Monorchiids (Digenea, Trematoda) of fishes in the Yucatán Peninsula, Mexico, with the description of three new species based on morphological and molecular data.

Adult specimens of monorchiids (Digenea) were collected from the intestines of the white grunt, Haemulon plumierii Lacepède (Haemulidae), and the white mullet, Mugil curema Valenciennes (Mugilidae) from five localities off the Yucatán Peninsula and one locality in the Gulf of Mexico. Some specimens were photographed and sequenced for two molecular markers, the large subunit (LSU) of nuclear rDNA and the cytochrome c oxidase subunit 1 (cox1) of mitochondrial DNA. Other specimens were processed for morphological analyses. Newly generated sequences were aligned with other sequences available in GenBank. Bayesian inference and maximum likelihood analyses were implemented using the data sets of LSU and cox1 independently. Reciprocal monophyly evidenced through phylogenetic analyses, sequence divergence values for both molecular markers, and detailed morphological analyses, including scanning electron microscopy photomicrographs, revealed three new genetic lineages, i.e., species, as parasites of M. curema. The three new species are Sinistroporomonorchis mexicanus n. sp., Sinistroporomonorchis yucatanensis n. sp., and Sinistroporomonorchis minutus n. sp. Two additional species of monorchiids were sampled, characterised molecularly, and re-described, namely Sinistroporomonorchis glebulentus (Overstreet, 1971) from the white mullet, and Alloinfundiburictus haemuli (Overstreet, 1969), from the white grunt.

Title: Monorchiidae (Digenea, Trematoda) de poissons de la péninsule du Yucatán, Mexique, avec description de trois nouvelles espèces sur la base de données morphologiques et moléculaires. Abstract: Des spécimens adultes de Monorchiidae (Digenea) ont été collectés dans les intestins de la gorette blanche, Haemulon plumierii Lacepède (Haemulidae), et du mulet blanc, Mugil curema Valenciennes (Mugilidae) de cinq localités au large de la péninsule du Yucatán et d'une localité dans le Golfe du Mexique. Certains spécimens ont été photographiés et séquencés pour deux marqueurs moléculaires, la grande sous-unité (LSU) de l'ADNr nucléaire et la sous-unité 1 de la cytochrome c oxydase (cox1) de l'ADN mitochondrial. D'autres spécimens ont été traités pour des analyses morphologiques. Les séquences nouvellement générées ont été alignées avec d'autres séquences disponibles dans GenBank. L'inférence bayésienne et les analyses de vraisemblance maximale ont été mises en œuvre en utilisant les ensembles de données de LSU et cox1 indépendamment. La monophylie réciproque mise en évidence par des analyses phylogénétiques, des valeurs de divergence de séquence pour les deux marqueurs moléculaires et des analyses morphologiques détaillées, y compris des photomicrographies au microscope électronique à balayage, a révélé trois nouvelles lignées génétiques, c'est-à-dire des espèces, qui sont parasites de M. curema. Les trois nouvelles espèces sont Sinistroporomonorchis mexicanus n. sp., Sinistroporomonorchis yucatanensis n. sp. et Sinistroporomonorchis minutus n. sp. (Monorchiidae). Deux espèces supplémentaires de Monorchiidae ont été échantillonnées, caractérisées moléculairement et redécrites, à savoir Sinistroporomonorchis glebulentus (Overstreet, 1971) du mulet blanc et Alloinfundiburictus haemuli (Overstreet, 1969) de la gorette blanche.

Genetic structure and historic demography of endangered unarmoured threespine stickleback at southern latitudes signals a potential new management approach.

Habitat loss, flood control infrastructure, and drought have left most of southern California and northern Baja California's native freshwater fish near extinction, including the endangered unarmoured threespine stickleback (Gasterosteus aculeatus williamsoni). This subspecies, an unusual morph lacking the typical lateral bony plates of the G. aculeatus complex, occurs at arid southern latitudes in the eastern Pacific Ocean and survives in only three inland locations. Managers have lacked molecular data to answer basic questions about the ancestry and genetic distinctiveness of unarmoured populations. These data could be used to prioritize conservation efforts. We sampled G. aculeatus from 36 localities and used microsatellites and whole genome data to place unarmoured populations within the broader evolutionary context of G. aculeatus across southern California/northern Baja California. We identified three genetic groups with none consisting solely of unarmoured populations. Unlike G. aculeatus at northern latitudes, where Pleistocene glaciation has produced similar historical demographic profiles across populations, we found markedly different demographics depending on sampling location, with inland unarmoured populations showing steeper population declines and lower heterozygosity compared to low armoured populations in coastal lagoons. One exception involved the only high elevation population in the region, where the demography and alleles of unarmoured fish were similar to low armoured populations near the coast, exposing one of several cases of artificial translocation. Our results suggest that the current "management-by-phenotype" approach, based on lateral plates, is incidentally protecting the most imperilled populations; however, redirecting efforts toward evolutionary units, regardless of phenotype, may more effectively preserve adaptive potential.

