Symbiosis maintenance in the facultative coral, Oculina arbuscula, relies on nitrogen cycling, cell cycle modulation, and immunity.

Symbiosis with unicellular algae in the family Symbiodiniaceae is common across tropical marine invertebrates. Reef-building corals offer a clear example of cellular dysfunction leading to a dysbiosis that disrupts entire ecosystems in a process termed coral bleaching. Due to their obligate symbiotic relationship, understanding the molecular underpinnings that sustain this symbiosis in tropical reef-building corals is challenging, as any aposymbiotic state is inherently coupled with severe physiological stress. Here, we leverage the subtropical, facultatively symbiotic and calcifying coral Oculina arbuscula to investigate gene expression differences between aposymbiotic and symbiotic branches within the same colonies under baseline conditions. We further compare gene ontology (GO) and KOG enrichment in gene expression patterns from O. arbuscula with prior work in the sea anemone Exaiptasia pallida (Aiptasia) and the salamander Ambystoma maculatum-both of which exhibit endophotosymbiosis with unicellular algae. We identify nitrogen cycling, cell cycle control, and immune responses as key pathways involved in the maintenance of symbiosis under baseline conditions. Understanding the mechanisms that sustain a healthy symbiosis between corals and Symbiodiniaceae algae is of urgent importance given the vulnerability of these partnerships to changing environmental conditions and their role in the continued functioning of critical and highly diverse marine ecosystems.

Level of UV-B radiation influences the effects of glyphosate-based herbicide on the spotted salamander.

Glyphosate-based herbicides are the number one pesticide in the United States and are used commonly around the world. Understanding the affects of glyphosate-based herbicides on non-target wildlife, for example amphibians, is critical for evaluation of regulations pertaining to the use of such herbicides. Additionally, it is important to understand how variation in biotic and abiotic environmental conditions, such as UV-B light regime, could potentially affect how glyphosate-based herbicides interact with non-target species. This study used artificial pond mesocosms to identify the effects of generic glyphosate-based herbicide (GLY-4 Plus) on mortality, cellular immune response, body size, and morphological plasticity of larvae of the spotted salamander (Ambystoma maculatum) under conditions that reflect moderate (UV(M)) and low (UV(L)) UV-B light regimes. Survival within a given UV-B level was unaffected by herbicide presence or absence. However, when herbicide was present, survival varied between UV-B levels with higher survival in UV(M) conditions. Herbicide presence in the UV(M) treatments also decreased body size and reduced cellular immune response. In the UV(L) treatments, the presence of herbicide increased body size and affected tail morphology. Finally, in the absence of herbicide, body size and cellular immune response were higher in UV(M) treatments compared to UV(L) treatments. Thus, the effects of herbicide on salamander fitness were dependent on UV-B level. As anthropogenic habitat modifications continue to alter landscapes that contain amphibian breeding ponds, salamanders may increasingly find themselves in locations with reduced canopy cover and increased levels of UV light. Our findings suggest that the probability of surviving exposure to the glyphosate-based herbicide used in this study may be elevated in more open canopy ponds, but the effects on other components of fitness may be varied and unexpected.

Toxicity of coal-tar pavement sealants and ultraviolet radiation to Ambystoma Maculatum.

Polycyclic aromatic hydrocarbons (PAHs) can affect amphibians in lethal and many sublethal ways. There are many natural and anthropogenic sources of PAHs in aquatic environments. One potentially significant source is run off from surfaces of parking lots and roads that are protected with coal tar sealants. Coal tar is 50% or more PAH by wet weight and is used in emulsions to treat these surfaces. Break down of sealants can result in contamination of nearby waters. The toxicity of PAHs can be greatly altered by simultaneous exposure to ultraviolet radiation. This study exposes larvae of the spotted salamander (Ambystoma maculatum) to determine if coal tar sealant can have negative effects on aquatic amphibians and if coal tar toxicity is influenced by ultraviolet radiation. Spotted salamanders were exposed to 0, 60, 280 and 1500 mg coal tar sealant/kg sediment for 28 days. Half of the animals were exposed to conventional fluorescent lighting only and half were exposed to fluorescent lighting plus ultraviolet radiation. No significant mortality occurred during the experiment. Exposure to sealants resulted in slower rates of growth, and diminished ability to swim in a dose-dependent fashion. Exposure to ultraviolet radiation affected the frequencies of leukocytes and increased the incidence of micronucleated erythrocytes. There was an interactive effect of sealant and radiation on swimming behavior. We conclude that coal-tar sealant and ultraviolet radiation increased sublethal effects in salamanders, and may be a risk to salamanders under field conditions.

