IntroductionThe ability to defend oneself from parasites and pathogens is essential for living organisms. Animals have evolved fundamental defence mechanisms, referred to as innate immune systems, which are often classified into three categories: physical, cellular and humoral immunities1. The humoral immunity consists of recognition of enemies, signal transduction, and production of immune effector molecules such as antimicrobial peptides. Initially, the humoral immune pathways were investigated using the fruit fly Drosophila melanogaster and several other model insects, thereby establishing the classical view that IMD pathway targeting Gram-negative bacteria and Toll pathway targeting Gram-positive bacteria constitute the humoral immune cascades to regulate antimicrobial effector molecules against a variety of microbial parasites and pathogens2,3. Some core components of the innate immune pathways turned out to be shared between insects and mammals, demonstrating that primitive forms of humoral immunity must have arisen in the common ancestor of invertebrates and vertebrates4,5,6,7.However, genomic and functional studies on diverse insects and other invertebrates uncovered that the conventional view of conservation and functional differentiation of IMD pathway and Toll pathway is not always true, which was highlighted, for example, by the genome sequencing of the pea aphid Acyrthosiphon pisum8. In the aphid genome, the majority of IMD pathway genes were not found, demonstrating that the innate immune pathways are not always conserved but sometime drastically modified8,9. Thus far, accumulation of genomic and transcriptomic data of diverse insects and other invertebrates have uncovered variation in innate immune pathways, particularly the IMD pathway, among insects belonging to the order Hemiptera10,11,12.The Hemiptera is a hemimetabolous insect order characterized by a needle-like sucking mouthpart, consisting of such major groups as Sternorrhyncha (aphids, mealybugs, whiteflies, etc.), Auchenorrhyncha (cicadas, leafhoppers, planthoppers, etc.) and Heteroptera (stinkbugs, bedbugs, waterbugs, etc.)13. In the Sternorrhyncha, not only aphids but also whiteflies, mealybugs and psyllids appear to lack not only the immune deficiency (Imd) gene but also several other IMD pathway genes14,15. In the Auchenorrhyncha, by contrast, cicadas, leafhoppers and planthoppers retain Imd gene and most other IMD pathway component genes except Fas-associated protein with Death Domain (FADD)12,16,17. In the Heteroptera, interestingly, presence/absence of Imd gene was polymorphic: Imd gene was identified in the milkweed bug Oncopeltus fasciatus, the water strider Gerris buenoi, the water scorpion Ranatra linearis, the kissing bug Rhodnius prolixus, the brown-winged green stinkbug Plautia stali, and others10,11,12,18,19,20, whereas Imd was not detected in the bedbug Cimex lectularius21,22 or the brown marmorated stinkbug Halyomorpha halys23. These findings highlight that the Heteroptera is an interesting insect group for understanding the diversity and the evolution of innate immune pathways.On the other hand, our previous work showed that, in the stinkbug P. stali, the Imd gene was present and, though its sequence similarity to known insect Imd genes was very low, the gene was certainly functioning in the IMD immune pathway, and other IMD pathway genes such as FADD and Relish were also present, indicating that the IMD pathway is intact and functioning in this stinkbug species10. Notably, however, in the bedbug C. lectularius and the stinkbug H. halys, although Imd gene was not detected, other IMD pathway genes were retained21,23. Here, a question arises: Are C. lectularius and H. halys really devoid of the Imd gene?In this study, we surveyed the genomic data of C. lectularius and H. halys using the Imd gene sequence of P. stali as a query, and identified Imd-like gene sequences in C. lectularius and H. haly. By functional, structural and evolutionary analyses, we demonstrate that these Imd-like genes are certainly functioning in the IMD pathway of the bedbug and the stinkbug, and we discusse how Imd gene has evolved in the diversification of hemipteran insects.Materials and methodsInsectsFor experimental analyses, adult insects of H. halys were collected in Tsukuba, Japan, and used to establish an inbred strain, which were maintained on raw peanuts and water supplemented with 0.05% ascorbic acid in plastic containers. A laboratory strain of C. lectularius was provided by the Japan Environmental Sanitation Center (JESC) and reared on purchased rabbit blood using an artificial feeding system as described24.For molecular phylogenetic and evolutionary analyses, P. stali