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. Author manuscript; available in PMC: 2020 Dec 18.
Published in final edited form as: Gen Comp Endocrinol. 2020 Sep 9;299:113609. doi: 10.1016/j.ygcen.2020.113609

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

Andrew E Christie 1, Cindy D Rivera 2, Catherine M Call 2, Patsy S Dickinson 3, Elizabeth A Stemmler 2, J Joe Hull 4,*
PMCID: PMC7747469  NIHMSID: NIHMS1650435  PMID: 32916171

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 decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues 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, eyestalk 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.

Keywords: in silico transcriptome mining, RT-PCR, mass spectrometry, nervous system, testis, hepatopancreas

1. Introduction

Over the past decade, considerable effort has been put into the development of molecular resources (genomes and deep transcriptomes) for arthropod species, including many crustaceans (e.g., Armstrong et al., 2019; Christie et al., 2017, 2018a, 2018b; Havird and Santos, 2016; He et al., 2012; Huerlimann et al., 2018; Li et al., 2015; Lv et al., 2015; Manfrin et al., 2015; Northcutt et al., 2016; Oliphant et al., 2018; Rahi et al., 2019; Santos et al., 2018; Souza et al., 2018; Sun et al., 2014; Tom et al., 2014; Verbruggen et al., 2015; Wang et al., 2019; Xu et al., 2015). Although these datasets were initially developed to serve a variety of functions, they have proven to be powerful resources for a wide array of gene and, by proxy, protein discoveries. Crustacean genomes and transcriptomes have been extensively exploited to facilitate the identification of the molecular components (genes and proteins) of peptidergic signaling systems, including peptide precursors (e.g., Bao et al., 2015; Christie and Hull, 2019; Christie and Pascual, 2016; Christie and Yu, 2019; Christie et al., 2015; Nguyen et al., 2016; Oliphant et al., 2018; Veenstra, 2015, 2016), peptide processing enzymes (Christie et al., 2018c), and peptide receptors (e.g., Bao et al., 2018; Buckley et al., 2016; Christie and Hull, 2019; Christie and Yu, 2019; Christie et al., 2015; Dickinson et al., 2019; Oliphant et al., 2018; Tran et al., 2019; Veenstra, 2016).

In silico mining of publicly accessible crustacean genomes and transcriptomes for peptide precursors has led to the identification of new isoforms for known crustacean peptide families as well as peptide groups either previously unknown or poorly represented in this taxon. Among the latter is the agatoxin-like peptide (ALP) family, which was first identified in the honey bee (Apis mellifera) corpora cardiaca (Sturm et al., 2016). Peptides in this family share structural similarities with a class of peptide toxins (agatoxins) initially isolated from the American funnel-web spider Agelenopsis aperta (Skinner et al., 1989; Bindokas and Adams, 1989; Adams et al., 1990). ALPs have since been identified in a number of arthropod genomic/transcriptomic datasets (Sturm et al., 2016; Veenstra, 2016). The ALP prepropeptide typically consists of a signal peptide, two to three precursor peptides, and an ~ 40 amino acid ALP region that contains a C-terminal amidation signal and eight highly conserved Cys residues that comprise two Cys motifs commonly found in spider toxins. The N-terminal principal structural motif is characterized by a Cys spacing pattern of CX6CX6CC (X = any amino acid) with the dual Cys residues (CC) four amino acid residues upstream of the C-terminal second extra structural motif, which follows a CXCX6CXC pattern. Although alternatively spliced variants of the ALP prepropeptide have been identified (Sturm et al., 2016; Veenstra, 2016; von Reumont et al., 2013), the splice variants typically affect the precursor peptides rather than the ALP region itself, suggesting evolutionary pressure to conserve functionality. While the biological role of ALPs has yet to be described, the structurally related spider agatoxins have been reported to affect neurotransmitter release via modification of various receptor-activated and/or voltage-gated channels at insect neuromuscular junctions (Adams 2004). These structural similarities and ALP expression in diverse venom glands (Bouzid et al., 2014; Liu et al., 2015; Torres et al., 2014; von Reumont et al., 2013) could indicate a similar toxin-like function; however, ALPs have been identified in transcriptomic and genomic datasets from a number of non-venomous arthropods (Bao et al., 2020; Christie 2020; Liessem et al., 2018; Oliphant et al., 2020; Sturm et al., 2016; Veenstra 2016). Furthermore, direct detection of ALPs in the neuroendocrine systems (corpora cardiaca, brain, and stomatogastric nervous system) of the honey bee (A. mellifera), the American cockroach (Periplaneta americana), and the firebrat (Thermobia domestica) suggest non-toxic functional roles (Sturm et al., 2016).

Although ALPs have been reported in members of the Crustacea (Sturm et al., 2016; Veenstra, 2016), numerous questions remain concerning these peptides in crustaceans. For example, to what extent are the ALPs phylogenetically and structurally conserved in the taxon? Similarly, what tissues produce ALPs in crustaceans, and what physiological roles do they play in members of this arthropod subphylum? Here, publicly accessible transcriptomes were used to address these questions in members of the Decapoda, a crustacean order that is of high economic importance due to its role in commercial fisheries and aquaculture, and whose members have been widely used as models for investigating peptidergic control of physiology and behavior.

2. Materials and methods

2.1. Animals

Lobsters, H. americanus, were purchased from local (Brunswick, Maine, USA) seafood distributors. All animals used were adults of ~500g and included both males and females. Lobsters were housed in recirculating natural seawater aquaria at 10–12°C and were fed chopped squid weekly.

2.2. In silico transcriptome mining and peptide structural prediction

2.2.1. In silico transcriptome mining

Searches of publicly accessible decapod transcriptomic datasets for putative ALP precursor-encoding transcripts were conducted on or before July 19, 2019 using a well-established protocol. In brief, the database of the online program tblastn (National Center for Biotechnology Information, Bethesda, MD; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was set to Transcriptome Shotgun Assembly (TSA) and restricted to data from the Penaeoidea (taxid:111520), Sergestoidea (taxid:111521), Caridea (taxid:6694), Astacidea (taxid:6712), Achelata (taxid:6730), or Brachyura (taxid:6752). An A. mellifera ALP precursor (Accession No. XP_003249809; unpublished direct GenBank submission) was used as the query sequence for all BLAST searches.

