Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Endocrine, immune and renal toxicity in male largemouth bass after chronic exposure to glyphosate and Rodeo®.

Glyphosate is the most used herbicide worldwide, with no historical comparison. It is used for genetically modified crops, and particularly in Florida, it is used as a sugar cane ripener. An aquatic formulation (Rodeo®) is used to treat aquatic weeds in waterbodies and drainage canals. Because of its extended use, glyphosate can run off or be sprayed directly into waterbodies, and chronically expose aquatic wildlife. Exposure in animal models has been associated with kidney and liver damage and glyphosate has been suggested as an endocrine disruptor. We exposed adult male largemouth bass for 21 days to two doses of glyphosate and Rodeo® (chemically equivalent concentration of glyphosate) at 0.5 mg L-1 and 10 mg L-1 and to a clean water control (n=4 fish/tank in quadruplicate). Concentrations during the experiment were corroborated with UHPLC-MS/MS. Total RNA was isolated from the trunk kidney and head kidney. RNA-seq was performed for the high doses compared to controls. Transcripts were analyzed with fish and mammalian pathway analyses software. Transcripts mapped to Zebrafish metabolic pathways using PaintOmics showed steroid hormone biosynthesis in the trunk kidney as the most significantly enriched pathway. Steroid hormones were measured in plasma by UHPLC-MS/MS. Total androgens were significantly reduced at 0.5 mg L-1 of glyphosate and at equivalent concentrations in Rodeo® compared to controls. 11-ketotestosterone and estrone concentrations were significantly reduced in all doses. A gene involved in the conversion of testosterone to 11-ketotestosterone was down-regulated by glyphosate. Using the mammalian pathway analysis algorithm, cellular processes associated with T-cell activation/development and intracellular pH were significantly enriched in the trunk kidney by glyphosate and Rodeo® exposure. Endocrine disruption was corroborated at the hormone and gene expression levels. Rodeo® and glyphosate share gene expression pathways, however, Rodeo® had more pronounced effects in largemouth bass.

Historic variation of trace elements in pinnipeds with spatially segregated trophic habits reveals differences in exposure to pollution.

Marine mammals and the ecological functions they provide to coastal and pelagic ecosystems are increasingly threatened by the intensification of anthropogenic impacts. The Uruguayan coastline throughout the 20th century, like other coastal environments worldwide, has been the sink of a variety of trace metals derived from the rapid urbanization and industrialization of related land areas. This coastline is inhabited by two species of pinnipeds trophically and spatially segregated. Otaria byronia feeds in coastal environments while Arctocephalus australis preys mainly offshore. The present study aimed to analyze historic changes in concentrations of trace elements in teeth of both species from 1941 to the present day. We analyzed the dentin of 94 canine teeth using stable isotope analysis (δ13C) and ICP-MS to determine their feeding areas and the concentration of 10 trace elements (Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) respectively. The concentration of Cr was significantly higher during '70-'80s, in both species coinciding with tannery industry development. Both species of pinnipeds have been differentially exposed to trace elements depending on their feeding area. A pelagic diet, possibly based on squid, increased the concentration of Cd in A. australis, while O. byronia has been more exposed to anthropogenic Pb and Cu associated to a costal and more benthic diet. Our results highlight dentin as a reliable matrix for historic studies on the exposure to trace elements. In light of our results, the O. byronia's declining population could be the result of the synergistic effects of trace elements together with other ecological pressures faced in their environment.

Chronic exposure to glyphosate in Florida manatee.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate's and AMPA's concentrations in Florida manatee plasma (n = 105) collected during 2009-2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees' plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees' immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

Antineoplastic Agents: Environmental Prevalence and Adverse Outcomes in Aquatic Organisms.

Cancer is the second leading cause of death worldwide, with 9.6 million cancer-related deaths in 2018. Cancer incidence has increased over time, and so has the prescription rate of chemotherapeutic drugs. These pharmaceuticals, known as antineoplastic agents, enter the aquatic environment via human excretion and wastewater. The objectives of the present critical review were to investigate the risk of antineoplastics to aquatic species and to summarize the current state of knowledge regarding their levels in the environment, because many antineoplastics are not adequately removed during wastewater treatment. We conducted 2 separate literature reviews to synthesize data on the global environmental prevalence and toxicity of antineoplastics. The antineoplastics most frequently detected in the environment included cyclophosphamide, ifosfamide, tamoxifen, methotrexate, and 5-fluorouracil; all were detectable in multiple water sources, including effluent and surface waters. These antineoplastics span 3 different mechanistic classes, with cyclophosphamide and ifosfamide classified as alkylating agents, tamoxifen as a hormonal agent, and methotrexate and 5-fluorouracil as antimetabolites. Studies that characterize the risk of antineoplastics released into aquatic environments are scarce. We summarize the biological impacts of the most environmentally prevalent antineoplastics on aquatic organisms and propose an adverse outcome pathway for cyclophosphamide and ifosfamide, 2 widely prescribed drugs with a similar immunotoxic mode of action. Acute and chronic ecotoxicity studies using aquatic models are needed for risk characterization of antineoplastics. Environ Toxicol Chem 2020;39:967-985. © 2020 SETAC.