subspecies stali was obtained from Tsukuba, and the subspecies sakishimensis was obtained from Ishigaki Island, Japan25, and kept in laboratory as described26. These two species were recently classified as two species based on morphological differences in their copulatory organs27. However, based on our observations that they can interbreed and produce fertile offspring, we treated them as subspecies in this paper. Plautia himetyabane (previously known as P. splendens)27 was obtained from Okinawa Island, Japan. Cimex hemipterus were provided by JESC. Cimex japonicus was collected in Centennial Forest Park, Hokkaido, Japan.RNA extractionFor H. halys, P. stali stali, P. stali sakishimaensis, C. lectularius and C. hemipterus, RNA samples were extracted from insects reared in laboratory for several generations. For P. himetyabane and C. japonicus, RNA samples were prepared from field-collected insects. Total RNA samples were extracted from fat bodies for stinkbugs and from whole bodies for bedbugs using Trizol reagent (Invitrogen, Waltham, MA, USA), and then purified by using RNeasy Mini Kit (Qiagen, Hilden, Germany).RNA sequencingLibrary preparation and RNA sequencing were performed using Illumina HiSeq 4000 or 2500 with paired-end 101 bp by Macrogen Japan Corp (Kyoto, Japan). The generated raw reads were deposited to DNA Data Bank of Japan with accession numbers DRR118501–DRR118507 and DRR635863-DRR635867.The obtained reads were de novo assembled using Trinity v2.11.028 after quality-trimming using Trimmomactic v3.629. The protein sequences were deduced using Transdecoder v5.7.130.The sequences of immune-related genes were obtained from these RNA sequencing data of stinkbugs and bedbugs. For Drosophila, the sequences were obtained from Flybase31,32 and National Center for Biotechnology Information (NCBI).RNA interference experimentsTo knockdown the mRNA levels of humoral immunity-related genes (Imd, Relish and MyD88), adult insects within 1 week of ecdysis were injected with 100 ng/µl double-stranded RNA (dsRNA) solution into the ventral septum between thoracic and abdominal segments. The amounts of dsRNA solution injected per individual were 3 µl for H. halys and 1 µl for C. lectularius. dsRNA was synthesized using Ribo Max Large Scale RNA Production System (Promega Madison, USA) from a PCR product using primers listed in Table S1. Three days after dsRNA injection, some insects were subjected to RNA extraction to examine the effects of RNAi. Other insects were injected with peptidoglycan (PGN) of Escherichia coli or Micrococcus luteus (Invivogen, San Diego, CA, USA). The PGN solution was adjusted to 1 ng/µl and injected as described above. One day after PGN injection, the insects were subjected to RNA extraction as described above, but the final purification using RNeasy Mini Kit was not performed. The extracted RNA samples were subjected to quantitative RT-PCR to evaluate the expression levels of antimicrobial peptides (AMPs).Quantitative RT-PCRTotal RNA samples were reverse-transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Life Technologies Japan Ltd, Tokyo, Japan). Quantitative RT-PCR of immunity-related genes and ribosomal protein L32 as an endogenous standard was conducted with primer pairs designed on the basis of RNA sequencing data (Table S1) using LightCycler 480 and LightCycler 480 SYBR Green Master (Roche Diagnostics, Basel, Switzerland).Prediction of protein 3D structureFor each deduced Imd protein sequence, the intrinsically disordered region was predicted by IUPred333. After removing the intrinsically disordered region, the remaining ordered region was subjected to 3D structure modelling using AlphaFold34,35. The results were analyzed in Dali server36,37 and visualized using UCSF Chimera38.Molecular evolutionary analysisBUSTED (Branch-site Unrestricted Statistical Test for Episodic Diversification)39,40, aBSREL (An adaptive branch-site random effects likelihood test for episodic diversification)41,42 implemented in Datamonkey43 were used to evaluate the levels of diversifying selection on DNA and protein sequences.ResultsDetection of Imd-like gene sequences from C. lectularius and H. halysWe conducted BLASTp searches against annotated protein coding gene sequences of the bedbug C. lectularius (accession no. GCA_000648675.3) and the stinkbug H. halys (accession no. GCA_000696795.3), which were reported to be devoid of Imd gene21,23, using Imd gene sequence of the brown-winged green stinkbug P. stali (accession no. LC384129) as a query. Then, we detected Imd-like genes with significant similarity indices from C. lectularius (LOC106664617; e-value 1 × 10− 11) and H. halys (LOC106687624; e-value 2 × 10− 62). LOC106687624 