Gonad development in seeded Haliotis laevigata: growth environment determines initial reproductive investment.

ABSTRACT Cultured Haliotis laevigata (Donovan), 18 mo old, were tagged and seeded at either high or low densities on boulder reefs (4-6 m depth) at Cape Jervis, South Australia in September 2001. Surveys every 3 mo quantified growth history and a sample was sacrificed in September 2002, prior to the expected spawning season of H. laevigata from this area, to measure gonad development. This provided a unique opportunity to investigate the onset of reproductive investment in known age H. laevigata in the field. Forty-five percent of sampled abalone had developed gonad tissue by September 2002. Larger individuals were more likely to have initiated gametogenesis and tended to have larger gonads. Gonad development was most closely related to abalone weight in September 2002 and was also strongly correlated with length and early growth rate. A significant proportion of abalone migrated from cryptic positions within boulder habitats into more exposed adult habitat at the outer edge of reefs between April and June. Abalone that had emerged to adult habitat were significantly more likely to be mature than cryptic individuals. The overall proportion of abalone with developing gonads differed significantly between densities. This seems to be an indirect effect, arising because abalone at low density grew faster than those at high density. The onset of sexual maturation in H. laevigata was thus partly dependent on density and site characteristics, which probably reflect the relative availability and quality of food. The results suggest that onset of sexual maturity in H. laevigata can advance under conditions of fast growth. As sampled gonads contained ripe gametes and maturity appears to be related to growth rate, fast growing abalone may reproduce [+ or -] 1 y before smaller juveniles in the same cohort.

KEY WORDS: gonad development, growth, maturity, density, Haliotis laevigata

INTRODUCTION

Abalone exhibit wide variation in growth rate (reviewed in Day & Fleming 1992) and size at maturity (McShane & Naylor 1995a). Much of this variation is likely due to inherent variability in the environment, but other aspects may explain these patterns. Life history theory predicts that most organisms will mature at neither a fixed age nor at a fixed size, but along an age-size trajectory according to environmental constraints such that reproductive fitness is optimized (Stearns & Koella 1986). For abalone, factors such as density, previous growth history, and food availability (or quality) may promote large differences in growth rate and the timing of maturation. How these factors specifically relate to gonad development, however, remains poorly understood. The difficulty of monitoring cryptic juveniles in the field (McShane & Smith 1988) has largely limited research to laboratory studies.

Intraspecific competition in abalone, which are dependent on a limited supply of drift algae as food (Shepherd 1973), might be expected to result in reduced growth and/or fecundity at higher densities. Laboratory and aquaculture studies have shown that high stocking densities can have strong negative effects on the growth of juvenile abalone (Mgaya & Mercer 1995, Capinpin et al. 1999, Huchette et al. 2003). A field experiment involving adult H. iris in New Zealand showed that reproductive investment (measured as dry gonad weight) was independent of density (McShane & Naylor 1995b). Douros (1985) found that size-specific reproductive investment in H. cracherodii was increased at a site where density was reduced, relative to a control site. Density effects on reproduction have been shown in several terrestrial molluscs (Carter & Ashdown 1984, Baur & Baur 1992), but there seem to be no studies of these effects on the initial reproductive investment of animals as they approach maturity.

Strong size-dependent mortality rates in juvenile abalone (Shepherd & Breen 1992, Shepherd 1998) may shape selective pressure such that the onset of maturity is linked with the attainment of a threshold size (McShane & Naylor 1995a, Worthington & Andrew 1997). Nash (1990, 1992), however, concluded that sexual maturity is determined by reaching a certain age, rather than size, in H. rubra. This is also argued for other abalone species (Shepherd & Laws 1974, Shepherd et al. 1991) and has strong theoretical support (Stearns & Koella 1986). There is some evidence that the age of first maturity in abalone may be a plastic trait that is influenced by growth rate (McShane & Naylor 1995a) but this is yet to be demonstrated in a known-age population.

This field study investigated the effect of growth history (i.e., previous growth) and local density on the timing and degree of maturation in seeded abalone reaching first maturity. Practical difficulties associated with relying on natural recruitment in the field were avoided by using tagged, cultured juveniles that were all the same age.

