Assessment of Electrolytes and Metals Profile of the Faro Lake (Capo Peloro Lagoon,Sicily, Italy) and Its Impact on Mytilus galloprovincialis

Faro Lake is a coastal meromictic lagoon with singular characteristics in the Mediterranean (Messina, Sicily – Italy). It is part of the Natural Oriented Reserve of Capo Peloro (38° 15′ 57″ N; 15° 37′ 50″ E). In this area, traditional mollusc farming activity persists, producing ‘autochthonous’ mussels. This study reports of the Mytilus galloprovincialis haemolymph chemical profile and water variables determination of 1 year‐lasted survey (April 2016 – March 2017). The determinations of electrolytes (Na⁺, Cl^–, K⁺, Ca²⁺, Mg²⁺, P inorganic) and heavy metals in both Faro lake water and haemolymph have been carried out. Heavy metals are elements with high density and are quite toxic in low concentrations, such as Aluminum (Al), Arsenic (As), Cobalt (Co), Chrome (Cr), Copper (Cu), Iron (Fe), Magnesium (Mg), Manganese (Mn), Lead (Pb), Tin (Sn), Zinc (Zn). Heavy metals toxicity depends, principally, on bioaccumulation processes. M. galloprovincialis is a good bio‐indicator, ideal for assess levels of environmental pollution thanks to its biological, ecological and physiological characteristics. The results of this study showed a typical fluctuation range in haemolymph and water parameters, related to the water ones; chemical‐physical parameters affected the ions (electrolytes and metals) levels in some period of the year. The study reports the interactions between biotic (Mytilus galloprovincialis) and abiotic (water parameters) components of Faro Lake, and creates reference data for further future study on the same area or on similar ones.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Seasonal Dynamics of Prokaryotic Abundance and Activities in Relation to Environmental Parameters in a Transitional Aquatic Ecosystem (Cape Peloro,Italy)

This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by β-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a 2-year period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6 °C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover, the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effects of temperature and trophic conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.     

Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG)

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy).Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed.One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques.The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.     

Spatial and temporal distribution of virioplankton and bacterioplankton in a Brackish Environment (Lake of Ganzirri,Italy)

Temporal and spatial changes of viral and bacterial abundance were examined in relation to environmental factors and hydrography at five stations between May 2000 and July 2001 in the brackish lake of Ganzirri (Sicily, Italy). Virioplankton abundance ranged from 5.26 × 10⁴ to 7.54 × 10⁸VLP ml^–1 (on average 1.38 × 10⁸particles ml^–1) and was significantly higher at the three eutrophic stations located in the lake of Ganzirri (Stations 1, 2, and 3) than in the channel connecting the lake with the Straits of Messina. The virus-to-bacterium abundance ratio (VBR range, 0.4–117; average:14) showed the highest values in channel connecting the lake of Ganzirri with the meromictic lake of Faro. VBR values <1.0 were found in summer 2000 in relation with peculiar hydrographic constraints. Virioplankton distribution was dependent on salinity, and on dilution of the oligotrophic waters flowing from the Straits. Virioplankton was closely related with bacterioplankton indicating a close coupling between viruses and host cell abundance.     

Genetics and taxonomy of Chilean smooth-shelled mussels, Mytilus spp. (Bivalvia: Mytilidae)

