Bioinformatic Prohormone Discovery in Basal Metazoans: Insights from Trichoplax.

Experimental discovery of neuropeptides and peptide hormones is a long and tedious task. Mining the genomic and transcriptomic sequence data with robust secretory peptide prediction tools can significantly facilitate subsequent experiments. We describe the application of various in silico neuropeptide discovery methods for the placozoan Trichopax adhaerens as an illustrated example and a powerful experimental paradigm for cellular and evolutionary biology. In total, 33 placozoan (neuro)peptide-like hormone precursors were found using homology-based BLAST search and repeat-based and comparative evolutionary methods. Some of the discovered precursors are homologous to insulins and RFamide precursors from Cnidaria and other animal phyla.

Long-Term Culturing of Placozoans (Trichoplax and Hoilungia).

The phylum Placozoa remains one of the least explored among early-branching metazoan lineages. For over 130 years, this phylum had been represented by the single species Trichoplax adhaerens-an animal with the simplest known body plan (three cell layers without any organs) but complex behaviors. Recently, extensive sampling of placozoans across the globe and their subsequent genetic analysis have revealed incredible biodiversity with numerous cryptic species worldwide. However, only a few culture protocols are available to date, and all are for one species only. Here, we describe the breeding of four different species representing two placozoan genera: Trichoplax adhaerens, Trichoplax sp. H2, Hoilungia sp. H4, and Hoilungia hongkongensis originating from diverse biotopes. Our protocols allow to culture all species under comparable conditions. Next, we outlined various food sources and optimized strain-specific parameters enabling long-term culturing. These protocols can facilitate comparative analyses of placozoan biology and behaviors, which together will contribute to deciphering general principles of animal organization.

Amino acids integrate behaviors in nerveless placozoans.

Placozoans are the simplest known free-living animals without recognized neurons and muscles but a complex behavioral repertoire. However, mechanisms and cellular bases of behavioral coordination are unknown. Here, using Trichoplax adhaerens as a model, we described 0.02-0.002 Hz oscillations in locomotory and feeding patterns as evidence of complex multicellular integration; and showed their dependence on the endogenous secretion of signal molecules. Evolutionary conserved low-molecular-weight transmitters (glutamate, aspartate, glycine, GABA, and ATP) acted as coordinators of distinct locomotory and feeding patterns. Specifically, L-glutamate induced and partially mimicked endogenous feeding cycles, whereas glycine and GABA suppressed feeding. ATP-modified feeding is complex, first causing feeding-like cycles and then suppressing feeding. Trichoplax locomotion was modulated by glycine, GABA, and, surprisingly, by animals' own mucus trails. Mucus triples locomotory speed compared to clean substrates. Glycine and GABA increased the frequency of turns. The effects of the amino acids are likely mediated by numerous receptors (R), including those from ionotropic GluRs, metabotropic GluRs, and GABA-BR families. Eighty-five of these receptors are encoded in the Trichoplax genome, more than in any other animal sequenced. Phylogenetic reconstructions illuminate massive lineage-specific expansions of amino acid receptors in Placozoa, Cnidaria, and Porifera and parallel evolution of nutritional sensing. Furthermore, we view the integration of feeding behaviors in nerveless animals by amino acids as ancestral exaptations that pave the way for co-options of glutamate, glycine, GABA, and ATP as classical neurotransmitters in eumetazoans.

Expanding of Life Strategies in Placozoa: Insights From Long-Term Culturing of Trichoplax and Hoilungia.

Placozoans are essential reference species for understanding the origins and evolution of animal organization. However, little is known about their life strategies in natural habitats. Here, by maintaining long-term culturing for four species of Trichoplax and Hoilungia, we extend our knowledge about feeding and reproductive adaptations relevant to the diversity of life forms and immune mechanisms. Three modes of population dynamics depended upon feeding sources, including induction of social behaviors, morphogenesis, and reproductive strategies. In addition to fission, representatives of all species produced "swarmers" (a separate vegetative reproduction stage), which could also be formed from the lower epithelium with greater cell-type diversity. We monitored the formation of specialized spheroid structures from the upper cell layer in aging culture. These "spheres" could be transformed into juvenile animals under favorable conditions. We hypothesize that spheroid structures represent a component of the innate immune defense response with the involvement of fiber cells. Finally, we showed that regeneration could be a part of the adaptive reproductive strategies in placozoans and a unique experimental model for regenerative biology.

Evolution of glutamatergic signaling and synapses.

