Cell biology of deep-sea multicellular organisms

Establishing tissue cultures derived from deep-sea multicellular organisms has been extremely difficult because of the serious damage they sustain upon decompression and exposure to the high temperature of surface seawater. We developed a novel pressure-stat aquarium system for the study of living deep-sea multicellular organisms under pressure. Using this system, we have succeeded in maintaining a variety of deep-sea multicellular organisms under pressure and atmospheric conditions after gradual, slow decompression. Furthermore, we successfully cultivated and freeze-stocked pectoral fin cells of the deep-sea eel Simenchelys parasiticus collected at a depth of 1,162 m under atmospheric pressure conditions. This review describes novel capture and maintenance devices for deep-sea organisms and cell culture studies of the organisms under atmospheric and pressure conditions.     

Larvae of the deep-sea Nematocarcinidae (Crustacea: Decapoda: Caridea) from the Southern Ocean

The early larval stages of the deep-sea Nematocarcinidae, Nematocarcinus longirostris Bate, 1888, from the south-western Atlantic Ocean, and N. lanceopes Bate, 1888, from the high Antarctic Weddell Sea, were obtained from plankton catches, described and illustrated. Furthermore, field collected larvae of N. lanceopes were compared with larvae hatched and reared under constant laboratory conditions. The morphology of larvae in both species clearly indicates a planktotrophic and extended mode of larval development. This is an outstanding feature in deep-sea and especially in high-latitudinal caridean shrimp species, and the consequence of such reproductive trait for life history adaptations to both deep-sea and polar environments is discussed.This revised version was published online in November 2004 with corrections to the figure part lettering.     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Tissue culture of the deep-sea bivalve Calyptogena soyoae

Tissue culture for the deep-sea clam Calyptogena soyoae (C. soyoae) has been examined. Mantle tissue was cultured in Dulbecco's modified Eagle medium that was prepared using artificial seawater supplemented with fetal bovine serum (FBS) and the body fluid of C. soyoae. The mantle cells were viable in culture for at least 13 days at 4°C and atmospheric pressure on a polylysine-coated dish, although no cells attached in the body fluid-free culture medium. It was found that mantle cells synthesized DNA and seemed to proliferate under atmospheric conditions. Received: June 1, 2000 / Accepted: October 4, 2000     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Bubble formation in crustaceans following decompression from hyperbaric gas exposures

In vivo bubble formation was studied in various crustaceans equilibrated with high gas pressures and rapidly decompressed to atmospheric pressure. The species varied widely in susceptibility to bubble formation, and adults were generally more susceptible than larval stages. Bubbles did not form in early brine shrimp larvae unless equilibration pressures of at least 175 atm argon or 350 atm helium were used; for adult brine shrimp, copepods, and the larvae of crabs and shrimps, 100-125 atm argon or 175-225 atm helium were required. In contrast, bubbles formed in the leg joints of megalopa and adult crabs following decompression from only 3-10 atm argon; stimulation of limb movements increased this bubble formation, whereas inhibition of movements decreased it. High hydrostatic compressions applied before gas equilibration or slow compressions did not affect bubble formation. We concluded that circulatory systems, musculature, and storage lipids do not necessarily render organisms susceptible to bubble formation and that bubbles do not generally originate as preformed nuclei. In some cases, tribonucleation appears to be the cause of the bubbles.     

Growth and maturation of a North American fairy shrimp, Streptocephalus seali (Crustacea; Anostraca): a laboratory study

SUMMARY.

