Using Plasma Vitellogenin in Loggerhead Sea Turtles to Assess Reproductive Maturation and Estrogen-Like Contaminant Exposure.

Vitellogenin (VTG), an egg yolk precursor, is abnormally produced by male and juvenile oviparous species after exposure to estrogens. Plasma VTG in loggerhead sea turtles (Caretta caretta) helped us understand their reproductive maturation and investigate it as a biomarker of contaminant exposure. The presence of VTG was screened in plasma from 404 loggerheads from the northwestern Atlantic Ocean using a freshwater turtle antibody in western blots. The concentrations of VTG were semiquantified using band intensities calibrated to results from a loggerhead antibody enzyme-linked immunoassay. The detection and concentrations of VTG were in (from highest to lowest): nesting females, in-water adult females, subadult females, smaller females, unknown sex, and males. Loggerheads from this region begin vitellogenesis at ≅77 cm straight carapace length. We classified VTG expression as abnormal in nine male or juvenile turtles. Organochlorine contaminant (OC) concentrations were measured in blood and/or fat biopsies of some turtles. One abnormal VTG female had the second highest fat polychlorinated biphenyl (PCB) and 4,4'-dichlorodiphenyldichloroethylene concentrations compared among 43 VTG-negative juveniles. The nine VTG-abnormal turtles had average blood PCB concentrations 8.5% higher, but not significantly different, than 46 VTG-negative juveniles (p = 0.453). In turtles less than 77 cm, blood PCB concentrations were significantly, but weakly, correlated with semiquantified VTG concentrations (tau = 0.1, p = 0.004). Greater blood OC concentrations were found in adult females than in males, which motivated the creation of a conceptual model of OC, VTG, and hormone concentrations across a reproductive cycle. A decision tree is also provided incorporating VTG as a sexing tool. Abnormal VTG expression cannot conclusively be linked to endocrine disruption caused by these OC concentrations. Studies should further investigate causes of abnormal VTG expression in wild sea turtles. Environ Toxicol Chem 2023;42:1309-1325. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

NMR Metabolomic Analysis of Skeletal Muscle, Heart, and Liver of Hatchling Loggerhead Sea Turtles (Caretta caretta) Experimentally Exposed to Crude Oil and/or Corexit.

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolic impacts of crude oil, Corexit 5900A, a dispersant, and a crude oil Corexit 5900A mixture exposure on skeletal muscle, heart, and liver physiology of hatchling loggerhead sea turtles (Caretta caretta). Tissue samples were obtained from 22 seven-day-old hatchlings after a four day cutaneous exposure to environmentally relevant concentrations of crude oil, Corexit 5900A, a combination of crude oil and Corexit 9500A, or a seawater control. We identified 38 metabolites in the aqueous extracts of the liver, and 30 metabolites in both the skeletal and heart muscle aqueous extracts, including organic acids/osmolytes, energy compounds, amino acids, ketone bodies, nucleosides, and nucleotides. Skeletal muscle lactate, creatines, and taurine concentrations were significantly lower in hatchlings exposed to crude oil than in control hatchlings. Lactate, taurine, and cholines appeared to be the basis of some variation in hatchling heart samples, and liver inosine, uracil, and uridine appeared to be influenced by Corexit and crude oil exposure. Observed decreases in concentrations of lactate and creatines may reflect energy depletion in skeletal muscle of oil-exposed animals, while decreased taurine concentrations in these animals may reflect higher oxidative stress.

1H-NMR metabolomic study of whole blood from hatchling loggerhead sea turtles (Caretta caretta) exposed to crude oil and/or Corexit.

