Immunochemical Detection of Modified Cytosine Species in Lampbrush Chromatin.

The lampbrush chromosomes found in the giant nucleus or germinal vesicle (GV) of amphibian oocytes provide unique opportunities for discrete closed and open chromatin structural domains to be directly observable by simple light microscopy. Moreover, the method described here for preparing spreads of lampbrush chromatin for immunostaining enables a straightforward approach to establishing the distributions of modified nucleotides within and between structurally and functionally distinctive chromatin domains.

Insights into the evolution of IG genes in Amphibians and reptiles.

Immunoglobulins are essential proteins of the immune system to neutralize pathogens. Gene encoding B cell receptors and antibodies (Ig genes) first appeared with the emergence of early vertebrates having a jaw, and are now present in all extant jawed vertebrates, or Gnathostomata. The genes have undergone evolutionary changes. In particular, genomic structural changes corresponding to genes of the adaptive immune system were coincident or in parallel with the adaptation of vertebrates from the sea to land. In cartilaginous fish exist IgM, IgD/W, and IgNAR and in bony fish IgM, IgT, IgD. Amphibians and reptiles witnessed significant modifications both in the structure and orientation of IG genes. In particular, for these amphibians and Amniota that adapted to land, IgM and IgD genes were retained, but other isotypes arose, including genes for IgA(X)1, IgA(X)2, and IgY. Recent progress in high throughput genome sequencing is helping to uncover the IG gene structure of all jawed vertebrates. In this work, we review the work and present knowledge of immunoglobulin genes in genomes of amphibians and reptiles.

Reviewing the effects of food provisioning on wildlife immunity.

While urban expansion increasingly encroaches on natural habitats, many wildlife species capitalize on anthropogenic food resources, which have the potential to both positively and negatively influence their responses to infection. Here we examine how food availability and key nutrients have been reported to shape innate and adaptive immunity in wildlife by drawing from field-based studies, as well as captive and food restriction studies with wildlife species. Examples of food provisioning and key nutrients enhancing immune function were seen across the three study type distinctions, as were cases of trace metals and pharmaceuticals impairing the immunity of wildlife species. More generally, food provisioning in field studies tended to increase innate and adaptive responses to certain immune challenges, whereas patterns were less clear in captive studies. Mild food restriction often enhanced, whereas severe food restriction frequently impaired immunity. However, to enable stronger conclusions we stress a need for further research, especially field studies, and highlight the importance of integrating nutritional manipulation, immune challenge, and functional outcomes. Despite current gaps in research on this topic, modern high throughput molecular approaches are increasingly feasible for wildlife studies and offer great opportunities to better understand human influences on wildlife health.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Genomic evidence of gene duplication and adaptive evolution of Toll like receptors (TLR2 and TLR4) in reptiles.

Toll-like receptors (TLRs) encoded by the TLR multigene family play an important role in initial pathogen recognition in vertebrates. Among the TLRs, TLR2 and TLR4 may be of particular importance to reptiles. In order to study the evolutionary patterns and structural characteristics of TLRs, we explored the available genomes of several representative members of reptiles. 25 TLR2 genes and 19 TLR4 genes from reptiles were obtained in this study. Phylogenetic results showed that the TLR2 gene duplication occurred in several species. Evolutionary analysis by at least two methods identified 30 and 13 common positively selected codons in TLR2 and TLR4, respectively. Most positively selected sites of TLR2 and TLR4 were located in the Leucine-rich repeat (LRRs). Branch model analysis showed that TLR2 genes were under different evolutionary forces in reptiles, while the TLR4 genes showed no significant selection pressure. The different evolutionary adaptation of TLR2 and TLR4 among the reptiles might be due to their different function in recognizing bacteria. Overall, we explored the structure and evolution of TLR2 and TLR4 genes in reptiles for the first time. Our study revealed valuable information regarding TLR2 and TLR4 in reptiles, and provided novel insights into the conservation concern of natural populations.

Evolutionary analysis of two complement C4 genes: Ancient duplication and conservation during jawed vertebrate evolution.

The complement C4 is a thioester-containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D-type C4 of bony fish except for mammals formed a cluster, termed D-lineage. The D-lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D-lineage before speciation, but D-type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody-dependent classical pathway.

Multiple IgH Isotypes Including IgD, Subclasses of IgM, and IgY Are Expressed in the Common Ancestors of Modern Birds.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two µ genes (µ1, µ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the µ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order µ1-δ-α-µ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Genomic structure and expression of immunoglobulins in Squamata.

The Squamata order represents a major evolutionary reptile lineage, yet the structure and expression of immunoglobulins in this order has been scarcely studied in detail. From the genome sequences of four Squamata species (Gekko japonicus, Ophisaurus gracilis, Pogona vitticeps and Ophiophagus hannah) and RNA-seq datasets from 18 other Squamata species, we identified the immunoglobulins present in these animals as well as the tissues in which they are found. All Squamata have at least three immunoglobulin classes; namely, the immunoglobulins M, D, and Y. Unlike mammals, however, we provide evidence that some Squamata lineages possess more than one Cµ gene which is located downstream from the Cδ gene. The existence of two evolutionary lineages of immunoglobulin Y is shown. Additionally, it is demonstrated that while all Squamata species possess the λ light chain, only Iguanidae species possess the κ light chain.

