Dolores V. Baxa

Anthropogenic debris in seafood: Plastic debris and fibers from textiles in fish and bivalves sold for human consumption

The ubiquity of anthropogenic debris in hundreds of species of wildlife and the toxicity of chemicals associated with it has begun to raise concerns regarding the presence of anthropogenic debris in seafood. We assessed the presence of anthropogenic debris in fishes and shellfish on sale for human consumption. We sampled from markets in Makassar, Indonesia, and from California, USA. All fish and shellfish were identified to species where possible. Anthropogenic debris was extracted from the digestive tracts of fish and whole shellfish using a 10% KOH solution and quantified under a dissecting microscope. In Indonesia, anthropogenic debris was found in 28% of individual fish and in 55% of all species. Similarly, in the USA, anthropogenic debris was found in 25% of individual fish and in 67% of all species. Anthropogenic debris was also found in 33% of individual shellfish sampled. All of the anthropogenic debris recovered from fish in Indonesia was plastic, whereas anthropogenic debris recovered from fish in the USA was primarily fibers. Variations in debris types likely reflect different sources and waste management strategies between countries. We report some of the first findings of plastic debris in fishes directly sold for human consumption raising concerns regarding human health.

Sublethal dietary effects of microcystin producing Microcystis on threadfin shad, Dorosoma petenense

The presence of the toxic cyanobacterium Microcystis in the upper San Francisco Estuary (SFE) since 1999 is a potential but unquantified threat to the health and survival of aquatic organisms such as fish and zooplankton. The microcystins (MCs) predominantly in the LR-form (MC-LR) produced by Microcystis is hepatotoxic and a potential threat to the fishery. Concurrently, in the SFE significant declines in pelagic fish, known as the Pelagic Organism Decline (POD), has been recognized by state and federal agencies since 2000. In 2005, the presence of the toxic algal bloom, Microcystis has been hypothesized as a link to the POD by the Interagency Ecology Program Management Team. This study aims to characterize the toxic effects of Microcystis in one of the POD species, threadfin shad (Dorosoma petenense) by exposure to diets containing Microcystis harvested from the SFE. The diets contained Microcystis with 4.4 (D5) and 10.0 (D10) μg g⁻¹ MC-LR that was fed to threadfin shad for 57 days. The treatments were compared to the control diet, 0 μg g⁻¹ MC-LR (D0). Results showed that ingested Microcystis was localized in the gut by in situ hybridization and MCs were localized in the tissues of the gut, kidney and liver. Condition factor (CF) and liver and gonadal lesions were sensitive to MC exposure. There was a significant inverse relationship between CF and MC-LR with exposed fish exhibiting severe cachexia. Liver lesions of sinusoidal congestion and glycogen depletion significantly increased with increasing MC-LR concentrations, indicating hemorrhaging in the liver and poor nutritional status, respectively. In females, there was a significant increase in severe ovarian necrosis with increasing MC-LR concentration, indicating loss of reproductive potential. The results indicate that MC-LR from Microcystis significantly impairs the health and reproductive potential of threadfin shad has a potential negative effect on populations in the SFE.

Impacts of the 2014 severe drought on the Microcystis bloom in San Francisco Estuary

The increased frequency and intensity of drought with climate change may cause an increase in the magnitude and toxicity of freshwater cyanobacteria harmful algal blooms (CHABs), including Microcystis blooms, in San Francisco Estuary, California. As the fourth driest year on record in San Francisco Estuary, the 2014 drought provided an opportunity to directly test the impact of severe drought on cyanobacteria blooms in SFE. A field sampling program was conducted between July and December 2014 to sample a suite of physical, chemical, and biological variables at 10 stations in the freshwater and brackish reaches of the estuary. The 2014 Microcystis bloom had the highest biomass and toxin concentration, earliest initiation, and the longest duration, since the blooms began in 1999. Median chlorophyll a concentration increased by 9 and 12 times over previous dry and wet years, respectively. Total microcystin concentration also exceeded that in previous dry and wet years by a factor of 11 and 65, respectively. Cell abundance determined by quantitative PCR indicated the bloom contained multiple potentially toxic cyanobacteria species, toxic Microcystis and relatively high total cyanobacteria abundance. The bloom was associated with extreme nutrient concentrations, including a 20-year high in soluble reactive phosphorus concentration and low to below detection levels of ammonium. Stable isotope analysis suggested the bloom varied with both inorganic and organic nutrient concentration, and used ammonium as the primary nitrogen source. Water temperature was a primary controlling factor for the bloom and was positively correlated with the increase in both total and toxic Microcystis abundance. In addition, the early initiation and persistence of warm water temperature coincided with the increased intensity and duration of the Microcystis bloom from the usual 3 to 4 months to 8 months. Long residence time was also a primary factor controlling the magnitude and persistence of the bloom, and was created by a 66% to 85% reduction in both the water inflow and diversion of water for agriculture during the summer. We concluded that severe drought conditions can lead to a significant increase in the abundance of Microcystis and other cyanobacteria, as well as their associated toxins.