We used proton nuclear magnetic resonance spectroscopy (1H-NMR) to evaluate metabolic impacts of environmentally relevant crude oil and Corexit exposures on the physiology of hatchling loggerhead sea turtles (Caretta caretta). Sample extraction and data acquisition methods for very small volume whole blood samples and sources of variation between individual hatchlings were assessed. Sixteen unclotted, whole blood samples were obtained from 7-day-old hatchlings after a 4-day cutaneous exposure to either control seawater, crude oil, Corexit 9500A or a combination of crude oil and Corexit 9500A. After extraction, one- and two-dimensional 1H-NMR spectra of the samples were obtained, and 17 metabolites were identified and confirmed in the whole blood spectra. Variation among samples due to the concentrations of metabolites 3-hydroxybutyrate, lactate, trimethylamine oxide and propylene glycol did not statistically correlate with treatment group. However, the characterization of the hatchling loggerhead whole blood metabolome provides a foundation for future metabolomic research with sea turtles and a basis for the study of tissues from exposed hatchling sea turtles.

Control of Histamine-Producing Bacteria and Histamine Formation in Fish Muscle by Trisodium Phosphate.

Scombrotoxin fish poisoning remains the primary cause of seafood poisoning outbreaks despite preventive guidelines. The purpose of this study was to investigate the use of pH for the control of growth and histamine formation by histamine-producing bacteria in fish muscle. We examined pH effects on growth and histamine formation in tuna fish infusion broth and in inoculated tuna and mahi-mahi fish muscle. Histamine production was significantly less for all bacterial strains at pH 8.5 compared to pH 5.5 in tuna fish infusion broth with no significant difference in growth. Elevated pH due to phosphate treatment of fish muscle tissues significantly reduced histamine formation with no effect on the growth of histamine-producing bacteria. This study revealed that phosphate treatment of mahi-mahi and tuna fish muscle resulted in significantly lower histamine production over 4 d of storage at 10 °C. Phosphate treatment of fish muscle may serve as a secondary barrier in addition to FDA recommended time and temperature controls for reducing public health concerns of scombrotoxin fish poisoning.

Dietary CdSe/ZnS quantum dot exposure in estuarine fish: bioavailability, oxidative stress responses, reproduction, and maternal transfer.

Continued development, use, and disposal of quantum dots (QDs) ensure their entrance into aquatic environments where they could pose a risk to biological organisms as whole nanoparticles or as degraded metal constituents. Reproductive Fundulus heteroclitus were fed a control diet with lecithin, diets containing 1 or 10 µg of lecithin-encapsulated CdSe/ZnS QD/day, or a diet containing 5.9 µg CdCl2/day for 85 days. Cadmium concentrations in liver, intestine, and eggs were quantified with inductively coupled plasma mass spectrometry. In fish fed 10 µg QD/day, QDs or their degradation products traversed the intestinal epithelia and accumulated in the liver. Less than 0.01% of the QD's cadmium was retained in the liver or intestinal tissues. This compares to 0.9% and 0.5% of the cadmium in the intestine and liver, respectively of fish fed a CdCl2 diet. Cadmium was also detected in the eggs from parents fed 10 µg QD/day. No significant changes in hepatic total glutathione, lipid peroxidation, or expression of genes involved in metal metabolism or oxidative stress were observed. While QDs in the diet are minimally bioavailable, unusual levels of vitellogenin transcription in male fish as well as declining fecundity require further investigation to determine if endocrine disruption is of environmental concern.

Development of molecular-based methods for determination of high histamine producing bacteria in fish.

Histamine (or scombroid) fish poisoning is a significant cause of food borne disease in the United States. In this study, we describe the development of a molecular-based technique which uses digoxigenin (DIG) labeled DNA probes for the detection of gram negative bacteria producing high amounts of histamine (>1000 ppm). A cocktail of PCR amplification fragments corresponding to a 709 bp fragment of the histidine decarboxylase (hdc) gene of four high producing bacteria (Morganella morganii, Enterobacter aerogenes, Raoultella planticola and Photobacterium damselae) was DIG-labeled and screened against a strain bank of 152 gram negative bacteria isolated from scrombroid fish and their harvest environment. The probe cocktail reacted specifically (100%) with the high histamine producing strains but failed to react with low histamine producers and non-producers. To further evaluate the feasibility of the approach, fish homogenate inoculated with known concentrations of four high histamine producing bacterial strains was plated on modified Niven's medium (culture method) and trypticase soy agar supplemented with 2% NaCl (for colony lift hybridization). The colony lift hybridization counts did not differ significantly from the level of the initial inoculum (p>0.05), while the modified Niven's counts were significantly lower (p<0.05) than either inoculum or colony lift counts. The use of digoxigenin (DIG) labeled DNA probes with colony lift hybridization shows promise for accurate and specific enumeration of histamine producing bacteria in scombroid fish.

