Project description:To investigate a Florida manatee (Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, a RNA-Seq experiment was conducted. Cell processes such as immune response, cell proliferation and differentiation and apoptosis were among the most affected by red tide. These were involved in potential diseases such as neoplasms, inflammation, and wounds and injuries, among others. There were both up-regulated and down-regulated genes, but the highest fold changes relative to controls were for genes that were down-regulated. Piccolo presynaptic cytomatrix protein (PCLO) gene, the one most down-regulated (fold change -9.93; p-value 0.0009) is associated with neurotransmitter release, cognitive functioning, neuronal loss, and neuronal synapse function. Another gene that has a similar function, ankyrin 2, neuronal, transcript variant 1 (ANK2) was also down-regulated (fold change -8.66; p-value 0.0023). ANK2 is associated with the stability of neuron synapses. Two immune genes, interleukin 6 (interferon, beta 2) (IL6) and zinc finger protein 804B (ZNF804B), were down-regulated (fold change -9.31; p-value 0.000003 and fold change -8.90; p-value 0.0164, respectively). Interleukin 6 encodes proteins involved in acute phase response, inflammation, and autoimmune response. ZNF804B is associated with neuronal chemokine and cytokine regulation, autoimmune response, and immune activation. The family with sequence similarity 186, member A (FAM186A) gene was down-regulated (fold change -8.79; p-value 0.0143). FAM186A gene mutation is associated with tumor metastasis in colorectal cancer tumors. Among the most up-regulated genes, CCAAT/enhancer binding protein (C/EBP) is involved in granulocytic differentiation and also involved with the immune system. Determining the differentially expressed genes associated with red tide enhances our understanding of manatee immune response to red tide toxins and aids in the development of red tide biomarkers. This information will better assist clinicians and researchers in diagnosing and treating future illnesses.
Project description:In 2013, two large-scale Florida manatee (Trichechus manatus latirostris) mortality episodes were reported on separate coasts of Florida. The east coast mortality episode was associated with an unknown etiology in the Indian River Lagoon (IRL). The west coast mortality episode was attributed to a persistent Karenia brevis algal bloom or ‘red tide’ centered in Southwest Florida. To investigate these two mortality episodes, proteomic experiments using two-dimensional difference gel electrophoresis (2D-DIGE) followed by protein identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were conducted, along with a separate gel-free analysis using isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS. In comparison to the control group, manatees from the IRL, an area associated with an unknown mortality episode, displayed increased levels of several proteins in their serum samples. These increased proteins, which were identified in the iTRAQ experiment, included kininogen-1 isoform 1 (average ratio 1.38), protein AMBP (1.38), histidine-rich glycoprotein (1.34), properdin (1.30), and complement C4-A isoform 1 (1.25). In the red tide group, ceruloplasmin (2.32), pyruvate kinase isozymes M1/M2 isoform 3 (2.29), angiotensinogen (2.08), complement C4-A isoform 1 (1.83), and complement C3 (1.42) were increased. The proteins kininogen-1 isoform 1, histidine-rich glycoprotein, complement C4-A isoform 1, angiotensinogen, and complement C3 were also identified in increased levels in the 2D-DIGE experiment (Table 2b). These proteins are associated with acute-phase response, amyloid formation and accumulation, copper and iron homeostasis, the complement cascade pathway, and other important cellular functions. The increased level of complement C4 protein observed in both the red tide and unknown mortality episode groups was confirmed through the use of Western Blot.