2.2.2. Peptide structural prediction

The putative mature structures of decapod ALPs (as well as ALP precursor-related peptides [ALP-PRPs]) were predicted using a well-vetted workflow. Specifically, all hits returned by a given BLAST search were translated using the ExPASy Translate tool (http://web.expasy.org/translate/) and assessed for completeness. Proteins listed as full-length exhibit a functional signal sequence (including a start methionine) and are flanked on their carboxyl (C)-terminus by a stop codon. Proteins listed as C-terminal fragments lack a start methionine. Each full-length precursor was assessed for the presence of a signal peptide using the online program SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/; Bendtsen et al., 2004). Prohormone cleavage sites were identified based on information presented in Veenstra (2000). The sulfation state of tyrosine residues was predicted using the online program Sulfinator (http://www.expasy.org/tools/sulfinator/; Monigatti et al., 2002). Disulfide bonding between cysteine residues was assessed using the online programs DiANNA (http://clavius.bc.edu/~clotelab/DiANNA/; Ferrè and Clote, 2005) and DISULFIND (http://disulfind.dsi.unifi.it/; Ceroni et al., 2006). Other post-translational modifications, i.e., cyclization of N-terminal glutamine/glutamic acid residues and C-terminal amidation at glycine residues, were predicted by homology to known arthropod peptides. All protein and peptide alignments were done using either the online program MAFFT version 7 (http://mafft.cbrc.jp/alignment/software/; Katoh and Standley, 2013) or default MUSCLE (Edgar, 2004) settings in Geneious v10.1.3 (Biomatters Ltd., Auckland, New Zealand; Kearse et al., 2012). Percent identities were calculated from either pairwise alignments or multiple sequence alignments using MUSCLE.

2.3. Assessment of phylogenetic relationships among agatoxin-like precursor proteins

The phylogenetic relationships of selected decapod ALP precursor proteins were inferred from a multiple protein sequence alignment constructed with MUSCLE. Evolutionary analyses were conducted in MEGA 7 (Kumar et al., 2016) using the maximum likelihood method based on the Jones-Taylor-Thorton matrix model (Jones et al., 1992). Initial trees for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances and then selecting the topology with the highest log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 0.8139]). The analysis involved 21 amino acid sequences. All positions with less than 95% site coverage were eliminated; consequently, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. The final dataset consisted of a total of 110 positions. Analyses included bootstrap support for 1000 iterations. Phylogenetic inferences made using neighbor joining (Saitou and Nei, 1987) and minimum evolution (Rzhetsky and Nei, 1992) approaches generated trees with similar topologies. Accession numbers for sequences used in the phylogenetic analyses are provided in Supplemental Table 2.

2.4. Cloning of agatoxin-like peptide precursors from the lobster Homarus americanus

Total RNAs were purified from freshly dissected individual H. americanus supraoesophageal ganglia (brains) using TRI Reagent (Life Technologies, Carlsbad, CA) and Direct-zol RNA MiniPrep spin columns (Zymo Research, Irvine, CA, USA). First‐strand complementary DNAs (cDNAs) were generated from 500 ng of DNase I‐treated total RNA using Superscript III Reverse Transcriptase (Life Technologies) and custom made random pentadecamers (IDT, San Diego, CA, USA). The complete open reading frame for the putative H. americanus agatoxin-like peptide precursor was PCR amplified using primers (sense – 5’-ATGGGTAGCAAGGTGTTGG; antisense – 5’-TTATTTCCCCCACTGCTGG) constructed to encompass the predicted start and stop sites in conjunction with Sapphire Amp Fast PCR Master Mix (Takara Bio USA Inc., Mountain View, CA). The primers were designed based on alignment of the H. americanus transcriptomic sequences GFDA01040082.1, GFUC01090606.1, and GFUC01090606.1. PCR was performed on a Biometra TRIO multiblock thermocycler (Biometra, Göttingen, Germany) in a 20-μL reaction volume containing 0.5 μL cDNA template and 0.2 μM of each primer. The thermocycler conditions consisted of: 95°C for 2 min followed by 37 cycles at 95°C for 20 s, 56°C for 20 s, 72°C for 30 s, and a final extension at 72°C for 5 min. The resulting products were separated on a 1.5% agarose gel using a tris/acetate/EDTA buffer system with SYBR Safe (Life Technologies). The reaction products were subcloned into pCR2.1‐TOPO TA (Life Technologies) and sequenced at the Arizona State University DNA Core Laboratory (Tempe, AZ).

2.5. RT-PCR profiling of agatoxin-like peptide precursor expression in specific Homarus americanus nervous system regions

To examine neural expression of the agatoxin-like peptide precursor-encoding transcripts, RNAs were purified from three biological replicates of H. americanus brain (one brain per replicate), eyestalk ganglia (one eyestalk pair per replicate), and cardiac ganglion (ten cardiac ganglia per replicate). cDNAs were generated as described above from ~100 ng (cardiac ganglia) or 500 ng (brain and eyestalk ganglia) of DNase I-treated total RNAs. RT-PCR was performed in 20‐μL reaction volumes containing 0.5 μL of each cDNA template and primers designed to amplify the complete agatoxin-like peptide precursor open reading frame (see above) as well as a 503-bp fragment of H. americanus actin (sense – 5’- GGTCGTACCACCGGTATT; antisense – 5’- CATCCTGTCGGCAATTCC). Thermocycler conditions were as described above. RT-PCR was performed in triplicate. The resulting products were visualized on 3% agarose gels with images obtained using an AlphaImager Gel Documentation System (ProteinSimple, San Jose, CA) and processed in Photoshop CS6 v13.0 (Adobe Systems Inc., San Jose, CA).

2.6. Mass spectrometry

2.6.1. Tissue isolation and sample preparation

Lobsters were anaesthetized on ice for ~30 min, after which brains from individual lobsters (n=8) were isolated via manual microdissection in chilled (8–10°C) physiological saline (composition in mM: NaCl, 479.12; KCl, 12.74; CaCl2, 13.67; MgSO4, 20.00; Na2SO4, 3.91; Trizma base, 11.45; maleic acid, 4.82 [pH, 7.45]) and placed in 58 μL of LCMS water (Fisherbrand; Life Technologies Corp.) in a 1.5 mL low retention tube. Samples were heated at 100°C for 5 min to deactivate proteolytic enzymes (Stemmler et al., 2013), then cooled to room temperature for immediate analysis or stored at −20°C.