It has been previously established that native smooth-shelled mussels in southern South America possess close evolutionary affinities with Northern-Hemisphere Mytilus edulis L. 1758 (McDonald et al. (1991) [5]). This result has since been challenged by authors claiming that Chilean mussels should be considered a local subspecies of M. galloprovincialis Lmk. 1819. Moreover, morphological, physiological, ecotoxicological and molecular genetic studies on Chilean smooth-shelled mussels still frequently refer to ‘M. chilensis’ Hupé 1854, even though the previous discovery of alien M. galloprovincialis and considerable heterogeneity in shell morphology among samples collected along the Chilean shores raise concerns that different Mytilus spp. species might have been included under ‘M. chilensis’. Here we reviewed the molecular and morphological data available on smooth-shelled mussels from Chile in an attempt to clarify both their genetic composition and their taxonomic status. Using multivariate analysis on sample × allozyme-frequency matrices, we confirmed the widespread occurrence of the Southern-Hemisphere form of M. edulis along the shores from the North Patagonia region of Chile to the southern tip of the South American continent. The populations sampled in southern central Chile showed some evidence of slight introgression from Southern-Hemisphere M. galloprovincialis. Morphological characterization of a sample from Dichato in southern central Chile was consistent with its previous genetic identification as Mediterranean M. galloprovincialis. The occurrence of Southern-Hemisphere M. galloprovincialis in Punta Arenas at the southern tip of the South American continent was also reported. Southern-Hemisphere M. edulis, including native Chilean smooth-shelled Mytilus, should be assigned subspecific rank and named M. edulis platensis d’Orbigny 1846.     

A population genetic study of the hybrid zone of Mytilus trossulus Gould, 1850 and an introduced species,M. galloprovincialis Lamarck, 1819, (Bivalvia: Mytilidae) in peter the great bay in the Sea of Japan

This paper examines the genetic variability of the Pacific mussel Mytilus trossulus and an introduced Atlantic species, M. galloprovincialis, in the northwestern Sea of Japan (Peter the Great Bay and Kievka Bay). The genotyping of individuals from eight populations was carried out using eight polymorphic enzyme loci and two nuclear DNA markers (Me-5 and ITS-1,2); the occurrence frequency of parent species and their hybrids was determined. The enzyme and nuclear markers demonstrated concordant genetic variation. The genotypes of the native species M. trossulus were predominant in the samples studied. The frequency of the introduced species M. galloprovincialis in the total material was relatively low; however, it reached 42 ± 2% in samples that were collected in Possjet Bay near the town of Zarubino in a zone of active international navigation. In this area the greatest number of hybrids was found as well. It is concluded that the invasion of M. galloprovincialis in the northwestern Sea of Japan is continuing; permanent populations of this mussel appeared in Possjet Bay that were not recorded here previously.     

Associations between Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms and Non-Alcoholic Fatty Liver Disease (NAFLD) Risk: A Meta-Analysis

BackgroundC677T and A1298C are the most common allelic variants of Methylenetetrahydrofolate Reductase (MTHFR) gene. The association between MTHFR polymorphisms and the occurrence of non-alcoholic fatty liver disease (NAFLD) remains controversial. This study was thus performed to examine whether MTHFR mutations are associated with the susceptibility to NAFLD.MethodsA first meta-analysis on the association between the MTHFR polymorphisms and NAFLD risks was carried out via Review Manager 5.0 and Stata/SE 12.0 software. The on-line databases, such as PubMed, EMBASE, CENTRAL, WOS, Scopus and EBSCOhost (updated to April 1^st, 2016), were searched for eligible case-control studies. The odd radio (OR), 95% confidence interval (CI) and P value were calculated through Mantel-Haenszel statistics under random- or fixed-effect model.ResultsEight articles (785 cases and 1188 controls) contributed data to the current meta-analysis. For C677T, increased NAFLD risks were observed in case group under homozygote model (T/T vs C/C, OR = 1.49, 95% CI = 1.03~2.15, P = 0.04) and recessive model (T/T vs C/C+C/T, OR = 1.42, 95% CI = 1.07~1.88, P = 0.02), but not the other genetics models, compared with control group. For A1298C, significantly increased NAFLD risks were detected in allele model (C vs A, OR = 1.53, 95% CI = 1.13~2.07, P = 0.006), homozygote model (C/C vs A/A, OR = 2.81, 95% CI = 1.63~4.85, P = 0.0002), dominant model (A/C+C/C vs A/A, OR = 1.60, 95% CI = 1.06~2.41, P = 0.03) and recessive model (C/C vs A/A+A/C, OR = 2.08, 95% CI = 1.45~3.00, P<0.0001), but not heterozygote model.ConclusionT/T genotype of MTHFR C677T polymorphism and C/C genotype of MTHFR A1298C are more likely to be associated with the susceptibility to NAFLD.     

