Improved genome assembly of the whiteleg shrimp Penaeus (Litopenaeus) vannamei using long- and short-read sequences from public databases.

The Pacific whiteleg shrimp Penaeus (Litopenaeus) vannamei is a highly relevant species for the world's aquaculture development, for which an incomplete genome is available in public databases. In this work, PacBio long-reads from 14 publicly available genomic libraries (131.2 Gb) were mined to improve the reference genome assembly. The libraries were assembled, polished using Illumina short-reads, and scaffolded with P. vannamei, Feneropenaeus chinensis, and Penaeus monodon genomes. The reference-guided assembly, organized into 44 pseudo-chromosomes and 15,682 scaffolds, showed an improvement from previous reference genomes with a genome size of 2.055 Gb, N50 of 40.14 Mb, L50 of 21, and the longest scaffold of 65.79 Mb. Most orthologous genes (92.6%) of the Arthropoda_odb10 database were detected as "complete," and BRAKER predicted 21,816 gene models; from these, we detected 1,814 single-copy orthologues conserved across the genomic references for Marsupenaeus japonicus, F. chinensis, and P. monodon. Transcriptomic-assembly data aligned in more than 99% to the new reference-guided assembly. The collinearity analysis of the assembled pseudo-chromosomes against the P. vannamei and P. monodon reference genomes showed high conservation in different sets of pseudo-chromosomes. In addition, more than 21,000 publicly available genetic marker sequences were mapped to single-site positions. This new assembly represents a step forward to previously reported P. vannamei assemblies. It will be helpful as a reference genome for future studies on the evolutionary history of the species, the genetic architecture of physiological and sex-determination traits, and the analysis of the changes in genetic diversity and composition of cultivated stocks.

Variabilidad genética del pez Lutjanus guttatus (Perciformes: Lutjanidae) mediante el uso de microsatélites en Costa Rica / Genetic variability of the fish Lutjanus guttatus (Perciformes: Lutjanidae) using microsatellites in Costa Rica

Introducción: El pargo mancha es un pez marino de alto consumo e interés comercial en Costa Rica que está sometido a una fuerte presión pesquera, la cual puede afectar la diversidad genética y generar problemas por depresión endogámica. Objetivo: Evaluar el estado genético de la población de Lutjanus guttatus mediante el uso microsatélites. Métodos: Se recolectaron muestras entre el 2018 y 2019 y se estudiaron 44 individuos de cada una de las localidades del Golfo de Nicoya y Golfo Dulce. Se realizó la extracción de ADN y la amplificación de diez loci con microsatélites mediante PCR, para la determinación del genotipo, análisis de diversidad genética y estructura poblacional. Resultados: Los parámetros de diversidad indican un elevado polimorfismo asociado con un alto número de alelos obtenidos por locus, pero con bajos niveles de heterocigosidad observada en comparación con la esperada (Ho= 0.774 y 0.800 y He= 0.948 y 0.954 para Golfo de Nicoya y Golfo Dulce, respectivamente). No hay evidencia suficiente para decir que las dos poblaciones son distintas (FST= 0.00264, P > 0.05). La desviación del Equilibrio de Hardy-Weinberg indica la posible mezcla de organismos de origen distinto a los del medio silvestre. Conclusiones: L. guttatus tiene niveles altos de diversidad genética, no hay evidencia de diferenciación en subpoblaciones genéticas, lo que en manejo pesquerías se considera una sola población panmíctica. La posible mezcla de individuos de origen distinto al silvestre sugiere la presencia de organismos de un programa de repoblación o de cultivos comerciales en la región. El uso de marcadores genéticos se recomienda para el monitoreo, además, en programas de repoblación y evaluar su efecto.

Introduction: The spotted snapper is a high-consumption and commercially important marine fish in Costa Rica, subjected to heavy fishing pressures, which can affect genetic diversity and generate problems due to inbreeding depression. Objective: To evaluate the genetic status of the population of Lutjanus guttatus using microsatellites. Methods: Samples were collected between 2018 and 2019, and 44 individuals from each of the localities of the Gulf of Nicoya and the Gulf of Dulce were studied. DNA extraction and amplification of ten loci with microsatellites using PCR were performed, followed by genotyping, analysis of genetic diversity, and population structure. Results: Diversity parameters indicate a high polymorphism associated with a high number of alleles obtained per locus, but with low levels of observed heterozygosity compared to expected (Ho= 0.774 and 0.800, and He= 0.948 and 0.954 for the Gulf of Nicoya and Gulf of Dulce, respectively). There is not enough evidence to say that the two populations are distinct (FST= 0.00264, P > 0.05). Deviation from Hardy-Weinberg equilibrium was recorded, indicating possible mixing of organisms of different origin from the wild environment. Conclusions: L. guttatus presents high levels of genetic diversity, without evidence of differentiation in genetic subpopulations. For fisheries management purposes, they would be considered a single panmictic population. The possible mixing with wild individuals suggests the presence of organisms derived from a restocking or commercial cultivation program carried out in the region. The use of genetic markers is recommended to maintain monitoring, follow up on restocking programs and evaluate their effect.

