Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development.

Nitric oxide (NO) is an ancestral key signalling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission and immunity. Although our knowledge of NO synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore nos gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that nos expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes' roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

The bowfin genome illuminates the developmental evolution of ray-finned fishes.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin's importance for illuminating vertebrate biology and diversity in the genomic era.

Morphological, Molecular, and Histopathological Data for Sebekia mississippiensis Overstreet, Self, and Vliet, 1985 (Pentastomida: Sebekidae) in the American Alligator, Alligator mississippiensis Daudin, and the Spotted Gar, Lepisosteus oculatus Winchell.

Novel molecular data from both mitochondrial ( cytochrome c oxidase subunit 1) and ribosomal regions ( 18S, ITS1-5.8S, ITS2, and 28S) are provided for Sebekia mississippiensis Overstreet, Self, & Vliet, 1985 , a pentastome infecting the American alligator, Alligator mississippiensis Daudin, 1801, and the spotted gar, Lepisosteus oculatus Winchell, 1864. Adult and nymphal pentastomes are described from the lungs and liver of the type host, A. mississippiensis, collected from Mississippi, while additional nymphs are described from the esophageal lining of L. oculatus specimens collected from Louisiana. This sequencing data will facilitate more accurate identification of various life cycle stages of S. mississippiensis, enabling future work to resolve many ambiguities in the literature regarding this species. Additionally, histopathological data are provided from both the definitive and intermediate hosts.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Connectivity of vertebrate genomes: Paired-related homeobox (Prrx) genes in spotted gar, basal teleosts, and tetrapods.

Teleost fish are important models for human biology, health, and disease. Because genome duplication in a teleost ancestor (TGD) impacts the evolution of teleost genome structure and gene repertoires, we must discriminate gene functions that are shared and ancestral from those that are lineage-specific in teleosts or tetrapods to accurately apply inferences from teleost disease models to human health. Generalizations must account both for the TGD and for divergent evolution between teleosts and tetrapods after the likely two rounds of genome duplication shared by all vertebrates. Progress in sequencing techniques provides new opportunities to generate genomic and transcriptomic information from a broad range of phylogenetically informative taxa that facilitate detailed understanding of gene family and gene function evolution. We illustrate here the use of new sequence resources from spotted gar (Lepisosteus oculatus), a rayfin fish that diverged from teleosts before the TGD, as well as RNA-Seq data from gar and multiple teleost lineages to reconstruct the evolution of the Paired-related homeobox (Prrx) transcription factor gene family, which is involved in the development of mesoderm and neural crest-derived mesenchyme. We show that for Prrx genes, the spotted gar genome and gene expression patterns mimic mammals better than teleosts do. Analyses force the seemingly paradoxical conclusion that regulatory mechanisms for the limb expression domains of Prrx genes existed before the evolution of paired appendages. Detailed evolutionary analyses like those reported here are required to identify fish species most similar to the human genome to optimally connect fish models to human gene functions in health and disease.

Comparative age and growth of common snook Centropomus undecimalis (Pisces: Centropomidae) from coastal and riverine areas in Southern Mexico.

Common snook Centropomus unidecimalis is an important commercial and fishery species in Southern Mexico, however the high exploitation rates have resulted in a strong reduction of its abundances. Since, the information about its population structure is scarce, the objective of the present research was to determine and compare the age structure in four important fishery sites. For this, age and growth of common snook were determined from specimens collected monthly, from July 2006 to March 2008, from two coastal (Barra Bosque and Barra San Pedro) and two riverine (San Pedro and Tres Brazos) commercial fishery sites in Tabasco, Mexico. Age was determined using sectioned saggitae otoliths and data analyzed by von Bertalanffy and Levenberg-Marquardt among others. Estimated ages ranged from 2 to 17 years. Monthly patterns of marginal increment formation and the percentage of otoliths with opaque rings on the outer edge demonstrated that a single annulus was formed each year. The von Bertalanffy parameters were calculated for males and females using linear adjustment and the non-linear method of Levenberg-Marquardt. The von Bertalanffy growth equations were FLt = 109.21(1-e-0.2(t+0.57)) for Barra Bosque, FLt = 94.56(1-e-027(t+0.485)) for Barra San Pedro, FLt = 97.15(1-e 0.17(t + 1.32)) for San Pedro and FLt = 83.77(1-e-026(t + 0.49)) for Tres Brazos. According to (Hotelling's T2, p < 0.05) test growth was significantly greater for females than for males. Based on the Chen test, von Bertalanffy growth curves were different among the study sites (RSS, p < 0.05). Based on the observed differences in growth parameters among sampling sites (coastal and riverine environments) future research need to be conducted on migration and population genetics, in order to delineate the stock structure of this population and support management programs.

