Theses @ Nicholls State University

Chromium (Cr), one of the prevalent trace metal contaminants in aquatic environments, is reportedly present in crustacean exoskeleton. Using the blue crab, Callinectes sapidus, as the model crustacean, this study sought to address during which phase of the molting cycle Cr is deposited to the exoskeleton and how Cr in different valencies this such a process and exoskeletal mineralization. It was hypothesized that due to Cr II and calcium (Ca) being both divalent, Cr II (Cr2+) would promote Cr incorporation into the shell during post-ecdysial mineralization, thereby adversely affecting mineralization, more than either Cr III or Cr VI. One-day post-ecdysial blue crabs were injected with 0 and 2 μg Cr/g wet weight in chromium chloride (Cr II), chromium potassium sulfate (Cr III) or sodium chromate (Cr VI) at day 0 (one-day post-ecdysis) and day 2 (three-day post-ecdysis). At day 4 (five-days post-ecdysis) exoskeletal Cr, Ca, and magnesium (Mg) content was analyzed using ICP-OES. Cr III and Cr VI treatments were found to result in a 2.53- and 2.96-fold increase in exoskeletal Cr content, respectively, while Cr II treatment had no effect. Additionally, both Cr III and Cr VI, not Cr II, significantly reduced the Ca and Mg content per unit shell area, suggesting that both Cr III and Cr VI inhibit the flux of Ca and Mg to the shell during mineralization, accounting for Cr III- and Cr VI-induced decreases in exoskeletal weight. These results are not supportive of the working hypothesis. Discussion on the mechanism driving the effects of Cr III and Cr VI is presented. Significant Cr accumulation was found in the gill, hepatopancreas, and hemolymph following treatment with Cr II, Cr III or Cr VI. The muscle also exhibited enhanced Cr accumulation after treatment with either Cr II or Cr VI. The significant incorporation of Cr in the exoskeleton of blue crab following Cr III and Cr VI treatment suggests that exoskeletal Cr can be used as a biomarker for aquatic Cr pollution.

 
Abstract
Bowfin, Amia calva, is the only extant species in the family Amiidae. In Louisiana, Bowfin are an important commercial fish, are harvested for meat and roe for use as caviar, and are top predators and key components in the trophic dynamics of aquatic systems. Previous research has concentrated on Bowfin diet and potential effects on fish assemblages. The objectives of this study were to measure linear home ranges of Bowfin in the upper Barataria Estuary (UBE), determine if Bowfin move onto the inundated floodplain of the UBE following large rain events and compare dissolved oxygen concentrations and Bowfin movement rates to evaluate how Bowfin respond to hypoxic conditions. Thirty-four Bowfin were fitted with external radio transmitters and released at capture sites in 2015. Bowfin were tracked and water quality was recorded every two weeks from 6 July 2015 to 26 June 2016. Bowfin in the UBE had small linear home ranges from 21.55 to 319.35 m with an average of 163.54 ± 17.90 m. Bowfin were observed in both hypoxic and normoxic water and there was no correlation between dissolved oxygen concentrations and Bowfin movement rates. There were 22 observations of Bowfin moving onto inundated floodplain habitat. Bowfin are well adapted to hypoxic conditions in the UBE due to the ability to use atmospheric oxygen to supplement oxygen intake by the gills. This study was the first to quantify movement of Bowfin in the UBE and can lead to a better understanding of how Bowfin have adapted to altered floodplains such as the UBE.

