MEES Theses and Dissertations
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Item Impact of salinity on morphology, growth, and pigment profiles of Scenedesmus obliquus HTB1 under ambient air and elevated CO2 (10%) conditions(2024) Jiao, Fanglue; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Certain microalgal species tolerate high CO2 concentrations and proliferate faster with elevated CO2 than with ambient air. This feature makes them attractive for carbon sequestration, a tool for mitigating climate change due to increasing atmospheric CO2. Scenedesmus species are among these microalgae. Scenedesmus obliquus strain HTB1 is a microalgal strain isolated from the Baltimore Inner Harbor (brackish water) and has shown a faster growth with 10% CO2 compared to air. However, how HTB1 grows under different salinity and if the salt response is affected by elevated CO2 remains elusive. Two experiments were set up to address these questions. The first experiment tested the impact of salinity gradient (0, 17.5, 20, 22.5, 25, 27.5, and 30 ppt) on HTB1 under ambient air. With increasing salinity, HTB1 cells became smaller, and the cultures changed color from green to brown, yellowish brown, and then to pale white. The pigment analysis showed that HTB1 reduced several pigments (i.e. zeaxanthin, lutein, chlorophyll b) in response to salt stress. However, HTB1 produced higher concentrations of canthaxanthin under the salt stress. The growth of HTB1 decreased with increasing salinity and was inhibited when the salinity was greater than 22.5 ppt. In the second experiment, we compared the impact of salinity (0, 10, and 20 ppt) on HTB1 under air and 10% CO2, respectively. HTB1 cultures showed little color change with increasing salinity under 10% CO2. In contrast, the change of culture color from dark green to brown was observed with increasing salinity when HTB1 was grown with air. Interestingly, the growth of HTB1 was less inhibited with salt under 10% CO2 than with air, suggesting that elevated CO2 mitigates the salt stress of HTB1. Lutein and canthaxanthin increased with increasing salinity when HTB1 was grown with 10% CO2. Our results indicate that increased salinity affects the growth of Scenedesmus obliquus HTB1 more with air than with 10% CO2. This study provides insight into the impact of salt stress on algal morphology, growth, and pigment composition.Item Climate Change and Vibrio species: Investigation of Environmental Parameters Associated with Occurrence and Transmission(2023) Brumfield, Kyle David; Colwell, Rita R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Climate change, associated with shifts in the geographical range of biological species, has become increasingly important in emergence and re-emergence of disease. Vibrio spp., native to aquatic ecosystems, are commonly associated with aquatic invertebrates, notably crustaceans and zooplankton. Some species of the genus Vibrio cause infection in humans, of which Vibrio cholerae, the etiological agent of pandemic cholera, is the most documented. Pathogenic non-cholera Vibrio spp., namely Vibrio parahaemolyticus and Vibrio vulnificus, cause gastroenteritis and also septicemia and extra-intestinal infections. They are responsible for a large number of public health emergencies in developed countries, including the United States. As sea temperatures rise and salinity profiles are altered, a pattern of poleward spreading of non-cholera Vibrio spp. has been observed globally, demonstrating significant geographic expansion of these bacterial populations, corroborated by an associated increase in the number of reported vibriosis cases. Since Vibrio spp., including pathogenic vibrios, play an important role in the degradation of polymeric substances, such as chitin, and in biogeochemical processes, they cannot be eradicated. Hence, routine monitoring and an early warning system are needed for public health preparedness. Since the 1960’s, ongoing research has focused on environmental factors linked with occurrence and distribution of clinically relevant Vibrio spp. and their role in disease transmission. We have reported that lack of, or damage to, water, sanitation, and hygiene (WASH) infrastructure, coupled with elevated air temperatures, and followed by above average rainfall promotes exposure of a population to contaminated water, hence increases the risk of an outbreak of cholera. Global predictive intelligence models applicable to diseases caused by non-cholera Vibrio spp. are in development. The research reported here describes results of intensive sampling to detect and characterize Vibrio spp. in the Chesapeake Bay, Maryland, and the Florida Gulf Coast, the latter an area significantly impacted by Hurricane Ian, September 2022, with a spike in confirmed vibriosis cases and deaths during weeks following the storm. Results of this study provide confirmation of environmental predictors for Vibrio spp. and document long-term increase and extended seasonality of Vibrio populations in the Chesapeake Bay. Using satellite remote sensing data, we demonstrate the impact of extreme heat, precipitation, and other key environmental and geophysical factors (e.g., temperature, salinity, and chlorophyll) on prevalence of pathogenic Vibrio spp. in aquatic systems. This