Comparison of antibacterial activity in the hemolymph of marine bivalves from Galicia (NW Spain)

A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative).     

Microgeographic allozyme differentiation in the hybrid zone ofMytilus galloprovincialis Lmk. andM. edulis L. on the continental European coast

The EuropeanMytilus galloprovincialis Lmk. andM. edulis L. coexist and hybridize in different proportions in extended areas of the British and Atlantic French coasts.M. galloprovincialis typical allozymes seem to predominate in wave exposed areas, at high levels of attachment and in larger mussels in the British hybrid zone. Mussel samples from exposed and sheltered areas, 200 m apart, and from high and low levels of attachment were collected from a location of the French hybrid zone in 1988–92. PureM. galloprovincialis andM. edulis populations were also taken as controls. Diagnostic enzyme loci for bothMytilus (EST-D ^*, LAP-1^*, MPI^*, ODH^*) andAP-1 ^*, LAP-2^* andPGM ^* loci were studied. The frequencies of theM. galloprovincialis typical alleles were significantly greater in exposed populations than in sheltered samples (e.g. 0.729 to 0.803 vs 0.192 to 0.581 forEST-D ^*90), and at high level of attachment than at low level for the sheltered area (e.g. 0.581 vs 0.192 forEST-D ^*90). PutativeM. galloprovincialis was more abundant on the exposed coast (0.591 and 0.702) than on the sheltered shore, where it predominated at the high shore but not at the low shore location (0.371 vs 0.045). Significantly positive correlations between shell length and typicalM. galloprovincialis compound allele frequencies were found only for populations from exposed areas. Relationships between theMytilus genetic differentiation and ecological factors are discussed.     

Genetic analysis of two Portuguese populations of <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> by RAPD profiling

The clam Ruditapes decussatus is commercially important in the south of Portugal. The random amplified polymorphic DNA (RAPD) technique was applied to assess the genetic diversity and population structure of two Portuguese populations occurring in the Ria Formosa (Faro) and the Ria de Alvor, respectively. Twenty-five individuals of each population were investigated by RAPD profiles. Genetic diversity within populations, measured by the percentage of polymorphic loci (%P), varied between 68.57% (Alvor) and 73.88% (Faro). Shannon’s information index (H) and Nei’s gene diversity (h) were 0.281 and 0.176, respectively, for the Alvor population and 0.356 and 0.234 for the Faro population. Overall, genetic variation within R. decussatus populations was high. The total genetic diversity (H _T) was explained by a low variation between populations (G _ST = 0.145), which is consistent with high gene flow (N _m = 2.9). The analysis of molecular variance (AMOVA) showed that 65% of variability is within populations and 35% between populations (Φ_PT = 0.345; P ≥ 0.001). The value of Nei’s genetic distance was 0.0881, showing a low degree of population genetic distance, despite the different geographic origin. This is the first study on the population genetics of R. decussatus by RAPD technique. The results may be useful for restocking programs and aquaculture.     

Haemolymph characteristics of a copper-tolerant decapod Orconectes virilis (Hagen) (Astacidae, Crustacea)

The mean haemolymph volume in a copper-tolerant, intermoult (C2-C4) adult (more than a year old) Orconectes virilis, as determined by [14C-carboxylic] inulin dilution method, ranged from 61.17 microliters in females (mean wet body weight = 4.34 g; mean carapace length = 4.43 cm) to 61.79 microliters in males (mean wet weight = 5.73 g; mean carapace length = 4.49 cm). The mean haemolymph volume per cent varied from 14.5 (in females) to 31.1 (in males). Haemolymph from males had an average pH of 7.35, osmotic pressure of 414.1 mmol/kg H2O, and a specific gravity of 1.0603, while that from females averaged a pH of 7.48, osmotic pressure of 433.6 mmol/kg H2O and a specific gravity of 1.0469. Mean total haemocyte counts ranged from 3,434/mm3 (1,000/mm3 halinocytes, 1,125/mm3 hemi-granulocytes and 1,309/mm3 granulocytes) in males to 4,905/mm3 (1,525/mm3 halinocytes. 1,490/mm3 hemi-granulocytes and 1,890/mm3 granulocytes) in females.     

