Frederico M. Batista

Identification of molecular and physiological responses to chronic environmental challenge in an invasive species: the Pacific oyster, Crassostrea gigas

Understanding the environmental responses of an invasive species is critical in predicting how ecosystem composition may be transformed in the future, especially under climate change. In this study, Crassostrea gigas, a species well adapted to the highly variable intertidal environment, was exposed to the chronic environmental challenges of temperature (19 and 24°C) and pH (ambient seawater and a reduction of 0.4 pH units) in an extended 3-month laboratory-based study. Physiological parameters were measured (condition index, shell growth, respiration, excretion rates, O:N ratios, and ability to repair shell damage) alongside molecular analyses. Temperature was by far the most important stressor, as demonstrated by reduced condition indexes and shell growth at 24°C, with relatively little effect detected for pH. Transcriptional profiling using candidate genes and SOLiD sequencing of mantle tissue revealed that classical “stress” genes, previously reported to be upregulated under acute temperature challenges, were not significantly expressed in any of the treatments, emphasizing the different response between acute and longer term chronic stress. The transcriptional profiling also elaborated on the cellular responses underpinning the physiological results, including the identification of the PI3K/AKT/mTOR pathway as a potentially novel marker for chronic environmental challenge. This study represents a first attempt to understand the energetic consequences of cumulative thermal stress on the intertidal C. gigas which could significantly impact on coastal ecosystem biodiversity and function in the future.

Effect of dietary supplementation of vitamins C and E on maternal performance and larval quality of the prawn Macrobrachium rosenbergii

Abstract The effects of vitamin C (ascorbic acid, AA) and vitamin E (tocopherol) on the maternal performance and offspring quality of the freshwater prawn Macrobrachium rosenbergii were investigated. Prawn females were fed four diets containing different levels of 2-ascorbyl- l -polyphosphate (ApP) and α-tocopherol acetate (α-TA) during 155 days. Three diets contained increasing AA levels (59, 121 and 918 μg g −1 DW) and a basal level of α-TA (around 300 μg g −1 DW), while a fourth diet contained comparatively higher α-TA levels (899 μg g −1 DW) combined with 957 μg AA g −1 DW. Higher dietary levels of AA and α-TA did not affect moulting, growth, or mortality rates of the broodstock. Also, breeding frequency and fecundity were not related to the dietary treatments. However, the contents of AA and α-tocopherol in the midgut gland, ovary, eggs, and newly hatched larvae increased along with higher dietary levels of these vitamins. Larvae from females fed higher levels of AA and α-TA tended to present an increased tolerance when exposed to increasing ammonia concentrations. The present results therefore suggest that broodstock diets containing around 60 μg AA g −1 DW and 300 μg α-TA g −1 DW are sufficient to ensure proper reproduction and offspring viability. However, feeding M. rosenbergii females higher dietary levels of both AA and α-TA (each around 900 μg g −1 DW) might increase larval quality, as demonstrated in this study by the higher tolerance to the exposure to ammonia.

Acidification effects on biofouling communities: winners and losers

Abstract How ocean acidification affects marine life is a major concern for science and society. However, its impacts on encrusting biofouling communities, that are both the initial colonizers of hard substrata and of great economic importance, are almost unknown. We showed that community composition changed significantly, from 92% spirorbids, 3% ascidians and 4% sponges initially to 47% spirorbids, 23% ascidians and 29% sponges after 100 days in acidified conditions (pH 7.7). In low pH, numbers of the spirorbid Neodexiospira pseudocorrugata were reduced ×5 compared to controls. The two ascidians present behaved differently with Aplidium sp. decreasing ×10 in pH 7.7, whereas Molgula sp. numbers were ×4 higher in low pH than controls. Calcareous sponge (Leucosolenia sp.) numbers increased ×2.5 in pH 7.7 over controls. The diatom and filamentous algal community was also more poorly developed in the low pH treatments compared to controls. Colonization of new surfaces likewise showed large decreases in spirorbid numbers, but numbers of sponges and Molgula sp. increased. Spirorbid losses appeared due to both recruitment failure and loss of existing tubes. Spirorbid tubes are comprised of a loose prismatic fabric of calcite crystals. Loss of tube materials appeared due to changes in the binding matrix and not crystal dissolution, as SEM analyses showed crystal surfaces were not pitted or dissolved in low pH conditions. Biofouling communities face dramatic future changes with reductions in groups with hard exposed exoskeletons and domination by soft‐bodied ascidians and sponges.