Glutamate (Glu) is the primary excitatory transmitter in the mammalian brain. But, we know little about the evolutionary history of this adaptation, including the selection of l-glutamate as a signaling molecule in the first place. Here, we used comparative metabolomics and genomic data to reconstruct the genealogy of glutamatergic signaling. The origin of Glu-mediated communications might be traced to primordial nitrogen and carbon metabolic pathways. The versatile chemistry of L-Glu placed this molecule at the crossroad of cellular biochemistry as one of the most abundant metabolites. From there, innovations multiplied. Many stress factors or injuries could increase extracellular glutamate concentration, which led to the development of modular molecular systems for its rapid sensing in bacteria and archaea. More than 20 evolutionarily distinct families of ionotropic glutamate receptors (iGluRs) have been identified in eukaryotes. The domain compositions of iGluRs correlate with the origins of multicellularity in eukaryotes. Although L-Glu was recruited as a neuro-muscular transmitter in the early-branching metazoans, it was predominantly a non-neuronal messenger, with a possibility that glutamatergic synapses evolved more than once. Furthermore, the molecular secretory complexity of glutamatergic synapses in invertebrates (e.g., Aplysia) can exceed their vertebrate counterparts. Comparative genomics also revealed 15+ subfamilies of iGluRs across Metazoa. However, most of this ancestral diversity had been lost in the vertebrate lineage, preserving AMPA, Kainate, Delta, and NMDA receptors. The widespread expansion of glutamate synapses in the cortical areas might be associated with the enhanced metabolic demands of the complex brain and compartmentalization of Glu signaling within modular neuronal ensembles.

Hidden cell diversity in Placozoa: ultrastructural insights from Hoilungia hongkongensis.

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly seven major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species-Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission.

Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans-one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.

Sodium action potentials in placozoa: Insights into behavioral integration and evolution of nerveless animals.

Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched metazoan lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals.

Coding palindromes in mitochondrial genes of Nematomorpha.

Inverted repeats are common DNA elements, but they rarely overlap with protein-coding sequences due to the ensuing conflict with the structure and function of the encoded protein. We discovered numerous perfect inverted repeats of considerable length (up to 284 bp) embedded within the protein-coding genes in mitochondrial genomes of four Nematomorpha species. Strikingly, both arms of the inverted repeats encode conserved regions of the amino acid sequence. We confirmed enzymatic activity of the respiratory complex I encoded by inverted repeat-containing genes. The nucleotide composition of inverted repeats suggests strong selection at the amino acid level in these regions. We conclude that the inverted repeat-containing genes are transcribed and translated into functional proteins. The survey of available mitochondrial genomes reveals that several other organisms possess similar albeit shorter embedded repeats. Mitochondrial genomes of Nematomorpha demonstrate an extraordinary evolutionary compromise where protein function and stringent secondary structure elements within the coding regions are preserved simultaneously.

Microbiomes of gall-inducing copepod crustaceans from the corals Stylophora pistillata (Scleractinia) and Gorgonia ventalina (Alcyonacea).

Corals harbor complex and diverse microbial communities that strongly impact host fitness and resistance to diseases, but these microbes themselves can be influenced by stresses, like those caused by the presence of macroscopic symbionts. In addition to directly influencing the host, symbionts may transmit pathogenic microbial communities. We analyzed two coral gall-forming copepod systems by using 16S rRNA gene metagenomic sequencing: (1) the sea fan Gorgonia ventalina with copepods of the genus Sphaerippe from the Caribbean and (2) the scleractinian coral Stylophora pistillata with copepods of the genus Spaniomolgus from the Saudi Arabian part of the Red Sea. We show that bacterial communities in these two systems were substantially different with Actinobacteria, Alphaproteobacteria, and Betaproteobacteria more prevalent in samples from Gorgonia ventalina, and Gammaproteobacteria in Stylophora pistillata. In Stylophora pistillata, normal coral microbiomes were enriched with the common coral symbiont Endozoicomonas and some unclassified bacteria, while copepod and gall-tissue microbiomes were highly enriched with the family ME2 (Oceanospirillales) or Rhodobacteraceae. In Gorgonia ventalina, no bacterial group had significantly different prevalence in the normal coral tissues, copepods, and injured tissues. The total microbiome composition of polyps injured by copepods was different. Contrary to our expectations, the microbial community composition of the injured gall tissues was not directly affected by the microbiome of the gall-forming symbiont copepods.

A new minute ectosymbiotic harpacticoid copepod living on the sea cucumber Eupentacta fraudatrix in the East/Japan Sea.