Polymorphism of alternative splicing of major histocompatibility complex transcripts in wild tiger salamanders.

Alternative splicing (AS) of mRNA transcripts is increasingly recognized as a source of transcriptome diversity. To date, most AS studies have focused either on comparisons across taxa or on intragenomic comparisons across gene families. We generated a novel data set that represents one of the first population genetic comparisons of AS across individuals. In ambystomatid salamanders, AS of the major histocompatibility complex (MHC) class IIbeta gene (Amti-DAB) produces two transcripts, one full-length and one truncated. The full-length transcript is functional, but the truncated transcript is missing the critical beta1 domain that forms half of the peptide binding region in the intact MHC class II molecule. We captured wild salamander larvae (Ambystoma tigrinum tigrinum) and genotyped them at Amti-DAB via DNA sequencing. From these same larvae, we extracted RNA from gill and spleen and evaluated the relative expression level of Amti-DAB in each tissue. Across individuals, 21% of the transcripts were truncated (alternatively spliced), and the absolute level of alternative transcript expression was higher in gill. The high level of nucleotide variation among seven Amti-DAB alleles provides the ability to detect substitutions (or linked DNA polymorphisms) that might have influenced AS. The data reveal no correlation between AS and haplotype, allele, or zygosity. However, indirect evidence (comparative expression patterns across 3 million years of evolution) suggests that the truncated Amti-DAB transcript may be functional and maintained by natural selection.

Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens.

Skin peptides were collected from living Ambystoma tigrinum larvae and adults and tested against two emerging pathogens, Batrachochytrium dendrobatidis and the Ambystoma tigrinum virus (ATV), as well as bacteria isolated from A. tigrinum. Natural mixtures of skin peptides were found to inhibit growth of B. dendrobatidis, Staphylococcus aureus, and Klebsiella sp., but activity against ATV was unpredictable. Skin peptides collected from salamanders held at three environmentally relevant temperatures differed in activity against B. dendrobatidis. Activity of the A. tigrinum skin peptides was found to be strongly influenced by pH.

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum).

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, trans-species evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relic of their unique historical demography, not their phylogenetic legacy.

Evaluation of two methods for measuring nonspecific immunity in tiger salamanders (Ambystoma tigrinum).

Study of amphibian immunotoxicology is a growing area of research, but very little information is available on how environmental contaminants affect disease resistance in urodele amphibians. Urodele amphibians lack the more highly evolved aspects of the specific immune system that are present in anurans, birds, and mammals. Instead, these animals rely more heavily on innate defense mechanisms than do anurans to provide rapid, nonspecific protection from pathogens. Thus, it is prudent that immunotoxicologic research with urodele amphibians includes an evaluation of effects of contaminant exposure on nonspecific immunity. The objectives of this study were to measure the phagocytic and oxidative-burst activity of peritoneal neutrophils collected from a urodele, the tiger salamander (Ambystoma tigrinum), and to evaluate the use of these assays in immunotoxicologic research using urodele amphibians. Using tiger salamanders collected in August 2000, phagocytosis and oxidative-burst assays modified from mammalian protocols were conducted through October 2001. Results indicated that large numbers of peritoneal neutrophils for use in immunotoxicologic tests can be collected from salamanders injected with thioglycollate. Moreover, these neutrophils readily engulfed foreign material (phagocytic activity) and produced measurable amounts of hydrogen peroxide (oxidative-burst activity). Phagocytosis was effectively inhibited by incubating cells with sodium azide (P<0.001), and quantification of phagocytosis using flow cytometry was well correlated with manual counts (r=0.84, P<0.001). Dexamethasone treatment reduced phagocytic activity as measured by manual counts (P<0.02), suggesting that this test is useful for detecting alteration by immunosuppressive agents. In contrast, oxidative function was unaffected by dexamethasone treatment, and results from the oxidative-burst assay were generally less consistent than those from the phagocytosis assay. Based on these results, phagocytic activity of peritoneal neutrophils may be a useful endpoint in immunotoxicologic studies to evaluate the impact of environmental contaminants on innate defense mechanisms in urodele amphibians.

