Isolation and cross-amplification of microsatellites in pink abalone (Haliotis corrugata)

Ten novel microsatellite loci were isolated in pink abalone, Haliotis corrugata, using (GT)₁₅ and (CT)₁₅ enriched genomic libraries. Two previously reported Haliotis kamtschatkana microsatellites cross‐amplified in H. corrugata. A set of 12 polymorphic microsatellites were evaluated in a wild population sample (N = 49). The number of alleles ranged from two to 55, and the observed and expected heterozygosities ranged from 0.104 to 0.939 and from 0.213 to 0.982, respectively. Significant deviations from Hardy–Weinberg equilibrium at three loci and no linkage disequilibrium were observed. Haliotis corrugata microsatellites cross‐amplified in other abalone species, two in H. fulgens, and seven in H. rufescens.     

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker–centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72‐h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second‐division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y‐values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y‐values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.     

Isolation and characterization of microsatellite markers in the South African abalone (Haliotis midae)

We report the isolation and characterization of 11 polymorphic microsatellite loci in the South African abalone Haliotis midae. These loci showed a range of five to 21 alleles per locus and observed heterozygosities ranging from 0.14 to 0.93 in a wild population of 32 individuals. All loci except four conformed to Hardy–Weinberg expectations and did not show linkage disequilibrium. The polymorphism exhibited at these loci indicate that they would be useful in determining levels of genetic variability in natural and commercial Haliotis midae populations as well as in parentage and Quantitative Trait Loci (QTL) analysis in hatchery reared abalone.     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

Genetic diversity and species identification in the endangered white abalone (<Emphasis Type="Italic">Haliotis sorenseni</Emphasis>)

In 2001, the white abalone Haliotis sorenseni became the first marine invertebrate in United States waters to receive federal protection as an endangered species. Prior to the endangered species listing, 20 abalone were collected as potential broodstock for a captive rearing program. Using DNA from these animals, we have developed genetic markers, including five nuclear microsatellite loci and partial sequences of one nuclear (VERL) and two mitochondrial (COI and CytB) genes, to assess genetic variability in the species, aid in species identification, and potentially track the success of future outplanting of captive-reared animals. All five microsatellite loci were polymorphic and followed expectations of simple Mendelian inheritance in laboratory crosses. Each of the wild-caught adult abalone exhibited a unique composite microsatellite genotype, suggesting that significant genetic variation remains in natural populations. A combination of nuclear and mitochondrial gene sequencing demonstrated that one of the original wild-caught animals was, in fact, not a white abalone, but H. kamtschatkana (possibly subspecies assimilis). Similarly, another animal of uncertain identity accidentally collected by dredging was also shown to be H. kamtschatkana. Inclusion of these two animals as broodstock could have resulted in unintentional hybridizations detrimental to the white abalone recovery program. Molecular genetic identifications will be useful both in preventing broodstock contamination and as markers for future restocking operations.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.     

Geographic range determinants of two commercially important marine molluscs

Aim We modelled the spatial abundance patterns of two abalone species (Haliotis rubra Donovan 1808 and H. laevigata Leach 1814) inhabiting inshore rocky reefs to better understand the importance of current sea surface temperature (SST) (among other predictors) and, ultimately, the effect of future climate change, on marine molluscs. Location Southern Australia. Methods We used an ensemble species distribution modelling approach that combined likelihood‐based generalized linear models and boosted regression trees. For each modelling technique, a two‐step procedure was used to predict: (1) the current probability of presence, followed by (2) current abundance conditional on presence. The resulting models were validated using an independent, spatially explicit dataset of abalone abundance patterns in Victoria. Results For both species, the presence of reef was the main driver of abalone occurrence, while SST was the main driver of spatial abundance patterns. Predictive maps at c. 1‐km resolution showed maximal abundance on shallow coastal reefs characterized by mild winter SSTs for both species. Main conclusions Sea surface temperature was a major driver of abundance patterns for both abalone species, and the resulting ensemble models were used to build fine‐resolution predictive range maps (c. 1 km) that incorporate measures of habitat suitability and quality in support of resource management. By integrating this output with structured spatial population models, a more robust understanding of the potential impacts of threatening human processes such as climate change can be established.     