Variation in toxicity of copper pyrithione among populations and families of the barnacle, Balanus amphitrite.

Inter- and intra-population variation in the toxicity of the antifouling biocide copper pyrithione (CuPT) was examined for nauplius larvae of the barnacle Balanus amphitrite. Nauplii were collected from brooding adults from four sites within the Newport River estuary (NC), chosen based on an initial estimation of recent and historical human activities that affect local contamination levels. Each site was characterized for the presence of polycyclic aromatic hydrocarbons and for the frequency of gastropod imposex, an indicator of contamination by organotins. Sensitivity of nauplii to CuPT varied significantly across the sites/populations, with LC(50) values ranging from 4.0 microg l(-1) to 6.1 microg l(-1). Larvae from the most contaminated site were the most sensitive to CuPT. Intrapopulation variation in toxicity was investigated by exposing nauplius larvae from 15 maternal families to a fixed concentration of CuPT (6.1 microg l(-1)). Variation in larval mortality among the families was significant, ranging from 15.1% to 98.9%.

Detection of gram-negative histamine-producing bacteria in fish: a comparative study.

Poisoning due to ingestion of foods with elevated levels of biogenic amines (histamine, putrescine, cadaverine, and tyramine) is well documented. Histamine fish poisoning largely is due to growth of naturally occurring bacteria associated with scombroid fish species. A rapid and reliable method is needed to screen for the presence of histamine-forming bacteria in fish. This study included a comparison of three methods for the detection of histamine-producing bacteria. A total of 152 histamine-producing and non-histamine-producing bacteria from multiple sources were screened using a modified Niven's agar method, a potentiometric method, and a PCR-based assay targeting a 709-bp fragment of the histidine decarboxylase gene. Histamine production by bacterial isolates was confirmed by high-performance liquid chromatography (HPLC). Bacterial strains were categorized as producing high amounts of histamine, low amounts of histamine, or no histamine. Of the 152 strains tested, 128 (84%) were positive with the Niven's agar method, 73 (48%) were positive with the potentiometric technique, and 74 (49%) were positive with the PCR assay. Overall, a 38% false-positive rate was observed with the modified Niven's agar method, although this method detected both low-histamine and high-histamine strains. There was a high degree of concordance (> 99%) between results of the potentiometric and PCR methods, but neither of these methods detected low-histamine bacteria. These observations support the need for a simple and straightforward yet sensitive method for detecting histamine-producing bacteria in seafood and environmental samples.

Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments.

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as polychlorinated biphenyls (PCBs) and pesticides, modulate immune responses in rodents, wildlife, and humans. In the present study we examined the effects of OCs on immunity in free-ranging loggerhead sea turtles (Caretta caretta). Mitogen-induced lymphocyte proliferation responses, lysozyme activity, and OC concentrations were measured from blood samples. Mitogens chosen in the lymphocyte proliferation assay were phytohemagglutinin (PHA) and concanavalin A (ConA) for T-lymphocyte stimulation, and lipopolysaccharide (LPS) and phorbol 12,13-dibutyrate (PDB) for B-lymphocyte stimulation. Lysozyme activity was significantly and negatively correlated with whole-blood concentrations of 4,4 -dichlorodiphenyldichloroethylene (4,4 -DDE) and the sum of chlordanes. Lymphocyte proliferation responses stimulated by PHA, LPS, and PDB were significantly and positively correlated with concentrations of the sum of PCBs measured in whole blood. LPS- and PDB-induced proliferation were also significantly and positively correlated with 4,4 -DDE blood concentrations. These correlative observations in free-ranging turtles suggest that current, chronic exposure to OCs may suppress innate immunity and enhance certain lymphocyte functions of loggerhead sea turtles. To further test this hypothesis, lymphocyte proliferation was measured after in vitro exposure of peripheral blood leukocytes from 16 turtles to Aroclor 1254 (0-13.5 microg/mL) or 4,4 -DDE (0-13.4 microg/mL). Both contaminants increased PHA- and PDB-induced proliferation at concentrations below those that affected cell viability. Moreover, the concentrations that enhanced PDB-induced proliferation in vitro were similar to concentrations measured in turtles with the highest proliferative responses. The similarities between the in vitro experiments and the correlative field study suggest that OC exposure modulates immunity in loggerhead turtles.

