EFECTOS ADVERSOS MORFOLÓGICOS EN EXPOSICIÓN HIPERAGUDA A LODOS DE PERFORACIÓN EN BASE AGUA EN Hydractinia symbiolongicarpus (Familia: Hydractiniidae)

Los desechos derivados del proceso de perforación en las plataformas marinas petroleras pueden dejar hasta 200 000 toneladas de residuos de perforación en los ecosistemas marinos. El principal agente contaminante son los lodos de perforación petrolera (LPPs), que son usados para enfriar y lubricar la broca de perforación, además de contrarrestar fuerzas de presión y posibles fugas de hidrocarburos. Se sabe que los LPPs tienen un efecto contaminante y tóxico observándose alteraciones en los ecosistemas y efectos adversos en organismos invertebrados marinos, debidos principalmente a la presencia de barita y trazas de metales pesados. En este trabajo se realizaron experimentos de exposición hiperagudos con mezclas completas de lodos de perforación base agua (WBM) a máximos niveles de concentración en colonias de Hydractinia symbiolongicarpus. Los resultados mostraron cambios significativos (p < 0,001) en la morfología de los pólipos inmediatamente después del contacto con WBM, con un incremento 1,5 veces en el diámetro del pólipo y una retracción en la longitud de los tentáculos del 75 %. Después de la exposición (fase de recuperación), se observó una disminución de biomasa a las 72 h con pérdida del 50 % de los pólipos y una reducción de la mata estolonal cercana al 50 % (p < 0,01). Efectos similares han sido reportados en otras especies de cnidarios, como los corales, donde se observó retracción de pólipos y zonas expuestas de exoesqueleto. La exposición a WBM genera irritación tisular en exposición directa y en casos severos pérdida de biomasa.

New self-identities evolve via point mutation in an invertebrate allorecognition gene

SummaryMany organisms use genetic self-recognition systems to distinguish themselves from other members of their species. To understand how new self-identities evolve, we studied Allorecognition 2 (Alr2), a self-recognition gene from the colonial cnidarian, Hydractinia symbiolongicarpus. Alr2 encodes a highly polymorphic transmembrane protein that discriminates self from non-self by selectively binding across cell membranes to other Alr2 proteins with identical or very similar sequences. Here, we show that new Alr2 proteins evolve by amino acid substitutions that immediately create isoforms with entirely novel binding specificities, or through intermediates with relaxed binding specificities. Our results also suggest a topology for homophilic interactions between Alr2 proteins. These results provide direct evidence for the generation and maintenance of functional variation at an allorecognition locus and reveal that one-component and two-component self-recognition systems evolve via different mechanisms.

Cryopreservation of Hydractinia symbiolongicarpus sperm to support community-based repository development for preservation of genetic resources

Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. However, one limitation of this model system is the lack of long-term storage for genetic resources. Our goal in this study was to establish a generalizable approach to sperm cryopreservation that would support future repository development and could be applied to many species according to available resources. Our approach was to: 1) Assess sperm characteristics and standardize collection and processing; 2) Assess acute toxicity to cryoprotectants, and 3) Evaluate and refine freezing conditions to permit post-thaw fertilization and produce viable offspring. By following this approach, we found that Hydractinia sperm incubated in 5% DMSO, equilibrated at 4°C for 20 min, and cooled at a rate of 20°C/min to - 80°C at a cell concentration of 108-109/mL in 0.25-mL aliquots were able to fertilize 150-300 eggs which yielded offspring that could metamorphose into juvenile polyps. In addition, improvements were made for processing sperm using a customized 3-D printed collection system. Other opportunities for improvement include optimizing the volumetric sperm-to-egg ratio for fertilization. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, this application-based approach highlights the long-term value of establishing repository-level resources that can be expanded to fit community needs.

Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Performing gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Biofilms and Extracts from Bacteria Producing "Quorum Sensing" Signaling Molecules Promote Chemotaxis and Settlement Behaviors in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa) Larvae

Many sessile marine invertebrates have life cycles involving the development of larvae that settle on specific substrates to initiate metamorphosis to juvenile forms. Although is recognized that bacterial biofilms play a role in this process, the responsible chemical cues are beginning to be investigated. Here, we tested the role of substrate-specific bacteria biofilms and their Quorum Sensing Signaling Molecule (QSSM) extracts on chemotaxis and settlement of larvae from Hydractinia symbiolongicarpus, a hydroid that grows on gastropod shells occupied by hermit crabs. We isolated and taxonomically identified by 16S rDNA sequencing, 14 bacterial strains from shells having H. symbiolongicarpus. Three isolates, Shigella flexneri, Microbacterium liquefaciens, and Kocuria erythromyxa, were identified to produce QSSMs using biosensors detecting N-acyl-L-homoserine lactones. Multispecies biofilms and QSSM extracts from these bacteria showed a positive chemotactic effect on H. symbiolongicarpus larvae, a phenomenon not observed with mutant strains of E. coli and Chromobacterium violaceum that are unable to produce QSSMs. These biofilms and QSSMs extracts induced high rates of larval attachment, although only 1 % of the attached larvae metamorphosed to primary polyps, in contrast to 99 % of larvae incubated with CsCl, an artificial inductor of attachment and metamorphosis. These observations suggest that bacterial QSSMs participate in H. symbiolongicarpus substrate selection by inducing larval chemotaxis and attachment. Furthermore, they support the notion that settlement in cnidarians is decoupled into two processes, attachment to the substrate and metamorphosis to a primary polyp, where QSSMs likely participate in the former but not in the latter.

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

BackgroundHydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs.ResultsHere, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies.ConclusionsThis is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.