encodes transcript XM_067047805 from H. halys, which corresponds to transcript XM_014431605.1 from LOC106687624 per the Hhal_1.0 annotation as described in the previous study23; expression levels for this transcript are presented in Additional File 2 from that study and are generally consistent across sample types. This transcript also corresponds to GDCO01104976.1 from an earlier RNA-Seq assembly44 and, although the authors explored immune-stimulated genes, because this Imd-like transcript had no compelling matches in NCBI NR at the time, it was not placed among “gold-tier” transcripts and thus not quantitatively interrogated (Michael Sparks, personal communication).RNAi knockdown of the Imd-like genes verified their functionality in the IMD pathway of C. lectularius and H. halysWe hypothesized that Imd-like genes identified in the genomes of C. lectularius and H. halys actually function like the canonical Imd gene. To test this, we conducted RNAi knockdown of the Imd-like genes and examined phenotypic consequences of the gene suppression in C. lectularius and H. halys. The insects were initially injected with dsRNA of Imd gene, then injected with peptidoglycan (PGN) preparations of a Gram negative bacterium (E. coli) or a Gram positive bacterium (M. luteus) 3 days after dsRNA injection, and the next day, RNA extraction and quantitative RT-PCR measurement of expression levels of downstream antimicrobial protein genes as effector molecules. As control RNAi target genes, dsRNA of the following genes were injected instead of dsRNA of Imd gene: EGFP as a negative control gene; Relish as an IMD pathway component gene, and MyD88 as a Toll pathway component gene (see Fig. S1).In C. lectularius, we confirmed that RNAi suppression efficiently worked for the Imd-like gene (LOC106664617), Relish (LOC106667016) and MyD88 (LOC106662592) (Fig. S2). As possible downstream genes, Defensin 1 (LOC106661793), Defensin 2 (LOC106661792)45 and Prolixicin 1 (LOC106664366)46 were examined. We found that, for all the antimicrobial protein genes and irrespective of Gram-negative or Gram-positive PGN injection, RNAi knockdown of Imd-like expression significantly suppressed host’s immune responses upon septic shock, RNAi knockdown of Relish expression also significantly suppressed host’s immune responses upon septic shock (except for Prolixicin 1 by septic shock with M. luteus PGN), and by contrast, RNAi knockdown of MyD88 expression did not suppress host’s immune responses upon septic shock (Fig. 1). These results suggested that, in C. lectularius, (i) the Imd-like gene is certainly functioning as expected for Imd, (ii) the antimicrobial protein genes Defensin 1, Defensin 2 and Prolixicin 1 are under the control of the IMD pathway, (iii) the antimicrobial protein genes may not be under the control of the Toll pathway, and (iv) both Gram-negative and Gram-positive bacteria can elicit antimicrobial protein production via the IMD pathway. The immune response is not much different between Gram-negative and Gram-positive bacteria, as has been shown in the previous paper analyzing AMP expression47. However, these results should be confirmed by further research.Fig. 1Effects of RNAi knockdown of immune pathway component genes on the septic shock-induced upregulation of immune-responsive effector genes in the bedbug C. lectularius. (a) Defensin 1. (b) Defensin 2. (c) Prolixicin 1. The expression levels of the immune effector genes were standardized to the expression levels of ribosomal protein L32 (rpL32). Adult females were injected with PGN of E. coli or M. luteus 3 days after dsRNA injection and subjected to RNA extraction from fat bodies on the following day. Different letters show statistically significant differences (Steel–Dwass test: P 8) are regarded as indicating strong structural resemblance37.Full size tablePhylogenetic relationship of Imd and Imd-like proteinsFigure 5 shows the phylogenetic relationship of the Imd-like proteins of C. lectularius and H. halys together with Imd proteins of diverse insects inferred from alignable 722 amino acid sites. Because of limited data size, bootstrap values supporting nodes were generally low, but the phylogenetic patterns showed that insects of the same order cluster together, plausibly reflecting general conservation of Imd, a core component of the IMD innate immune pathway, in the course of insect evolution.Fig. 5Phylogenetic relationship of Imd proteins of H. halys, C. lectularius and other insects based on deduced amino acid sequences. A maximum-likelihood phylogeny inferred from 722 aligned amino acid positions under the LG + G + I + F model is shown with bootstrap probabilities based on 1000 replications on the nodes. Accession