Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements

The establishment of cell lines from marine invertebrates has been encountered with obstacles. Contrary to insects and arachnids where the development of a variety of cell lines has become routine, there is no single established cell line from marine invertebrates. This review examines the activity in the field of marine invertebrate cell cultures within the last decade (1988–1998). During this period, attempts (90 peer reviewed studies in addition to many other abstracts, chapters in books, symposia presentations and reports) were limited to a few species within only six phyla (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, Urochordata; in addition to freshwater/terrestrial annelids and platyhelminths). These studies which are summarized here, on one hand indicated ubiquitous problems and on the other, unique characterizations to each phylum studied. Only one-third of the studies revealed cultures of 1 month or longer but most of these were long-term cultures found or suspiciously considered to be contaminated by other unicellular eukaryotic organisms, mainly by thraustochytrids. Three unique approaches/obstacles for marine invertebrate cell cultures (source of cell, cryopreservation and eukaryotic contaminants) are further discussed. The overall impact of recent improvements and developed protocols raises the suggestion for testing different, novel routes in the establishment of cell cultures from marine invertebrates.     

Bioactive peptides from marine organisms: a short overview

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.     

Natural products from marine invertebrates against <Emphasis Type="Italic">Leishmania</Emphasis> parasites: a comprehensive review

Parasitic infections by Leishmania parasites remains a severe public health problem, especially in developing countries where it is highly endemic. Chemotherapy still remains a first option for the treatment of those diseases, despite the fact that available drugs exhibit a variety of shortcomings. Thus, innovative, less toxic more affordable and effective antileishmanial agents are urgently needed. The marine environment holds an immeasurable bio- and chemical diversity, being a valuable source of natural products with therapeutic potential. As invertebrates comprise about 60 % of all marine organisms, bioprospecting this class of organisms for antileishmanial properties may unravel unique and selective hit molecules. In this context, this review covers results on the literature of marine invertebrate extracts and pure compounds evaluated against Leishmania parasites mainly by in vitro methods. It comprises results obtained from the phyla Porifera, Cnidaria, Bryozoa (Ectoprota), Mollusca, Echinodermata, Annelida, Cetnophora, Platyhelminthes, sub phyla Crustacea (phylum Arthropoda) and Tunicata (phylum Chordata). Moreover, structure–activity relationships and possible mechanisms of action are mentioned, whenever available information is provided. About 70 species of marine invertebrates belonging to seven different phyla are included in this work. Besides a variety of crude extracts, a total of 140 pure compounds was tested against different Leishmania species. Although the research on the antileishmanial potential of marine invertebrates is in its early beginnings, promising results have been achieved that encourage further research. As more extracts and compounds are being screened, the possibility of finding active and selective antileishmanial molecules increases, rising new hope in the search for new treatments against leishmaniases.     

Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues

1. Using a specific HPLC assay, a wide variety of marine invertebrate connective tissues was screened for the 3-hydroxypyridinium amino acids that are prominent intermolecular cross-linking residues in the collagens of many vertebrate connective tissues. 2. One or both of the two structural forms that exist, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), was found in organisms from the following phyla: coelenterata, Annelida, Echinodermata, Mollusca and Arthropoda. 3. Neither amino acid was found in tissues from representative species of Porifera and Chordata. 4. Of special note was an unusually high ratio of LP to HP in Limulus polyphemus gill cartilage.     

One "different kind of gentleman": Alfred Merle Norman (1831–1918), invertebrate zoologist

Alfred Merle Norman (1831–1918), an active clergyman in rural Co. Durham, England, came from an ancient Somerset family. He began to study marine biology during the early 1850s and during the 1860s was an important participant in John Gwyn Jeffreys' expeditions to the Shetland Islands. Norman's field collecting all over the British Isles was extensive and he made important trips to Norwegian fjords for dredging. His best known discoveries were Rhabdopleura and Synagoga , but he published major work on Protozoa, Porifera, Coelenterata, Mollusca, Crustacea, Echinodermata and other invertebrates. The publication Museum Normanianum summarized Norman's collection of 11,086 species, which was acquired by the British Museum (Natural History). His library, which incorporated John Gwyn Jeffreys' library on molluscs, is now in the Department of Zoology, Cambridge University. Norman was primarily a taxonomist secondarily interested in zoogeography who avoided Darwinian controversies.
A bibliography of Norman's 218 publications is included.     