study lays the groundwork for a predictive intelligence system for Vibrio spp. and other pathogens under varying climatic scenarios.Item Viromics and biogeography of estuarine virioplankton(2021) Sun, Mengqi; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Viruses are the most abundant biological entity in the ocean, and they can influence microbial mortality, evolution and biogeochemical cycles in marine ecosystems. Virioplankton communities in oceans have been studied extensively using viral metagenomics (viromics), but the estuarine viromes remain relatively unexplored. Estuaries are a complex and dynamic ecosystem. My dissertation is dedicated to understanding the composition and distribution of the virioplankton community in the Delaware Bay and Chesapeake Bay by investigating 16 viromes collected from these two bays. A total of 26,487 viral populations (contigs > 5kb) were identified in the two bays, establishing a high quality viromic dataset. The vast majority of the dominant viral populations are unclassified viruses. Viral sequences obtained from marine single cell genomes or long read single molecule sequencing comprised 13 of the top 20 most abundant viral populations, suggesting that we are still far from understanding the diversity of viruses in estuaries. Abundant viral populations (top 5,000) are significantly different between the Delaware Bay and Chesapeake Bay, indicating a strong niche adaptation of the viral community to each estuary. Surprisingly, no clear spatiotemporal patterns were observed for the viral community based on water temperature and salinity. The composition of known viruses (i.e. phages infecting Acinetobacter, Puniceispirillum, Pelagibacter, Synechococcus, Prochlorococcus, etc.) appeared to be relatively consistent across a wide range of salinity gradients and different seasons. Overall, the estuarine viral community is distinct from that in the ocean according to the composition of known viruses. N4-like viruses belong to a newly established viral family and have been isolated from diverse bacterial groups. Marine N4-like viruses were first found in the Chesapeake Bay, but little is known about their biogeographic pattern in the estuarine environment. N4-like viruses were confirmed to be rare in the estuary, and relatively more abundant in the samples from lower water temperature. Viruses which infect SAR11 bacteria (pelagiphage) are one of most abundant viral groups in the open ocean. We found that the abundance and community profile of pelagiphage in the estuaries is similar to that in the open ocean, and has no correlation with environmental factors.Item TOXIN-ANTITOXIN SYSTEMS AND OTHER STRESS RESPONSE ELEMENTS IN PICOCYANOBACTERIA AND THEIR ECOLOGICAL IMPLICATIONS.(2020) Fucich, Daniel Christopher Ehlers; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Picocyanobacteria (mainly Synechococcus and Prochlorococcus) contribute significantly to oceanic primary production. Unlike Prochlorococcus, which is mainly constrained to the warm and oligotrophic ocean, Synechococcus has a ubiquitous distribution. Synechococcus is present in freshwater, estuarine, coastal, and open ocean habitats. They have also been found in polar regions and hot springs. Endemic to the hot and the cold, the saline and the fresh, and every condition in between, Synechococcus appears to have the capability to adapt and tolerate nearly any environment and climate. This ability to adapt to any aquatic environment is possible through their genome plasticity, a character that is not present in the Prochlorococcus.Due to the differential distribution of the genera, Synechococcus is considered a generalist and Prochlorococcus is considered a specialist in ecological theory. More than 400 picocyanobacterial genomes have now been sequenced, and this large genomic resource enables comprehensive genome mining and comparison. One possibility is to study the prevalence of Toxin-Antitoxin (TA) systems in picocyanobacterial genomes. TA systems are present in nearly all bacteria and archaea and are involved in cell growth regulation in response to environmental stresses. However, little is known about the presence and complexity of TA systems in picocyanobacteria. By querying 77 complete genomes of freshwater, estuarine, coastal and ocean picocyanobacteria, Type II TA systems (the most well studied TA family) were predicted in 27 of 33 (81%) Synechococcus strains, but no type II TA genes were predicted in any of the 38 Prochlorococcus strains. The number of TA pairs varies from 0 to 80 in Synechococcus strains, with a trend for more type II TA systems being predicted in larger genomes. A linear correlation between the genome size and the number of putative TA systems in both coastal and freshwater Synechococcus was established. In general, open ocean Synechococcus contain no or few TA systems, while coastal and freshwater Synechococcus contain more TA systems. The type II TA systems inhibit microbial translation via ribonucleases and allow cells to enter the “dormant” stage in adverse environments. Inheritance of more TA genes in freshwater and coastal Synechococcus could confer a recoverable persister state which would be an important mechanism to survive in variable environments. Different genotypes of