MATERIALS AND METHODS

Experimental Design

This study was carried out near Cape Jervis, South Australia (138[degrees]06'E, 35[degrees]36'S) between September 2001 and September 2002. Eighteen-month-old cultured abalone from Kangaroo Island Abalone hatchery with mean shell length 28 mm (SE = 3 mm) were tagged with polyethylene tags (Hallprint, South Australia). Abalone were allowed to recover from the tagging procedure for 5 days before being transported to the study area in ice boxes filled with damp hessian sacks.

Four sites at depths of 4-6 m were selected according to their proximity to seagrass habitat (Amphibolis and Posidonia spp.) and the presence of adult H. laevigata in low densities. Sites were semiexposed areas of flat sand substratum surrounded by seagrass with high epiphyte loads and were situated 20-30 m from natural abalone reefs. At each site, six 6 [m.sup.2] rectangular experimental reefs were assembled, arranged in a 12 m x 9 m grid, each 3 m apart. Each experimental reef consisted of two layers of medium-sized boulders (20-40 cm across) collected from the surrounding area with intact natural faunal assemblages associated with them. Six stocking densities (9, 14, 18, 24, 30, and 45 abalone per [m.sup.2]) were randomly allocated among the experimental reefs at each site. The two highest densities at each site were designated as high-density treatments and the remaining four densities were designated as low-density treatments. Tagged abalone were seeded onto experimental reefs in open bags of plastic mesh from which they could easily disperse. The methods used during the seeding process are dealt with more fully in Dixon et al. (2005).

At 3, 6, 9, and 12 mo postseeding, surveys were made to quantify growth and mortality. The reefs were progressively dismantled and reassembled by divers, and an exhaustive search undertaken to find all abalone from each reef. The tag number and length (to the nearest 1 mm) were recorded in situ. to calculate growth over each 3-mo period (growth increment). The position of each recovered abalone was also recorded to determine if the individual was more likely to have been exposed to higher flow rates on the tops of rocks or more food at the outer edges of reefs. During surveys, we chose not to remove predators from experimental sites as we sought to create near-natural conditions.

In early September 2002 (Austral spring), a number of abalone that had moved off reefs at one site were collected to determine an appropriate sampling regimen. These animals were removed from the shell and visually assessed for the presence or absence of gonad tissue. In this pilot study, the minimum size of abalone with differentiated gonad was ~60 mm. Following the results of the pilot study, we visited each site for sampling in late September 2002, just before spawning is expected in H. laevigata from this area (Shepherd 1992). We targeted our collection to recover 30 individuals from high and low-density reefs. After an exhaustive search of each reef, we sampled animals that were larger than 60 mm and represented a range of sizes present at each site. An additional 14 abalone >60 mm were randomly collected from high and low-density reefs at one site for histologic examination of gonad tissue.

Following sampling, abalone were weighed (drained live-weight) to the nearest 0.1 g and shell length was measured to the nearest 0.2 mm. Following measurement, the soft tissues were shucked and photographed in dorsal view using a SLR camera with a macro lens. The photographs were used to score a visual gonad index (VGI) based on the progression of the early stages of gonad development following the criteria in Table 1. In developing a scoring system for the VGI, it was important to include a wide range of possible scores to allow good estimates of the error variance in the data. The VGI differs from other visual indices in this respect, since these systems are based on much coarser (4 or 5 point) scales (e.g. Kikuchi & Uki 1975, Ault 1986). A fine scale VGI was also better able to distinguish between samples at the early stages of gonad development.

Histologic samples of gonad tissue were stained with Mallory Triple Stain and examined under a high power microscope for the presence of well-developed gametes (after Giorgi & DeMartini 1977).

Statistical Analyses

The visual gonad index used to quantify gonad development in maturing abalone cannot be considered an interval scale, and so could not be used as a dependent variable in statistical parametric analyses. To study the relationships between length, growth rate, and gonad development, simple regressions were performed against VGI. Although there is no reason to expect a linear relationship against the index scale in these analyses, the regressions measure the strength of a trend.

Because a large proportion of sampled abalone had a VGI of zero, parametric analyses of these data were not appropriate. To study the effects of density and site on gonad development, abalone were grouped by size. The proportion of abalone in each group with a VGI > 0 and the mean length of the group were measured as new variables. The proportion of mature abalone per group with some gonad could then be analyzed as the dependent variable in an analysis of covariance with mean length of the group as the covariate and treatment and site as factors. Similarly, an analysis of variance was performed with the mean size of the group as the response variable. One site was excluded from these analyses because only one individual at that site had developed gonad.

P < 0.05 was regarded as significant in all statistical tests (0.001, highly significant).