• 1 We evaluated survival, growth and time to maturation of the fairy shrimp, Streptocephalus seali Ryder, in the laboratory at various combinations of temperature and water hardness.
• 2 Both independent factors affected survival and growth of S. seali. Multiple regression analysis and response surface modelling predict that after 4 days, over 80% survival is obtained at temperatures from 14 to 28°C and water hardnesses from 60 to 130 mg CaCO₃ 1-^?1.
• 3 Growth rates of larvae were often maximum at physicochemical conditions other than those which had promoted maximum rates of survival. For example, after 12 days mean total body length was almost 12 mm in larvae which had been maintained at 34°C (80 mg CaCO₃ 1-^?1): the maximum survival rate had been obtained at 19°C. Total length was directly correlated with temperature at the lowest hardness tested, but not at the other two hardnesses (100 and 120 mg CaCO₃ 1-^?1). At the latter water hardnesses, total length was significantly less at 34°C than at 32°C on all three sampling occasions (4, 8 and 12 days post-hatch).
• 4 Similarly, developmental stage of larvae correlated well with temperature but larvae reared at 34°C did not develop more quickly than those reared at 32°C. After 12 days, most larvae at the two highest temperature treatments had developed at least to Stage 14 and many were nearly mature; at 17°C most larvae were still at Stage 10.
• 5 During our study of maturation rate of females we noted that egg production was initiated after completion of fourteen or fifteen moults. Mean time to maturation at 27°C (17.3±2.8 days) exceeded that at 32°C (12.3±2.6days). The minimum time to maturation of a shrimp was 9 days at 32°C.

     

Rupture of the Cell Envelope by Decompression of the Deep-Sea Methanogen Methanococcus jannaschii

The effect of decompression on the structure of Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen, was studied in a novel high-pressure, high-temperature bioreactor. The cell envelope of M. jannaschii appeared to rupture upon rapid decompression (ca. 1 s) from 260 atm of hyperbaric pressure. When decompression from 260 atm was performed over 5 min, the proportion of ruptured cells decreased significantly. In contrast to the effect produced by decompression from hyperbaric pressure, decompression from a hydrostatic pressure of 260 atm did not induce cell lysis.     

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

Cultivation and biomass production of the diatom Thalassiosira weissflogii as a live feed for white-leg shrimp in hatcheries and commercial farms in Vietnam

This study investigated the biomass production process from the laboratory to the pilot scale in order to use the nutrient-rich biomass of the diatom Thalassiosira weissflogii as live feed for white-leg shrimp (Litopenaeus vannamei) at larval stages (zoeal, mysis, and postlarval) and in commercial production in hatcheries in Vietnam. Our results showed that T. weissflogii was successfully cultured in 1–2 L Erlenmeyer flasks, 0.2–3.5 m³ composite tanks, and 6.5 m³ tubular photobioreactors, with the highest cell density of 1.6 × 10⁶ cells mL^?1 reached after 6 days of culture. Under optimal culture conditions, the protein, lipid, and carbohydrate contents in this algal biomass were 13.2%, 20.0%, and 10.0% of dry cell weight, respectively. The fatty acid composition contains high amount of palmitic acid (C16:0, 43.11% of total fatty acid), and polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5ω-3), approximated 16.5% of total fatty acid. In a 50 L larval rearing tank, at the optimal stocking density of 125 nauplii L^?1, the survival percentage (75.55%), the total body length (from 5.376 ± 0.007 to 10.860 ± 0.030 mm), and weight (at from PL₁ to PL₁₂ stages) (from 0.145 ± 0.002 to 1.158 ± 0.005 g) of the white-leg shrimp larvae reached the highest values but the metamorphosis time (234 h) was shortest compared with the other stocking densities. Further, adding living T. weissflogii biomass to the diet of white-leg shrimp larvae at the nauplii 6 stage led to an increase in the body length, weight, and survival percentage of white-leg shrimp larvae of 21.17%, 35.7%, and 33% higher compared with those of larvae fed the control diet (without the addition of T. weissflogii), respectively. At the same time, the metamorphosis time of larvae (from Z₁ to PL₁) decreased by 4 h compared to the control group. In intensive ponds (area of 6400 m² pond^?1), using seed stocks at the postlarvae 12 stage that had been fed T. weissflogii, the final weight, yield, and survival percentage of the shrimp were increased by 7.3%, 14.2%, and 16.3%, respectively, compared with those of the control group. There were no statistically significant differences in the protein and carbohydrate contents in the shrimp flesh among the experimental and control group (p > 0.05). The lipid, omega-3, omega-6, and omega-9 fatty acid contents of shrimp flesh in experiment formula (per 100 g shrimp) were 1.21 g, 72.9 mg, 114 mg, and 86.1 mg, 11%, 29%, 21.6%, and 17.7% higher than that those in control, respectively. The obtained results show the great potential of using T. weissflogii as live feed on white-leg shrimp farms in Vietnam.

