Gut bacteria induce heterologous immune priming in Rhodnius prolixus encompassing both humoral and cellular immune responses

Gut bacteria induce heterologous immune priming in Rhodnius prolixus encompassing both humoral and cellular immune responses | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Gut bacteria induce heterologous immune priming in Rhodnius prolixus encompassing both humoral and cellular immune responses Carissa A. Gilliland , View ORCID Profile Kevin J. Vogel doi: https://doi.org/10.1101/2025.01.31.635857 Carissa A. Gilliland 1 Entomology Department, The University of Georgia , Athens, GA, 30602 2 Present Address: Department of Entomology, The University of California , Riverside, Riverside, CA,92521 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin J. Vogel 1 Entomology Department, The University of Georgia , Athens, GA, 30602 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kevin J. Vogel For correspondence: kjvogel{at}uga.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Insects lack the adaptive, antibody mediated responses of vertebrates, yet they possess a robust innate immune system capable of defending the host against pathogens. Immune priming has been observed in multiple insect species, wherein exposure to a pathogen provides protection against subsequent infections by the pathogen. Less frequently, heterologous immune priming has been observed where exposure to one bacterial species provides protection against other species. We determined that Rhodococcus rhodnii , a gut symbiont of the kissing bug Rhodnius prolixus, induces a strong heterologous immune priming effect, while axenic bugs lacking any gut bacteria are highly susceptible to pathogens in their hemolymph. Commensal Escherichia coli provides a less robust protective effect than R. rhodnii . R. rhodnii must be alive within the insect as dead bacteria do not stimulate immune priming and pathogen resistance. Removal of R. rhodnii from the gut reduces resistance to pathogens while restoring it to otherwise axenic bugs improves resistance to pathogens, though not completely. R. rhodnii and E. coli activate both the Imd and Toll pathways, indicating cross-activation of the pathways and demonstrating the canonical Drosophila immune response has diverged in Hemiptera. Silencing of either pathway leads to a loss of the protective effect. Several antimicrobial peptides are induced in the fat body by presence of gut bacteria. When E. coli is in the gut, expression of antimicrobial peptides is often higher than when R. rhodnii , though R. rhodnii stimulates proliferation of hemocytes and induce a stronger melanization response. Hemolymph from R. rhodnii bugs has a greater ability to convert the melanin precursor DOPA to melanization products than axenic or E. coli -harboring bugs. These results demonstrate that R. rhodnii’s benefits to its host extend beyond nutritional provisioning, playing an important role in the host immune system. Author Summary Insects often form beneficial relationships with bacteria allowing them to eat nutritionally deficient diets. In insects that only consume blood, symbionts are necessary to provide B vitamins absent in the host diet. There is a growing appreciation that in some of these symbiotic associations, the bacteria provide services beyond nutrition. We show that in kissing bugs, which feed exclusively on vertebrate blood and require bacterial symbionts for development, these symbiotic bacteria are important in activating the insect immune system. Insects with no gut bacteria are highly susceptible to infection and cannot mount an effective immune response. The bacteria reside exclusively in the insect gut yet protect against infections in the rest of the insect’s body. The bacteria must be alive to prime the immune system, and the response is dependent on the species of bacteria in the gut, with symbiotic bacteria providing stronger protection against infection and inducing a broader array of immune responses than commensal bacteria. This study expands our understanding of the role of beneficial bacteria in insect immunity and demonstrates that immune systems differ between major groups of insects. Introduction Though they lack the adaptive, acquired immunity seen in vertebrates, insects possess a robust and well-developed innate immune system [ 1 ] allowing them to mount a strong and specific defense against pathogens [ 2 – 6 ]. An immune response is initiated when an insect pattern recognition receptor recognizes a conserved motif or microbially-associated molecular pattern (MAMP) that is present in microbes but not found in insects [ 7 ]. Pathogen recognition triggers the activation of signal transduction pathways that amplify immune responses and activates immune cascades corresponding to the specific pathogen [ 8 ]. Immune effector mechanisms include production of antimicrobial peptides, phagocytosis, melanization, encapsulation, lysis, and others [ 4 , 8 – 10 ]. Insect immunity is often characterized into two response systems, humoral and cellular, though this division is for the ease of discussion as these systems are largely connected. Humoral defenses consist mainly of soluble effector molecules mainly derived from the fat body, an organ analogous to the vertebrate liver with both nutritional and immunological functions [ 11 – 14 ]. The humoral immune cascade can be activated via three different pathways: the Toll pathway, which recognizes Gram-positive bacteria and fungi, the immune deficiency (Imd) pathway, which recognizes Gram-negative bacteria, and the Jak/Stat pathway, which recognizes viruses [ 4 ]. Cellular defenses are mediated by hemocytes circulating in the hemolymph [ 10 ]. These cells can sequester and kill microbes via phagocytosis, encapsulation, or deposition of toxic melanin (melanization). The insect gut is considered a crucial organ in insect immune defense as it is exposed to environmental microbes, including pathogens, through feeding [ 15 ]. When an insect ingests a pathogen, the immune pathways may be activated and produce different AMPs to maintain gut homeostasis [ 16 ]. In addition to encountering pathogenic bacteria, insects often maintain beneficial associations with microbes they rely upon for successful development and reproduction. Some symbiotic bacteria have been shown to benefit their host through enhancing the immune response to pathogens. Tsetse flies harbor intracellular Wigglesworthia in bacteriocytes which provide essential B vitamins to the host. Flies cleared of Wigglesworthia are more likely to succumb to infection with normally non-pathogenic Escherichia coli compared to their symbiotic counterparts [ 17 ]. In the bean bug, Riptortus pedestris, their symbiont Caballeronia and other commensal bacteria increase host immune competence when challenged with an entomopathogen [ 18 , 19 ]. The microbiota of honeybees, Apis mellifera , have also been shown to be an essential component of the host immune response to pathogens [ 20 ]. The role of bacterial symbionts in the immune responses of kissing bugs (Hemiptera: Reduviidae) has not been well studied. Like all other obligately and exclusively hematophagous arthropods, kissing bugs rely on microbes to provide essential B vitamins that are deficient in vertebrate blood, and these microbes are essential for host development [ 21 , 22 ]. The kissing bug, Rhodnius prolixus, was discovered to house a free-living, Gram-positive bacterium in the lumen of the gut, Rhodococcus rhodnii [ 23 – 25 ]. This bacterium was demonstrated to be beneficial to the host as its removal via surface sterilization of eggs results in increased mortality, increased development time, and failure to reach adulthood [ 22 , 25 , 26 ]. Surveys utilizing 16S rDNA amplicon sequencing of kissing bug gut microbiomes highlight that other microbes are present in wild populations and that Rhodococcus is not ubiquitous across species, especially in the genus Triatoma [ 27 – 37 ]. These studies have shown that kissing bug microbiomes comprise