Increasing comparability among coral bleaching experiments.

Coral bleaching is the single largest global threat to coral reefs worldwide. Integrating the diverse body of work on coral bleaching is critical to understanding and combating this global problem. Yet investigating the drivers, patterns, and processes of coral bleaching poses a major challenge. A recent review of published experiments revealed a wide range of experimental variables used across studies. Such a wide range of approaches enhances discovery, but without full transparency in the experimental and analytical methods used, can also make comparisons among studies challenging. To increase comparability but not stifle innovation, we propose a common framework for coral bleaching experiments that includes consideration of coral provenance, experimental conditions, and husbandry. For example, reporting the number of genets used, collection site conditions, the experimental temperature offset(s) from the maximum monthly mean (MMM) of the collection site, experimental light conditions, flow, and the feeding regime will greatly facilitate comparability across studies. Similarly, quantifying common response variables of endosymbiont (Symbiodiniaceae) and holobiont phenotypes (i.e., color, chlorophyll, endosymbiont cell density, mortality, and skeletal growth) could further facilitate cross-study comparisons. While no single bleaching experiment can provide the data necessary to determine global coral responses of all corals to current and future ocean warming, linking studies through a common framework as outlined here, would help increase comparability among experiments, facilitate synthetic insights into the causes and underlying mechanisms of coral bleaching, and reveal unique bleaching responses among genets, species, and regions. Such a collaborative framework that fosters transparency in methods used would strengthen comparisons among studies that can help inform coral reef management and facilitate conservation strategies to mitigate coral bleaching worldwide.

Identification of biomarkers indicative of barotrauma and recovery in black rockfish Sebastes melanops.

A Sebastes-specific complementary DNA (cDNA) microarray was developed to identify potential biomarkers involved in the capture stress and recovery of Sebastes species if they are assisted in returning to their original depth of capture following barotrauma. Black rockfish Sebastes melanops were exposed to simulated decompression from 450 kPa (c. 35 m depth) (which resulted in barotrauma) and subsequent recompression. Sebastes melanops were sampled for liver tissue at days 3, 15 and 31 post-barotrauma. Potential candidate genes were identified from the microarray and then quantitative real-time PCR (qrt-PCR) was used to validate expression levels in biological replicates. Six potential biomarkers associated with the innate immune system were identified that were up-regulated in liver tissue at 3 days post-barotrauma: complement C1q-like protein 2, complement component C3, complement regulatory plasma protein, serum amyloid A-5, c-type lysozyme and hepcidin precursor type I. In addition, complement c1q was correlated to the presence of a ruptured swimbladder, providing further support that this gene may be a good biomarker of injury and recovery. Immune genes were no longer up-regulated at day 31 post-barotrauma, a good indication of recovery in S. melanops.

Comparative proteomics of symbiotic and aposymbiotic juvenile soft corals.

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral-algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association.

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.

Symbiosis-enhanced gene expression in cnidarian-algal associations: cloning and characterization of a cDNA, sym32, encoding a possible cell adhesion protein.

Mutualistic endosymbioses between two partners are complex associations that are regulated by the genetic interactions of the partners. One important marine symbiosis is that between various cnidarians, such as corals and anemones, and their photosynthetic algal symbionts. We have been interested in characterizing cnidarian host genes that are expressed as a function of the symbiotic state, using the temperate sea anemone Anthopleura elegantissima as a model. In this study, we report on symbiosis-enhanced expression and synthesis of sym32 in anemones. We characterized the full-length sym32 cDNA, obtained by RT-PCR, and demonstrated, by semi-quantitative RT-PCR, that sym32 transcript was much more abundant in symbiotic than in non-symbiotic host anemone RNA. Further, using immunoblots, we determined that an antibody made to a sym32 fusion protein labeled a 32 kD band much more strongly in symbiotic compared to non-symbiotic anemone protein homogenates. Databank searches revealed that the sym32 deduced amino acid sequence shares significant homology with the fasciclin I (Fas I) family of homophilic cell adhesion proteins, present in a variety of organisms ranging from bacteria to humans. This strong homology with the Fas I family suggests that sym32 is involved in regulation of the symbiosis by mediating cell-cell interactions.

Carbonic anhydrase expression and synthesis in the sea anemone Anthopleura elegantissima are enhanced by the presence of dinoflagellate symbionts.

Endosymbiotic dinoflagellates resident within cnidarian hosts are extremely productive primary producers. This high productivity may be due in part to an inorganic carbon transport system, present in host tissue, that accelerates carbon delivery to the algae. The enzyme carbonic anhydrase (CA; EC 4.2.1.1) has been shown to be important in this transport system in a variety of tropical symbiotic cnidarians. This study extends the examination of CA to a temperate anemone, Anthopleura elegantissima, and documents symbiosis-enhanced production of CA at the biochemical and molecular level. Depending on light availability, A. elegantissima can occur naturally with (symbiotic) or without (aposymbiotic) dinoflagellates, making it an ideal study organism for symbiosis-enhanced gene expression. We compared (1) CA activities, (2) quantities of CA using an anti-human CA immunoprobe, and (3) quantities of transcript using a semiquantitative PCR in symbiotic versus aposymbiotic A. elegantissima host tissue. Amounts of activity, enzyme, and transcript were greatly enhanced in symbiotic animals compared with aposymbiotic animals. This is the first direct evidence that the presence of symbionts affects the expression of a host cnidarian gene. In addition, we report a full-length A. elegantissima CA cDNA sequence, obtained from subcloned reverse transcriptase-PCR products, and its relatedness to alpha-CAs from a variety of other metazoa, including higher vertebrates.