 
Abstract
Because of activities of oil and gas production in the northern Gulf of Mexico, marine and coastal organisms are subject to petroleum contamination. In order to obtain a molecular biomarker for polycyclic aromatic hydrocarbon (PAH) pollution, the cytochrome P450 (CYP) system in the blue crab, Callinectes sapidus, was explored using a degenerate primer approach. A partial CYP4 cDNA sequence from the hepatopancreas of Callinectes sapidus, consisting of 412 bp and with an open reading frame of 136 amino acids, was acquired. CYP enzymes are responsible for metabolizing hormones, such as ecdysteroids, and xenobiotics, such as PAHs. The responsiveness of the blue crab CYP4 gene to the molting hormone 20-hydroxyecdysone (20E) and a representative PAH chrysene was determined using tissue culture and quantitative real-time PCR (qPCR). Hepatopancreatic tissues from blue crabs were exposed to three concentrations of 20E, encompassing ecdysteroid concentrations found during the molting cycle of Callinectes sapidus, and three concentrations of chrysene. It was found that CYP4 gene expression decreased with increasing 20E concentrations, with 10 and 100 nM 20E significantly increasing the relative abundance of CYP4 mRNA while 1 μM 20E resulted in the absence of CYP4 expression. Such a pattern of changes in CYP4 gene expression is consistent with the previous finding that the peak activity of microsomal ethoxyresorufin O-deethylase (EROD) in the hepatopancreas of Uca pugilator occurs in postmolt when ecdysteroid titers in the hemolymph are depressed. This result lends support to the notion that CYP activity in the hepatopancreas is used to prevent untimely rises in ecdysteroid concentrations in the hemolymph. Increasing concentrations of chrysene led to decreasing amounts of CYP4 mRNA, with chrysene at 0.2, 2 and 20 μM all significantly suppressed CYP4 gene expression. The inhibitory effect of chrysene on CYP4 mRNA in cultured hepatopancreatic tissues may indicate iv that chrysene exposure can induce cytotoxicity, thereby compromising the transcriptional machinery. Overall, the results show that the CYP4 gene acquired in the present study appears to be involved in ecdysteroid regulation and is not suitable for use as a biomarker for petroleum contamination.

 
Abstract
Ridges in the Mississippi River delta are unique estuarine ecotone areas. Bottomland hardwood forests, swamps, mangrove forests, marshes, and open water habitats occur in close proximity to each other along the elevation and salinity gradients of ridges. However, due to sea level rise, subsidence, and a lack of sediment input many of these ridges are shrinking and disappearing. Although it is documented that wetlands reduce predation and act as nursery grounds for aquatic fauna, including many commercially harvested species, there has been little research on ridge influences on aquatic faunal communities. Therefore, the purpose of this research was to examine ridge edge habitat use by aquatic fauna and compare the marsh edge and open water aquatic faunal communities at Isle de Jean Charles, a degraded natural estuarine ridge in Terrebonne Basin. Water quality and fish and crustacean size, abundance, and diversity at marsh edge and open water sample locations were sampled monthly from July 2022 to June 2023. Sample efforts employed multiple passive sampling methods at marsh edge and open water locations including minnow traps, crab traps, and gill nets. Catch per unit effort (CPUE), species richness, diversity, and overall size in aquatic fauna between marsh edge and open water ridge sites were compared. The marsh edge habitat had a higher species richness (n = 31) compared to the open water location (n = 17). Furthermore, total species richness was higher at the marsh edge sample location on every sample event and mean monthly species richness was significantly higher (F1,22 = 23.59, P < 0.001) at the marsh edge sample location (9.1 ± 0.8) compared to the open water location (4.4 ± 0.6). Although not significant, Shannon-Weiner diversity was higher at the marsh edge location (0.61 ± 0.09) compared to the open water location (0.43 ± 0.07). Additionally, fish (F1,22 = 81.28, P < 0.001), crustacean (F1,22 = 28.22, P < 0.001), and total (F1,22 = 61.58, P < 0.001) CPUE in minnow traps were all significantly higher at
iv
the marsh edge location. Commercially important Blue Crabs Callinectes sapidus collected in marsh edge crab traps had a significantly smaller mean carapace width (F1,245 = 12.03, P < 0.001) which was an artifact of the higher percentage of juveniles (carapace width < 125 mm) collected at the marsh edge location (63.8%) compared to the open water (29.4%). Examination of the ecological function of a degraded natural ridge can help inform conservation and preservation efforts for Louisiana’s estuarine and wetland habitats and future ridge restoration efforts.