a limited diversity of bacteria that can range from dozens to hundreds of members. Although kissing bugs can house multiple species of bacteria it has been demonstrated that R. rhodnii is necessary and sufficient to support development and reproduction in R. prolixus [ 27 ]. When specifically looking at members of the genus Rhodnius , others have found that R. rhodnii is ubiquitous among the genus, suggesting it is an important co-evolved symbiont of this group [ 38 ]. In addition to R. rhodnii , other bacteria have been found in guts of Rhodnius sp. [ 39 – 41 ] though their specific roles are not known. The diversity of microbes within and amongst these studies suggests that the Rhodnius microbiome contains at least some commensal organisms. Our previous work indicated that non- Rhodococcus species including Escherichia coli could at least temporarily colonize the gut of R. prolixus , and that some proportion of these insects were able to successfully reach adulthood and reproduce, though not as many as insects harboring R. rhodnii [ 1 ]. Insects harboring exclusively E. coli did not exhibit signs of infection, and E. coli remained at a much lower titer in the gut than R. rhodnii . These results suggested that in the gut of R. prolixus , E. coli could exist as a non-pathogenic resident. The observation that R. prolixus can harbor multiple species of bacteria in its gut led us to wonder how the host immune system is impacted by the presence of different microbes in the gut. Using our ability to produce axenic insects (germ free) and those with a defined microbiota (gnotobiotic), we compared the host’s immune response to pathogens in these two states and explored the mechanisms, if any, underlying resistance to pathogens. Our results highlight how symbionts and commensals can play distinct roles in shaping the host immune response. Results The Presence of Bacteria Protects R. prolixus Against Bacterial Infection To investigate the effects of gut bacteria on host immune function, we removed all bacteria through surface sterilization of eggs using our previously described protocol [ 28 ]. Bugs were either reared in axenic conditions (Rpro Axn ), or experimentally infected through a blood meal with E. coli MG1655 (Rpro Ec ), or their symbiont R. rhodnii ATCC 35071 (Rpro Rr ). These bugs were injected with 10 6 CFUs of E. coli , R. rhodnii , the Gram-positive Micrococcus luteus or sterile saline into the hemolymph of unfed 4 th instar nymphs and survival after infection was monitored. Almost all Rpro Axn individuals died within 5 days after injection with either E. coli (9% survival, Fig. 1A ) or M. luteus (17% survival, Fig. 1A ). In contrast, Rpro Rr bugs showed significantly higher survival after E. coli or M. luteus infection (79% and 68% survival, respectively) than Rpro Axn (p < 0.0001 for both pathogens, log-rank test). Rpro Ec bugs had significantly higher survival rates compared to Rpro Axn individuals for both challenges (52% survival, p < 0.0001, and 50% survival, p = 0.003, respectively, log-rank test, Fig. 1A ) and lower survival relative to Rpro Rr challenged with E. coli (p = 0.018, log-rank test) but not when challenged with M. luteus (p = 0.07, log-rank test). To test if the mortality we saw was due to bacterial infection or due to wounding alone, we also injected Rpro Axn , Rpro Ec , and Rpro Rr insects with sterile saline. All insects subject to sterile saline injection had high survival and were not significantly different from each other (p > 0.05, log-rank test, Fig. 1A ). Download figure Open in new tab Figure 1: Gut bacteria promote immune priming against bacterial infection in R. prolixus . (A) Survival curves of Rpro Axn , Rpro Ec , and Rpro Rr after injection with 10 6 CFU of E. coli, M. luteus, or sterile saline into their hemocoel. Rpro Rr and Rpro Ec had significantly higher survival than Rpro Axn regardless of the bacteria used for challenge (p < 0.0001, log-rank test), while there was no difference in survival of bugs injected with sterile saline (p ≥ 0.15, log-rank test) indicating that the presence of gut microbes plays a critical role in insect defense against pathogens. When challenged with E. coli there was a significant difference in survival (26.8%) between Rpro Rr and Rpro Ec (p = 0.018, log-rank test). The difference in survival between Rpro Rr and Rpro Ec was smaller (18%) and approached but did not reach significance when challenged with M. luteus (p = 0.072, log-rank test). Lines connected by different letters are significantly different (p < 0.05, log-rank test). (B) Gnotobiotic R. prolixus limit the growth of E. coli in the hemocoel. Boxplots of E. coli CFUs in R. prolixus hemolymph collected at 1 and 5 DPI. Points represent individual bugs. Rpro Rr bugs had fewer E. coli CFU at both 1 and 5 DPI than Rpro Ec or Rpro Axn . Rpro Ec had fewer E. coli CFU at both 1 and 5 DPI than Rpro Axn (** p < 0.002, * p < 0.05, Wilcoxon test). (C) Hemolymph from Rpro Rr bugs suppresses growth of both E. coli and M. luteus in vitro more than Rpro Ec or Rpro Axn bugs. ***p < 0.001, Tukey’s HSD. Our survival curves indicate that bugs reared with bacteria in their gut can survive infection with E. coli or M. luteus while nearly all Rpro Axn die after infection. Insects can overcome infection through either resistance - killing or clearing of a pathogen – or through tolerance – minimizing fitness costs of infection without reducing the pathogen load. To determine if the observed difference in survival was due to tolerance or resistance, we measured E. coli titer in the hemolymph after infection over time. Because both the E. coli and M. luteus treatment groups had similar mortality rates we chose to further investigate only E. coli for this experiment. We injected bugs with 10 6 CFU of live, kanamycin-resistant E. coli, then collected hemolymph at 1-and 5-days post injection. Hemolymph was spread onto plates containing kanamycin to calculate CFUs of the kanamycin-resistant E. coli present in the hemolymph. The presence of bacteria in the gut had a significant impact on the number of Kan R E. coli hemolymph (F 2,32 = 123.2, p < 0.0001, aligned-rank transformed ANOVA). One day post injection with E. coli , Rpro Rr insects had on average 1.6 x 10 3 CFUs of E. coli , Rpro Ec had 1.82 x 10 4 , and Rpro Axn individuals had 4.6 x 10 5 CFUs, higher than either gnotobiotic treatment (p = 0.002, Wilcoxon signed-rank test, Fig. 1B ). Five days after injection with E. coli most Rpro Axn had died; those who remained alive had on average 4.3 x 10 6 CFUs of E. coli. Rpro Ec bugs had significantly fewer E. coli than Rpro Axn bugs (1.8 x 10 4 CFUs, p = 0.014, Wilcoxon signed-rank test, Fig. 1B ), while Rpro Rr bugs had the least E. coli in their hemolymph of any treatment (2 x 10 2 CFUs, p = 0.014, Wilcoxon signed-rank test). The identity of the gut bacteria influences the extent to which the gnotobiotic bugs can clear the bacteria from their hemolymph, with Rpro Rr bugs having significantly fewer bacteria at day 5 than Rpro Ec bugs (p = 0.014, Wilcoxon signed-rank test). Though neither gnotobiotic treatment completely cleared E. coli from their hemolymph during the observation window, they appear to suppress the pathogen to a level that is survivable. We next sought to determine if the decrease in bacteria in the hemocoel of challenged Rpro Axn , Rpro Ec , and Rpro Rr bugs was due to bacterial killing via antimicrobial factors in the hemolymph. To explore if presence of gut bacteria results in differences in antimicrobial activity in the bugs’ hemolymph, we conducted a zone of inhibition assay [ 42 , 43 ]. We injected Rpro Axn , Rpro Ec , and Rpro Rr 4 th instar bugs with 10 6 cells of either heat-killed E. coli or M. luteus. We used heat-killed bacteria for this assay to avoid bacterial growth on the plates that could potentially confound our zone of inhibition measurements. Rpro Rr and Rpro Ec bugs had significantly larger zones of inhibition when infected with E. coli than Rpro Axn bugs (p < 0.0001, Tukey’s HSD, Fig. 