Nocardiosis in Mediterranean bivalves: First detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy)

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.     

Species status and population structure of mussels (Mollusca: Bivalvia: Mytilus spp.) in the Wadden Sea of Lower Saxony (Germany)

Three species of mussel (genus Mytilus) occur in Europe: M. edulis (Linnaeus 1758), M. galloprovincialis (Lamarck 1819) and M. trossulus (Gould, Boston Society of Natural History 3: 343?C348, 1850). Although these species are indigenous to the North Sea, the Mediterranean and the Baltic Sea, respectively, they form an extended patchy species complex along the coasts of Europe (??the Mytilus edulis complex??) and are able to hybridize where their distributions overlap. Recent studies examining the taxonomic status and genetic composition of Mytilus populations in the Netherlands and the British Isles have revealed introgressive hybridization processes within this species complex, with hints of an invasion of nonindigenous M. galloprovincialis into the North Sea. Furthermore, an extensive international mussel fishery industry in Europe (i.e., Great Britain, the Netherlands, Denmark, and Germany) is also in discussion for a possibly anthropogenically induced bioinvasion of nonindigenous Mytilus traits into the Wadden Sea area. Although it is assumed that the Wadden Sea of Germany comprises M. edulis only, this has never been confirmed in a molecular genetic study. To assess the situation for the Wadden Sea of Lower Saxony, we conducted the first molecular study of the Mytilus genus in the region. Taxonomic identification of 504 mussels from 13 intertidal mussel banks using the nDNA marker Me15/16 revealed a population composition of 99% M. edulis and 1% M. edulis X M. galloprovincialis hybrids. Hence, the Wadden Sea population is unaffected by range expansion of nonindigenous Mytilus traits. The genetic structure of the M. edulis populations was investigated using the phylogenetic and population genetics analyses of the mitochondrial DNA cytochrome-c-oxidase subunit I (COI) and the first variable domain of the control region (VD1), which were sequenced for >120 female individuals. These results showed a heterogeneous, panmictic population due to unrestricted gene flow. This can be attributed to extensive larval dispersal linked to the tidal circulation system in the back barrier basins of the Wadden Sea.     

Haematological and serum protein profiles of Mugil cephalus: effect of two different habitats

The aim of this study was to assess the influence of two different habitats, Faro Lake (group A) and Tyrrhenian Sea (group B), on the haematological and serum protein profiles of Mugil cephalus. Our results showed significant differences of white blood cells, total proteins, prealbumin, albumin and α-globulins between groups A and B. These findings suggest that changes in haematological and serum protein profiles are important indices in monitoring the effects of aquatic habitat changes, representing an adaptive physiological response to different habitats of M. cephalus.     

Interspecific differences in prey items in relation to morphological characteristics among four lutjanid species (Lutjanus decussatus, L. fulviflamma, L. fulvus and L. gibbus)

Clarifying interspecific differences in prey items in relation to morphological characteristics is a fundamental aspect to understand the mechanism enabling the diversity of feeding ecology of fishes. The aim of the present study was to clarify the relationship between prey items and body shape variation, teeth and mandible characteristics for four lutjanid species: Lutjanus decussatus, L. fulviflamma, L. fulvus and L. gibbus. Stomach contents analysis revealed that the main prey items of L. decussatus were fishes, L. fulviflamma were crabs and fishes, and L. fulvus and L. gibbus was crabs. Body shape analysis revealed that L. decussatus and L. fulviflamma had a shallower body depth whereas L. fulvus and L. gibbus had a deeper body depth. The two species with a shallower body had long teeth whereas the other two species with a deeper body depth had shorter teeth. The jaw-lever mechanics were compared and L. decussatus and L. fulviflamma have a faster mouth opening–closing mechanism. Canonical correspondence analysis revealed that fishes was the major prey item for the species having a shallower body depth, higher teeth length and lower ratios of in-lever to out-lever of mandibles, whereas crabs was the major prey item for the species having a deeper body depth, lower teeth length and higher ratios of in-lever to out-lever of mandibles. It is suggested that the interspecific differences in main prey items among the four species are directly related to behavioral differences based on body shape, teeth characteristics and jaw-lever mechanics.     

