Faculty Bibliography

Cytochrome P4501A1 (CYP1A1) mRNA is not inducible in Atlantic tomcod from the Hudson River that are treated with halogenated aromatic hydrocarbons (HAHs). In contrast, CYP1A1 mRNA is inducible in Hudson River tomcod that are treated with polycyclic aromatic hydrocarbons (PAHs) and in tomcod that are collected from cleaner rivers and treated with HAHs or PAHs. We hypothesize that CYP1A1 transcription is inhibited in Hudson River tomcod because of down-regulation of the aromatic hydrocarbon receptor (AhR) pathway and that separate molecular pathways modulate CYP1A1 transcription in fish treated with HAHs and PAHs. We initially evaluated levels of hepatic nuclear protein binding at enhancer elements (DREs) in the regulatory region of tomcod CYP1A1. No difference in levels of protein binding was observed between tomcod from the Hudson and Miramichi (cleaner) rivers that were untreated or were treated with benzo[a]pyrene. In contrast, levels of protein binding were lower in tomcod from the Hudson River that were treated with TCB than in similarly treated fish from the Miramichi River, suggesting differences between the populations in the structure or expression of AhR pathway molecules. To address this possibility, AhR DNA sequences were characterized from tomcod cDNA and genomic DNA libraries. In tomcod and mammals, AhR is represented by 11 exons, overall peptide sizes are similar, and amino acid sequences at basic, helix-loop-helix, PAAS A, and PAAS B domains are highly conserved. In contrast, little similarity was observed between tomcod and mammals in the sizes or sequences of AhR exons 10 and 11, including the absence in tomcod of glutamine-rich domains. No differences in levels of hepatic AhR mRNA were observed between the two populations or treatment groups when tomcod were untreated or were treated with aromatic hydrocarbons. In contrast, variation in levels of AhR mRNA expression was observed among tomcod tissues; however, no relationship was observed between levels of AhR mRNAs and CYP1A1 mRNAs in tissues from chemically or vehicle control-treated fish. RFLP analysis revealed extensive variation in exons 10 and 11 of AhR cDNA among tomcod from different rivers. Our results suggest that variation between tomcod populations in CYP1A1 mRNA inducibility is reflected by differences in levels of inducible hepatic protein binding to DREs. However, levels of hepatic AhR mRNA are not down-regulated in the Hudson River population, are not affected by AH treatments, and levels of AhR mRNA expression are not responsible for the differential inducibility of CYP1A1 transcription

We used a factorial experimental design to determine whether the antimicrobial Baytril (enrofloxacin) had an effect on induction of cytochrome P4501A (CYP1A) mRNA in Atlantic tomcod. Fish were injected with corn oil, Baytril and corn oil, Baytril and 3,3',4,4'-tetrachlorobiphenyl (PCB) dissolved in corn oil, or just PCB in corn oil. Analysis of gene induction in both sexes considered together showed significant induction by PCB treatment and a significant effect of sex (males higher than females). Because the experiment was carried out just before spawning, our results provide additional evidence for pretranslational suppression of CYP1A induction in spawning females. Separate analysis of the sexes showed that in both males and females, PCB had a significant effect on induction. However, in analyzing the males we also observed significant induction by Baytril alone. In addition, the BaytrilPCB combination resulted in the highest induction in any group. These findings demonstrate the potential for confounding results due to exposure of sampled fish to antibiotics, either in a laboratory or field situation

Striped bass (Morone saxatilis) stocks comingle along the northeastern United States and Canadian coasts and support mixed-stock fisheries in which stock compositions fluctuate widely. Many approaches to stock analysis of these populations have been tried. The recent use of mitochondrial DNA (mtDNA) haplotype frequency data showed promising results, despite low levels of mtDNA variation; to improve resolution, we used a single-copy nuclear DNA (nDNA) probe with mio mtDNA markers (major length variants and Tag I variants), alone or in combination. Striped bass reference collections were from the Hudson River and Chesapeake Bay, and mixed-stock collections (1989 and 1991) were from eastern Long Island, New York. The combination of the nDNA and mtDNA major length variant data provided lower but still quite high resolution potential (D-st = 0.417) in mixed-stock analysis (1991 collection) than the combination of all three markers (D-st = 0.552). However, unlike the Hudson River stock, the Chesapeake Bay stock is composed of multiple substocks that vary significantly in the frequencies of Taq I variants; this among-substock variation destabilizes the Chesapeake Bay reference data set and the resultant mixed-stock estimates. Thus, we recommend an approach based on composite nDNA and mtDNA major length variant markers

Sturgeon and paddlefish populations worldwide have declined because of anthropogenic influences. The structure and magnitude of genetic diversity of natural populations serves to buffer these fishes against environmental variation and should be maintained. Modern molecular biological techniques provide the ability to sensitively characterize and quantify the extent of genetic variation in natural populations. We provide a summary of those problems in sturgeon population biology that are amenable to investigation with DNA approaches, and their applications to date. These have included genetic identification and discrimination of taxa, identification of hybrids, stock identification, mixed-stock analysis, and estimation of gene flow and homing fidelity. To date, almost all studies have been restricted to North American fauna. Improvements to these technologies, including nondestructive sampling, should permit more widespread application of molecular approaches to problems of acipenseriform conservation. We suggest that the use of more sensitive molecular tools such as analyses of hypervariable repetitive and non-coding single copy nuclear DNA may assist management even in those taxa which exhibit overall low levels of genetic diversity

Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FI, (3) Choctawbatchee River, FL and (4) Apalachicola Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids

One of the few remaining fisheries for Atlantic sturgeon Acipenser oxyrinchus takes place during spring and fall in the New York Eight, but no information on the stock composition of this fishery is available. We used data from mitochondrial DNA (mtDNA) restriction fragment length polymorphism analysis to estimate the relative contributions of source stocks of Atlantic sturgeon to a New York Eight fishery sample (N = 112) collected in 1993 and 1994. Composite mtDNA haplotype frequencies of source populations were first characterized with five informative restriction enzymes: Bgl I, Msp I, EcoR V, HinfI, and Hinc II. All St. Lawrence River, Quebec, and St. John River, New Brunswick, specimens had an identical haplotype (genotypic diversity = 0.0); for the purposes of mixed-stock analysis, both populations were pooled as the ''Canadian stock.'' Genotypic diversity ranged between 0.483 and 0.750 among samples from the Hudson River in New York, the Edisto River in South Carolina, and the Ogeechee, Altamaha, and Satilla rivers in Georgia. Chi-square analyses indicated that the Edisto, Savannah (Georgia), Ogeechee, Altamaha, and Satilla River samples should be grouped as the ''southeastern stock,'' and that haplotype frequencies of the three source stocks (Canadian, Hudson River, southeastern) were highly heterogeneous (P = 0.0000). Mixed-stock analysis with a constrained least-squares approach under the conditional method indicated a 97.2% Hudson River contribution, a 2.8% contribution by the southeastern stock, and a 0.0% contribution by the Canadian stock; the unconditional method provided estimates of 99.1%, 0.9%, and 0.0%, respectively. Frequencies of mtDNA haplotypes of subadults (N = 30) from a seasonal aggregation of Atlantic sturgeon from the lower Delaware River were intermediate between those of the Hudson River and southeastern stocks. This finding, together with ancillary information, suggests that the Delaware River aggregation is primarily either a mixture of both the Hudson River and southeastern stocks or of the Hudson River stock and a relict Delaware River stock

Large, recreationally or commercially important populations of Atlantic sturgeon (Acipenser oxyrinchus), American shad (Alosa sapidissima), and striped bass (Morons saxatilis) occur in the Hudson River. Members of the Hudson River populations of these fishes also occur over a broad range along the Atlantic coast where they mix with conspecifics from other anadromous populations. For management purposes, it is imperative to be able to discriminate among individual stocks so that weak stocks may be protected and harvest may be allocated equitably. Because of their sensitivity and resistance to environmentally-induced temporal variation, molecular approaches have been increasingly employed in stock identification studies. However, post-Pleistocene recolonization of the Hudson River must have occurred less than 10,000 years ago-a relatively brief period for genetic divergence among populations. We tested whether various measures of DNA variation between Hudson River populations and adjacent populations of Atlantic sturgeon, American shad, and striped bass were sufficient to discriminate among their conspecific populations. American shad populations surveyed for mtDNA variation were highly diverse genotypically, but genotypic frequencies among the populations of the Connecticut, Hudson, and Delaware rivers were statistically homogeneous (p > 0.05). In contrast, Atlantic sturgeon (surveyed for mtDNA variation) and striped bass (surveyed for mtDNA and nuclear DNA variation) populations of the Hudson River were not genotypically diverse, but they were differentiated from northern and southern populations. Our results suggest higher gene flow (and lesser homing fidelity) among American shad populations in comparison with the two other species

Quantification of levels of cytochrome P4501A (CYP1A) gene expression in sentinel species of fishes has been proposed as a management tool to evaluate contamination of aquatic systems. Based on preliminary studies, we hypothesized that differences in CYP1A mRNA inducibility among individuals, populations, or species might lead to spurious conclusions when using this approach in environmental monitoring programs. To address this possibility, we quantitated and compared CYP1A mRNA induction levels in four species of common Atlantic Coast estuarine fish: smooth flounder, hogchoker, striped bass, and Atlantic tomcod, which were treated with model chemicals (beta naphthoflavone (beta-NF), or benzo[a]pyrene at 10 ppm) known to induce CYP1A mRNA, or were exposed to contaminated environments. Species-specific CYP1A DNA probes were generated from PCR (polymerase chain reaction) amplification of genomic DNA using conserved oligonucleotide primers, and, along with cloned rainbow trout and Atlantic tomcod CYP1A cDNA probes were used to quantify CYP1A mRNA levels in northern blot analyses. Successful PCR amplification of CYP1A hybridizable DNA fragments was observed for all four species. Results from northern blot analyses showed large differences in CYP1A mRNA induction among species; only Atlantic tomcod exhibited significant induction of CYP1A mRNA for both chemically treated (97-fold) and environmentally exposed fish (34-fold). Significant, although lower, levels of induction were observed in beta-NF treated (14-fold) smooth flounder, but not in environmentally exposed smooth flounder. Only low levels (not significant) of CYP1A gene induction were detected in hogchokers and striped bass. We conclude that CYP1A mRNA inducibility differed significantly among fish taxa perhaps due to differences in regulation of gene expression, suggesting that careful selection of sentinel species should be exercised prior to the use of CYP1A mRNA induction in environmental monitoring programs. However, the significance of differences in CYP1A mRNA inducibility in relation to higher level biological endpoints has yet to be determined