1C ). Interestingly, only Rpro Rr bugs had significantly larger zones of inhibition after being infected with M. luteus (p < 0.0001, Tukey’s HSD, Fig. 1C ). These results indicate that Rpro Rr and Rpro Ec bugs have higher antimicrobial activity in their hemolymph when challenged with Gram-negative E. coli while only Rpro Rr bugs had higher antimicrobial activity after infection with the Gram-positive M. luteus, possibly due to activation of the Toll pathway in Rpro Rr . Naïve individuals did not show a significant difference in antimicrobial activity from each other, suggesting this is an induced response ( Fig. S1 ). These data demonstrate the presence of gut bacteria promotes resistance to pathogens in R. prolixus through a reduction in the number of bacteria in the hemolymph of gnotobiotic bugs but not Rpro Axn bugs. Download figure Open in new tab Figure S1: Zone of inhibition assay from naive insects. No inhibition of growth was observed in any gnoto-biotic background. The Protective Effect of R. rhodnii Requires Live Bacteria and is Lost Upon Removal Insects sense bacteria through detection of MAMPs such as peptidoglycan, lipopolysaccharides, and lipoteichoic acid, which activate signaling cascades via serine proteases. We therefore asked whether the immune priming effect of R. rhodnii was primarily due to the presence of MAMPs, and so we fed Rpro Axn bugs blood meals that were spiked with an amount of heat-killed R. rhodnii that was equivalent to 10 8 CFUs/bug (Rpro Axn+HK Rr ) from the first blood meal until they developed into 4 th instars, then challenged them as before with injection of E. coli . All Rpro Axn+HK Rr bugs died within the 10-day window and their survival was not statistically different from the survival of Rpro Axn bugs (p = 1.00, Cox proportional hazards model, Fig. 2A ). From these results we conclude that live bacteria are necessary for immune priming in this system. Download figure Open in new tab Figure 2: R. prolixus requires live bacteria to mount an effective immune response. Within a panel, lines not connected by the same letter are significantly different (p < 0.001, log-rank test). (A) Survival after bacterial injection of 4 th instar Rpro Axn nymphs fed heat-killed R. rhodnii throughout development (Rpro Axn + HK Rr ) was similar to 4 th instar Rpro Axn nymphs not fed bacteria (p = 0.69, log-rank test). Survival of Rpro Rr was significantly higher (p < 0.0001, log-rank test). (B) Survival after immune challenge of Rpro Axn bugs after restoration of R. rhodnii (Rpro Axn+ Rr ). Restoring R. rhodnii via a blood meal significantly increases survival relative to axenic nymphs (p < 0.0001, log-rank test) but there was still significantly lower survival in the Rpro Axn + Rr than in Rpro Rr (p < 0.0001, log-rank test). (C) Clearance of R. rhodnii from Rpro Rr significantly reduced survival following immune challenge with E. coli . Rpro Rr-KanR bugs treated with kanamycin retained their immune priming effect. Different letters represent significant differences between treatments (p < 0.001, log-rank test). The main function of R. rhodnii is thought to be supplementation of B vitamins to the host [ 22 , 44 ]. These nutrients are important in numerous essential processes in the host and their absence throughout Rpro Axn development may underlie the higher survival of Rpro Rr following immune challenge. Alternatively, the protective effect of R. rhodnii may be independent of its nutrient provisioning services. If the effect were nutritional, we would expect that removal of R. rhodnii from Rpro Rr bugs shortly before an immune challenge would not have a dramatic effect on survival, while if the effect were primarily immune priming, addition of R. rhodnii to Rpro Axn bugs shortly before challenge would lead to increased survival relative to Rpro Axn individuals. To investigate this, we fed 3 rd instar Rpro Axn nymphs a blood meal containing 10 6 CFU/mL of R. rhodnii . Nymphs developed into 4 th instars and a subset were sacrificed and qPCR was used to confirm that sacrificed nymphs harbored R. rhodnii ( Fig. S2 ). Two weeks after molting, insects were injected with live E. coli as previously outlined. Rpro Axn+Rr bugs had higher survival than Rpro Axn bugs ( Fig. 2B , p = 0.0047, Cox proportional hazards) but still suffered increased mortality compared to Rpro Rr individuals. Interestingly, when we removed R. rhodnii from Rpro Rr bugs by feeding kanamycin to 3 rd instar insects via the blood meal (Rpro Rr + Kan), they also suffered from increased mortality compared to bugs that were fed kanamycin but had been inoculated with kanamycin resistant R. rhodnii (Rpro Rr-KanR + Kan, Fig. 2C ). Thus, we conclude that the nutritional role of R. rhodnii in R. prolixus is not sufficient to explain the observed immune priming effects, but that nutrient provisioning may contribute to the protective effects of R. rhodnii . We did not test this in Rpro Ec bugs, as their B vitamin synthesis capabilities are similar to R. rhodnii , and we do not anticipate that nutritional provisioning by E. coli would be able to rescue immune priming when R. rhodnii could not. Download figure Open in new tab Figure S2: R rhodnii titer in Rpro Rr and Rpro Rr KanR insects treated with kanamycin (Rpro Rr + Kan) and Rpro Axn fed R. rhodnii. Titer was determined using qPCR to measure the number of gyrB copies per insect using extractions of whole-body DNA. Immune Priming by Gut Bacteria is Dependent on Both Toll and Imd Pathways Immune responses to bacteria in insects are thought to be mediated by two major pathways, Toll and Imd [ 44 ]. Our zone of inhibition assays suggested that some factor(s) in the hemolymph are involved in suppressing bacterial proliferation, which may be regulated by Toll or Imd. To investigate whether the Toll or Imd pathways are essential to protecting kissing bugs against bacterial infection, we first measured expression of two transcription factors, dorsal and relish , which drive expression of Toll and Imd response genes including antimicrobial peptides (AMPs). For both genes, we assessed expression via qPCR in the fat body of naïve insects and insects that were injected with live E. coli or M. luteus as described above. We tested Rpro Rr , Rpro Ec , and Rpro Axn bugs, and found that naïve, Rpro Axn bugs had low expression of both dorsal and relish ( Fig. 3A, B ). Download figure Open in new tab Figure 3: Immune priming by gut bacteria is dependent on Toll and Imd pathways. (A) Expression of the Toll pathway transcription factor dorsal in Rpro Rr , Rpro Ec , and Rpro Axn in naïve bugs or bugs challenged with E. coli or M. luteus injection. Bars connected by different letters are significantly different (p < 0.05, Tukey’s HSD). (B) Expression of the Imd pathway transcription factor relish in Rpro Rr , Rpro Ec , and Rpro Axn in naïve bugs or bugs challenged with E. coli or M. luteus injection. Bars connected by different letters are significantly different (p < 0.05, Tukey’s HSD). (C) Survival curves of Rpro Rr bugs treated with dsRNA against dorsal, relish , or a control egfp sequence demonstrate that silencing of relish or dorsal via RNAi significantly reduced the survival of Rpro Rr bugs, indicating that these pathways are necessary for R. rhodnii -mediated immune priming. Different letters indicate treatments with significantly different survival (p < 0.0001, log-rank test). (D) Silencing dorsal or relish reduces the bacteriostatic factors in hemolymph of Rpro Rr . Different letters indicate treatments with significantly different survival (p < 0.0001, Tukey’s HSD). For each gene, there was a highly significant effect of the gnotobiotic state of the bugs, the immune challenge, and the interaction between state and challenge (see Table S2 for statistical details). Naïve Rpro Ec bugs had higher expression of dorsal and relish than Rpro Axn , indicating that E. coli in the gut stimulates expression of both transcription factors independent of immune challenge via injection. Naïve Rpro Rr bugs had low expression