Genetic, including genotypic, diversity is positively associated with traits important for the maintenance of seagrass populations, while clonal reproduction has been shown to be correlated with an increased probability of survival, especially in stressful environments. We performed a pilot study to investigate the relationship between the intrusion of hydrogen sulfide ("sulfide"), an environmental phytotoxin, with genotypic diversity and clone size in the seagrass Halodule wrightii. Isotope ratio mass spectrometry (IRMS) was used to collect sulfur isotopic (delta 34S) data to measure sulfide intrusion in 143 root, rhizome, and leaf tissue samples from three sites in the western Gulf of Mexico. A series of microsatellite markers and gridded coordinates were used for genotyping, clonal type (single-vs multi-ramet) and size estimation. While individual genotypes varied for sulfide intrusion, multi-way ANOVA identified location to be a more important factor. Environmental attributes appear to have a more significant role than genotypic identity, clonal type, or size for sulfide uptake and distribution in this species.

The transport of passively dispersed organisms across tropical margins remains poorly understood. Hypotheses of oceanographic transportation potential lack testing with large scale empirical data. To address this gap, we used the seagrass species, Halodule wrightii, which is unique in spanning the entire tropical Atlantic. We tested the hypothesis that genetic differentiation estimated across its large-scale biogeographic range can be predicted by simulated oceanographic transport. The alternative hypothesis posits that dispersal is independent of ocean currents, such as transport by grazers. We compared empirical genetic estimates and modelled predictions of dispersal along the distribution of H. wrightii. We genotyped eight microsatellite loci on 19 populations distributed across Atlantic Africa, Gulf of Mexico, Caribbean, Brazil and developed a biophysical model with high-resolution ocean currents. Genetic data revealed low gene flow and highest differentiation between (1) the Gulf of Mexico and two other regions: (2) Caribbean-Brazil and (3) Atlantic Africa. These two were more genetically similar despite separation by an ocean. The biophysical model indicated low or no probability of passive dispersal among populations and did not match the empirical genetic data. The results support the alternative hypothesis of a role for active dispersal vectors like grazers.

Abstract Hydrogen sulfide (H2S, “sulfide”) is a naturally occurring component of the marine sediment. Eutrophication of coastal waters, however, can lead to an excess of sulfide production that can prove toxic to seagrasses. We used stable sulfur isotope ratio (δ34S) measurements to assess sulfide intrusion in the seagrass Halodule wrightii, a semi-tropical species found throughout the Gulf of Mexico, Caribbean Sea, and both western and eastern Atlantic coasts. We found a gradient in δ34S values (−5.58 ± 0.54‰+13.58 ± 0.30‰) from roots to leaves, in accordance with prior observations and those from other species. The results may also represent the first values reported for H. wrightii rhizome tissue. The presence of sulfide-derived sulfur in varying proportions (15–55%) among leaf, rhizome, and root tissues suggests H. wrightii is able to assimilate sedimentary H2S into non-toxic forms that constitute a significant portion of the plant’s total sulfur content.

Seagrass conservation and management plans are placing increased emphasis on the collection of data related to seagrass bed "condition" such as vegetative characteristics, seed production and genetic diversity. One important aspect of genetic diversity in species that reproduce both sexually and asexually is genotypic diversity. Genotypic (clonal) diversity is usually described as the proportion of unique genets within a population (richness), but it can also be characterized in other ways such as the degree to which individual ramets are distributed among genets (evenness) or the spatial arrangement of genets relative to one another (architecture). Genotypic richness, evenness, and architecture have the potential to influence sexual reproduction by affecting the proximity of genets, a key feature in dioecious species where pollen dispersal is limited and clones can vary greatly in size. They may also differ substantially according to the scale at which they are measured. This study examined genotypic richness, evenness and architecture across multiple spatial scales in the seagrass Halodule wrightii, and its association with seedbank density from three sites in the northwest Gulf of Mexico. While the magnitude of diversity estimates differed, the overall patterns remained consistent across scales. Seedbank density ranged from 19 +/- 9-188 +/- 30 seeds m(-2), following a gradient from north to south. The highest and most consistent production of seeds occurred at a site where richness (R: 0.24 - 0.42) and evenness (ED: 0.67 - 0.93) were uniformly high across spatial scales, and clonal architecture (A(c): 0.20 - 0.36) was represented by a high degree of intermingling.

