High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.

Reproductive periodicity, spawning induction, and larval metamorphosis of the hemichordate acorn worm Ptychodera flava.

The indirect-developing enteropneust acorn worm Ptychodera flava has been used as a hemichordate model system for studying the developmental evolution of deuterostome body plans and the origins of chordate characteristics. However, research progress has been hindered by the limited accessibility of its embryonic materials and metamorphosing larvae. In this study, we identified an abundant population of P. flava in Penghu, Taiwan, and examined the feasibility of using this animal for developmental studies. Through histological examination, we established that the reproductive season of this population is between September and December, with a peak breeding period in October and November. In addition, we have developed new procedures that can induce P. flava spawning at any time of the day during the breeding season, with a higher successful rate than that achieved using a previously published method. Moreover, the culturing system we developed enables rearing of P. flava larvae through various planktonic stages and eventual metamorphosis into benthic juveniles, all under laboratory conditions. We anticipate that the animal resources and new technical procedures reported here will further facilitate the use of P. flava as a model organism for evolutionary and developmental biology research.

Identification of an intact ParaHox cluster with temporal colinearity but altered spatial colinearity in the hemichordate Ptychodera flava.

BACKGROUND: ParaHox and Hox genes are thought to have evolved from a common ancestral ProtoHox cluster or from tandem duplication prior to the divergence of cnidarians and bilaterians. Similar to Hox clusters, chordate ParaHox genes including Gsx, Xlox, and Cdx, are clustered and their expression exhibits temporal and spatial colinearity. In non-chordate animals, however, studies on the genomic organization of ParaHox genes are limited to only a few animal taxa. Hemichordates, such as the Enteropneust acorn worms, have been used to gain insights into the origins of chordate characters. In this study, we investigated the genomic organization and expression of ParaHox genes in the indirect developing hemichordate acorn worm Ptychodera flava. RESULTS: We found that P. flava contains an intact ParaHox cluster with a similar arrangement to that of chordates. The temporal expression order of the P. flava ParaHox genes is the same as that of the chordate ParaHox genes. During embryogenesis, the spatial expression pattern of PfCdx in the posterior endoderm represents a conserved feature similar to the expression of its orthologs in other animals. On the other hand, PfXlox and PfGsx show a novel expression pattern in the blastopore. Nevertheless, during metamorphosis, PfXlox and PfCdx are expressed in the endoderm in a spatially staggered pattern similar to the situation in chordates. CONCLUSIONS: Our study shows that P. flava ParaHox genes, despite forming an intact cluster, exhibit temporal colinearity but lose spatial colinearity during embryogenesis. During metamorphosis, partial spatial colinearity is retained in the transforming larva. These results strongly suggest that intact ParaHox gene clustering was retained in the deuterostome ancestor and is correlated with temporal colinearity.

Somite Compartments in Amphioxus and Its Implications on the Evolution of the Vertebrate Skeletal Tissues.

Mineralized skeletal tissues of vertebrates are an evolutionary novelty within the chordate lineage. While the progenitor cells that contribute to vertebrate skeletal tissues are known to have two embryonic origins, the mesoderm and neural crest, the evolutionary origin of their developmental process remains unclear. Using cephalochordate amphioxus as our model, we found that cells at the lateral wall of the amphioxus somite express SPARC (a crucial gene for tissue mineralization) and various collagen genes. During development, some of these cells expand medially to surround the axial structures, including the neural tube, notochord and gut, while others expand laterally and ventrally to underlie the epidermis. Eventually these cell populations are found closely associated with the collagenous matrix around the neural tube, notochord, and dorsal aorta, and also with the dense collagen sheets underneath the epidermis. Using known genetic markers for distinct vertebrate somite compartments, we showed that the lateral wall of amphioxus somite likely corresponds to the vertebrate dermomyotome and lateral plate mesoderm. Furthermore, we demonstrated a conserved role for BMP signaling pathway in somite patterning of both amphioxus and vertebrates. These results suggest that compartmentalized somites and their contribution to primitive skeletal tissues are ancient traits that date back to the chordate common ancestor. The finding of SPARC-expressing skeletal scaffold in amphioxus further supports previous hypothesis regarding SPARC gene family expansion in the elaboration of the vertebrate mineralized skeleton.

Deeply conserved synteny resolves early events in vertebrate evolution.

Although it is widely believed that early vertebrate evolution was shaped by ancient whole-genome duplications, the number, timing and mechanism of these events remain elusive. Here, we infer the history of vertebrates through genomic comparisons with a new chromosome-scale sequence of the invertebrate chordate amphioxus. We show how the karyotypes of amphioxus and diverse vertebrates are derived from 17 ancestral chordate linkage groups (and 19 ancestral bilaterian groups) by fusion, rearrangement and duplication. We resolve two distinct ancient duplications based on patterns of chromosomal conserved synteny. All extant vertebrates share the first duplication, which occurred in the mid/late Cambrian by autotetraploidization (that is, direct genome doubling). In contrast, the second duplication is found only in jawed vertebrates and occurred in the mid-late Ordovician by allotetraploidization (that is, genome duplication following interspecific hybridization) from two now-extinct progenitors. This complex genomic history parallels the diversification of vertebrate lineages in the fossil record.

Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota.