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

• 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
• 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
• 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
• 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

     

Structure and dynamics of the community associated to cultivated Gracilariopsis tenuifrons (Gracilariacea) in Chacopata, Sucre, Venezuela. I: Faunistic inventory

The associated fauna of Gracilariopsis tenuifrons (Gracilariaceae, Rhodophyta) cultures was collected between October 1994 and December 1996 in Chacopata (Sucre State, Venezuela) and preserved in 10% formaldehyde. The species list includes 17 Crustacea, 14 Mollusca and six Polychaeta, the remaining taxa added ten species (total 47 species in eight Phyla). Grazing by mesoherbivores affected the algae and the mollusk Aplysia protea damaged new cultures significantly. The abundance of tubes built by the amphipod Euricthonius brasiliensis impairs algal aspect and facilitates colonization by other organisms.     

Abundance and diversity of littoral invertebrates in the Socorro Island,Revillagigedo Archipelago,Mexico

Composition, abundance, diversity and distribution of the littoral benthic invertebrates of Socorro Island with transects (parallel to the coast and with 1 m2 quadrats) were analyzed. During the spring of 1991 and 1992 samples were taken from the upper and middle levels of the intertidal zone in Vargas Lozano, Braithwaite, Blanca SW, Blanca NE, Binners, Grayson, Academia and Norte bays. The 161 species found belong to Porifera, Cnidaria, Platyhelmintha, Nemertina, Sipunculida, Annelida, Mollusca, Anthropoda and Echinodermata. Crustaceans and mollusks were the richest groups in both years, as in other rocky shores. Highest total density was found in Blanca NE bay in both samplings, with 281 orgs./m2 in the first and 172 orgs./m2 in the second. Most frequently found species were Isognomon janus (Mollusca, Pelecypoda), Littorina pullata (Mollusca, Gastropoda), Hipponix panamensis (Mollusca, Gastropoda), Pachygrapsus transversus (Crustacea, Grapsoidea) and Turbo funiculosus (Mollusca, Gastropoda). Because of the complexity of the habitat structure, Vargas Lozano was the bay with the highest specific richness (83 species), greatest diversity (4.7 bits/individual) and lowest dominance (0.065). Most species were classified as accidentals with the Dajoz's frequency classification, while the dominant species were accessories and only I. janus, in the spring 1991, was a constant species. Two kinds of bays were distinguished: those with some dominant species (density) and those in which there was no evident dominance by a particular species. Consequently, the diversity and evenness values were set apart: homogeneous communities (Vargas Lozano and Binners) and heterogeneous communities (Grayson bay and others), characterized by intermediate evenness values. The Jaccard similarity index identified two regions: one formed by bays found mainly in the southwest part of the island (Binners, Vargas Lozano, Braithwaite, Grayson and Blanca SW) and the other found in the northern region that included Blanca NE, Academia and Norte; however, Blanca NE had the most diverse species composition because of its physiographic particularities and because the sediment retained by the filamentous algae on the flat stones allow many species to settle down. Isognomon janus, Mitrella baccata (Mollusca, Gastropoda), P. transversus and Xanthodius cooksoni (Crustacea, Xanthoidea) were the most common species in all bays in both expeditions. The dominance-diversity relationship, the high specific richness and the low dominance of coastal invertebrates from the different bays, are indicative that the communities in Socorro Island are little disturbed; their specific richness are similar to those of coral reefs.     

Barcoding Antarctic Biodiversity: current status and the CAML initiative,a case study of marine invertebrates

The Census of Antarctic Marine Life (CAML) aims to collate DNA barcode data for Antarctic marine species. DNA barcoding is a technique that uses a short gene sequence from a standardised region of the genome as a diagnostic ‘biomarker’ for species. This study aimed to quantify genetic data currently available in GenBank in order to establish whether a representative cross-section of Antarctic marine taxa and bio-geographic areas has been sequenced and to propose priorities for barcoding, with a particular emphasis on marine invertebrate species. It was found that, amongst marine invertebrate fauna, sequence information covers a limited range of taxa and areas—mainly Crustacea, Annelida and Mollusca from the Weddell Sea and the Antarctic Peninsula. Only 15% of genes sequenced in Antarctic marine invertebrates were the standard barcode gene cytochrome c oxidase subunit 1 (CO1), the majority were other nuclear and mitochondrial genes. There is an urgent need for more in-depth genetic barcoding and species identification studies in Antarctic science, from a range of taxa and areas, given the rate of climate-driven habitat changes that might lead to extinctions in the region. CAML hopes to redress the balance, by collecting and sequencing over the circum-Antarctic area, using material from voyages that occurred during 2008 and 2009, within the framework of the International Polar Year (IPY).     