: Seagrass cover has declined in many areas of the world in a trend that has acceler-ated over the past several decades. This raises concern for both the impact the decline in cover has on coastal ecosystems and the effect it may have on seagrass evolutionary potential, as genotypic and genomic variation is lost. We used 8 microsatellite loci to investigate genetic diversity, structure, and connectivity in the seagrass Halodule wrightii from the Gulf of Mexico (Texas, USA) and western Atlantic (Bermuda). We examined how estimates correlated with changes in H. wrightii abundance and distribution on the Texas Gulf coast over the past 50 yr. Results show that, compared to other species, H. wrightii from this region exhibits variable clonal diversity ( R = 0.02−0.81), moderate allelic diversity (mean A R = 4.09), and relatively high heterozygosity (mean H e = 0.56). The patterns of genetic diversity and structure, however, do not entirely coincide with either geography or recent historical trends in seagrass distribution in this region. Results from a basin in which seagrasses have recently been expanding were consistent with expectations, as they were for an isolated site near the limit of H. wrightii ’s range. Results from basins in which seagrasses have been experiencing decline and/or fragmentation, however, were mixed. Genetic structure on the Texas coast was relatively weak and coincided more strongly with tidal range than with geographic barriers or distance. Rapid expansion and the discovery of identical multilocus genotypes at several sites raises the possibility of migration via drifting vegetative fragments, as the geographic distance among certain multi-locus genotypes cannot be explained by rhizome growth models.

Sequencing genomic samples utilizing conserved primers to the cytochrome oxidase subunit 1 (COI) gene is the most reliable and popular way to obtain information from PCR fragments for seafood identification. There are times when this procedure cannot be reliably implemented to identify a seafood sample; including when samples are mixed, two species within a genera are very closely related such as various tuna and snapper species, and when samples are highly processed resulting in the DNA being fairly degraded. This chapter discusses other molecular methods such as species-specific PCR primers, random-amplified polymorphic DNA fragments, and other approaches that can successfully be implemented to identify target species from more problematic samples when it is not conducive to sequence a sample using the conserved primers to COI.

Halodule wrightii is a seagrass widely distributed in tropical areas, particularly in the western Atlantic. We have developed eight microsatellite markers from H. wrightii for use in genetic studies. The number of alleles ranged from 2 to 12 and the observed and expected heterozygosity ranged from 0.080 to 0.88, and from 0.077 to 0.86, respectively. No locus pair showed significant linkage disequilibrium or deviation from Hardy–Weinberg proportions after Bonferroni correction (P < 0.05). These markers should prove useful for elucidating genetic structure, phylogenetic relationships, and ecological processes in this foundational coastal species.

We report here the effect of one form of disturbance, boat propeller scarring, on genetic variation in the subtropical seagrass Halodule wrightii. We developed an amplified fragment length polymorphism assay to measure genetic variation in plots representing four levels of scarring intensity: reference (0% scarring), low (1-5%), moderate (5-15%) and severe (> 15%). Although we found severely scarred plots to have the lowest, and moderately scarred plots to have the highest, mean genetic diversity estimates (H-e, P), differences among scarring levels were found to be non-significant (alpha = 0.05). Analysis of molecular variance also showed no significant effect of scarring intensity. While propeller scarring can cause significant habitat loss, scarring intensities of up to 20% may not yet have seriously affected those factors (population size, flowering density, recruitment, gene flow) that strongly influence population genetic variation. The relatively recent occurrence of this type of disturbance, however, could mean that any long-term effects have yet to be detected.