Evolution of V genes from the TRV loci of mammals.

Information concerning the evolution of T lymphocyte receptors (TCR) can be deciphered from that part of the molecule that recognizes antigen presented by major histocompatibility complex (MHC), namely the variable (V) regions. The genes that code for these variable regions are found within the TCR loci. Here, we describe a study of the evolutionary origin of V genes that code for the α and ß chains of the TCR loci of mammals. In particular, we demonstrate that most of the 35 TRAV and 25 TRBV conserved genes found in Primates are also found in other Eutheria, while in Marsupials, Monotremes, and Reptiles, these genes diversified in a different manner. We also show that in mammals, all TRAV genes are derived from five ancestral genes, while all TRBV genes originate from four such genes. In Reptiles, the five TRAV and three out of the four TRBV ancestral genes exist, as well as other V genes not found in mammals. We also studied the TRGV and TRDV loci from all mammals, and we show a relationship of the TRDV to the TRAV locus throughout evolutionary time.

Analysis of the reptile CD1 genes: evolutionary implications.

CD1, as the third family of antigen-presenting molecules, is previously only found in mammals and chickens, which suggests that the chicken and mammalian CD1 shared a common ancestral gene emerging at least 310 million years ago. Here, we describe CD1 genes in the green anole lizard and Crocodylia, demonstrating that CD1 is ubiquitous in mammals, birds, and reptiles. Although the reptilian CD1 protein structures are predicted to be similar to human CD1d and chicken CD1.1, CD1 isotypes are not found to be orthologous between mammals, birds, and reptiles according to phylogenetic analyses, suggesting an independent diversification of CD1 isotypes during the speciation of mammals, birds, and reptiles. In the green anole lizard, although the single CD1 locus and MHC I gene are located on the same chromosome, there is an approximately 10-Mb-long sequence in between, and interestingly, several genes flanking the CD1 locus belong to the MHC paralogous region on human chromosome 19. The CD1 genes in Crocodylia are located in two loci, respectively linked to the MHC region and MHC paralogous region (corresponding to the MHC paralogous region on chromosome 19). These results provide new insights for studying the origin and evolution of CD1.

The immunoglobulins of cold-blooded vertebrates.

Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more "conventional" mammalian species.

Genomic V exons from whole genome shotgun data in reptiles.

Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

Life without TTP: apparent absence of an important anti-inflammatory protein in birds.

Both innate and adaptive immunity in birds are different from their mammalian counterparts. Understanding bird immunity is important because of the enormous potential impact of avian infectious diseases, both in their role as food animals and as potential carriers of zoonotic diseases in man. The anti-inflammatory protein tristetraprolin (TTP) is an important component of the mammalian innate immune response, in that it binds to and destabilizes key cytokine mRNAs. TTP knockout mice exhibit a severe systemic inflammatory syndrome, and they are abnormally sensitive to innate immune stimuli such as LPS. TTP orthologs have been found in most vertebrates studied, including frogs. Here, we attempted to identify TTP orthologs in chicken and other birds, using database searches and deep mRNA sequencing. Although sequences encoding the two other widely expressed TTP family members, ZFP36L1 and ZFP36L2, were identified, we did not find sequences corresponding to TTP in any bird species. Sequences corresponding to TTP were identified in both lizards and alligators, close evolutionary relatives of birds. The induction kinetics of Zfp36l1 and Zfp36l2 mRNAs in LPS-stimulated chicken macrophages or serum-stimulated chick embryo fibroblasts did not resemble the normal mammalian TTP response to these stimuli, suggesting that the other two family members might not compensate for the TTP deficiency in regulating rapidly induced mRNA targets. Several mammalian TTP target transcripts have chicken counterparts that contain one or more potential TTP binding sites, raising the possibility that birds express other proteins that subsume TTP's function as a rapidly inducible regulator of AU-rich element (ARE)-dependent mRNA turnover.

Granulocytes of reptilian sauropsids contain beta-defensin-like peptides: a comparative ultrastructural survey.

