Domitília Matias

Spawning of Hexaplex (Trunculariopsis) trunculus (Gastropoda: Muricidae) in the laboratory: description of spawning behaviour, egg masses, embryonic development, hatchling and juvenile growth rates

Summary Some authors have studied and described different aspects of the reproductive cycle of Hexaplex (Trunculariopsis) trunculus, but most data are quite ancient and fragmented, lacking information in important respects on the reproductive cycle of this species. Based on several individual and collective spawns deposited in laboratory aquaria, this study provides additional and more detailed information on the spawning behaviour and egg-laying pattern, description of the general morphology and dimensions of the spawns, egg capsules, eggs, embryos and early post-metamorphic juveniles, as well as the first data available on the growth rate of T. trunculus hatchlings and juveniles (until 4 months old). Females deposited an average of 118 ± 89 tongue-shaped egg capsules per individual spawn, measuring on average 5.5 mm length x 4.7 mm width x 2.6 mm thickness. These egg capsules contained 723 ± 66 eggs with an average diameter of 240 ± 8 μm. T. trunculus is a direct developer species (metamorphosed hatchlings) with an incubation period of approximately 1 month. At hatching, individuals measured 1.64 ± 0.22 mm shell length and presented a growth rate of 2.5 mm/month at the end of 4 months. The breeding habits, embryonic development, hatchling and juvenile growth rates are discussed in terms of their implications for the management of the artisanal fishery for T. trunculus in the Ria Formosa lagoon and the assessment of the potential of this muricid species for molluscan aquaculture.

A Microarray-Based Analysis of Gametogenesis in Two Portuguese Populations of the European Clam Ruditapes decussatus

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Insights into Molecular Features of Venerupis decussata Oocytes: A Microarray-Based Study

The production of Venerupis decussata relies on wild seed collection, which has been recently compromised due to recruitment failure and severe mortalities. To address this issue and provide an alternative source of seed, artificial spawning and larval rearing programs were developed. However, hatchery-based seed production is a relatively new industry and it is still underdeveloped. A major hurdle in the European clam seed production is the control of spawning and reproduction, which is further hindered by the impossibility of obtaining fertile gametes by gonadal “stripping”, as meiosis re-initiation is constrained to a maturation process along the genital ducts. In the present study, oocytes were collected from 15 females and microarray analyses was performed to investigate gene expression profiles characterizing released and stripped ovarian oocytes. A total of 198 differentially expressed transcripts between stripped and spawned oocytes were detected. Functional analysis carried out on these transcripts highlighted the importance of a few biological processes, which are most probably implicated in the control of oocyte competence. Significant differences were observed for transcripts encoding proteins involved in meiosis progression (e.g. dual specificity phosphatase CDC25), WNT signalling (e.g. frizzled class receptor 8, wingless-type MMTV integration site family member 4), steroid synthesis (e.g. progestin and adipoQ receptor family member 3, cytochrome P450-C17), mRNA processing (e.g. zinc finger protein XlCOF28), calcium regulation (e.g. regucalcin, calmodulin) and ceramide metabolism (ceramidase B, sphingomyelinase). This study provides new information on transcriptional profiles putatively associated with ovarian egg infertility, and suggests potential mechanisms regulating early oocyte development in clams. Genes which were differentially expressed between stripped and spawned oocytes might have a pivotal role during maturation process in the gonadal duct and could be interesting targets for further functional studies aiming to make ovarian oocytes fertilizable.

Genetic diversity of two Portuguese populations of the pullet carpet shell Venerupis senegalensis, based on RAPD markers: contribution to a sustainable restocking program

The pullet carpet shell Venerupis senegalensis (=V. pullastra) is a commercially important species in Portugal, Spain, France, and Italy. In Portugal, this species was once abundant in the Ria Formosa (southern Portugal). However, in the early 1980s, its abundance declined dramatically due to overfishing. In order to reverse this negative trend, the genetic sustainable management of the wild stocks of V. senegalensis should be performed by promoting successful restocking actions and the development of an aquaculture commercial production program of this species. In order to find the best broodstock for aquaculture purposes and therefore minimize the deleterious effects of hatchery practices, we analyzed the genetic diversity of the natural population to be restocked (Ria Formosa) but also of another potential genetically close population (Ria de Aveiro) by RAPD. Similar and substantive percentage of polymorphic loci, effective number of alleles, Nei’s gene diversity, and Shannon’s diversity index was found within both populations. This high genetic variability within populations suggests that they might have a gene pool with sufficient genetic plasticity to support changes in the environmental conditions. Analyses of population genetic structure also revealed a small genetic differentiation between the two populations. The high genetic variability of the natural population to be restocked makes it the preferential broodstock for aquaculture purposes. However, the Ria de Aveiro population could also be a viable alternative, due to its genetic plasticity and the genetic similarity of both populations. The results of this study can be useful to the sustainable management of wild stocks as well as in promoting successful restocking actions based on aquaculture production.

RESTRICTION ENZYME DIGESTION CHROMOSOME BANDING ON TWO COMMERCIALLY IMPORTANT VENERID BIVALVE SPECIES: RUDITAPES DECUSSATUS AND CERASTODERMA EDULE

Abstract Reliable banding techniques are a major necessity for the genetic research in marine bivalves. Restriction enzyme banding (HaeIII) was performed, in this study, on chromosomes of two commercially important species of veneroid bivalves: the clam Ruditapes decussatus (Adams and Reeve) and the cockle Cerastoderma edule. Identification of the nineteen individual chromosome pairs was obtained for both species. The cytogenetic studies made in marine molluscs have recently experienced a very fast development caused by the introduction of new molecular techniques mainly fluorescence in situ hybridization (FISH). Recently it has been shown in mammalian chromosomes that restriction enzyme banding is compatible with FISH, allowing simultaneous banding, and consequent accurate identification of the localization of the probes and unambiguously identification of the chromosome(s) carrier(s). As far as we know this is the first RE-banding obtained in karyotypes of veneroid species. The application of restriction enzyme chromosome banding in veneroids are diverse and this study can constitute a fundamental step for future gene mapping on this commercially important group of bivalves and could offer a new approach to specific problems in veneroid taxonomy and genetics.