numbers in NCBI or Vector Base (R. prolixus) are indicated in brackets. Insect orders are shown on the right side.Full size imageGenomic location and gene structure of Imd and Imd-like genesFigure S5 shows exon-intron structure and flanking genes of the Imd-like genes of C. lectularius and H. halys in comparison with the Imd genes of the fruit fly D. melanogaster. The number of exons and flanking genes were not conserved among these insects, which may be indicative of high molecular evolutionary activity at Imd and Imd-like gene loci.Positive selection detected in hemipteran Imd-like and Imd genesThe coexistence of structural conservation and sequence divergence in the Imd-like proteins of C. lectularius and H. halys suggested the possibility of accelerated molecular evolution due to positive selection acting on the immunity-related genes of the hemipteran insects. To test this hypothesis, we conducted comparative sequence analyses for five fruit fly species, four stinkbug species and three bedbug species. aBSREL analysis detected significant positive selection in stinkbugs (in P. himechabane; ω1 = 0.00, ω2 = 1790) and bedbugs (in C. japonica; ω1 = 0.00, ω2 = 100000), but not in fruit flies (Fig. S6). BUSTED analysis revealed strong diversifying selection acting on the Death domain sequences in stinkbugs and bedbugs, but not in fruit flies (Fig. S7).Other component genes of IMD pathway in C. lectularius and H. halysUsing the genes of the stinkbug P. stali as a query, we also found FADD and death-related ced-3/Nedd2-like protein (Dredd) in the bedbud C. lectularius and the stinkbug H. halys (Table S3). Further studies such as expression analysis or RNAi effects are needed, but it is likely that the candidate genes found in this study would be functional components of the IMD pathway. These results suggest that these heteropteran insects retain a complete IMD pathway (Fig. 6).Fig. 6A schematic phylogenetic tree of Hemipteran insects on which presence/absence of humoral immunity-related genes is mapped. Black and white squares indicate the presence and absence of humoral immunity-related genes, respectively. The phylogeny was drawn on the basis of the previous studies12,23.Full size imageDiscussionPrevious studies reported that the bedbug C. lectularius and the stinkbug H. halys lack Imd gene in their genomes21,22,23. In this study, however, we identified Imd-like gene in their genomes, and experimentally verified its functionality as Imd gene (Figs. 1 and 2). The reason why Imd gene was overlooked in C. lectularius and H. halys is that the gene sequences are highly divergent from previously reported Imd genes like those of fruit flies – we were able to detect the hemipteran Imd genes by using the Imd gene sequence of the hemipteran stinkbug P. stali as a query. In general, accelerated molecular evolution tends to be observed with genes related to innate immune systems52,53, and in this study, high levels of sequence divergence and positive selection were detected in the Imd genes of the hemipteran insects (Fig. 3; Fig. S6; Fig. S7).In C. lectularius and H. halys, Imd-like genes and other IMD pathway component genes were found in this study. These results suggest that heteropteran insects (stinkbugs, bedbugs, kissingbugs, waterbugs) may generally retain a complete IMD pathway. Although previous studies12,16,17 reported that FADD gene is absent in the Auchenorrhyncha (cicadas, leafhoppers and planthoppers), the possibility cannot be excluded that Auchenorrhyncha actually possesses functional FADD gene but it cannot be recognized due to too divergent nucleotide/protein sequences. Besides the hemipteran insects, absence of Imd and IMD component genes have been reported for the genomes of llouse54 (an insect); of chelicerates, including spiders55, scorpions56 and mites57,58,59,60. Further studies are also anticipated for these cases.While Imd gene sequences were highly divergent among different insect lineages (Fig. 3), 3D structures of the Imd proteins were very similar to each other (Fig. 4). Strikingly, even human’s RIPK1 was structurally similar to Imd proteins of the fruit fly, the bedbug and the stinkbug (Fig. 4). To account for the structural similarity despite the sequence divergence, the possibility of convergent evolution seems unlikely, though not completely excluded, on the ground that the phylogenetic relationship of Imd genes of diverse insects reflected the insect systematic wherein insect species belonging to the same insect order cluster together (Fig. 5). We suppose that shared ancestry and accelerated molecular evolution rather than parallel convergent evolution better account for the evolutionary patterns observed with the