Strong impact of orally administered BCR4 defensin on aphid survival, embryo development and symbiotic cells in three Acyrthosiphon pisum parthenogenetic lines

Strong impact of orally administered BCR4 defensin on aphid survival, embryo development and symbiotic cells in three Acyrthosiphon pisum parthenogenetic lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Strong impact of orally administered BCR4 defensin on aphid survival, embryo development and symbiotic cells in three Acyrthosiphon pisum parthenogenetic lines Hugo Terrasson, Karen Gaget, Garance Lapetoule, Isabelle Rahioui, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5386556/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Journal of Pest Science → Version 1 posted 12 You are reading this latest preprint version Abstract Aphids are major crop pests capable of colonizing the main plants grown for human consumption. They have specialized cells, the bacteriocytes, which house the obligatory symbionts Buchnera aphidicola that provide them with essential nutrients missing from their diet. Bacteriocyte-specific cysteine-rich peptides (BCRs) are encoded by a defensin gene family exclusively present in aphids and specifically expressed in bacteriocyte. One BCR family member, BCR4, has been shown to have insecticidal properties against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). In the present study, we exposed the pea aphid to different doses of BCR4 and examined the impact on aphid survival, mass, anatomy, fecundity, as well as on bacterial symbiosis. As different pea aphid lines with various symbiotic status may be differently affected by stress, we investigated the effect of BCR4 ingestion on three different A. pisum lines: LL01 and YR2-amp, that are mono-infected with B. aphidicola , and YR2- Ri , that is genetically identical to YR2-amp but also contains the extracellular facultative symbionts Regiella insecticola . Our results show a strong dose-response effect of BCR4 on LL01 survival and a more moderate effect on both YR2 lines, while an impact on the mass was observed in the three lines. Histological analyses revealed severe embryonic developmental defects due to the treatment. Finally, BCR4 treatment reduced symbiont quantity, with B. aphidicola being more affected than R. insecticola . This study supports the idea that BCR4 could act as a key regulator of aphid symbiosis and development, and highlights its potential as a candidate bioinsecticide for pest control. Aphididae bacteriocyte-specific cysteine rich peptide bio-insecticidal peptide symbiont pest management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message BCR4 belongs to a new defensin family of peptides whose role in aphid symbiosis remains elusive BCR4 ingestion negatively impacts aphid survival and mass, and alters embryonic development, in a dose-dependent manner. Susceptibility of aphids to BCR4 varies with the genetic background and symbiotic status BCR4 significantly lowers symbiont populations, affecting Buchnera aphidicola to a greater extent than Regiella insecticola . BCR4 is a promising insecticide candidate for aphid management 1. Introduction Aphids are major crop pests, causing global losses estimated at hundreds of millions of dollars each year (Gula 2023 ). They make up to 26% of pests on important food crops in temperate regions, including maize, wheat, potatoes, sugar beets, barleys, and tomatoes (Calevro et al. 2019 ; Dedryver et al. 2010 ). Aphids have also been reported as the most efficient and important vectors of plant viruses, transmitting nearly 300 different virus species (Hogenhout et al. 2008 ), which corresponds to 30% of the total number of plant pathogenic viruses identified to date. Aphids' ecological success is largely due to their symbiotic relationship with the γ-proteobacterium Buchnera aphidicola , an obligate ‘primary’ endosymbiont that provides them with essential nutrients poorly represented in their diet (Wernegreen 2012 , Hansen & Moran 2014 ). This primary symbiont is vertically transmitted and lives within specialized host cells known as bacteriocytes (Baumann, 2005 ) that are the interface of metabolic exchanges (Smith & Moran 2020 ), regulate symbiont populations (Simonet et al. 2018 ) and are involved in their vertical transmission (Koga et al. 2012 ). Aphids can also host various facultative ‘secondary’ endosymbionts, including Spiroplasma , Wolbachia , Rickettsia species, as well as Enterobacteria such as Regiella insecticola , Hamiltonella defensa , and Serratia symbiotica . These secondary symbionts, located in the haemolymph, secondary bacteriocytes and other organs (sheath cells, oenocytes, digestive tract) (Tsuchida et al. 2014 ), can be transmitted both vertically and horizontally (Darby & Douglas, 2003 ; Li et al. 2018 ; Pons et al. 2019a , b ; Renoz et al. 2019 ). They have been associated with benefits like thermal resistance (Montllor et al. 2002 ), protection against natural enemies (Oliver et al. 2003 ; Scarborough et al. 2005 ; Frago et al. 2017 ), and adaptation to different host plants (Renoz 2024 ; Tsuchida et al. 2004 ). Current aphid control strategies rely essentially on chemical treatments, whose extensive use contributes to environmental pollution and the development of insect resistance (Goulson 2018 ). Consequently, there is an urgent need for the development of environmentally friendly alternatives. Small disulfide-rich proteins (DRPs) from plants or arthropods are promising candidates to be used instead of, or in combination with low doses of, chemical insecticides, due to their wide range of biological activities related to host defense, as well as their stability and resistance to enzymatic degradation (Ho et al. 2023 ). A specific class of DRPs, known as bacteriocyte-specific cysteine-rich (BCR) peptides, has been identified in the pea aphid, Acyrthosiphon pisum (Shigenobu & Stern 2013 ). These peptides, encoded by seven orphan genes, are expressed in the bacteriocytes of both embryonic and adult aphids, suggesting a role in the maintenance of bacteriocyte homeostasis and the control of endosymbionts (Shigenobu & Stern 2013 ). Previous research has shown that BCR4, in particular, exhibits insecticidal effects when orally delivered to the A. pisum line LL01, reducing its survival and mass (Loth et al. 2022 ). This investigation aimed to determine which are the developmental and anatomical bases of BCR4 toxicity, whether this toxicity could be explained by a direct effect on symbiotic cells, and whether those vary depending on the aphid genetic background and/or the presence of facultative symbionts. The effects of ingestion of sublethal doses of BCR4 were investigated using a panel of complementary approaches (life history traits, histological and FISH analyses, flow cytometry) applied to three A. pisum lines: LL01, YR2-amp, and YR2- Ri . LL01 and YR2 lines are genetically different. Moreover, the first two lines only harbour the obligatory symbiont B. aphidicola , while the third also hosts the facultative symbiont R. insecticola . 2. Material and Methods 2.1. Aphid lines and rearing All the experiments were performed using is the pea aphid, Acyrthosiphon pisum . Three distinct lines were used: LL01, YR2 -Ri and YR2-Amp. LL01 corresponds to a natural line (Rahbé & Febvay 1993 ) harboring only the obligate primary symbiont B. aphidicola . YR2 -Ri naturally harbors both B. aphidicola and the facultative symbiont R. insecticola (Simon et al. 2011 ). YR2-Amp, was produced from YR2 -Ri line, after treatment with antibiotics to eliminate R. insecticola (Koga et al. 2007 ; Simon et al. 2011 ). The presence of the latter and the absence of other secondary symbionts that may be found in pea aphids ( i.e., Hamiltonella defensa , cd. Fukatsuia symbiotica , Rickettsia sp, Rickettsiella sp, Serratia symbiotica and Spiroplasma sp.) was verified by a PCR-based diagnostic developed by Peccoud et al. ( 2014 ). The three lines were maintained as strictly parthenogenetic matrilines on young broad bean plants ( Vicia faba , L. cv. Aquadulce), with a photoperiod of 16 h light-8 h dark, at 21°C with ~ 60% of humidity. To obtain synchronized aphids, around 100 reproductive females (9 to 11-day- old) per line were left on plants for 24–72 hours (pre-synchronization). The adults were then removed, and newborn nymphs were kept on the plant for 9 days (= adult stage), then transferred to a new plant for 24 hours (synchronization). The resulting synchronized first instar nymph (< 1day old) were then placed on artificial diets containing different concentrations of the BCR4 peptide for the different experiments. The complete experimental design is illustrated in Fig. S1 . 2.2. Synthesis and purification of BCR4 The BCR4 peptide sequence was synthesized and folded according to the optimized procedure described in a previous study (Loth et al. 2022 ). Briefly, BCR4 was generated by native chemical ligation (NCL) of two peptide segments, followed by oxidative folding to form the three disulfide bridges. Both peptide segments were purified using C18 reverse phase (RP) HPLC. The purified peptides were then chemoselectively coupled and purified under standard NCL conditions to yield the pure reduced form of BCR4. This form was then subjected to in vitro oxidative folding followed by purification to homogeneity using C18 RP-HPLC. Analysis of the final folded product by ESI-HRMS confirmed the presence of three disulfide bridges. 2.3. Survival and growth monitoring following BCR4 ingestion To test the effect of BCR4 ingestion on aphid survival and growth, the synthetic peptide was serially diluted in AP3 artificial diet (Rahbé et al. 1988 ; Calatayud 2000 ) at the following concentrations (in µg/ml): 0 (control), 50, 100, 250, 375, 500, 750 and 1000, and prepared in ad hoc feeding chamber as previously described (Rahbé et al. 1988 ). For each aphid line and each concentration, three groups of ten first instar nymphs were collected and fed on independent feeding chambers. Survival was monitored daily for seven days. At the end of the experiment, ten surviving aphids per group were randomly selected and individually weighed on a Mettler AE163 analytical microbalance (Mettler Toledo, Columbus, OH, United States). To confirm the specificity of the observed effect, i.e. that it is due to BCR4, the same experimental design was repeated, using a negative control peptide at the highest concentration only (1000 µg/ml). The peptide used (U 11 ; for details, see Barassé et al. 2023 ) comes from the ant Tetramorium bicarinatum , possesses a disulfide bond and a molecular weight of 4,0 kDa, close to the 5,9 kDa of BCR4. 2.4. Analysis of the effect of BCR4 ingestion on aphid morphology and anatomy To study the effect of BCR4 ingestion on aphid morphology and anatomy, the previous experimental design was repeated with aphids fed either on a control AP3 diet or on a diet supplemented with the dose of 1000 µg/ml of BCR4 for seven days. Dissection and histological preparations were carried out as described below. 2.4.1. Dissection and macroscopic observations After seven days of treatment, at least six aphids per condition and per line were randomly selected, and photographed with a Moticam S6 camera (MoticEurope, Barcelona, Spain) attached to a Leica MZ FLIII stereomicroscope (Leica Microsystems, Wetzkar, Germany) to compare their size, shape and color. They were then dissected in isoosmotic buffer A (0.025 M KCl, 0.01 M MgCl2, 0.25 M Sucrose, and 0.035 M Tris–HCl, pH 7.5) by opening the abdomen with microdissection tweezers (Dumont type 5 Dumoxel; Electron Microscopy Science, Hatfield, UK) as previously described (Ribeiro Lopes et al. 2021 ). The digestive tract and the embryonic chains were isolated and photographed using the same camera. 2.4.2. Aphid section preparation For histological staining and FISH (Fluorescent In-Situ Hybridization) of treated and control aphids, four individuals per condition were randomly selected after seven days of treatment, and sections were prepared as previously described (Ribeiro Lopes et al. 2022 ). Briefly, antennae and legs were removed from aphids with microdissection tweezers, before immersing them in a solution of PBS containing 4% paraformaldehyde and 0.1% Triton X-100 at 4°C for 24 h. Aphids were then transferred in a solution of PBS with 4% paraformaldehyde. After several washing steps in PBS, aphids were embedded in 1.3% agar. The resulting samples were dehydrated through a series of ethanol solutions ranging from 70–100%, and transferred in 1-butanol at 4°C for at least 24 h. In order to prepare aphid sections, aphids were embedded in Paraplast/1-butanol (v/v) for 10 h and then in 100% Paraplast (Paraffine Paraplast Plus Mc Cormick Scientific Dutscher). Resulting Paraplast blocks were sectioned at 3 µm thickness using a HM340E rotary microtome (ThermoScientific, Waltham, MA, United States). Sections were positioned on polylysine coated slides and conserved at 4°C until staining. 