Use of Transcriptomic Analysis for the Design of Antibodies to Serve as Neuronal and Synaptic Markers in Macrobrachium crustaceans

Abstract

Crustaceans are major constituents of aquatic ecosystems and, as such, changes in their behavior and the structure and function of their bodies can serve as indicators of alterations in their immediate environment, such as those associated with climate change and anthropogenic contamination. We want to develop markers of crustacean neural circuits and terminal synaptic structure that can be used for quantifiable characterization with confocal microscopy. When compared with other invertebrate species such as Drosophila and C. elegans, little is known regarding crustacean genomic information, particularly of the animal's nervous system. We have used bioinformatics and a de novo transcriptome assembly approach to identify potential targets for developing specific antibodies to serve as nervous system markers for freshwater prawns of the Macrobrachium spp. These species of prawn are found in rivers of Puerto Rico and are being used to study the impact of exposure to emergent contaminants, such as phthalates and metabolites of pharmaceuticals, in natural sources of freshwater near urban areas.Total RNA was extracted from brain ganglia of Macrobrachium carcinus freshwater prawns and Illumina Next Generation Sequencing was performed using an Eel Pond mRNA Seq Protocol to construct a de novo transcriptome. Transcript sequences related to glutamate receptors were ran through multiple sequence conservation analysis using the MUSCLE webserver. Conserved sequences were analyzed for antigenicity to design specific antibodies. Raw data from the Illumina Sequencing yielded 97,202,662 sequences: 47,630,546 paired and 1,941,570 singletons. Assembly with Trinity resulted in 197,898 assembled sequences, of which 30,576 were annotated: 9,600 annotated by orthology, 17,197 annotated by homology, and 3,779 annotated by transcript families. In the search for glutamate receptor sequences, we found 138 sequences related to ionotropic receptors, 32 sequences related to metabotropic receptors, and 18 sequences to unidentified receptors. Using this information, we were able to develop antibodies for prawn AMPA ionotropic glutamate receptor 1 (GluRIA1), metabotropic glutamate receptor subunit 1 and 4 (mGluRG1S1 and mGluRG3S4), and NMDA ionotropic receptor 2B (GluRNMDA2B). Immunohistochemistry of the ventral nerve cord has thus far shown both punctuate and cell body staining of mGluRG1S1 located throughout all the central nervous system, including the eyestalks, brain, and the circumesophageal, subesophageal, thoracic and abdominal ganglia. The other antibodies are presently being tested in the prawn central nervous system as well as in peripheral synapses such as the neuromuscular junction.Support or Funding InformationNSF DBI‐1337284NSF CREST NSF HRD‐1736019NIH NIMHD 8G12‐MD007600 (RCMI)NIGMS‐RISE R25 GM061838 NSF (USA): CREST HRD‐1137725This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.