     

Analysis of the diet of the tadpole shrimp, Triops sinensis, in paddy fields of Shouchang River watershed

The tadpole shrimp, Triops sinensis, was discovered in the paddy fields of the Shouchang River watershed. The gut contents of the shrimp from these field populations were analyzed in order to quantify their dietary patterns. Some species of tadpole shrimps were found to prey on mosquito larvae as well as uproot weeds. Mosquito larvae and weeds were seldom found in the paddy fields which the tadpole shrimp inhabited in the Shouchang River watershed. The results further showed that the larvae of these tadpole shrimp mainly feed on algae and rice lamina. The tadpole shrimp could feed on animal food including protozoa, nematodes, rotifers, crustaceans, and aquatic insect larvae, though the animal food was subsidiary.     

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Abstract

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 μg ml^?1.     

A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85°C (T _opt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%–93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that P _habitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained. Received: May 14, 1999 / Accepted: July 30, 1999     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

New records of two deep-sea eels collected from the Western Pacific Ocean based on COI and 16S rRNA genes

Background

Two deep-sea eels collected from the Western Pacific Ocean are described in this study. Based on their morphological characteristics, the two deep-sea eel specimens were assumed to belong to the cusk-eel family Ophidiidae and the cutthroat eel family Synaphobranchidae.

Methods and results

To accurately identify the species of the deep-sea eel specimens, we sequenced the mitochondrial genes (cytochrome c oxidase subunit I [COI] and 16S ribosomal RNA [16S rRNA]). Through molecular phylogenetic analysis based on mtDNA COI and 16S rRNA gene sequences, these species clustered with the genera Bassozetus and Synaphobranchus, suggesting that the deep-sea eel specimens collected are two species from the genera Bassozetus and Synaphobranchus in the Western Pacific Ocean, respectively.

Conclusions

This is the first study to report new records of the genera Bassozetus and Synaphobranchus from the Western Pacific Ocean based on COI and 16S rRNA genes

     

Étude biométrique de deux espèces de crevettes profondes exploitées en Guyane française

Values for selected morphometric relationships are extremely important for shrimp fisheries management. This information, however, does not exist for some deep-sea shrimp species which are of interest to fisheries. A variety of weight-weight, length-length and length-weight relationships have been established, therefore, for two commercially important species of deep-sea shrimp from the continental slope off French Guiana (western central Atlantic), i.e. the solenocerid shrimp Solenocera acuminata (orange shrimp) caught on the upper slope (about 200 m) and the aristeid shrimp Plesiopenaeus edwardsianus (scarlet prawn) widely distributed on the middle slope (from 400 to 900 m). This paper represents the first contribution on the morphometric relationships of these two deep-sea shrimp species.     

Presence and development of populations of the introduced brown alga Sargassum muticum in the southwest Netherlands

Of the three Brachionus species used in aquaculture, Brachionus rubens, B. calycilorus and B. plicatilis, the latter is most widely used in raising marine fish and shrimp larvae due to its tolerance to the marine environment. In freshwater aquaculture the use of B. rubens and B. calycilorus is limited, probably because inert food products are readily available as feed for freshwater larvae.The rotifer Brachionus plicatilis is used in large numbers as the first food organism in intensive cultures of marine fish and shrimp larvae. An adequate supply of these rotifers relies on mass cultures. The reproductive rate of rotifers in these cultures depends on food quality and quantity, salinity, temperature and pH of the medium. Removal of waste products from culture tanks leads to higher and more efficient production of rotifers over extended periods of time. Rotifers have to be enriched with polyunsaturated fatty acids, which are essential for proper development and survival of marine fish and shrimp larvae.The future use of preserved rotifers and their resting eggs may help to overcome unforeseen failures of live cultures and may lead to more efficient use of these organisms in raising freshwater and marine fish and shrimp larvae.     