RESULTS

The mean density of abalone on experimental reefs a year after release (September 2002 in situ survey) was 16 abalone per [m.sup.2] at high density (range 15-17) and 7 abalone per [m.sup.2] at low density (range 5-11). A t-test showed that these densities had remained significantly different after 1 y ([t.sub.7] = 4.708, P = 0.002). Table 2 shows the size distribution of our overall sample from high and low-density reefs. Overall, 45% of the individuals sampled after 1 y had developed visible gonad tissue (48% at low-density, 40% at high-density). The mean length and weight of these was 74.1 mm (SE = 4.3 mm) and 47.5 g (SE = 8.8 g) respectively.

Examination of the 14 abalone collected for histology showed that those with discernable gonad development had gametes present in all cases and that females with a VGI > 4 had eggs in advanced stages of vitellogenesis (large, rounded oocytes, >100 [micro]m). All male gonads appeared to contain viable sperm, spermatozoa being densely packed in the lumen of tubules.

A regression of length at capture after 1 y against VGI gave a significant positive relationship ([r.sup.2] = 0.196, n = 244, slope = 0.443, P < 0.001; Fig. 1). Thus abalone with a higher VGI tended to be larger and, conversely, those that had not developed gonad or had small gonads tended to be smaller. Regressions were performed to relate growth history and size of these abalone to gonad development (Table 3). Density was not considered in these models.

[FIGURE 1 OMITTED]

Over all size classes, more abalone were mature at low density than at low density (48% and 40% respectively, Fig. 2). Animals at high density, however, formed a larger percentage of mature animals in the medium size classes (e.g. up to 13% more by the 72-mm class). In the analysis of covariance (described in the Methods section), density treatment showed no significant effect on gonad development, but the covariate of length was highly significant (ANCOVA: [F.sub.1,23] = 38.195, P < 0.001). Furthermore, density had a significant effect on the mean size of abalone across all sites (2-way ANOVA: [F.sub.1,24] = 6.774, P = 0.016).

[FIGURE 2 OMITTED]

There was a marked increase at all sites in the percentage of abalone that were found on the edge of experimental reefs (i.e. adjacent to sand and seagrass) between March and June (Fig. 3). Abalone on the edge of the reef in June were also more likely to have developed gonad by September (2-way table: [[chi].sub.1.sup.2] = 5.881; P = 0.015). Being on the edge of the reef at other survey times did not correlate with gonad development. Abalone found on the tops of rocks during the study were also no more likely to develop gonad than other animals. A smaller proportion of individuals were on the edge of reefs at high density compared with low-density reefs (Fig. 3) and this difference was significant in June (2-way table: [X.sub.1.sup.2] = 5.14, P = 0.023).

[FIGURE 3 OMITTED]

DISCUSSION

This study was carried out using 18-mo-old cultured juveniles (28.1 mm mean length) that were seeded into a near-natural field environment. Growth rates for these animals while in the hatchery (i.e. prior to release) were very similar to those reported for wild juveniles of the same age at West Island (a nearby site with similar characteristics). Shepherd (1988) showed that wild juveniles consistently reached 25-29 mm in their first 18 mo in a study of 5 separate cohorts at West Island, whereas Shepherd and Hearn (1983) recorded growth rates of up to 39 mm in the first year at the same location. The experimental reefs were assembled from natural substrates complete with predators and interspecific competitors. Treatment densities, in terms of the number of abalone per area of suitable habitat, may also be compared with densities of wild adult H. laevigata found by Dixon and Day. The growth rates we observed in the field for our study (~40 mm per year) were comparable to those seen in wild juveniles of a similar size (Shepherd & Hearn 1983, 33 mm per year; Shepherd 1992, 43.4 mm per year). The mortality rates recorded in our experiment (see Dixon et al. 2005) were also similar to those found in wild juvenile populations (Shepherd 1998). With all of these considerations, we are confident that our results provide valuable and relevant information for wild stocks.

The size of abalone, in terms of length or weight, seems to be the most important determinant of maturation in H. laevigata. Considering the known age of all abalone released into the field (30 mo in September 2002), we can conclude that, in a maturing population, larger abalone would become reproductive one or more years before smaller abalone of the same age. This is an important finding in the light of other studies that suggest maturity in wild abalone is genetically determined on reaching a certain age (Shepherd & Laws 1974, Prince et al. 1988, Nash 1990, Shepherd et al. 1991, Nash 1992). Because larger abalone were the faster growing individuals over the year, our results strongly suggest that there is significant plasticity in the age at which H. laevigata become mature and that this age varies with growth rate.