 
Abstract
In the Lower Mississippi River floodplain, distributary ridges formed by riverine sediment serve as some of the only areas of terrestrial habitat. Ridges provide ecosystem services in the form of storm surge mitigation and forested habitat that supports native fauna and migratory avifauna. At the elevation gradient from the crown of a ridge to the neighboring marsh, an ecotone exists that supports plant species with different tolerances to inundation. The Mississippi River deltaic plain is heavily degraded due to anthropogenic activities and natural processes, and remaining natural ridges are subsiding due to a lack of sediment input. Ridge restoration is a relatively new technique with uncertainty regarding the elevation, slope, and size of ridge construction and what should be planted on and around them. In this study, we characterize the communities found on the slopes and associated marshes of eight elevated coastal landforms, including natural ridges, restored ridges, and spoil banks. Emergent and submergent plant community composition, elevation, and soil characteristics were documented at fine spatial scales in summer 2022 and 2023. Hierarchical cluster analysis was used to identify plant communities, and those communities were then quantitatively assessed in terms of associated abiotic variables. Communities found in higher elevation salt marshes generally did not differ significantly among abiotic variables, nor did communities found in lower elevation salt marshes. Soil salinity and percent inundation were found to be the most important drivers of community composition, and sites with more available habitat at a wide range of elevations had increased community diversity. Random Forest was used to predict community change up to 50 years in the future based on relative sea-level rise estimates, and at saline sites there is a predicted shift to dominance of Spartina alterniflora and Avicennia germinans communities as higher elevation marshes are increasingly exposed to inundation with salt water. Evidence was found that suggests the marshes associated with restored ridges may have insufficient hydrologic connectivity, which can have negative implications for resilience and health of marsh plant communities. Ridge slopes appear to be eroding faster than ridge crowns, limiting upslope migration potential.

 
Abstract
The Atchafalaya River Basin (ARB) is the largest bottomland hardwood river-floodplain system in North America and produces 90% of the wild crayfish harvest in Louisiana. However, anthropogenic modifications to the natural hydrology in the ARB have altered historic riverfloodplain connectivity and reduced water circulation and flow patterns that facilitate extensive areas of hypoxia for several months throughout the annual flood pulse. Although Red Swamp Crayfish Procambarus clarkii can tolerate relatively low dissolved oxygen concentrations, chronic environmental hypoxia can negatively affect P. clarkii population characteristics. The purpose of this project was to compare P. clarkii life history, fecundity, and trophic position between chronically hypoxic and normoxic areas in the ARB. Crayfish were sampled every two weeks at 14 sample sites in the eastern ARB during the 2020 and 2021 crayfish seasons. Water quality and CPUE were recorded at all sites on every sample date and sex and carapace length were recorded for all captured crayfish. Additionally, P. clarkii hemolymph samples were collected from ten intermolt individuals at all sample locations on each sample date to determine hemolymph protein concentration. Procambarus clarkii oocyte number and maturation stage were compared between individuals from hypoxic and normoxic sites. Trophic position of P. clarkii in hypoxic and normoxic sites was examined using stable isotope analyses of δ 15N and δ 13C. Procambarus clarkii CPUE was significantly lower in chronically hypoxic locations compared to normoxic in 2020 (U = 1718, P = 0.027) and 2021 (U = 483, P < 0.001). Mean hemolymph protein concentration was also significantly lower in hypoxic locations in 2020 (F1,118 = 19.32, P < 0.001) and 2021 (F1,374 = 5.80, P = 0.017). Female P. clarkii from hypoxic locations produced significantly fewer total oocytes in both 2020 (F1,36 = 6.80, P = 0.013) and 2021 (F1,141 = 4.77, P = 0.031). Furthermore, P. clarkii mean annual maturation index was lower in hypoxic locations compared to normoxic locations during both sample years. Trophic position was significantly lower in P. clarkii from hypoxic locations in 2020 (F1,13 = 313.81, P < 0.001), however, no significant difference was observed in 2021. The results of this study demonstrate that chronic environmental hypoxia in the ARB can adversely influence P. clarkii life history, fecundity, and trophic characteristics. This information can be applied to future ARB management decisions and is critical to stakeholders and resource managers as efforts to improve water quality and reduce the severity, extent, and duration of ARB hypoxia move forward.

