Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity.     

SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes

The lower deuterostomes, including the echinoderms, possess an innate immune system that includes a subsystem with similarities to the vertebrate complement system. A homologue of the central component of this system, C3, has recently been identified in the purple sea urchin, Strongylocentrotus purpuratus, and is called SpC3. We determined previously that coelomocytes specifically express the SpC3 gene (Sp064); however, the sea urchin has at least four different types of coelomocytes: amoeboid phagocytes, red spherule cells, colorless spherule cells, and vibratile cells. To determine which of these subpopulations expresses Sp064 and produces SpC3, coelomocytes were separated by discontinuous gradient density centrifugation. Relatively homogenous fractions were obtained consisting of the four major cell types in addition to two types of amoeboid phagocytes with different densities and distinct morphologies. Analysis of proteins from separated cell subpopulations by Western blot and analysis of gene expression by RT-PCR revealed that phagocytes express the gene and contain the protein. Immunolocalization showed that SpC3+ phagocytes are present as subsets of both the low- and high-density subpopulations of phagocytes; however, the subcellular localization of SpC3 is different in these two subpopulations.     

The 185/333 gene family is a rapidly diversifying host-defense gene cluster in the purple sea urchin Strongylocentrotus purpuratus

The genome of the purple sea urchin contains numerous large gene families with putative immunological functions. One gene family, known as 185/333, is characterized by extraordinary molecular diversity resulting from single nucleotide polymorphisms and the presence or the absence of 27 large blocks of sequences known as elements. The mosaic composition of elements, known as element patterns, that is present within the members of this gene family is encoded entirely in the second of two exons. Many of the elements correspond to one of six types of repeats that are present throughout the genes. The sequence diversity and variation in element patterns led us to investigate the evolution of the 185/333 gene family. The work presented here suggests that the element patterns are the result of both recombination and duplication and/or deletion of intragenic repeats. Each element is composed of a limited number of similar but distinct sequences, and their distribution among the 185/333 genes suggests frequent recombination within this gene family. Phylogenetic analyses of five 185/333 elements and two regions of the intron were performed using two tests: incongruence length difference and incongruence permutation. Results indicated that each pair of sequence segments was incongruent, suggesting that recombination occurs frequently along the length of the genes, including both the intron and the second exon, and that recombination is not restricted to intact elements. Paradoxically, the high level of similarity among the elements indicated that the 185/333 genes appear to be the result of a recent diversification. These results add to the growing body of evidence suggesting that invertebrate immune systems are not simple and static, but are dynamic and highly complex, and may employ group-specific mechanisms for diversification.     

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.     

Agrobacterium-mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.     

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.     

Patterns of Mass Mortality among Rocky Shore Invertebrates across 100 km of Northeastern Pacific Coastline

Mass mortalities in natural populations, particularly those that leave few survivors over large spatial areas, may cause long-term ecological perturbations. Yet mass mortalities may remain undocumented or poorly described due to challenges in responding rapidly to unforeseen events, scarcity of baseline data, and difficulties in quantifying rare or patchily distributed species, especially in remote or marine systems. Better chronicling the geographic pattern and intensity of mass mortalities is especially critical in the face of global changes predicted to alter regional disturbance regimes. Here, we couple replicated post-mortality surveys with preceding long-term surveys and historical data to describe a rapid and severe mass mortality of rocky shore invertebrates along the north-central California coast of the northeastern Pacific Ocean. In late August 2011, formerly abundant intertidal populations of the purple sea urchin (Strongylocentrotus purpuratus, a well-known ecosystem engineer), and the predatory six-armed sea star (Leptasterias sp.) were functionally extirpated from ~100 km of coastline. Other invertebrates, including the gumboot chiton (Cryptochiton stelleri) the ochre sea star (Pisaster ochraceus), and subtidal populations of purple sea urchins also exhibited elevated mortality. The pattern and extent of mortality suggest the potential for long-term population, community, and ecosystem consequences, recovery from which may depend on the different dispersal abilities of the affected species.     

Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus

Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases.     

Echinoid phagocytes in vitro

A method is described for obtaining pure monolayers of phagocytes from the sea urchin Strongylocentrotus droebachiensis in vitro. The coelomic fluid contains four types of cells. About 67% of the cells are phagocytes, the rest is comprised of the red and white morula cells and the vibratile cells. The different cell types could be separated by centrifugation on a discontinuous gradient of sodium metrizoate. Release of granula from the vibratile cells was found to be responsible for rapid and extensive clotting of the coelomic fluid immediately after its removal from the animal. Clotting was prevented by adding a mixture of 50 mM mercaptoethanol, 3 mM caffeine and 2 mM TAME (p-tosyl- -arginine methyl ester) to the coelomic fluid. The phagocytes were isolated from other cell types by their attachment to glass, and were grown at 10 °C in a simple peptone-sea water medium. The phagocytes are very motile cells and spread rapidly on glass, accompanied by a complete change of their morphology to flattened cells with peripheral ruffling. After few hours in vitro the cells fuse to form monolayer-syncytia, and later still cell clusters and free floating balls of cells are formed. During a culture period of 10 days there was no change in the DNA content per culture, while a small increase in protein was found.     

Manipulation of Developing Juvenile Structures in Purple Sea Urchins (Strongylocentrotus purpuratus) by Morpholino Injection into Late Stage Larvae

Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.     

Primary cell cultures from sea urchin ovaries: a new experimental tool

In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.