Intertidal Snail Research Articles

Access to multiple mates increases fecundity but does not affect per-offspring maternal investment in a marine gastropod

Females of many organisms mate more than once and with more than one male, suggesting that polyandry confers some advantage to the female or her offspring. However, variation in maternal investment in response to mate choice and mate number can confound efforts to determine if there are benefits of polyandry. Access to multiple mates could increase maternal investment in offspring via a number of different mechanisms. Few studies have determined if investment is influenced by mate choice and number, and data are particularly lacking for marine invertebrates. This study was designed to determine if maternal investment and offspring size increase with access to increasing numbers of mates in the protandrous intertidal slipper snail Crepidula cf. marginalis. Virgin female slipper limpets were exposed to one, three, or five potential mates and their fecundity, egg size, and hatchling size were measured for multiple clutches. Treatment had a significant effect on fecundity, with fecundity increasing with the number of potential mates. Treatment did not have an effect on the size of eggs or hatchlings, on the variation in egg size or hatchling size within broods, or on the frequency of oviposition. Treatment did alter the variation in average offspring size among females, but not in the way predicted by theory. The main result, that access to multiple mates does not have an effect on per offspring maternal investment, makes C. cf. marginalis an ideal candidate to study the effects of polyandry on offspring fitness without having to take into account confounding effects of variation in maternal investment.

Taken to the limit — Is desiccation stress causing precocious encystment of trematode parasites in snails?

When hosts experience environmental stress, the quantity and quality of resources they provide for parasites may be diminished, and host longevity may be decreased. Under stress, parasites may adopt alternative strategies to avoid fitness reductions. Trematode parasites typically have complex life cycles, involving asexual reproduction in a gastropod first intermediate host. A rare phenomenon, briefly mentioned in the literature, and termed ‘precocious encystment’ involves the next stage in the parasites' life cycle (metacercarial cyst) forming within the preceding stage (redia), while still inside the snail. In the trematode Parorchis sp. NZ using rocky shore snails exposed to long periods outside water, we hypothesised that this might be an adaptive strategy against desiccation, preventing parasite emergence from the snail. To test this, we first investigated the effect of prolonged desiccation on the survival of two species of high intertidal snails. Secondly, we measured the reproductive output (cercarial production) of the parasite under wet and dry conditions. Finally, we quantified the influence of desiccation stress on the occurrence of precocious encystment. Snail mortality was higher under dry conditions, indicating stress, and it was somewhat exacerbated for infected snails. Parasite reproductive output differed between wet and dry conditions, with parasites of snails kept in dry conditions producing more cercariae when placed in water. Little variation was observed in the occurrence of precocious encystment, although some subtle patterns emerged. Given the stresses associated with living in high intertidal environments, we discuss precocious encystment as a possible stress response in this trematode parasite.

Patterns of parasite transmission in polar seas: Daily rhythms of cercarial emergence from intertidal snails

Trematodes are common parasites in intertidal ecosystems. Cercariae, their dispersive larvae, ensure transmission of infection from the first intermediate molluscan host to the second intermediate (invertebrates and fishes) or the final (fishes, marine birds and mammals) host. Trematode transmission in polar seas, while interesting in many respects, is poorly studied. This study aimed to elucidate the patterns of cercarial emergence from intertidal snails at the White Sea and Barents Sea. The study, involving cercariae of 12 species, has provided the most extensive material obtained so far in high latitude seas (66–69° N). The experiments were conducted in situ. Multichannel singular spectral analysis (MSSA) used for processing primary data made it possible to estimate the relative contribution of different oscillations into the analysed time series and to separate the daily component from the other oscillatory components and the noise. Cercarial emergence had pronounced daily rhythms, which did not depend on the daily tidal schedule but were regulated by thermo- and photoperiod. Daily emergence maximums coincided with periods favourable for infecting the second intermediate hosts. Cercarial daily emergence rhythms differed in species using the same molluscan hosts which can be explained by cercarial host searching behaviour. Daily cercarial output (DCO) correlated negatively with larval volume and positively with that of the molluscan host except in cercariae using ambuscade behaviour. In the Barents Sea cercariae emerged from their molluscan hosts at lower temperatures than in the warmer White Sea but the daily emergence period was prolonged. Thus, DCO of related species were similar in these two seas and comparable with DCO values reported for boreal seas. Local temperature adaptations in cercarial emergence suggests that in case of Arctic climate warming trematode transmission in coastal ecosystems is likely to be intensified not because of the increased summer temperature but because of the prolongation of the warm season favouring cercarial emergence (transmission window).