Drivers for genetic structure at different geographic scales for Pacific red snapper (Lutjanus peru) and yellow snapper (Lutjanus argentiventris) in the tropical eastern Pacific.

The tropical eastern Pacific (TEP) is a highly dynamic region and a model system to study how habitat discontinuities affect the distribution of shorefishes, particularly for species that display ontogenetic habitat shifts, including snappers (Lutjanidae). To evaluate the genetic structure of the Pacific red snapper (Lutjanus peru) and the yellow snapper (Lutjanus argentiventris) throughout their distribution range along the TEP, 13 and 11 microsatellite loci were analysed, respectively. The genetic diversity of L. peru (N = 446) and L. argentiventris (N = 170) was evaluated in 10 and 5 localities, respectively, showing slightly higher but non-significant values in the Gulf of California for both species. The genetic structure analysis identified the presence of significant genetic structure in both species, but the locations of the identified barriers for the gene flow differed between species. The principal driver for the genetic structure at large scales >2500 km was isolation by distance. At smaller scales (<250 km), the habitat discontinuity for juveniles and adults and the environmental differences throughout the distribution range represented potential barriers to gene flow between populations for both species.

Population genomics reveals a mismatch between management and biological units in green abalone (Haliotis fulgens).

Effective fishery management strategies should be based on stock delimitation and knowledge of the spatial scale at which species are distributed. However, a mismatch often occurs between biological and management units of fishery resources. The green abalone (Haliotis fulgens) supports an important artisanal fishery in the west coast of the Baja California Peninsula (BCP), Mexico, which has shown a declining tendency despite the several management measures. Thus, the aim of this study was to characterize the spatial patterns of neutral genomic variation of green abalone along the BCP to test whether the genomic structure patterns support the current green abalone management areas. To test this hypothesis, a set of 2,170 putative neutral single nucleotide polymorphisms discovered by a double digest restriction-site associated DNA approach was used on 10 locations along the BCP. The results revealed a population structure with three putative groups: Guadalupe Island and northern and southern BCP locations. The contemporary gene flow might be explained by local oceanographic features, where it is bidirectional within the southern region but with a predominant southward flow from the northern region. These findings indicated that the administrative areas did not match the biological units of H. fulgens fishery; hence, the stock assessment and management areas should be revised.

SNP markers for the genetic characterization of Mexican shrimp broodstocks.

Selective breeding of shrimp has major potential to enhance production traits, including growth and disease resistance. Genetic characterization of broodstock populations is a key element of breeding programs, as it enables decisions on inbreeding restrictions, family structure, and the potential use of genomic selection. Single Nucleotide Polymorphisms (SNPs) are suitable genetic markers for this purpose. A set of SNPs was developed to characterize commercial breeding stocks in Mexico. Individuals from local and imported lines were selected for sequencing using the nextRAD technique, resulting in the identification of 2619 SNPs. Genetic structure analysis showed three to five genetic groups of Ecuadorian and Mexican origins. A subset of 1231 SNPs has potential for stock identification and management. Further, three SNPs were identified as candidate sex-linked markers. The role of SNPs possibly associated with genes related to traits of importance to shrimp farming, such as growth and immune response, should be further investigated.