To extract peptides, 142 μL of extraction solvent A (90.1% methanol; Fisher Scientific; LCMS-grade and 9.9% glacial acetic acid; SigmaAldrich, St. Louis, MO, USA; reagent grade; > 99%) was added to the tube. The brain/solvent mixture was homogenized using a motor-driven tissue grinder (Argos Technologies; Vernon Hills, IL) equipped with a polypropylene pestle (SigmaAldrich) for 2–3 min, sonicated for 5 min and centrifuged at 14.5 krpm (Mini-Spin Plus, Eppendorf; Hauppauge, NY, USA) for 5 min. The supernatant was transferred to a clean 1.5 mL tube and the remaining tissue pellet was re-suspended and re-homogenized in 50 μL of extraction solvent B (30% water; 65% methanol; 5% glacial acetic acid), sonicated for 5 min, and centrifuged at 14.5 krpm for 5 min; the supernatant was again removed. The combined supernatants were filtered through a 30 kDa molecular weight cut-off (MWCO) filter (Amicon Ultra-0.5 mL; Millipore, Burlington, MA, USA), which had been prewashed with two-200 μL volumes of extraction solvent B. MWCO filters were centrifuged at 14.5 krpm for up to 20 min, and the flow-through (approximately 200 μL) was collected for further processing. Samples were then subjected to delipidation with chloroform (n=4) or high pH fractionation (n=4). For delipidation, chloroform (NMR-grade 13CDCl3; Cambridge Isotope Laboratories, Tewksbury, MA, USA; 200 μL) was added to the MWCO filter flow-through solution. The two-phase mixture was vigorously shaken or sonicated for ~1 min. The bottom organic layer was removed and discarded. Chloroform (200 μL) was added and the extraction was repeated. A portion of the top aqueous layer (30 μL) was mixed with 0.1% formic acid in water (LCMS-grade; Fisherbrand; 70 μL) for analysis by LCMS. With the goal of fractionating peptides and removing salts and phospholipids, brain extracts (n=4) were fractionated using PierceTM High pH Reversed-Phase Peptide Fractionation Kits (Thermo Fisher Scientific). In preparation for fractionation, the MWCO filter flow-through was dried and reconstituted in 300 μL of 0.1% TFA (Sigma Aldrich; ≥ 99.7%) in water (Fisher Scientific; LCMS-grade). The SPE columns were prepared following the manufacturer instructions; after sample loading and washing, peptides were eluted into 10 fractions using 300 μL steps of: 5%, 7%, 10%, 12.5%, 15%, 17.5%, 20%, 50%, 70%, and 80% acetonitrile, all in 0.1% trimethylamine in water (Thermo Fisher Scientific). The eluted peptides were dried and reconstituted in 40 μL of 4.5% formic acid (Thermo Fisher Scientific; 99+%) and 13% acetonitrile in water.

2.6.2. Liquid chromatography/mass spectrometry

Liquid chromatographic/mass spectrometric (LC/MS) analyses of individual H. americanus brain extracts (delipidated or fractionated) were performed using a 6530 quadrupole time-of-flight (Q-TOF) mass analyzer (Agilent Technologies, Santa Clara, CA, USA). Mass spectra (MS and MS/MS) were collected in positive ion mode; the ionization voltage ranged from 1850–1975 V and the ion source temperature was held at 350°C. Spectra were internally calibrated using dibutyl phthalate (C16H22O4) and hexakis(1H, 1H, 4H-hexafluorobutyloxy)phosphazine (HP-1221; C24H18O6N3P3F36) continuously infused and detected as [M+H]+. Collision induced dissociation (CID)-MS/MS experiments were executed with precursor ions subjected to CID using nitrogen as the target gas. Chromatographic separation and nano-electrospray ionization (nanoESI) were performed using a 1260 Chip Cube system (Agilent Technologies) using a ProtID-chip with a 40 nL enrichment column and a 150 mm x 75 μm analytical column (Agilent Technologies). The enrichment and analytical columns were packed with 300 Å, 5 μm particles with Zorbax 300SB-C18 stationary phase. The mobile phases were 0.1% formic acid/water (A) and 0.1% formic acid/2% water/acetonitrile (B). Samples (1–16 μL) were loaded on the enrichment column using 99:1 (A:B) at 4 μL/min. Samples were analyzed with the 150-mm analytical column using either a linear gradient of 99:1 (A:B) for 1 min to 65:35 (A:B) at 40 min, to 30:70 (A:B) at 45 min and 0:100 (A:B) at 50.0 min or a longer linear gradient of 98:2 (A:B) for 1 min to 65:35 (A:B) at 130 min, to 40:60 (A:B) at 133 min and 0:100 (A:B) at 140.0 min using a flow rate of 0.3 μL/min. LC/MS figures were generated by exporting Mass Hunter (Agilent Technologies) chromatograms or mass spectral data as text files, graphing the data using Prism 7 (GraphPad Software, San Diego, CA), and importing these graphics into CorelDRAW X4 (Corel Corporation, Austin, TX, USA) for final figure production.

3. Results and Discussion

3.1. Transcriptomic data suggest agatoxin-like peptides are broadly conserved in the Decapoda

To assess the extent to which the presence of ALPs is conserved in members of the Decapoda, the publicly accessible TSA datasets for the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimp), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs) were searched for putative ALP precursor-encoding transcripts using an ALP precursor from the honey bee, A. mellifera, as the query protein. These searches resulted in the identification of putative ALP-encoding sequences from 22 species, including one or more members of each of the abovementioned taxa (Table 1). For the Penaeoidea, putative ALP precursor-encoding transcripts were found in Litopenaeus vannamei, Penaeus monodon, Metapenaeus bennettae, and Fenneropenaeus penicillatus transcriptomes, while for the Sergestoidea, putative ALP-encoding sequences were identified from an Acetes chinensis assembly. For the Caridea, putative ALP precursor-encoding transcripts were identified from Macrobrachium australiense, Macrobrachium koombooloomba, Macrobrachium novaehollandiae, Macrobrachium tolmerum, Metabetaeus lohena, Halocaridina rubra, Halocaridinides trigonophthalma, Neocaridina denticulate, and Antecaridina lauensis datasets. For the Astacidea, transcripts encoding putative ALP precursors were identified in Homarus americanus (sequences confirmed via RT-PCR; Accession Nos. MN304943-MN304945), Cherax quadricarinatus, Pontastacus leptodactylus, and Procambarus clarkii assemblies, while for the Achelata, transcripts encoding putative ALPs were found in a Jasus edwardsii transcriptome. For the Brachyura, ALP precursor-encoding sequences were found in Carcinus maenas, Eriocheir sinensis, and Portunus trituberculatus datasets. These transcriptomic data strongly suggest that the presence of the ALP family is highly, and likely ubiquitously, conserved in the major taxa of the Decapoda.

Table 1.

Agatoxin-like peptide (ALP) precursor transcript/protein discovery in decapod crustaceans