The ectosymbiotic copepods, Vostoklaophonte eupenta gen. & sp. nov. associated with the sea cucumber Eupentacta fraudatrix, was found in the subtidal zone of Peter the Great Bay, East/Japan Sea. The new genus, Vostoklaophonte, is similar to Microchelonia in the flattened body form, reduced mandible, maxillule and maxilla, but with well-developed prehensile maxilliped, and in the reduced segmentation and setation of legs 1-5. Most appendages of the new genus are more primitive than those of Microchelonia. The inclusion of the symbiotic genera Microchelonia and Vostoklaophonte gen. nov. in Laophontidae, as well as their close phylogenetic relationships, are supported by morphological observations and molecular data. This is the third record of laophontid harpacticoid copepods living in symbiosis with sea cucumbers recorded from the Korean and Californian coasts.

Lack of host specificity of copepod crustaceans associated with mushroom corals in the Red Sea.

The radiation of symbiotic copepods (Crustacea: Copepoda) living in association with stony corals (Cnidaria: Scleractinia) is considered host-specific and linked to the phylogenetic diversification of their hosts. However, symbiotic copepods are poorly investigated, occurrence records are mostly anecdotal, and no explicit analysis exists regarding their relationship with the hosts. Here, we analysed the occurrence of symbiotic copepods on different co-occurring and phylogenetically closely related scleractinian corals. We used an innovative approach of DNA extraction from single microscopic specimens that preserves the shape of the organisms for integrative morphological studies. The rationale of the study involved: (i) sampling of mushroom corals (Fungiidae) belonging to 13 species and eight genera on different reefs along the Saudi coastline in the Red Sea, (ii) extraction of all the associated copepods, (iii) morphological screening and identification of copepod species, (iv) use of DNA taxonomy on mitochondrial and nuclear markers to determine species boundaries for morphologically unknown copepod species, (v) reconstruction of phylogenies to understand their evolutionary relationships, and (vi) analysis of the ecological drivers of the occurrence, diversity and host specificity of the copepods. The seven species of coral-associated copepods, all new to science, did not show any statistically significant evidence of host-specificity or other pattern of ecological association. We thus suggest that, contrary to most assumptions and previous anecdotal evidence on this coral-copepod host-symbiont system, the association between copepods and their host corals is rather labile, not strict, and not phylogenetically constrained, changing our perception on evolutionary patterns and processes in symbiotic copepods.

Genomic exaptation enables Lasius niger adaptation to urban environments.

BACKGROUND: The world is rapidly urbanizing, and only a subset of species are able to succeed in stressful city environments. Efficient genome-enabled stress response appears to be a likely prerequisite for urban adaptation. Despite the important role ants play in the ecosytem, only the genomes of ~13 have been sequenced so far. Here, we present the draft genome assembly of the black garden ant Lasius niger - the most successful urban inhabitant of all ants - and we compare it with the genomes of other ant species, including the closely related Camponotus floridanus. RESULTS: Sequences from 272 M Illumina reads were assembled into 41,406 contigs with total length of 245 MB, and N50 of 16,382 bp, similar to other ant genome assemblies enabling comparative genomic analysis. Remarkably, the predicted proteome of L. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, DNA repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. With respect to environmental stress, we note a proliferation of various detoxification genes, including glutatione-S-transferases and those in the cytochrome P450 families. Notably, the CYP9 family is highly expanded with 19 complete and 21 nearly complete members - over twice as many compared to other ants. This family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. Gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation. CONCLUSIONS: Our results suggest that the success of L. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the CYP9 cytochrome family due to coevolution with parasitic fungi, the diversification of DNA repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. Diversification of cytochromes and DNA repair systems along with reduced odorant communication are the basis of L. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources.

Mitochondrial Genomes of Kinorhyncha: trnM Duplication and New Gene Orders within Animals.

Many features of mitochondrial genomes of animals, such as patterns of gene arrangement, nucleotide content and substitution rate variation are extensively used in evolutionary and phylogenetic studies. Nearly 6,000 mitochondrial genomes of animals have already been sequenced, covering the majority of animal phyla. One of the groups that escaped mitogenome sequencing is phylum Kinorhyncha-an isolated taxon of microscopic worm-like ecdysozoans. The kinorhynchs are thought to be one of the early-branching lineages of Ecdysozoa, and their mitochondrial genomes may be important for resolving evolutionary relations between major animal taxa. Here we present the results of sequencing and analysis of mitochondrial genomes from two members of Kinorhyncha, Echinoderes svetlanae (Cyclorhagida) and Pycnophyes kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules approximately 15 Kbp in size. The kinorhynch mitochondrial gene sequences are highly divergent, which precludes accurate phylogenetic inference. The mitogenomes of both species encode a typical metazoan complement of 37 genes, which are all positioned on the major strand, but the gene order is distinct and unique among Ecdysozoa or animals as a whole. We predict four types of start codons for protein-coding genes in E. svetlanae and five in P. kielensis with a consensus DTD in single letter code. The mitochondrial genomes of E. svetlanae and P. kielensis encode duplicated methionine tRNA genes that display compensatory nucleotide substitutions. Two distant species of Kinorhyncha demonstrate similar patterns of gene arrangements in their mitogenomes. Both genomes have duplicated methionine tRNA genes; the duplication predates the divergence of two species. The kinorhynchs share a few features pertaining to gene order that align them with Priapulida. Gene order analysis reveals that gene arrangement specific of Priapulida may be ancestral for Scalidophora, Ecdysozoa, and even Protostomia.