Opposing selective pressures decouple pattern and process of parasitic infection over small spatial scale

Species face multiple selective pressures that may require opposing responses to mitigate. On rocky shorelines, fitness of the intertidal snail Littorina littorea is determined by both parasitism and predation. We experimentally demonstrated that L. littorea was at greatest risk of infection from trematode parasites high in the intertidal zone where it was in closest proximity to abundant gull feces (the vector for the snail's parasites). However, because of extreme, size‐selective predation pressure at low tidal elevations, small snails often live high in the intertidal until they have grown sufficiently large. By prolonging their exposure to infection higher on the shore, ontogenetic responses to predation risk accentuate parasite risk. Counterintuitively, snails exhibited the highest trematode prevalence at the lowest tidal elevations where they had almost no risk of contracting infection. By carrying contracted infections into the lowest tidal zones, the larger, predation‐resistant snails invert hotspots of infection risk and prevalence, underscoring that size‐dependent selection pressures can decouple infection process and pattern even over small scales.

RNA-seq reveals regional differences in transcriptome response to heat stress in the marine snail Chlorostoma funebralis.

To investigate the role of gene expression in adaptation of marine ectotherms to different temperatures, we examined the transcriptome-wide thermal stress response in geographically separated populations of the intertidal snail Chlorostoma funebralis. Snails from two southern (heat tolerant) and two northern (heat sensitive) populations were acclimated to a common thermal environment, exposed to an environmentally relevant thermal stress and analysed using RNA-seq. Pooling across all populations revealed 306 genes with differential expression between control and heat-stressed samples, including 163 significantly upregulated and 143 significantly downregulated genes. When considered separately, regional differences in response were widely apparent. Heat shock proteins (Hsps) were upregulated in both regions, but the magnitude of response was significantly greater in northern populations for most Hsp70s, while the southern populations showed greater upregulation for approximately half of the Hsp40s. Of 177 stress-responsive genes in northern populations, 55 responded to heat stress only in northern populations. Several molecular chaperones and antioxidant genes that were not differentially expressed in southern populations showed higher expression under control conditions compared with northern populations. This suggests that evolution of elevated expression of these genes under benign conditions preadapts the southern populations to frequent heat stress and contributes to their higher thermal tolerance. These results indicate that evolution has resulted in different transcriptome responses across populations, including upregulation of genes in response to stress and preadaptation of genes in anticipation of stress (based on evolutionary history of frequent heat exposure). The relative importance of the two mechanisms differs among gene families and among populations.

Feeding behaviour of an intertidal snail: Does past environmental stress affect predator choices and prey vulnerability?

Predation is one of the most important factors in determining structure and dynamics of communities on intertidal rocky shores. Such regulatory role may be of special relevance in novel communities resulting from biological invasions. Non-indigenous species frequently escape natural predators that limit their distribution and abundance in the native range. However, biological interactions also can limit the establishment and spread of non-native populations. There is a growing concern that climate change might affect predator–prey interactions exacerbating the ecological impacts of non-indigenous species. However, mechanisms underlying such interactions are poorly understood in marine ecosystems. Here, we explored if past environmental stress, i.e., increasing temperature and decreasing pH, could affect the vulnerability of two mussel prey, the native Mytilus galloprovincialis and the non-indigenous Xenostrobus securis, to predation by the native dogwhelk Nucella lapillus. In addition, we evaluated the consequences on the feeding behaviour of N. lapillus. First, we exposed monospecific assemblages of each mussel species to combined experimental conditions of increasing temperature and decreasing pH in mesocosms for 3weeks. Then assemblages were placed on a rocky shore and were enclosed in cages with dogwhelks where they remained for 3weeks. Despite the lack of preference, consumption was much greater on the native than on the invasive mussels, which barely were consumed by dogwhelks. However, this trend was diverted when temperature increased. Thus, under a coastal warming scenario shifts in dogwhelks feeding behaviour may help to contain invader's populations, especially in estuarine areas where these predators are abundant.

Thermal stress and predation risk trigger distinct transcriptomic responses in the intertidal snail Nucella lapillus.