Transcriptomic response in thermally challenged seahorses Hippocampus erectus: The effect of magnitude and rate of temperature change.

Hippocampus erectus inhabiting the shallow coastal waters of the southern Gulf of Mexico are naturally exposed to marked temperature variations occurring in different temporal scales. Under such heterogeneous conditions, a series of physiological and biochemical adjustments take place to restore and maintain homeostasis. This study investigated the molecular mechanisms involved in the response of H. erectus to increased temperature using transcriptome analysis based on RNA-Seq technology. Data was obtained from seahorses after 0.5-h exposure to combinations of different target temperatures (26 °C: control, and increased to 30 and 33 °C) and rates of thermal increase (abrupt: < 5 min; gradual: 1-1.5 °C every 3 h). The transcriptome of seahorses was assembled de novo using Trinity software to obtain 29,211 genes and 30,479 transcripts comprising 27,520,965 assembled bases. Seahorse exposure to both 30 and 33 °C triggered characteristic processes of the cellular stress response, regardless of the rate of thermal change. The transcriptomic profiles of H. erectus suggest an arrest of muscle development processes, the activation of heat shock proteins, and a switch to anaerobic metabolism within the first 0.5 h of exposure to target temperatures to ensure energy supply. Interestingly, apoptotic processes involving caspase were activated principally in gradual treatments, suggesting that prolonged exposure to even sublethal temperatures results in the accumulation of deleterious effects that may eventually terminate in cellular death. Results herein validate 30 °C and 33 °C as potential upper limits of thermal tolerance for H. erectus at the southernmost boundary of its geographic distribution.

De novo assembly and characterization of the liver transcriptome of Mugil incilis (lisa) using next generation sequencing.

Mugil incilis (lisa) is an important commercial fish species in many countries, living along the coasts of the western Atlantic Ocean. It has been used as a model organism for environmental monitoring and ecotoxicological investigations. Nevertheless, available genomic and transcriptomic information for this organism is extremely deficient. The aim of this study was to characterize M. incilis hepatic transcriptome using Illumina paired-end sequencing. A total of 32,082,124 RNA-Seq read pairs were generated utilizing the HiSeq platform and subsequently cleaned and assembled into 93,912 contigs (N50 = 2,019 bp). The analysis of species distribution revealed that M. incilis contigs had the highest number of hits to Stegastes partitus (13.4%). Using a sequence similarity search against the public databases GO and KEGG, a total of 7,301 and 16,967 contigs were annotated, respectively. KEGG database showed genes related to environmental information, metabolism and organismal system pathways were highly annotated. Complete or partial coding DNA sequences for several candidate genes associated with stress responses/detoxification of xenobiotics, as well as housekeeping genes, were employed to design primers that were successfully tested and validated by RT-qPCR. This study presents the first transcriptome resources for Mugil incilis and provides basic information for the development of genomic tools, such as the identification of RNA markers, useful to analyze environmental impacts on this fish Caribbean species.

Effects of water-accommodated fraction of diesel fuel on seahorse (Hippocampus reidi) biomarkers.

The present work aimed to investigate the effects of acute (12, 24, 48 and 96 h) and subchronic (168 and 336 h) exposure of seahorse, Hippocampus reidi to water-accommodated fraction (WAF) of diesel fuel on biotransformation parameters, antioxidant defenses and DNA integrity. In addition, a recovery experiment was performed, where the organisms remained in absence of the contaminant for 336 h, after WAF exposure for 168 h (totaling 504 h). At the end of each experimental protocol, the concentration of pyrene-, benzo(a)pyrene- and naphthalene-type metabolites in bile, hepatic activity of glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), as well as lipid peroxidation (LPO) levels in hepatocytes, were analyzed, in addition to the DNA damage and the micronucleus (MN) test in the peripheral blood. It was observed that both acute and subchronic WAF exposure affected the investigated parameters in different ways. In general, the exposed groups presented higher mean values for the investigated parameters if compared with their respective controls. After the recovery experiment, the mean values of PAH metabolites, LPO, DNA damage and MN frequency were significantly lower than those of animals exposed for 168 h, indicating that the recovery period was appropriately long for the evaluated biomarkers return to the control levels. The results indicated that the selected H. reidi biomarkers proved to be adequate and complementary tools in determining the first impacts of acute and subchronic exposure caused by WAF of diesel fuel in fish, as well as their recovery in clean water.