 
Abstract
Lead (Pb), a toxic, non-essential metal, gets into aquatic ecosystems through a wide range of natural and anthropogenic sources, and has been frequently found in tissues of aquatic crustaceans. The post-ecdysial mineralization in crustaceans involves the deposition of carbonate salts, such as calcium carbonate, to the organic matrix. Due to the resemblance between Pb2+ and Ca2+, this study was carried out to investigate whether Pb is incorporated into the new shell during post-ecdysial mineralization using the blue crab, Callinectes sapidus, as the model crustacean. It was hypothesized that injected Pb would be deposited in the shell via calcium transporters in the epidermis during the mineralization process. Post-ecdysial blue crabs were injected with two doses of 5 µg Pb/g wet weight each in lead acetate and then Pb, Ca and Mg content were analyzed in the exoskeleton, while only Pb bioaccumulation was quantified for the hepatopancreas, gills, muscles, and hemolymph. Results showed a statistically non-significant increase in exoskeletal Pb content in Pb-treated crabs compared to control. Interestingly, Pb was found present in the exoskeleton of control crabs as well as in the exuviae, indicating that these crabs were exposed to lead in their previous habitat. There was a significant decrease in Ca content in Pb-treated crabs, suggesting that lead hindered the deposition of Ca to crab exoskeleton, thereby obstructing calcification. A trend of decrease in exoskeletal Mg was also observed in Pb-treated crabs. There was a significant increase in Pb content found in the gills, hepatopancreas, muscle, and hemolymph in Pb-treated crabs. The rank of the Pb level amongst three soft tissues in a decreasing order is: hepatopancreas > gill > muscle. This study is the first to present evidence that lead disrupts post-ecdysial exoskeletal calcification in a crustacean.

 
Abstract
To mitigate flood damage, Louisiana has implemented extensive hydrological modifications that have disconnected floodplains such as the upper Barataria Estuary (UBE) from the mainstem of the Mississippi River. Another Mississippi River floodplain, the Atchafalaya River Basin (ARB) remains connected to the Mississippi River. Adult Spotted Gar Lepisosteus oculatus were collected seasonally from May 2022 through December 2022 from the ARB and UBE to evaluate if connection to the Mississippi River influences adult Spotted Gar reproductive potential and body condition. Overall mean total lengths (TL ± SE; mm) and weights (WW ± SE; g) of the ARB female (n = 50; TL = 602 ± 9; WT = 930 ± 51) and male (n = 45; TL = 527 ± 7; WT = 578 ± 31) Spotted Gar were larger than UBE female (n = 30; TL = 532 ± 8; WT = 572 ± 31) and male (n = 27; TL = 485 ± 9; WT = 453 ± 27; P < 0.05) Spotted Gar. Mean seasonal gonadosomatic index (GSI) was higher for UBE female Spotted Gar than ARB female Spotted Gar for spring and summer while ARB female Spotted Gar GSI was higher than UBE female Spotted Gar GSI for fall (P < 0.05). Egg diameters were greater for ARB female Spotted Gar than UBE female Spotted Gar across all seasons (P < 0.05) except winter (P > 0.05). Mean seasonal condition factor (CF) of ARB female Spotted Gar was higher than UBE female Spotted Gar for spring and summer (P < 0.05) and similar for fall and winter (P > 0.05). Mean seasonal GSI was similar between ARB and UBE male Spotted Gar across all seasons (P > 0.05). Mean seasonal CF of ARB male Spotted Gar was higher than UBE male Spotted Gar for spring (P < 0.05) and similar for summer, fall and winter (P > 0.05). Overall mean fecundity was higher for ARB Spotted Gar (8,472 ± 1,661) than UBE Spotted Gar (3,775 ± 967; P < 0.05). Overall mean δ13C values were depleted in ARB Spotted Gar compared to UBE Spotted Gar (P < 0.05). Trophic position of ARB Spotted Gar was higher than UBE Spotted Gar for spring and summer while trophic position of UBE Spotted Gar was higher than ARB Spotted Gar for fall (P < 0.05). Mean DNA methylation amount of Spotted Gar between basins and sexes were similar (P > 0.05). Although, Spotted Gar were larger from the ARB compared to the UBE, seasonal differences in reproductive and body condition variables of Spotted Gar between basins suggests that basin connection to the mainstem of the Mississippi River may not impact Spotted Gar reproductive potential and body condition.

