Pisum

The assemblies of C. floridanus (version 3.5) and H. saltator (version 3.5) were downloaded from the Hymenoptera Genome Database [89] (link). Protein sequences of reported chemosensory gene were also collected from Apis mellifera, Acyrthosiphon pisum, Drosophila melanogaster, Nasonia vitripennis, L. humile, and P. barbatus[15] (link), [26] (link), [28] , [47] (link), [48] (link), [50] (link), [54] (link). An in-house bioinformatics pipeline was developed to identify candidate chemosensory genes in C. floridanus and H. saltator. First, all collected chemosensory gene sequences were searched against the two ant genomes using TBLASTN [90] (link) with an e-value cutoff of 1e-5. Resulting High-scoring Segment Pairs (HSPs) were sorted by their blast bit-scores, and an average bit-score of the top 75% HSPs were calculated. Any HSPs with a bit-score less than 25% of the average was discarded. Chains of HSPs were than created from retained HSPs. Two HSPs were chained together if the following criteria were met: 1) they are derived from the same query; 2) they are located within 3 kb on the same strand of a scaffold/contig; and 3) the corresponding query region of the upstream HSPs must also be N-terminal to that of the downstream HSPs. The third criterion was applied to avoid artificial concatenation of neighboring chemosensory genes. Genomic regions covered by HSPs chains were considered putative chemosensory gene coding regions. For each putative gene, we then selected the query corresponding to the highest scoring HSPs at that region as reference sequence for homology-based gene prediction using GeneWise (version 2.2.0) [91] (link). All predictions were sorted by ORF length and the lowest 25% was filtered. This pipeline was iterated by adding results of previous run to input until no additional genes were found.
Multiple sequence alignments (MSAs) of predicted OR/GR/IRs were constructed using MUSCLE (version 3.8) [92] (link) and manually inspected. Attempts to improve annotations were made whenever an obvious problem was identified (e.g. missing exon, incorrect exon-exon junction). In addition, in the OR and GR families, we observed many fragmented gene models, likely due to pseudogenization and incomplete genome assembly. For the convenience of subsequent analyses, a minimum size cutoff of 300 amino acids was used for the ORs and GRs. For IRs, we screened all predicted protein sequences with InterProScan (V4.8) [93] (link) and filtered the ones without characteristic domains of IR (PF10613 and PF00060) [26] (link).

To facilitate the annotation of innate immunity genes in insects, we initially created an Insect Immunity Database (IIID) composed of the published immune repertoires of four insect models spanning several different orders: Drosophila melanogaster, Diptera [18] (link), [19] (link), Anopheles gambiae, Diptera [16] (link), [20] (link), Apis mellifera, Hymenoptera [17] (link), [21] (link), and Acrythosiphon pisum, Hemiptera [22] (link). Our criteria for inclusion were that the species have a complete, publicly-available genome sequence, that the innate immune genes have been previously identified in computational or molecular studies, and that each species has an extensive review of its global immune pathways available as a resource. Sequence information was obtained through NCBI for the 105 immunity genes described for Acrythosiphon pisum[22] (link), 317 genes for Anopheles gambiae[20] (link), [23] (link), 379 genes for Drosophila melanogaster[18] (link), [19] (link), and 174 genes for Apis mellifera[17] (link), [21] (link). In total, 975 genes were included in the dataset used to analyze the Nasonia genomes. Each gene was categorized into its primary, secondary and tertiary pathways of putative function (i.e. Toll pathway, IMD pathway, humoral response, JAK/STAT, and cell cycle regulation) and into finite classes of function based upon its putative role in an immune response. Such classes include recognition (identifying potential pathogens and stressors), signaling (communicating between recognition and response), and response (molecules that interact with the pathogen or stressor).

Construction of CWBI-2.3^T-GFP was described previously (Elston et al., 2021 (link)). HT115-GFP was built using pGRG36::PA1-GFP, a plasmid derived from pGRG-36 (Phillips & Cooper, 2021 (link)). To apply this system to HT115, we adapted methods from McKenzie & Craig (2006) (link). The donor E. coli strain, MFDpir, and the recipient HT115 strain were first grown overnight, then washed twice with 145 mM NaCl saline solution, mixed to a 1:2 ratio of donor:recipient, and 50 µL of the mix was spot-plated on LB + DAP agar. After a day of growth, the spot was scraped up and washed with saline as before. Dilutions of these bacteria were plated on LB + Carb agar and grown at 30 °C to select for transconjugants that had acquired the plasmid. Then, colonies were picked and re-grown on LB + Cam agar at 37 °C to select for transposition of the GFP cassette into the chromosome and loss of the temperature-sensitive plasmid. The resulting colonies were picked and regrown at 37 °C in liquid LB with Cam or Carb to confirm loss of the plasmid and successful integration.
The design of our dsRNA expression vector is based on a system for smRNAi in honey bees (Leonard et al., 2020 (link); Lariviere et al., 2022 (link)). In brief, the plasmid has a low to medium copy RSF1010-based origin of replication, spectinomycin resistance, and a dsRNA target sequence flanked by two identical inverted CP25 promoters. Each target for the RNAi experiments was first amplified from A. pisum LSR1 cDNA with “GGA” primers specified in Table S2, then cloned into the dsRNA site on the pBTK800 expression vector (Lariviere et al., 2022 (link)) with BsaI Golden Gate Assembly. The choice of amplified gene fragment was selected to replicate previous publications (Mutti et al., 2006 (link); Vellichirammal et al., 2017 (link); Chung et al., 2018 (link)). Assembled plasmids were conjugated or electroporated into CWBI-2.3^T-GFP and HT115-GFP for aphid feeding as described previously (Elston et al., 2021 (link)), except the dsNuc1-800 plasmid, which was tested in wild-type CWBI-2.3^T and HT115 hosts. Plasmids used in this study are shown in Table S3.

To prepare for injection of EcR dsRNA, colonies of A. pisum LSR1 were reared at low density (5–7 aphids per plant) for at least two generations to decrease colony stress and the numbers of alate adults used in the experiments. Then, 45 10-day-old adult aphids were injected with ~0.1 μL of 2 μg/μL EcR or non-targeting E2-Crimson dsRNA. The injected aphids were allowed to recover overnight in petri dishes, then placed onto fava beans with five aphids per plant. Every 24 h the adults were transferred to fresh plants for a total of 3 days (Vellichirammal et al., 2017 (link)). The offspring were then reared to the 4th instar and scored for wing bud development.