2.4.3. Histological preparation and observation Sections were prepared for Hematoxylin and Eosin (H&E) staining with RAL products (RAL Diagnostics, Martillac, France) using a protocol adapted from Sapountzis et al. ( 2014 ). Briefly, sections were deparaffinated in methylcyclohexane (2x 10 min) and rehydrated through an ethanol series to PBS. Nucleus and cytoplasm were stained in Mayer Hematoxylin for 3 min and Eosin for 2 min, respectively, with a water rinse in between. Sections were then washed in water and treated through an ethanol series from 70–100%. After a rapid bath in Diasolv (Diapath, Martinengo, Italia) solution, sections were mounted with Diamount mounting medium (Diapath, Martinengo, Italia). Histological observation was performed under transmitted light, on a Thunder Imager 3D Live Cell™ microscope (Leica, Wetzlar, Germany) with 4X or 20X magnification. 2.4.4. FISH analysis FISH procedure was adapted from Simonet et al. ( 2018 ). Sections were deparaffinated using methylcyclohexane (2x 10 min), followed by two 10 min baths in 100% ethanol, and then air-dried. Deproteinization was conducted with 0.01 M hydrochloric acid and 10 mg/mL pepsin at 37°C for 10 minutes. Sections were rinsed in PBS and dehydrated through a series of ethanol solutions ranging from 70–100%. Prehybridization took place at 45°C for 30 minutes in a prehybridization buffer consisting of 79% hybridization buffer (0.9 M NaCl, 20 mM Tris, 5 mM EDTA, pH 7.2), 20% Denhardt’s solution (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g BSA in 500 mL water), and 1% SDS solution (10% SDS). In situ hybridization was performed by incubating the slides at 45°C for 3 hours, shielded from light, with 5 µl of 100 µM oligonucleotide probes specifically targeting B. aphidicola and R. insecticola 16S rRNAs (Table S1 ). Probes were prepared in prehybridization buffer and their 5' end was labeled with Alexa Fluor 488 and Alexa Fluor 594, respectively. Sections were washed twice in washing solution (99% hybridization buffer and 1% SDS 10%), rinsed in PBS and finally mounted using PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific) containing DAPI (3 µg/mL; Vector Laboratories). Positive fluorescent signals were analyzed using a Thunder Imager 3D Live Cell™ microscope (Leica) equipped with the appropriate emission filters. 2.5. Analysis of the effect of BCR4 ingestion on aphid symbiont The BCR4 bioassay experiment was repeated with only aphids fed either a control AP3 diet or a diet supplemented with of 1000 µg/µl BCR4 for seven days. To assess the impact of the treatment on the quantity of the primary symbiont B. aphidicola and the secondary symbiont R. insecticola , a flow cytometry approach, that proved to be effective to obtain the absolute numbers of bacteria in this model, was used (Simonet et al. 2016 ). For each condition, symbiotic bacteria from 12 whole aphids were purified as previously described (Simonet et al. 2016 ). Aphids were gently crushed with a Potter homogenizer in 3 ml of ice-cold buffer A and the homogenate was consecutively filtered through nylon net filters with 60, 30 and 10 µm pore sizes (Merck Millipore, Tullagreen, Ireland). Following centrifugation (4,000 × g, 5 min, 4°C), the pellet of endosymbiotic bacteria was re-suspended in 200µl for aphids reared on AP3 + BCR4 (1000 µg/ml) medium and 400 µl for aphids reared on AP3 of NaCl 0.85% supplemented with 20% glycerol as a cryoprotective agent, and stored at − 80°C until flow cytometry analysis. Immediately prior to flow cytometry analyses, samples were thawed for 3 min at 42°C, vortexed and diluted to 1:20 with a NaCl 0.85% solution to reach the optimal cell concentration for flow cytometry analysis (< 2,000 events/sec). All solutions were filter sterilized at 0.22 µm prior to use to avoid contamination with exogenous bacteria. For bacterial staining, 0.5 µl of the nucleic acid probe SYTO9 (3.34 mm; Molecular Probes Inc, Eugene, OR, United States) was added to 300 µl of the diluted bacterial suspensions. The mixture was then vortexed and incubated in the dark for 15 min at room temperature, following the manufacturer’s instructions. The stained samples were analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States) equipped with a blue laser (488 nm, air-cooled, 20 mW solid state) and cell green fluorescence was acquired with a photomultiplier tube detector and a 530 nm band-pass filter (503–563 nm). Flow cytometry measurements were run at low flow rate (14 µl/min) and the core stream was allowed to stabilize for 30 s prior to the 2 min acquisition. Cellular data were collected and processed using BD Accuri C6 software (version 1.0.264.21, BD Biosciences). The analyses were performed using logarithmic gains and specific detector settings, adjusted on non-stained samples to eliminate endosymbiont autofluorescence. Then, the endosymbiont population was identified by screening samples on FSC-W vs. FSC-H (Forward Scatter Width vs. Height) and SSC-W vs. SSC-H (Side Scatter Width vs. Height) dot plots. B. aphidicola populations were identified using the same parameter than previously described (Simonet et al. 2016 ). A second population of SYTO9-positive event, of different size and granularity was identified in YR2- Ri samples, and used to count R. insecticola symbionts (Fig. S2 ). For each condition, four biological replicates were processed. 2.6. Statistical analysis Statistical analysis and graphical representations were conducted using the R software (v4.3.1) (R Core Team, 2023 ). R-packages « survival » and « survminer » (Therneau, 2024 ) were used to analyze survival data, comparing curves with a Wilcoxon-Gehan rank test. To estimate the lethal time taken for 20% of the aphid population to die (LT 20 ) when fed on artificial diet containing 1000 µg/µl of BCR4, generalized linear models (glm) with a probit link transformation were fitted for each aphid line and compared with a ratio test, using the R-package “ecotox” (Hlina et al. 2021 ). Dose-response curves of the effect of BCR4 ingestion on aphid mass were fitted with the « drc » package (Ritz et al. 2015 ) using a log-logistic model with 3 parameters (with the lowest limit fixed to 0) and box-cox transformation, allowing to predict 95% confidence intervals (CI 95 %) and the effective dose that affect 50% of the mass compared to control (ED 50 ). For the flow cytometry analyses, the absolute numbers of symbionts obtained for each aphid pool were converted into “symbiont numbers per aphid”. A linear regression model was applied on data. Pairwise comparisons were performed to investigate significant differences between modalities using the eammeans function of the R package “emmeans”. 3. Results 3.1. BCR4 treatment reduces survival and mass of three aphid lines The dose-response effect of BCR4 ingestion on survival was first compared between the three aphid lines LL01, YR2-amp and YR2- Ri . In the absence of BCR4, the three lines showed comparable survival rates on the artificial diet, with a 7-day survival of 93%, 97% and 97% for LL01, YR2-amp and YR2- Ri respectively. Survival of LL01 and YR2-amp lines was significantly affected by BCR4 ingestion (log-rank test: p < 0.001), but not that of YR2- Ri (log-rank test: p = 0.113) (Fig. 1 ). However, both YR2 lines survived better than the LL01 line (log-rank test LL01 vs YR2-amp or YR2- Ri : p < 0.001). Similar results were obtained when focusing solely on the treatment with the highest concentration of BCR4 (1000 µg/µl): (i) the 7-day survival is of 77% and 83% for YR2-amp and YR2- Ri , respectively, and only 50% for LL01; (ii) the estimated time to reach 20% of the mortality (LT 20 ) for LL01 (3.5 days) is significantly different from the LT 20 of both YR2-amp (6.8 days; ratio-test: p = 0.002) and YR2- Ri (8,6 days; ratio-test: p < 0.001). In contrast, no mortality was observed in aphid fed with 1000 µg/ml of U11 peptide (Fig. S3 ). The results show that there is a dose-response effect of BCR4 on the mass of each line (lack-of-fit test: p < 0.05; Fig. 2 a). Feeding on BCR4 had a significant, negative impact on aphids’ mass, with an average reduction of 78%, 65% and 75% for LL01, YR2-amp, and YR2- Ri respectively, compared to the control. The BCR4-concentration that would cause a 50% reduction in aphid mass (ED 50 ) was predicted, revealing a more potent effect on the LL01 line (ED 50 ± SD of 383.8 ± 39.6 µg/ml; 535.3 ± 75.6 µg/ml and 445.2 µg/ml ± 52.1 for LL01, YR2-amp, and YR2- Ri respectively). Discoloration of the aphid cuticle was also observed for each line, with a more noticeable effect on YR2-amp and - Ri compared to LL01 probably due to their more contrasted color (Fig. 2 b-d). 3.2. Impact of BCR4 on the anatomy of aphids and their embryos To assess whether the reduction in mass following BCR4 treatment is accompanied by a change in aphid tissue organization, dissections, histological and FISH analysis of individuals treated with the highest concentration of BCR4 were carried out. 3.2.1. Macroscopic observations Macroscopic observations of dissected aphids revealed no obvious differences between the digestive tracts of control and treated individuals (Fig. S4 ). However, significant differences were observed in the development of embryonic chains (Fig. 3 ). In control aphids, the classic two ovaries, each containing seven ovarioles, are clearly visible. These ovarioles consist of chains of embryos at different developmental stages. In these aphids, the most developed embryos exceed 500 µm in length and are pigmented. The pair of red eyes and the beginning of thorax segmentation are also visible (Fig. 3 ). In contrast, the embryonic chains of treated individuals are more crumbly and fragile. They contain fewer and smaller embryos, with the largest ones reaching only 300 µm. In addition, no coloration, no eyes and no segmentation was noticeable, whatever the aphid line (Fig. 3 ). 3.2.2. Histological analysis with hemalum and eosin staining Histological analysis confirmed the adverse effect of BCR4 on aphid development. In particular, numerous well-developed embryos can be distinguished in sections from control aphids, a developed central nervous system (CNS) and visible limbs (Fig. 4 a’, c’, e’). Bacteriocytes are cellularized and form two lobes located in proximity of the developing digestive tract. While embryos are still visible in treated aphids, they are negatively impacted by the treatment: their organs are undistinguishable and the bacteriocytes, though present, appear acellularized, with no apparent cell membrane (Fig. 4 b’, d’, e’). In all the embryos that could be observed, the bacteriocyte mass was found at the posterior pole, which suggests that these embryos are undergoing a developmental delay. BCR4-ingestion also shows a strong effect on the morphology of the mother’s bacteriocytes (Fig. 4 a”-f”). Although the symbiotic cells are presents in both control and treated aphid, bacteriocytes from the latter are deformed, appearing crushed between organs, and sometimes difficult to distinguish from each other (Fig. 4 b”, d”, e”). In addition, observation of whole insect section suggests a reduction of bacteriocyte number (Fig. 4 a-f). 3.2.3. FISH analyses FISH analyses were carried out to assess the effect of BCR4 on symbiont localization and the structure of bacteriocytes. Observation of control aphids (reared on AP3 artificial diet) shows that the localization of B. aphidicola and R. insecticola is the same as previously described in other aphid lines (Moran et al. 2005 ; Tsuchida et al. 2005 ): B. aphidicola is exclusively found in maternal and embryonic bacteriocytes (Fig. 5 a-c), while R. insecticola is observed extracellularly, in the hemolymph and around the bacteriocytes, and intracellularly, in sheath cells attached to the bacteriocytes, resulting in a signal that completely surrounds the bacteriocyte clusters (Fig. 5 c). After BCR4 treatment, the localization of the primary symbiont is not affected, and it is strictly confined to maternal and embryonic bacteriocytes. Nevertheless, consistently with H&E observations, maternal bacteriocytes appear deformed and less numerous (Fig. 5 a’-c’). Moreover, in embryos, bacteriocytes appear to be packed in the posterior region, contrary to their classical localization around the digestive tract (Fig. 5 d-f). Regarding R. insecticola , the signal is also detected in treated aphids at the same localization as in the control (Fig. 5 f). However, a reduced signal is observed in the hemolymph and around the bacteriocytes and the signal surrounding the bacteriocyte clusters is not continuous as in the controls (Fig. 5 c, f). 3.3. BCR4 significantly reduces symbiont populations To assess whether the observed effect of BCR4 ingestion on bacteriocyte morphology and numbers translates into a change in the density of symbiont populations, a flow cytometry analysis using a single-cell counter cytometer was performed to determine the absolute number of symbionts per aphid (Simonet et al. 2016 ). The mean total number of events corresponding to B. aphidicola decreased drastically from 1.07x10 6 to 1.48x10 5 for LL01, from 6.67x10 5 to 1.82x10 5 for YR2-amp, and from 7.04x10 5 to 1.10x10 5 for YR2 -Ri . This corresponds to reductions of 86%, 73% and 84%, respectively. The BCR4 treatment (F 1,17 = 120.172, p < 0.001) and the interaction between line and treatment (F 2,17 = 4.224, p = 0.03) significantly impact B. aphidicola count while the line alone do not (F 2,17 = 3.327, p = 0.06). The mean total number of events corresponding to R. insecticola in YR2 -Ri line decreased more modestly, from 1.79x10 6 to 7.57x10 5 , a significant reduction of 58% (F 1,5 = 50.336, p < 0.001) (Fig. 6 ). 