Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans

Authors: CHRISTIE, ANDREW - University Of Hawaii; RIVERIA, CINDY - Bowdoin College; CALL, CATHERINE - Bowdoin College; DICKINSON, PATSY - Bowdoin College; STEMMLER, ELIZABETH - Bowdoin College; Hull, Joe

Submitted to: General and Comparative Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2020
Publication Date: 12/1/2020
Citation: Christie, A.E., Riveria, C.D., Call, C.M., Dickinson, P.S., Stemmler, E.A., Hull, J.J. 2020. Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans. General and Comparative Endocrinology. 299. https://doi.org/10.1016/j.ygcen.2020.113609.
DOI: https://doi.org/10.1016/j.ygcen.2020.113609

Interpretive Summary: Considerable progress has been made in the generation and distribution of genomic and transcriptomic resources for arthropod species, including many crustaceans. Although these datasets were initially developed to serve specific functions, they have proven to be powerful resources for a number of unrelated gene discoveries including the identification of previously uncharacterized or unknown peptides. One example of this are the agatoxin-like peptides, which share structural similarities to a spider venom peptide known as agatoxin. To examine questions regarding the prevalence of agatoxin-like peptides in crustaceans and to provide insights into the potential physiological roles the peptides play, publicly accessible transcriptomes representing diverse crustacean species were searched for sequences exhibiting similarity with agatoxin. Transcripts encoding agatoxin-like peptides were identified in all datasets examined including a number of tissue specific assemblies. The presence of the sequence in the American lobster was confirmed by PCR and mass spectrometric analysis of dissected brain tissue. The presence of agatoxin-like peptide sequences in crustacean nervous system-specific and liver/pancreas-like specific transcriptome assemblies suggests potential roles in neuromodulation, reproduction, stress responses, detoxification, and/or innate immunity. These findings provide potential new avenues areas of research and can be used to guide future studies on the biochemical and physiological roles of agatoxin-like peptides in crustaceans.

Technical Abstract: Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of dec- apod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine re- sidues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eye- stalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.