Thermal stress and predation risk have profound effects on rocky shore organisms, triggering changes in their feeding behaviour, morphology and metabolism. Studies of thermal stress have shown that underpinning such changes in several intertidal species are specific shifts in gene and protein expression (e.g. upregulation of heat-shock proteins). But relatively few studies have examined genetic responses to predation risk. Here, we use next-generation RNA sequencing (RNA-seq) to examine the transcriptomic (mRNA) response of the snail Nucella lapillus to thermal stress and predation risk. We found that like other intertidal species, N.lapillus displays a pronounced genetic response to thermal stress by upregulating many heat-shock proteins and other molecular chaperones. In contrast, the presence of a crab predator (Carcinus maenas) triggered few significant changes in gene expression in our experiment, and this response showed no significant overlap with the snail's response to thermal stress. These different gene expression profiles suggest that thermal stress and predation risk could pose distinct and potentially additive challenges for N.lapillus and that genetic responses to biotic stresses such as predation risk might be more complex and less uniform across species than genetic responses to abiotic stresses such as thermal stress.

The smell of sex: water-borne and air-borne sex pheromones in the intertidal gastropod Littorina littorea

We investigated whether individuals of the intertidal snail Littorina littorea may be attracted by waterborne and air-borne pheromones from conspecifics. The potential relative role of water-borne and air-borne pheromone in the behavioural and reproductive biology of L. littorea was assessed through chemotactic assays. These involved a series of choice experiments with snails, snail smell and snail mucus as stimuli in a T-maze under conditions of immersion and emersion, and during the nonmating and mating seasons. During the nonmating season, L. littorea did not choose the snail-conditioned side of the T-maze significantly more frequently than the control side under conditions of both immersion and emersion. During the mating season, under condition of immersion both males and females chose the side of the T-maze conditioned with individual of the opposite sex significantly more frequently than the control side, and did not show any preference for the side conditioned with individuals of the same sex. Under conditions of emersion, only males were significantly more attracted by cues from the opposite sex than by the control. These results indicate that L. littorea are not attracted by conspecifics during the nonmating season; the cues of conspecifics are then unlikely to play a role in the orientation or the aggregation behaviour of this species. The results also suggest that males and females are attracted by the odour of conspecifics of the opposite sex, which contains both water-borne and air-borne pheromones in females and only water-borne pheromones in males. Also the similar behaviours observed with snails, water-borne and air-borne snail smell, and snail mucus, as stimuli indicate that male and female sex pheromones are both present in mucus. Temperature manipulations conducted during both seasons did not affect any of the observed behavioural changes, hence suggesting that the chemoreceptive abilities of L. littorea may be driven by exogenous and/or endogenous factors that are decoupled from direct control by temperature. This is in sharp contrast to the locomotor activity of L. littorea, which consistently increased with temperature. We also report the possibility of mate-tracking in females, a behaviour that has yet to be reported in gastropods, since males are typically believed to be the active sex that searches for females. Our results build on previous evidence of the function played by mucus-trail following in the reproductive success of intertidal gastropods, through the ability of L. littorea to remotely detect the water-borne and air-borne sex-specific cues that are incorporated in their mucus.

Broad geographic analyses reveal varying patterns of genetic diversity and host specificity among echinostome trematodes in New Zealand snails.

Host specificity is a fundamental component of a parasite's life history. However, accurate assessments of host specificity, and the factors influencing it, can be obscured by parasite cryptic species complexes. We surveyed two congeneric species of intertidal snail intermediate hosts, Zeacumantus subcarinatus and Zeacumantus lutulentus, throughout New Zealand to identify the number of genetically distinct echinostome trematodes infecting them and determine the levels of snail host specificity among echinostomes. Two major echinostome clades were identified: a clade consisting of an unidentified species of the subfamily Himasthlinae and a clade consisting of five species of the genus Acanthoparyphium. All five Acanthoparyphium species were only found in a single snail species, four in Z. subcarinatus and one in Z. lutulentus. In contrast, the Himasthlinae gen. sp. was found in both hosts, but was more prevalent in Z. lutulentus (97 infections) than Z. subcarinatus (10 infections). At least two of the Acanthoparyphium spp. and the Himasthlinae gen. sp. are widespread throughout New Zealand, and can therefore encounter both snail species. Our results suggest that host specificity is determined by host-parasite incompatibilities, not geographic separation, and that it can evolve in different ways in closely related parasite lineages.