A random amplified polymorphic DNA assay was used to assess genetic variation in populations of the seagrass Halodule wrightii (Ascherson) from the western Gulf of Mexico. This region includes one of the world's few hypersaline lagoons (Laguna Madre) and contains the vast majority of seagrasses on the Texas coast. Results indicate a moderate amount of genetic diversity among populations. The highest level (Hc= 0.33) was found in a population from Nueces Bay, a disturbed site in the Coastal Bend area, whereas the lowest was found in a Lower Laguna Madre population (Hc = 0.15). Genetic differentiation generally followed an isolation-by-distance model. The Nueces Bay population also showed the greatest degree of differentiation, whereas the Redflsh Bay and Lower Laguna Madre populations were relatively similar (φST = 0.091). Combined with previous results, we now have a rudimentary picture of genetic variation in this species from the Texas Gulf Coast.

Hypoxic conditions in estuaries are one of the major factors responsible for the declines in habitat quality. Previous studies examining effects of hypoxia on crustacea have focused on individual/population-level, physiological or molecular responses but have not considered more than one type of response in the same study. The objective of this study was to examine responses of grass shrimp, Palaemonetes pugio, to moderate (2.5 ppm DO) and severe (1.5 ppm DO) chronic hypoxia at both the molecular and organismal levels. At the molecular level we measured hypoxia-induced alterations in gene expression using custom cDNA macroarrays containing 78 clones from a hypoxiaresponsive suppression subtractive hybridization cDNA library. Grass shrimp exposed to moderate hypoxia show minimal changes in gene expression. The response after short-term (3 d) exposure to severe hypoxia was up-regulation of genes involved in oxygen uptake/transport and energy production, such as hemocyanin and ATP synthases. The major response by day 7 was an increase of transcription of genes in the mitochondrial genome (16S rRNA, cytochrome b, cytochrorne c oxidase I and III), and up-regulation of genes encoding proteins involved in iron metabolism. By day 14 a dramatic reversal was seen, with a significant down-regulation of both mitochondrial and Fe-metabolism genes. Validation of the macroarray results with q-PCR showed similar up- or down-regulation at multiple time points for 9 genes. At the organismal level, our studies showed condition factor of grass shrimp exposed to severe chronic hypoxia was lower than normoxic controls during the first 7 days of the experiment, but there were no differences after that time point, or in grass shrimp exposed to moderate hypoxia. Surprisingly, chronic hypoxia appeared to enhance grass shrimp reproduction; females exposed to moderate hypoxia. had higher fecundities and a greater percentage produced first, second and third broods than normoxic shrimp. The hypoxic shrimp took longer to produce their first brood than the normoxic controls, although starved larvae from hypoxia-exposed mothers lived longer than normoxic control larvae. Shrimp exposed to severe hypoxia also had higher fecundity than nonnoxic controls, although embryos from hypoxia-exposed mothers took longer to hatch than normoxic control embryos. ne gene expression and reproductive results suggest that expression levels of genes encoding proteins involved in oxygen and electron transport, energy, and iron metabolism may be useful molecular indicators of both short term (< 7 d) and moderate (14 d) exposure to severe hypoxia, and that chronic hypoxia, may have population-level impacts on grass shrimp. (c) 2006 Elsevier B.V. All fights reserved.

Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000‐gene oligonucleotide microarray for the fathead minnow Pimephales promelas , a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17β‐estradiol, a well‐characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17β‐estradiol (40 or 100 ng/L) in a flow‐through system, and gene expression in liver samples was characterized. Seventy‐one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.

Graphical systems models are powerful tools that can help facilitate hypothesis-driven ecotoxicogenomic research and aid in mechanistic interpretation of results. This paper describes a novel graphical model of the teleost brain-pituitary-gonadal (BPG) axis designed for ecotoxicogenomics research on endocrine-disrupting chemicals using small fish models. The model incorporates six compartments representing the major organs involved in the fish reproductive axis and depicts the interactions of over 105 proteins and 40 simple molecules, transcriptional regulation of 25 genes, and over 300 different reactions/ processes. Application of the model is illustrated in the context of a study examining effects of the competitive aromatase inhibitor, fadrozole, on gene expression in gonad, brain, and liver tissue of fathead minnows. Changes in mRNA transcript abundance were measured using a fathead minnow oligonucleotide microarray and quantitative real-time polymerase chain reaction. Gene expression changes observed in the ovaries of females exposed to 6.3 microg fadrozole/L for7 d were functionally consistent with fadrozole's mechanism of action, and expected compensatory responses of the BPG axis to fadrozole's effects. Furthermore, microarray results helped identify additional elements (genes/ proteins) that could be included in the model to potentially increase its predictive capacity. With proper recognition of