Marine invertebrate cytochrome P450: emerging insights from vertebrate and insects analogies

Cytochrome P450 enzymes (P450s) are responsible for the oxidative metabolism of a plethora of endogenous and exogenous substrates. P450s and associated activities have been demonstrated in numerous marine invertebrates belonging to the phyla Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata. P450s of marine invertebrates and vertebrates show considerable sequence divergence and the few orthologs reveal the selective constraint on physiologically significant enzymes. P450s are present in virtually all tissues of marine invertebrates, although high levels usually are found in hepatic-like organs and steroidogenic tissues. High-throughput technologies result in the rapid acquisition of new marine invertebrate P450 sequences; however, the understanding of their function is poor. Based on analogy to vertebrates and insects, it is likely that P450s play a pivotal role in the physiology of marine invertebrates by catalyzing the biosynthesis of signal molecules including steroids such as 20-hydroxyecdysone (the molting hormone of crustaceans). The metabolism of many exogenous compounds including benzo(a)pyrene (BaP), pyrene, ethoxyresorufin, ethoxycoumarin and aniline is mediated by P450 enzymes in tissues of marine invertebrates. P450 gene expression, protein levels and P450 mediated metabolism of xenobiotics are induced by PAHs in some marine invertebrate species. Thus, regulation of P450 enzyme activity may play a central role in the adaptation of animals to environmental pollutants. Emphasis should be put on the elucidation of the function and regulation of the ever-increasing number of marine invertebrate P450s.     

Components of the cytochrome P450-dependent monooxygenase system and 'NADPH-independent benzo[a]pyrene hydroxylase' activity in a wide range of marine invertebrate species

Levels of components of the cytochrome P450 (CYP)-dependent monooxygenase system were characterised in microsomes of major biotransformation tissues, or whole bodies, of 33 species from six phyla of aquatic invertebrates. The phylogenetic distribution of benzo[a]pyrene hydroxylase (BPH) activity in the absence of added NADPH (so-called 'NADPH-independent BPH activity') and presence of NADPH was also examined. Microsomal protein yield was higher in individual tissues than whole tissues. The main components (total CYP and cytochrome b5; NADPH-dependent cytochrome c (CYP) reductase, NADH-dependent cytochrome c reductase and NADH-dependent ferricyanide (b5) reductase activities) were found in most species of the Porifera, Cnidaria, Mollusca, Polychaeta, Crustacea and Echinodermata examined. The so-called '418-peak' of the carbon-monoxide difference spectrum of reduced microsomes was found in all species, indicating the wide distribution of this protein. Total CYP levels (pmol mg(-1) protein; mean+/-SEM) were similar in molluscs (50+/-7), crustaceans (61+/-11) and echinoderms (56+/-9), with the exception of high levels (223-266) in two crustacean species. NADPH-dependent BPH activity (pmol min(-1) mg(-1) protein) was found in 32 species, being lowest in Porifera and Cnidaria (3-4), intermediate in Mollusca (7.8+/-1.3), and highest in Crustacea (25+/-4) and Echinodermata (15+/-4). NADPH-independent BPH activity was evident in 13 out of 15 molluscan species examined, with the addition of NADPH either stimulating (8 species) or inhibiting (5 species) the activity. NADPH-independent BPH activity was also seen in two poriferan species and indicated in three crustacean species, suggesting that the phenomenon is not solely restricted to the Mollusca.     

Endocrine mechanisms in marine invertebrates

The objective of this minireview is to summarize the recent advances made in the area of marine invertebrate endocrinology, with special emphasis on the literature published in 1972 and 1973. Most of the pertinent publications in these two years have dealt with crustaceans. Nevertheless, significant contributions have also been made with representatives of other groups of marine invertebrates.     

