Characterization and localization of primordial germ cells in Totoaba macdonaldi.

The totoaba, Totoaba macdonaldi, is an endangered fish of the Gulf of California with high economic and ecological potential. Therefore, our purpose was to characterize the Primordial Germ Cells (PGCs) of this Sciaenid with two objectives: (1) to provide the basis for PGCs cryopreservation to preserve the genetic resources and (2) to take the first step to know the gonadal genesis and sex differentiation of totoaba. Immunofluorescence analysis performed from 2-cell stage to 8-day after hatch (DAH) shows that Vasa protein is specific for PGCs. These cells were first observed in the peripheral and dorsal regions of the blastodisc at approximately the 50%-epiboly stage and migrated to both sides of embryo body during the development. Finally, at 7 DAH the PGCs of the hatching embryo reached the place where the gonad will be developed. Histology analysis of larvae showed a genital ridge with enclosed PGCs on the dorsal side of the peritoneum at 9 DAH, gonadal primordium growth was observed at 11 DAH as a result of the interaction between PGCs and somatic cells derived from the peritoneum. Results of qPCR showed that vasa expression was restricted to the embryonic and early larval development, highest values were observed in 2-cell and mid-blastula stage suggesting the maternal inheritance of vasa mRNA. These findings support the hypothesis of preformation in T. macdonaldi PGCs. The migration pattern of PGCs allow us to recommend the isolation and subsequent cryopreservation of these cells before 7 DAH when the embryonic and larval development is given at 21 °C.

Monomorphic pathogens: The case of Candidatus Xenohaliotis californiensis from abalone in California, USA and Baja California, Mexico.

Withering syndrome (WS) is a chronic wasting disease affecting abalone species attributed to the pathogen Candidatus Xenohaliotis californiensis (CXc). Wild populations of blue (Haliotis fulgens) and yellow (H. corrugata) abalone have experienced unusual mortality rates since 2009 off the peninsula of Baja California and WS has been hypothesized as a possible cause. Currently, little information is available about the genetic diversity of CXc and particularly the possible existence of strains differing in pathogenicity. In a recent phylogenetic analysis, we characterized five coding genes from this rickettsial pathogen. Here, we analyze those genes and two additional intergenic non-coding regions following multi-locus sequence typing (MLST) and multi-spacer typing (MST) approaches to assess the genetic variability of CXc and its relationship with blue, yellow and red (H. rufescens) abalone. Moreover, we used 16S rRNA pyrosequencing reads from gut microbiomes of blue and yellow abalone to complete the genetic characterization of this prokaryote. The presence of CXc was investigated in more than 150 abalone of the three species; furthermore, a total of 385 DNA sequences and 7117 16S rRNA reads from Candidatus Xenohaliotis californiensis were used to evaluate its population genetic structure. Our findings suggest the absence of polymorphism in the DNA sequences of analyzed loci and the presence of a single lineage of CXc infecting abalone from California (USA) and Baja California (Mexico). We posit that the absence of genetic variably in this marine rickettsia may be the result of evolutionary and ecological processes.

Multigenetic characterization of 'Candidatus Xenohaliotis californiensis'.

'Candidatus Xenohaliotis californiensis' (or Ca.Xc) is the aetiological agent of withering syndrome, a chronic wasting disease affecting most if not all North American species of abalone, and has been described as a Rickettsiales-like prokaryote. Genetic data regarding this species are limited to the 16S rRNA gene. The inability to grow it axenically has hindered its genetic and genomic characterization and, in consequence, a thorough analysis of its systematics. Here, we amplified and sequenced five genes (16S rRNA, 23S rRNA, ftsZ, virD4 and virB11) of Ca.Xc from infected abalone to analyse its phylogenetic position. Phylogenies from concatenated DNA and amino acid sequences with representative genera of most Rickettsiales unequivocally place Ca.Xc in the family Anaplasmataceae. Furthermore, the family has two reciprocally monophyletic lineages: one leading to (Neorickettsia, Ca.Xc) and the other to ((Ehrlichia, Anaplasma), Wolbachia)). A molecular-clock Bayesian reconstruction places Ca.Xc as the most basal lineage in Anaplasmataceae. These phylogenetic hypotheses shed light on patterns of host evolution and of ecological transitions. Specifically, Neorickettsia and Ca.Xc inhabit aquatic hosts whereas the remaining Anaplasmataceae are found in terrestrial hosts. Additionally, our evolutionary timeline places the directly transmitted marine Ca.Xc as the basal Anaplasmataceae, ancestral to both freshwater and terrestrial species with adaptations leading to more complex life cycles involving intermediate vectors or reservoir species; this supports the hypothesis of a marine origin for this bacterial family.

Entire mitochondrion genome sequence of the Desert Pupfish, Cyprinodon macularius Baird & Girard, 1853.

The complete mitochondrial genome sequence of the Desert Pupfish, Cyprinodon macularius (Gene accession number KM985373) has a length of 16,940 bp, and the arrangement consisted of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA, which are similar to other known mitogenomes for the family Cyprinodontidae.

The complete mitochondrial DNA of the endemic shortfin silverside, Chirostoma humboldtianum (Valenciennes, 1835).

The shortfin silverside Chirostoma humboldtianum, is an endemic fish from the Mesa Central of Mexico, it is considered the "ancestral" species of the "peces blancos" and plays an important role as a potential species for aquaculture. Here we sequence its mitogenome (Genbank accession number KJ921739), which has a total length of 16,447 bp, and the arrangement consist of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA similar to other Atheriniformes. This mitogenome will be useful for phylogenetic, population and phylogeographic studies of this and other important atherinopsid species.