Synechococcus are present in the Chesapeake Bay in winter and summer. Winter isolates of Synechococcus have shown high tolerance to cold conditions and other stressors. To explore their potential genetic capability, complete genomes of five representative winter Synechococcus strains CBW1002, CBW1004, CBW1006, CBW1107, and CBW1108 were fully sequenced. These five winter strains share many homologs that are unique to them and not shared with pelagic Synechococcus. Winter Synechococcus genomes are enriched with particular desaturases, chaperones, and transposases. Similar amino acid sequences and annotated features were not found in distantly related Synechococcus from Subcluster 5.1. These shared genomic features between the winter strains imply that maintaining membrane fluidity, protein stability, and genomic plasticity are important to cold adaption of Synechococcus. The winter strains also contain genes that are not traditionally considered with the canonical bacterial cold shock response. They contain a particularly high abundance of Type II TA pairs with complex association networks. They feature promiscuous toxins, like VapC, that pair with multiple antitoxins, which support the mix and match hypothesis. Winter strains also contain more monogamous toxins, such as BrnT, which tend to pair with their traditionally named antitoxin, BrnA. Expression of certain TA transcripts in response to environmental stress has been observed in the model strain CB0101, and the activity of one TA pair in CB0101 for growth arrest has been experimentally confirmed via heterologous expression in E. coli. My thesis work has identified interesting genetic systems related to niche partitioning of picocyanobacteria, particularly among the Chesapeake Bay Synechococcus. Future studies are paramount to understand the functional role of TA systems, desaturases, chaperons, and transposases of picocyanobacteria under various environmental stressors.Item Diet and Stomach Microbiota of Gulf Menhaden, a key forage filter feeding fish species(2020) Hanif, Ammar Wali; Jagus, Rosemary; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Menhaden represent a family of important filter feeding forage fish that serves as a trophic link between plankton and piscivorous predators in the marine environment. Dietary analysis is difficult because diet items are small and >80 % of the stomach content is amorphous material. DNA metabarcoding combines mass-amplification of short DNA sequences (barcodes) with high-throughput sequencing. This application allows the simultaneous identification of many taxa within the same environmental sample, as well as the analysis of many samples simultaneously, providing a comprehensive assessment of diet items and gut microbiota. Here we present a methodological approach using DNA metabarcoding suitable for a small filter feeding fish to identify the stomach contents of juvenile Gulf menhaden (Brevoortia patronus), collected within Apalachicola Bay, Florida. I describe the optimization of DNA extraction, comparison of two primers and sequencing protocols, estimation of menhaden DNA contamination, quality filtering of sequences, post-sequence processing and taxonomic identification of recovered sequences. I characterized the prokaryotic community using 16S universal ribosomal RNA (rRNA) gene sequencing primers in the V3-V4 hypervariable regions. Using two different sequencing protocols employing different “universal” 16S rRNA gene sequencing primers. Although no difference in overall operational taxonomic units (OTUs) was found, the two sequencing protocols gave differences in the relative abundancies of several bacterial classes. The dominant OTUs resulting from 16S rRNA gene sequencing at the phylum level were assigned to Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi and included oil eating bacteria consistent with the Gulf of Mexico location. Stomach microbiota and diet were compared in juvenile Gulf menhaden, Brevoortia patronus, caught at two locations, Two Mile Channel and St. Vincent Sound, in Apalachicola Bay, FL in May and July of 2013. The stomach microbiota of samples from both locations showed a predominance of Proteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria and Actinobacteria, although significant differences in composition at the class level were seen. The stomach microbiota from fish from Two-Mile Channel showed a higher level of taxonomic richness and there was a strong association between the microbiota and sampling location, correlating with differences in salinity. Approximately 1050 diet items were identified, although significant differences in the species represented were found in samples from the two locations. Members of the Stramenopile/ Alveolate/Rhizaria (SAR) clade accounted for 66 % representation in samples from Two Mile Channel, dominated by the diatoms Cyclotella and Skeletonema, as well as the ciliate Oligotrichia. In contrast, Metazoa (zooplankton) dominated in samples from St. Vincent Sound, accounting for over 80 % of the reads. These are mainly Acartia copepods. Since ciliates are considered to be microzooplankton, this means there is just over 60 % representation of phytoplankton in samples from Two Mile Channel and over 90 % representation of zooplankton in samples