Species Transcript Deduced protein
Taxonomic groupa Species Accession No. Tissue source Name Length Type
Penaeoidea Litopenaeus vannamei GFRP01012298 Mixed Litva-prepro-ALP-v1 112 F
GGQV01059635 Eyestalk Litva-prepro-ALP-v2 108 F
GGKO01019727 Mixed Litva-prepro-ALP-v3 99 F
GFRP01012297 Mixed Litva-prepro-ALP-v3 99 F
Penaeus monodon GGLH01104228 Mixed Penmo-prepro-ALP-v1 108 F
GGLH01049277 Mixed Penmo-prepro-ALP-v2 99 F
Metapenaeus bennettae GHDJ01038348 Hepatopancreas Metbe-prepro-ALP-v1 112 F
GHDJ01038347 Hepatopancreas Metbe-prepro-ALP-v2 99 F
Fenneropenaeus penicillatus GFRT01015650 Mixed Fenpe-prepro-ALP 46 C
Sergestoidea Acetes chinensis GGVZ01058550 Mixed Acech-prepro-ALP-v1 106 F
GGVZ01005325 Mixed Acech-prepro-ALP-v1 106 F
GGVZ01058552 Mixed Acech-prepro-ALP-v2 104 F
GGVZ01005326 Mixed Acech-prepro-ALP-v2 104 F
GGVZ01058551 Mixed Acech-prepro-ALP-v3 95 F
GGVZ01005324 Mixed Acech-prepro-ALP-v3 95 F
Caridea Macrobrachium australiense GHDT01074459 Mixed Macau-prepro-ALP-I-v1 117 F
GHDT01074458 Mixed Macau-prepro-ALP-I-v2 111 F
GHDT01074460 Mixed Macau-prepro-ALP-I-v3 106 F
GHDT01074462 Mixed Macau-prepro-ALP-I-v4 100 F
GHDT01074464 Mixed Macau-prepro-ALP-II-v1 117 F
GHDT01074461 Mixed Macau-prepro-ALP-II-v2 111 F
GHDT01074463 Mixed Macau-prepro-ALP-II-v3 100 F
Macrobrachium koombooloomba GHDU01066920 Mixed Macko-prepro-ALP-I-v1 111 F
GHDU01066922 Mixed Macko-prepro-ALP-I-v2 100 F
GHDU01066919 Mixed Macko-prepro-ALP-II-v1 111 F
GHDU01066918 Mixed Macko-prepro-ALP-II-v2 100 F
Macrobrachium novaehollandiae GHDW01093390 Mixed Macno-prepro-ALP-I 111 F
GHDW01093388 Mixed Macno-prepro-ALP-II-v1 117 F
GHDW01093389 Mixed Macno-prepro-ALP-II-v2 111 F
Macrobrachium tolmerum GHDQ01089244 Mixed Macto-prepro-ALP-I-v1 111 F
GHDQ01089242 Mixed Macto-prepro-ALP-I-v2 100 F
GHDQ01089241 Mixed Macto-prepro-ALP-II 111 F
GHDQ01089243 Mixed Macto-prepro-ALP-II 111 F
Metabetaeus lohena GHAP01062116 Mixed Metlo-prepro-ALP-I-v1 111 F
GHAP01062115 Mixed Metlo-prepro-ALP-I-v2 100 F
GHAP01062117 Mixed Metlo-prepro-ALP-II-v1 111 F
GHAP01062114 Mixed Metlo-prepro-ALP-II-v2 100 F
Halocaridina rubra GHBK01056446 Mixed Halru-prepro-ALP-I-v1 117 F
GHBK01056445 Mixed Halru-prepro-ALP-I-v2 106 F
GHBK01108229 Mixed Halru-prepro-ALP-II 53 C
Halocaridinides trigonophthalma GHBI01088024 Mixed Haltr-prepro-ALP-I 108 C
Neocaridina denticulata GGXN01030525 Mixed Neode-prepro-ALP-I 102 F
Antecaridina lauensis GHBJ01037781 Mixed Antla-prepro-ALP-I 103 F
GHBJ01037782 Mixed Antla-prepro-ALP-I 103 F
Astacidea Homarus americanus GFDA01040082 Eyestalk ganglia Homam-prepro-ALP-v1 113 F
GGPK01031489 Cardiac ganglion Homam-prepro-ALP-v1 113 F
GFUC01090608 Brain Homam-prepro-ALP-v1 113 F
GGPK01031491 Cardiac ganglion Homam-prepro-ALP-v2 109 F
GFUC01090606 Brain Homam-prepro-ALP-v2 109 F
GFDA01040083 Eyestalk ganglia Homam-prepro-ALP-v3 100 F
GGPK01031490 Cardiac ganglion Homam-prepro-ALP-v3 100 F
GEBG01002733 Nervous system Homam-prepro-ALP-v3 100 F
Cherax quadricarinatus HACK01031533 Mixed Chequ-prepro-ALP-v1 113 F
HACK01031534 Mixed Chequ-prepro-ALP-v2 109 F
HACB02001107 Mixed Chequ-prepro-ALP-v3 100 F
HACK01031532 Mixed Chequ-prepro-ALP-v3 100 F
Pontastacus leptodactylus GBEI01092752 Mixed Ponle-prepro-ALP 113 F
Procambarus clarkii GARH01001590 Eyestalk Procl-prepro-ALP 129 F
Achelata Jasus edwardsii GGHM01043346 Mixed Jased-prepro-ALP-v1 113 F
GGHM01043347 Mixed Jased-prepro-ALP-v2 100 F
Brachyura Carcinus maenas GFYV01055236 Mixed Carma-prepro-ALP-v1 113 F
GFYW01063051 Mixed Carma-prepro-ALP-v1 61 C
GFYW01063050 Mixed Carma-prepro-ALP-v1 61 C
GFYW01063049 Mixed Carma-prepro-ALP-v1 61 C
GFXF01149802 Mixed Carma-prepro-ALP-v2 109 F
GBXE01084435 Mixed Carma-prepro-ALP-v2 109 F
GFYV01055237 Mixed Carma-prepro-ALP-v3 100 F
Eriocheir sinensis GBUF01003221 Mixed Erisi-prepro-ALP-v1 113 F
GFBL01086394 Mixed Erisi-prepro-ALP-v1 113 F
GFBL01086391 Mixed Erisi-prepro-ALP-v1 113 F
GBZW01010384 Eyestalk Erisi-prepro-ALP-v2 109 F
JR775346 Testis Erisi-prepro-ALP-v2 52 C
HAAX01020856 Mixed Erisi-prepro-ALP-v3 100 F
GGQO01017109 Mixed Erisi-prepro-ALP-v3 100 F
HAAX01020856 Mixed Erisi-prepro-ALP-v3 100 F
GBUF01003220 Mixed Erisi-prepro-ALP-v3 100 F
GFBL01086390 Mixed Erisi-prepro-ALP-v3 100 F
GFBL01086392 Mixed Erisi-prepro-ALP-v3 100 F
Portunus trituberculatus GFFJ01041078 Eyestalk Portr-prepro-ALP 109 F
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a

Taxonomic groups: Penaeoidea, a superfamily within the suborder Dendrobranchiata; Sergestoidea, a superfamily within the suborder Dendrobranchiata; Caridea, an infraorder within the suborder Pleocyemata; Astacidea, an infraorder within the suborder Pleocyemata; Achelata, an infraorder within the suborder Pleocyemata; Brachyura, an infraorder within the suborder Pleocyemata.