 
Abstract
Metribuzin is a triazine herbicide used in sugarcane fields to control for broadleaf weeds. Metribuzin acts as a photosystem-II inhibitor which prevents the energy gain of electrons while also allowing oxidative damage to cellular membranes. Metribuzin first saw use in Germany and the United States as pre-emergent herbicide for roadleaf weeds in soybean fields. Use of metribuzin has since expanded into sugarcane and wheat fields. Metribuzin that is washed into waterways as runoff can be linked to hermaphroditism in frogs as well as algal and macrophyte death. Therefore, metribuzin degradation by bacteria is essential to reduce the half-life of metribuzin in the environment. The objective of this study was to determine metribuzin degradation by various bacterial consortia developed from an agricultural soil and from an anaerobic digester of Thibodaux sewage treatment plant. Soil from the USDA/ARS, Sugarcane Research Unit’s Ardoyne Research Farm in Schriever and sludge from the anaerobic digester of the Thibodaux sewage plant were exposed to metribuzin along with a pure culture of Rhodococcus rhodochrous under various electron acceptor conditions including, aerobic, anaerobic, nitrate reducing, sulfate reducing, and mixed (nitrate and sulfate) electron acceptor conditions with the commercial formulation of metribuzin, Sencor. Bacterial enrichment cultures were developed in basic mineral salt (BMS) medium. Once the enrichment cultures were developed, detailed studies were conducted under various conditions and the duration of each experiment for each condition was 14 days. The results showed that the 80% pure metribuzin was not degraded in most of the conditions from various bacterial enrichments. The only conditions that showed significant degradation of metribuzin were the treatment condition in which metribuzin was used the sole source of carbon under mixed electron acceptor conditions and in co-metabolic nitrate reducing conditions. In both conditions, the enrichment came from iv the sludge of the anaerobic digester of the Thibodaux sewage treatment plant. The co-metabolic nitrate reducing bacterial consortium showed the best result of 50.9% reduction of 99.99% pure metribuzin. The major metabolites identified in metribuzin degradation were desamino-metribuzin, diketo-metribuzin, and desamino-diketo-metribuzin. The bacterial consortia developed from the agricultural soil and the pure culture, Rhodococcus rhodochrous was unable to degrade metribuzin. This indicates that more time is needed for the soil bacteria to evolve necessary enzymes to degrade metribuzin. Repeated exposure to metribuzin may help the soil bacteria to adapt and evolve metribuzin degrading enzymes.