Seascape genomics reveals adaptive divergence in a connected and commercially important mollusc,the greenlip abalone (Haliotis laevigata), along a longitudinal environmental gradient

Populations of broadcast spawning marine organisms often have large sizes and are exposed to reduced genetic drift. Under such scenarios, strong selection associated with spatial environmental heterogeneity is expected to drive localized adaptive divergence, even in the face of connectivity. We tested this hypothesis using a seascape genomics approach in the commercially important greenlip abalone (Haliotis laevigata). We assessed how its population structure has been influenced by environmental heterogeneity along a zonal coastal boundary in southern Australia linked by strong oceanographic connectivity. Our data sets include 9,109 filtered SNPs for 371 abalones from 13 localities and environmental mapping across ~800 km. Genotype–environment association analyses and outlier tests defined 8,786 putatively neutral and 323 candidate adaptive loci. From a neutral perspective, the species is better represented by a metapopulation with very low differentiation (global F_ST = 0.0081) and weak isolation by distance following a stepping‐stone model. For the candidate adaptive loci, however, model‐based and model‐free approaches indicated five divergent population clusters. After controlling for spatial distance, the distribution of putatively adaptive variation was strongly correlated to selection linked to minimum sea surface temperature and oxygen concentration. Around 80 candidates were annotated to genes with functions related to high temperature and/or low oxygen tolerance, including genes that influence the resilience of abalone species found in other biogeographic regions. Our study includes a documented example about the uptake of genomic information in fisheries management and supports the hypothesis of adaptive divergence due to coastal environmental heterogeneity in a connected metapopulation of a broadcast spawner.     

Molecular genetics to inform spatial management in benthic invertebrate fisheries: a case study using the Australian Greenlip Abalone

Hierarchical sampling and subsequent microsatellite genotyping of >2300 Haliotis laevigata (greenlip abalone) from 19 locations distributed across five biogeographic regions have substantially advanced our knowledge of population structure and connectivity in this commercially important species. The study has found key differences in stock structure of H. laevigata compared with the sympatric and congeneric Haliotis rubra (blacklip abalone) and yielded valuable insights into the management of fisheries targeting species characterized by spatial structure at small scales (i.e. S‐fisheries). As with H. rubra, H. laevigata comprise a series of metapopulations with strong self‐recruitment. However, the spatial extent of H. laevigata metapopulations (reefal areas around 30 km²; distances of up to 135 km are effective barriers to larval dispersal) was substantially greater than that identified for H. rubra (Miller et al. 2009). Differences in the dynamics and scale of population processes, even between congeneric haliotids as made evident in this study, imply that for S‐fisheries, it is difficult to generalize about the potential consequences of life history commonalities. Consequently, species‐specific management reflective of the population structure of the target species remains particularly important. This will likely require integration of information about stock structure and connectivity with data on life history and population dynamics to determine the necessary input (e.g. number of fishers, fishing effort) and output (e.g. minimum legal size, total allowable catch) controls to underpin their sustainable management.     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Population dynamics can be more important than physiological limits for determining range shifts under climate change

Evidence is accumulating that species' responses to climate changes are best predicted by modelling the interaction of physiological limits, biotic processes and the effects of dispersal‐limitation. Using commercially harvested blacklip (Haliotis rubra) and greenlip abalone (Haliotis laevigata) as case studies, we determine the relative importance of accounting for interactions among physiology, metapopulation dynamics and exploitation in predictions of range (geographical occupancy) and abundance (spatially explicit density) under various climate change scenarios. Traditional correlative ecological niche models (ENM) predict that climate change will benefit the commercial exploitation of abalone by promoting increased abundances without any reduction in range size. However, models that account simultaneously for demographic processes and physiological responses to climate‐related factors result in future (and present) estimates of area of occupancy (AOO) and abundance that differ from those generated by ENMs alone. Range expansion and population growth are unlikely for blacklip abalone because of important interactions between climate‐dependent mortality and metapopulation processes; in contrast, greenlip abalone should increase in abundance despite a contraction in AOO. The strongly non‐linear relationship between abalone population size and AOO has important ramifications for the use of ENM predictions that rely on metrics describing change in habitat area as proxies for extinction risk. These results show that predicting species' responses to climate change often require physiological information to understand climatic range determinants, and a metapopulation model that can make full use of this data to more realistically account for processes such as local extirpation, demographic rescue, source‐sink dynamics and dispersal‐limitation.     

