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Spotting disease infects a variety of sea urchin species across many different marine locations. The disease is characterized by discrete lesions on the body surface composed of discolored necrotic tissue that cause the loss of all surface appendages within the lesioned area. A similar, but separate disease of sea urchins called bald sea urchin disease (BSUD) has overlapping symptoms with spotting disease, resulting in confusions in distinguishing the two diseases. Previous studies have focus on identifying the underlying causative agent of spotting disease, which has resulted in the identification of a wide array of pathogenic bacteria that vary based on location and sea urchin species. Our aim was to investigate the spotting disease infection by characterizing the microbiomes of the animal surface and various tissues.

We collected samples of the global body surface, the lesion surface, lesioned and non-lesioned body wall, and coelomic fluid, in addition to samples from healthy sea urchins. 16S rRNA gene was amplified and sequenced from the genomic DNA. Results show that the lesions are composed mainly of Cyclobacteriaceae, Cryomorphaceae, and a few other taxa, and that the microbial composition of lesions is the same for all infected sea urchins. Spotting disease also alters the microbial composition of the non-lesioned body wall and coelomic fluid of infected sea urchins. In our closed aquarium systems, sea urchins contracted spotting disease and BSUD separately and therefore direct comparisons could be made between the microbiomes from diseased and healthy sea urchins.

Results show that spotting disease and BSUD are separate diseases with distinct symptoms and distinct microbial compositions.

 

Bald sea urchin disease (BSUD) is most likely a bacterial infection that occurs in a wide range of sea urchin species and causes the loss of surface appendages. The disease has a variety of additional symptoms, which may be the result of the many bacteria that are associated with BSUD. Previous studies have investigated causative agents of BSUD, however, there are few reports on the surface microbiome associated with the infection. Here, we report changes to the surface microbiome on purple sea urchins in a closed marine aquarium that contracted and then recovered from BSUD in addition to the microbiome of healthy sea urchins in a separate aquarium. 16S rRNA gene sequencing shows that microhabitats of different aquaria are characterized by different microbial compositions, and that diseased, recovered, and healthy sea urchins have distinct microbial compositions, which indicates that there is a correlation between microbial shifts and recovery from disease.

 

The SpTransformer ( SpTrf ) gene family encodes a set of proteins that function in the sea urchin immune system. The gene sequences have a series of internal repeats in a mosaic pattern that is characteristic of this family. This mosaic pattern necessitates the insertion of large gaps, which has made alignments of the deduced protein sequences computationally difficult such that only manual alignments have been reported previously. Because manual alignments are time consuming for evaluating newly available SpTrf sequences, computational approaches were evaluated for the sequences reported previously. Furthermore, because two different manual alignments of the SpTrf sequences are feasible because of the multiple internal repeats, it is not known whether additional alternative alignments can be identified using different approaches. The bioinformatic program, PRANK, was used because it was designed to align sequences with large gaps and indels. The results from PRANK show that the alignments of the internal repeats are similar to those done manually, suggesting multiple feasible alignments for some regions. GUIDANCE based analysis of the alignments identified regions that were excellent and other regions that failed to align. This suggests that computational approaches have limits for aligning the SpTrf sequences that include multiple repeats and that require inserted gaps. Furthermore, it is unlikely that alternative alignments for the full-length SpTrf sequences will be identified. 

The sea urchin, Strongylocentrotus purpuratus has seven described populations of distinct coelomocytes in the coelomic fluid that are defined by morphology, size, and for some types, by known functions. Of these subtypes, the large phagocytes are thought to be key to the sea urchin cellular innate immune response. The concentration of total coelomocytes in the coelomic fluid increases in response to pathogen challenge. However, there is no quantitative analysis of how the respective coelomocyte populations change over time in response to immune challenge. Accordingly, coelomocytes collected from immunoquiescent, healthy sea urchins were evaluated by flow cytometry for responses to injury and to challenge with either heat-killed Vibrio diazotrophicus , zymosan A, or artificial coelomic fluid, which served as the vehicle control. Responses to the initial injury of coelomic fluid collection or to injection of V. diazotrophicus show significant increases in the concentration of large phagocytes, small phagocytes, and red spherule cells after one day. Responses to zymosan A show decreases in the concentration of large phagocytes and increases in the concentration of small phagocytes. In contrast, responses to injections of vehicle result in decreased concentration of large phagocytes. When these changes in coelomocytes are evaluated based on proportions rather than concentration, the respective coelomocyte proportions are generally maintained in response to injection with V. diazotrophicus and vehicle. However, this is not observed in response to zymosan A and this lack of correspondence between proportions and concentrations may be an outcome of clearing these large particles by the large phagocytes. Variations in coelomocyte populations are also noted for individual sea urchins evaluated at different times for their responses to immune challenge compared to the vehicle. Together, these results demonstrate that the cell populations in sea urchin immune cell populations undergo dynamic changes in vivo in response to distinct immune stimuli and to injury and that these changes are driven by the responses of the large phagocyte populations. 

Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.