Transcriptional Responses of Flavin-Containing Monooxygenase Genes in Scallops Exposed to PST-Producing Dinoflagellates Implying Their Involvements in Detoxification

Flavin-containing monooxygenase (FMO) is one of the most prominent xenobiotic metabolic enzymes. It can catalyze the conversion of heteroatom-containing chemicals to polar, readily excretable metabolites and is considered an efficient detoxification system for xenobiotics. Bivalves can accumulate paralytic shellfish toxins (PSTs) produced by dinoflagellates, especially during outbreaks of harmful algal blooms. Exploring FMO genes in bivalves may contribute to a better understanding of the adaptation of these species and the mechanisms of PSTs bioavailability. Therefore, through genome screening, we examined the expansion of FMO genes in two scallops (Patinopecten yessoensis and Chlamys farreri) and found a new subfamily (FMO_like). Our expression analyses revealed that, in both scallops, members of the FMO_N-oxide and FMO_like subfamilies were mainly expressed from the D-stage larvae to juveniles, whereas the FMO_GS-OX subfamily genes were mainly expressed at and prior to the trochophore stage. In adult organs, higher expressions of FMOs were observed in the kidney and hepatopancreas than in other organs. After exposure to PST-producing algae, expression changes in FMOs occurred in hepatopancreas and kidney of both scallops, with more members being up-regulated in hepatopancreas than in kidney for Alexandrium catenella exposure, while more up-regulated FMOs were found in kidney than in hepatopancreas of C. farreri exposed to A. minutum. Our findings suggest the adaptive functional diversity of scallop FMO genes in coping with the toxicity of PST-producing algae.

Maintained larval growth in mussel larvae exposed to acidified under-saturated seawater

Ocean acidification (OA) is known to affect bivalve early life-stages. We tested responses of blue mussel larvae to a wide range of pH in order to identify their tolerance threshold. Our results confirmed that decreasing seawater pH and decreasing saturation state increases larval mortality rate and the percentage of abnormally developing larvae. Virtually no larvae reared at average pHT 7.16 were able to feed or reach the D-shell stage and their development appeared to be arrested at the trochophore stage. However larvae were capable of reaching the D-shell stage under milder acidification (pHT ≈ 7.35, 7.6, 7.85) including in under-saturated seawater with Ω a as low as 0.54 ± 0.01 (mean ± s. e. m.), with a tipping point for normal development identified at pHT 7.765. Additionally growth rate of normally developing larvae was not affected by lower pHT despite potential increased energy costs associated with compensatory calcification in response to increased shell dissolution. Overall, our results on OA impacts on mussel larvae suggest an average pHT of 7.16 is beyond their physiological tolerance threshold and indicate a shift in energy allocation towards growth in some individuals revealing potential OA resilience.

Effect of increased <i>p</i>CO₂ on early shell development in great scallop (<i>Pecten maximus</i> Lamarck) larvae

As a result of high anthropogenic emission of CO2, partial pressure of carbon dioxide (pCO2) in the oceans has increased causing a drop in pH, known as ocean acidification (OA). Numerous studies have shown negative effects on marine invertebrates, and that the early life stages are the most sensitive to OA. We studied the effects on embryo and larvae of great scallop (Pecten maximus L.), using mean pCO2-levels of 477 (ambient), 821, 1184, and 1627 ppm. OA affected both survival and shell growth negatively after seven days. Growth was reduced with 5–10% when pCO2 increased from ambient 477 ppm to 1627 ppm, and survival based on egg number was reduced from 40.4% in the ambient group to 10.7% in the highest pCO2-group. Larvae/embryos stained with calcein one day after fertilization, showed fluorescence in the newly formed shell area indicating calcification of the shell already at the trochophore stage. Shell hinge deformities were observed at elevated pCO2-levels in trochophore larvae after two days. After seven days, deformities in both shell hinge and shell edge were observed in veliger larvae at elevated pCO2-levels. Although the growth showed a moderate reduction, survival rate and increased amount of deformed larvae indicates that P. Maximus larvae are affected by elevated pCO2 levels within the range of what is projected for the next century.