Axolotl MHC class II beta chain: predominance of one allele and alternative splicing of the beta1 domain.

The axolotl MHC is composed of multiple polymorphic class I loci linked to class II B loci. In this report, evidence of the existence of one class II B locus (Amme-DAB) that codes for two different transcripts is given. A 2.1-kb transcript is translated to a complete beta chain and a shorter transcript of 1.8 kb encodes a molecule lacking the beta1 domain. For two complete class II B mRNA synthesized, up to one mRNA devoid of the beta1 domain is synthesized. Alternative splicing involving a peptide binding domain at a class II B locus evidenced in axolotl (Ambystoma mexicanum) is also observed for A. tigrinum, the tiger salamander. Very little variability is found among various axolotl MHC class II B cDNA sequences, and the same allele is obtained from inbred and wild axolotls. The transcription of one MHC class B locus in two class II B isoforms in thymic cells and in splenic lymphocytes may shed new light on the well-known deficient immune responder state of the axolotl.

Axolotl MHC architecture and polymorphism.

The MHC of the urodele amphibian Ambystoma mexicanum consists of multiple polymorphic class I loci linked, so far as yet known, to a single class II B locus. This architecture is very different from that of the anuran amphibian Xenopus. The number of class I loci in the axolotl can vary from 6 to 21 according to the haplotypes as shown by cDNA analysis and Southern blot studies in families. These loci can be classified into seven sequence groups with features ranging from the class Ia to the class Ib type. All individuals express genes from at least three of the seven groups, and all individuals possess the class Ia-like type.

Axolotl (Ambystoma mexicanum) lymphocytes produce and are growth-inhibited by transforming growth factor-beta.

Recombinant (r)TGF-beta 5, an isoform of TGF-beta thus far identified only in the frog, Xenopus' laevis, inhibited phytohemagglutinin (PHA)-induced mitogenesis of salamander (axolotl) splenocytes and thymocytes, and T cell growth factor-induced proliferation of splenic lymphoblasts. This inhibition could be reversed by incubating (r)TGF-beta 5 with an anti-TGF-beta 5 antibody, but not with an antibody directed against TGF-beta 2, another Xenopus-produced TGF-beta isoform. Acid-treated supernatants from cultures of PHA-stimulated axolotl splenocytes (PHA-SNs) inhibited proliferation of mink lung fibroblasts (MLF), cells whose growth is sensitive to all known isoforms of TGF-beta. This inhibition was reversed by incubating the supernatants with the anti-pan-specific TGF-beta antibody which neutralizes TGF-beta 1, TGF-beta 2, TGF-beta 3 and TGF-beta 5. This inhibition was unaffected by antibodies that specifically neutralize either TGF-beta 1, TGF-beta 2, TGF-beta 3 or TGF-beta 5. Acid-treated axolotl PHA-SNs also inhibited proliferation of PHA-stimulated freshly harvested axolotl spleen cells. Once again, this inhibition could be reversed by treating supernatants with an anti-pan-specific TGF-beta antibody but not with anti-TGF-beta 2 or anti-TGF-beta 5 antibodies. All these data are consistent with the hypothesis that axolotl lymphocytes secrete a previously unknown TGF-beta isoform.

Wide tissue distribution of axolotl class II molecules occurs independently of thyroxin.

