Analysis of genetic structure of blacklip abalone (Haliotis rubra) populations using RAPD, minisatellite and microsatellite markers

 We investigated the utility of three polymerase chain-reaction (PCR)-based DNA molecular markers in analysing genetic structure of the populations of the blacklip abalone Haliotis rubra (Leach) of Victoria, Australia. The DNA markers included 84 randomly amplified polymorphic DNA (RAPD) bands amplified using six random primers, two minisatellites, GHR (putative growth-hormone-gene-repeat) and MIPR (putative mollusca-insulin-like peptide-gene-repeat), and three microsatellites, RUBGT1 [containing (GT)_n repeats], RUBCA1 [containing (CA)_n repeats] and RUBGACA1 [containing (GACA)_n repeats]. All three types of DNA markers revealed significant subdivision in the H. rubra populations along the coastline. This is postulated as being related to the abalone's relatively short pelagic period and limited dispersion. Further analysis revealed that a Point Cook population sampled from within the semi-enclosed Port Phillip Bay was distinct from two other central zone populations (Apollo Bay and Cape Schanck). The genotypes of microsatellites indicated excessive homozygotes across all the populations at all three microsatellite loci, and possible causes such as larval recruitment pattern and asynchronous spawning are discussed. The excessive homozygotes recorded for the three microsatellite loci contrast with those observed in the minisatellite loci GHR and MIPR, the heterozygosities of which were at Hardy–Weinberg equilibrium. Received: 17 March 1999 / Accepted: 24 November 1999     

Tissue and sub-cellular distribution of Fe,Cu, Zn and 210Po in the abalone Haliotis rubra

The tissue and sub-cellular distribution of Fe, Cu, Zn and the naturally occurring radionuclide polonium-210 was determined in the gastropod mollusc Haliotis rubra collected from Western Port Bay, Australia, between March and July 1988. The highest concentrations of the metals, with the exception of Cu, were found in the digestive gland. Copper was more uniformly distributed, with tissues that are more vasculated having higher concentrations. Ultrastructural examination of the digestive gland, gill and kidney showed dense membrane-bound granules within the cytoplasm. Elemental analysis of the granules by electron probe x-ray microanalysis indicated that the granules in the digestive gland and gill contained high concentrations of iron, with small amounts of copper and zinc. In contrast, the metal-containing granules in the kidney were predominantly composed of iron, copper and phosphorus, with variable contributions of sodium potassium, and calcium. Homogenisation and fractionation of the digestive gland by differential centrifugation confirmed that approximately 80, 10, 90 and 50% of the total homogenate Fe, Cu, Zn and ²¹⁰Po, respectively, sedimented at 1200xg. In the haemolymph, all the elements studied were associated with the soluble high molecular weight component of the serum, not with the amoebocytes. ²¹⁰Po was present in the mucus-secreting hypobranchial glands at about half the concentration found in gill tissue.     

Sublethal effects of zinc and municipal effluents on larvae of the red abalone Haliotis rufescens

Experiments were conducted to develop a sensitive sublethal toxicity test protocol to determine the toxicity of municipal wastewater effluents to larvae of the red abalone Haliotis rufescens. In multiple tests, fertilized abalone embryos were exposed for 48 h to dilutions of a reference toxicant, zinc sulfate, and to dilutions of primary-and secondary-treated effluents. The resulting veliger larvae were examined microscopically for larval shell abnormalities. In a longer flowthrough experiment, abalone were exposed for the entire larval phase, from the two-cell stage through metamorphosis, to compare zinc effects on metamorphosis with zinc effects on short-term larval shell development. Dissolved oxygen, pH, salinity and temperature were measured daily in test solutions, and zinc concentrations were verified by chemical analysis. No observed effect concentrations (NOECs) for zinc were 39±2.1 g l^-1 in three 48 h exposures, and 19 g l^-1 for the 9 d exposure through metamorphosis. Median effect concentrations (EC₅₀s) were 68±6.9 g l^-1 in 48 h tests and 50 g l^-1 in the 9 d test. Abalone larvae were affected at lower concentrations of primary than of secondary effluent.     