Comparative age and growth of common snook Centropomus undecimalis (Pisces: Centropomidae) from coastal and riverine areas in Southern Mexico

Common snook Centropomus unidecimalis is an important commercial and fishery species in Southern Mexico, however the high exploitation rates have resulted in a strong reduction of its abundances. Since, the information about its population structure is scarce, the objective of the present research was to determine and compare the age structure in four important fishery sites. For this, age and growth of common snook were determined from specimens collected monthly, from July 2006 to March 2008, from two coastal (Barra Bosque and Barra San Pedro) and two riverine (San Pedro and Tres Brazos) commercial fishery sites in Tabasco, Mexico. Age was determined using sectioned saggitae otoliths and data analyzed by von Bertalanffy and Levenberg-Marquardt among others. Estimated ages ranged from 2 to 17 years. Monthly patterns of marginal increment formation and the percentage of otoliths with opaque rings on the outer edge demonstrated that a single annulus was formed each year. The von Bertalanffy parameters were calculated for males and females using linear adjustment and the non-linear method of Levenberg-Marquardt. The von Bertalanffy growth equations were FLt=109.21(1-e-0.21(t+0.57)) for Barra Bosque, FLt=94.56(1-e-0.27(t+0.48)) for Barra San Pedro, FLt=97.15(1-e-0.17(t+1.32)) for San Pedro and FLt=83.77(1-e-0.26(t+0.49)) for Tres Brazos. According to (Hotelling’s T², p<0.05) test growth was significantly greater for females than for males. Based on the Chen test, von Bertalanffy growth curves were different among the study sites (RSS, p<0.05). Based on the observed differences in growth parameters among sampling sites (coastal and riverine environments) future research need to be conducted on migration and population genetics, in order to delineate the stock structure of this population and support management programs.

El robalo blanco Centropomus undecimalis representa un ingreso monetario significativo y un recurso alimentario para todas las comunidades rurales cercanas a su distribución. Se determinó la edad y crecimiento de esta especie. Los organismos se recolectaron mensualmente en los desembarcos de la pesca artesanal de las cooperativas de mayor contribución en la zona costera (Barra Bosque y San Pedro) y ribereña (San Pedro y Tres Brazos) entre julio 2006 y marzo 2008. La edad se determinó mediante otolitos seccionados. La edad estimada fue de 2 a 17 años. Mensualmente se estableció la formación anillos opacos y traslúcidos. Para ambas zonas de estudio agrupadas, se validó el retrocálculo por comparación de tres métodos (Fraser-Lee, Dahl-Lea y Whitney-Carlander), se encontraron diferencias significativas (ANCOVA, p<0.05) entre el promedio de la longitud observada y la longitud retrocalculada. Las constantes de la ecuación de von Bertalanffy fueron calculadas para cada sexo y combinados, se empleó el método no lineal de Levenberg-Marquardt’s. La edad estimada para el robalo blanco fue de 2 a 17 años. Se encontraron diferencias significativas en el crecimiento entre sexos (T² Hotelling, p<0.05). Los parámetros de crecimiento para ambos sexos fueron, zona costera: Barra Bosque Lf t=109.21(1-e-0.21(t+0.57)), Barra San Pedro Lf t=94.56(1-e-0.27(t+0.48)), y para la zona ribereña: San Pedro Lf t=97.15(1-e-0.17(t+1.32)) y Tres Brazos Lf t=83.77(1- e-0.26(t+0.49)). Se encontraron diferencias significativas en las curvas de crecimiento de von Bertalanffy entre las poblaciones comparadas (RSS, p<0.05).

Genome evolution and meiotic maps by massively parallel DNA sequencing: spotted gar, an outgroup for the teleost genome duplication.

Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F(1) offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F(1) dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing.

Chloride inhibition of nitrite uptake for non-teleost Actinopterygiian fishes.

Fish that transport environmental chloride with a gill uptake mechanism (gill epithelial Cl(-)/HCO(3)(-)cotransport exchange system), also transport nitrite into plasma through the same mechanism. Because of the relationship between nitrite uptake and the gill chloride uptake mechanism, nitrite uptake can provide insight regarding the method of chloride uptake for fish. This study was designed to determine if non-teleost fishes concentrate nitrite in their plasma, and to determine if chloride inhibits nitrite uptake in non-teleost fish. To determine if bowfin Amia calva, spotted gar Lepisosteus oculatus, alligator gar Atractosteus spatula, and paddlefish Polyodon spathula concentrate environmental nitrite in their plasma, individuals were exposed to concentrations of 0, 1, 10, or 100 mg/L nitrite-N. After exposure, all species had plasma nitrite-N concentrations greater than environmental levels. To determine if chloride inhibits nitrite uptake for spotted gar, alligator gar, and paddlefish, fish were exposed to 1 mg/L nitrite-N and 20 mg/L chloride as calcium chloride, or to 1 mg/L nitrite-N only. Chloride effectively prevented nitrite from being concentrated in the plasma of all species. It appears that non-teleost fish concentrate nitrite in their plasma via their chloride uptake mechanism and that this is an ancestral characteristic for teleost.