4. Discussion This study investigated the impact of BCR4 peptide ingestion on three A. pisum lines, LL01, YR2-amp and YR2- Ri , differing in their genetic background (LL01 or YR2) and by the absence (LL01 and YR2-amp) or presence (YR2- Ri ) of the secondary symbiont R. insecticola . Overall, we showed an insecticidal effect of BCR4, with a strong impact on survival, fresh weight, embryo development, bacteriocyte morphology, and symbiont and numbers. Our findings corroborate previous results obtained by Loth et al. 2022 , which showed a dose-response effect of BCR4 on aphid survival and weight. However, the latter study was carried out on a single genetic line (LL01), carrying only the primary symbiont B. aphidicola , whereas several works have shown a significant impact of the genotype on aphid response to biotic stresses, such as host plant changes (Ferrari et al. 2007 ), parasitoid wasp attacks (Libbrecht et al. 2007 ; Martinez et al. 2018 ), or temperature increase (Jahan et al. 2023 ), highlighting the importance of considering different genetic lines. In the present study, we showed a contrasting effect of BCR4 on aphids’ survival depending on their genetic background : at equivalent concentration, LL01 individuals died faster and to a greater extent than YR2-amp and YR2-Ri. Similarly, BCR4 ingestion reduced aphid mass in a dose-dependent manner, with the LL01 line being the most affected. While the insecticidal mode of action of BCR4 is unknown to date, our findings on the impact of BCR4 on aphid survival are similar to previous results about the use of other known antimicrobial peptides on aphids. Indeed, ingestion of indolicidin (an AMP produced by bovine neutrophils) reduces the survival of Myzus persicae (Le-Feuvre et al. 2007 ) and Luna-Ramirez et al. ( 2017 ) showed that ingestion of certain scorpion AMPs exerts insecticidal activity against A. pisum . Presence of R. insecticola had no impact on A. pisum survival, however, the symbiont aggravated the negative impact of BCR4 on aphid weight. Secondary symbionts have been reported to positively or negatively affect aphids’ fitness, depending on the environment or the stress to which the insect is subjected, including xenobiotic exposure (Lemoine et al. 2020 , Zytynska et al. 2021 ). For instance, infection by Serriatia symbiotica in A. pisum (Skaljac et al. 2018 ) and Rickettsia in the whitefly Bemisia tabaci (Kontsedalov et al. 2008 ), is associated with increased susceptibility to chemical insecticides, while the infection by Hamiltonella defensa is associated with reduced susceptibility in the aphid Sitobion miscanthi (Li et al. 2021 ). In A. pisum , infection by R. insecticola is associated with reduced aphid survival during heat stress (Russel and Moran, 2005) or bacterial infection (Luo et al. 2021 ). Conversely, the symbiont confers protection against fungal (Scarborough et al. 2005 ; Parker et al. 2013 ; Parker et al. 2017 ) or viral infections (Higashi et al. 2023 ), indicating that the bacterium could contribute to host defences in different ways. Although symbionts have been reported to increase their hosts’ sensitivity to insecticides (Kontsedalov et al. 2008 , Skaljac et al. 2018 ), the underlying mechanisms remain unknown. Hosting secondary symbionts is a finely-tuned cost-benefit trade-off for aphids, as symbionts are associated with a permanent physiological cost, acting as sinks for metabolites (Zytynska et al. 2021 ). Harboring the R. insecticola symbiont has been shown to negatively impact the host fitness (Sochard et al. 2020; Man et al. 2023 ). Here, the cost of hosting R. insecticola may be exacerbated when A. pisum experiences stressful conditions such as exposure to BCR4. Similarly, specific symbiont combinations may aggravate the whitefly B. tabaci performance on unfavourable host plants (Benhamou et al. 2021 ). The negative impact of BCR4 on survival and weight of A . pisum could be direct, by affecting aphid cells, or indirect, by targeting symbionts and disrupting the obligate nutritional relationship between the insect and its symbiotic bacteria. Concerning the direct cytotoxic effet of BCR4 on aphid cells, it is important to underline that this could not be excluded, as it have been shown that high concentration of certain AMPs could exhert a cytotoxic activity, in vitro , against mammalian cells, by distrupting cell membranes (Vaucher et al. 2009; Greco et al. 2020 ). Nevertheless, Greco et al. 2020 have shown that when inoculated in rats, AMPs lose their cytotoxic potential. Moreover, eukaryotic cells are generally resistant to AMPs. Contrary to prokaryotes, eukaryotic cell membranes contain sterol and mainly zwitteronic phospholipids that stabilize the membrane and confer a protection against AMPs (Mason et al. 2007 ; Anderson et al. 2016; Almeida et al. 2021 ). To gain an in-depth understanding of the effects of BCR4 ingestion on aphids and their symbionts, dissection and histological observations were performed. These analyses revealed a similar and significant negative effect on the embryos of each aphid line. Following treatment, embryonic chains were more fragile and contained smaller and fewer embryos, for which it was impossible to distinguish appendages or organs on histological sections. Histological and FISH observations revealed that the bacteriocytes and the B. aphidicola they contain were mostly grouped at the posterior pole of the embryo, a localization that is usually limited to the early stages of embryo development (Miura et al. 2003 , Braendle et al. 2003 ). This, combined with the lack of bacteriocytes organization in distinct cells, demonstrates at least a delay, if not a disruption, of embryonic development of bacteriocytes morphogenesis. Whereas previous studies using indolicidin (Le-Feuvre et al. 2007 ) or scorpion AMPs (Luna-Ramirez et al. 2017 ) have shown an adverse impact of these peptides on aphid fecundity, our study presents for the first time a disruption of aphid embryonic development caused by the ingestion of an AMP. Moreover, analyses of FISH and H&E staining revealed an alteration in maternal bacteriocytes. While bacteriocytes in the control group are numerous and round-shaped, most bacteriocytes from treated aphids appear fewer and distorted in the three aphid lines. The effect on aphid bacteriocytes is similar to the one observed by Le-Feuvre et al. ( 2007 ) in M. persicae , who reported distortion of bacteriocyte morphology and reduction of bacteriocyte numbers after treatment with indolicidin. With FISH, we also show that the R. insecticola signal appears to be less intense around the bacteriocytes than in the rest of the body. Taken together, the disturbance in the anatomical organization of bacteriocytes and the reduction of R. insecticola signal suggests an effect of BCR4 on both symbiotic populations. To test this hypothesis, we used flow cytometry to get insights in symbiotic bacterial numbers. This showed a massive reduction (72–84%) of B. aphidicola numbers in BCR4-treated aphids. This method also showed that BCR4 has a strong, but lower effect (58%) on R. insecticola numbers. Comparable results have been obtained by Luna-Ramirez et al. ( 2017 ), who showed that ingestion of scorpion AMPs reduces the symbiotic load ( B. aphidicola and Serratia symbiotica) in A. pisum , and argue that insecticidal effects of those AMPs are due to a direct effect on symbionts. Importantly, previous studies have shown that BCR4 is bactericidal in vitro against E. coli , a Gram-negative bacterium closely related to B. aphidicola (Uchi et al. 2019 ; Loth et al. 2022 ). Taken together, these results suggest that BCR4 could similarly have a in vivo bactericidal effect on A. pisum symbiont, thereby having a negative, indirect impact on the growth and survival of the aphid. Interestingly, B. aphidicola symbionts appear to be more sensitive to BCR4 than R. insecticola . Defensins typically target bacterial membranes, often leading to membrane disruption or pore formation (Cociancich et al. 1993 ; Shai 2002 ; Wimley, 2010 ; Wu et al. 2018 ; Agadi et al. 2022 ) and it has been shown that BCR4 alter E. coli morphology and membrane permeability (Uchi et al. 2019 ). It has been demonstrated that the B. aphidicola outer membrane lacks lipopolysaccharides (LPS) (Shigenobu et al. 2000 ; Charles et al. 2011 ), which may explain its greater vulnerability compared to R. insecticola . As for other AMPs, one could propose that BCR4 exhibits a selective activity, being more specific against one symbiont (here B. aphidicola ) than another (here R. insecticola ). For instance, in the bean bug Riptortus pedestris , several AMPs called Crypt-specific Cysteine-Rich peptides (CCRs) are expressed in the specialized posterior midgut region M4, prior to the acquisition of orally transmitted microbiota. This results in a selective barrier against unwanted bacteria, that facilitates the colonization by its beneficial symbiont Caballeronia insecticola (formerly known as Burkholderia insecticola ) that is resistant to host CCRs (Lachat et al. 2024 ). Once established, beneficial C. insecticola becomes vulnerable to host AMPs due to an alteration in its LPS, which allows the host to regulate its gut symbionts (Kim et al. 2015 ). Similarly, in Drosophila , AMPs expressed in the gut have been shown to exhibit high selectivity depending on the invading pathogen (Hanson et al. 2019 ). As an example, the commensal, Lactiplantibacillus plantarum , is resistant to host AMPs during infection phases, allowing it to stably colonize the fly gut (Arias-Rojas et al. 2023 ). Further investigations are needed to explore the potential of orally administered BCRs in selectively targeting different facultative symbionts in A. pisum , and more generally in aphids. 5. Conclusion In conclusion, this study highlights the detrimental effect of BCR4 on aphids when administered orally. Further research is needed to understand the functions of the BCR-family in aphids and their potential use as bioinsecticides. Targeting insect symbionts in the context of pest management is increasingly being discussed (Arora & Douglas 2017 , Gonella et al. 2020 ; Noman et al. 2020 ; Sinno et al. 2020 ; Gonella & Alma 2023 ; Rupawate et al. 2023 ). Combined with advances in plant bioengineering (Bisht et al. 2020; Suhag et al. 2020; Mateos Fernández et al. 2022 ; Komal et al. 2023 ), and because they are restricted to the aphid lineage (Loth et al. 2022 ), BCR delivery could be a powerful tool to specifically control aphid pest through their interference with their symbionts’ populations. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Mélanie Ribeiro-Lopes, Federica Calevro and Pedro Da Silva conceived and designed research. Chrystèle Jouve and Gabrielle Duport prepared the AP3 medium. Catherine Sivignon and Vincent Auchard produces the BCR4 peptide. Hugo Terrasson, Isabelle Rahioui and Chrystèle Jouve conducted bioassays experiments. Hugo Terrasson and Sylvain Benhamou performed the survival and mass monitoring and analyses the data. Hugo Terrasson, Karen Gaget and Garance Lapetoule conducted histological preparations and observations. Hugo Terrasson, Karen Gaget and Garance Lapetoule conducted dissections and macroscopical observations. Karen Gaget and Garance Lapetoule performed flow cytometry experiments and Karen Gaget analyses the data. Jean-Christophe Simon provide both YR2 A. pisum lines. Hugo Terrasson prepared the figures and wrote the first draft. François Renoz, Sylvain Benhamou, Jean-Christophe Simon Mélanie Ribeiro-Lopes Federica Calvero and Pedro Da Silva reviewed and corrected the draft. All authors read and approved the manuscript. Consent to participate Not applicable. Consent for publication Not applicable. Ethics approval No international, national and institutional guidelines for the care and use of animals were needed and followed. Funding This work was supported by INSA Lyon (Institut National des Sciences Appliquées de Lyon), INRAE (Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement) and the French ANR BIOFAMILY project (ANR-19-CE11-0004). Author Contribution M.R.L., F.C. and P.D.S. conceived and designed research. C.J. and G.D. prepared the AP3 medium. C.S. and V.A. produces the BCR4 peptide. H.T., I.R., and C.J. conducted bioassays experiments. H.T. and S.B. performed the survival and mass monitoring and analyses the data. H.T., K.G. and G.L. conducted histological preparations and observations. H.T., K.G. and G.L. conducted dissections and macroscopical observations. K.G. and G.L. performed flow cytometry experiments