from St. Vincent Sound. Overall, I demonstrate the diversity of juvenile menhaden stomach contents that supports a characterization of menhaden as environmental samplers.Item THE PROBIOTICS OF BIOFUEL: A METAGENOMIC STUDY OF MICROALGAE GROWN FOR FUEL PRODUCTION(2018) Major, Samuel; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ponds in Frederick, MD were fertilized with chicken manure to increase the nutrient load in the water and stimulate microalgal growth. Nutrient analyses indicate that fertilization results in significant increases in the DOC, TDN, and TDP. The bacterial and eukaryotic microalgal communities were analyzed using 16S and 18S rRNA gene sequencing, respectively. Communities were analyzed pre-fertilization and for 15 days following fertilization. Molecular data reveals a decrease in diversity as microalgal blooms form. The microalgal density increased following fertilization, with enrichment for the Chlamydomonadales order. Prior to fertilization the bacterial communities were dominated by five phyla: Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria, and Verrucomicrobia. Dominant bacterial genera post-fertilization included Flavobacterium, Limnohabitans, and Polynucleobacter. Bacteria isolated from the ponds were screened for effects on Scenedesmus sp. HTB1 to identify bacteria that either enhance or inhibit microalgal growth. The growth-promoting bacteria were closely related to bacteria found to be enriched during microalgal bloom formation.Item Influence of prey density and dietary supplementation on the growth and development of the blue crab, Callinectes sapidus(2017) Maurer, Leah Marie; Chung, Sook; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The early developmental stages of arthropods often vary and appear to be influenced by dietary conditions. It is hypothesized that food availability and dietary components may affect the number and length of developmental stages of the blue crab, Callinectes sapidus, specifically those that are reared in aquaculture settings. This hypothesis was examined with C. sapidus 1) larvae and 2) juveniles. 1) During the zoeal period, development from stage 1 to megalopae was monitored under a full factorial experiment with treatments: high and low prey density coupled with and without poly-β-hydroxybutyrate (PHB) supplementations. Our data showed that prey density influences variation in the zoeal development of C. sapidus by increasing stage skipping, reducing the number of the stages from 7-8 to 5-8. Additionally, a high prey density coupled with PHB supplementation caused increasing instances of stage skipping. 2) During the juvenile period, the growth was monitored for three molting events (57-165 days) under the following treatments: 0, 5, 10, and 20% chitin supplemented diets and squid control. Our data showed that chitin supplementation did not affect the growth of the juvenile C. sapidus (molt increment or interval).Item SPONGE MUTUALISM IN THE FACE OF CLIMATE CHANGE(2016) Vicente, Jan; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three epizoic symbioses between new sponge species of the genera Plakortis, Haliclona, and Xestospongia deweerdtae are reported here. Barcoding of the cytochrome oxidase subunit 1, 28S rRNA and 18S rRNA genes allowed me to formally describe the Plakortis spp. as P. deweerdtaephila and P. symbiotica. Both Plakortis spp. are obligate hosts of the sponge X. deweerdtae. Unlike Plakortis spp., X. deweerdtae can have a free-living lifestyle. This discovery motivated me to: 1) Use next-generation sequencing to ask whether microbial symbionts are playing a role in shaping these sponge associations; 2) Evaluate how top-down factors influence these associations by analyzing crude extracts of each species by LCMS and determine their palatability to fish to test if chemical defenses from Plakortis spp. translocate into the Xestospongia tissue, and protect it from predation, and 3) Test whether the X. deweerdtae and P. deweerdtaephila sponge pairs in Panama are more resilient than free-living X. deweerdtae in the face of climate change. My results on bacterial and sponge cell counts revealed that Plakortis spp. are high microbial abundance sponges and that X. deweerdtae and H. plakophila are low microbial abundance sponges. Diversity indices showed no differences in microbial richness but a higher Simpson’s index (D) for Plakortis spp. than both epibionts. Microbial community shifts in X. deweerdtae epibionts not observed in the free-living lifestyle were a consequence of the presence of microbial phyla found in the Plakortis spp. basibiont, suggesting the possibility of horizontal transfer of symbionts from the basibiont to the epibiont. Crude extracts from tissues of both free-living and associated lifestyles of X. deweerdtae confer chemical defense. These results suggest that top-down predation pressures from reef fish do not influence the associated life-style of X. deweerdtae. Exposure to high pCO2 and warmer temperature revealed that acidification had an ameliorating effect against necrosis caused by high temperatures in free-living and associated individuals of X. deweerdtae as well as their P. deweerdtaephila basibiont. The X. deweerdtae epibiont was more resistant to temperature increments than P. deweerdtaephila. I