Genetic tracing of farmed shrimp (Decapoda: Penaeidae) in wild populations from a main aquaculture region in Mexico / Rastreo genético de camarón de cultivo (Decapoda: Penaeidae) en poblaciones silvestres de la principal región de cultivo de México

Abstract Release or escapes of aquaculture organisms may impact the genetic composition and variability of wild populations, leading to diverse issues that may compromise long-term wild stock fitness. Therefore, it is relevant to determine if farmed stocks are currently interacting with wild populations. Shrimp farming is an aquaculture activity taking place along the tropical Pacific coast of the Americas, and represents the most important culture business of Northwestern Mexico. In this study, wild and farmed whiteleg shrimp Litopenaeus vannamei from the State of Sinaloa were genetically evaluated to determine admixture levels. A newly developed set of 14 microsatellite markers (mean number of alleles per locus 11.8, and 0.836 expected heterozygosity) was obtained by Next Generation Sequencing to characterize samples. Sampling consisted of 32 wild shrimps collected during three years (2002, 2012, and 2013) and three different sites, and two hatchery stocks from 2007. No significant differences were observed among years in the wild samples, but cluster analyses showed that hatchery-produced individuals were different from wild specimens. Deviations from Hardy-Weinberg Equilibrium and genotype assignment tests indicated that a fraction from each sample could contain individuals from hatchery origin. Even though the estimated fraction of escaped farmed individuals in the most recent samples (2012-2013; mean = 7.1 %) is considered of low genetic risk, management recommendations for hatcheries and farms were provided. Besides, the reasons that explain the intended and unintended farmed shrimp release into the wild were discussed. Rev. Biol. Trop. 66(1): 381-393. Epub 2018 March 01.

Resumen La liberación o escape de lotes de cultivo pueden impactar la composición y variabilidad genética de las poblaciones silvestres, dando lugar a diversos problemas que pueden comprometer la eficacia biológica a largo plazo. Por lo tanto, es relevante determinar si las poblaciones de cultivo se encuentran actualmente interactuando con las poblaciones silvestres. El cultivo de camarón es una actividad de acuicultura que tiene lugar a lo largo de la costa del Pacífico tropical de América, y es la más importante en el noroeste de México. En este estudio, el camarón blanco Litopenaeus vannameisilvestre y de cultivo proveniente del Estado de Sinaloa, México, fueron evaluados genéticamente para determinar los niveles de mezcla. Se desarrolló un lote de 14 marcadores microsatélites nuevos (número de alelos promedio por locus de 11.8 y heterocigosidad esperada promedio de 0.836), mediante secuenciación de nueva generación, para la caracterización de las muestras. El muestreo consistió en camarón silvestre recolectado durante tres años (2002, 2012 y 2013) y dos lotes de unidades productoras de larva del 2007. No se observaron diferencias significativas entre años en las muestras silvestres, pero el análisis de agrupamiento indicó que los lotes de las unidades productoras de larva fueron distintos a los ejemplares silvestres. Desviaciones del equilibrio de Hardy-Weinberg y los análisis de asignación de genotipos indicaron que una fracción de cada una de las muestras silvestres podría contener individuos originados del larvicultivo. Se discuten las razones que explican la liberación de camarón de cultivo intencional y no intencional al medio silvestre. Aun cuando la fracción estimada de individuos de origen de cultivo en las muestras silvestres más recientes (2012-2013; promedio = 7.1 %) se considera de bajo riesgo, se dan recomendaciones de manejo para unidades de larvicultura y granjas de cultivo.

Proposed synonymy for Micropogonias altipinnis (Günther 1864), Micropogonias ectenes (Jordan & Gilbert 1882), and Micropogonias megalops (Gilbert 1890).

Within the Sciaenidae family, the genus Micropogonias is composed of three recognized species along the Pacific coast of Mexico: Micropogonias altipinnis, M. ectenes, and M. megalops. These species exhibit overlapping diagnostic characters, which make species identification difficult. This study ties morphological differences (meristic, morphometry of body, and otolith) with DNA sequences (CO1 and 16S fractions of mtDNA and 28S of nDNA) among Micropogonias species in the Pacific. Meristic analysis showed a latitudinal variation among the three species in the number of rays, the number of gill rakers, and length of the longest spine of the dorsal fin. Discriminant analysis of morphometric characters (body and otolith) showed three morphological entities (p < 0.001). However, the mean genetic divergences among the three species with partial sequences of mtDNA (CO1 and 16S), and nuclear (28S) were lower than those reported at the interspecific level (>2%). Genetic results suggest that the three species are one species and that the differences in meristics and morphometry could be the result of phenotypic plasticity or incipient speciation. In this sense, M. ectenes and M. megalops are proposed as junior synonyms of M. altipinnis.

Morphological description of genetically identified Cortez bonefish (Albula gilberti, Pfeiler and van der Heiden 2011) leptocephali from the southern Gulf of California.