A02 Session 1: Translational Hibernation : Stress response and adaptation: A new molecular toolkit for the 21st century

Molecular studies of cold and frozen systems “be they naturally cold-hardy animals, cryopreserved cells/tissues/organs, or tissues ablated by cryosurgery” all have one thing in common. This is a need to identify and quantify biochemical markers of stress, injury and survival. Physical assessment methods (cell rupture, enzyme leakage, etc.) have long been used as have “bulk” protective strategies (e.g. cryoprotectant infusion). However, in-depth knowledge of the key biochemical parameters of cell systems under freezing stress, the negative versus positive responses that can constitute molecular markers of stress versus endurance, and the features that differentiate natural versus man-made freezing survival still leave much to be determined. Assessing complex cell functions has traditionally been difficult and time consuming absorbing many grad student careers for energy-intensive methods like gene screening, PCR, immunoblotting and enzyme assay. However, new technologies have recently emerged that are re-focusing research efforts and allowing huge amounts of data to be gathered from very small cell/tissue samples. Among these, accelerated rates of genome sequencing are giving us access to genomic tools to find inherent differences in genes between species and tools for screening a stress-responsive transcriptome (e.g. RNA seq.; the RT2 profiler array technology) or proteome are becoming easier and cheaper. This is providing ways to identify signatures of cells/organisms under stress. A relatively new analytical tool, multiplex suspension array technology (Luminex), is also radically changing biomedical analytical capabilities for both research and diagnostic purposes by providing a way to quantify panels of multiple analytes simultaneously in single small samples in a very short time. For example, this approach can allow multiple members of integrated signal transduction pathways to be quantified in a single sample. Critical assessments of metabolic status can also be derived from analyzing the patterns of posttranslational modification of macromolecules including the phosphoproteome or the epigenome. Quick ‘snapshots’ of transcriptional and translational control using state-of-the-art methodologies can identify derangements as varied as changes in cell cycle control, the status of apoptosis and autophagy, or microRNA involvement in stress and adaptation. Most of these technologies are now available in kit formats (that can often be customized) to screen for large numbers of targets simultaneously at an ever-decreasing cost to researchers. To illustrate the power of these new methodologies, a variety of examples of their use by my lab in probing the molecular adaptations for low temperature function in hibernating mammals, freeze tolerant frogs, and freeze/anoxia tolerant intertidal snails will be provided. For comparative and cryo-biochemistry the future is bright as we now have the capacity to apply the latest molecular technologies to analyze stress and adaptation.

Ecological Speciation and the Intertidal Snail Littorina saxatilis

In recent decades biologists studying speciation have come to consider that the process does not necessarily require the presence of a geographical barrier. Rather, it now seems to be possible for reproductive barriers to evolve within what was hitherto a single ‘‘species.’’ The intertidal snail Littorina saxatilis has been the focus of a considerable amount of work in this context, and it is now thought of as a good case study of ‘‘ecological speciation.’’ We review some of this work and briefly consider prospects for future developments.

Differential response to ocean acidification in physiological traits of Concholepas concholepas populations

Phenotypic adaptation to environmental fluctuations frequently occurs by preexisting plasticity and its role as a major component of variation in physiological diversity is being widely recognized. Few studies have considered the change in phenotypic flexibility among geographic populations in marine calcifiers to ocean acidification projections, despite the fact that this type of study provides understanding about how the organism may respond to this chemical change in the ocean. We examined the geographic variation in CO2 seawater concentrations in the phenotype and in the reaction norm of physiological traits using a laboratory mesocosm approach with short-term acclimation in two contrasting populations (Antofagasta and Calfuco) of the intertidal snail Concholepas concholepas. Our results show that elevated pCO2 conditions increase standard metabolic rates in both populations of the snail juveniles, likely due to the higher energy cost of homeostasis. Juveniles of C. concholepas in the Calfuco (southern) population showed a lower increment of metabolic rate in high-pCO2 environments concordant with a lesser gene expression of a heat shock protein with respect to the Antofagasta (northern) population. Combined these results indicate a negative effect of ocean acidification on whole-organism functioning of C. concholepas. Finally, the significant Population×pCO2 level interaction in both studied traits indicates that there is variation between populations in response to high-pCO2 conditions.