The initial development of gangliar rudiments in a posterior position in mytilus galloprovincialis (Mollusca: Bivalvia)

Histochemical methods for cholinesterase activities were applied to embryos and early larvae of the marine mussel Mytilus galloprovincklis Lamarck (Mollusca: Bivalvia: Mytilidae) and demonstrated that the cerebral ganglia, which are generally accepted as the first differentiating ganglia, appear after two bilaterally symmetrical nerve rudiments have developed in a posterior position prior to the trochophore stage. They are intimately associated with non-nervous superficial cells in two structures which have features of sensory organs.

Sexual reproduction and larval development of Rhaphidrilus nemasoma Monticelli, 1910 (Polychaeta: Ctenodrilidae)

Adult specimens and egg masses of Rhaphidrilus nemasoma were collected in the low intertidal zone from Execution Rock, Bamfield, Vancouver Island, British Columbia, in June of 1986. Each egg mass contained about 1000 eggs. The eggs were green, spherical, and measured 125–145 μm in diameter. Larval development took place within the egg mass until the three-or four-setiger stage, at which time they emerged from the egg mass. Newly emerged larvae crawled on the bottom of the culture beakers and fed on benthic diatoms. Metamorphosis took place soon after emergence and was completed within 2 weeks. Paddle cilia were observed at the early trochophore stage, and their possible function, and the extremely high fecundity of this polychaete, are discussed.

Development of the gut and segmentation of newly settled stages of Ridgeia (Vestimentifera): implications for relationship between Vestimentifera and Pogonophora

Young vestimentiferans, ranging from 0.15 to 10 mm in length, were examined by light and electron microscopy (SEM and TEM). The newly settled stages have a functional gut and traces of larval ciliation, which suggests that they may have developed from a planktonic trochophore stage. The larval mouth becomes an elongated siphon and the ciliated gut persists for some time after the development of the bacterial symbiosis in the trophosome. The segmentation of the body in the earliest stages is similar to that in postlarval Pogonophora, and it is concluded that Vestimentifera and Pogonophora are closely related.

Scanning electron microscopic observations of the larval development of the reef-building polychaete Phragmatopoma lapidosa

The scanning electron microscope (SEM) was used in a study of the larval development of the reef-building sabellariid polychaete Phragmatopoma lapidosa. Mature sperm are modified from the 'primitive' polychaete plan by possessing a tapered and bent acrosome. The outer surface of the irregularly shaped, freshly spawned egg envelope is granular in appearance. SEM observations of various stages in development from the early trochophore through larval metamorphosis and the early juvenile stages suggest that the egg envelope serves as the cuticle through the trochophore stage but is then replaced by another cuticle penetrated by microvilli. SEM has revealed the presence of 'sensory tufts' on the dorsal surface of the larval tentacles which may play a role in larval substrate selection.

Further Investigations on Transcription and Translation in Limnaea Embryos

14C-amino acid incorporation in uncleaved Limnaea eggs is slight but it slowly increases up to the trochophore stage followed by a rapid rise during the veliger stage when 32P incorporation is falling. There is a peak in the 10 S region of the sucrose density gradient profile of morula RNA. Presumably this morula RNA (messenger RNA) is necessary for final development and hatching of the veliger. The trochophore 10 S peak is the largest, stable and strongly depressed by actinomycin; on the contrary, the veliger 10 S RNA peak is insensitive to actinomycin and seems to be meant for immediate translation.Actinomycin treatment of the uncleaved eggs prevents cleavage in a certain percentage of eggs but in the rest the treatment is not effective until the trochophore stage.

The Development of the Egg of Limnaea Stagnalis L. From the First Cleavage Till the Troghophore Stage, With Special Reference To Its "Chemical Embryology"