Genomic damage in Mugil curema (Actinopterygii: Mugilidae) reveals the effects of intense urbanization on estuaries in northeastern Brazil.

The largest metropolitan centers in northeastern region of Brazil are all located near the coast, and industrial, tourist, and agro-industrial activities are the principal causes of water contamination due to discharges of untreated sewage. Adverse environmental conditions can often be detected by analyzing the genetic material of organisms exposed to pollutants, and furnish an overview of environmental quality. We evaluated possible damage to the DNA of one of the fish resources most widely consumed and commercialized by coastal communities in northeastern Brazil, Mugil curema ("tainha"). Erythrocytes from M. curema were analyzed by the presence of micronuclei and by comet assay (single cell gel electrophoresis, SCGE). Statistical comparisons to both tests revealed considerably greater genomic damage in polluted estuaries than in the control site (p < 0.05), suggesting strong genotoxic impacts on the specimens evaluated, principally among those taken near localities with dense demographic and industrial development.

Exposure to E2 and EE2 environmental concentrations affect different components of the Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes bonariensis).

The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.

Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis.

Sex hormone binding globulin (Shbg) is a plasma glycoprotein that binds and transports steroids in the blood of all vertebrate classes apart from birds. In the present study we characterized shbg from pejerrey, a fish species with a well characterized temperature-dependent sex determination. The pejerrey shbg mRNA comprises 1185bp encoding for a 395 amino acid Shbg precursor protein that includes a leader sequence for secretion. Relative quantification of shbg transcript abundance revealed expression early in development coinciding with the sex-determining period and probably in association with temperature leading to male determination. The hepatopancreas was the main site of shbg expression, which varied according to the sex cycle in females. It was also expressed in gills, gonads, gut and taste buds during both larval stages and in adult fish. The presence of Shbg in organs in close contact with the environment such as gills, pseudobranchs, gut and taste buds suggests that these are potential sources of uptake or release of steroids/xenosteroids to and from the aquatic environment.

Development and characterization of microsatellite markers of the eastern keelback mullet (Liza affinis).

Twenty-four polymorphic microsatellite loci were isolated and characterized for Liza affinis using a (GT)13-enriched genomic library. The number of alleles per locus ranged from 3 to 9, with a mean number of 6.250. The observed and expected heterozygosities ranged from 0.417 to 1.000 and from 0.550 to 0.861, with an average of 0.859 and 0.779, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Evidence of null alleles was found at two loci. These markers will be useful in further studies investigating the genetic variation and population structure of this species, and may provide insights into the maintenance and efficient management of eastern keelback mullet resources.

Growth hormone receptors in the atherinid Odontesthes bonariensis: characterization and expression profile after fasting-refeeding and growth hormone administration.

In order to improve the understanding of pejerrey Odontesthes bonariensis, growth hormone (Gh)-insulin-like growth factor-1(Igf1) axis, O. bonariensis growth hormone receptor type 1 (ghr1) and type 2 (ghr2) mRNA sequences were obtained. Both transcripts were ubiquitously expressed except in kidney, encephalon and anterior intestine. Alternative transcripts of both receptors were found in muscle. Interestingly, two different ghr2 transcripts with alternative polyadenylation (APA) sites located in the long 3' untranslated region (UTR-APA) were also found in liver. Hepatic ghr1, ghr2 and insulin-like growth factor type 1 (igf1) transcript levels were examined under two different metabolic conditions. In the first experimental condition, fish were fasted for 2 weeks and then re-fed for another 2 weeks. Despite igf1 mRNA relative expression did not show significant differences under the experimental period of time examined, both ghr transcripts decreased their expression levels after the fasting period and returned to their control levels after re-feeding. In the second treatment, recombinant O. bonariensis growth hormone (r-pjGh) was orally administered once a week. After 4 weeks of treatment, liver igf1, ghr1 and ghr2 mRNA relative expression increased (13, 4·5 and 2·1 fold, P < 0·05) compared to control values. These results add novel information to the growth hormone-insulin-like growth factor system in teleosts.

Surfing among species, populations and morphotypes: Inferring boundaries between two species of new world silversides (Atherinopsidae).