Imd genes.Why is the evolutionary rate of Imd gene so high and entailing positive selection? The rapid evolution of immune-related genes is thought to be the evolution of counter response to microbial parasites and pathogens61,62. It is notable that some viruses are known to hijack innate immune pathways of their host insects. For example, Sindbis virus was reported to disrupt the IMD signaling pathway63, and Ank proteins of polydnaviruses were shown to inhibit Relish, a key gene in the IMD pathway64. While the major roles of the innate immune pathways are recognized as defense against bacterial and fungal enemies3, the innate immune pathways were also reported to function against viruses, although the mechanisms are not fully understood65,66.Conclusion and perspectiveIn this study, we focused on the diversity of innate immune pathways in hemipteran insects, uncovered the presence of the cryptic Imd gene in the bedbug and the stinkbug that were overlooked for divergent nucleotide/protein sequences but certainly functioning in the IMD pathway of the insects, and highlighted the limitations of relying on alignment methods using only nucleotide or protein primary structures (i.e., sequences) for identifying “absent/lost genes” among genomic data. As for Imd and other IMD pathway genes of the hemipteran insects, several fundamental issues are still to be addressed. Why do Imd and other IMD pathway genes tend to exhibit divergent nucleotide/protein sequences? Is the nucleotide/protein sequence divergence actually due to accelerated molecular evolution of these immune-related genes? Is the nucleotid/protein sequence divergence relevant to evolutionary arms race against parasites/pathogens? In the Sternorrhyncha including aphids, whiteflies and mealybugs, the reported loss of Imd and other IMD pathway genes may be true, but the possibility of the presence of highly-divergent orthologous genes should be seriously considered. Taken together, the Hemiptera is an interesting insect group for understanding the diversity and the evolution of innate immune pathways, to which future studies should be directed to.Data availabilityThe raw reads were deposited to DNA Data Bank of Japan (DDBJ) with accession numbers DRR118501–DRR118507 and DRR635863-DRR635867. The sequences used in this study are also deposited to DDBJ and shown in the body text and figures.ReferencesGillespie, J. P., Kanost, M. 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Virol. 103, 001741 (2022).CAS Google Scholar Download referencesAcknowledgementsWe thank Masae Takashima and Maria Murakami for technical assistance. H. halys was provided by Dr. Toyomi Kotaki and C. lectularius and C. hemipterus were provided by Japan Environmental Sanitation Center. The sample collection of C. japonicus was supported by Dr. Taro Nojiri and Centennial Forest Park, Kutchan-cho.FundingThis study was supported by the JSPS KAKENHI Grant JP22K19352 to Y.N. and the Japan Science and Technology Agency (JST) ERATO Grant Numbers JPMJER1803 to T.F.Author informationAuthors and AffiliationsInstitute of Agrobiological Sciences Ohwashi, National Agriculture and Food Research Organization (NARO), Tsukuba, 305-8634, JapanYudai Nishide, Daisuke Kageyama & Yuki YoshidaNational Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8566, JapanMinoru Moriyama & Takema FukatsuDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, JapanTakema FukatsuGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305- 8572, JapanTakema FukatsuAuthorsYudai NishideView author publicationsYou can also search for this author inPubMed Google ScholarDaisuke KageyamaView author publicationsYou can also search for this author inPubMed Google ScholarYuki YoshidaView author publicationsYou can also search for this author inPubMed Google ScholarMinoru MoriyamaView author publicationsYou can also search for this author inPubMed Google ScholarTakema FukatsuView author publicationsYou can also search for this author inPubMed Google ScholarContributionsY.N. and Y.Y. performed analysis of 3D structure of Imd. M.M. analyzed RNA sequencing data. Y.N. performed all the other experiments. Y.N. analyzed experimental data. Y.N. wrote the draft manuscript. Y.N., D.K., Y.Y., M.M. and T.F. conceived and designed the experiments. D.K. and T.F. revised the manuscript. All authors discussed the results and contributed to the article writing.Corresponding authorsCorrespondence to Yudai Nishide or Takema Fukatsu.Ethics declarationsCompeting interestsThe authors declare no competing interests.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary materialBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. 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