Repeatability of physiological traits in juvenile Pacific abalone, Haliotis discus hannai

Several studies on individual physiological traits assume that past records may predict future performance. Marine mollusks, as other animals, show a wide range of between- and within-individual variation of physiological traits. However, in this group, almost nothing is known about the relative influence of genetic factors on that variation. Repeatability (R) is a measure of the consistency of the variation of a trait, which includes its genetic variance and represents the maximum potential value of its heritability (h ²). Traits that show high inter-individual variation and high repeatability levels could potentially evolve through selection (natural or artificial). We estimated the repeatability [using intra-class (τ) and Pearson-moment (r) correlation coefficients] of several physiological traits related to energy acquisition and allocation in juvenile Pacific abalone Haliotis discus hannai, maintained under controlled environment growing systems. In order to estimate the range of the R values and the effect of the time elapsed between measurements on these estimates, we measured these traits monthly during 6 months for each individual. Among the physiological traits, those related to energy allocation like oxygen consumption (standard metabolic rate, SMR) and ammonium excretion rates, and oxygen/nitrogen ratio (O/N), showed intermediate levels of repeatability (0.48, 0.55 and 0.39, respectively), when this was estimated by τ coefficient. However, the r estimation showed that SMR and O/N repeatability were significant and high (0.6–0.7 and 0.5–0.7, respectively) during the first 5 consecutive measurements, decreasing strongly (0.3 and 0.2, respectively) during the last measurement. For ammonia excretion, although repeatability (r) decreased from 0.8 to 0.5 during the 6 consecutive measurements, they remain significant during the experimental period. Therefore, our results indicate that for H. discus hannai juveniles, physiological traits like SMR, ammonia excretion and O/N are significantly repeatable (i.e. good predictors of future measurements) during a period of 4–5 months. These significant repeatability values suggest an important genetic control upon the phenotypic variation of these physiological traits, and could potentially respond to natural or artificial selection, and be used in genetic improvement programs. By contrast, those traits related to energy acquisition (i.e. ingestion, absorption and assimilation) and physiological efficiencies (i.e. net growth and scope for growth) showed very low levels of repeatability (0–0.07). This indicates that the phenotypic variation of these traits would be more influenced by environment rather than by genetic factors.     

Molecules from cyanobacteria and red algae that induce larval settlement and metamorphosis in the mollusc Haliotis rufescens

Potent inducers of metamorphosis of planktonic larvae of the gastropod mollusc Haliotis rufescens have been found in the following phycobiliprotein-producing cyanobacteria. Synechococcus spp. (one marine and one freshwater strain). Synechocystis spp. (one hypersaline and one freshwater strain) and Spirulina platensis (a freshwater strain). No inducers were detected in the bacterium Escherichia coli. Inducers from one of the cyanobacteria (S. platensis) were partially purified and compared to inducers from the foliose red macroalga Porphyra sp. and the crustose coralline red alga Lithothamnium californicum. In all three species the inducers can be largely separated from the biliproteins, with which they appear to be associated, by high-resolution gel-filtration chromatography. The molecular weights of the relatively small inducing molecules resolved by these procedures from cyanobacteria and red algac are similar, falling in the range of 640 to 1 250 daltons. The amenability of the cyanobacteria to largescale cultivation, and to physiological and genetic manipulation, make them useful for production of metamorphic inducers of marine invertebrate larval metamorphosis, and for further studies of the synthesis, structure and mechanism of action of such inducing molecules.     

Variability in grazer-mediated defensive responses of green and red macroalgae on the south coast of South Africa

Variabilities in the responses of several South African red and green macroalgae to direct grazing and the responses of one green alga to cues from grazers were tested. We used two feeding experiments: (1) testing the induced responses of three red and one green algae to direct grazing by mesograzers and (2) a multi-treatment experiment, in which the direct and indirect effects of one macrograzer species on the green alga Codium platylobium were assessed. Consumption rates were assessed in feeding assays with intact algal pieces and with agar pellets containing non-polar extracts of the test algae. Defensive responses were induced for intact pieces of Galaxaura diessingiana, but were not induced in pellets, suggesting either morphological defence or chemical defence using polar compounds other than polyphenols. In contrast, exposure to grazing stimulated consumption of Gracilaria capensis and Hypnea spicifera by another grazing species. In the multi-treatment experiment, waterborne cues from both grazing and non-grazing snails induced defensive algal traits in C. platylobium. We suggest that inducible defences among macroalgae are not restricted to brown algae, but that both the responses of algae to grazers and of grazers to the defences of macroalgae are intrinsically variable and complex.     