A Review of the Chemical Ecology of Antarctic Marine Invertebrates

The interdisciplinary field of marine invertebrate chemicalecology is relatively young, and particularly so in polar marineenvironments. In this review we present evidence that the incidenceof chemical defense in antarctic benthic marine invertebratephyla is widespread. Mechanisms of chemical defense have beendetected in antarctic representatives of the Porifera, Cnidaria,Brachiopoda, Tunicata, Nemertea, Mollusca and Echinodermata.This argues against earlier bio-geographic theories that predicteda low incidence of chemical defense in polar waters where levelsof fish predation are low. Selection for chemical defense inbenthic sessile and sluggish marine invertebrates is likelya response to an environmentally stable community shown to bestructured primarily by biotic factors such as predation andcompetition. Holoplankton and the eggs, embryos and larvae ofboth benthic and planktonic antarctic macroinvertebrates mayalso employ chemical defense to offset mortality during characteristicallyslow development and long life span where susceptibility topredation is seemingly high. While most research to date hasfocused on the role of secondary metabolites in mediating predation,it is likely that bioactive compounds in antarctic marine invertebratesalso serve roles as antifoulants and allelochemics. The diversityof bioactive metabolites detected to date in antarctic marineinvertebrates sets the stage both for continuing and for broadeningefforts to evaluate their functional and ecological significance.     

The Paramyxea Levine 1979: An original example of evolution towards multicellularity

The Paramyxea are parasitic in marine invertebrates. Their development is a sporulation involving the differentiation within a stem cell of several sporonts which produce spores made of cells enclosed inside each other. Three genera are recognized according to the number of spores and sporal cells, and the taxonomic position of the host (Polychaeta, Mollusca, Crustacea).The Paramyxea exhibit both protistan and metazoan characters. Their nine singlets centrioles are observed in different Protoctists whereas the fact that their sporal cells acquire distinctive cytological features may be interpreted as an evolution towards multicellularity.     

Decrease in anaerobe-related bacteraemias and increase in Bacteroides species isolation rate from 1998 to 2007: A retrospective study

Conflicting data have accumulated in recent years regarding the incidence of anaerobic bacteraemias. The aim of this study was to determine the prevalence of bacteraemias due to anaerobic bacteria and evaluate the importance of anaerobic blood cultures in a university hospital in Israel. A retrospective survey which focused on anaerobic blood culture bottles was performed on blood cultures received in our laboratory during the decade from January 1998 to December 2007. Anaerobic-related bacteraemias decreased during that period, whereas a significant increase was observed in Bacteroides species isolated from the blood cultures (from 18% during 1998–2002 to 43% during 2003–2007). Comparison of the medical records of 54 patients with Bacteroides-related bacteraemia during the two end periods (1998–1999 and 2006–2007) revealed a marked increase in complex underlying diseases. Hypertension and diabetes mellitus type II were found in 29% of the patients in 1998–1999 and increased to 43–45% of the patients in 2006–2007. Ischemic heart disease also increased from 14% of the patients in 1998–1999 to 43% in 2006–2007. We conclude that although positive anaerobic blood cultures account for a small percentage of positive blood samples, the growing involvement of Bacteroides species-related bacteraemias together with an increase in complex underlying diseases in these patients emphasize the importance of anaerobic blood cultures, particularly in patients with co-morbidities.     

Methods of synthesis of glycosyl fluorides

In recent years glycosyl fluorides have been utilized as versatile sugar donors in the synthesis of natural products and carbohydrates. This paper provides an update on the advances made in the preparation of glycosyl fluorides during the last decade (1988-1998).     

Recovery of Benthic Megafauna from Anthropogenic Disturbance at a Hydrocarbon Drilling Well (380 m Depth in the Norwegian Sea)