Mammalian gut microbiota are integral to host health. However, how this association began remains unclear. We show that in basal chordates the gut space is radially compartmentalized into a luminal part where food microbes pass and an almost axenic peripheral part, defined by membranous delamination of the gut epithelium. While this membrane, framed with chitin nanofibers, structurally resembles invertebrate peritrophic membranes, proteome supports its affinity to mammalian mucus layers, where gut microbiota colonize. In ray-finned fish, intestines harbor indigenous microbes, but chitinous membranes segregate these luminal microbes from the surrounding mucus layer. These data suggest that chitin-based barrier immunity is an ancient system, the loss of which, at least in mammals, provided mucus layers as a novel niche for microbial colonization. These findings provide a missing link for intestinal immune systems in animals, revealing disparate mucosal environment in model organisms and highlighting the loss of a proven system as innovation.

Diversity and Universality of Endosymbiotic Rickettsia in the Fish Parasite Ichthyophthirius multifiliis.

Although the presence of endosymbiotic rickettsial bacteria, specifically Candidatus Megaira, has been reported in diverse habitats and a wide range of eukaryotic hosts, it remains unclear how broadly Ca. Megaira are distributed in a single host species. In this study we seek to address whether Ca. Megaira are present in most, if not all isolates, of the parasitic ciliate Ichthyophthirius multifiliis. Conserved regions of bacterial 16S rRNA genes were either PCR amplified, or assembled from deep sequencing data, from 18 isolates/populations of I. multifiliis sampled worldwide (Brazil, Taiwan, and USA). We found that rickettsial rRNA sequences belonging to three out of four Ca. Megaira subclades could be consistently detected in all I. multifiliis samples. I. multifiliis collected from local fish farms tend to be inhabited by the same subclade of Ca. Megaira, whereas those derived from pet fish are often inhabited by more than one subclade of Ca. Megaira. Distributions of Ca. Megaira in I. multifiliis thus better reflect the travel history, but not the phylogeny, of I. multifiliis. In summary, our results suggest that I. multifiliis may be dependent on this endosymbiotic relationship, and the association between Ca. Megaira and I. multifiliis is more diverse than previously thought.

A new copepod with transformed body plan and unique phylogenetic position parasitic in the acorn worm Ptychodera flava.

Symbiotic copepods compose one-third of the known copepod species and are associated with a wide range of animal groups. Two parasitic copepods endoparasitic in acorn worms (Hemichordata), Ive balanoglossi and Ubius hilli, collected in the Mediterranean Sea and Australian waters, respectively, were described a century ago. Here we report a new parasitic copepod species, Ive ptychoderae sp. nov., found in Ptychodera flava, a widespread acorn worm in the Indo-Pacific Ocean and an emerging organism for developmental and evolutionary studies. The female of I. ptychoderae is characterized by having a reduced maxilliped and five pairs of annular swellings along the body that are morphologically similar but distinguishable from those in the two previously described parasitic copepods in acorn worms. Phylogenetic analysis based on the 18S rDNA sequence shows that I. ptychoderae may belong to Poecilostomatoida but represent a new family, which we name Iveidae fam. nov. Ive ptychoderae is commonly found in the acorn worm population with an average prevalence of 42% during the collecting period. The infection of the parasite induces the formation of cysts and causes localized lesions of the host tissues, suggesting that it may have negative effects on its host. Interestingly, most cysts contain a single female with one or multiple male copepods, suggesting that their sex determination may be controlled by environmental conditions. The relationships between the parasitic copepods and acorn worms thus provide a platform for understanding physiological and ecological influences and coevolution between parasites and hosts.

Sequencing and analysis of the transcriptome of the acorn worm Ptychodera flava, an indirect developing hemichordate.

Hemichordates are the sister group of echinoderms, and together they are closely related to chordates within the deuterostome lineage. Therefore, hemichordates represent an important animal group for the understanding of both the evolution of developmental mechanisms in deuterostome animals and the origin of chordates. Recently, the majority of studies investigating hemichordates have focused on the direct-developing enteropneust hemichordate Saccoglossus kowalevskii; few have focused on the indirect-developing hemichordates, partly because of the lack of extensive genomic resources in these animals. In this study, we report the sequencing and analysis of a transcriptome from an indirect-developing enteropneust hemichordate Ptychodera flava. We sequenced a mixed cDNA library from six developmental stages using the Roche GS FLX Titanium System to generate more than 879,000 reads. These reads were assembled into 17,990 contigs with an average length of 1316bp. We found that 60% of the assembled contigs, along with 28% of the unassembled singleton reads, had significant hits to sequences in the NCBI database by a BLASTx search, and we also annotated these sequences and obtained Gene Ontology (GO) terms for 6744 contigs and 5802 singletons. We further identified candidate P. flava transcripts corresponding to genes involved in major developmental signaling pathways, including the Wnt, Notch and TGF-ß signaling pathways. Using available genome/transcriptome datasets from the direct-developing hemichordate S. kowalevskii, the echinoderm Strongylocentrotus purpuratus and the chordate Branchiostoma floridae, we found that 90%, 80% and 73% of the annotated protein sequences in these respective species matched our P. flava transcriptome in a homology search. We also constructed a database for the P. flava transcriptome, and researchers can easily access this dataset online. Our dataset significantly increases the amount of available P. flava sequence data and can serve as a reference transcriptome for future studies using this species.