Aggregation of Sea Urchin Phagocytes Is Augmented In Vitro by Lipopolysaccharide

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only similar to 10% of the cells were positive for Sp185/333 proteins. At 24 hr, similar to 90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures

El primer genoma mitocondrial completo de Diadema antillarum (Diadematoida, Diadematidae)

The mitochondrial genome of the long-spined black sea urchin, Diadema antillarum, was sequenced using Illumina next-generation sequencing technology. The complete mitogenome is 15,708 bp in length, containing two rRNA, 22 tRNA and 13 protein-coding genes, plus a noncoding control region of 133 bp. The nucleotide composition is 18.37% G, 23.79% C, 26.84% A and 30.99% T. The A + T bias is 57.84%. Phylogenetic analysis based on 12 complete mitochondrial genomes of sea urchins, including four species of the family Diadematidae, supported familial monophyly; however, the two Diadema species, D. antillarum and D. setosum were not recovered as sister taxa.El genoma mitocondrial del erizo de mar negro de espinas largas, Diadema antillarum, se secuenció utilizando la tecnología de secuenciación de nueva generación de Illumina. El mitogenoma completo tiene un tamaño de 15,708 pb, que contiene dos ARNr, 22 ARNt y 13 genes codificadores de proteínas, además de una región de control no codificante de 133 pb. La composición de nucleótidos es 18.37% G, 23.79% C, 26.84% A y 30.99% T. El sesgo A+T es del 57.84%. El análisis filogenético basado en 12 genomas mitocondriales completos de erizos de mar, incluyendo cuatro especies de la familia Diadematidae, apoya la monofilia familiar. Sin embargo,  las dos especies de Diadema en este estudio,  D. antillarum y D. setosum no fueron identificadas como taxones hermanos

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology

Aggregation of sea urchin phagocytes is augmented in vitro by lipopolysaccharide.

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ~10% of the cells were positive for Sp185/333 proteins. At 24 hr, ~90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures

Viability of phagocytes from the purple sea urchin in short-term cultures.

<p>Viability decreases from 91.6 to 57.0% over six days. Standard error of the mean (SEM) are shown.</p

The percentage of nuclei associated with Sp185/333 proteins increases when phagocytes are exposed to LPS in culture.

<p>Coelomocytes from Iq animal #2 were settled for 1 hr onto culture well plates and incubated with 0 µg (A) 10 µg (B) or 100 µg (C) LPS/ml ECCM for different times. D shows combined data from A, B and C. There is a significant increase in the proportion of nuclei associated with Sp185/333 proteins within syncytia-like structures vs. non-aggregated Sp185/333⁺ phagocytes after 3 h and 24 hr exposure to 10 (B) or 100 (C) µg LPS/ml, compared to results for 1 hr (asterisks; <i>P<</i>0.05). Significant differences are shown in D where data points are marked with a different number (1 or 2). Lines in D with the same number are not significantly different. SEM are shown.</p

A syncytium-like structure contains both tightly packed and more evenly dispersed nuclei at 24 hr.

<p>Settled phagocytes were processed for immunocytology and stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061419#pone-0061419-g003" target="_blank">Figure 3</a>. Merged images are shown in D and H. Arrows in A mark the direction of actin cables that cross the structure. Sp185/333 proteins (red) associated with syncytia-like structures are present in the center of B. Tightly packed nuclei (smaller circle in C) and more evenly dispersed nuclei (larger circle in C) are both present within a syncytium-like structure. Sp185/333 proteins are associated with the clustered nuclei in a syncytium-like structure (E–H). Scale bars are 10 µm.</p

Syncytia-like structures are present after 5 hr of incubation.

<p>Settled phagocytes were processed for immunocytology and stained and labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061419#pone-0061419-g003" target="_blank">figure 3</a>. Structures contain clustered nuclei associated with Sp185/333 proteins. Arrows in B indicate Sp185/333 proteins (red) in syncytia-like structures. In C, a group of tightly packed nuclei is circled, which are associated with Sp185/333 proteins (D, merged). Scale bar is 10 µm.</p

Phagocytes aggregate into syncytia-like structures after 3 hr of incubation.

<p>Settled phagocytes were incubated for 3 hr in ECCM, fixed and labeled for actin (green, A, E), Sp185/333 (red, B, F) and DNA (blue, C, G). Merged images are shown in D and H. The morphology of different types of phagocytes is no longer recognizable (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061419#pone-0061419-g002" target="_blank">Figure 2</a>). Sp185/333 proteins (arrow in B, F) within syncytia-like structures are presumably in perinuclear vesicles, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061419#pone.0061419-Brockton1" target="_blank">[24]</a>. Scale bars are 10 µm.</p

The proportion of nuclei associated with Sp185/333 proteins in cultured phagocytes increases after exposure to LPS.

<p>Phagocytes from Iq animals (<i>n</i> = 4) were incubated with 0 µg (A), 10 µg (B), 50 µg (C), or 100 µg (D) LPS/ml in ECCM over time. The percentage of nuclei associated with Sp185/333 proteins is significantly higher after incubation with 100 µg LPS/ml (D) compared to 0 (A), 10 (B) or 50 (C) µg LPS/ml (<i>P<</i>0.05). There is a significant increase in the percentages of nuclei associated with Sp185/333 proteins when cultures are incubated with LPS (all concentrations) for ON to 48 hr compared to cultures incubated for shorter periods of up to 3 hr (brackets with asterisks; <i>P<</i>0.05). ON indicates 16–21 hr. SEM are shown.</p