and K.G. analyses the data. J.C.S. provide both YR2 A. pisum lines. H.T. prepared the figures and wrote the first draft F.R., S.B., J.C.S., M.R.L., F.C. and P.D.S. reviewed and corrected the draft. All authors read and approved the manuscript. Acknowledgement Authors would like to thank Hubert Charles for his suggestions about statistical analysis, and Aurélie Herbomez for secretarial assistance. This work was supported by INSA Lyon (Institut National des Sciences Appliquées de Lyon), INRAE (Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement) and the French ANR BIOFAMILY (ANR-19-CE11-0004) program grant. Data availability The data supporting the findings of this study are available in the supplementary materials References Agadi N, Maity A, Jha AK, et al (2022) Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides. Biochimica et Biophysica Acta (BBA) - Biomembranes 1864:184047. https://doi.org/10.1016/j.bbamem.2022.184047 Almeida CV, de Oliveira CFR, dos Santos EL, et al (2021) Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes. Biochimica et Biophysica Acta (BBA) - General Subjects 1865:129937. https://doi.org/10.1016/j.bbagen.2021.129937 Andersson DI, Hughes D, Kubicek-Sutherland JZ (2016) Mechanisms and consequences of bacterial resistance to antimicrobial peptides. Drug Resistance Updates 26:43–57. https://doi.org/10.1016/j.drup.2016.04.002 Arias-Rojas A, Frahm D, Hurwitz R, et al (2023) Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila . Proceedings of the National Academy of Sciences 120. https://doi.org/10.1073/pnas.2305649120 Arora AK, Douglas AE (2017) Hype or opportunity? Using microbial symbionts in novel strategies for insect pest control. Journal of Insect Physiology 103:10–17. https://doi.org/10.1016/j.jinsphys.2017.09.011 Barassé V, Jouvensal L, Boy G, et al (2023) Discovery of an insect neuroactive helix ring peptide from ant venom. Toxins 15:600. https://doi.org/10.3390/toxins15100600 Baumann P (2005) Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annual Review of Microbiology 59:155–189. https://doi.org/10.1146/annurev.micro.59.030804.121041 Benhamou S, Rahioui I, Henri H, et al (2021) Cytotype affects the capability of the whitefly Bemisia tabaci MED species to feed and oviposit on an unfavorable host plant. mBio 12:e0073021. https://doi.org/10.1128/mBio.00730-21 Bisht DS, Bhatia V, Bhattacharya R (2019) Improving plant-resistance to insect-pests and pathogens: the new opportunities through targeted genome editing. Seminars in Cell & Developmental Biology 96:65–76. https://doi.org/10.1016/j.semcdb.2019.04.008 Braendle C, Miura T, Bickel R, et al (2003) Developmental origin and evolution of bacteriocytes in the aphid– Buchnera symbiosis. PLOS Biology 1. https://doi.org/10.1371/journal.pbio.0000021 Calatayud P-A (2000) Influence of linamarin and rutin on biological performances of Phenacoccus manihoti in artificial diets. Entomologia Experimentalis et Applicata 96:81–86. https://doi.org/10.1046/j.1570-7458.2000.00681.x Calevro F, Tagu D, Callaerts P (2019) Acyrthosiphon pisum . Trends in Genetics 35:781–782. https://doi.org/10.1016/j.tig.2019.07.003 Charles H, Balmand S, Lamelas A, et al (2011) A genomic reappraisal of symbiotic function in the aphid/ Buchnera symbiosis: reduced transporter sets and variable membrane organisations. PLOS ONE 6. https://doi.org/10.1371/journal.pone.0029096 Cociancich S, Ghazi A, Hetru C, et al (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus . Journal of Biological Chemistry 268:19239–19245. https://doi.org/10.1016/S0021-9258(19)36505-6 Darby AC, Douglas AE (2003) Elucidation of the transmission patterns of an insect-borne bacterium. Applied and Environmental Microbiology 69:4403–4407. https://doi.org/10.1128/AEM.69.8.4403-4407.2003 Dedryver C-A, Le Ralec A, Fabre F (2010) The conflicting relationships between aphids and men: a review of aphid damage and control strategies. Comptes Rendus Biologies 333:539–553. https://doi.org/10.1016/j.crvi.2010.03.009 Febvay G, Delobel B, Rahbé Y (1988) Influence of the amino acid balance on the improvement of an artificial diet for a biotype of Acyrthosiphon pisum (Homoptera: Aphididae). Can J Zool 66:2449–2453. https://doi.org/10.1139/z88-362 Ferrari J, Scarborough CL, Godfray HCJ (2007) Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids. Oecologia 153:323–329. https://doi.org/10.1007/s00442-007-0730-2 Frago E, Mala M, Weldegergis BT, et al (2017) Symbionts protect aphids from parasitic wasps by attenuating herbivore-induced plant volatiles. Nat Commun 8:1860. https://doi.org/10.1038/s41467-017-01935-0 Gonella E, Alma A (2023) The role of symbiont-targeted strategies in the management of Pentatomidae and Tephritidae pests under an integrated vision. Agronomy 13:868. https://doi.org/10.3390/agronomy13030868 Gonella E, Orrù B, Marasco R, et al (2020) Disruption of host-symbiont associations for the symbiotic control and management of pentatomid agricultural pests—a review. Front Microbiol 11:547031. https://doi.org/10.3389/fmicb.2020.547031 Goulson D, 232 signatories (2018) Call to restrict neonicotinoids. Science 360:973–973. https://doi.org/10.1126/science.aau0432 Greco I, Molchanova N, Holmedal E, et al (2020) Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides. Sci Rep 10:13206. https://doi.org/10.1038/s41598-020-69995-9 Gula LT (2023) Researchers helping protect crops from pests. NIFA. https://www.nifa.usda.gov/about-nifa/blogs/researchers-helping-protect-crops-pests . Accessed 26 Jun 2024 Hansen AK, Moran NA (2014) The impact of microbial symbionts on host plant utilization by herbivorous insects. Molecular Ecology 23:1473–1496. https://doi.org/10.1111/mec.12421 Hanson MA, Dostálová A, Ceroni C, et al (2019) Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach. eLife 8. https://doi.org/10.7554/eLife.44341 Higashi CHV, Nichols WL, Chevignon G, et al (2023) An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen. Molecular Ecology 32:936–950. https://doi.org/10.1111/mec.16801 Hlina BL, Birceanu O, Robinson CS, et al (2021) The relationship between thermal physiology and lampricide sensitivity in larval sea lamprey ( Petromyzon marinus ). Journal of Great Lakes Research 47–S284. https://doi.org/10.1016/j.jglr.2021.10.002 Ho TNT, Turner A, Pham SH, et al (2023) Cysteine-rich peptides: from bioactivity to bioinsecticide applications. Toxicon 230:107173. https://doi.org/10.1016/j.toxicon.2023.107173 Hogenhout SA, Ammar E-D, Whitfield AE, Redinbaugh MG (2008) Insect vector interactions with persistently transmitted viruses. Annual Review of Phytopathology 46:327–359. https://doi.org/10.1146/annurev.phyto.022508.092135 Jahan H, Khudr MS, Arafeh A, Hager R (2023) Exposure to heat stress leads to striking clone-specific nymph deformity in pea aphid. PLOS ONE 18. https://doi.org/10.1371/journal.pone.0282449 Kim JK, Son DW, Kim C-H, et al (2015) Insect gut symbiont susceptibility to host antimicrobial peptides caused by alteration of the bacterial cell envelope. Journal of Biological Chemistry 290:21042–21053. https://doi.org/10.1074/jbc.M115.651158 Koga R, Tsuchida T, Sakurai M, Fukatsu T (2007) Selective elimination of aphid endosymbionts: effects of antibiotic dose and host genotype, and fitness consequences. FEMS Microbiology Ecology 60:229–239. https://doi.org/10.1111/j.1574-6941.2007.00284.x Koga R, Meng X-Y, Tsuchida T, Fukatsu T (2012) Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte–embryo interface. Proceedings of the National Academy of Sciences 109:E1230–E1237. https://doi.org/10.1073/pnas.1119212109 Komal J, Desai HR, Samal I, et al (2023) Unveiling the genetic symphony: harnessing CRISPR-Cas genome editing for effective insect pest management. Plants 12:3961. https://doi.org/10.3390/plants12233961 Kontsedalov S, Zchori-Fein E, Chiel E, et al (2008) The presence of Rickettsia is associated with increased susceptibility of Bemisia tabaci (Homoptera: Aleyrodidae) to insecticides. Pest Management Science 64:789–792. https://doi.org/10.1002/ps.1595 Lachat J, Lextrait G, Jouan R, et al (2024) Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts. Proceedings of the National Academy of Sciences 121. https://doi.org/10.1073/pnas.2401802121 Le-Feuvre RR, Ramírez CC, Olea N, Meza-Basso L (2007) Effect of the antimicrobial peptide indolicidin on the green peach aphid Myzus persicae (Sulzer). Journal of Applied Entomology 131:71–75. https://doi.org/10.1111/j.1439-0418.2006.01117.x Lemoine MM, Engl T, Kaltenpoth M (2020) Microbial symbionts expanding or constraining abiotic niche space in insects. Current Opinion in Insect Science 39:14–20. https://doi.org/10.1016/j.cois.2020.01.003 Li Q, Fan J, Sun J, et al (2018) Plant-mediated horizontal transmission of Hamiltonella defensa in the wheat aphid Sitobion miscanthi . J Agric Food Chem 66:13367–13377. https://doi.org/10.1021/acs.jafc.8b04828 Li Q, Sun J, Qin Y, et al (2021) Reduced insecticide susceptibility of the wheat aphid after infection by the secondary bacterial symbiont Hamiltonella defensa . Pest Management Science 77:1936–1944. https://doi.org/10.1002/ps.6221 Libbrecht R, Gwynn DM, Fellowes MDE (2007) Aphidius ervi preferentially attacks the green morph of the pea aphid, Acyrthosiphon pisum . J Insect Behav 20:25–32. https://doi.org/10.1007/s10905-006-9055-y Loth K, Parisot N, Paquet F, et al (2022) Aphid BCR4 structure and activity uncover a new defensin peptide superfamily. International Journal of Molecular Sciences 23:12480. https://doi.org/10.3390/ijms232012480 Luna-Ramirez K, Skaljac M, Grotmann J, et al (2017) Orally delivered scorpion antimicrobial peptides exhibit activity against pea aphid ( Acyrthosiphon pisum ) and its bacterial symbionts. Toxins 9:261. https://doi.org/10.3390/toxins9090261 Luo C, Belghazi M, Schmitz A, et al (2021) Hosting certain facultative symbionts modulates the phenoloxidase activity and immune response of the pea aphid Acyrthosiphon pisum . Insect Science 28:1780–1799. https://doi.org/10.1111/1744-7917.12888 Man Y, Li D, Wang M, et al (2023) Indirect and direct interactions between grain aphid and parasitoid in the presence of symbiont Regiella insecticola . CABI Agriculture and Bioscience 4:59. https://doi.org/10.1186/s43170-023-00202-1 Mason AJ, Marquette A, Bechinger B (2007) Zwitterionic phospholipids and sterols modulate antimicrobial peptide-induced membrane destabilization. Biophysical Journal 93:4289–4299. https://doi.org/10.1529/biophysj.107.116681 Martinez AJ, Doremus MR, Kraft LJ, et al (2018) Multi-modal defences in aphids offer redundant protection and increased costs likely impeding a protective mutualism. Journal of Animal Ecology 87:464–477. https://doi.org/10.1111/1365-2656.12675 Mateos Fernández R, Petek M, Gerasymenko I, et al (2022) Insect pest management in the age of synthetic biology. Plant Biotechnology Journal 20:25–36. https://doi.org/10.1111/pbi.13685 Miura T, Braendle C, Shingleton A, et al (2003) A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 295B:59–81. https://doi.org/10.1002/jez.b.3 Montllor CB, Maxmen A, Purcell AH (2002) Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress. Ecological Entomology 27:189–195. https://doi.org/10.1046/j.1365-2311.2002.00393.x Moran NA, Russell JA, Koga R, Fukatsu T (2005) Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Applied and Environmental Microbiology 71:3302–3310. https://doi.org/10.1128/AEM.71.6.3302-3310.2005 Noman MS, Liu L, Bai Z, Li Z (2020) Tephritidae bacterial symbionts: potentials for pest management. Bulletin of Entomological Research 110:1–14. https://doi.org/10.1017/S0007485319000403 Oliver KM, Russell JA, Moran NA, Hunter MS (2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proceedings of the National Academy of Sciences 100:1803–1807. https://doi.org/10.1073/pnas.0335320100 Parker BJ, Hrček J, McLean AHC, Godfray HCJ (2017) Genotype specificity among hosts, pathogens, and beneficial microbes influences the strength of symbiont-mediated protection. Evolution 71:1222–1231. https://doi.org/10.1111/evo.13216 Parker BJ, Spragg CJ, Altincicek B, Gerardo NM (2013) Symbiont-mediated protection against fungal pathogens in pea aphids: a role for pathogen specificity? Applied and Environmental Microbiology 79:2455–2458. https://doi.org/10.1128/AEM.03193-12 Peccoud J, Bonhomme J, Mahéo F, et al (2014) Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes. Insect Science 21:291–300. https://doi.org/10.1111/1744-7917.12083 Pons I, Renoz F, Noël C, Hance T (2019a) Circulation of the cultivable symbiont Serratia symbiotica in aphids is mediated by plants. Front Microbiol 10:. https://doi.org/10.3389/fmicb.2019.00764 Pons I, Renoz F, Noël C, Hance T (2019b) New insights into the nature of symbiotic associations in aphids: infection process, biological effects, and transmission mode of cultivable Serratia symbiotica bacteria. Applied and Environmental Microbiology 85. https://doi.org/10.1128/AEM.02445-18 R Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/ Rahbé Y, Febvay G (1993) Protein toxicity to aphids: an in vitro test on Acyrthosiphon pisum . Entomologia Experimentalis et