of dorsal , equivalent to Rpro Axn bugs but had higher expression of relish than Rpro Axn . In bugs challenged with injection of E. coli , both dorsal and relish expression were elevated in their respective gnotobiotic bugs relative to Rpro Axn . M. luteus also stimulated robust expression of relish and dorsal , with Rpro Rr bugs having the strongest expression of dorsal while relish expression was high in both Rpro Rr and Rpro Ec . View this table: View inline View popup Download powerpoint Table SI: Primer sequences used in this study View this table: View inline View popup Download powerpoint Table S2: Statistical details for gene expression experiments From our expression analysis of dorsal and relish , it is clear that there is significant cross-activation of the Toll and Imd pathways, with both Gram-negative and Gram-positive bacteria activating each. The Toll pathway, and by extension dorsal , is thought to primarily respond to Gram-positive bacteria while Imd and relish are thought to be activated by Gram-negative bacteria, but significant crosstalk between these pathways has been observed in other hemipterans [ 45 ]. Absence of gut bacteria does not eliminate the ability of bugs to mount an immune response, but gnotobiotic bugs almost always have higher expression of these genes. In all bugs tested, both dorsal and relish can be induced by the presence of bacteria in the hemolymph, but we were surprised to see that Rpro Ec often induces higher expression of dorsal and relish than Rpro Rr , given the latter’s stronger protective effect. We next asked whether the immune priming effect of gut bacteria is dependent on the Toll or Imd pathway. We suppressed each pathway via RNAi knockdown of the transcription factors relish (Imd) or dorsal (Toll). RNAi of relish or dorsal was confirmed via qPCR revealing over a 90% reduction in dorsal or relish transcripts after injection with dsRNA ( Fig. S3 ). After injection with dsRNA, 10 6 CFUs of E. coli were injected into the bugs hemocoel, and survival rates were measured ( Fig. 3C ). Rpro Rr treated with either dorsal or relish dsRNA succumbed to E. coli infection at a higher rate than Rpro Rr individuals treated with ds egfp (25% and 34% survival respectively, p = 0.002, p = 0.0004, respectively, log-rank test). Download figure Open in new tab Figure S3: qPCR confirmation of dsRNA knockdown of relish and dorsal . RNA was extracted from either gut or fatbody. Asterisks indicate signficiant differences **** p < 0.001, *** p < 0.001, independent contrast test. We then examined the change in antimicrobial activity in relish or dorsal -silenced R. prolixus by comparing the inhibitory effects of hemolymph on microbial growth using the zone of inhibition assay described above, though we only tested the effect of hemolymph from dsRNA-treated bugs against E. coli . There was a significant effect of silencing either relish or dorsal as silenced bugs had smaller zones of inhibitions compared to the control ds egfp -injected group ( Fig. 3D, p < 0.0007, p < 0.0001, Tukey’s HSD). The decrease in survival after challenge with E. coli in either relish or dorsal knockdown bugs suggests immune pathway crosstalk in R. prolixus , as silencing of the Toll pathway transcription factor dorsal led to significantly higher mortality following challenge with Gram-negative bacteria. The reduction in antimicrobial activity in hemolymph following knockdown of dorsal or relish suggests that the activation of the Toll and Imd pathways by R. rhodnii has functional consequences for host immune responses. Expression of Immune-Related Genes is Influenced by the Gut Microbiome Our experiments demonstrated that dorsal and relish were important mediators of the immune priming effect seen in Rpro Rr . We next wanted to see if antimicrobial peptides were upregulated in gnotobiotic insects. To test this, we evaluated the fat body expression profiles of AMPs in 4 th instar Rpro Axn , Rpro Ec , and Rpro Rr bugs that were either uninfected or 1-day post-inoculation with either E. coli or M. luteus . We measured the expression of the AMPs prolixicin , two defensins (RPRC012182 and RPRC012184, subsequently referred to as defensin82 , and defensin84 ), and a lysozyme (RPRC015442 subsequently referred to as lysozyme 42 , Fig. 4 ). Download figure Open in new tab Figure 4: Antimicrobial peptide (AMP) genes are differentially expressed among naïve, Rpro Ec , and Rpro Rr bugs. Within each AMP panel, bars connected by different letters are significantly different (p < 0.05, Tukey’s HSD). The interaction of gnotobiotic state x injection was highly significant for all genes tested (p < 0.0001, aligned ranks transformation ANOVA for non-parametric interactions). Our expression analysis of immune genes revealed several interesting patterns. First, Rpro Axn insects often had lower expression of AMP genes regardless of the immune challenge ( Fig. 5 ), which may be a consequence of lower relish and dorsal expression in Rpro Axn bugs ( Fig. 3A, B ). The overall reduced expression of immune genes may partially explain the heightened susceptibility of Rpro Axn to pathogens. A second, surprising pattern is that Rpro Ec bugs often have higher expression of AMPs than Rpro Rr bugs, despite Rpro Rr having a significantly higher survival rate when challenged with pathogens. In the Rpro Ec bugs challenged with E. coli , expression of all immune genes except defensin84 were significantly higher than when Rpro Rr bugs were challenged with E. coli , indicative of a strong immune priming effect of E. coli in the gut against E. coli in the hemolymph ( Fig. 4 , p < 0.05, Tukey’s HSD). Rpro Rr bugs exhibited strong induction of immune gene expression in response to bacterial challenge relative to naïve Rpro Rr or Rpro Axn , apart from defensin84 , suggesting that humoral immune responses are important in the heterologous immune priming seen in Rpro Rr following pathogen challenge. Download figure Open in new tab Figure 5: Rpro Rr bugs have higher baseline and induced numbers of hemocytes relative to Rpro Ec and Rpro Axn bugs. (A) Hemocyte counts from hemolymph extracted at 0 h post-challenge with E. coli . There was no significant difference in the number of hemocytes between Rpro Axn and Rpro Ec but Rpro Rr had significantly more than either (p = 0.0001, p = 0.02, Tukey’s HSD). (B) At 4h post challenge, all bugs had more hemocytes but Rpro Rr still had significantly more than Rpro Ec or Rpro Axn , which were not significantly different from one another (p = 0.0008, p = 0.001, p = 0.23 respectively, Tukey’s HSD). (C) Fluorescent latex beads are consumed by a hemocyte (arrow) in Rpro Rr . Top panel: light microscopy, middle panel: 395 nm, bottom panel: merge. (D) Hemocytes are essential for R. rhodnii -mediated immune effects. Inactivation of hemocytes by injection of latex beads dramatically reduced survival of Rpro Rr bugs after challenge with E. coli (**** p < 0.0001, log-rank test). The third pattern we observed was further evidence of crosstalk between the Toll and Imd immune pathways, consistent with the earlier experiments examining dorsal and relish ( Fig. 3 ). Defensins, AMPs which act primarily against Gram-positive bacteria [ 46 ], were highly expressed in Rpro Ec in response to challenge with Gram-negative bacteria ( Fig. 5 , defensin 84, 82 ). Likewise, lysozyme42 expression was also induced by Gram-negative bacteria ( Fig. 5 ) despite canonically being considered an AMP against Gram-positive bacteria [ 47 ]. A similar pattern was seen with expression of prolixicin , an ortholog of Drosophila diptericin, as it was induced in Rpro Rr bugs after challenge with M. luteus . This is despite Diptericin having been shown to be active against Gram-negative bacteria [ 48 ]. Our data supports and expands on earlier work in the stink bug Plautia stali which demonstrated that expression of relish and several immune effectors can be induced by both E. coli and M. luteus in Hemiptera [ 45 ]. R. rhodnii Influences the Cellular Immune System Since the