Evidencia morfológica y molecular de especiación críptica en morfotipos de colores simpátricos de Mycale (Carmia) cecilia (Porifera: Poecilosclerida) del Pacífico mexicano

Identifying cryptic species is pivotal for understanding marine biodiversity and optimizing strategies for its conservation. A robust understanding of poriferan diversity is a complex endeavour. It has also been extremely hampered by the high phenotypic plasticity and the limited number of diagnostic characters. Mycale (Carmia) cecilia has different body colours, even among individuals living together. We tested whether the colour variation could be due to polymorphism, phenotypic plasticity or cryptic speciation. Phylogenetic reconstructions of nuclear and mitochondrial loci were congruent. Individuals of different body colour did not cluster together and had high levels of genetic divergence. Furthermore, the green morphotype clustered in almost all reconstructions with Mycale (C.) phyllophila, as both showed higher gene similarity at the transcriptomic level (public transcriptome). Morphologically, the green individuals consistently showed discrepancies from the red ones. These results suggest that all individuals with the same body colour, either red or green, correspond to the same species, while individuals with different body colours probably belong to different species. These results reveal high levels of morphologic and genetic diversity, which could have important implications for what is known as M. (C.) cecilia and the Mycalidae systematics.Identificar especies crípticas es fundamental para comprender la biodiversidad marina y optimizar estrategias para su conservación. Una comprensión sólida de la diversidad de los poríferos es una tarea compleja; puesto que ha sido extremadamente obstaculizada debido a la alta plasticidad fenotípica y al número limitado de caracteres diagnósticos. Mycale (Carmia) cecilia tiene diferentes colores corporales incluso en individuos que viven uno al lado del otro. Probamos si la variación de color podría deberse a polimorfismos, plasticidad fenotípica o especiación críptica. Las reconstrucciones filogenéticas de loci nucleares y mitocondriales fueron congruentes. Los individuos de diferente color corporal no se agrupaban y tenían altos niveles de divergencia genética. El morfotipo verde se agrupó en casi todas las reconstrucciones con Mycale (C.) phyllophila, mostrando también una elevada similitud genética a nivel transcriptómico (transcriptoma público). Morfológicamente, los individuos verdes mostraron consistentemente discrepancias con los individuos rojos. Estos resultados sugieren que todos los individuos con el mismo color corporal ya sea rojo o verde corresponden a la misma especie, mientras que los individuos con diferentes colores corporales probablemente pertenecen a especies diferentes. Estos resultados revelan altos niveles de diversidad morfológica y genética, lo que podría tener implicaciones importantes para lo que se conoce como M. (C.) cecilia y la sistemática Mycalidae

Identification of a tubulin-α gene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus.

International audienceFor non-model species, as many used for aquaculture, with minimal or no genomic information, relative quantification of gene expression studies requires preliminary research including the isolation of potential reference genes and the identification of those stably expressed under the biological conditions of interest. Here we report on the isolation of five partial gene sequences from gonad tissue cDNA in the functional hermaphrodite scallop Nodipecten subnodosus to be evaluated as reference genes: 18S-rRNA, riboprotein l8 (rp-l8), actin-β (act-β), elongation factor 1α (ef-1α) and alpha-tubulin-α (tub-α). We found that 18S-rRNA was stably expressed independently of the priming method used to reverse transcribe RNA to cDNA, oligo-dT or random hexamer. Stability analysis for the five putative reference genes with geNorm and NormFinder indicated that 18S together with rp-l8 were the most stable genes for normalization of gene expression during gonad development in both, male and female sexual regions of the hermaphrodite N. subnodosus. The least stable gene was tub-α, showing a biased expression profile between sexual regions of the gonad, therefore this gene was analyzed thereafter as a target gene together with vitellogenin (vit) and a DEAD-box RNA helicase (dbx) gene. Relative expression, estimated by normalization with the combination of 18S and rp-l8 as reference genes, indicated that as gonad development advanced two of the target genes were up-regulated, tub-α in the male region and vit in the female region. Whereas an increased expression was expected during development for vit for its known role in vitellogenesis, the increased expression of tub-α in the male sexual region was unexpected, and pointed toward this gene being a testis-specific α-tubulin isotype. Further analyses of gene expression among tissues indicated that tub-α is specifically and highly expressed in the male gonad, although expression in adductor muscle was also observed at significantly lower levels. The existence of testis specific α- and β-tubulins has been previously reported in other taxa, relating their function to sperm axoneme formation. Tissue-specific tubulin genes, particularly their promoters, have recently found an application as native promoters for transgene tissue-specific expression in research and reproductive control of insect plagues. The third target gene, a putative member of the DEAD-box RNA helicase family (dbx), showed no changes in expression during gonad development or between sexual regions, therefore it was chosen to discuss the different statistical inferences resulting from the arbitrary use of 'randomly chosen' reference genes when normalizing gene expression

Co-expression and regulation of ovarian vitellogenins in the Pacific oyster Crassostrea gigas

International audienceIn addition to the reported single cDNA sequence encoding vitellogenin in the Pacific oyster Crassostrea gigas (referred as Cg-Vtg1), we isolated and characterized the 3′ end of a novel vitellogenin (Vtg) in C. gigas, named Cg-Vtg2. The transcript size is up to 6 kb long and includes a D-type von Willebrand domain, characteristic of most vitellogenins, but not occurring in Cg-Vtg1. The expression of Cg-Vtg2 was found restricted to follicular cells in maturing and ripe female gonads. Quantitative expression analysis of both vitellogenins showed a closely correlated transcription pattern during the female gametogenic cycle, with the highest expression during the ripe (mature) stage, and a declining expression, to almost undetectable levels, in post-spawning (resting) stage. Levels of vitellogenin mRNA associates with the quantity of ingested food. Mature female oysters fed either a 2% or a 12% algal biomass per female biomass, showed a 2.9-fold increase in expression of Cg-Vtg1 and a 3.7-fold increase of Cg-Vtg2 expression levels in those females fed the higher level of algae, suggesting that Vtg's could be used as quantitative indicator of vitellogenesis. Our results suggest that vitellogenins are synthesized during the late period of vitellogenesis, when most of the yolk has already been incorporated into maturing oocytes

transcriptome assembly and identification of G-Protein-Coupled-Receptors (GPCRs) in two species of monogenean parasites of fish

Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites

Reproductive stages evaluated in this study.

<p>Microphotographs of the reproductive stages evaluated in this study: inactive stage at pre-gametogenesis (A) and after spawning (B); early maturation of the ovary (C) and testis part (D); Late maturation of the ovary (E) and testis part (F). Tissues or cell types are denoted as follows: connective tissue (ct), empty acinus (ea), muscle fibers (m), oogonia (ovog), previtellogenic oocytes (previt), primary vitellogenic oocytes (vit-I),secondary vitellogenic oocytes (vit-II), spermatogonia (spg), spermatocytes (spc), spermatid (spm), spermatozoa (spz).</p

Meiosis-associated genes expressed in the maturing testis library, with GO terms related to meiosis/meiotic, recombination, repair, checkpoint.

<p>Meiosis-associated genes expressed in the maturing testis library, with GO terms related to meiosis/meiotic, recombination, repair, checkpoint.</p

Relative gene expression from the maturing testis library transcripts.

<p>Relative expression of selected genes from the maturing testis library over different gametogenic conditions (n = 6 individuals for each stage): late and early oogenesis on the female part, inactive gonad, and early and late maturing testis part. Different letters denote statistical significant differences (<i>P</i><0.05; Fisher post-hoc test for differences between groups).</p