First study in cryopreserved Crassostrea angulata sperm

Sperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition.

Changes of paralytic shellfish toxins in gills and digestive glands of the cockle Cerastoderma edule under post-bloom natural conditions.

Concentrations of the paralytic shellfish toxins C1+2, C3+4, GTX5, GTX6, dcGTX2+3, dcSTX, dcNEO, GTX2+3, GTX1+4, STX and NEO were determined by LC-FLD in composite samples of digestive glands and gills of Cerastoderma edule cockle. The specimens were sampled in Aveiro lagoon, Portugal, under natural depuration conditions (days 0, 8, 12, 14, 19, 21 and 25) after exposure to a bloom of Gymnodinium catenatum. Individual paralytic shellfish toxins indicated different pathways of elimination and biotransformation in digestive gland and gills. Toxin concentrations in gills were lower than in digestive gland. Most of the quantified toxins in digestive gland decreased during the 25 days of observation according to negative exponential curves, and only GTX5, GTX6 and NEO showed slight irregularities with time. Concentrations of C1+2, C3+4 and dcGTX2+3 in gills decreased progressively, however GTX5, GTX6 and dcSTX showed pronounced increases. Higher concentrations of those toxins in days 8 and 12 in comparison to the initial value (day 0) indicate conversion of other toxins into GTX5, GTX6 and dcSTX during those periods. It appears that inter-conversion of toxins occurs as G. catenatum cells are retained in gills before being transferred to other compartments.

Larval hatching and development of the wedge shell ( Donax trunculus L.) under increased CO 2 in southern Portugal

Noticeable changes in global temperatures, climate and ocean carbon chemistry are the result of carbon dioxide increase in the atmosphere. This increase has been mitigated by the oceans capacity to absorb one-fourth of the carbon dioxide in the atmosphere, although this CO2 intake affects oceans carbonate chemistry [i.e., ocean acidification—(OA)]. The detrimental effect of OA in the development and shell formation has been studied in several species of bivalves, although no information is available on the wedge shell Donaxtrunculus, a gastronomically appreciated species and an important economical resource in several southern European countries. We evaluated the effect of pCO2 increase on hatching and early life stages of D.trunculus, considering two ocean acidification scenarios (ΔpH −0.3 and ΔpH −0.6). Our results showed that elevated pCO2 caused a delay in hatching into D-larvae and reduced larvae survival. In the extreme scenario (ΔpH −0.6), some trochophore larvae persisted to day 9 of the experiment and more abnormal larvae were produced than in the ΔpH −0.3 and control treatments. At day 5, normal veligers under extreme acidification were smaller than in other treatments, but by day 9, these differences were attenuated and the average size of normal D-larvae varied inversely to the pH gradient. Possible underlying mechanisms for these complex response patterns are discussed, including the existence of phenotypic plasticity or genetic pre-adaptive capacity in this D.trunculus population to cope with future environmental changes.

Reproductive effort of the European clam Ruditapes decussatus (Linnaeus, 1758): influence of different diets and temperatures

Abstract Ruditapes decussatus is a species of importance to aquaculture. For hatcheries to consistently produce spat it is essential to develop broodstock conditioning techniques. Food and temperature are the main factors that regulate the timing and rate of energy storage and reproduction in bivalves. This study was designed to evaluate the effects of different diets and temperatures on reproductive output of R. decussatus and express the evolution of the different lipid classes during sexual maturation. Broodstock clams were conditioned at 20 ± 1 °C under four nutritional regimes: unfed, two mono-specific diets, Isochrysis galbana clone T-ISO and Chaetoceros calcitrans and, a mixture of these microalgae. Another group of clams was conditioned at 22 ± 1 °C and was fed the same mixture of microalgae. Gametogenesis, energy storage and spawning success were all influenced by the nutritional value of the diet received, as evidenced by the differences in reproductive effort among the single and combined supplemental diets. Temperature must be carefully managed to improve the reproductive conditioning of bivalves: high temperature throughout gametogenesis shortens the time to full ripeness but does not produce better reproductive output. The combination diet at 20 ± 1 °C is best for R. decussatus broodstock conditioning.

Relationships between broodstock condition, oocyte quality, and 24 h D-larval survival during the spawning season of the pullet carpet shell Venerupis corrugata (Gmelin, 1791)

Abstract Venerupis corrugata is commercially exploited in Europe. Over-fishing and recruitment failure is causing the decline of its populations and stock sustainability. Knowledge of this species reproduction is paramount to establish hatchery production of juveniles for restoring natural beds. This work aimed to find a relationship between broodstock condition, oocyte quality, and viability of 24 h D-larvae. Adult specimens were induced to spawn by thermal stimulation. From each female, oocytes were taken for biochemical analyses (proteins, total lipids, and carbohydrates), and the remaining oocytes were fertilized. The 24 h D-larval yield was calculated after embryo incubation. Spawning in the hatchery with ‘wild’ broodstock was possible for a long period, however, subsequent larval viability varied according to oocyte quality. Two distinct periods of spawning were recorded: in January/March, with a higher number of oocytes released, and in June/July with a lower response to the spawning stimulation, however with greater success in 24 h D-larval survival. The condition index of broodstock and the total lipids of oocytes released can be used as benchmarks for estimating the success of D veliger larvae.