microbiome enhanced the humoral immune response and provided protection against bacterial infections, we wondered if gut bacteria-mediated immune priming extended to cellular immune responses. We first measured the number of circulating hemocytes in the hemolymph of Rpro Rr , Rpro Ec , and Rpro Axn bugs. Fourth instar nymphs were immune challenged with E. coli and hemolymph was collected at 0 and 4 hours post-injection and a hemocytometer was used to count the number of hemocytes present. Immediately after injection Rpro Rr bugs had a significantly higher number of circulating hemocytes compared to Rpro Axn bugs (p = 0.0001, Fig 5A ) and Rpro Ec bugs (p = 0.02, Tukey’s HSD) and there was no significant difference in hemocyte counts between Rpro Axn and Rpro Ec (p = 0.11, Tukey’s HSD). These results demonstrate that Rpro Rr bugs have a higher initial number of hemocytes than Rpro Ec or Rpro Axn . Four hours after injection, all hemocyte titers had increased ( Fig. 4B, p < 0.001 for all comparisons, Tukey’s HSD), but Rpro Rr insects still had significantly higher hemocyte counts than both Rpro Axn and Rpro Ec bugs (p = 0.0008 and p = 0.001, respectively, Tukey’s HSD). To investigate whether the differences in hemocyte counts among the treatments was a major factor in infection outcome, we suppressed hemocyte phagocytosis by pre-injecting fluorescent latex beads into the hemocoel of Rpro Rr bugs [ 49 ]. The beads were visualized to be phagocytosed by the hemocytes in vitro ( Fig. 5C ). Twenty-four hours after bead injection, E. coli was injected into the hemolymph and survival was monitored as previously described. Insects with suppressed cellular immunity (Rpro Rr/bead ) succumbed to bacterial infection faster than Rpro Rr/control insects (p < 0.05, log-rank test, Fig. 5D ), indicating that R. rhodnii -mediated cellular immunity plays an important role in immune priming, but that E. coli in the gut does not induce as strong of a cellular response. Melanization and Phenol Oxidase Activity are Dependent on the Presence of R. rhodnii We observed throughout our experiments that Rpro Axn individuals exhibited reduced wound healing. Rpro Axn bugs did not develop a robust, dark melanization scar at injection sites but rather a light, thin scar, while Rpro Rr developed a characteristic think, dark, scar ( Fig. 6A ). Interestingly we also saw a lack of dark, thick scar tissue in Rpro Ec bugs. The dark scars following wounding is due to deposition of melanin produced by the action of phenol oxidases and other enzymes in the hemolymph which convert tyrosine to melanin [ 49 ]. The cascade leading to melanization can be triggered by both wounding and MAMPs [ 50 ]. Based on these observations and the role of melanization in insect immunity, we decided to further investigate differences in melanization by measuring phenol oxidase activity in Rpro Rr , Rpro Ec , and Rpro Axn insects. Download figure Open in new tab Figure 6: R. rhodnii enables successful melanization in R. prolixus. (A) Melanization of wounds is impaired in Rpro Axn and Rpro Ec relative to Rpro Rr . Wound area is outlined. (B) Hemolymph DOPA conversion assay of Rpro Axn , Rpro Ec , and Rpro Rr performed at 1 or 24 h post-challenge with E. coli (circles) or M. luteus (triangles). At 0 h, there was no significant difference in conversion of DOPA to melanin between the bugs or bacterial challenges (F2,44 = 0.11 p = 0.74, ANOVA). At 24 h, there was a significant increase in DOPA conversion in all treatments relative to 0 h (p < 0.05, Tukey’s HSD). Rpro Rr bugs had higher DOPA conversion than Rpro Axn and Rpro Ec bugs (p 0.05, Tukey’s HSD). As with the 0 h time point, there was no significant effect of the bacterial species used in the challenge (F1,44 = 0.21, p = 0.648, ANOVA). (C) Wounding induces activation of the melanization response at 24h post-wounding. Rpro Rr had higher DOPA conversion in response to wounding than Rpro Axn . Bars connected by different letters are significantly different (p < 0.05, Tukey’s HSD). We measured phenol oxidase activity via a dihydroxyphenylalanine (DOPA) conversion assay [ 51 ] to assess how the presence of gut microbes influences melanization. DOPA is a precursor metabolite that is converted into melanin via the action of phenol oxidases. Hemolymph DOPA conversion was measured at 1 and 24 hours post injection with either E. coli or M. luteus . There was a highly significant effect of the gnotobiotic state ( Fig. 6B , F 2,54 = 23.8, p = 3.88 x 10 -8 , ANOVA) and time (F 1,54 = 681, p < 2 x 10 -16 , ANOVA) on the amount of DOPA conversion. Surprisingly, there was not an effect of the microbe injected, as both E. coli and M. luteus injection triggered similar melanization among the different gnotobiotic states ( Fig. 6B , F 3,54 = 0.09, p = 0.96). There was no significant difference in DOPA conversion at 1h post-injection, but at 24 h post-injection there was a significant increase after injection of either E. coli or M. luteus . Compared to Rpro Ec and Rpro Axn , Rpro Rr had greater DOPA conversion regardless of challenge. We also investigated whether a stab wound alone was sufficient to trigger an increase in melanization. We stabbed Rpro Axn and Rpro Rr bugs with a sterile insulin syringe, then collected hemolymph at 1 and 24 h post-stab and measured DOPA conversion as before. We found that similar to injections with E. coli or M. luteus , stabbing with a sterile needle induced the ability to convert DOPA in both Rpro Axn and Rpro Rr hemolymph at 24 h ( Fig. 6C, p < 0.001, Tukey’s HSD) but not at 1 h (p = 0.99, Tukey’s HSD), with a larger increase in conversion seen in the Rpro Rr bugs (p = 0.014, Tukey’s HSD). Our DOPA conversion assays suggest that R. rhodnii is important for successful melanization in R. prolixus . Discussion We demonstrate that the presence of gut microbes is integral to a functional immune system in R. prolixus and key to their ability to overcome infection with facultatively pathogenic microbes. We provide multiple lines of evidence that gut microbes in R. prolixus induce a stronger immune response, or “primes” the immune system by influencing humoral and cellular immunity, and that symbiotic R. rhodnii induce a stronger priming and protective effect than commensal E. coli . Bugs that harbored a microbe in their gut exhibited a strong humoral immune response by expressing significantly higher levels of antimicrobial peptides than microbe-free Rpro Axn individuals, that corresponded to higher antimicrobial activity in their hemolymph, and subsequently an increased ability to reduce the number of infecting microbes compared to Rpro Axn bugs. Our results suggest that different gut microbes prime the immune system in different ways and to different extents. R. rhodnii produces a stronger effect and modulates both cellular and humoral immunity while E. coli mainly stimulates humoral immune factors and does not have as strong of a priming effect. Immune priming has been revealed to be a broadly important facet in insect immunity even though insects lack an adaptive immune system as seen in vertebrates. Despite the lack of antibody-mediated immune memory, in many insect species prior exposure to a nonlethal dose of a pathogen or pathogen derived material results in an elevated immune response which can be seen in the form of increased amount of AMPs and circulating hemocytes [ 52 ]. This elevated immune response results in the insect being resistant to a subsequent infection [ 18 , 53 – 56 ]. Immune priming has primarily been described in scenarios where an insect is exposed to an initial, non-lethal infection followed by subsequent exposure to the initial pathogen, known as homologous immune priming [ 57 ]. More recently, heterologous immune priming, in which one pathogen induces a protective immune response towards a different secondary pathogen has been observed [ 53 ]. Our results suggest that both the Gram-positive symbiotic R. rhodnii and Gram-negative commensal E. coli are capable of stimulating heterologous immune-priming