Lionfish, Pterois volitans Linnaeus 1758, the complete mitochondrial DNA of an invasive species.

The lionfish, Pterois volitans, native from the Indo-Pacific, has been found in Atlantic and Caribbean waters and is considered as an invasive species. Here we sequence its mitogenome (Genbank accession number KJ739816), which has a total length of 16,500 bp, and the arrangement consist of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA similar to other Pteroinae subfamily (family Scorpaenidae). This mitogenome will be useful for phylogenetic and population genetic studies of this invasive species.

The complete mitochondrial DNA of the tropical gar (Atractosteus tropicus).

The mitogenome of the tropical gar, Atractosteus tropicus, (GeneBank accession number KJ531198) has a total length of 16,280 bp, and the arrangement consist of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA similar to other Lepisosteidae family mitogenomes.

The complete mitochondrial DNA of endemic Eastern Pacific coral (Porites panamensis).

The mitogenome of the endemic coral Porites panamensis (Genbank accession number KJ546638) has a total length of 18,628 bp, and the arrangement consist of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 2 transfer RNA (tRNA) genes. Gene order was equal to other scleractinian coral mitogenomes.

Complete mitochondrial genome of the beaubrummel Damselfish, Stegastes flavilatus (Pisces: Perciformes, Pomacentridae).

The mitogenome of the beaubrummel damselfish, Stegastes flavilatus Gill, 1862 (Genebank accession number KP136922), has a total length of 16,816 bp. It encodes 13 protein-coding, two ribosomal RNAs (rRNAs), and 22 transfer RNA (tRNA) genes. Base composition is 28.6% A, 26.0% T, 29.8% C, and 15.7% G and 45.5% GC content. The gene arrangement was found to be the same of other pomacentrid mitogenomes.

The complete mitochondrial DNA of the bay snook, Petenia splendida, a native Mexican cichlid.

The mitogenome of the tenguayaca, Petenia splendida (GenBank accession number KJ914664) has a total length of 16,518 bp, and the arrangement consist of 15 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA (tRNA) genes. Gene order was equal to the mitogenomes of other new world cichlids.

Characterization of the growth-related transcriptome in California red abalone (Haliotis rufescens) through RNA-Seq analysis.

One of the largest detriments in the abalone aquaculture industry is the inherently low growth rate of this marine gastropod. In order to confront this issue, greater molecular knowledge is needed on growth traits. Therefore, transcriptome analyses were performed using RNA-Seq for groups of California red abalones (Haliotis rufescens) cultured under the same conditions, but with high growth rates (HGR) or low growth rates (LGR). De novo assembly generated 44312 contigs used as references for RNA-Seq analysis. Results showed a total of 1437 differentially expressed contigs, among which, 435 were up-regulated in the HGR group and 1002 in LGR individuals. Overall, LGR abalones evidenced a greater number of exclusive transcripts and differentially transcribed genes. These results provide a valuable resource of novel transcripts in this species and further understandings of the molecular bases regulating growth traits in H. rufescens.

BACTERIA IN CRYOPRESERVED SPERM MASS OF THE WHITE SHRIMP Penaeus vannamei.

BACKGROUND: Cryopreservation and global trading of P. vannamei sperm will become a potential and important biotechnological tool. Nevertheless, information of the possible transfer of bacteria in cryopreserved shrimp sperm has not been registered yet. OBJECTIVE: The objective of this work was to determine the type of bacteria that could be cryopreserved together with white shrimp sperm masses. MATERIALS AND METHODS: Sixteen sperm masses were cryopreserved in 10% DMSO and 0.5 M trehalose and sixteen fresh sperm masses were used for bacterial analysis. Bacterial colonies were isolated and selected for sequencing. RESULTS: Strains were seawater borne and facultative aerobic bacteria from the genera Bacillus, Micrococcus, Paracoccus, Ruegeria and Staphylococcus. Most of them have been related with benefits to its host. None were pathogenic for P. vannamei. CONCLUSION: Cryopreservation implies preserving pathogenic or beneficial bacteria together with the sample. Therefore, it is possible to enhance cryopreserved samples or disperse pathogenic bacteria, which needs to be prevented.

Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822).

The red abalone Haliotis rufescens is one of the most important species for aquaculture in Baja California, México, and despite this, few gene expression studies have been done in tissues such as gill, head and gonad. For this purpose, reverse transcription and quantitative real time PCR (RT-qPCR) is a powerful tool for gene expression evaluation. For a reliable analysis, however, it is necessary to select and validate housekeeping genes that allow proper transcription quantification. Stability of nine housekeeping genes (ACTB, BGLU, TUBB, CY, GAPDH, HPRTI, RPL5, SDHA and UBC) was evaluated in different tissues of red abalone (gill, head and gonad/digestive gland). Four-fold serial dilutions of cDNA (from 25 ngµL(-1) to 0.39 ngµL(-1)) were used to prepare the standard curve, and it showed gene efficiencies between 0.95 and 0.99, with R(2)=0.99. geNorm and NormFinder analysis showed that RPL5 and CY were the most stable genes considering all tissues, whereas in gill HPRTI and BGLU were most stable. In gonad/digestive gland, RPL5 and TUBB were the most stable genes with geNorm, while SDHA and HPRTI were the best using NormFinder. Similarly, in head the best genes were RPL5 and UBC with geNorm, and GAPDH and CY with NormFinder. The technical variability analysis with RPL5 and abalone gonad/digestive gland tissue indicated a high repeatability with a variation coefficient within groups ≤ 0.56% and between groups ≤ 1.89%. These results will help us for further research in reproduction, thermoregulation and endocrinology in red abalone.