The south Texas Gulf coast is a unique ecosystem that contains a number of different bay systems. We used random amplification of polymorphic DNA (RAPD) markers to assess genetic diversity, differentiation and genetic distance between populations from two different bays that differed significantly in terms of flowering rate and disturbance. We found that while each bay contained a number of unique RAPD profiles, the average genetic diversity in each population was low. Genetic distance between the two populations was also low (Fst=0.084) and the majority (92%) of the genetic variation was attributed to differences between individuals within populations. The population from the Laguna Madre location, however, was polymorphic for a larger number of markers, had a higher average genetic diversity and a larger number of unique RAPD profiles. The higher level of flowering at this location most likely accounts for the higher diversity.

Using subtractive hybridization, we have identified 17 genes that are either up- or down-regulated in the hepatopancreas (Hp) of the lobster, Homarus americanus, by acute exposure to the juvenile hormone analog methoprene. The expression of some of the genes obtained from the subtraction libraries was confirmed by real time Q-PCR experiments. These genes encode several different classes of proteins including: structural, enzymatic and regulatory polypeptides. Enzymes represent the predominant genes up-regulated by methoprene. Included in this group are betaine-homocysteine S-methyltransferase (BHMT) and two other enzymes of the methionine cycle. Increased expression of a translation factor (eIF2), as well as of cytosolic (aldose reductase), structural (beta-tubulin, L5A) and plasma membrane (CD42d) proteins was observed. In addition, a major feature of altered gene expression in methoprene treated Hp was increased levels of enzymes associated with protein turnover, including trypsin, ubiquitin conjugating enzyme and ubiquitin carboxyl terminal hydrolase. Down-regulation of the members of the hemocyanin family was observed. Assays confirmed elevated levels of trypsin in the Hp of lobsters after 24 h exposure to methoprene. Our findings suggest a wide variety of cellular targets are altered by methoprene.

This chapter reviews current methods to study the transcriptome and the proteome (the set of proteins encoded by a genome) and indicates approaches that can be taken for non-model species. The chapter briefly explains genomics and proteomics. The ultimate goal of genomic experiments is to link-induced gene expression patterns to detrimental effects, harmless effects, or even protective effects. The term "proteomics" refers to the large-scale study of proteins in cells or tissues and also understanding the complex interactions among proteins that occur within cells. These interactions include formation of functional complexes as well as interactions with other cellular components such as nucleic acids, lipids, and carbohydrates. Proteome research has been subdivided into two categories: (1) expression proteomics and (2) interaction proteomics.

In this study male largemouth bass (LMB) were exposed to the naturally occurring androgens, dihydrotestosterone (DHT) or 11-ketotestosterone (11-KT) in order to identify genes that are differentially regulated by these steroid hormones. Using subtractive hybridization on livers of fish treated with DHT against vehicle control, many novel LMB genes were cloned. These genes were added to our gene library and arrayed. Six genes were up-regulated and five were down-regulated by both androgens. But, each androgen also regulated specific genes. One gene that was identified as a potential androgen marker was spermidine-spermine-N(1)-acetyltransferase that was up-regulated by both androgens. Determining which genes are responsive to natural androgens will help to identify biochemical pathways that are impacted.

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.

Starch structure and functionality have a significant impact on the utilization of cereal grains as food and feed. Starch viscosity characteristics are used to characterize rice cooking, processing and eating quality. In order to examine the genetics of viscosity characteristics, we developed molecular markers for five of the major enzymes involved in starch synthesis in the endosperm: granule bound starch synthase, soluble starch synthase, rice branching enzymes 1 and 3 and starch debranching enzyme. These markers were polymorphic in a cross between specialty rice varieties of diverging amylose content and viscosity characteristics. Our results indicate that the Waxy locus, encoding the gene for granule bound starch synthase, has a significant effect on peak viscosity, hot paste viscosity, cool paste viscosity, breakdown and setback viscosity. We estimate that the tightly linked (5–10 cm)locus for starch synthase may have a lesser, additive effect.