Bonefish leptocephali of the genus Albula are difficult to identify to the species level due to morphological similarities between two different species present in the Northeastern Pacific Ocean, A. esuncula and A. gilberti. In this study, 22 bonefish leptocephali (premetamorphic and early metamorphic), collected from two locations in the southern Gulf of California were identified as Albula gilberti by comparing 459 bp of their mitochondrial cytochrome b gene sequences to those of four other species of bonefish. The characteristics of these A. gilberti leptocephali were compared to those of previously described bonefish leptocephali in the region. No distinctive morphological characteristics (meristic and pigmentation) were found that differentiate premetamorphic leptocephali of A. gilberti from those of other Albula species, making species identification by molecular-genetics a necessity. In early metamorphic leptocephali some differences in horizontal eye diameter-head length ratio, number of rays of pelvic and anal fins and myomere of pelvic-fin origin may help to differentiate A. gilberti from A. esuncula.

Molecular identification and morphological description of Micropogonias megalops, Cynoscion othonopterus, C. reticulatus and Menticirrhus nasus larvae, collected in the upper Gulf of California during Summer 2012.

Sciaenidae fish larvae were collected from the upper Gulf of California during September 2012 using a conical net (505 µm) through surface tows. These were pre-classified into four larval morphotypes, based on external characteristics (mainly meristic and pigmentation). Partial sequences of cytochrome c oxidase, subunit 1 and 16S rRNA (16S) genes of mitochondrial DNA, were used in molecular genetic identification from each larval morphotype. Genetic results indicated the identification of four larval morphotypes as Micropogonias megalops, Cynoscion othonopterus, C. reticulatus and Menticirrhus nasus. Pigmentation patterns of larvae described after molecular genetic identification made it possible to distinguish between M. megalops, M. nasus and C. othonopterus (postflexion). However, pigmentation was not reliable for differentiating between preflexion larvae of C. othonopterus and C. reticulatus. From these results, both morphological and genetic approaches were proposed as complementary tools in taxonomic studies of ichthyoplankton, particularly in early fish larvae identification of congeneric species with similar morphological characteristics.

Genotyping WSSV isolates from northwestern Mexican shrimp farms affected by white spot disease outbreaks in 2010-2012.

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010-2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.

Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico.

This study investigated whether white spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus, can survive in wild invertebrates and vertebrates in the environment surrounding shrimp farms along the Pacific coast of Mexico. The evidences imply that both viruses have a potential of persisting in crabs, blue, white and brown shrimps. The most prevalent virus, IHHNV was present in 19.5% (344/1736) followed by WSSV in 3.6% (65/1736). Coinfection of WSSV and IHHNV was also detected in crabs, blue and white shrimps. This is the first prevalence report of WSSV and IHHNV associated with wildlife species in Mexico.

Geographical variation in the genetic diversity and composition of the endangered queen conch Strombus gigas (Mesogastropoda: Strombidae) from Yucatán, México.

In Mexico and elsewhere in the Caribbean, the queen conch Strombus gigas is an endangered species. Understanding the genetic connectivity of their populations will support management strategies for long-term conservation of the species. Genetic diversity and population differentiation was assessed from samples collected at Banco Chinchorro and Isla Cozumel in the Mexican Caribbean and at Arrecife Alacranes in the Gulf of Mexico. Samples were obtained from the commercial capture at Banco Chinchorro (n = 50) and Isla Cozumel (n = 40) on March 2004. On November 2004, a non-invasive method for the Arrecife Alacranes sampling was applied, taking the hemolymph of live animals (n = 65) and releasing them to the wild. The mitochondrial DNA variation at two genes (COI and Cyt-b) was analyzed. Genetic diversity at the three locations ranged between 0.55-0.65 in COI and 0.87-0.94 in Cyt-b, showing no bottleneck evidences. A non-significant fixation index (F(ST) = 0.019, p = 0.161) and a Maximum Parsimony Network tree that did not show particular clades associated with any of the geographical locations, suggested a lack of statistically significant genetic differentiation among populations. Nevertheless, the cline patterns observed in both genetic diversity and haplotypic frequencies from Banco Chinchorro through Arrecife Alacranes, and the larger genetic distance between these locations from those between Isla Cozumel, Banco Chinchorro and Arrecife Alacranes, suggest the possibility of a pattern of isolation-by distance. The role of the main current systems over the potential genetic differences in S. gigas populations along the Mexican Caribbean, and the conservation management of S. gigas at these locations as discrete units is discussed.