I. The development of Limnaea stagnalis from the first cleavage till the trochophore stage has been studied with various cytochemical methods. 2. The uncleaved egg is surrounded by a vitelline membrane. At cleavage this membrane is carried inward with the cleavage furrows; it forms a partition wall separating the blastomeres. The cleavage cavity arises by a splitting of this wall. Apparently, the vitelline membrane of Limnaea is a layer of living protoplasm forming the outer layer of the egg cortex. 3. The cleavage cavity, which is formed by the coalescence of several lenticular spaces between the blastomeres, widens by the secretion of fluid into this cavity by special secretion cones on the adjacent sides of the blastomeres. The fluid is expulsed to the exterior at regular intervals. In this way, the cleavage cavity plays an important part in the water regulation of the egg. 4. Prior to the 3d cleavage, the subcortical plasm of the egg, which formed a subcortical layer of uniform thickness at earlier stages, concentrates at the animal side and fuses with the animal pole plasm and the perinuclear protoplasm to a common mass of dense protoplasm; the rest of the egg consists of vacuolar protoplasm. Both substances are distributed unequally over the cells at further cleavage; the dense protoplasm, forming the ectoplasm of all cells, is most abundant in the animal blastomeres and decreases in a vegetative direction, whereas the vacuolar protoplasm, which forms the endoplasm, is most abundant at the vegetative side. The ectoplasm is rich in α-granules (mitochondria) and β-granules; the endoplasm in γ-granules and fat droplets. 5. During cleavage and gastrulation, the nuclei have a constant position at the boundary between ectoplasm and endoplasm. The nucleoli show an intense activity; intranucleolar vacuoles are formed, which are extruded, apparently, into the nucleoplasm. An extrusion of entire nucleoli into the cytoplasm is indicated. The nucleoli are rich in ribonucleic acids, sulfhydril compounds, iron and benzidine peroxidase. The cytoplasmic ribonucleic acids and glutathione are, in many cells, most concentrated in the neighbourhood of the nuclear membrane. These facts point to an active role of the nucleus, and especially the nucleolus, in cell metabolism. 6. During later cleavage stages, a transformation of the proteid yolk occurs; the β-granules are dissolved in the cytoplasm, the y-granules increase in number. At the same time, albumen is ingested by the cells from the surrounding egg capsule fluid, and laid down in numerous albumen vacuoles in the ectoplasm. Probably, the nucleus plays a part in these transformation processes ; the same holds true of the Golgi bodies. During further development, the γ-granules decrease in size; ultimately, they disappear altogether. The ingestion of albumen is restricted more and more to the albumen cells of the gut. Both extracellular and intracellular digestion of albumen can be observed. 7. Probably, no direct continuity exists between the Golgi bodies of the uncleaved egg and those found in the cells of the embryo. 8. In the course of development, there is a marked increase of the thymonucleic acid contents of the nuclei. 9. Particular granules, rich in ribonucleic acid, accumulate at the central end of the macromeres, where they fuse into single dark bodies; these bodies are transmitted into the cells of the 4th quartette at the next division; at later stages, they disappear. 10. Glycogen is accumulated in the "central plasm" at the 24-cell stage; at later stages, especially the cells 4a-4c and their descendants are rich in glycogen. 11. Four equatorial groups of thickened ringlets are formed on cells of the 2d quartette during cleavage. 12. With basic vital dyes, in early stages a weak granular staining of the yolk occurs. At later stages, the basic dyes are accumulated in the albumen vacuoles of the cells which may be considered to represent a "vacuome" in the sense of PARAT. Nile blue hydrochloride stains, furthermore, the yolk granules and mitochondria; brilliant cresyl violet gives a diffuse staining of the cytoplasm. Methylene blue and Janusgreen do not penetrate into intact cells. 13. Probably, there exists a relation between the composition of the cells and their further development; especially, the proportion between ecto- and endoplasm seems to be important for the fate of the cells. 14. At the late gastrula stage, the velum and head vesicle show an elective indophenol oxidase reaction; at still later stages, the reaction is especially localized in the shell anlage and mantle fold and in special subepidermal cells of the mesenchyme. 15. At the late gastrula stage, a strong elective benzidine peroxidase reaction of 2 pairs of cells at the lateral angles of the embryo, representing, probably, the descendants of the anterior trochoblasts, occurs; the velum and head vesicle react more weakly. At the late trochophore stage, the gut shows also a rather strong reaction; still later, it is localized in the mantle fold, the shell, and particular mesenchyme cells in the interior of the embryo. 16. The histological differentiation is accompanied with the appearence of considerable differences in chemical composition between the cells. This leads in each of the 3 germ layers to the formation of 2 types of cells: I. large cells, without cell division, with albumen vacuoles, many Golgi bodies, mitochondria and fat droplets, rich in glycogen and iron, but poor in thymo- and ribonucleic acid; they form the larval organs (ciliary cells, albumen cells, protonephrideum); 2. small, dividing cells, without albumen vacuoles, poor in Golgi substance, mitochondria, fat, glycogen and iron, but rich in thymo- and ribonucleic acids; they have to build up the body of the adult snail. These two cell types can be regarded as representatives of 2 different directions of cell life.