Comparative studies on the nutrition of two species of abalone,Haliotis tuberculata Linnaeus and Haliotis discus hannai Ino I. Effects of algal diets on growth and biochemical composition

Summary

This study was conducted to examine the nutritional value of eight algal diets for two species of abalone, Haliotis tuberculata and Haliotis discus hannai, by measuring biochemical composition of the algae and relating this to feeding rate, growth and biochemical composition of the animals. Nutritional value of algal diets can be divided into three categories for each species of abalone. For H. tuberculata the best performance was on the mixed diet and Palmaria palmata intermediate was Alaria esculenta, Ulva lactuca and Laminaria digitata, and lowest growth was on Laminaria saccharina and Chondrus crispus. For H. discus hannai, best performance was on A. esculenta, P. palmata and the mixed diet; intermediate was on L. saccharina and L. digitata and lowest was on U. lactuca. It is generally accepted that high “balanced” levels of protein (>15%), lipid (3–5%) and carbohydrate (20–30%), with no detrimental substances in natural algae are essential for optimal growth performance of these abalone. The fact that A. esculenta, L. saccharina and U. lactuca had different dietary values for the two abalone species indicates specific nutritive requirements and/or digestive physiology. Overall, H. tuberculata grew faster, had higher food conversion efficiencies and muscle yield than H. discus hannai. Generally abalone fed on the highest category diets, had higher muscle yields and levels of protein, visceral lipids and muscle carbohydrate. Viscera and foot muscle are reservoirs for lipid and carbohydrate, respectively. The effect of algal diet on sexual maturation is similar to that on somatic growth.     

Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

Identification of a female spawn‐associated Kazal‐type inhibitor from the tropical abalone Haliotis asinina

Abalone (Haliotis) undergoes a period of reproductive maturation, followed by the synchronous release of gametes, called broadcast spawning. Field and laboratory studies have shown that the tropical species Haliotis asinina undergoes a two‐week spawning cycle, thus providing an excellent opportunity to investigate the presence of endogenous spawning‐associated peptides. In female H. asinina, we have isolated a peptide (5145 Da) whose relative abundance in hemolymph increases substantially just prior to spawning and is still detected using reverse‐phase high‐performance liquid chromatography chromatograms up to 1‐day post‐spawn. We have isolated this peptide from female hemolymph as well as samples prepared from the gravid female gonad, and demonstrated through comparative sequence analysis that it contains features characteristic of Kazal‐type proteinase inhibitors (KPIs). Has‐KPI is expressed specifically within the gonad of adult females. A recombinant Has‐KPI was generated using a yeast expression system. The recombinant Has‐KPI does not induce premature spawning of female H. asinina when administered intramuscularly. However it displays homomeric aggregations and interaction with at least one mollusc‐type neuropeptide (LRDFVamide), suggesting a role for it in regulating neuropeptide endocrine communication. This research provides new understanding of a peptide that can regulate reproductive processes in female abalone, which has the potential to lead to the development of greater control over abalone spawning. The findings also highlight the need to further explore abalone reproduction to clearly define a role for novel spawning‐associated peptide in sexual maturation and spawning. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Segregation of Microsatellite Alleles in Gynogenetic Diploid Pacific Abalone (Haliotis discus hannai)

Inheritance of 9 microsatellite loci was examined in 3 families of gynogenetic Pacific abalone Haliotis discus hannai produced by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division. The proportion of heterozygous progeny was used to estimate marker-centromere (M-C) distances. All loci conformed to Mendelian segregation in the control crosses when null alleles were accounted for. The absence of paternal alleles confirmed the gynogenetic origin of the offspring and indicated 100% success for 3 families. Estimated recombinant frequencies ranged from 0.10 to 0.60, which is lower than those observed in other gynogenetic diploid animals. The mean recombination frequency was 0.22, corresponding to a fixation index of 0.78 in one generation. This is 3.12 times the increase in homozygosity expected after one generation of sib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapid inbreeding in the abalone. M-C map distances for the 9 loci varied between 5 and 30 cM under the assumption of complete interference. The information about M-C distances will be useful for future gene mapping in H. discus hannai.     