Characterization of a novel alloherpesvirus from Atlantic cod (Gadus morhua).

Alloherpesviruses affect freshwater and marine fish species. The aim of the current study was to characterize a novel alloherpesvirus in Atlantic cod (Gadus morhua). Samples were processed for histopathology, transmission electron microscopy (TEM), virus isolation, molecular characterization, and in situ hybridization (ISH). Histopathology revealed that the infection was restricted to the gills and that it induced cytomegaly in infected cells. By TEM, numerous viral particles with morphology compatible with a herpesvirus were observed inside the cytomegalic cells. To characterize this new agent, polymerase chain reaction amplified regions of the ATPase subunit of the terminase, and DNA polymerase genes were sequenced. Phylogenetic analysis revealed strongest similarity with alloherpesviruses belonging to the genus Ictalurivirus and Salmonivirus. The ISH showed specific labeling of nuclear inclusions in the cytomegalic cells. While virus isolation was unsuccessful, the results obtained through different diagnostic tests in the present study confirm the discovery of a new alloherpesvirus affecting Atlantic cod. The authors propose the formal species designation Gadid herpesvirus 1 (GaHV-1) to be considered for approval by the International Committee on the Taxonomy of Viruses.

Susceptibility to Myxobolus cerebralis among Tubifex tubifex Populations from Ten Major Drainage Basins in Colorado Where Cutthroat Trout Are Endemic

Establishment of Myxobolus cerebralis (Mc) resulted in declines of wild Rainbow Trout Oncorhynchus mykiss populations in streams across Colorado during the 1990s. However, the risk for establishment and spread of this parasite into high-elevation habitats occupied by native Cutthroat Trout O. clarkii was unknown. Beginning in 2003, tubificid worms were collected from all major drainages where Cutthroat Trout were endemic and were assayed by quantitative PCR to determine the occurrence and distribution of the various lineages of Tubifex tubifex (Tt) oligochaetes. Over a 5-year period, 40 groups of Tt oligochaetes collected from 27 streams, 3 natural lakes, 2 private ponds, and a reservoir were evaluated for their relative susceptibility to Mc. Exposure groups were drawn from populations of pure lineage III Tt, mixed-lineage populations where one or more of the highly resistant (lineage I) or nonsusceptible lineages (V or VI) were the dominant oligochaete and susceptible lineage III worms were the subdominant worm, or pure lineage VI Tt. Experimental replicates of 250 oligochaetes were exposed to 50 Mc myxospores per worm. The parasite amplification ratio (total triactinomyxons [TAMs] produced / total myxospore exposure) was very high among all pure lineage III Colorado exposure groups, averaging 363 compared with 8.24 among the mixed-lineage exposure groups. Lineage III oligochaetes from Mt. Whitney Hatchery in California, which served as the laboratory standard for comparative purposes, had an average parasite amplification ratio of 933 among 10 exposed replicates over a 5-year period. Lineage I oligochaetes were highly resistant to infection and did not produce any TAMs. Lineages V and VI Tt did not become infected and did not produce any TAMs. These results suggest that the risk of establishment of Mc is high for aquatic habitats in Colorado where Cutthroat Trout and lineage III Tt are sympatric.

Henneguya sp. in yellowfin goby Acanthogobius flavimanus from the San Francisco Estuary