Phylogenomic analyses reveal latitudinal population structure and polymorphisms in heat stress genes in the North Atlantic snail Nucella lapillus

North Atlantic rocky intertidal species have been shaped by repeated glaciations and strong latitudinal temperature gradients, making them an excellent system to study postglacial phylogeography and thermal tolerance. Population genetics data from northwestern Atlantic species, however, often show patterns inconsistent with the prediction that high dispersal should generate weaker genetic structure among populations. Here, we used next-generation sequencing restriction-associated DNA tags (RAD-seq) and a transcriptome assembled from RNA-seq data to analyse the genetic structure of northwestern Atlantic populations of the low-dispersal intertidal snail Nucella lapillus. Although previous studies in this region have detected almost no genetic structure in N. lapillus, our phylogenomic approach identified a well-supported split between northern and southern clades. By comparing RAD-seq data and our transcriptome assembly, we identified thousands of fixed single-nucleotide polymorphisms (SNPs) between these latitudinal clades that map to protein-coding genes, including genes associated with heat stress tolerance. These fixed SNPs might represent loci under selection for different thermal regimes in the northwestern Atlantic.

How to best smash a snail: the effect of tooth shape on crushing load

Organisms that are durophagous, hard prey consumers, have a diversity of tooth forms. To determine why we see this variation, we tested whether some tooth forms break shells better than others. We measured the force needed with three series of aluminium tooth models, which varied in concavity and the morphology of a stress concentrating cusp, to break a shell. We created functionally identical copies of two intertidal snail shells: the thicker shelled Nucella ostrina and the more ornamented Nucella lamellosa using a three-dimensional printer. In this way, we reduced variation in material properties between test shells, allowing us to test only the interaction of the experimental teeth with the two shell morphologies. We found that for all tooth shapes, thicker shells are harder to break than the thinner shells and that increased ornamentation has no discernible effect. Our results show that for both shell morphologies, domed and flat teeth break shells better than cupped teeth, and teeth with tall or skinny cusps break shells best. While our results indicate that there is an ideal tooth form for shell breaking, we do not see this shape in nature. This suggests a probable trade-off between tooth function and the structural integrity of the tooth.

Size-specific maternal effects in response to predator cues in an intertidal snail

Size-specific maternal effects in response to predator cues in an intertidal snail

Nutritional status affects the capacity of the snail Concholepas concholepas to synthesize Hsp70 when exposed to stressors associated with tidal regimes in the intertidal zone

Synthesis of the heat shock protein Hsp70 is one of the most important physiological mechanisms that intertidal organisms possess to counteract damage to macromolecules caused by stressors associated with the tidal cycle. However, the synthesis and activity of Hsp70 involves an elevated energetic cost. We evaluated the effect of the nutritional status (fed vs. starved for 2 weeks) of juvenile Concholepas concholepas mollusc on their capacity to synthesize Hsp70 during emersion (i.e. low tide) and immersion (i.e. high tide) at high temperatures (24 °C, e.g. summer conditions) and at low temperatures (7 °C, e.g. winter conditions). In addition, we evaluated whether Hsp70 is induced directly upon exposure to stress (emersion) or during recovery (re-immersion). Starvation decreased the content of stored energy substrates of juveniles as well as their ability to synthesize Hsp70 during emersion under thermal stress, especially at high temperatures. Additionally, analysis of environmental factors associated with laboratory simulation of tidal regimes indicated that juveniles in starvation, in contrast to fed juveniles, did not significantly increase their levels of Hsp70 during cold emersion (7 °C) or warm emersion (24 °C) or upon re-immersion. Induction of Hsp70 occurred during exposure to stress (low-tide conditions) and not when juveniles returned to “normal” conditions (high-tide conditions). Thus, the synthesis of Hsp70 for the juveniles of this intertidal snail species was coordinated and adapted to the tidal cycle, and the species responds in a similar way to hot and cold emersion conditions. The observed levels of Hsp70 reflect the ability of the individual to synthesize these proteins, which is dependent on the nutritional status of the individual.

Species and gene divergence in Littorina snails detected by array comparative genomic hybridization.