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.

A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.

Organochlorine contaminants in sea turtles: correlations between whole blood and fat.

Monitoring toxic organochlorine (OC) compounds is an important aspect in wildlife studies, especially in protected species such as sea turtles. The goal of this study was to determine whether blood OC concentrations can predict those in adipose tissue of sea turtles. Blood offers many benefits for monitoring OCs. It can be collected nondestructively from live turtles and can be sampled repeatedly for continuous monitoring. Organochlorine concentrations in blood may better represent the exposure levels of target tissues, but blood concentrations may fluctuate more than those in fatty tissues following recent dietary exposure or lipid mobilization. Paired fat and blood samples were collected from 44 live, juvenile loggerhead sea turtles and 10 juvenile Kemp's ridley sea turtle carcasses. Organochlorines were analyzed using gas chromatography with electron capture detection and mass spectrometry. Lipid-normalized OC concentrations measured in the blood significantly correlated to levels found in the fat samples of both species. This result suggests that sea turtle blood is a suitable alternative to fatty tissues for measuring OCs because blood concentrations reasonably represent those observed in the paired fat samples. However, blood OC concentrations calculated on a wet-mass basis were significantly and inversely correlated to lipid content in the fat samples. Therefore, caution should be used when monitoring spatial or temporal trends, as OC levels may increase in the blood following mobilization of fat stores, such as during long migrations, breeding, or disease events.

Associations between organochlorine contaminant concentrations and clinical health parameters in loggerhead sea turtles from North Carolina, USA.

Widespread and persistent organochlorine (OC) contaminants, such as polychlorinated biphenyls (PCBs) and pesticides, are known to have broad-ranging toxicities in wildlife. In this study we investigated, for the first time, their possible health effects on loggerhead sea turtles (Caretta caretta). Nonlethal fat biopsies and blood samples were collected from live turtles for OC contaminant analysis, and concentrations were compared with clinical health assessment data, including hematology, plasma chemistry, and body condition. Concentrations of total PCBs (Sigma PCBs), Sigma DDTs, Sigma chlordanes, dieldrin, and mirex were determined in 44 fat biopsies and 48 blood samples. Blood concentrations of Sigma chlordanes were negatively correlated with red blood cell counts, hemoglobin, and hematocrit, indicative of anemia. Positive correlations were observed between most classes of OC contaminants and white blood cell counts and between mirex and Sigma TCDD-like PCB concentrations and the heterophil:lymphocyte ratio, suggesting modulation of the immune system. All classes of OCs in the blood except dieldrin were correlated positively with aspartate aminotransferase (AST) activity, indicating possible hepatocellular damage. Mirex and Sigma TCDD-like PCB blood concentrations were negatively correlated with alkaline phosphatase (ALP) activity. Significant correlations to levels of certain OC contaminant classes also suggested possible alteration of protein (increasing blood urea nitrogen, decreasing albumin:globulin ratio), carbohydrate (decreasing glucose), and ion (increasing sodium, decreasing magnesium) regulation. These correlations suggest that OC contaminants may be affecting the health of loggerhead sea turtles even though sea turtles accumulate lower concentrations of OCs compared with other wildlife.