Abalone in this experiment were of a similar size when released, so differences in the growth rate of individuals are responsible for whether these abalone matured in 2002 and their degree of gonad development. Significantly, juvenile somatic growth during the first 3 mo of the study was most important in determining both final length and the amount of gonad developed by September, whereas growth in subsequent periods was not strongly correlated to the VGI. The strong influence of initial growth rate on subsequent gonad development might be attributable to stress induced during seeding (Schiel 1993), such that less stressed individuals that grew well immediately following release had a significant advantage over other abalone. It is possible, therefore, that growth and the timing of maturation in abalone will be sensitive to other environmental stresses.

Another intriguing possibility is that seasonal growth (i.e. in Spring) when food is more abundant is especially important for maturing abalone. Another hypothesis is that individual juvenile abalone may have differing inherent growth rate potentials by virtue of some aspect of body condition. We were unable to separate these hypotheses in our experiment, but suggest that stress is likely the dominating influence.

It remains to be determined whether early growth performance in a natural cohort of juveniles would be a strong determinant of the timing of maturation. It is believed, however, that nutritive differences very early in life will shape growth and maturity patterns as animals grow (Stearns 1992). Indeed, early growth in abalone is often associated with later growth performances in the hatchery (Huchette 2003).

Abalone at high-density grew more slowly than those at low density, as reported in aquaculture studies (Mgaya & Mercer 1995, Capinpin et al. 1999, Huchette et al. 2003) and in the field (Dixon & Day 2004). Density also seems to affect the timing and degree of gonad development in H. laevigata. Abalone at high-density seemed to initiate gonad development at smaller sizes than those at low-density (Fig. 2). This tendency amongst slow growing populations of organisms is well documented in life history theory (Stearns & Koella 1986) but this study constitutes direct evidence of this in abalone.

The finding that, overall, a higher percentage of abalone from low density reefs were mature is clearly an indirect effect, related to the size of abalone at low density. That is, abalone at low density grew faster. The indirect effect of density on maturation is important because in years of low recruitment to an abalone population, compensatory increases in the growth of young individuals are likely (if levels of interspecific competition are the same) to increase the average individual egg production from that cohort.

It has been suggested that abalone populations with fast growth tend to mature at a younger age than slower growing populations (Wells & Mulvay 1995, Shepherd & Triantafillos 1997). Our results correspond with this since large, faster growing H. laevigata began to show signs of maturity at least 1 y before slower growing individuals. This suggests that the timing of maturation in abalone is a plastic trait in response to growth rate and that maturation is not genetically determined to commence at a given age. Empirical evidence for this plasticity was provided by a study on H. iris by McShane and Naylor (1995a). Based on length-at-age data, McShane and Naylor found that abalone that were exposed to higher flow rates (and hence more food) near headlands tended to grow faster, and mature earlier, than those in sheltered bays. Our study is the first to demonstrate plasticity in the timing of maturation within a local abalone population and our results are consistent with findings in other marine invertebrates (Thompson 1979, Franz 1996) and in fish (Stearns & Crandall 1984, Reznick 1990).

Anecdotal evidence from aquaculture studies suggests that small H. laevigata (50-60 mm) can be successfully induced to spawn in the hatchery (John Hall, pers. comm.). This may represent a size limit in H. laevigata, below which individuals are physiologically incapable of spawning. Histologic examination of gonad tissue from Cape Jervis abalone (all of which were >60 mm) suggested that larger individuals with more gonad would be capable of spawning but, because of their relatively small size, gamete production would be low.

If these abalone gain one or more extra spawning seasons due to their earlier maturation, this could be significant in the management of the fishery because the reproductive output of a cohort will vary considerably as individuals approach sexual maturity. It is possible that larger abalone at Cape Jervis actually spawned in 2002. The fact that these abalone had already developed gonads at 30 too, and were also the fastest growing individuals in the cohort, might allow them to produce larger gonads in future spawning seasons than other abalone. It is also possible that abalone with faster early growth may reach a higher asymptotic length (and hence higher reproductive capacity, Steams 1992), but our data cannot test this hypothesis. In either case, abalone that mature earlier have a substantial reproductive advantage over slower growing abalone that have remained small (Roff 2002).