Late Larval Development and Onset of Symbiosis in the Scleractinian Coral Fungia scutaria.

Many corals that harbor symbiotic algae (zooxanthellae) produce offspring that initially lack zooxanthellae. This study examined late larval development and the acquisition of zooxanthellae in the scleractinian coral Fungia scutaria, which produces planula larvae that lack zooxanthellae. Larvae reared under laboratory conditions developed the ability to feed 3 days after fertilization; feeding behavior was stimulated by homogenized Artemia. Larvae began to settle and metamorphose 5 days after fertilization. In laboratory experiments, larvae acquired experimentally added zooxanthellae by ingesting them while feeding. Zooxanthellae entered the gastric cavity and were phagocytosed by endodermal cells. As early as 1 h after feeding, zooxanthellae were observed in both endodermal and ectodermal cells. Larvae were able to form an association with three genetically distinct strains of zooxanthellae. Both zooxanthellate and azooxanthellate larvae underwent metamorphosis, and azooxanthellate polyps were able to acquire zooxanthellae from the environment. Preliminary evidence suggests that the onset of symbiosis may influence larval development; in one study symbiotic larvae settled earlier than aposymbiotic larvae. Protein profiles of eggs and larvae throughout development revealed a putative yolk protein doublet that was abundant in eggs and 1-day-old larvae and was absent by day 6. This study is the first to examine the onset of symbiosis between a motile cnidarian host and its algal symbiont.

A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism.

Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking. In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri. Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils. MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ. We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity. An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis. These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.

Abundant mRNAs in the squid light organ encode proteins with a high similarity to mammalian peroxidases.

A library derived from mRNA in the bacterial light organ of the squid, Euprymna scolopes, contained an unexpectedly high proportion of cDNAs that encode proteins with approximately 30% similarity to a family of mammalian peroxidases (PO) including myelo-PO, eosinophil PO, and thyroid PO (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7). Two nearly full-length cDNAs were determined to encode putative PO of nearly 93 kDa each that are 97% identical in amino acid sequence to each other. Each contains four potential glycosylation sites, and His416, believed to be within the active site of the human PO, is conserved in the putative PO from the squid light organ. The mRNAs for the putative squid PO were approximately 250 times more abundant in the tissue housing the bacterial symbiont than in the ocular lens or mantle and were undetectable in the light organ lens. By analogy with the bacteriocidal function of PO in mammalian neutrophils, the putative squid PO may be important for modulating or limiting the population of bacteria within the light organ. The possibility that the squid light organ contains a high concentration of PO raises the possibility that the light organ lens is under oxidative stress, providing a possible rationale for the recruitment of its aldehyde dehydrogenase-like crystallin.

Enhanced Production of ALDH-Like Protein in the Bacterial Light Organ of the Sepiolid Squid Euprymna scolopes.

We localized one or more aldehyde dehydrogenase (ALDH)-like proteins in the bacterially bioluminescent light organ of the sepiolid squid Euprymna scolopes, and determined the temporal changes in expression through normal light organ development. Our previous studies have revealed that 70% of the total protein in the light organ lens of adult animals is comprised of an ALDH-like protein, which we called L-crystallin. In the present study, antibodies raised to this protein were used in immunocytochemical analyses which showed that, in adult light organ lens cells, ALDH-like protein was localized to the cytoplasm, but not to the nuclei or mitochondria. Labeling in adult tissue was also found in moderate abundance in the ciliated duct epithelium, a tissue that is in direct contact with the bacterial symbionts. To determine the spatial and temporal onset of expression of ALDH-like protein(s), we examined light organs from juveniles at developmental stages before and after the differentiation of lens cells, which begins approximately 7-10 days after hatching. In 5-day symbiotic juvenile light organs, ALDH-like protein was not detected at levels significantly above those in non-symbiotic tissue of the same animals. However, expression of ALDH-like protein began within 10 days after hatching, seen first in a few cells of the ciliated duct, adjacent to the symbiont-containing tissue and in a few differentiated cells of the anterior presumptive light organ lens. These data suggest that, during normal development, induction of one or more ALDH-like proteins occurs simultaneously in both the lens and ciliated duct soon after the differentiation of lens cells.

The Induction of Carbonic Anhydrase in the Symbiotic Sea Anemone Aiptasia pulchella.

The activity and nature of carbonic anhydrase (CA, EC 4.2.1.1 .) was measured and described in the tropical sea anemone Aiptasia pulchella. The hypothesis that high CA activity in animal tissue is induced by the presence of symbiotic algae was tested. CA activity was positively correlated with the number of symbiotic dinoflagellates (zooxanthellae) present. CA activity in aposymbiotic anemone tissue was 2.5 times lower than that in control symbiotic animals or in aposymbiotic animals repopulated with algae. Polyclonal antisera against human CA were used to probe for the presence of CA in both symbiotic and aposymbiotic anemone tissue, and in freshly isolated and cultured zooxanthellae. The resulting immunoblots showed one band with a molecular weight of 30 kDa in symbiotic animal tissue and control mammalian CA lanes, no bands in the aposymbiotic animal lanes, and one band at a molecular weight of 22.5 kDa in freshly isolated and cultured zooxanthellae lanes. Because no 22.5 kDa band was detected in the symbiotic animal tissue lanes, the high CA activity found in symbiotic animal tissue is considered to be due to the induction of animal enzyme by the presence of algae. The lack of any band in the aposymbiotic lanes further supports the hypothesis that CA activity in A. pulchella is induced by the presence of algae.