3.2. Most decapods appear to have two isoforms of agatoxin-like peptide, both derived from a single gene by alternative splicing

Translation of the putative decapod ALP precursor-encoding sequences suggests the presence of a single ALP gene that can be alternatively spliced to generate (typically) three variants in nearly all species (Fig. 1 and Supplemental Fig. 1). The one possible exception is in the Caridea, in which a duplication of either the ancestral ALP gene (named ALP-I in members of this infraorder) or one of the terminal exons appears to have resulted in a paralog (caridean ALP-II) that also undergoes alternative splicing (Fig. 1B and Supplemental Fig. 1). Comparison of the caridean ALP-I and -II open reading frames across the first 198 nucleotides revealed ~88% sequence identity that was independent of paralog type, whereas percent identity across the remaining 138 nucleotides segregated based on paralog type with identity > 95% (Supplemental Fig. 2A). When compared with the same region of the H. americanus and P. clarkii sequences, sequence identity was highest with the type I paralogs. Alignment of this region of the caridean ALP-I and -II sequences revealed clear areas of conservation (Supplemental Fig. 2B) that are consistent with a caridean-specific exon duplication event. In this scenario, the respective variants could be generated from homologous equivalent exon substitution (Supplemental Fig. 2C), a prevalent alternative splice mechanism that substitutes one homologous, possibly duplicated, exon for another (Abascal et al., 2015; Ezukurdia et al., 2012). Comparison of selected decapod ALP precursor sequences, including caridean ALP-I and ALP-II proteins, shows that the caridean ALP-II precursors cluster in a clade distinct from the ALP-I sequences, providing additional support for their paralog status (Fig. 2, Supplemental Fig. 3). The functional relevance of the caridean-specific ALP-I and -II sequences remains to be determined.

Figure 1.

Figure 1.

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MAFFT alignments of selected decapod agatoxin-like peptide (ALP) precursor proteins. (A) Alignment of Litopenaeus vannamei ALP precursor variants. (B) Alignment of Acetes chinensis ALP precursor variants. (C) Alignment of Macrobrachium australiense ALP-I and II precursor variants. (D) Alignment of Homarus americanus ALP precursor variants. (E) Alignment of Jasus edwardsii ALP precursor variants. (F) Alignment of Carcinus maenas ALP precursor variants. In each protein, the signal peptide is shown in gray, the ALP isoform is shown in red, and the linker/precursor-related peptides are shown in blue. In the line immediately below each sequence grouping, the symbol “*” indicates amino acids that are identical in all proteins, while “.” and “:” denote amino acids that are similar in structure among all sequences.

Figure 2.

Figure 2.

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Phylogenetic relationship of putative decapod agatoxin-like peptide (ALP) precursors with those of other arthropod species. The evolutionary history was inferred using the maximum likelihood method with the highest log likelihood (−1307.53) tree shown. Boostrap support (1000 iterations) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Type II ALP sequences are indicated in bold. Species abbreviations are: Macau, Macrobrachium australiense; Macno, Macrobrachium novaehollandiae; Macko, Macrobrachium koombooloomba; Macto, Macrobrachium tolmerum; Metlo, Metabetaeus lohena; Jased, Jasus edwardsii; Homam, Homarus americanus; Chequ, Cherax quadricarinatus; Procl, Procambarus clarkii; Ponle, Pontastacus leptodactylus; Erisi, Eriocheir sinensis; Carma, Carcinus maenas; Litva, Litopenaeus vannamei; Metbe, Metapenaeus bennettae; Acech, Acetes chinensis; and Apime, Apis mellifera. The tree was rooted with Apis mellifera U8-agatoxin-Ao1a isoform X1 (Accession No. XP_003249809). Accession numbers for sequences used in the phylogenetic tree are listed in Supplemental Table 2.

Regardless of the number of precursor variants, it appears that the collective set of preprohormones gives rise to two distinct isoforms of ALP in any given species (except carideans), which are 44 and 42 amino acids in length (Fig. 3). In members of the Caridea, four isoforms of ALP appear to be the rule, with 44 and 42 amino acid peptides derived from both the ALP-I and ALP-II preprohormones (Fig. 3). All decapod ALPs appear to be C-terminally amidated. All also possess eight identically positioned cysteine residues (positions 4, 11, 18, 19, 24, 26, 33, and 35 in the 44 amino acid peptides, including those derived from the caridean ALP-II precursors; Fig. 3), which are theorized to give rise to four identically-positioned disulfide bridges; the positioning of these bonds remains unclear as predictions of their locations by DiANNA and DISULFIND showed considerable variability both between programs and among ALP isoforms (data not shown). Comparison of the amino acids at other positions in decapod ALP isoforms (those derived from ALP-I for the carideans) reveals extremely high levels of residue conservation in these peptides, with substitutions/deletions seen at just nine positions (Fig. 3A), positions 1 and 2 in the 44 amino acid peptides (tryptophan-arginine in all isoforms), which are deleted in the 42 amino acid peptides, and substitutions at positions 5, 8, 10, 16, 28, 29, and 40 (numbering based on the 44 amino acid isoforms). Four of the substitutions are conservative (isoleucine or valine at position 5, asparagine or histidine at position 16, leucine or phenylalanine at position 28, and isoleucine or leucine at position 40), with the remaining three being non-conservative (methionine, asparagine, serine, or glycine at position 8, alanine, valine, serine, threonine, methionine, or proline at position 10, and tryptophan or leucine at position 29). The ALPs derived from the caridean ALP-II precursors also show extremely high levels of amino acid identity among species, varying at just eight positions, i.e., insertions/deletions at positions 1 and 2 and variable residues at positions 3, 8, 10, 16, 21, and 36, all of which are conservative substitutions (Fig. 3B). Interestingly, there is more variation between the ALP-I- and ALP-II-derived peptides from a given caridean species (e.g., Fig. 1B) than there is among the caridean ALP-I-derived isoforms and those from members of other decapod superfamilies/infraorders (Fig. 3A).

Figure 3.

Figure 3.

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MAFFT alignments of all predicted decapod agatoxin-like peptide (ALP) isoforms. (A) Alignment of ALPs derived from decapod type-I precursors. (B) Alignment of ALPs derived from caridean type-II precursors. In the line immediately below each sequence grouping, the symbol “*” indicates amino acids that are identical in all proteins, while “.” and “:” denote amino acids that are similar in structure among all sequences. All peptides are predicted to possess amidated carboxyl-termini (not shown in the alignments). All peptides are also predicted to possess four identically positioned disulfide bridges (also not shown in the alignments). The conserved Cys motifs are indicated: PSM - principal structural motif; ESM - extra structural motif. Species identifications for A: Penaeoidea-1, Litopenaeus vannamei and Metapenaeus bennettae; Penaeoidea-2, L. vannamei, M. bennettae, Penaeus monodon, and Fenneropenaeus penicillatus; Sergestoidea-1, Acetes chinensis; Sergestoidea-2 A. chinensis; Caridea-1a, Macrobrachium australiense, Macrobrachium koombooloomba, Macrobrachium novaehollandiae, and Macrobrachium tolmerum; Caridea-1b, Metabetaeus lohena; Caridea-1c, Halocaridina rubra; Caridea-2a, M. australiense, M. koombooloomba, and M. tolmerum; Caridea-2b, M. lohena; Caridea-2c, H. rubra, Halocaridinides trigonophthalma, and Antecaridina lauensis; Caridea-2d, Neocaridina denticulata; Astacidea-1a, Cherax quadricarinatus, Pontastacus leptodactylus, and Procambarus clarkii; Astacidea-1b, Homarus americanus; Astacidea-2a, C. quadricarinatus; Astacidea-2b, H. americanus; Achelata-1, Jasus edwardsii; Achelata-2, J. edwardsii; Brachyura-1, Carcinus maenas and Eriocheir sinensis; Brachyura-2a, C. maenas and E. sinensis; Brachyura-2b, Portunus trituberculatus. Species identifications for B: Macxx-1, M. australiense, M. koombooloomba, M. novaehollandiae, and M. tolmerum; Macxx-2, M. australiense and M. koombooloomba; Metlo-1, M. lohena; Metlo-2, M. lohena; Halru-2, H. rubra.