Myxozoan spores were observed in yellowfin goby Acanthogobius flavimanus collected from Suisun Marsh, San Francisco Estuary (SFE). Although histopathological changes associated with the parasite were not observed, the spores formed plasmodia that partially blocked the gastric and intestinal mucosa and gut lumen and may affect the perfomance and survival of the yellowfin goby. Morphological features of the spores resembled Henneguya sp. and molecular analysis of the 18S ribosomal DNA (Domain III) confirmed close similarity to H. rhinogobii and H. pseudorhinogobii isolated from the Japanese freshwater goby. The yellowfin goby myxozoan however, is likely an undescribed species based on phylogenetic analysis and morphologic features. Detailed description of vegetative and spore stages are currently lacking for proposal to a new species of Henneguya. A specific PCR test was developed, which confirmed a 100% prevalence of the parasite among randomly collected gobies in group 1 (N = 30) and group 2 (N = 15) at termination of the study at one month in captivity. The myxozoan was also detected from 18 gobies (12%) that died in the first group within two weeks in captivity. Apparently healthy gobies that served as controls did not reveal the presence of the myxozoan by PCR. This study documents the occurrence of a potentially new species of myxozoan in the yellowfin goby and underscores the detection of a parasitic infection in an introduced fish in the SFE. Although the pathogenesis of the myxozoan was not assessed and the prevalence as reported here is restricted to a comparatively small collection site in Suisun slough, the reemergence, identification, and ecological relevance of the parasite on goby populations in the SFE may be investigated in the future using the specific diagnostic tool developed in this study.

Identification of harmful cyanobacteria in the Sacramento-San Joaquin Delta and Clear Lake, California by DNA barcoding

Accurate identification of cyanobacteria using traditional morphological taxonomy is challenging due to the magnitude of phenotypic plasticity among natural algal assemblages. In this study, molecular approach was utilized to facilitate the accurate identification of cyanobacteria in the Sacramento-San Joaquin Delta and in Clear Lake in Northern California where recurring blooms have been observed over the past decades. Algal samples were collected from both water bodies in 2011 and the samples containing diverse cyanobacteria as identified by morphological taxonomy were chosen for the molecular analysis. The 16S ribosomal RNA genes (16S rDNA) and the adjacent internal transcribed spacer (ITS) regions were amplified by PCR from the mixed algal samples using cyanobacteria generic primers. The obtained sequences were analyzed by similarity search (BLASTN) and phylogenetic analysis (16S rDNA) to differentiate species sharing significantly similar sequences. A total of 185 plasmid clones were obtained of which 77 were successfully identified to the species level: Aphanizomenon flos-aquae, Dolichospermum lemmermannii (taxonomic synonym: Anabaena lemmermannii), Limnoraphis robusta (taxonomic synonym: Lyngbya hieronymusii f. robusta) and Microcystis aeruginosa. To date, Dolichospermum and Limnoraphis found in Clear Lake have only been identified to the genus lavel by microscopy. During the course of this study, morphological identification and DNA barcoding confirmed A. flos-aquae as the predominant cyanobacterium in the Sacramento-San Joaquin Delta indicating a shift from M. aeruginosa that have dominated the blooms in the past decade. Lastly, the species-specific identification of Limnoraphis robusta in Clear Lake is another significant finding as this cyanobacterium has, thus far, only been reported in Lake Atitlan blooms in Guatemala.

A novel and versatile flash-freezing approach for evaluating the health of Delta Smelt

A common approach used to assess environmental impacts in aquatic environments is to measure indicators of stress (biomarkers) and condition of fish within ecosystems. Particularly in estuarine ecosystems with multiple stressors, it is often desirable to quantify a suite of biological endpoints that (1) reflect fish condition at several levels of biological organization and time scales and (2) are sensitive to a range of environmental stressors. However, established methods of preservation and processing of fish for specific endpoints are often incompatible. Here, we developed a novel flash-freezing approach for assessing the health of a small, sensitive fish, the endangered Delta Smelt (Hypomesus transpacificus) after collections from the San Francisco Estuary (SFE). We assess whether flash-freezing the entire fish ensures effective preservation of multiple tissues for subsequent biomarker analyses by comparing measurements of fresh to frozen tissue. Tissues included brain, gill, and liver for enzyme activity, kidney and spleen for pathogens, and gills, liver, and gonads for histopathology and reproduction. Although flash-freezing in liquid nitrogen altered the length, weight, and condition factor of Delta Smelt, the percent changes were small (<1.5%). Histological analyses of the cellular morphology of gills, liver, and gonads were similar between both methods. Freezing artefacts were observed in ovaries, but did not hinder the identification and interpretation of cell types and oocyte stages. Freezing did not alter bacterial isolation or the activities of ethoxyresorufin-0-deethylase (EROD) or acetylcholinesterase (AChE), but had a small, negative influence on sodium potassium adenosine triphosphatase (ATPase) activity. Thus, flash-freezing in the field is a versatile preservation method for Delta Smelt, allowing for multiple tissue collections and bioassays from an individual tiny fish exposed to a wide range of natural and anthropogenic stressors. Similar methodology may be applicable to other species for which a range of biological endpoints and histopathology data are needed.