The ability of lizards to withstand infections after wounding or amputation of the tail or limbs has suggested the presence of antimicrobial peptides in their tissues. Previous studies on the lizard Anolis carolinensis have identified several beta-defensin-like peptides that may potentially be involved in protection from infections. The present ultrastructural immunocytochemical study has analyzed tissues in different reptilian species in order to localize the cellular source of one of the more expressed beta-defensins previously sequenced in lizard indicated as AcBD15. Beta-defensin-like immunoreactivity is present in some of the larger, nonspecific granules of granulocytes in two lizard species, a snake, the tuatara, and a turtle. The ultrastructural study indicates that only heterophilic and basophilic granulocytes contain this defensin while other cell types from the epidermis, mesenchyme, and dermis, muscles, nerves, cartilage or bone are immunonegative. The study further indicates that not all granules in reptilian granulocytes contain the beta-defensin peptide, suggesting the presence of granules with different content as previously indicated for mammalian neutrophilic leucocytes. No immunolabeling was instead observed in granulocytes of the alligator and chick using this antibody. The present immunocytochemical observations suggest a broad cross-reactivity and conservation of beta-defensin-like sequence or steric motif across lepidosaurians and likely in turtles while archosaurian granulocytes may contain different beta-defensin-like or other peptides.

A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods.

In the past several decades, immunoglobulin (Ig) genes have been extensively characterized in many tetrapod species. This review focuses on the expressed Ig isotypes and the diversity of Ig genes in mammals, birds, reptiles, and amphibians. With regard to heavy chains, five Ig isotypes - IgM, IgD, IgG, IgA, and IgE - have been reported in mammals. Among these isotypes, IgM, IgD, and IgA (or its analog, IgX) are also found in non-mammalian tetrapods. Birds, reptiles, and amphibians express IgY, which is considered the precursor of IgG and IgE. Some species have developed unique isotypes of Ig, such as IgO in the platypus, IgF in Xenopus, and IgY (ΔFc) in ducks and turtles. The κ and λ light chains are both utilized in tetrapods, but the usage frequencies of κ and λ chains differ greatly among species. The diversity of Ig genes depends on several factors, including the germline repertoire and recombinatorial and post-recombinatorial diversity, and different species have evolved distinct mechanisms to generate antibody diversity.

Immunoglobulin genes of the turtles.

The availability of reptile genomes for the use of the scientific community is an exceptional opportunity to study the evolution of immunoglobulin genes. The genome of Chrysemys picta bellii and Pelodiscus sinensis is the first one that has been reported for turtles. The scanning for immunoglobulin genes resulted in the presence of a complex locus for the immunoglobulin heavy chain (IGH). This IGH locus in both turtles contains genes for 13 isotypes in C. picta bellii and 17 in P. sinensis. These correspond with one immunoglobulin M, one immunoglobulin D, several immunoglobulins Y (six in C. picta bellii and eight in P. sinensis), and several immunoglobulins that are similar to immunoglobulin D2 (five in C. picta belli and seven in P. sinensis) that was previously described in Eublepharis macularius. It is worthy to note that IGHD2 are placed in an inverted transcriptional orientation and present sequences for two immunoglobulin domains that are similar to bird IgA domains. Furthermore, its phylogenetic analysis allows us to consider about the presence of IGHA gene in a primitive reptile, so we would be dealing with the memory of the gene that originated from the bird IGHA. In summary, we provide a clear picture of the immunoglobulins present in a turtle, whose analysis supports the idea that turtles emerged from the evolutionary line from the differentiation of birds and the presence of the IGHA gene present in a common ancestor.

Snakes antibodies.

Immunoglobulins are basic molecules of the immune system of vertebrates. In previous studies we described the immunoglobulins found in two squamata reptiles, Anolis carolinensis and Eublepharis macularius. Snakes are squamata reptiles too but they have undergone an extreme evolutionary process. We therefore wanted to know how these changes affected their immunoglobulin coding genes. To perform this analysis we studied five snake transcriptomes and two genome draft sequences. Sequences coding for immunoglobulin M (IgM), immunoglobulin D (IgD) and two classes of immunoglobulin Y (IgY - named IgYa and IgYb-) were found in all of them. Moreover, the Thamnophis elegans transcriptome and Python molurus genome draft sequences showed a third class of IgY, the IgYc, whose constant region only presents three domains and lacks the CH2. All data suggest that the IgYb is the evolutionary origin of this IgYc. An exhaustive search of the light chains were carried out, being lambda the only light chain found in snakes. The results provide a clear picture of the immunoglobulins present in the suborder Serpentes.

Identification of the pre-T-cell receptor alpha chain in nonmammalian vertebrates challenges the structure-function of the molecule.

In humans and mice, the early development of αß T cells is controlled by the pre-T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor ß (TCRß) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRß chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling.

Understanding the vertebrate immune system: insights from the reptilian perspective.

Reptiles are ectothermic amniotes, providing the key link between ectothermic anamniotic fishes and amphibians, and endothermic amniotic birds and mammals. A greater understanding of reptilian immunity will provide important insights into the evolutionary history of vertebrate immunity as well as the growing field of eco-immunology. Like mammals, reptile immunity is complex and involves innate, cell-mediated and humoral compartments but, overall, there is considerably less known about immune function in reptiles. We review the current literature on each branch of the reptilian immune system, placing this information in context to other vertebrates. Further, we identify key areas that are prime for research as well as areas that are lagging because of lack of reagents in non-model systems.