Perkinsosis in the clams Ruditapes decussatus and R. philippinarum in the Northeastern Atlantic and Mediterranean Sea: A review

Perkinsosis is a disease of gastropod and bivalve molluscs caused by protozoan parasites of the Perkinsus genus. These parasites have been responsible for mass mortalities worldwide, with a significant impact in both fisheries and aquaculture, and resulting in severe economic losses. This review focuses mainly on current knowledge of diagnostic methods and on the distribution of Perkinsus spp. in the Northeastern Atlantic and Mediterranean Sea, which infect the grooved carpet shell clam Ruditapes decussatus and the Japanese carpet shell clam Ruditapes philippinarum. The association between perkinsosis and high mortality rates of R. decussatus and R. philippinarum in southern European countries such as Portugal and Italy is discussed as is the role of environmental factors in those mortality outbreaks. The putative introduction of Perkinsus olseni into the Northeastern Atlantic and Mediterranean Sea is also discussed, as are management strategies that could be used to mitigate the impact of perkinsosis in production of R. decussatus and R. philippinarum.

Evidence of vertical transmission of ostreid herpesvirus 1 in the Portuguese oyster Crassostrea angulata

In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.

Cytogenetic Characterization of the Dwarf Oyster Ostrea stentina (Mollusca: Bivalvia) and Comparative Karyological Analysis within Ostreinae

ABSTRACT Regardless of the high economic value and large geographical distribution of oysters, the current knowledge of oyster taxonomic relationships and systematics is still limited, particularly for flat oysters. In this study, the molecular cytogenetic characterization of mitotic chromosomes of the Provence flat oyster or dwarf oyster, Ostrea stentina, was performed through Giemsa staining, chromosome measurements, in situ restriction endonuclease banding, C-banding, fluorescence in situ hybridization with major ribosomal RNA genes (ITS1), and telomeric sequence (TTAGGG)n. The karyotype (2n = 20) consisted of 6 metacentric (1, 3, 4, 6, 8, and 10) and 4 submetacentric (2, 5, 7, and 9) chromosome pairs. Chromosome treatment with HaeIII produced specific banding patterns for all chromosomal pairs, confirming the efficiency of this restriction enzyme for chromosome banding in oysters. Results for C-banding revealed the presence of heterochromatin in the telomeric regions of the short arms: on a large metacentric chromosomal pair and on a submetacentric chromosomal pair. In situ hybridization with telomeric sequence revealed bright hybridization signals in the telomeres of all chromosomes. The location of the major ribosomal rDNA (ITS1) displayed the presence of 2 signals: in the telomeric regions of the short arms of the largest metacentric chromosome and in a submetacentric chromosome. The cytogenetic data obtained was used to perform a comparative karyological analysis within the subfamily Ostreinae. It is also important to highlight that this type of work can provide new insights on major genomic changes at the chromosome level in the flat oysters.

Occurrence and characterization of pearls from oysters of the genus Crassostrea

The occurrence of pearls in the ‘true’ oysters, the Ostreioidea, is poorly documented despite being the most produced mollusc species in the world. Oysters of the Crassostrea genus were collected in two different sites in southern Portugal where both Crassostrea angulata and C. gigas are present, namely in: (1) the Ria Formosa lagoon where pearls were not observed (N = 446); and (2) the Guadiana estuary where pearls were found in 12 out of the 798 oysters analysed. The pearls were located mainly at the edge of the right mantle lobe in the inhalant chamber and their maximum length ranged from 0.9 to 5.5 mm. Almost all the pearls had a white-cream colouration with the exception of two pearls that had a black-brown colour. X-ray diffraction analysis of one pearl showed that it was entirely calcitic with no traces of either aragonite or vaterite. The pearls observed were therefore non-nacreous pearls. Scanning electron microscopy (SEM) revealed a diversity of microstructures including prismatic, foliae-like sheets and blocky textures, i.e. highly reminiscent of the host oyster shell microstructures. Parasites (e.g. parasitic copepods, Haplosporidium-like plasmodia) and signs of diseases (e.g. foot disease) were observed in some of the oysters analysed, but they were not associated with the occurrence of pearls. The present work is one of the few studies on the occurrence of natural pearls in ‘true’ oysters and to our knowledge the first description of their microstructure by SEM.

Detection of Marteilia refringens infecting the European flat oyster Ostrea edulis and the dwarf oyster Ostrea stentina in southern Portugal and Spain.

In the present study, Marteilia sp. was detected by histological examination and in situ hybridisation in Ostrea edulis and Ostrea stentina collected in southern Iberian Peninsula. Marteilia refringens DNA was detected by PCR in O. edulis (collected in southern Portugal) and O. stentina (collected in southern Spain and Portugal). Sequencing analysis revealed the presence of M. refringens type O in O. edulis, and type O and M in O. stentina. This is the first confirmed detection of M. refringens in Portugal and the first report on the occurrence of M. refringens infecting O. stentina in Europe.