A morphological and structural study of the larval shell from the abalone <Emphasis Type="Italic">Haliotis tuberculata</Emphasis>

The larval shell of the marine gastropod Haliotis tuberculata was investigated by polarised light microscopy, scanning electron microscopy, Raman microspectroscopy and infra-red spectroscopy. Trochophore and veliger larval sections were used for histological examination of the growing shell and each larval stage was related to the shell development and the appearance of calcified formations. We determined the stage of initial calcification by specific staining combined with polarised light examination. The shell of 30-h-old pre-veliger larvae was found to be mineralized, confirming that calcification occurred before larval torsion. Using both infra-red and Raman spectroscopy, we showed that CaCO₃ deposition occurred at the pre-veliger stage and that the mineral phase initially deposited was essentially composed of aragonite.     

Reproductive cycle and gametogenesis in the black abalone Haliotis cracheroidii (Gastropoda: Prosobranchiata)

Seasonal changes in gonald size and stages of gametogenesis in the black abalone Haliotis cracheroidii were related to changes in environmental parameters. H. cracheroidii showed an annual reproductive cycle terminating in a synchronized spawning in late summer. Gametogenesis was initiated immediately after spawning. Gametes were present in the gonad through the winter months. Gametogenesis was initiated a second time in the spring months. Maximal gonad growth (to a gonad index of 20%) occurred during summer months prior to spawning. Changes in gonad size andperiods of initiation of gametogenesis revealed no apparent correlation with changes in seasonal water temperature. Changes in gonad size showed no apparent relation to change in day length. Total polysaccharide levels in foot tissue changed seasonally, indicating that food availability is probably not a factor in directly regulating gonad growth. Gonad index data for the chiton Katharina tunicata (collected over a 10 year period) showed no apparent correlation to seasonal change in water temperature.     

Using opportunistic green macroalgae as indicators of nitrogen supply and sources to estuaries.

Nutrient inputs to estuaries are increasing worldwide, and anthropogenic contributions are increasingly complex and difficult to distinguish. Measurement of integrated effects of salinity and nutrient changes simultaneously can help ascertain whether N sources of similar magnitude and stable isotope (sigma15N) signatures are river dominated. We used Enteromorpha spp., an opportunistic macroalga, to obtain integrated measures of salinity, nutrient supply, and nutrient source in estuaries. We outplanted cultured algae in the field along spatial gradients within three southern California estuaries for 24 hours in wet and dry seasons. Tissue was analyzed for potassium (K+) to measure osmoregulatory changes, total nitrogen to examine changes in nutrient supply, and sigma15N to assess nutrient sources. Discrete measures of water salinity correlated with tissue K+ content; however, there was significant variability in the relationship, suggesting that the algae were subject to considerable variation in salinity over a tidal cycle. Tissue total N was not always related to snapshot measures of water column dissolved inorganic nitrogen (DIN), suggesting that integrated measures may be better at capturing the temporal and spatial complexity of nutrient availability. The combination of tissue K+, total N, and sigma15N measures revealed that inflowing rivers delivered N from watershed sources to Mugu Lagoon and Carpinteria Salt Marsh, while both the inflowing river and a mid estuary source were important sources of high sigma15N N in Upper Newport Bay. These experiments revealed complex patterns of supply and sources of N and demonstrate the usefulness of macroalgal indicators over water sampling to detect these patterns.     

Waterborne chemical compounds in tropical macroalgae: positive and negative cues for larval settlement