Prince et al. (1988) and Nash (1992) suggested that the commencement of maturation and end of exclusively somatic growth also coincided with the emergence of H. rubra to less cryptic positions on rocks, characteristic of adult habitat. The results for H. laevigata at Cape Jervis correspond with this because many of the abalone that developed gonad were found to be on the edge of plots in June where they sat on rocks bordered by sand and seagrass. This position would give abalone better access to drift algae (Shepherd 1973) and is characteristic of adult H. laevigata habitat. It is important to note that in our experiment, densities of adult H. laevigata were low on experimental reefs such that adult habitat was largely vacant. In populations where adult densities are higher, juveniles may face stronger competition for positions on the edge of reefs.

The emergence of large numbers of abalone to the edge of plots between March and June seems to precede the development of gonad tissue, which is believed to take place between June and September (Shepherd & Laws 1974, Shepherd & Hearn 1983). This may reflect a delay between when more food becomes available and the energetic allocation of resources to growth or reproduction (Day & Fleming 1992, Donovan & Carefoot 1998).

Abalone that were on the edge of reefs in June would have access to more abundant food and were subsequently likely to have developed gonad. This suggests that the amount or quality of food available to juvenile abalone may be important in the initiation of gonad development. Nutrition is known to play an important role in the timing of maturation and is strongly correlated with growth rate in other invertebrates (Lucas & Lawes 1998, Boyd et al. 1986) and in fish (Reznick 1990). Thus differences in growth rate and maturation between high and low-density abalone presumably reflect the more basic difference of increased nutrition.

Across many taxa (including humans), poorly nourished individuals mature much later than well-fed individuals (Stearns & Koella 1986). Our study shows that this is also true for abalone, because animals with greater access to food resources (e.g. those at lower experimental density and/or residing on the edge of reefs) were likely to mature in 2002 whereas abalone with poor access to food would likely not mature for another year.

The age at first maturity in H. laevigata seems to be a highly plastic trait in response to different growth rates and/or nutrition levels. The results of our study indicate that the onset of maturation is not fixed at a certain age within a population but can be advanced under conditions of fast growth.

TABLE 1.

Criteria for assessment of the Visual gonad index (VGI) in H.
laevigata from Cape Jervis, South Australia.

VGI                             Criteria

 0     No gonad visible, immature.
 1     Gonad membrane apparent, beginnings of gonad on
         under-side of conical appendage.
 2     Some eonad membrane on proximal side of stomach and
         visceral spine.
 3     Gonad tissue apparent on conical appendage.
 4     Gonad tissue apparent on visceral spire.
 5     Gonad tissue thickening around visceral spire and
         proximal side of stomach.
 6     Gonad tissue thickening on conical appendage.
 7     Thick gonad tissue on proximal side of stomach.
 8     Approximately 50% of visceral spire covered in gonad
         tissue.
 9     Visceral spire covered in gonad tissue.
10     Approximately 50% of conical appendage covered in
         gonad tissue.
11     Thick gonad tissue covering entire visceral spire, stomach
         and conical appendage.

TABLE 2.

Size distribution of H. laevigata sampled for gonad analysis in
September, 2002.

                    Size Class (mm)

Density   60-65   65-70   70-75   75-80   80+   Mean Size (mm)

 HIGH       9      41      40      12      1         70.2
 LOW        1      36      53      40     11         72.4

TABLE 3. Regression for growth, length and weight measurements against
visual gonad index for H. laevigata from Cape Jervis,
South Australia.

            Measurement                 [r.sup.2]    Slope

Length
September 2001                            0.006       --
December 2001                             0.135      0.367
March 2002                                0.139      0.373
June 2002                                 0.165      0.407
September 2002                            0.196      0.443

Length Increment (growth)
September to December 2001 (Spring)       0.132      0.363
December 2001 to March 2002 (Summer)      0           --
March to June 2002 (Autumn)               0           --
June to September 2002 (Winter)           0.032      0.180

Weight
September 2002                            0.224      0.473

ACKNOWLEDGMENTS

The authors thank all those who helped with survey diving and collection, especially James Brooks. We also thank Dr. Scoresby Shepherd and two anonymous reviewers for their helpful comments on earlier drafts of the manuscript.

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LUKE A. MCAVANEY, * ROBERT W. DAY, CAMERON D. DIXON AND SYLVAIN M. HUCHETTE

Zoology Department, University of Melbourne, Victoria, 3010, Australia

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