BackgroundArray comparative genomic hybridization (aCGH) is commonly used to screen different types of genetic variation in humans and model species. Here, we performed aCGH using an oligonucleotide gene-expression array for a non-model species, the intertidal snail Littorina saxatilis. First, we tested what types of genetic variation can be detected by this method using direct re-sequencing and comparison to the Littorina genome draft. Secondly, we performed a genome-wide comparison of four closely related Littorina species: L. fabalis, L. compressa, L. arcana and L. saxatilis and of populations of L. saxatilis found in Spain, Britain and Sweden. Finally, we tested whether we could identify genetic variation underlying “Crab” and “Wave” ecotypes of L. saxatilis.ResultsWe could reliably detect copy number variations, deletions and high sequence divergence (i.e. above 3%), but not single nucleotide polymorphisms. The overall hybridization pattern and number of significantly diverged genes were in close agreement with earlier phylogenetic reconstructions based on single genes. The trichotomy of L. arcana, L. compressa and L. saxatilis could not be resolved and we argue that these divergence events have occurred recently and very close in time. We found evidence for high levels of segmental duplication in the Littorina genome (10% of the transcripts represented on the array and up to 23% of the analyzed genomic fragments); duplicated genes and regions were mostly the same in all analyzed species. Finally, this method discriminated geographically distant populations of L. saxatilis, but we did not detect any significant genome divergence associated with ecotypes of L. saxatilis.ConclusionsThe present study provides new information on the sensitivity and the potential use of oligonucleotide arrays for genotyping of non-model organisms. Applying this method to Littorina species yields insights into genome evolution following the recent species radiation and supports earlier single-gene based phylogenies. Genetic differentiation of L. saxatilis ecotypes was not detected in this study, despite pronounced innate phenotypic differences. The reason may be that these differences are due to single-nucleotide polymorphisms.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-687) contains supplementary material, which is available to authorized users.

Isolation and characterization of microsatellite loci in the intertidal snail Batillaria multiformis (Mollusca Gastropoda)

Intertidal snails of the genus Batillaria are a common group in tidelands throughout Japan. Recently, population sizes of Batillaria multiformis have been reduced due to coastal area reclamation. Using next generation sequencing, we developed 11 microsatellite markers to reveal the genetic diversity and gene flow among populations and combined them into a single multiplex panel. Evaluating polymorphisms in two populations showed that the numbers of alleles per locus ranged from 2 to 17 and 2 to 15, and the expected heterozygosity ranged from 0.274 to 0.936 and 0.455 to 0.934 in Mangokuura estuary and Kasari Bay, respectively.

Sex-specific temperature dependence of foraging and growth of intertidal snails

Predicting the biological impacts of climate change requires an understanding of how temperature alters organismal physiology and behavior. Given differences in reproductive physiology between sexes, increases in global temperature may be experienced differently by the males and females of a species. This study tested for sex-specific effects of increased air temperature on foraging, growth, and survival of an intertidal snail, Nucella ostrina (San Juan Island, Washington, 48–30′44″N, 123–08′43″W). Snails exhibited periodic peaks in foraging. Subjecting snails to elevated low tide air temperatures did not alter the timing or magnitude of this pattern. Despite similar temporal patterns in foraging, females foraged more than males, even when the risk of thermal stress was high. While males and females appear to have a similar body temperature threshold for optimal growth, females were more likely to cross that threshold resulting in a loss of body mass when exposed to daily increases in air temperature. These results suggest that the consequences of a warming climate in the short term may be different for males and females of N. ostrina, but also imply longer-term costs of reduced reproductive output, abundance, and distribution of this ubiquitous intertidal predator. Generally, this study points to the possible significance of sex-specific responses in an increasingly warm world.

Characterization of new EST-linked microsatellites in the rough periwinkle (Littorina saxatilis) and application for parentage analysis