Atherinopsidae are widespread freshwater and shallow marine fish with singular economic importance. Morphological, genetical and life cycles differences between marine and estuarine populations were already reported in this family, suggesting ongoing speciation. Also, coexistence and interbreeding between closely related species were documented. The aim of this study was to infer boundaries among: (A) Odontesthes bonariensis and O. argentinensis at species level, and intermediate morphs; (B) the population of O. argentinensis of Mar Chiquita Lagoon and its marine conspecifics. To achieve this, we integrated, meristic, Geometrics Morphometrics and DNA Barcode approaches. Four groups were discriminated and subsequently characterized according to their morphological traits, shape and meristic characters. No shared haplotypes between O. bonariensis and O. argentinensis were found. Significative-meristic and body shape differences between the Mar Chiquita and marine individuals of O. argentinensis were found, suggesting they behave as well differentiated populations, or even incipient ecological species. The fact that the Odontesthes morphotypes shared haplotypes with both, O. argentinensis and O. bonariensis, but also possess meristic and morphometric distinctive traits open new questions related to the origin of this morphogroup.

Effects of 5α-dihydrotestosterone on expression of genes related to steroidogenesis and spermatogenesis during the sex determination and differentiation periods of the pejerrey, Odontesthes bonariensis.

Sex steroid hormones are important players in the control of sex differentiation by regulating gonadal development in teleosts. Although estrogens are clearly associated with the ovarian differentiation in teleosts, the effects of androgens on early gonadal development are still a matter of debate. Traditionally, 11-ketotestosterone (11-KT) is considered the major androgen in fish; however, 5α-dihydrotestosterone (5α-DHT), the most potent androgen in tetrapods, was recently found in fish testis and plasma, but its physiological role is still unknown. In this context, the expression of genes associated with steroidogenesis and spermatogenesis, body growth and sex differentiation were assessed in Odontesthes bonariensis larvae fed with food supplemented with two doses of 5α-DHT (0.1 and 10µg/g of food) from hatching to 6weeks of age. At the lowest dose, 5α-DHT treated larvae showed an estrogenic gene expression pattern, with low hsd11b2 and high cyp19a1a and er2 expression levels with no differences in sex ratio. At the highest dose, 5α-DHT produced a male-shifted sex ratio and the larvae exhibited a gene expression profile characteristic of an advancement of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No differences were observed in somatic growth. These results suggest that in this species, 5α-DHT could have a role on sex differentiation and its effects can differ according to the dose.

Genetic evidence and new morphometric data as essential tools to identify the Patagonian seahorse Hippocampus patagonicus (Pisces, Syngnathidae).

A genetic study to support morphometric analyses was used to improve the description and validate the Patagonian seahorse Hippocampus patagonicus (Syngnathidae) on the basis of a large number of specimens collected in the type locality (San Antonio Bay, Patagonia, Argentina). DNA sequence data (from the cytochrome b region of the mitochondrial genome) were used to differentiate this species from its relatives cited for the west Atlantic Ocean. Both phylogenetic and genetic distance analyses supported the hypothesis that H. patagonicus is a species clearly differentiated from others, in agreement with morphometric studies. Hippocampus patagonicus can be distinguished from Hippocampus erectus by the combination of the following morphometric characteristics: (1) in both sexes and all sizes of H. patagonicus, the snout length is always less than the postorbital length, whereas the snout length of H. erectus is not shorter than the postorbital length in the largest specimens; (2) in both sexes of H. patagonicus, the trunk length:total length (LTr :LT ) is lower than in H. erectus (in female H. patagonicus: 0·27-0·39, H. erectus: 0·36-0·40 and in male H. patagonicus: 0·24-0·34, H. erectus: 0·33-0·43) and (3) in both sexes, tail length:total length (LTa :LT ) in H. patagonicus is larger than in H. erectus (0·61-0·78 v. 0·54-0·64).

Population structure of the Gulf pipefish in and around Mobile Bay and the northern Gulf of Mexico.