Applicata 67:149–160. https://doi.org/10.1111/j.1570-7458.1993.tb01663.x Rahbé Y, Febvay G, Delobel B, Bournoville R (1988) Acyrthosiphon pisum performance in response to the sugar and amino acid composition of artificial diets, and its relation to lucerne varietal resistance. Entomologia Experimentalis et Applicata 48:283–292. https://doi.org/10.1111/j.1570-7458.1988.tb01175.x Renoz F (2024) The nutritional dimension of facultative bacterial symbiosis in aphids: current status and methodological considerations for future research. Current Research in Insect Science 5:100070. https://doi.org/10.1016/j.cris.2023.100070 Renoz F, Pons I, Vanderpoorten A, et al (2019) Evidence for gut-associated Serratia symbiotica in wild aphids and ants provides new perspectives on the evolution of bacterial mutualism in insects. Microb Ecol 78:159–169. https://doi.org/10.1007/s00248-018-1265-2 Ribeiro Lopes M, Gaget K, Renoz F, et al (2022) Bacteriocyte plasticity in pea aphids facing amino acid stress or starvation during development. Front Physiol 13:. https://doi.org/10.3389/fphys.2022.982920 Ribeiro Lopes M, Simonet P, Duport G, et al (2021) Isolation of insect bacteriocytes as a platform for transcriptomic analyses. Methods Mol Biol 2170:185–198. https://doi.org/10.1007/978-1-0716-0743-5_13 Ritz C, Baty F, Streibig JC, Gerhard D (2015) Dose-response analysis using R. PLOS ONE 10. https://doi.org/10.1371/journal.pone.0146021 Rupawate PS, Roylawar P, Khandagale K, et al (2023) Role of gut symbionts of insect pests: a novel target for insect-pest control. Front Microbiol 14:. https://doi.org/10.3389/fmicb.2023.1146390 Russell JA, Moran NA (2005) Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures. Proceedings of the Royal Society B: Biological Sciences 273:603–610. https://doi.org/10.1098/rspb.2005.3348 Sapountzis P, Duport G, Balmand S, et al (2014) New insight into the RNA interference response against cathepsin-L gene in the pea aphid, Acyrthosiphon pisum : molting or gut phenotypes specifically induced by injection or feeding treatments. Insect Biochemistry and Molecular Biology 51:20–32. https://doi.org/10.1016/j.ibmb.2014.05.005 Scarborough CL, Ferrari J, Godfray HCJ (2005) Aphid protected from pathogen by endosymbiont. Science 310:1781–1781. https://doi.org/10.1126/science.1120180 Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Peptide Science 66:236–248. https://doi.org/10.1002/bip.10260 Shigenobu S, Stern DL (2013) Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont. Proceedings of the Royal Society B: Biological Sciences 280:20121952. https://doi.org/10.1098/rspb.2012.1952 Shigenobu S, Watanabe H, Hattori M, et al (2000) Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407:81–86. https://doi.org/10.1038/35024074 Simon J-C, Boutin S, Tsuchida T, et al (2011) Facultative symbiont infections affect aphid reproduction. PLOS ONE 6. https://doi.org/10.1371/journal.pone.0021831 Simonet P, Duport G, Gaget K, et al (2016) Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis. Sci Rep 6:19967. https://doi.org/10.1038/srep19967 Simonet P, Gaget K, Balmand S, et al (2018) Bacteriocyte cell death in the pea aphid/ Buchnera symbiotic system. Proceedings of the National Academy of Sciences 115:E1819–E1828. https://doi.org/10.1073/pnas.1720237115 Sinno M, Bézier A, Vinale F, et al (2020) Symbiosis disruption in the olive fruit fly, Bactrocera oleae (Rossi), as a potential tool for sustainable control. Pest Management Science 76:3199–3207. https://doi.org/10.1002/ps.5875 Skaljac M, Kirfel P, Grotmann J, Vilcinskas A (2018) Fitness costs of infection with Serratia symbiotica are associated with greater susceptibility to insecticides in the pea aphid. Pest Management Science 74:1829–1836. https://doi.org/10.1002/ps.4881 Sochard C, Le Floch M, Anton S, et al (2021) Limited influence of gain and loss of symbionts on host plant selection in specialized pea aphid genotypes. Entomologia Generalis 39–47. https://doi.org/10.1127/entomologia/2020/1076 Smith TE, Moran NA (2020) Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera . Proceedings of the National Academy of Sciences 117:2113–2121. https://doi.org/10.1073/pnas.1916748117 Suhag A, Yadav H, Chaudhary D, et al (2021) Biotechnological interventions for the sustainable management of a global pest, whitefly ( Bemisia tabaci ). Insect Science 28:1228–1252. https://doi.org/10.1111/1744-7917.12853 Therneau T (2024). A Package for Survival Analysis in R. R package version 3.7-0, https://CRAN.R-project.org/package=survival . Tsuchida T, Koga R, Fukatsu T (2004) Host plant specialization governed by facultative symbiont. Science 303:1989–1989. https://doi.org/10.1126/science.1094611 Tsuchida T, Koga R, Meng XY, et al (2005) Characterization of a facultative endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum . Microb Ecol 49:126–133. https://doi.org/10.1007/s00248-004-0216-2 Tsuchida T, Koga R, Fujiwara A, Fukatsu T (2014) Phenotypic effect of “ Candidatus Rickettsiella viridis ,” a facultative symbiont of the pea aphid ( Acyrthosiphon pisum ), and its interaction with a coexisting symbiont. Applied and Environmental Microbiology 80:525. https://doi.org/10.1128/AEM.03049-13 Uchi N, Fukudome M, Nozaki N, et al (2019) Antimicrobial activities of cysteine-rich peptides specific to bacteriocytes of the pea aphid Acyrthosiphon pisum . Microbes and Environments 34:155–160. https://doi.org/10.1264/jsme2.ME18148 Vaucher RA, Teixeira ML, Brandelli A (2010) Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells. Curr Microbiol 60:1–5. https://doi.org/10.1007/s00284-009-9490-z Wernegreen JJ (2012) Endosymbiosis. Current Biology 22:R555–R561. https://doi.org/10.1016/j.cub.2012.06.010 Wimley WC (2010) Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol 5:905–917. https://doi.org/10.1021/cb1001558 Wu Q, Patočka J, Kuča K (2018) Insect antimicrobial peptides, a mini review. Toxins 10:461. https://doi.org/10.3390/toxins10110461 Zytynska SE, Tighiouart K, Frago E (2021) Benefits and costs of hosting facultative symbionts in plant-sucking insects: A meta-analysis. Molecular Ecology 30:2483–2494. https://doi.org/10.1111/mec.15897 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Journal of Pest Science → Version 1 posted Editorial decision: Revision requested 11 Feb, 2025 Reviews received at journal 11 Feb, 2025 Reviewers agreed at journal 09 Feb, 2025 Reviewers agreed at journal 05 Feb, 2025 Reviews received at journal 16 Jan, 2025 Reviewers agreed at journal 10 Jan, 2025 Reviewers agreed at journal 11 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers invited by journal 18 Nov, 2024 Editor assigned by journal 09 Nov, 2024 Submission checks completed at journal 09 Nov, 2024 First submitted to journal 04 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5386556","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381701799,"identity":"b0bd3ae5-e052-47aa-9942-56177898b32b","order_by":0,"name":"Hugo Terrasson","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"","lastName":"Terrasson","suffix":""},{"id":381701800,"identity":"8622cda8-871e-49e1-b593-5a7ee257a8da","order_by":1,"name":"Karen Gaget","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"","lastName":"Gaget","suffix":""},{"id":381701801,"identity":"68c83cc3-9a7f-4bb5-a8a9-999ebf1f839b","order_by":2,"name":"Garance Lapetoule","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Garance","middleName":"","lastName":"Lapetoule","suffix":""},{"id":381701802,"identity":"37ef38d0-5e02-48ae-a526-2d6051b29f9c","order_by":3,"name":"Isabelle Rahioui","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Rahioui","suffix":""},{"id":381701803,"identity":"107d2a04-3453-4dbd-9534-ab11e1a122cc","order_by":4,"name":"François Renoz","email":"","orcid":"","institution":"UCLouvain, Biodiversity Research Centre, Earth and Life Institute, Louvain-la-Neuve","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"Renoz","suffix":""},{"id":381701804,"identity":"3936e336-3466-4a1a-afb7-82bc3c26659f","order_by":5,"name":"Sylvain Benhamou","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Sylvain","middleName":"","lastName":"Benhamou","suffix":""},{"id":381701805,"identity":"71d05a97-816e-4980-9b45-e73c1d81e9f3","order_by":6,"name":"Chrystele Jouve","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Chrystele","middleName":"","lastName":"Jouve","suffix":""},{"id":381701806,"identity":"ba0e3cb5-5498-4078-b366-e526c8a8fcca","order_by":7,"name":"Catherine Sivignon","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Sivignon","suffix":""},{"id":381701807,"identity":"614298d9-689a-4c36-a190-6daf132b19c8","order_by":8,"name":"Gabrielle Duport","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"","lastName":"Duport","suffix":""},{"id":381701808,"identity":"ae3992a1-1c02-46b8-bd6d-76276eb0de9f","order_by":9,"name":"Vincent Aucagne","email":"","orcid":"","institution":"Centre de Biophysique Moléculaire, CNRS UPR 4301, 45071 Orléans","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Aucagne","suffix":""},{"id":381701809,"identity":"77881128-cf55-43a5-8041-baba50eb1ecd","order_by":10,"name":"Jean-Christophe Simon","email":"","orcid":"","institution":"INRAE, UMR IGEPP, Institut Agro, Université de Rennes, Le Rheu","correspondingAuthor":false,"prefix":"","firstName":"Jean-Christophe","middleName":"","lastName":"Simon","suffix":""},{"id":381701810,"identity":"54de1da4-5013-4863-83a1-c77f615f408a","order_by":11,"name":"Mélanie Ribeiro-Lopes","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Mélanie","middleName":"","lastName":"Ribeiro-Lopes","suffix":""},{"id":381701811,"identity":"60d091b5-a2fe-43bf-aadb-046aa3c234ad","order_by":12,"name":"Federica Calevro","email":"","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Calevro","suffix":""},{"id":381701812,"identity":"99cfd4b8-0342-4f96-aa90-01301038d005","order_by":13,"name":"Pedro Da Silva","email":"data:image/png;base64,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","orcid":"","institution":"INSA Lyon, INRAE, BF2I, UMR203, 69621 Villeurbanne","correspondingAuthor":true,"prefix":"","firstName":"Pedro","middleName":"Da","lastName":"Silva","suffix":""}],"badges":[],"createdAt":"2024-11-04 09:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5386556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5386556/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10340-025-01923-0","type":"published","date":"2025-06-18T15:57:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70045094,"identity":"92ee79e3-c3d6-4898-8b20-95e8af6bafcb","added_by":"auto","created_at":"2024-11-27 19:09:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":523952,"visible":true,"origin":"","legend":"\u003cp\u003eDose-response effect of BCR4 ingestion on survival of three \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e lines. Survival curves of aphid fed on artificial diets (AP3) containing different concentrations of BCR4 peptide during seven days. Left, central and right panels correspond to survival curves of LL01, YR2-amp and YR2\u003cem\u003e-Ri\u003c/em\u003e lines, respectively. n=30 for each BCR4-concentration group for each line. Data were analyzed using the Wilcoxon-Gehan Test.\u003c/p\u003e","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/8fb6b7a0811d204f2012ece4.jpeg"},{"id":70045095,"identity":"4500d5cb-ec82-47a2-ad78-ac1ea09d9267","added_by":"auto","created_at":"2024-11-27 19:09:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4137354,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCR4 ingestion on fresh mass, size and body color of three \u003cem\u003eAcyrthosiphon pisum \u003c/em\u003elines\u003cstrong\u003e. a\u003c/strong\u003e Dose-reponse curves of aphids’ mass following 7 days of feeding on artificial diets containing different concentrations of BCR4 peptide. Results for the LL01 line, YR2-amp line and YR2\u003cem\u003e-Ri\u003c/em\u003eline, are presented in green, yellow and red respectively. For each BCR4 concentration, dots with error bar correspond to mean mass with standard deviation. For each line, dose response effect of BCR4 ingestion was modeled with a log-logistic model with three parameters followed by box-cox transformation and displayed with its predicted confidence interval. Dash lines correspond to the estimated BCR4-concentration that leads to a 50% reduction in mass compared with aphids fed on AP3 medium without BCR4 (ED50). n=10 aphids for each BCR4-concentration group for each line\u003cstrong\u003e. b-d \u003c/strong\u003ePictures of 7-days-old aphids fed on artificial diets (AP3) complemented or not with 1000 µg/ml of BCR4 during seven days. Panels b, c and d correspond to the LL01 line, YR2-amp line and YR2\u003cem\u003e-Ri\u003c/em\u003e line, respectively. Scale bar: 1 mm.