against a different bacterial species, though to varying degrees. Immune priming in insects has been thoroughly examined in several lineages including Drosophila melanogaster [ 58 , 59 ], Aedes aegypti [ 60 ], Anopheles sp. [ 61 – 63 ] , Tenbrio [ 64 ], Gallaria mellonella [ 65 ], Tribolium castaneum [ 66 ] and others. Inoculation of D. melanogaster with sub-lethal doses of Streptococcus pneumoniae led to life-long resistance to subsequent S. pneumoniae infection, which, similar to our observations required Toll activation and was mediated by hemocytes [ 64 ], however dead S. pneumoniae were sufficient to stimulate priming in Drosophila while dead R. rhodnii are not sufficient for R. prolixus priming. In Drosophila and Aedes mosquitos, Wolbachia has been demonstrated to upregulate immune-related genes of hosts which then confers protection against a variety of pathogens [ 67 – 71 ]. The mosquito gut symbiont Asaia has been shown to activate AMP production in the gut of Anopheles stephensi mosquitos [ 72 ]. We also observe an increase in expression of various immune genes when we alter the gut microbiome of kissing bug nymphs, though the identity and degree of expression varies based on the identity of the gut microbe. The repeated evolution of symbiosis between insects and bacteria, in conjunction with diverse pathogenic threats, has resulted in a diverse array of mechanisms underpinning immune priming. In the bean bug, Riptortus pedestris, their gut symbiont Caballeronia strongly contributes to the activation ofs host immunity by influencing AMP activity and hemocyte number [ 65 ]. Other bacteria have been demonstrated to contribute to R. pedestris immune priming. A soil-derived Burkholderia colonizes R. pedestris and escapes the gut where it primes the immune system through influencing AMP expression and hemocyte number. This immune priming directly results in increased survival of Burkholderia -harboring insects compared to bugs who harbor Caballeronia alone [ 68 ]. We did not detect R. rhodnii outside of the R. prolixus gut, suggesting that a different mechanism is at work in kissing bugs. Tsetse flies ( Glossina sp.), which are also obligately exclusively hematophagous, harbor an intracellular symbiont, Wigglesworthia glossinidia . The symbiont is essential for proper immune function in Glossina morsitans , as removal of the symbiont leads to disruption of the immune response and melanization [ 17 , 73 , 74 ]. Like R. prolixus , absence of symbiotic bacteria leads to a reduction in AMP expression, loss of melanization response, and fewer circulating hemocytes. Interestingly, the melanization response appears to be linked to the activity of an odorant binding protein [ 74 ]. The loss of Wigglesworthia reduces expression of a peptidoglycan recognition protein, pgrp-lb , which in turn leads to activation of Imd signaling, while RNAi-mediated suppression of pgrp-lb in flies with their native Wigglesworthia also leads to Imd activation and AMP production, followed by a reduction in Wigglesworthia titers in the fly [ 75 ]. In R. prolixus , we observe higher expression of AMPs in Rpro Rr bugs, and suppression of the Imd or Toll pathway leads to higher mortality after immune challenge, suggesting that while similarities exist between these two systems, the immune priming effect of R. rhodnii in R. prolixus differs in important ways from that of Glossina and Wigglesworthia . Several mechanisms by which symbiotic bacteria protect their hosts from pathogens have been described. Competitive exclusion is one mechanism, as seen in honeybees where their native gut bacteria prevent opportunistic pathogens from colonizing the gut [ 76 ]. Symbionts can also directly interfere with pathogens by producing toxins that act against them [ 77 ]. We doubt this is a plausible mechanism in R. prolixus , as our experiments tested an immune response to bacteria directly injected into the hemocoel, bypassing the gut altogether and preventing direct contact between the microbes. The effect of symbionts on immunity may be indirect, potentially mediated by nutritional factors. Rpro Axn individuals lack symbiont-derived B vitamins, which are important for general homeostasis and may be essential for immune processes. R. prolixus relies on its microbiome to supplement B vitamins, key nutrients that are lacking in vertebrate blood [ 22 , 78 ]. Rpro Axn individuals that were reared in axenic conditions suffer from increased development time, increased mortality, and failure to reach the reproductive adulthood stage [ 22 , 25 , 79 ]. The decrease in fitness seen in Rpro Axn bugs potentially contributes in some way to their compromised immune function. Immune function has been linked to nutrition in many different insects, starving or rearing insects on nutritionally poor diets alters the humoral and cellular immunity leading to increased mortality after infection [ 80 – 82 ]. Diet has been shown to interact with the immune response in R. prolixus as well. Thirty days of starvation post-ecdysis resulted in increased mortality after infection due to changes in the cellular immune system, and similar results were found when bugs were fed an incomplete diet of plasma alone [ 80 ]. All bugs used in our experiments were two weeks post-molt to control for any effects starvation has on survival after infection. We attempted to disentangle the impacts of nutrition on survival by clearing Rpro Rr with a blood meal containing antibiotics. These cleared bugs were confirmed to have no R. rhodnii present and were challenged with E. coli . Interestingly, these bugs suffered high mortality rates, though not as high as Rpro axn bugs, suggesting that R. rhodnii ’ s influence on the immune system is not entirely mediated by nutritional factors. Conversely, axenic individuals that were fed a blood meal containing R. rhodnii at their 3 rd instar then challenged with E. coli shortly after had higher survival than Rpro axn but significantly lower survival than our Rpro Rr group. These results taken together suggest that nutritional supplementation via the microbiome may play some role in immunity, but likely other factors are contributing to this interaction. Direct experiments with B vitamin supplementation and B vitamin auxotrophic R. rhodnii will be necessary to fully resolve the role of symbiont-provisioned nutrients in R. prolixus immunity. Another indirect mechanism of immune priming is the activation of humoral immune responses by gut bacteria. In the current study we demonstrated that the presence of the gut microbiome influenced these induced humoral immune responses. Naïve, unchallenged Rpro Rr individuals had higher expression of both relish and defensin82 compared to Rpro axn individuals, suggesting that the presence of R. rhodnii is inducing an immune response. Interestingly, Rpro Ec individuals had higher expression of all genes tested when compared to Rpro axn bugs and higher expression than Rpro Rr bugs for multiple genes tested. This suggests that different gut microbes activate the humoral immune system, and that some bacteria can induce humoral immunity more than the symbiont R. rhodnii alone. This does not translate to equivalent survival upon pathogen challenge, suggesting that other factors beyond expression of immune genes are playing key roles in symbiont-mediated immune priming. The heterologous priming phenomenon seen in R. prolixus may be due to non-canonical activation or cross-talk between the Imd and Toll pathways. Immune signaling in insects was initially described in Drosophila as a linear response where different classes of pathogens triggered different immune pathways: Gram-negative bacteria activated the Imd pathway, Gram-positive bacteria and fungi activate the Toll pathway [ 83 ]. Yet as insect immune studies have moved beyond Drosophila and into other insects, it appears that crosstalk between immune pathways may be more common than previously thought. Work in the hemipteran stink bug, Plautia stali , revealed that not only are both Imd and