Settlement sites of marine invertebrate larvae are frequently influenced by positive or negative cues, many of which are chemical in nature. Following from the observation that many shallow-water, Hawai'ian marine macroalgae are free of fouling by sessile invertebrates, we predicted that the algae are chemically protected and dependent on either surface-bound or continuously released soluble compounds to deter settling invertebrate larvae. To address the importance of waterborne algal compounds, we experimentally determined whether larvae of two of Hawai'i's dominant hard-surface fouling organisms, the polychaete tube worm Hydroides elegans and the bryozoan Bugula neritina, would settle in the presence of waters conditioned by 12 species of common Hawai'ian macroalgae (representing the Phaeophyta, Chlorophyta, Rhodophyta and Cyanophyta). The results included a full spectrum of biological responses by each larval species to waterborne algal compounds. Larval responses to conditioned water were consistent for each algal species, but the outcomes were not predictable based on the taxonomic relationships of the algae. For example, among the species of Phaeophyta examined, different conditioned waters were: (1) toxic, (2) inhibited settlement, (3) simulated settlement, or (4) had no effect, compared to larvae in control dishes containing filtered seawater. Additionally, larval responses to aged (24 h) conditioned waters could not be predicted from the results of assays run with conditioned waters utilized immediately after preparation. Finally, settlement by larvae of one species did not predict outcomes of tests for the other species. Four of 12 shallow-reef Hawai'ian macroalgae tested released compounds into surrounding waters that immediately killed or inhibited settlement by both H. elegans and B. neritina (toxic: Dictyota sandvicensis; inhibitory: Halimeda discoidea, Sphacelaria tribuloides, Ulva reticulata); the remaining 8 algal species prevented settlement by one of these fouling organisms but for the other had no effect or, in some cases, even stimulated settlement     

Green abalone, <Emphasis Type="Italic">Haliotis fulgens</Emphasis> infected with the agent of withering syndrome do not express disease signs under a temperature regime permissive for red abalone, <Emphasis Type="Italic">Haliotis rufescens</Emphasis>

All California abalone species have been shown to be susceptible to infection with the bacterial agent of abalone withering syndrome (WS), although expression of signs of the disease may vary between species and with environmental conditions. We examined thermal modulation of WS expression in green abalone Haliotis fulgens at temperatures mimicking El Niño (18.0°C) and La Niña (14.2°C) events in southern California. In contrast to results obtained from previous experiments with red abalone, H. rufescens, the higher temperature did not result in higher infection intensities of the causative agent of the disease nor increase in clinical signs of disease. These results demonstrate clear differences in thermal regulation of disease expression between abalone species, and provide further data suggesting that green abalone should be a target species of recovery efforts in southern California, where WS is endemic.     

Convergent development of ecological,genetic, and morphological traits in native supercolonies of the red ant Myrmica rubra

Ant supercolonies (large networks of interconnected nests) represent the most extreme form of multi-queen breeding (polygyny) and have been found across ant lineages, usually in specific long-term stable populations. Many studies on the genetic population structure and demography of ant supercolonies have been done in recent decades, but they have lacked multicolonial control patches with separated colonies headed by a single or few queens so the origin of the supercolonial trait syndrome has remained enigmatic. Here, we set out to compare sympatric supercolonial and multicolonial patches in two natural Danish populations of the common red ant Myrmica rubra. We used DNA microsatellites to reconstruct genetic colony/population structure and obtained morphological and density measurements to estimate life history and ecology covariates. We found that supercolonies in both populations completely dominated their patches whereas colonies in multicolonial patches coexisted with other ant species. Supercolony patches had very low genetic differentiation between nests, negligible relatedness within nests, and lower inbreeding than multicolonial patches, but there were no significant morphological differences. One population also had nests that approached true outbred monogyny with larger workers and males but smaller queens than in the two other social nest types. Our results suggest that once smaller colonies start to adopt additional queens, they also gain the potential to ultimately become supercolonial when the habitat allows rapid expansion through nest budding. This is relevant for understanding obligate polygyny in ants and for appreciating how and why introduced North American populations of M. rubra have recently become invasive.     

Comparing community structure on shells of the abalone Haliotis midae and adjacent rock: implications for biodiversity

This paper concerns the effects on biodiversity of depletion of the South African abalone Haliotis midae, which is a long-lived species with a large corrugated shell that provides a habitat for diverse benthic organisms. We compared community structure on H. midae shells with that on adjacent rock at three sites (Cape Point and Danger Point sites A and B) and at two different times of the year at one of these sites. Shells of H. midae consistently supported communities that were distinctly different from those on rock. In particular, three species of non-geniculate (encrusting) corallines, Titanoderma polycephalum, Mesophyllum engelhartii and Spongites discoideus, were all found either exclusively or predominantly on shells, whereas another non-geniculate coralline, Heydrichia woelkerlingii, occurred almost exclusively on adjacent rock. The primary rocky substratum, however, supported a higher number of species than abalone shells. Possible reasons for the differences between the two substrata include the relative age, microtopography and hardness of the substrata; the abundance of grazers on them; and the relative age of different zones of the abalone shell, which support communities at different stages of succession. Diversity on shells was lowest in zones that were either very young or very old, in keeping with the intermediate disturbance hypothesis. The distinctiveness of shell epibiota will increase β diversity despite having a lower α diversity than that of adjacent rock. Decimation of H. midae by overfishing therefore has implications for biodiversity conservation.     