In addition to ALP, the decapod prepro-ALPs also contain one or more linker/precursor-related peptide (e.g., Fig. 1). In their predicted mature forms, these linker/precursor-related sequences show greater variability within and between species than do the ALP isoforms, though there is considerable amino acid sequence conservation across species, and even across members of different superfamilies/infraorders (e.g., Figs. 1). The putative mature structures of all predicted PRPs derived from decapod ALP precursors are provided in Supplemental Table 1.

To determine if the predicted mature forms of the two ALP isoforms were present in H. americanus (Fig. 4), we extracted brains from individual lobsters (n=8) and analyzed the extracts using a chip-based nanoLC-QTOF-MS/MS instrument. Give the methods used, we expected the mature ALP isoforms to be challenging peptides to detect and characterize because of their size (~5 kDa; Table 2), which results in a reduction in signal-to-noise associated with increasing dispersal of signal across a broader distribution of isotopic combinations and charge states (Compton, et al. 2011). Furthermore, the use of MS/MS for structural confirmation of putative ALPs would be impeded by the presence of disulfide bonds, which restrict formation of fragment ions under our low energy CID conditions and makes structural confirmation more challenging. In the eight brain samples examined, we were unable to unambiguously confirm that native forms of the peptides were present. To circumvent the challenges associated with analysis of the native peptides, we reduced, alkylated and trypsinized three brain extracts to apply a proteomic strategy to the peptide analysis (data not shown). This approach also failed to produce MS support for the direct detection of the two ALP isoforms.

Figure 4.

Figure 4.

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Theoretical processing schemes for the three lobster, Homarus americanus, agatoxin-like peptide (ALP) precursors. In this figure, the predicted structures of mature ALP isoforms are shown in red, with those of mature precursor-related peptides (PRPs) shown in blue. In the mature ALPs, the presence of cysteine residues that putatively participate in the formation of disulfide bridges are indicated by “c”. In the mature structure of the first PRP present in each precursor (the sole PRP in present in the precursor shown in panel C), a sulfated tyrosine residue is indicated by “Y(SO3H)”, while the presence of an amino-terminal pyroglutamic acid residue is indicated by “pQ”.

Table 2.

Summary of agatoxin-like peptide (ALP) and ALP precursor-related peptide (ALP-PRP) isoforms detected in H. americanussupraoesophageal ganglion (brain) extracts

Peptide Precursor variant contained within Structure Predicted mass (Da)a Error (ppm)b Detection Summaryc
ALP isoforms
ALP-1 1 WRScIRRGGMcDHRPNDccYNSScRcNLWGTNcRcQRMGIFQQWamide 5290.1906 NA 0/8
ALP-2 2, 3 ScIRRGGMcDHRPNDccYNSScRcNLWGTNcRcQRMGIFQQWamide 4948.0102 NA 0/8
ALP-PRP isoforms
ALP-PRP-1a 1, 2, 3 pQPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ 3814.79542 3.9 7/8
ALP-PRP-1b 1, 2, 3 pQPLLEEGREEDGVQQAEPDYAADLLERLLARTQ 3734.83862 2.0 4/8
ALP-PRP-1c 1, 2, 3 QPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ 3831.82197 0.1 4/8
ALP-PRP-1d 1, 2, 3 QPLLEEGREEDGVQQAEPDYAADLLERLLARTQ 3751.86517 0.3 6/8
ALP-PRP-2 1 DDVAGSDPI 887.38715 −1.2 7/8
ALP-PRP-3 2 SSYIYLF 891.43775 −2.4 8/8
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a

Monoisotopic mass for [M+H]+

b

Error (ppm) = ((Mmeaured-Mpredicted)*106/Mpredicted)

c

Number of brain tissues where peptide was detected/total number of brain tissues analyzed

In contrast, we were able to detect all three of the predicted precursor/linker peptides (Table 2 and Figs. 4–6). Homam-ALP-PRP-1, common to all three variants of the ALP-preprohormone and having a mature structure predicted to be pQPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ (ALP-PRP-1a), ionized to produce abundant [M+4H]4+ and [M+3H]3+ ions. The MS/MS spectrum of the [M+3H]3+ ion yielded an abundant product ion resulting from the neutral loss of SO3, which supported the presence of sulfation in the sequence. While the facile loss of SO3 precluded our ability to verify the localization of the sulfate group to the single tyrosine (Y) residue, the MS/MS spectrum of the [M+4H]4+ ion (Fig. 5A) supported the amino acid sequence assignment through the detection of b-type ions, which included the N-terminus, and y-type ions, which included the C-terminus. We also detected and used MS/MS to characterize three additional immature forms of ALP-PRP-1, pQPLLEEGREEDGVQQAEPDYAADLLERLLARTQ (ALP-PRP-1b; Fig. 5C), QPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ (ALP-PRP-1c; Fig. 5E), and QPLLEEGREEDGVQQAEPDYAADLLERLLARTQ (ALP-PRP-1d; Fig. 5G). ALP-PRP-1a, the mature form of the peptide, was most strongly retained by the column and was detected with the highest signal intensity (Fig. 5B); ALP-PRP-1d, the most immature form of the peptide (no N-terminal pyroglutamate and no sulfation) was least strongly retained by the column and was detected with the lowest signal intensity (Fig. 5H). We also detected and characterized DDVAGSDPI (Homam-ALP-PRP-2; Table 2 and Fig. 6A), and SSYIYLF (Homam-ALP-PRP-3; Table 2 and Fig. 6B). These PRPs were specific to variant 1 and 2, respectively, of the Homarus ALP preprohormone. Whether or not any of the H. americanus ALP-PRPs represent functionally important or bioactive peptides remains unknown. However, the mass spectral detection of them in their predicted mature forms in the lobster brain suggests that they are not subject to rapid degradation and is a finding that may indicate that these peptides play a role as bioactive modulators or as peptides playing a functional role in preprohormone or peptide processing.

Figure 6.

Figure 6.

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Mass spectrometric identification of agatoxin-like peptide precursor-related peptide (ALP-PRP)-2 and −3 from Homarus americanus brain extract. (A) MS/MS spectrum for the m/z 888.39, [M+H]+ ion from DDVAGSDPI (ALP-PRP-2) at a collision energy of 28.7 eV. (B) MS/MS spectrum for the m/z 892.45, [M+H] + ion from SSYIYLF (ALP-PRP-3) at a collision energy of 28.8 eV. The assigned sequences were supported by N-terminus containing b-type product ions, many of which lost CO to produce a-type ions. C-terminus containing y-type ions provided additional sequence support, as did the detection of internal product ions and immonium ions. Ions that have lost H2O are shown with an open circle. Monoisotopic masses are displayed.