\u003c/p\u003e","description":"","filename":"Fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/99d52bfa7d459378cbc9369d.jpeg"},{"id":70045228,"identity":"29eb1748-9618-4844-8dc2-c7ff8d83a3df","added_by":"auto","created_at":"2024-11-27 19:17:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150469,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCR4 ingestion on dissected embryonic chain in the three \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e lines\u003cstrong\u003e. \u003c/strong\u003eLeft panels correspond to embryonic chain dissected from LL01, YR2-amp and YR2\u003cem\u003e-Ri\u003c/em\u003e lines fed on AP3 medium (control) for seven days. Right panels correspond to embryonic chain dissected from LL01, YR2-amp and YR2\u003cem\u003e-Ri\u003c/em\u003e fed on BCR4 diluted in AP3 medium (treated) for seven days. Scalebar: 200 µm\u003c/p\u003e","description":"","filename":"Fig3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/d1eeb78126e73190dbd334b0.jpeg"},{"id":70045091,"identity":"488bd67a-bff5-45d3-b623-8e8021b9e80a","added_by":"auto","created_at":"2024-11-27 19:09:09","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":738626,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCR4 ingestion on embryos and bacteriocytes in three \u003cem\u003eAcyrthosiphon pisum \u003c/em\u003elines. \u003cstrong\u003ea-f\u003c/strong\u003e H\u0026amp;E staining of 7-day-old aphids belonging to the LL01, YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e \u003cem\u003eAcyrthosiphon pisum \u003c/em\u003elines fed on artificial diets (AP3) complemented or not with 1000 µg/ml of BCR4 during seven days. \u003cstrong\u003ea’-f’\u003c/strong\u003eHigher magnification of embryos. The green and red dotted lines surround the bacteriocytes and the central nervous system of embryos, respectively. The black arrows indicate a visible limb (antenna or leg). \u003cstrong\u003ea’’-f’’\u003c/strong\u003e Higher magnification of mother bacteriocytes indicated by the black arrowheads. Scalebars: 100 µm in \u003cem\u003ea-f\u003c/em\u003e, 20 µm in \u003cem\u003ea’-f’\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e20 µm in \u003cem\u003ea’’-f’’\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/8e5ef9b019873f38dbd2f397.jpeg"},{"id":70045097,"identity":"970d869a-a699-415e-905e-3a1deb6c3190","added_by":"auto","created_at":"2024-11-27 19:09:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2599350,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCR4 treatment on symbiont localization. FISH observation of 7-day-old aphids belonging to the LL01, YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e \u003cem\u003eAcyrthosiphon pisum \u003c/em\u003elines fed on artificial diets (AP3) only (\u003cstrong\u003ea-c\u003c/strong\u003e) or complemented with 1000 µg/ml of BCR4 during seven days (\u003cstrong\u003ed-f\u003c/strong\u003e). \u003cem\u003eBuchnera aphidicola\u003c/em\u003e is labelled in green and \u003cem\u003eRegiella insecticola\u003c/em\u003ein red, respectively. Maternal bacteriocytes are indicated with white arrows. In YR2-\u003cem\u003eRi \u003c/em\u003eindividuals (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e) higher magnification shows maternal bacteriocytes filled with \u003cem\u003eB. aphidicola\u003c/em\u003e and surrounded by \u003cem\u003eR. insecticola\u003c/em\u003e. White arrowhead indicates \u003cem\u003eR. insecticola\u003c/em\u003e signal in a sheath cell. Scale: 200 µm and 50 µm for whole body images and zoom on bacteriocytes, respectively\u003c/p\u003e","description":"","filename":"Fig5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/3d2e1a2d3f1eecf23e93fd52.jpeg"},{"id":70045229,"identity":"3bdbadc6-c09b-43a1-b6d5-2e96f08615cf","added_by":"auto","created_at":"2024-11-27 19:17:09","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":224133,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCR4 ingestion on \u003cem\u003eBuchnera \u003c/em\u003eand \u003cem\u003eRegiella \u003c/em\u003enumbers in three \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e lines. Number of bacteria per aphid fed on AP3 (blue) or in AP3+BCR4 (1000 µg/ml) (yellow) for \u003cstrong\u003ea\u003c/strong\u003e \u003cem\u003eBuchnera\u003c/em\u003e from different lines (\u003cem\u003ei.e.\u003c/em\u003e LL01, YR2-amp and YR2\u003cem\u003e-Ri\u003c/em\u003e) and \u003cstrong\u003eb\u003c/strong\u003e the \u003cem\u003eRegiella\u003c/em\u003efrom YR2\u003cem\u003e-Ri\u003c/em\u003e line. (pwc = pairwise comparison with emmeans test: ****: p\u0026lt;0.0001; ***: p\u0026lt;0.001. n=4 per aphid, per line and per treatment)\u003c/p\u003e","description":"","filename":"Fig6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/a4563a8a05c7cd8fda821cb9.jpeg"},{"id":85231464,"identity":"b66c152e-0b8e-4625-9b22-b4864000d4a1","added_by":"auto","created_at":"2025-06-23 16:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8580448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/018f8486-52d3-4e6b-ac54-66647587519a.pdf"},{"id":70045098,"identity":"89ba6daa-9d82-4d12-91c9-c551d1c50a2b","added_by":"auto","created_at":"2024-11-27 19:09:09","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2998319,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5386556/v1/5612ffe2e34fec98cd59716b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Strong impact of orally administered BCR4 defensin on aphid survival, embryo development and symbiotic cells in three Acyrthosiphon pisum parthenogenetic lines","fulltext":[{"header":"Key Message","content":"\u003cul\u003e\n \u003cli\u003eBCR4 belongs to a new defensin family of peptides whose role in aphid symbiosis remains elusive\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBCR4 ingestion negatively impacts aphid survival and mass, and alters embryonic development, in a dose-dependent manner.\u003c/li\u003e\n \u003cli\u003eSusceptibility of aphids to BCR4 varies with the genetic background and symbiotic status\u003c/li\u003e\n \u003cli\u003eBCR4 significantly lowers symbiont populations, affecting \u003cem\u003eBuchnera aphidicola\u0026nbsp;\u003c/em\u003eto a greater extent\u003cem\u003e\u0026nbsp;\u003c/em\u003ethan\u003cem\u003e\u0026nbsp;Regiella insecticola\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eBCR4 is a promising insecticide candidate for aphid management\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eAphids are major crop pests, causing global losses estimated at hundreds of millions of dollars each year (Gula \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They make up to 26% of pests on important food crops in temperate regions, including maize, wheat, potatoes, sugar beets, barleys, and tomatoes (Calevro et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dedryver et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Aphids have also been reported as the most efficient and important vectors of plant viruses, transmitting nearly 300 different virus species (Hogenhout et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), which corresponds to 30% of the total number of plant pathogenic viruses identified to date.\u003c/p\u003e \u003cp\u003eAphids' ecological success is largely due to their symbiotic relationship with the γ-proteobacterium \u003cem\u003eBuchnera aphidicola\u003c/em\u003e, an obligate \u0026lsquo;primary\u0026rsquo; endosymbiont that provides them with essential nutrients poorly represented in their diet (Wernegreen \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Hansen \u0026amp; Moran \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This primary symbiont is vertically transmitted and lives within specialized host cells known as bacteriocytes (Baumann, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) that are the interface of metabolic exchanges (Smith \u0026amp; Moran \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), regulate symbiont populations (Simonet et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and are involved in their vertical transmission (Koga et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Aphids can also host various facultative \u0026lsquo;secondary\u0026rsquo; endosymbionts, including \u003cem\u003eSpiroplasma\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e species, as well as Enterobacteria such as \u003cem\u003eRegiella insecticola\u003c/em\u003e, \u003cem\u003eHamiltonella defensa\u003c/em\u003e, and \u003cem\u003eSerratia symbiotica\u003c/em\u003e. These secondary symbionts, located in the haemolymph, secondary bacteriocytes and other organs (sheath cells, oenocytes, digestive tract) (Tsuchida et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), can be transmitted both vertically and horizontally (Darby \u0026amp; Douglas, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pons et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Renoz et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). They have been associated with benefits like thermal resistance (Montllor et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), protection against natural enemies (Oliver et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Scarborough et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Frago et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and adaptation to different host plants (Renoz \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tsuchida et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent aphid control strategies rely essentially on chemical treatments, whose extensive use contributes to environmental pollution and the development of insect resistance (Goulson \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, there is an urgent need for the development of environmentally friendly alternatives. Small disulfide-rich proteins (DRPs) from plants or arthropods are promising candidates to be used instead of, or in combination with low doses of, chemical insecticides, due to their wide range of biological activities related to host defense, as well as their stability and resistance to enzymatic degradation (Ho et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA specific class of DRPs, known as bacteriocyte-specific cysteine-rich (BCR) peptides, has been identified in the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e (Shigenobu \u0026amp; Stern \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These peptides, encoded by seven orphan genes, are expressed in the bacteriocytes of both embryonic and adult aphids, suggesting a role in the maintenance of bacteriocyte homeostasis and the control of endosymbionts (Shigenobu \u0026amp; Stern \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Previous research has shown that BCR4, in particular, exhibits insecticidal effects when orally delivered to the \u003cem\u003eA. pisum\u003c/em\u003e line LL01, reducing its survival and mass (Loth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis investigation aimed to determine which are the developmental and anatomical bases of BCR4 toxicity, whether this toxicity could be explained by a direct effect on symbiotic cells, and whether those vary depending on the aphid genetic background and/or the presence of facultative symbionts. The effects of ingestion of sublethal doses of BCR4 were investigated using a panel of complementary approaches (life history traits, histological and FISH analyses, flow cytometry) applied to three \u003cem\u003eA. pisum\u003c/em\u003e lines: LL01, YR2-amp, and YR2-\u003cem\u003eRi\u003c/em\u003e. LL01 and YR2 lines are genetically different. Moreover, the first two lines only harbour the obligatory symbiont \u003cem\u003eB. aphidicola\u003c/em\u003e, while the third also hosts the facultative symbiont \u003cem\u003eR. insecticola\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Aphid lines and rearing\u003c/h2\u003e \u003cp\u003eAll the experiments were performed using is the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Three distinct lines were used: LL01, YR2\u003cem\u003e-Ri\u003c/em\u003e and YR2-Amp. LL01 corresponds to a natural line (Rahb\u0026eacute; \u0026amp; Febvay \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) harboring only the obligate primary symbiont \u003cem\u003eB. aphidicola\u003c/em\u003e. YR2\u003cem\u003e-Ri\u003c/em\u003e naturally harbors both \u003cem\u003eB. aphidicola\u003c/em\u003e and the facultative symbiont \u003cem\u003eR. insecticola\u003c/em\u003e (Simon et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). YR2-Amp, was produced from YR2\u003cem\u003e-Ri\u003c/em\u003e line, after treatment with antibiotics to eliminate \u003cem\u003eR. insecticola\u003c/em\u003e (Koga et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Simon et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The presence of the latter and the absence of other secondary symbionts that may be found in pea aphids (\u003cem\u003ei.e., Hamiltonella defensa\u003c/em\u003e, \u003cem\u003ecd. Fukatsuia symbiotica\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e sp, \u003cem\u003eRickettsiella\u003c/em\u003e sp, \u003cem\u003eSerratia symbiotica\u003c/em\u003e and \u003cem\u003eSpiroplasma\u003c/em\u003e sp.) was verified by a PCR-based diagnostic developed by Peccoud et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The three lines were maintained as strictly parthenogenetic matrilines on young broad bean plants (\u003cem\u003eVicia