Opposite shifts in size at metamorphosis in response to larval and metamorph predators

Predation risk can cause organisms to alter the timing of life history switch points. Theory suggests that increased risk in an early life stage should select for switching earlier and smaller, while increased risk in the subsequent stage should select for switching later and larger. This framework has frequently been applied to metamorphosis in amphibians, with mixed results. Few studies examining the effect of larval predation risk on metamorphosis have observed the predicted pattern, and no studies, to our knowledge, have examined the effect of increased risk during and after metamorphosis on the timing of this switch point. Here we examine the effect of larval and post-metamorphic predation risk on metamorphosis in the red-eyed treefrog, Agalychnis callidryas. We raised tadpoles in the presence or absence of cues from caged water bugs fed larvae and cues from spiders fed emerging metamorphs. Water bugs are effective larval predators, while spiders are poor larval predators but prey on metamorphs. Furthermore, since spiders forage on the water surface it is possible that tadpoles could assess future risk from this predator. Predators induced opposite shifts in life history. Tadpoles emerged smaller and less developed in response to water bugs, but later and larger in response to spiders. Interestingly, predator effects on larval duration were not independent; tadpoles delayed emerging in response to spiders, but only in the absence of water bugs.     

Elasticity analyses of size-based red and white abalone matrix models: management and conservation.

Prospective elasticity analyses have been used to aid in the management of fished species and the conservation of endangered species. Elasticities were examined for deterministic size-based matrix models of red abalone, Haliotis rufescens, and white abalone, H. sorenseni, to evaluate which size classes influenced population growth (lambda) the most. In the red abalone matrix, growth transitions were determined from a tag recapture study and grouped into nine size classes. In the white abalone matrix, abalone growth was determined from a laboratory study and grouped into five size classes. Survivorship was estimated from tag recapture data for red abalone using a Jolly-Seber model with size as a covariate and used for both red and white abalone. Reproduction estimates for both models used averages of the number of mature eggs produced by female red and white abalone in each size class from four-year reproduction studies. Population growth rate (lambda) was set to 1.0, and the first-year survival (larval survival through to the first size class) was estimated by iteration. Survival elasticities were higher than fecundity elasticities in both the red and white matrix models. The sizes classes with the greatest survival elasticities, and therefore the most influence on population growth in the model, were the sublegal red abalone (150-178 mm) and the largest white abalone size class (140-175 mm). For red abalone, the existing minimum legal size (178 mm) protects the size class the model suggests is critical to population growth. Implementation of education programs for novice divers coupled with renewed enforcement may serve to minimize incidental mortality of the critical size class. For white abalone, conservation efforts directed at restoring adults may have more of an impact on population growth than efforts focusing on juveniles. Our work is an example of how prospective elasticity analyses of size-structured matrix models can be used to quantitatively evaluate research priorities, fishery management strategies, and conservation options.     

Biomineralization markers during early shell formation in the European abalone <Emphasis Type=Italic>Haliotis tuberculata</Emphasis>, Linnaeus

Larval shell formation was investigated in the European abalone Haliotis tuberculata. Stages of mineralization as well as enzymatic and endocrine biomarkers were monitored throughout larval development, from hatching to post-larval stages. Polarized light microscopy and infrared spectroscopy analyses revealed the presence of crystallized calcium carbonate arranged in aragonite polymorphs from the late trochophore stage. A correlation between the main steps of shell formation and enzymatic activities of alkaline phosphatase and carbonic anhydrase was seen. The variations of these biologic activities were related to the onset of mineralization, the rapid shell growth, and the switch from larval to juvenile shell following metamorphosis. Furthermore, a strong increase in the level of calcitonin gene-related molecules was measured in post-larvae, suggesting that endocrine control takes place after metamorphosis. The changes measured for the three biomineralization markers together with mineralogical analysis allowed us to correlate physiologic mechanisms with early steps of abalone shell formation.     