Toll pathways present but there is a blurred functional differentiation, as immune challenge with Gram-negative or Gram-positive bacteria elicited expression of immune effector genes of both pathways [ 82 ]. We see similar patterns in our expression data as immune challenge with Gram-positive and Gram-negative trigger expression of both dorsal and relish immune transcription factors belonging to the Toll and Imd pathway respectively. Knockdown of either dorsal or relish also result in increased mortality after challenge with Gram-negative E. coli, and AMPs thought to be active against either Gram-positive or Gram-negative bacteria are expressed in response to both Gram-negative and positive bacteria. Crosstalk has been seen in other insects, including the beetle Tenebrio molitor [ 83 ], and other species of kissing bugs. In Triatoma pallidipennis, silencing of Toll pathway genes led to increased mortality when bugs were challenged with a Gram-negative bacteria [ 84 ]. Interestingly, silencing of relish in T. pallidipennis did not result in increased mortality. Even in Drosophila , gut bacteria can elicit immune priming against heterologous pathogens including fungi [ 84 ] and viruses [ 85 ]. Our results contribute to a growing understanding that Hemipteran immunity seems to function differently than many holometabolous model organisms such as Drosophila . Genomic data has revealed that many hemipteran genomes lack elements of the canonical insect immune pathways, including aphids [ 86 ], bedbugs [ 87 ], and scale insects [ 88 ]. The genome sequence of R. prolixus was initially thought to be missing key components of the Imd pathway [ 89 ], though subsequent analysis has revealed that despite lack of the genes imd and kenny , R. prolixus does indeed possess a functioning Imd pathway [ 90 ]. Our results support these earlier findings, as both R. rhodnii and E. coli stimulate expression of relish and Imd-associated AMPs, while inactivation of Imd signaling through RNAi silencing of relish lead to increased mortality of R. prolixus. The absence of imd and kenny suggests that Imd immune signaling functions differently in R. prolixus and more research is needed to further understand immune signaling in R. prolixus . Most insects possess an acellular chitinous and proteinaceous peritrophic matrix that lines the midgut epithelium and is responsible for protecting the midgut cells from direct contact with gut microbes. This protective barrier modulates immune activation by the gut microbiome [ 91 ]. Hemipterans, including kissing bugs, do not have a peritrophic matrix (PM), but rather a lipid-based structure called the perimicrovillar membrane (PMM). While the PMM forms a barrier between the gut lumen and its resident microbiota, it is possible that the PMM is not as significant a barrier to microbes or MAMPs, and may permit direct contact of gut microbes with the gut epithelia. Such direct contact could potentially activate host immune responses to a greater extent than if bacteria did not contact the epithelial cells directly [ 92 ]. Additional studies are necessary to understand the extent to which gut bacteria or MAMPs in R. prolixus encounter the epithelium, and how this influences immune responses. Kissing bugs are the vectors of Trypanosoma cruzi , the causative agent of Chagas disease. T. cruzi is a stercorarian parasite, residing exclusively in the gut of its triatomine host during the insect phase of its development. As a result, it may be directly or indirectly influenced by the presence of bacteria. The activation of the immune system by gut bacteria may have consequences for T. cruzi persistence and transmission, as has been seen in Anopheles mosquitoes and their gut bacteria [ 93 ], where presence of gut bacteria induces a strong immune priming effect via hemocyte differentiation following ookinete escape from the midgut, and subsequently reduces the survival of Plasmodium in the mosquito. In kissing bugs, the microbiome has been implicated in both interactions with the host immune system as well as antagonistic interactions with T. cruzi [ 94 – 99 ]. Batista et al found similar results to ours, with restoration of R. rhodnii to antibiotic-cleared R. prolixus leading to increased expression of AMPs. Likewise, inactivation of Imd signaling with a pharmacological agent, IMD-0354, lead to increased mortality in R. prolixus due to infection [ 95 ]. These experiments were focused on the outcomes of bacterial infection in the gut, while ours examined infection of the hemocoel. Together they suggest that the immune priming effect of symbiotic bacteria also likely occurs in the gut. Our results support and expand upon these previous studies and provide a framework for further understanding how gut microbiomes influence kissing bug immunity and T. cruzi transmission. Our study provides evidence that the gut microbiome in kissing bugs plays an essential role in activating the host immune system against pathogens in the hemocoel. The nature of the immune priming appears to vary based on the identity of the gut microbe in question, as symbiotic microbes provide a stronger protective effect than non-symbiotic commensals. Surprisingly, both commensal and symbiotic bacteria were able to activate both the Toll and Imd pathways, revealing that our understanding of insect immunity in kissing bugs and possibly other hemipterans, largely based on studies in Drosophila and other holometabolous insects, is not complete. Materials and Methods Insect Maintenance Rhodnius prolixus were obtained from the lab of Dr. Ellen Dotson at the Centers for Disease Control and Prevention through BEI Resources. Insects were reared at 28 °C with a photoperiod of 12 h of light and 12 h of dark and 80% relative humidity. General colony insects were kept in 1 L Nalgene containers and regularly fed defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA) inoculated with R. rhodnii bacteria in the exponential phase of growth through an artificial membrane feeder. Generation of Axenic and Gnotobiotic Nymphs R. prolixus eggs were collected 7 days after being laid then placed in a sterile cell collection basket and washed with 70% ethanol for 5 minutes followed by 3 minutes in 10% povidone-iodine solution, then another 5-minute wash in 70% ethanol, followed by three rinses in autoclaved deionized water. Sterilized eggs were then transferred to autoclaved glass containers enclosed in sterile Nalgene containers with gas-exchange tape covering an air hole. Sterility was validated by screening total genomic DNA from insects with PCR to amplify a 16S rDNA gene with the universal primers 27F and 1492R (supplementary table S1 ). No bands were observed in axenic nymphs. Gnotobiotic nymphs were generated by feeding axenic first instar nymphs a blood meal inoculated with 10 6 CFU/mL of R. rhodnii or E. coli . Nymphs were fed at every instar approximately 2 weeks after molting. Gnotobiotic states were confirmed through qPCR on DNA extracted from whole bodies of nymphs using primers specific to the gyrB sequence of each bacteria (supplementary table S1 ). Bacterial Strains Rhodococcus rhodnii (NRRL B-16535) was obtained from ATCC and grown at 28 °C in liquid Luria-Broth (LB). Escherichia coli MG1655 was a gift of Eric Stabb and was grown in liquid LB at 37 °C. Micrococcus luteus NCTC 2665 bacteria was a gift of Michael Strand and was grown in liquid LB at 37 °C. Bacterial titers were determined by measuring the OD 600 of cultures on a Beckman Coulter DU640 spectrophotometer and then plating out serial dilutions of culture on LB agar plates to correlate OD 600 with Colony Forming Units (CFU) counts. Bacterial Immune Challenge Bacterial immune challenge in kissing bugs was performed on either Rpro axn , Rpro Ec , or Rpro Rr 4 th instar nymphs that were two weeks post molt. Bacteria were injected intrathoracically with 2 μl of 10 8 CFU/ml of an overnight culture. Nymphs were challenged with either R. rhodnii , M. luteus , E. coli , or sterile Aedes saline. Bacteria were collected