Using molecular pedigree reconstruction to evaluate the long-term survival of outplanted hatchery-reared larval and juvenile northern abalone (<Emphasis Type="Italic">Haliotis kamtschatkana</Emphasis>)

The restoration of abalone (Haliotis spp.) populations through supplementation with the offspring of hatchery-spawned wild parents has been attempted in several species, with variable results. Between 2002 and 2005, the Bamfield Huu-ay-aht Community Abalone Project released 4.5 million larvae and 152,000 juveniles of the northern abalone (Haliotis kamtschatkana) into Barkley Sound, BC. The purpose of this study was to estimate the long-term survival of outplanted abalone 3–7 years after their release and thus determine their contribution to local population densities at three different outplanting sites. We identified outplanted abalone by genotyping epipodal tentacles sampled from wild-caught abalone for seven microsatellite loci. We then used three different pedigree reconstruction programs: one that used genotypes from hatchery-reared siblings (pedigree 2.2), one that used the wild parent genotypes that were available (cervus 3.0), and one that used both sources of information (colony 2.0). Each program identified different but partially overlapping subsets of hatchery-outplanted offspring. From this we inferred that up to 26% of the individuals sampled at the main outplanting site were from hatchery spawnings. Despite this large contribution of hatchery-reared stock, the density of mature abalone at each site was below the level required for successful fertilization. More intensive outplanting efforts might increase population densities of this broadcast spawner above this minimum sustainable level. However, for supplementation to be successful, other factors that could reduce outplanted juvenile survival, including the low genetic diversity of hatchery-produced offspring and the low habitat quality of some outplanting sites, need to be monitored.     

<Emphasis Type="Italic">Polydora uncinata</Emphasis> (Polychaeta: Spionidae) in Chile: an accidental transportation across the Pacific

A Polydora species was found boring in shells of the abalone Haliotis discus hannai cultivated in land-based tanks in Coquimbo, Chile. Spionid polychaetes of Polydora and related genera have been reported from Chile but no worms similar to those found in abalone have been described. The abalone pest corresponds in morphology to Polydora uncinata Sato-Okoshi, 1998, a shell-boring species which was originally described from Japan and never reported from outside the country. It is suggested that occurrence of the species in Chile resulted from its accidental transportation from Japan. Adult worms were most likely transported to Coquimbo with imported abalone brood stock. Prevalence of abalone infestation by worms in Coquimbo varied substantially among cultivation tanks, reaching values as high as 98.8%. Up to 42 worms were found in one shell. The worms often caused formation of nacreous blisters which covered up to 50% of the inner shell surface. Egg capsules with developing larvae were present in female burrows. Larval development was entirely lecithotrophic, with larvae feeding on numerous nurse eggs, staying inside egg capsules until 16–17-segment stage and hatching shortly before metamorphosis. Polydora uncinata is redescribed based on individuals from Coquimbo to alert zoologists in case of accidental release of worms into Chilean coastal waters. Regardless of how the species was transported to Chile, its release to the natural ecosystem may have negative unforeseen impacts on the native fauna.     

Isolation and characterization of eleven microsatellite loci in small abalone, Haliotis diversicolor Reeve

Eleven novel microsatellite markers were isolated from small abalone, Haliotis diversicolor Reeve. These loci were tested on 22 individuals from two different geographic populations. We identified a total of 162 alleles from the 11 microsatellite loci. All of the loci were highly polymorphic. Polymorphism information content (PIC) is ranging from 0.7276 to 0.9163. Observed and expected heterozygosities ranged from 0.2727 to 1.0000 and from 0.7738 to 0.9429, respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium. No pairs of loci displayed linkage disequilibrium. These polymorphic markers will be used to analyze population structure, genetic diversity and construct a genetic linkage map. Xin Zhan and Hai-Yan Hu contribute equally to this study.     

Construction of an Integrated Map of Haliotis diversicolor Using Microsatellite Markers

Small abalone, Haliotis diversicolor, is naturally distributed along the coastal waters of East Asia from Japan to the Philippines. It is an economically important maricultured species in southern China and Taiwan. Genetic linkage maps for small abalone were constructed using a total of 308 simple sequence repeat markers including 297 novel markers. Segregation data on 96 progeny were genotyped using a pseudo-testcross strategy. Sixteen linkage groups were identified in both female and male maps, consistent with the haploid chromosome number. The female linkage map covered 758.3 cM, with an average interval of 5.2 cM. The male linkage map spanned a total genetic distance of 676.2 cM, with an average interval of 4.5 cM. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in 16 linkage groups with a total of 762.1 cM. Genome coverage of the integrated linkage map was approximately 80.7%. The genetic linkage maps of small abalone may facilitate marker-assisted selection and quantitative trait loci mapping.