 
Abstract
Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are commonly found in Louisiana’s local waterways, specifically in the waterways of Bayou Lafourche and the Intracoastal Canal. In August of 2021, Hurricane Ida flooded levees in Larose, Louisiana, where the bayou and canal intersect, and deposited sediment contaminated with industrial chemicals, antibiotics, and ARB. Many multidrug resistant bacteria were isolated from sediment collected from Larose, including Bacillus cereus, Proteus mirabilis, Bordetella avium, Pseudomonas aeruginosa, and Alcaligenes faecalis. These isolates’ ability to resist multiple antibiotics made them likely candidates to degrade antibiotics as their sole carbon or nitrogen source. One of the bacterial isolates, Alcaligenes faecalis, was able to use sulfamethoxazole (SMX) as its sole nitrogen source and was resistant at concentrations of 500 mg/L of SMX. When this bacterium was grown on a basic mineral salt medium with 250 mg/L of SMX as the sole nitrogen source, as SMX contains amino groups, Alcaligenes faecalis was able to cleave the amino group. SMX was removed by 39.81% in the culture. HPLC analysis showed the production of a metabolite. LC/MS confirmed the identity of the metabolite as 3-hydroxy-5-methylisoxazole based on a standard. A. faecalis has deaminase enzymes that removed an amino group from SMX and used the ammonia as a nitrogen source only when there is no other nitrogen source in the medium. The isolate did not produce 3-hydroxy-5-methylisoxazole when SMX and ammonium salt were provided in a basic mineral salt medium. A. faecalis is also capable of degrading 3-amino-5-methylisoxazole as its sole nitrogen source. 3-Amino-5-methylisoxazole was identified as a transformation metabolite of SMX degradation which was further converted to 3-hydroxy-5-methylisoxazole. A biodegradation pathway for SMX degradation was constructed which included 3-hydroxy-5-methylisoxazole and 3-amino-5-methylisoxazole.

 
Abstract
The 2010 Deepwater Horizon (DWH) oil spill released an estimated 506 million liters of oil into the northern Gulf of Mexico, impacting shorelines from Louisiana to Florida. The salt marshes of the Mississippi River Delta were particularly impacted by the spill. Little attention has been given to the salt marsh-stabilizing interactions of the keystone species Geukensia granosissima (southern ribbed mussel) and Spartina alterniflora (smooth cordgrass). The mutualistic relationship between G. granosissima and S. alterniflora has been shown to promote salt marsh sustainability by facilitating enhanced productivity, soil shear strength, and vertical accretion of the marsh soil surface. The goals of this study were to assess DWH impacts on (1) the mussel populations of Barataria Bay, LA, (2) salt marsh function and stability by altering the facilitative interactions of S. alterniflora and G. granosissima, (3) feasibility of mussel transplantation into impacted salt marshes to mitigate hydrocarbon stress. During 2017, I conducted five mussel surveys at salt marsh sites in northern Barataria Bay that were exposed to either heavy, moderate, or no visible oiling as determined by the S.C.A.T technique immediately after the spill. Mussels within a 0.25 m2 quadrat placed at random within 1 m of the marsh edge were harvested, classified as either adult (> 60 mm) or juvenile, and the effects of oiling assessed on each size-class individually by determining mussel density, approximate size, and average mussel biomass. My results show that adult mussels had significantly smaller dimensions and tended to have less mass at moderately oiled sites. In contrast, juvenile mussels tended to be larger at heavily oiled sites, but their density, as well as total mussel density, followed a decreasing trend with increasing oil intensity. A field manipulation experiment was conducted at four reference and four heavily oiled salt marsh sites located within Barataria Bay, LA. Four 0.25 m2 plots were iv established at each site: two control plots with all G. granosissima removed, and two transplant plots with G. granosissima removed then added back at a density of 100 mussels m-2. Mussels transplanted into reference sites tended to positively effect total aboveground biomass during the second sampling year, with an overall negative trend of decreasing total aboveground biomass at heavily oiled sites. Transplanted mussels increased live rhizome standing crop at reference sites during the first year only, a similar trend occurred for live (root + rhizome) standing crop. On average, soil surface strength was greatest at heavily oiled sites and transplanted mussel sites. Oiling reduced mussel survivorship and recruitment, but not growth. Impacts on mussel populations and salt marsh structure, function, and stability indicate recovery is not complete at our sampled sites in Barataria Bay, and requires additional study.