by centrifugation at 8,000 x g for 5 minutes and resuspended in sterile Aedes saline. A group of nymphs received a stab wound without injection and a group of nymphs were unaltered and left as a naïve treatment group. Twenty-four hours after injection, whole guts and fat body were dissected out in sterile PBS and stored at-80 °C. Real-Time Quantitative PCR (qPCR) For analysis of gene expression, axenic and gnotobiotic individuals’ total RNA was isolated from homogenized tissues using the Direct-zol 96 RNA MagBead Kit (Zymo Research) and KingFisher Apex extraction system. Total RNA was subject to DNAse treatment with the Turbo DNA-free kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Purified, DNased RNA quantification and purity was validated using a NanoDrop spectrophotometer to measure absorbance ratios. One hundred nanograms of RNA was reverse transcribed using iScript Reverse Transcription Supermix (Bio-Rad). qPCR was performed on synthesized cDNA in quadruplicate using the QuantiNova SYBR Green master mix (Qiagen) in a total volume of 20 µl with 0.5 mM of each primer on a Roche LightCycler96 system or a Qiagen Rotor Gene system. For each gene tested, 4 technical replicates and 5 biological replicates were performed. Absolute quantification of genes was performed as previously described [ 94 ] using standard curves of pSCA plasmids containing qPCR products. All qPCR primer pairs had an efficiency of > 0.85. Survival Analysis E. coli , R. rhodnii , and M. luteus were grown overnight at 37 °C in LB broth to OD 600 = 1. Cells were centrifuged at 6,000 rpm and resuspended in sterile Aedes saline to a concentration of 5 x 10 7 CFU/ml. Fourth instar Rpro axn , Rpro Rr , or Rpro Ec nymphs 2 weeks post-molt were injected with 2 µl (10 6 CFU) of either E. coli , R. rhodnii , M. luteus , or sterile saline with a sterile Hamilton syringe using a Micro4 syringe pump controller (World Precision Instruments). Nymphs were placed individually into wells of a sterile 24-well polystyrene cell culture plates for observation and mortality was observed daily for 21 days post-injection. Bacterial Clearance in Hemolymph Bacterial abundance in hemolymph was measured by collecting hemolymph from 4 th instar nymphs after challenge with kanamycin resistant E. coli as described above. All legs were removed with forceps, then an individual was placed inside a sterilized, filtered p1000 pipette tip inserted into a 2 mL microcentrifuge tube, which was then centrifuged at 2000 RCF for 10 min at 15 °C, resulting in the collection of 2-3 µl of hemolymph from an individual. Hemolymph of 4 individuals per treatment was pooled, diluted in 20 µl of sterile PBS, and spread on LB agar plates with 50 µg/ml of kanamycin sulfate, then incubated for 24 h at 37°C and the number of CFUs were counted. For each treatment, three biological replicates were performed. Quantification of Antimicrobial Hemolymph Activity Antimicrobial activity of kissing bug hemolymph was measured using a zone of inhibition assay as described by [ 42 , 43 ]. A culture of M. luteus was grown overnight in LB at 37 °C, then 1 mL of culture was added to 10 mL of sterile, cooled liquid LB agar. The M. luteus -LB agar solution was mixed and poured into Petri plates. After solidifying, 1 mm diameter holes were created in the agar using a sterile glass Pasteur pipette. Hemolymph was collected from individual nymphs as described above, and 1 µl of hemolymph was placed in each hole and the plates were incubated overnight at 37 °C. The diameters of the individual zones of bacterial growth inhibited were measured using an ocular micrometer on a stereo dissecting microscope. Two independent trials were conducted, and each trial consisted of 10 bugs per treatment group. Antibiotic Clearing/Recolonization of R. rhodnii To investigate the impacts on survival of microbiome recolonization, we inoculated 3 rd instar Rpro axn bugs with R. rhodnii (Rpro Axn + Rr ) through a blood meal as previously described. Bugs were allowed to develop to the 4 th instar when the presence of R. rhodnii was confirmed in a subset of bugs via qPCR (Fig. S4). Two weeks after molting bugs were immune challenged with E. coli and survival was monitored as previously described. To determine the effects of removal of R. rhodnii on host immune function, 3 rd instar Rpro Rr were fed a bloodmeal containing either 150 µg/ml of kanamycin or a bloodmeal containing 150 µg/ml of kanamycin along with kanamycin resistant R. rhodnii (Rpro Rr + Kan and Rpro Rr-KanR + Kan). Bugs were confirmed to be removed of R. rhodnii or confirmed to still harbor R. rhodnii via qPCR as described above. All bugs were then allowed to molt to the 4 th instar then immune challenged as previously described and survival was monitored. RNAi-Mediated Immune Suppression PCR Primers containing the minimal T7 promoter sequence were designed to amplify 400-500bp of relish, dorsal, or egfp ( Table S1 ). Total RNA was extracted from the fat bodies of 4 th instar nymphs, DNased, and reverse transcribed as described above. The PCR product was subsequently cloned to the pSCA vector using the Strataclone PCR cloning kit (Agilent). Target DNA was amplified by PCR from isolated plasmid DNA. dsRNA was synthesized using the MEGAscript RNAi kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Synthesized dsRNA was precipitated with sodium acetate and ethanol, then resuspended to 2 μg/μl in Aedes saline. Fourth instar nymphs were injected with 1 μl of dsRNA, then allowed to recover for 1 week before immune challenge as described previously. Knockdown of relish or dorsal was confirmed by qPCR on cDNA extracted from treated nymphs as described previously. Hemocyte Quantification and Inactivation Hemolymph was collected from 4 th instar nymphs that were 2 weeks post-molt. Hemolymph was collected via perfusion of 100 µL of cold Aedes saline injected through the abdomen via insulin syringe. The samples were stored on ice until hemocyte numbers were counted using a Neubauer hemocytometer and an inverted stereo micrscope. Two counts per insect were conducted and the average of the two counts was used for each of 5 individuals per treatment. To reduce hemocyte activity, fluorescent latex microbeads (Polyscience, Fluoresbrite Microspheres 2.00 µm) were diluted to 10 8 beads/μl in Aedes saline and injected into the hemolymph of 4 th instar nymphs that were two weeks post-molt. Beads were confirmed to be engulfed by host hemocytes by observation with an epifluorescence microscope (Leica). Four hours after injection with beads, nymphs were challenged with injection of either E. coli or sterile Aedes saline then monitored for survival as previously described. DOPA Conversion Assay Hemolymph was collected as described above from Rpro axn , Rpro Ec , or Rpro Rr injected with either E. coli or M. luteus . DOPA conversion was measured as described in [ 100 ]. Briefly, 100 µl of perfused hemolymph was suspended into 100 µl of PBS containing 4 mg/ml DOPA, and added to the wells of a sterile 96 well plate. The plate was then incubated at 28 °C for 1 h in a humidified chamber and absorbance was read at 470 nm on a µQuant plate reader (BioTek). Four to six bugs per treatment were tested. Statistical Analyses Statistical analysis of insect survival was determined using a Cox-proportional hazards model followed by a log-rank test for pairwise comparisons via R package survminer and survival . Bacterial clearance was analyzed using a Wilcoxon rank-sum test with a Benjamini-Hochberg correction for multiple comparisons. Statistical analysis of gene expression and hemocyte counts was performed using an aligned-rank transformed ANOVA test followed by a Tukey post-hoc test using the R package ARTool [ 100 ]. DOPA conversion assay data was analyzed via ANOVA and Tukey post-hoc tests. Data files and R scripts used in the study are found in the supplemental online data. Funding This work was funded by National Science Foundation award number 2239595 to KJV. 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