = 15, P = 0.850; underneath the hide: U = 92.0, N = 15, P = 0.390; inner spot of the hide: U = 102.5, N = 15, P = 0.605). 4. Discussion The results of Experiment 1 clearly show that sheltering behavior in H. tuberculata is independent of the individuals' body size but it is highly affected by their rearing in artificial seawater. Although all individuals displayed a tendency to improve their position with respect to the shelter, only the abalones reared in natural seawater were able to reach and maintain a position in the hide. A recent study investigating the use of space by H. tuberculata in an artificial environment (Cenni et al., 2009) showed that each individual, independently of its sex, adopts one of two locomotion strategies, behaving as either a “wanderer” or a “sedentary”, with wanderers covering longer distances and occupying the hides, particularly their inner spots, more often than the sedentary individuals. Although the experimental animals were randomly extracted from a seemingly homogeneous sample, equilibrium was soon reached between the
Table 5 Experiment 2: differences among replicates in natural (NS), artificial (AS), and calcium-enriched artificial (CE) seawater for the parameters measured. (N = 3). NS
Statistics
AS
Statistics
CE
Statistics
Data
Mean (SE)
H
P
Mean (SE)
H
P
Mean (SE)
H
P
Length (mm) Daily sum of scores Chesson's α (out of the hide) Chesson's α (underneath the hide) Chesson's α (inner spot of the hide)
44.9 22.78 0.14 0.38 0.49
3.02 4.103 0.012 0.145 0.603
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
45.1 6.8 0.51 0.28 0.21
2.701 2.936 0.198 0.041 1.01
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
44.8 22.72 0.15 0.37 0.48
2.911 3.188 0.234 0.799 0.021
P N 0.1 P N 0.1 P N 0.1 P N 0.1 P N 0.1
(4.9) (7.07) (0.02) (0.03) (0.02)
(5.3) (1.98) (0.04) (0.01) (0.03)
(3.1) (5.11) (0.04) (0.05) (0.03)
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two behavioral strategies. Our results here showed that living in artificial seawater increased the number of sedentary animals (i.e. animals that did not change their position), suggesting some properties of the medium that might shift the equilibrium between the two behavioral strategies. The chemical analyses showed that the artificial seawater used had a lower content in calcium than natural seawater. Experiment 2 indicated that low calcium concentration may affect the behavior of H. tuberculata. In fact, abalones reared in calcium-enriched artificial seawater restored the behavior exhibited in natural seawater. There are several explanations to this phenomenon. Indeed, the lack of calcium may impede a number of functions in this taxon, including shell building, blood clotting, muscle function, sperm motility and metabolism, and nerve transmission (Azuma, 1976; Tash and Means, 1983; Lovell, 1989; Coote et al., 1996; Tan et al., 2001; Moulis, 2006). In turn, an affected neural transmission due to a lack of calcium may decrease locomotion rate and the ability to home in abalones (Nakamura and Soh, 1997). How calcium can be obtained by abalone is debated (Coote et al., 1996). Similarly to fish (e.g. Ogino and Takeda, 1978; Shim and Ho, 1989; but see Robinson et al., 1986, 1987), shrimp (Deshimaru et al., 1978) and other mollusks (Thomas and Lough, 1974; Coote et al., 1996), abalones are likely to obtain adequate calcium directly from the surrounding seawater, although the mechanisms of calcium absorption has not been yet established (Coote et al., 1996; Tan et al., 2001). These results may be supported by our second experiment, in which a progressive decrease in calcium was recorded. This result also suggests that in closed systems (as currently used in aquaculture), a calcium supply, together with periodical water changes, is constantly required, even when the used medium is natural seawater. At this point of the study, our suggestion is that, to farm this species in closed systems, natural seawater should be preferred over artificial seawater, and that this should be enriched with calcium to keep its concentration constant through time. Obviously, further experiments are needed to determine the optimal concentration of calcium for the on-farm management of H. tuberculata and of other abalone species. Acknowledgements We thank Laura Aquiloni, Leonardo Barsalini, Ilaria Galigani, Gianluca Giorgi, Riccardo Innocenti, Silvano Lancini, and Susanna Pucci for their help in the experiments and analyses. ARSIA (“Agenzia Regionale per lo Sviluppo e l'Innovazione nel Settore Agricoloforestale”) is acknowledged for having funded the supply of the study animals. References Azuma, N., 1976. Calcium sensitivity of abalone, Haliotis discus, myosin. Journal of Biochemistry 80, 187–189. Basuyaux, O., Mathieu, M., 1999. Inorganic nitrogen and its effect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck. Aquaculture 174, 95–107. Cenni, F., Parisi, G., Gherardi, F., 2009. Use of space and costs/benefits of locomotion strategies in the abalone, Haliotis tuberculata. Ethology Ecology & Evolution 21, 15–26.
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