Figure 5.

Figure 5.

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Mass spectrometric identification of mature and immature forms of agatoxin-like peptide precursor-related peptide 1 (ALP-PRP-1) from Homarus americanus brain extract. (A) MS/MS spectrum for the m/z 954.71, [M+4H]4+ ion from pQPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ (ALP-PRP-1a) at a collision energy of 30.6 eV; (B) Summed extracted ion chromatograms (EICs) for the [M+4H]4+ and [M+3H]3+ peaks from ALP-PRP-1a, b, c, d; ALP-PRP-1a eluted last and was detected with the highest signal intensity; (C) MS/MS spectrum for the m/z 934.72, [M+4H]4+ ion from pQPLLEEGREEDGVQQAEPDYAADLLERLLARTQ (ALP-PRP-1b) at a collision energy of 30.0 eV; (D) Summed extracted ion chromatograms (EICs) for the [M+4H]4+ and [M+3H]3+ peaks from ALP-PRP-1a, b, c, d; ALP-PRP-1b eluted third and was detected with the second highest signal intensity; (E) MS/MS spectrum for the m/z 958.96, [M+4H]4+ ion from QPLLEEGREEDGVQQAEPDY(SO3H)AADLLERLLARTQ (ALP-PRP-1c) at a collision energy of 30.8 eV; (F) Summed extracted ion chromatograms (EICs) for the [M+4H]4+ and [M+3H]3+ peaks from ALP-PRP-1a, b, c, d; ALP-PRP-1c eluted second and was detected with the third highest signal intensity; (G) MS/MS spectrum for the m/z 938.97, [M+4H]4+ ion from QPLLEEGREEDGVQQAEPDYAADLLERLLARTQ (ALP-PRP-1d) at a collision energy of 30.2 eV; (H) Summed extracted ion chromatograms (EICs) for the [M+4H]4+ and [M+3H]3+ peaks from ALP-PRP-1a, b, c, d; ALP-PRP-1d eluted first and was detected with the lowest signal intensity. The assigned sequences were supported by N-terminus containing b-type product ions, many of which lost CO to produce a-type ions. C-terminus containing y-type ions provided additional sequence support, as did the detection of internal product ions, including QQ, and immonium ions, including peaks for V, L, and Q. Ions that have lost NH3 are shown with a filled circle; ions that have lost H2O are shown with an open circle. Monoisotopic masses are displayed.

3.3. Possible functions of agatoxin-like peptides in members of the Decapoda

While the majority of the transcripts identified here as encoding putative ALP precursors come from transcriptomes generated using mixed tissues (either whole organism or selected multiple tissues) as the source of RNAs (e.g., Havird and Santos, 2016; Huerlimann et al., 2018; Li et al., 2015; Oliphant et al., 2018; Rahi et al., 2019; Santos et al., 2018; Souza et al., 2018; Sun et al., 2014; Tom et al., 2014; Verbruggen et al., 2015), some are from tissue or tissue region-specific assemblies (Armstrong et al., 2019; Christie et al., 2017, 2018a, 2018b; He et al., 2012; Northcutt et al., 2016). The transcripts identified from the latter datasets provide insight into the putative source(s) of ALP production in members of the Decapoda. Of particular note are the ALP-encoding sequences identified from lobster, H. americanus, where all were identified from nervous system-specific assemblies (Table 1), including several region-specific ones, i.e., those for the brain (Christie et al., 2018a), eyestalk ganglia (Christie et al., 2017), and cardiac ganglion (Christie et al., 2018b). Their presence in these portions of the lobster nervous system was confirmed by RT-PCR (Fig. 7). ALP-encoding transcripts were also identified from multiple eyestalk transcriptomes (Table 1), i.e., those for the shrimp, L. vannamei (Wang et al., 2019), the crayfish, P. clarkii (Manfrin et al., 2015), and the crabs, E. sinensis (Xu et al., 2015) and P. trituberculatus (Lv et al., 2017), for which the neural eyestalk ganglia undoubtedly represent the largest single tissue source. Detection of ALP precursor-encoding transcripts in neural-specific assemblies suggests that ALPs may serve as neuropeptides, though precisely how they function within the nervous system remains to be determined. It is possible that they may act as locally-released and/or circulating neuromodulators, as many peptides produced by the nervous systems of decapods have been shown to do (e.g., Christie, 2011; Christie et al., 2010), exerting their effects by binding to a G-protein coupled receptor or some other type of cell surface receptor, thereby modifying the properties of their neuronal targets. However, no ALP receptor has been identified in any arthropod species. Alternatively, ALPs might exert their action by directly interacting with ion channels, possibly neuronal calcium channels, leading to a reduction in calcium influx, and hence decreased neurotransmitter release, which is the mode of action of spider venom-derived agatoxins (e.g., Bindokas and Adams, 1989; Adams et al., 1990). Indeed, Sturm and co-workers (2016) postulated that the spider agatoxins and the venom gland ALPs may have been co-opted from the ancestral ALPs, which persist in most arthropods, to act as toxic ligands of previously targeted signaling proteins. Functional genomics and/or pharmacological-based studies, however, will be needed to clarify this issue.

Figure 7.

Figure 7.

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RT-PCR profiling of agatoxin-like peptide (ALP) precursor-encoding transcripts in Homarus americanus neural tissues. The three ALP variants (342, 330, and 303-bp) were amplified using a single primer set from all three tissues examined. As a positive control, a 503-bp fragment of H. americanus actin was likewise amplified. The 3% agarose gel image shown is representative of three independent reactions using different cDNA replicates. Abbreviations are: Br, brain; EG, eyestalk ganglia; CG, cardiac ganglia; NT, RT-PCR reaction without template.

In addition to their localization in neural-specific transcriptomes, ALP precursor-encoding transcripts were identified from a testis-specific assembly for the crab, E. sinensis (He et al., 2012), and a hepatopancreas-specific transcriptome for the shrimp, M. bennettae (Armstrong et al., 2019). Previous investigations have identified many “neuropeptides” in non-neuronal tissues, including the reproductive organs and hepatopancreas. For example, transcripts encoding adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), diuretic hormone 31, eclosion hormone, FMRFamide-like peptide, HIGSLYRamide, insulin-like peptide, inotocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, and tachykinin-related peptide precursors were recently identified from a testis-specific transcriptome for the crab, Scylla olivacea (Christie, 2016), with members of the CNMamide family recently identified from multiple decapod testis- and/or ovary-specific assemblies (Christie and Hull, 2019). The presence of ALPs in the testis suggests that members of this peptide family may be involved in male reproductive control and/or may signal male reproductive status to other regulatory systems.