Unlike most salamanders, the Mexican axolotl (Ambystoma mexicanum) fails to produce enough thyroxin to undergo anatomical metamorphosis, although a "cryptic metamorphosis" involving a change from fetal to adult hemoglobins has been described. To understand to what extent the development of the axolotl hemopoietic system is linked to anatomical metamorphosis, we examined the appearance and thyroxin dependence of class II molecules on thymus, blood, and spleen cells, using both flow cytometry and biosynthetic labeling followed by immunoprecipitation. Class II molecules are present on B cells as early as 7 weeks after hatching, the first time analyzed. At this time, most thymocytes, all T cells, and all erythrocytes lack class II molecules, but first thymocytes at 17 weeks, then T cells at 22 weeks, and finally erythrocytes at 26-27 weeks virtually all bear class II molecules. Class II molecules and adult hemoglobin appear at roughly the same time in erythrocytes. These data are most easily explained by populations of class II-negative cells being replaced by populations of class II-positive cells, and they show that the hemopoietic system matures at a variety of times unrelated to the increase of thyroxin that drives anatomical metamorphosis. We found that administration of thyroxin during axolotl ontogeny does not accelerate or otherwise affect the acquisition of class II molecules, nor does administration of drugs that inhibit thyroxin (sodium perchlorate, thiourea, methimazole, and 1-methyl imidazole) retard or abolish this acquisition, suggesting that the programs for anatomical metamorphosis and some aspects of hemopoietic development are entirely separate.

Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl.

The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2.5-, 3.5-, 10-, and 24-month-old animals were sequenced. Six JH segments were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Igh chains in the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolotls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid longer in 2-year-old adult animals (mean: 9.5 residues). Several pairs of identical VDJ/CDR3 sequences were shared between 3.5-month-old individually analyzed axolotls, or between groups of axolotl of different ages. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the probability that different 3.5-month-old axolotl larvae would produce identical junctions seems very low, considering their limited number of B cells (less than 10(5)). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens.

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.

Isolation and characterization of the third complement component of axolotl (Ambystoma mexicanum).

1. Using a monoclonal anti-human C3 antibody and a polyclonal anti-cobra venom factor antibody as probes, a protein homologous to the mammalian third complement component (C3) was purified from axolotl plasma and found to be axolotl C3. 2. Axolotl C3 consists of two polypeptide chains (Mr = 110,000 and 73,000) linked by disulfide bonds. An internal thiolester bond in the alpha chain was identified by the incorporation of [14C]methylamine and NH2-terminal sequence from the C3d fragment of C3. 3. Digestion of C3 by trypsin resulted in the cleavage of both the alpha and beta chains, generating fragments with a cleavage pattern similar to that of human C3. 4. The amino acid composition of axolotl C3 and the amino acid sequences of the thiolester site (and the surrounding amino acids), the cleavage site for the C3-convertase, and one of the factor I cleavage sites are similar to C3 from other vertebrates. 5. In contrast to human C3, which has concanavalin A binding carbohydrates on both the alpha and beta chains, only the beta chain of axolotl C3 contains such carbohydrates.

Characterization of an IgY-like low molecular weight immunoglobulin class in the Mexican axolotl.

The general thinking about the phylogenic distribution of vertebrate Ig classes is that fish and urodele amphibians are only able to synthesize polymeric IgM-like molecules and that the emergence of a new class of LMW Ig occurs for the first time in anouran species. Following immunization of the Mexican axolotl (Ambystoma mexicanum, Amphibia, Urodela) with TNP-SRBC, HMW anti-TNP antibody molecules are only detected. We have previously shown that these polymeric Ig are constituted of 76 kDa H-chains associated to 27-30 kDa L-chains, respectively recognized by MAbs 33.45.1 and 33.101.2. However, the euglobulin fraction purified from normal axolotl serum contains, beside HMW Ig, abundant 172 kDa molecules which are recognized by MAb 33.101.2 in Western blotting in non-reducing conditions but are not labelled with MAb 33.45.1. In the present work, we characterize this 172 kDa molecule as a LMW Ig which differs from the HMW Ig both at the level of the physicochemical and antigenic properties of their H-chain components. This new 11.9 S axolotl Ig presents some similarities with anouran IgY. The detection of IgY-like molecules in urodele amphibian extends the occurrence of at least two antigenically different H-chain isotypes to all the representative modern classes of the Tetrapoda superclass.

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations.

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS-PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66-68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66-68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (approximately 40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (approximately 20%) is labeled by mAb 33.39.2 specific for the 66-68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.

Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies.

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.

Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2,4-dinitrophenyl antibody responses.

Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP-SRBC) and anti-2,4-dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS-PAGE and IEF-separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti-mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti-DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4-5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti-DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti-DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti-DNP/TNP antibodies. The specific detection of the axolotl anti-DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti-DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody "specificity" in this primitive vertebrate.