faba\u003c/em\u003e, L. cv. Aquadulce), with a photoperiod of 16 h light-8 h dark, at 21\u0026deg;C with ~\u0026thinsp;60% of humidity. To obtain synchronized aphids, around 100 reproductive females (9 to 11-day- old) per line were left on plants for 24\u0026ndash;72 hours (pre-synchronization). The adults were then removed, and newborn nymphs were kept on the plant for 9 days (=\u0026thinsp;adult stage), then transferred to a new plant for 24 hours (synchronization). The resulting synchronized first instar nymph (\u0026lt;\u0026thinsp;1day old) were then placed on artificial diets containing different concentrations of the BCR4 peptide for the different experiments. The complete experimental design is illustrated in Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis and purification of BCR4\u003c/h2\u003e \u003cp\u003eThe BCR4 peptide sequence was synthesized and folded according to the optimized procedure described in a previous study (Loth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, BCR4 was generated by native chemical ligation (NCL) of two peptide segments, followed by oxidative folding to form the three disulfide bridges. Both peptide segments were purified using C18 reverse phase (RP) HPLC. The purified peptides were then chemoselectively coupled and purified under standard NCL conditions to yield the pure reduced form of BCR4. This form was then subjected to \u003cem\u003ein vitro\u003c/em\u003e oxidative folding followed by purification to homogeneity using C18 RP-HPLC. Analysis of the final folded product by ESI-HRMS confirmed the presence of three disulfide bridges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Survival and growth monitoring following BCR4 ingestion\u003c/h2\u003e \u003cp\u003eTo test the effect of BCR4 ingestion on aphid survival and growth, the synthetic peptide was serially diluted in AP3 artificial diet (Rahb\u0026eacute; et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Calatayud \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) at the following concentrations (in \u0026micro;g/ml): 0 (control), 50, 100, 250, 375, 500, 750 and 1000, and prepared in \u003cem\u003ead hoc\u003c/em\u003e feeding chamber as previously described (Rahb\u0026eacute; et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). For each aphid line and each concentration, three groups of ten first instar nymphs were collected and fed on independent feeding chambers. Survival was monitored daily for seven days. At the end of the experiment, ten surviving aphids per group were randomly selected and individually weighed on a Mettler AE163 analytical microbalance (Mettler Toledo, Columbus, OH, United States). To confirm the specificity of the observed effect, i.e. that it is due to BCR4, the same experimental design was repeated, using a negative control peptide at the highest concentration only (1000 \u0026micro;g/ml). The peptide used (U\u003csub\u003e11\u003c/sub\u003e; for details, see Barass\u0026eacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) comes from the ant \u003cem\u003eTetramorium bicarinatum\u003c/em\u003e, possesses a disulfide bond and a molecular weight of 4,0 kDa, close to the 5,9 kDa of BCR4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Analysis of the effect of BCR4 ingestion on aphid morphology and anatomy\u003c/h2\u003e \u003cp\u003eTo study the effect of BCR4 ingestion on aphid morphology and anatomy, the previous experimental design was repeated with aphids fed either on a control AP3 diet or on a diet supplemented with the dose of 1000 \u0026micro;g/ml of BCR4 for seven days. Dissection and histological preparations were carried out as described below.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Dissection and macroscopic observations\u003c/h2\u003e \u003cp\u003eAfter seven days of treatment, at least six aphids per condition and per line were randomly selected, and photographed with a Moticam S6 camera (MoticEurope, Barcelona, Spain) attached to a Leica MZ FLIII stereomicroscope (Leica Microsystems, Wetzkar, Germany) to compare their size, shape and color. They were then dissected in isoosmotic buffer A (0.025 M KCl, 0.01 M MgCl2, 0.25 M Sucrose, and 0.035 M Tris\u0026ndash;HCl, pH 7.5) by opening the abdomen with microdissection tweezers (Dumont type 5 Dumoxel; Electron Microscopy Science, Hatfield, UK) as previously described (Ribeiro Lopes et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The digestive tract and the embryonic chains were isolated and photographed using the same camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Aphid section preparation\u003c/h2\u003e \u003cp\u003eFor histological staining and FISH (Fluorescent In-Situ Hybridization) of treated and control aphids, four individuals per condition were randomly selected after seven days of treatment, and sections were prepared as previously described (Ribeiro Lopes et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, antennae and legs were removed from aphids with microdissection tweezers, before immersing them in a solution of PBS containing 4% paraformaldehyde and 0.1% Triton X-100 at 4\u0026deg;C for 24 h. Aphids were then transferred in a solution of PBS with 4% paraformaldehyde. After several washing steps in PBS, aphids were embedded in 1.3% agar. The resulting samples were dehydrated through a series of ethanol solutions ranging from 70\u0026ndash;100%, and transferred in 1-butanol at 4\u0026deg;C for at least 24 h. In order to prepare aphid sections, aphids were embedded in Paraplast/1-butanol (v/v) for 10 h and then in 100% Paraplast (Paraffine Paraplast Plus Mc Cormick Scientific Dutscher). Resulting Paraplast blocks were sectioned at 3 \u0026micro;m thickness using a HM340E rotary microtome (ThermoScientific, Waltham, MA, United States). Sections were positioned on polylysine coated slides and conserved at 4\u0026deg;C until staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Histological preparation and observation\u003c/h2\u003e \u003cp\u003eSections were prepared for Hematoxylin and Eosin (H\u0026amp;E) staining with RAL products (RAL Diagnostics, Martillac, France) using a protocol adapted from Sapountzis et al. (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, sections were deparaffinated in methylcyclohexane (2x 10 min) and rehydrated through an ethanol series to PBS. Nucleus and cytoplasm were stained in Mayer Hematoxylin for 3 min and Eosin for 2 min, respectively, with a water rinse in between. Sections were then washed in water and treated through an ethanol series from 70\u0026ndash;100%. After a rapid bath in Diasolv (Diapath, Martinengo, Italia) solution, sections were mounted with Diamount mounting medium (Diapath, Martinengo, Italia). Histological observation was performed under transmitted light, on a Thunder Imager 3D Live Cell\u0026trade; microscope (Leica, Wetzlar, Germany) with 4X or 20X magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. FISH analysis\u003c/h2\u003e \u003cp\u003eFISH procedure was adapted from Simonet et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Sections were deparaffinated using methylcyclohexane (2x 10 min), followed by two 10 min baths in 100% ethanol, and then air-dried. Deproteinization was conducted with 0.01 M hydrochloric acid and 10 mg/mL pepsin at 37\u0026deg;C for 10 minutes. Sections were rinsed in PBS and dehydrated through a series of ethanol solutions ranging from 70\u0026ndash;100%. Prehybridization took place at 45\u0026deg;C for 30 minutes in a prehybridization buffer consisting of 79% hybridization buffer (0.9 M NaCl, 20 mM Tris, 5 mM EDTA, pH 7.2), 20% Denhardt\u0026rsquo;s solution (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g BSA in 500 mL water), and 1% SDS solution (10% SDS). \u003cem\u003eIn situ\u003c/em\u003e hybridization was performed by incubating the slides at 45\u0026deg;C for 3 hours, shielded from light, with 5 \u0026micro;l of 100 \u0026micro;M oligonucleotide probes specifically targeting \u003cem\u003eB. aphidicola\u003c/em\u003e and \u003cem\u003eR. insecticola\u003c/em\u003e 16S rRNAs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Probes were prepared in prehybridization buffer and their 5' end was labeled with Alexa Fluor 488 and Alexa Fluor 594, respectively. Sections were washed twice in washing solution (99% hybridization buffer and 1% SDS 10%), rinsed in PBS and finally mounted using PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific) containing DAPI (3 \u0026micro;g/mL; Vector Laboratories). Positive fluorescent signals were analyzed using a Thunder Imager 3D Live Cell\u0026trade; microscope (Leica) equipped with the appropriate emission filters.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Analysis of the effect of BCR4 ingestion on aphid symbiont\u003c/h2\u003e \u003cp\u003eThe BCR4 bioassay experiment was repeated with only aphids fed either a control AP3 diet or a diet supplemented with of 1000 \u0026micro;g/\u0026micro;l BCR4 for seven days. To assess the impact of the treatment on the quantity of the primary symbiont \u003cem\u003eB. aphidicola\u003c/em\u003e and the secondary symbiont \u003cem\u003eR. insecticola\u003c/em\u003e, a flow cytometry approach, that proved to be effective to obtain the absolute numbers of bacteria in this model, was used (Simonet et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor each condition, symbiotic bacteria from 12 whole aphids were purified as previously described (Simonet et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Aphids were gently crushed with a Potter homogenizer in 3 ml of ice-cold buffer A and the homogenate was consecutively filtered through nylon net filters with 60, 30 and 10 \u0026micro;m pore sizes (Merck Millipore, Tullagreen, Ireland). Following centrifugation (4,000 \u0026times; g, 5 min, 4\u0026deg;C), the pellet of endosymbiotic bacteria was re-suspended in 200\u0026micro;l for aphids reared on AP3\u0026thinsp;+\u0026thinsp;BCR4 (1000 \u0026micro;g/ml) medium and 400 \u0026micro;l for aphids reared on AP3 of NaCl 0.85% supplemented with 20% glycerol as a cryoprotective agent, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until flow cytometry analysis. Immediately prior to flow cytometry analyses, samples were thawed for 3 min at 42\u0026deg;C, vortexed and diluted to 1:20 with a NaCl 0.85% solution to reach the optimal cell concentration for flow cytometry analysis (\u0026lt;\u0026thinsp;2,000 events/sec). All solutions were filter sterilized at 0.22 \u0026micro;m prior to use to avoid contamination with exogenous bacteria. For bacterial staining, 0.5 \u0026micro;l of the nucleic acid probe SYTO9 (3.34 mm; Molecular Probes Inc, Eugene, OR, United States) was added to 300 \u0026micro;l of the diluted bacterial suspensions. The mixture was then vortexed and incubated in the dark for 15 min at room temperature, following the manufacturer\u0026rsquo;s instructions. The stained samples were analyzed using a BD Accuri\u0026trade; C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States) equipped with a blue laser (488 nm, air-cooled, 20 mW solid state) and cell green fluorescence was acquired with a photomultiplier tube detector and a 530 nm band-pass filter (503\u0026ndash;563 nm). Flow cytometry measurements were run at low flow rate (14 \u0026micro;l/min) and the core stream was allowed to stabilize for 30 s prior to the 2 min acquisition. Cellular data were collected and processed using BD Accuri C6 software (version 1.0.264.21, BD Biosciences). The analyses were performed using logarithmic gains and specific detector settings, adjusted on non-stained samples to eliminate endosymbiont autofluorescence. Then, the endosymbiont population was identified by screening samples on\u003c/p\u003e \u003cp\u003eFSC-W vs. FSC-H (Forward Scatter Width vs. Height) and SSC-W vs. SSC-H (Side Scatter Width vs. Height)\u003c/p\u003e \u003cp\u003edot plots. \u003cem\u003eB. aphidicola\u003c/em\u003e populations were identified using the same parameter than previously described (Simonet et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A second population of SYTO9-positive event, of different size and granularity was identified in YR2-\u003cem\u003eRi\u003c/em\u003e samples, and used to count \u003cem\u003eR. insecticola\u003c/em\u003e symbionts (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). For each condition, four biological replicates were processed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis and graphical representations were conducted using the R software (v4.3.1) (R Core Team, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). R-packages \u0026laquo; survival \u0026raquo; and \u0026laquo; survminer \u0026raquo; (Therneau, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were used to analyze survival data, comparing curves with a Wilcoxon-Gehan rank test. To estimate the lethal time taken for 20% of the aphid population to die (LT\u003csub\u003e20\u003c/sub\u003e) when fed on artificial diet containing 1000 \u0026micro;g/\u0026micro;l of BCR4, generalized linear models (glm) with a probit link transformation