Genetic structure of natural populations of California red abalone (<Emphasis Type="Italic">Haliotis rufescens</Emphasis>) using multiple genetic markers

Mitochondrial cytochrome oxidase subunit one (COI) sequence, nuclear microsatellites, and amplified fragment length polymorphisms (AFLPs) were used to evaluate connectivity among nine red abalone (Haliotis rufescens) populations sampled between August 1998 and November 2003 along approximately 1,300 km of California coastline from Crescent City (41°46′N, 124°12′W) to San Miguel Island (34°02′N, 120°22′W). COI sequences and microsatellite genotypes did not show significant genetic divergence among nine sampled populations. A subset of five populations spanning the geographic range of the study was scored for 163 polymorphic AFLP markers. Of these, 41 loci showed significant divergence (P < 0.001) among populations. Still, no AFLP markers were diagnostic for any of the study populations, and assignment tests did not consistently assign individuals to the correct population. Although the AFLP data are the first to suggest there is significant genetic differentiation among California red abalone populations, the discordance between the different genetic markers needs further study before unambiguous conclusions can be drawn with respect to connectivity among the populations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fertilisation, embryogenesis and larval development in the tropical intertidal sand dollar Arachnoides placenta in response to reduced seawater pH

We examined the response of the tropical sand dollar Arachnoides placenta to reduced seawater pH in experiments spanning ca. 50 % of the planktonic larval duration. A. placenta inhabits intertidal sandy beaches where we observed a minimum in situ pH range 0.06 pH units (pH 8.10–8.16). The responses of gametes and larvae to seawater pH were tested in vitro in ambient (pH 8.14, pCO₂ = 525.7 μatm, total alkalinity = 2,651 μmol kg soln^?1) and three reduced pH seawater treatments (7.8–7.0). Percentage fertilisation decreased significantly with decreasing pH across a range of sperm/egg ratios (4:1 up to 4,000:1). A. placenta reached the advanced pluteus stage in 4 days, and during this time, we saw no difference in survival rate of larvae between the ambient (67 %) and pH 7.79 (72 %) treatments. Four-day survival was, however, reduced to 44 and 11 % in the pH 7.65 and 7.12 treatments, respectively. Larval development and morphometrics varied among pH treatments. Embryos reared in pH 7.12 exhibited arrested development. Larvae reared at pH 7.65 showed delayed development and greater mortality compared with those reared at pH 7.79 and 8.14. When larval morphometrics are compared among larvae of the same size, differences in larval width and total arm length between pH treatments disappear. These results suggest that variation in larval size among the three highest pH treatments at a given time are likely the result of slower development and apparent shrinkage of surviving larvae and not direct changes in larval shape. There were no differences in the percentage inorganic content (a proxy for calcification) in larvae reared in either an ambient or a pH 7.7 treatment. The responses of fertilisation and development to decreased pH/increased pCO₂ in A. placenta are within the range of those reported for other intertidal and subtidal echinoid species from colder latitudes.     

Using growth band autofluorescence to investigate large-scale variation in growth of the abalone <Emphasis Type="Italic">Haliotis midae</Emphasis>

Growth of the abalone, Haliotis midae, was investigated at Port Alfred, on the south coast of South Africa, using both new and established techniques. A new method for aging animals is described, which makes use of shell autofluorescence under UV light to visualise internal growth bands. The deposition of growth bands was validated using measurements from shells of known age and, at one site, comparing growth estimates to those from cohort analysis undertaken at the same site. The new technique is far less time consuming and labour intensive than previously described methods; it is also non-destructive and proved to have potential for the reliable and rapid assessment of growth in large-scale studies. Growth of H. midae was also investigated at nine other sites, incorporating the full distribution range of the species. Systematic geographic variation in growth was observed along the South African coastline. Statistically significant differences existed among sites in growth rates for animals <4 years and between 4 and 6 years and in the mean maximum sizes attained. Generally, H. midae from the south/southeast coast were found to have faster growth rates, smaller mean maximum sizes and were assumed to attain sexual maturity (determined in previous studies) earlier than those along the southwest/west coast. The geographic differences in estimates of growth observed have significant implications for future modelling approaches and indicate that present national management strategies are not appropriate as they fail to take regional variability into account.