A number of “neuropeptides” have likewise been predicted from transcriptomes derived from the hepatopancreas. These include transcripts encoding CHH family members identified in a shrimp, M. bennettae, hepatopancreas-specific transcriptome (Armstrong et al., 2019), as well as several “neuropeptides”, again including CHH family members, in a hepatopancreas-specific transcriptome derived from the shrimp, L. vannamei (A.E. Christie, unpublished observations). Given the various functions of the hepatopancreas, the identification of ALP-encoding sequences from it suggests that ALP family members may be involved in the regulation of stress response, detoxification, and/or innate immunity in members of the Decapoda.

4. Summary and Conclusions

In the study presented here, publicly accessible transcriptomes were used to investigate the phylogenetic and structural conservation, tissue localization, and putative functions of ALPs in decapod crustaceans. Transcripts encoding members of this peptide family were identified from one or more members of the Penaeoidea and Sergestoidea, two superfamilies of the suborder Dendrobranchiata, and one or more species from the Caridea, Astacidea, Achelata, and Brachyura, four infraorders of the suborder Pleocyemata. This diversity suggests that ALPs are broadly, and perhaps ubiquitously, conserved in decapods. Comparison of the mature structures of the ALPs predicted from the identified transcripts shows high levels of amino acid conservation, suggesting conserved function(s). The identification of ALP precursor-encoding transcripts in decapod nervous system-specific transcriptomes suggests that ALPs (and/or ALP-PRPs) may have a neuromodulatory role, while the presence of transcripts in the testis- and hepatopancreas-specific assemblies suggests that ALPs (and/or ALP-PRPs) may help regulate reproduction, stress response, detoxification, and/or innate immunity.

Supplementary Material

Supplementary Data 1
Supplementary Data 2

Supplemental Figure 1. Deduced amino acid sequences of decapod agatoxin-like peptide precursors identified via in silico transcriptome mining. In each protein, the signal peptide is shown in gray, the ALP isoform is shown in red, and the linker/precursor-related peptides are shown in blue. “+” indicated the presence of additional, unknown amino-terminal amino acids.

Supplementary Data 3

Supplemental Figure 2. Type-I and Type-II caridean agatoxin-like peptide (ALP) precursors. (A) Percent sequence identity heat maps of open reading frames from Type-I and Type-II caridean ALP precursors and closely related sequences. In the upper panel, the percent identity spanning the first 204 nucleotides (based on the Homarus americanus ALP precursor sequence) is largely uniform across all ALP precursor types. The lower panel, which depicts percent identity across the remaining nucleotides, shows clear evidence of sequence conservation that segregates based on ALP precursor type. Species abbreviations are as in Figure 2. (B) Multiple sequence alignment of the last 154 nucleotides in the respective open reading frames of Type-I and Type-II caridean ALP precursor and related sequences from other species. Absolute nucleotide identity among Type-I precursors and closely related sequences is shown in red, absolute nucleotide identity among Type-II precursors is shown in blue. Species abbreviations are as in Figure 2. (C) Schematic diagram of a potential alternative splicing mechanism. The observed variations in Type-I and Type-II caridean sequences are consistent with homologous equivalent exon substitution. Exons are indicated by boxes and splicing events by colored lines. Exons marked “A” and “B” represent putative exons that potentially encode nucleotides 1–189, whereas exon “I” encodes the terminal 154 nucleotide found in Type-I precursors and exon “II” those nucleotides in Type-II precursors.

Supplementary Data 4

Supplemental Figure 3. Additional phylogenetic relationship analyses of putative decapod agatoxin-like peptide (ALP) precursors with those of other arthropod species. The evolutionary histories were inferred using the minimum evolution (A) or neighbor joining (B) methods with the highest log likelihood tree shown. Boostrap support (1000 iterations) is shown at branch nodes. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Type II ALP sequences are indicated in bold. Species abbreviations are as in Figure 2.

Supplementary Data 5

Acknowledgements

Lisa Baldwin and Colin Brent are thanked for reading and commenting on an earlier version of this article. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture; the USDA is an equal opportunity provider and employer.

Funding

This work was supported by funds from the National Science Foundation (IOS-1353023 [to AEC]; IOS-1856307 [to AEC]; IOS-1354567 [to PSD]; IOS-1856433 [to PSD and EAS]; CHE-1126657 [to EAS]), the National Institutes of Health (INBRE grant 8P20GM103423-12), the Cades Foundation (to AEC), and base CRIS funding from the US Department of Agriculture (Project #2020-22620-022-00D; to JJH).

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Associated Data

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Supplementary Materials

Supplementary Data 1
NIHMS1650435-supplement-Supplementary_Data_1.xlsx (11.1KB, xlsx)
Supplementary Data 2

Supplemental Figure 1. Deduced amino acid sequences of decapod agatoxin-like peptide precursors identified via in silico transcriptome mining. In each protein, the signal peptide is shown in gray, the ALP isoform is shown in red, and the linker/precursor-related peptides are shown in blue. “+” indicated the presence of additional, unknown amino-terminal amino acids.

NIHMS1650435-supplement-Supplementary_Data_2.docx (17.9KB, docx)
Supplementary Data 3

Supplemental Figure 2. Type-I and Type-II caridean agatoxin-like peptide (ALP) precursors. (A) Percent sequence identity heat maps of open reading frames from Type-I and Type-II caridean ALP precursors and closely related sequences. In the upper panel, the percent identity spanning the first 204 nucleotides (based on the Homarus americanus ALP precursor sequence) is largely uniform across all ALP precursor types. The lower panel, which depicts percent identity across the remaining nucleotides, shows clear evidence of sequence conservation that segregates based on ALP precursor type. Species abbreviations are as in Figure 2. (B) Multiple sequence alignment of the last 154 nucleotides in the respective open reading frames of Type-I and Type-II caridean ALP precursor and related sequences from other species. Absolute nucleotide identity among Type-I precursors and closely related sequences is shown in red, absolute nucleotide identity among Type-II precursors is shown in blue. Species abbreviations are as in Figure 2. (C) Schematic diagram of a potential alternative splicing mechanism. The observed variations in Type-I and Type-II caridean sequences are consistent with homologous equivalent exon substitution. Exons are indicated by boxes and splicing events by colored lines. Exons marked “A” and “B” represent putative exons that potentially encode nucleotides 1–189, whereas exon “I” encodes the terminal 154 nucleotide found in Type-I precursors and exon “II” those nucleotides in Type-II precursors.

NIHMS1650435-supplement-Supplementary_Data_3.pdf (503.2KB, pdf)
Supplementary Data 4

Supplemental Figure 3. Additional phylogenetic relationship analyses of putative decapod agatoxin-like peptide (ALP) precursors with those of other arthropod species. The evolutionary histories were inferred using the minimum evolution (A) or neighbor joining (B) methods with the highest log likelihood tree shown. Boostrap support (1000 iterations) is shown at branch nodes. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Type II ALP sequences are indicated in bold. Species abbreviations are as in Figure 2.

NIHMS1650435-supplement-Supplementary_Data_4.pdf (422.1KB, pdf)
Supplementary Data 5
NIHMS1650435-supplement-Supplementary_Data_5.xlsx (10.6KB, xlsx)

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