Coccomyxa sp. (Chlorophyta: Chlorococcales), a new pathogen in mussels (Mytilus galloprovincialis) of Vigo estuary (Galicia, NW Spain)

In this work, we describe the occurrence of irregular shaped green aggregations in the mantle, gill filaments, adductor muscle, visceral mass and haemolymph of wild mussels (Mytilus galloprovincialis) collected from the Vigo estuary (Galicia, NW Spain). Microscopic examination of these masses revealed that they consist of intracellular green algae which are spherical to oval in shape, 5 μm in length and 3 μm in width, without flagella and with a smooth surface. The algal cells present a small single nucleus, a mitochondrion, 1-2 parietal chloroplasts and lack pyrenoids. Reproduction is by formation of 2-4 autospores or daughter cells. Pigment analysis reveals the presence of photopigments typical of green algae in addition to alloxanthin, diadinoxanthin and diatoxanthin. These carotenoids are noted for the first time in a parasitic chlorophyte. The signs of infection, together with the morphological observations, suggest that this parasitic algae may be Coccomyxa parasitica. However, further molecular studies are required for confirmation. This is the first report of Coccomyxa algae parasitizing the species M. galloprovincialis.     

Competition and facilitation between the alien mussel Mytilus galloprovincialis and indigenous species: Moderation by wave action

As the alien species that most dominates space along the South African coast, the Mediterranean mussel Mytilus galloprovincialis has radically altered community composition on invaded shores. We experimentally assessed interspecific interactions between this invasive species and dominant indigenous species in conjunction with considering how wave action moderates such interactions. The density of both M. galloprovincialis and the limpet Scutellastra granularis increased with wave action. Conversely, the tube-building polycheate Gunnarea capensis was negatively affected by wave exposure, being most abundant on sheltered shores. The influence of wave action on the indigenous mussel Aulacomya ater, however, remains unclear. M. galloprovincialis outcompeted both G. capensis and A. ater at moderate to high exposure levels, whereas it had both positive and negative effects on S. granularis. It outcompeted adult limpets on primary rock space on semi-exposed and exposed shores, reducing densities of this portion of the population. However, recruitment of S. granularis was facilitated by M. galloprovincialis, as greater numbers recruited to the secondary substratum offered by mussel shells. Again this interaction intensified with wave action. Due to the extremely high density of recruits on secondary space, the net effect of M. galloprovincialis on S. granularis was positive. Thus, wave action not only influences the abundance of individual species, but also mediates both positive and negative interspecific interactions in rocky shore communities, including the impact of alien species such as M. galloprovincialis.     

Histology of the reproductive tissue of the clam Ruditapes decussatus from the gulf of Gabès (Tunisia)

Summary

The gametogenic cycle of the clam Ruditapes decussatus was investigated during one year. Samples were collected each month in two different sites located at the gulf of Gabès area. Gametogenic activity was characterized on the basis of histological analysis of the gonads. A classification and a scoring system were then proposed. According to our study, Ruditapes decussatus gametogenesis can be divided in four major steps: (1) a sexual rest (December- February), (2) starting of gametogenesis (February-March), (3) development (March-May), (4) ripe (from April onwards). This study revealed that the gonad of the clam Ruditapes decussatus was surrounded by muscular tissue which could represent the principal storage tissue. Two kinds of hermaphroditism appeared since maturity of gonads: simultaneous one showing gonadal tubules of both males and females in regionally distinct and separate zones and a “superposed” one showing a gonad consisting essentially of mature male gametes and few residual oocytes.