Computational protocol: A Flight Sensory-Motor to Olfactory Processing Circuit in the Moth Manduca sexta

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Protocol publication

[…] To determine the amino acid sequence of the Manduca homolog of the HA B-type receptor (MsHisClB), we used the Manduca genome (Agricultural Pest Genomics Resource Database: to perform a forward protein BLAST analysis of the Drosophila melanogaster histamine B-type receptor (HisClB) amino acid sequence (ACA13298.1). The top match from the Manduca genome had an e-value of 0.0 (Msex2.04603-RA). We then reverse blasted this sequence from the Manduca genome into the Drosophila genome in NCBI and the first three matches were Drosophila HisClB isoforms (NP_650116.2, NP_731632.1, and NP_001163591.1), all of which had e-values of 0.0. The next highest match from the Drosophila genome was the HisClA receptor (otherwise known as “ora transientless”; NP_524406.1) which is the other of the two HA receptor types in Drosophila () and had e-values of 3e-148 which is consistent with both HA receptor types having high sequence homology (; ). To ensure that there were not two predicted amino acid sequences from the Manduca genome with high sequence homology to the Drosophila HisClB receptor, we took the amino acid sequence from the Manduca genome with the second highest e-value for the Drosophila MsHisClB (Msex2.04216-RA; e-value = 1e-119) and ran a BLAST analysis of this sequence in the Drosophila genome. The BLAST analysis resulted in an e-value of 7.37e-158 for the Drosophila ora transientless indicating that the Manduca protein with the next closest sequence similarity to Drosophila HisClA was likely not the MsHisClB homolog. Figure is a sequence alignment of the Manduca HisClB receptor (MsHisClB) with the sequences for known histamine B receptors from Drosophila melanogaster (ACA13298.1), Apis meliferia (ABG75740.1), and Nasonia vitripennis (ACZ51422.1) () using the EMBL-EBI Clustal omega tool ().Custom affinity purified antibodies were generated in rabbit (Bethyl laboratories) using Cys-VNPDIELPQLD as the immunogenic sequence. The immunogenic sequence was highly conserved across D. melanogaster, A. mellifera, and N. vitripennis (Figure ). For western blots, adult brains were placed in BoltTM LDS Sample Buffer (Life Technologies, B0007, Life Technologies) with protease inhibitor cocktail (Research Products International, P50900) and DNase I (Invitrogen, 18068-015) and kept on ice for homogenization with a pestle. Samples were heated in a water bath for 10 min at 95°C. We used the Novex® BoltTM Gel Electrophoresis System (Life Technologies) with Tris-Glycine SDS Running Buffer at 165V for 2.5 h and BoltTM 4–12% Bis-Tris Plus Precast Gels (BG04120BOX) to resolve proteins. We used the iBlot® Gel Transfer Device (Life Technologies, IB1001) program P0 (20 V for 1 min, 23 V for 4 min, 25 V for 2 min) to transfer proteins to nitrocellulose membranes (nitrocellulose iBlot® Transfer Stacks, Life Technologies, IB3010-01). The WesternBreeze® Chromogenic Western Blot Immunodetection Kit (WB7105, anti-rabbit) protocol was used to detect proteins. Images of membranes were taken with FluorChem Q using Alpha View Analysis Software. The amino acid sequence of the MsHisClB receptor has a predicted molecular weight of 36 kDa (ExPASy Bioinformatics Resource Portal) and the western blot resulted in a single band at the predicted molecular weight of 36 kDa (Figure ). HA is the primary neurotransmitter of arthropod photoreceptors (; ) and the HisClB receptor is expressed by glial cells in the lamina of Drosophila (). Consistent with this, we observed a band of MsHisClB labeling in the lamina (Figure ). Pre-adsorbing the MsHisClB antibody in a 10:1 antigenic peptide to antibody solution eliminated all labeling (Figure ). Pre-adsorption controls were run concurrently with samples incubated in antibody that had not been pre-absorbed with the antigenic peptide (Figure ), but otherwise treated identically. Scan settings were increased slightly for preadsorbed tissue so that autoflourescence outlined brain structures. Finally, RT-PCR of the insect HisClA showed no band at the predicted height for the receptor (data not shown) suggesting that the MsHisClB receptor is the only HA receptor expressed in AL tissue. […]

Pipeline specifications

Software tools DELTA-BLAST, Clustal Omega
Databases ExPASy
Application Amino acid sequence alignment
Organisms Manduca sexta
Diseases Epilepsy, Temporal Lobe
Chemicals Histamine