performed a similar experiment on an invasive Hawaiian sponge Mycale grandis and discovered that neither acidification nor temperature affect skeleton synthesis. Taken together these findings suggest that these symbioses are mutualistic in nature and that sponges are likely to survive the predicted temperature and pCO2 conditions for the end of the century.Item Adaptive Mechanisms of an Estuarine Synechococcus based on Genomics, Transcriptomics, and Proteomics(2016) Marsan, David Wilfred; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Picocyanobacteria are important phytoplankton and primary producers in the ocean. Although extensive work has been conducted for picocyanobacteria (i.e. Synechococcus and Prochlorococcus) in coastal and oceanic waters, little is known about those found in estuaries like the Chesapeake Bay. Synechococcus CB0101, an estuarine isolate, is more tolerant to shifts in temperature, salinity, and metal toxicity than coastal and oceanic Synechococcus strains, WH7803 and WH7805. Further, CB0101 has a greater sensitivity to high light intensity, likely due to its adaptation to low light environments. A complete and annotated genome sequence of CB0101 was completed to explore its genetic capacity and to serve as a basis for further molecular analysis. Comparative genomics between CB0101, WH7803, and WH7805 show that CB0101 contains more genes involved in regulation, sensing, and stress response. At the transcript and protein level, CB0101 regulates its metabolic pathways, transport systems, and sensing mechanisms when nitrate and phosphate are limited. Zinc toxicity led to oxidative stress and a global down regulation of photosystems and the translation machinery. From the stress response studies seven chromosomal toxin-antitoxin (TA) genes, were identified in CB0101, which led to the discovery of TA genes in several marine Synechococcus strains. The activation of the relB2/relE1 TA system allows CB0101 to arrest its growth under stressful conditions, but the growth arrest is reversible, once the stressful environment dissipates. The genome of CB0101 contains a relatively large number of genomic island (GI) genes compared to known marine Synechococcus genomes. Interestingly, a massive shutdown (255 out of 343) of GI genes occurred after CB0101 was infected by a lytic phage. On the other hand, phage-encoded host-like proteins (hli, psbA, ThyX) were highly expressed upon phage infection. This research provides new evidence that estuarine Synechococcus like CB0101 have inherited unique genetic machinery, which allows them to be versatile in the estuarine environment.Item CHARACTERIZATION OF THE EUKARYOTIC TRANSLATION INITIATION FACTOR 4E (eIF4E) FAMILY MEMBERS IN THE ZEBRAFISH (Danio rerio)(2015) Gillespie, Kathleen M.; Jagus, Rosemary; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The present study examines the six eIF4E cognates in zebrafish. In addition to the prototypical translation initiation factor eIF4E, eukaryotes have evolved eIF4E variants with distinct characteristics, some of which negatively regulate the recruitment of specific mRNAs. Metazoan eIF4E family members fall into three classes, with Class I containing the canonical translation initiation factor eIF4E-1. eIF4E-1 binds eIF4G to initiate translation, a process inhibited by eIF4E binding proteins such as the 4E-BPs and other eIF4E interactive proteins. Analysis of eIF4E sequences from the twenty fish genomes currently available, as well as those of echinoderm, tunicate and cephalocordate, has allowed a glimpse of the origins and evolution of the eIF4E family in vertebrates. All deuterostomes have one representative from each class of eIF4Es. Early deuterostomes such as sea urchins, tunicates, and lancelets have only one from each class; eIF4E-1, eIF4E-2 and eIF4E-3. The distribution of the subclasses of eIF4E-1 is consistent with the duplication of Class I prior to the teleost specific whole genome duplication, probably at one of the whole genome duplications at ~550 (1R) and 500 (2R) mya. Evidence of the duplication of Class I eIF4Es can be seen in elephant shark (Callorhinchus milii), coelacanth (Latimeria chalumnae) and basal ray-finned fish (Lepisosteus oculatus), which have eIF4E-1A, -1B, and -1C. eIF4E-1B has neofunctionalized to become a tissue specific regulator of mRNA recruitment. It has been retained in tetrapods, but lost in higher teleosts. eIF4E-1C, appears to have retained function as a prototypical initiation factor. A duplication of Class II eIF4Es occurred prior to the emergence of the tetrapod branch, becoming eIF4E-2A and -2B. The genes proximal to the eIF4E-2A locus appear to be conserved across teleosts and tetrapods, the eIF4E-2B genetic loci are more variable, suggesting that eIF4E-2A is the ancestral form. eIF4E-2B is retained by amphibians and teleosts, but has been lost in coelacanth and amniotes. Although 88 % identical, eIF4E-2B can be distinguished from eIF4E-2A by its ability to bind trimethyl GTP (TMG) and to complement a S. cerevisiae strain conditionally deficient in eIF4E. This study has shown that duplication within the different classes of eIF4E occurred early in vertebrate evolution with some neofunctionalization, as well as asymmetric losses in different vertebrate classes