The genetic structure of inshore aquatic populations can be influenced by a number of factors, including coastal configurations, flow rates, and local adaptation. Properties such as salinity and temperature can differ significantly along the coasts and into the bays and rivers that contribute to these systems. Within these environments, low migratory euryhaline species provide a unique system to examine how these factors influence population structure, even when these populations are continuously distributed. In this study, we utilized microsatellite data to assess the population structure of 7 Gulf pipefish populations located in and around Mobile Bay and the northern Gulf of Mexico. Global F (ST) values (F (ST) = 0.025) suggest moderate levels of genetic structure among the populations. Local genetic structure was present among all coastal pipefish populations. Significant levels of genetic structure were also observed between coastal and estuarine populations (P < 0.05), with bay populations being distinct from their coastal counterparts. Cluster analysis suggests 2 parental populations, with one consisting mainly of estuarine individuals and the other comprising mainly coastal individuals. However, although these populations were genetically distinct, the genetic data also suggested moderate levels of migration between coastal and estuarine areas. We suggest that the differentiation between these populations is likely due to the colonization of individuals from coastal populations followed by limited dispersal out of the bay. It is also possible that different selective pressures between coastal and estuarine habitats may be contributing to the genetic differences between these populations.

Cytogenetics of Synbranchiformes: a comparative analysis of two Synbranchus Bloch, 1795 species from the Amazon.

Synbranchidae belongs to the Synbranchiformes and occurs in Africa, Asia, Australia, Mexico, and Central and South America. This family comprises four genera: Synbranchus, Ophisternon, Monopterus, and Macrotrema. Only two are known from the neotropical region, Ophisternon and Synbranchus. According to current classification, Synbranchus has three valid species: S. marmoratus (Bloch 1795), S. madeirae (Rosen and Rumney 1972), and S. lampreia (Favorito, Zanata and Assumpção 2005). Thus the present research is aimed to cytogenetically characterize (by classical and molecular methods) two syntopic species-S. aff. lampreia and S. madeirae-from the central Amazon basin to validate the taxonomy of both species and provide a revisionary discussion on the cytogenetics of Synbranchiformes. Synbranchus aff. lampreia was found to possess 2n = 44 chromosomes (6 m + 2st + 36a, NF = 50), while S. madeirae had 2n = 46 chromosomes (6 m + 2st + 38a, NF = 52). Constitutive heterochromatin was dominant in the centromeric and terminal regions of most of the chromosomes in both species, although the precise distribution patterns were species-specific. The nucleolar organizing region was single in S. aff. lampreia and multiple in S. madeirae, as indicated by both AgNO(3) and hybridization using 18S rDNA probes. The 5S rDNA sites were located interstitially on the long arms of an acrocentric pair in both species, and the telomeric probe did not show any interstitial sites in either species. These data indicate the occurrence of interspecific karyotypic variability in Synbranchus and suggest that taxonomic review for this genus is necessary.

Feminization and altered gonadal gene expression profile by ethinylestradiol exposure to pejerrey, Odontesthes bonariensis, a South American teleost fish.

In pejerrey (Odontesthes bonariensis), ovarian differentiation has been associated with gonadal aromatase expression. It is also known that exposure of pejerrey larvae to estradiol (E(2)) produces all female populations. During the last few years, the presence of ethinylestradiol (EE(2)), a synthetic E(2) analogue, has been reported in water reservoirs of different parts of the world. In the present study, the effects of EE(2) were assessed on sex ratio bias and gene expression levels of gonadal aromatase (cyp19a1a), 11ß-hydroxysteroid dehydrogenase type 2 (hsd11b2), estrogens (erα, erß1), and androgen receptors (arα, arß). Pejerrey larvae were fed with commercial food containing EE(2) (0.1 and 1 µg/g) and E(2 ) (50 µg/g) as a positive control for six weeks after hatching. The gonadal histological analysis showed that 42 to 46% of the fish had clearly differentiated ovaries in both the EE(2) - and E(2) -treated groups, compared with 27% in the control group. Moreover, in the EE(2) - (1 µg/g) and E(2) -treated groups, no fish presented signs of testicular development compared with controls. In addition, expression of cyp19a1a and hsd11b2 was significantly up- and downregulated, respectively, by EE(2) and E(2) . The authors' results suggested that the feminization process driven by EE(2) depends on the positive balance of cyp19a1a in relation to hsd11b2. Thus, these genes can be used as early indicators of exposure to xenoestrogens in this species.

Development and characterization of polymorphic microsatellite markers in the gray mullet (Mugil cephalus).

Using an (AG)13 enriched genomic library of Mugil cephalus, 12 polymorphic microsatellite loci were isolated and characterized in a test population; the number of alleles ranged from 2 to 11. The observed and expected heterozygosities ranged from 0.2593 to 0.8966 and from 0.3047 to 0.8454, respectively. Two loci deviated from Hardy-Weinberg equilibrium; linkage disequilibrium among the 12 loci was non-significant. These polymorphic microsatellite loci will be useful for genetic diversity analysis and molecule-assisted breeding of the gray mullet.