The rough periwinkle, Littorina saxatilis (Olivi, 1792), has become a model of choice for studying processes of local adaptation and ecological speciation (e.g. Johannesson et al., 2010). Interestingly, this ovoviviparous intertidal snail displays extreme female promiscuity, since a female can carry offspring sired by at least 23 males (Panova et al., 2010). Microsatellite-based parentage analysis has proved valuable for the study of multiple paternity in L. saxatilis (Makinen, Panova & Andre, 2007; Panova et al., 2010). However, three of the five markers used in these two previous studies have shown problems such as heterozygote deficiencies, null alleles and variation in allele size not equal to repeat motif (Panova et al., 2008). In many invertebrates, including molluscs in general and L. saxatilis in particular, microsatellite flanking regions contain indels and mutations causing amplification failures (null alleles), as well as cryptic repetitive DNA leading to multiple bands in polymerase chain reaction (PCR) products (McInerney et al., 2011). Such issues may significantly affect the accuracy of parentage assignment by increasing the rate of genotyping errors. To avoid these problems, the present study aimed at developing expressed sequence tag (EST)-linked microsatellites, which are likely to have more conserved flanking regions (Bouck & Vision, 2007). Six new EST-linked microsatellites were developed and combined with two recently-published loci (McInerney et al., 2009), and the performance of this new set of markers was tested for parentage analysis. EST sequences were downloaded from the Littorina Sequence Database (Canback et al., 2012), and microsatellites were identified with Tandem Repeats Finder (Benson, 1999). The 57 best di-, trior tetranucleotide microsatellites, displaying a minimum of eight repeats, a minimum of 75% matches and sufficiently long flanking regions (.40 bp), were loaded in Geneious Pro v. 5.1.7 (Drummond et al., 2010; Biomatters Ltd), and the Primer3 plugin (Rozen & Skaletsky, 2000) was used to design primer pairs with the following parameters: optimal primer length 1⁄4 20 bp (range 1⁄4 18–25 bp), optimal GC content 1⁄4 50% (range 1⁄4 30–80%), optimal Tm 1⁄4 608C (range 1⁄4 55– 618C) with a Tm difference not larger than 28C between the forward and reverse primers from the same pair and product length 1⁄4 100–400 bp. Finally, 36 primer pairs were selected for further PCR amplification tests. A ‘universal tail’ was added to the 50-end of each forward primer to reduce the genotyping cost (Schuelke, 2000). Three distinct ‘universal tails’ were used (tail1, tail2 or tail4), as described by Real, Schmidt & Hughes (2009). A PIG-tail (50-GTTTCTT) was also added to the 50-end of each reverse primer to avoid ‘plus-A’ PCR artefacts (Brownstein et al., 1996). In addition, three microsatellites developed by McInerney et al. (2009), with perfect trinucleotide (Lsax18 and Lsax20) or tetranucleotide repeats (Lsax13), were included in the study. All 39 microsatellites were tested with a population sample of L. saxatilis collected on the western Swedish coast, on the island of Salto (588530N, 118100E). Individuals were stored in ethanol at 2208C. DNA was extracted using the EZNA Mollusc DNA Extraction kit following the manufacturer’s instructions. Amplification tests were performed on eight individuals for all 39 loci. Each EST-linked microsatellite was amplified by PCR in a 10-ml volume, containing 1 RBC reaction buffer (RBC Bioscience), 1.0–2.0 mM MgCl2, 0.1 mM each dNTP, 0.125 mM ‘universal’ primer end-labelled with a WellRED dye (D2, D3 or D4; Sigma-Aldrich), 0.0125 mM tailed forward primer, 0.125 mM reverse primer, 0.025 U ml RBC Taq DNA polymerase (RBC Bioscience) and 1 ml DNA template. The three microsatellites published by McInerney et al. (2009) were amplified following the same protocol as above but with 0.15 mM forward primer end-labelled with a WellRED dye (Sigma-Aldrich) and 0.15 mM reverse primer. PCR amplifications were performed in a Veriti Thermal Cycler (Life Technologies). A touchdown procedure was incorporated in the thermal cycling regime to increase the stringency of the PCRs: 948C for 3 min, Ta1 for 2 min, 728C for 1 min, (948C for 30 s, Ta1 218C for 30 s [218C per cycle until Ta2], 728C for 1 min) 9–12 cycles, (948C for 30 s, Ta2 for 30 s, 728C for 1 min) 22–30 cycles, 728C for 5 min. A total of 35 PCR cycles were run by default, but this was increased to 40 cycles when the peak intensity was too low. Details of the PCR protocol, i.e. MgCl2 concentration, number of PCR cycles and type of ‘universal’ tail, are provided in Table 1 for the successfully amplified loci. Amplification success was checked with electrophoresis on 1% agarose gel in 0.5 tris-borate-EDTA. Finally, PCR products were run on a Beckman Coulter CEQ 8000 capillary sequencer