Recovery from disturbance in deep water is poorly understood, but as anthropogenic impacts increase in deeper water it is important to quantify the process. Exploratory hydrocarbon drilling causes physical disturbance, smothering the seabed near the well. Video transects obtained by remotely operated vehicles were used to assess the change in invertebrate megafaunal density and diversity caused by drilling a well at 380 m depth in the Norwegian Sea in 2006. Transects were carried out one day before drilling commenced and 27 days, 76 days, and three years later. A background survey, further from the well, was also carried out in 2009. Porifera (45% of observations) and Cnidaria (40%) dominated the megafauna. Porifera accounted for 94% of hard-substratum organisms and cnidarians (Pennatulacea) dominated on the soft sediment (78%). Twenty seven and 76 days after drilling commenced, drill cuttings were visible, extending over 100 m from the well. In this area there were low invertebrate megafaunal densities (0.08 and 0.10 individuals m⁻²) in comparison to pre-drill conditions (0.21 individuals m⁻²). Three years later the visible extent of the cuttings had reduced, reaching 60 m from the well. Within this area the megafaunal density (0.05 individuals m⁻²) was lower than pre-drill and reference transects (0.23 individuals m⁻²). There was a significant increase in total megafaunal invertebrate densities with both distance from drilling and time since drilling although no significant interaction. Beyond the visible disturbance there were similar megafaunal densities (0.14 individuals m⁻²) to pre-drilling and background surveys. Species richness, Shannon-Weiner diversity and multivariate techniques showed similar patterns to density. At this site the effects of exploratory drilling on megafaunal invertebrate density and diversity seem confined to the extent of the visible cuttings pile. However, elevated Barium concentration and reduced sediment grain size suggest persistence of disturbance for three years, with unclear consequences for other components of the benthic fauna.     

A comparison of the relative contributions of temporal and spatial variation in the density of drifting invertebrates in a Dorset (U.K.) chalk stream

1. Invertebrate drift is commonly investigated in streams, with the majority of studies focussed on temporal (typically diel) variation. In comparison, few studies have investigated spatial variation in drift and there is little consensus among them. We tested the hypothesis that spatial variation in invertebrate drift is as important as temporal variation. 2. The density of drifting invertebrates in a chalk stream was sampled using an array of nets arranged to determine vertical, lateral and longitudinal variation. Samples were collected at dawn, during the day, at dusk and by night, on four separate monthly occasions. Insecta and Crustacea were analysed separately to identify the effect of differing life history strategies. The density of drifting debris was also recorded, to act as a null model. 3. Time of day and vertical position together explained the majority of the variance in invertebrate drift (79% for Insecta and 97% for Crustacea), with drift densities higher at dusk and night, and nearer the stream bed. Independently, time of day (38%, Insecta; 52%, Crustacea) and vertical position (41%, Insecta; 45%, Crustacea) explained a similar amount of the observed variance. Month explained some of the variance in insect drift (9%) but none for Crustacea. 4. Variation in the density of drifting debris showed little in common with invertebrate drift. There was little variation associated with time of day and only 27% of the observed variation in debris could be explained by the factors investigated here, with month explaining the largest proportion (20%). We suggest the difference in drifting debris and invertebrates provides further evidence for a strong behavioural component in invertebrate drift. 5. Spatial variation in invertebrate drift can be of the same order of magnitude as the much‐described diel temporal variation. The extent of this spatial variation poses problems when attempting to quantify invertebrate drift and we recommend that spatial replication should be incorporated into drift studies.     

Investigating the establishment of primary cell culture from different abalone (<Emphasis Type="Italic">Haliotis midae</Emphasis>) tissues

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.     

Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process developments and economics

Large-scale, renewable supplies of chemical constituents derived from marine invertebrates have limited development of potential new natural product drugs. This paper describes the development of two in-sea aquaculture systems designed and engineered for production of large quantities of biomass for two species of marine invertebrates desired for their natural product chemical constituents. The two invertebrates and their products were: (1) the cosmopolitan, arborescent bryozoan Bugula neritina (Phylum Bryozoa) for its anticancer chemical constituent bryostatin 1; and (2) Ecteinascidia turbinate (Phylum Tunicata) the source of anticancer ecteinascidin 743. For the third invertebrate Phylum Porifera, and its representative sponge Acanthella cavernosa (desired for its anti-parasitic and anti-infective kalihinols) in-sea systems were not developed in favor of controlled environment tank aquaculture systems. For the bryozoan and tunicate, projected economics for commercial-scale in-sea production proved cost effective. This was in contrast to the controlled environment sponge culture tank system, which did not prove to be economical due to inherent slow growth and low natural product yields of the sponge in culture. A non-destructive method for "milking" natural product chemicals from sponges was tested and is described.