Geographical variation in the genetic diversity and composition of the endangered Queen Conch Strombus gigas (Mesogastropoda: Strombidae) from Yucatán, México

In Mexico and elsewhere in the Caribbean, the queen conch Strombus gigas is an endangered species. Understanding the genetic connectivity of their populations will support management strategies for long term conservation of the species. Genetic diversity and population differentiation was assessed from samples collected at Banco Chinchorro and Isla Cozumel in the Mexican Caribbean and at Arrecife Alacranes in the Gulf of Mexico. Samples were obtained from the commercial capture at Banco Chinchorro (n=50) and Isla Cozumel (n=40) on March 2004. On November 2004, a non-invasive method for the Arrecife Alacranes sampling was applied, taking the hemolymph of live animals (n=65) and releasing them to the wild. The mitochondrial DNA variation at two genes (COI and Cyt-b) was analyzed. Genetic diversity at the three locations ranged between 0.55-0.65 in COI and 0.87-0.94 in Cyt-b, showing no bottleneck evidences. A non-significant fixation index (F ST=0.019, p=0.161) and a Maximum Parsimony Network tree that did not show particular clades associated with any of the geographical locations, suggested a lack of statistically significant genetic differentiation among populations. Nevertheless, the cline patterns observed in both genetic diversity and haplotypic frequencies from Banco Chinchorro through Arrecife Alacranes, and the larger genetic distance between these locations from those between Isla Cozumel, Banco Chinchorro and Arrecife Alacranes, suggest the possibility of a pattern of isolation-by distance. The role of the main current systems over the potential genetic differences in S. gigas populations along the Mexican Caribbean, and the conservation management of S. gigas at these locations as discrete units is discussed. Rev. Biol. Trop. 59 (3): 1115-1126. Epub 2011 September 01.

El caracol rosado Strombus gigas es una especie amenzada en México y otros sitios del Caribe. Su conservación a largo plazo requiere la comprensión de la conectividad entre sus poblaciones. En este estudio se evaluó la diversidad y diferenciación genética de muestras recolectadas en tres sitios del Caribe y Golfo de México adyacentes a la Península de Yucatán. Las muestras se obtuvieron de la captura comercial en Banco Chinchorro (n=50) e Isla Cozumel (n=40) en marzo de 2004. En noviembre de 2004 se obtuvieron muestras de Arrecife Alacranes (n=65) de animales vivos, mediante un método no invasivo diseñado para la obtención de hemolinfa; los organismos muestreados se liberaron de vuelta al medio natural. Se analizó la diversidad genética de dos genes del ADN mitocondrial (COI y Cyt-b). La diversidad genética en las tres localidades varió entre 0.55 - 0.65 en COI y 0.87 - 0.94 en Cyt-b no indicando reducción por cuello de botella. Un índice de fijación no significativamente diferente de cero (F ST=0.019, p=0.161) y un árbol en Red de Máxima Parsimonia que no mostró clados particulares asociados con localidades específicas, sugiere que no hay diferencias genéticas significativas entre sitios. Sin embargo, los patrones clinales observados en la diversidad genética y en las frecuencias haplotípicas, así como la mayor distancia genética registrada entre las localidades más alejadas (Banco Chinchorro y Arrecife Alacranes) sugiere un posible un patrón de aislamiento por distancia. Se discute el papel de los sistemas de corrientes principales del Caribe mexicano sobre la posible diferenciación genética de S. gigas. Asimismo, se discute el manejo de las localidades estudiadas como unidades discretas.

Genetic variability assessed by microsatellites in a breeding program of Pacific white shrimp (Litopenaeus vannamei).

Genetic diversity in a shrimp-breeding program was monitored for 2 generations by microsatellite DNA markers (Pvan1578 and Pvan1815) to establish levels of variation and proceed with a selection program. An increase in the number and frequencies of some alleles in both microsatellite loci from G0 to G2 was induced by foreign sire contributions. Most common alleles and high heterozygosities (around 70% in both loci) were maintained through the generations, indicating that there had not been a significant loss of genetic variability in the breeding program. However, when compared with variability in other wild and cultured stocks, the presence of 4 main alleles at both loci may be an indication that a certain reduction in variability already was present in the line used as founder stock (G0). Therefore, it is recommended that additional genetic variability be introduced to the breeding stock by crossing it with a different line.