Effects of omnivorous shrimp in a montane tropical stream: sediment removal,disturbance of sessile invertebrates and enhancement of understory algal biomass

Freshwater shrimp dominate the faunal biomass of many headwater tropical streams: however, their role in community organization is unclear. Enclosure/exclosure experiments in a montane Puerto Rican stream examined direct and indirect effects of two dominant taxa of atyid (Atyidae) shrimp, Atya lanipes Holthuis and Xiphocaris elongata Guerin-Meneville. Both shrimp taxa caused significant reductions in sediment cover on rock substrata, reducing sedimentation and enhancing algal biovolume on clay tiles in cages. When tiles incubated in shrimp exclosures for 2 wks were placed outside of cages, atyid shrimp removed 100% of the sediment cover within a 30 min observation period. Atyid shrimp appear to play an important role in stream recovery after high discharge events by rapidly removing sediments and detritus deposited on benthic substrata in pools. We evaluated the mechanism by which A. lanipes influences algae and benthic insects by comparing patterns of algal biomass, taxonomic composition, and insect abundance between shrimp-exclusion and shrimp-presence treatments both with and without manual sediment removal. The shrimp exclusion treatment without manual sediment removal bad significantly lower algal biomass and greater sedimentation than all other treatments. The treatment in which shrimp were excluded but sediment was manually removed, however, accrued almost the same algal biovolume as the shrimp enclosure treatment, supporting the hypothesis that sediment removal enhances the biovolume of understory algal taxa. Algal community composition was similar between stream bottom bedrock exposed to natural densities of shrimp and all experimental treatments for both Atya and Xiphocaris: a diatom community strongly dominated (78–95%) by the adnate taxon, Achnanthes lanceolata Breb ex. Kutz. Atyid shrimp are important in determining the distribution and abundance of benthic insects through both direct and indirect effects. Sessile, retreat-building chironomid larvae (Chironomidae: Diptera) are negatively affected by both A. lanipes and X. elongata, through direct removal by foraging activities and/or indirectly through depression of sediment resources available to larvae for the construction of retreats. In constrast, the mobile grazer, Cloeodes maculipes (Baetidae: Ephemeroptera) was not adversely affected and atyid shrimp have the potential to exert positive indirect effects on this taxon by facilitating its exploitation of algal resources and/or through enhancement of understory algal food resources through sediment removal.     

Novel alternative to antibiotics in shrimp hatchery: effects of the essential oil of Cinnamosma fragrans on survival and bacterial concentration of Penaeus monodon larvae

Aims: The activity of two essential oils (EOs) of Cinnamosma fragrans, an endemic plant to Madagascar (B8: linalool‐type and B143: 1,8‐cineole‐type), against bacterial isolates from a shrimp hatchery of Penaeus monodon and their effects on the survival and bacterial concentration of larvae were determined. Methods and Results: Minimum inhibitory concentrations were determined using a broth dilution technique. The bacterial concentrations of both larvae and water tank were assessed on Marine agar and Thiosulfate Citrate Bile Sucrose agar. The assays took place in OSO Farming’s shrimp hatchery in Madagascar. EOs were directly added to the water tank. Regarding the survival, the assays in larval culture (four replicates each of B8, B143, E and control) showed that B8 oil had a similar effect (P > 0·05) as the antibiotic (Erythromycin) and was more active than B143 (P < 0·05). A negative correlation was observed between the bacterial concentration and the survival of larvae for all assays. Conclusion: Both C. fragrans essential oils, as antibiotic, exhibited significantly higher survival rates and lower bacterial concentrations of the larvae than the control (oil and antibiotic free). Significance and impacts of the study: The potential of C. fragrans essential oil to control the bacterial load in in vivo conditions, thereby enhancing survival rate of P. monodon larvae, makes it a relevant option for developing a novel alternative to antibiotics in shrimp hatchery culture.     

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15 kDa, and it contained 0.96 mol of protoheme and 1.95 mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN⁻, and the catalytic activity of the enzyme was 50% inhibited by 4 μM CN⁻. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. Received: November 25, 1997 / Accepted: December 25, 1997