The Genome of Intoshia linei Affirms Orthonectids as Highly Simplified Spiralians.

Orthonectids are rare parasites of marine invertebrates [1] that are commonly treated in textbooks as a taxon of uncertain affinity [2]. Trophic forms of orthonectids reside in the tissues of their hosts as multinucleated plasmodia, generating short-lived, worm-like ciliated female and male organisms that exit into the environment for copulation [3]. These ephemeral males and females are composed of just several hundred somatic cells and are deprived of digestive, circulatory, or excretory systems. Since their discovery in the 19(th) century, the orthonectids were described as organisms with no differentiated cell types and considered as part of Mesozoa, a putative link between multicellular animals and their unicellular relatives. More recently, this view was challenged as the new data suggested that orthonectids are animals that became simplified due to their parasitic way of life [3, 4]. Here, we report the genomic sequence of Intoshia linei, one of about 20 known species of orthonectids. The genomic data confirm recent morphological analysis asserting that orthonectids are members of Spiralia and possess muscular and nervous systems [5]. The 43-Mbp genome of I. linei encodes about 9,000 genes and retains those essential for the development and activity of muscular and nervous systems. The simplification of orthonectid body plan is associated with considerable reduction of metazoan developmental genes, leaving what might be viewed as the minimal gene set necessary to retain critical bilaterian features.

Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea.

Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

Comparative study of genome divergence in salmonids with various rates of genetic isolation.

The aim of the study is a comparative investigation of changes that certain genome parts undergo during speciation. The research was focused on divergence of coding and noncoding sequences in different groups of salmonid fishes of the Salmonidae (Salmo, Parasalmo, Oncorhynchus, and Salvelinus genera) and the Coregonidae families under different levels of reproductive isolation. Two basic approaches were used: (1) PCR-RAPD with a 20-22 nt primer design with subsequent cloning and sequencing of the products and (2) a modified endonuclease restriction analysis. The restriction fragments were shown with sequencing to represent satellite DNA. Effects of speciation are found in repetitive sequences. The revelation of expressed sequences in the majority of the employed anonymous loci allows for assuming the adaptive selection during allopatric speciation in isolated char forms.

Effects of taxon sampling in reconstructions of intron evolution.

Introns comprise a considerable portion of eukaryotic genomes; however, their evolution is understudied. Numerous works of the last years largely disagree on many aspects of intron evolution. Interpretation of these differences is hindered because different algorithms and taxon sampling strategies were used. Here, we present the first attempt of a systematic evaluation of the effects of taxon sampling on popular intron evolution estimation algorithms. Using the "taxon jackknife" method, we compared the effect of taxon sampling on the behavior of intron evolution inferring algorithms. We show that taxon sampling can dramatically affect the inferences and identify conditions where algorithms are prone to systematic errors. Presence or absence of some key species is often more important than the taxon sampling size alone. Criteria of representativeness of the taxonomic sampling for reliable reconstructions are outlined. Presence of the deep-branching species with relatively high intron density is more important than sheer number of species. According to these criteria, currently available genomic databases are representative enough to provide reliable inferences of the intron evolution in animals, land plants, and fungi, but they underrepresent many groups of unicellular eukaryotes, including the well-studied Alveolata.

The origin of Metazoa: a transition from temporal to spatial cell differentiation.

For over a century, Haeckel's Gastraea theory remained a dominant theory to explain the origin of multicellular animals. According to this theory, the animal ancestor was a blastula-like colony of uniform cells that gradually evolved cell differentiation. Today, however, genes that typically control metazoan development, cell differentiation, cell-to-cell adhesion, and cell-to-matrix adhesion are found in various unicellular relatives of the Metazoa, which suggests the origin of the genetic programs of cell differentiation and adhesion in the root of the Opisthokonta. Multicellular stages occurring in the complex life cycles of opisthokont protists (mesomycetozoeans and choanoflagellates) never resemble a blastula. Here, we discuss a more realistic scenario of transition to multicellularity through integration of pre-existing transient cell types into the body of an early metazoon, which possessed a complex life cycle with a differentiated sedentary filter-feeding trophic stage and a non-feeding blastula-like larva, the synzoospore. Choanoflagellates are considered as forms with secondarily simplified life cycles.