were fitted for each aphid line and compared with a ratio test, using the R-package \u0026ldquo;ecotox\u0026rdquo; (Hlina et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Dose-response curves of the effect of BCR4 ingestion on aphid mass were fitted with the \u0026laquo; drc \u0026raquo; package (Ritz et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) using a log-logistic model with 3 parameters (with the lowest limit fixed to 0) and box-cox transformation, allowing to predict 95% confidence intervals (CI\u003csub\u003e95\u003c/sub\u003e%) and the effective dose that affect 50% of the mass compared to control (ED\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eFor the flow cytometry analyses, the absolute numbers of symbionts obtained for each aphid pool were converted into \u0026ldquo;symbiont numbers per aphid\u0026rdquo;. A linear regression model was applied on data. Pairwise comparisons were performed to investigate significant differences between modalities using the eammeans function of the R package \u0026ldquo;emmeans\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. BCR4 treatment reduces survival and mass of three aphid lines\u003c/h2\u003e \u003cp\u003eThe dose-response effect of BCR4 ingestion on survival was first compared between the three aphid lines LL01, YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e. In the absence of BCR4, the three lines showed comparable survival rates on the artificial diet, with a 7-day survival of 93%, 97% and 97% for LL01, YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e respectively. Survival of LL01 and YR2-amp lines was significantly affected by BCR4 ingestion (log-rank test: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not that of YR2-\u003cem\u003eRi\u003c/em\u003e (log-rank test: p\u0026thinsp;=\u0026thinsp;0.113) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, both YR2 lines survived better than the LL01 line (log-rank test LL01 vs YR2-amp or YR2-\u003cem\u003eRi\u003c/em\u003e: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar results were obtained when focusing solely on the treatment with the highest concentration of BCR4 (1000 \u0026micro;g/\u0026micro;l): (i) the 7-day survival is of 77% and 83% for YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e, respectively, and only 50% for LL01; (ii) the estimated time to reach 20% of the mortality (LT\u003csub\u003e20\u003c/sub\u003e) for LL01 (3.5 days) is significantly different from the LT\u003csub\u003e20\u003c/sub\u003e of both YR2-amp (6.8 days; ratio-test: p\u0026thinsp;=\u0026thinsp;0.002) and YR2-\u003cem\u003eRi\u003c/em\u003e (8,6 days; ratio-test: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, no mortality was observed in aphid fed with 1000 \u0026micro;g/ml of U11 peptide (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results show that there is a dose-response effect of BCR4 on the mass of each line (lack-of-fit test: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Feeding on BCR4 had a significant, negative impact on aphids\u0026rsquo; mass, with an average reduction of 78%, 65% and 75% for LL01, YR2-amp, and YR2-\u003cem\u003eRi\u003c/em\u003e respectively, compared to the control. The BCR4-concentration that would cause a 50% reduction in aphid mass (ED\u003csub\u003e50\u003c/sub\u003e) was predicted, revealing a more potent effect on the LL01 line (ED\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of 383.8\u0026thinsp;\u0026plusmn;\u0026thinsp;39.6 \u0026micro;g/ml; 535.3\u0026thinsp;\u0026plusmn;\u0026thinsp;75.6 \u0026micro;g/ml and 445.2 \u0026micro;g/ml\u0026thinsp;\u0026plusmn;\u0026thinsp;52.1 for LL01, YR2-amp, and YR2-\u003cem\u003eRi\u003c/em\u003e respectively). Discoloration of the aphid cuticle was also observed for each line, with a more noticeable effect on YR2-amp and -\u003cem\u003eRi\u003c/em\u003e compared to LL01 probably due to their more contrasted color (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Impact of BCR4 on the anatomy of aphids and their embryos\u003c/h2\u003e \u003cp\u003eTo assess whether the reduction in mass following BCR4 treatment is accompanied by a change in aphid tissue organization, dissections, histological and FISH analysis of individuals treated with the highest concentration of BCR4 were carried out.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Macroscopic observations\u003c/h2\u003e \u003cp\u003eMacroscopic observations of dissected aphids revealed no obvious differences between the digestive tracts of control and treated individuals (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). However, significant differences were observed in the development of embryonic chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In control aphids, the classic two ovaries, each containing seven ovarioles, are clearly visible. These ovarioles consist of chains of embryos at different developmental stages. In these aphids, the most developed embryos exceed 500 \u0026micro;m in length and are pigmented. The pair of red eyes and the beginning of thorax segmentation are also visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, the embryonic chains of treated individuals are more crumbly and fragile. They contain fewer and smaller embryos, with the largest ones reaching only 300 \u0026micro;m. In addition, no coloration, no eyes and no segmentation was noticeable, whatever the aphid line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Histological analysis with hemalum and eosin staining\u003c/h2\u003e \u003cp\u003eHistological analysis confirmed the adverse effect of BCR4 on aphid development. In particular, numerous well-developed embryos can be distinguished in sections from control aphids, a developed central nervous system (CNS) and visible limbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026rsquo;, c\u0026rsquo;, e\u0026rsquo;). Bacteriocytes are cellularized and form two lobes located in proximity of the developing digestive tract. While embryos are still visible in treated aphids, they are negatively impacted by the treatment: their organs are undistinguishable and the bacteriocytes, though present, appear acellularized, with no apparent cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026rsquo;, d\u0026rsquo;, e\u0026rsquo;). In all the embryos that could be observed, the bacteriocyte mass was found at the posterior pole, which suggests that these embryos are undergoing a developmental delay.\u003c/p\u003e \u003cp\u003eBCR4-ingestion also shows a strong effect on the morphology of the mother\u0026rsquo;s bacteriocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026rdquo;-f\u0026rdquo;). Although the symbiotic cells are presents in both control and treated aphid, bacteriocytes from the latter are deformed, appearing crushed between organs, and sometimes difficult to distinguish from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026rdquo;, d\u0026rdquo;, e\u0026rdquo;). In addition, observation of whole insect section suggests a reduction of bacteriocyte number (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. FISH analyses\u003c/h2\u003e \u003cp\u003eFISH analyses were carried out to assess the effect of BCR4 on symbiont localization and the structure of bacteriocytes. Observation of control aphids (reared on AP3 artificial diet) shows that the localization of \u003cem\u003eB. aphidicola and R. insecticola\u003c/em\u003e is the same as previously described in other aphid lines (Moran et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tsuchida et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2005\u003c/span\u003e): \u003cem\u003eB. aphidicola\u003c/em\u003e is exclusively found in maternal and embryonic bacteriocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c), while \u003cem\u003eR. insecticola\u003c/em\u003e is observed extracellularly, in the hemolymph and around the bacteriocytes, and intracellularly, in sheath cells attached to the bacteriocytes, resulting in a signal that completely surrounds the bacteriocyte clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eAfter BCR4 treatment, the localization of the primary symbiont is not affected, and it is strictly confined to maternal and embryonic bacteriocytes. Nevertheless, consistently with H\u0026amp;E observations, maternal bacteriocytes appear deformed and less numerous (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026rsquo;-c\u0026rsquo;). Moreover, in embryos, bacteriocytes appear to be packed in the posterior region, contrary to their classical localization around the digestive tract (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f). Regarding \u003cem\u003eR. insecticola\u003c/em\u003e, the signal is also detected in treated aphids at the same localization as in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). However, a reduced signal is observed in the hemolymph and around the bacteriocytes and the signal surrounding the bacteriocyte clusters is not continuous as in the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. BCR4 significantly reduces symbiont populations\u003c/h2\u003e \u003cp\u003eTo assess whether the observed effect of BCR4 ingestion on bacteriocyte morphology and numbers translates into a change in the density of symbiont populations, a flow cytometry analysis using a single-cell counter cytometer was performed to determine the absolute number of symbionts per aphid (Simonet et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The mean total number of events corresponding to \u003cem\u003eB. aphidicola\u003c/em\u003e decreased drastically from 1.07x10\u003csup\u003e6\u003c/sup\u003e to 1.48x10\u003csup\u003e5\u003c/sup\u003e for LL01, from 6.67x10\u003csup\u003e5\u003c/sup\u003e to 1.82x10\u003csup\u003e5\u003c/sup\u003e for YR2-amp, and from 7.04x10\u003csup\u003e5\u003c/sup\u003e to 1.10x10\u003csup\u003e5\u003c/sup\u003e for YR2\u003cem\u003e-Ri\u003c/em\u003e. This corresponds to reductions of 86%, 73% and 84%, respectively. The BCR4 treatment (F\u003csub\u003e1,17\u003c/sub\u003e = 120.172, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the interaction between line and treatment (F\u003csub\u003e2,17\u003c/sub\u003e = 4.224, p\u0026thinsp;=\u0026thinsp;0.03) significantly impact \u003cem\u003eB. aphidicola\u003c/em\u003e count while the line alone do not (F\u003csub\u003e2,17\u003c/sub\u003e = 3.327, p\u0026thinsp;=\u0026thinsp;0.06). The mean total number of events corresponding to \u003cem\u003eR. insecticola\u003c/em\u003e in YR2\u003cem\u003e-Ri\u003c/em\u003e line decreased more modestly, from 1.79x10\u003csup\u003e6\u003c/sup\u003e to 7.57x10\u003csup\u003e5\u003c/sup\u003e, a significant reduction of 58% (F\u003csub\u003e1,5\u003c/sub\u003e = 50.336, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study investigated the impact of BCR4 peptide ingestion on three \u003cem\u003eA. pisum\u003c/em\u003e lines, LL01, YR2-amp and YR2-\u003cem\u003eRi\u003c/em\u003e, differing in their genetic background (LL01 or YR2) and by the absence (LL01 and YR2-amp) or presence (YR2-\u003cem\u003eRi\u003c/em\u003e) of the secondary symbiont \u003cem\u003eR. insecticola\u003c/em\u003e. Overall, we showed an insecticidal effect of BCR4, with a strong impact on survival, fresh weight, embryo development, bacteriocyte morphology, and symbiont and numbers.\u003c/p\u003e \u003cp\u003eOur findings corroborate previous results obtained by Loth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, which showed a dose-response effect of BCR4 on aphid survival and weight. However, the latter study was carried out on a single genetic line (LL01), carrying only the primary symbiont \u003cem\u003eB. aphidicola\u003c/em\u003e, whereas several works have shown a significant impact of the genotype on aphid response to biotic stresses, such as host plant changes (Ferrari et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), parasitoid wasp attacks (Libbrecht et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Martinez et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or temperature increase (Jahan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), highlighting the importance of considering different genetic lines. In the present study, we showed a contrasting effect of BCR4 on aphids\u0026rsquo; survival depending on their genetic background : at equivalent concentration, LL01 individuals died faster and to a greater extent than YR2-amp and YR2-Ri. Similarly, BCR4 ingestion reduced aphid mass in a dose-dependent manner, with the LL01 line being the most affected. While the insecticidal mode of action of BCR4 is unknown to date, our findings on the impact of BCR4 on aphid survival are similar to previous results about the use of other known antimicrobial peptides on aphids. Indeed, ingestion of indolicidin (an AMP produced by bovine neutrophils) reduces the survival of \u003cem\u003eMyzus persicae\u003c/em\u003e (Le-Feuvre et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Luna-Ramirez et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) showed that ingestion of certain scorpion AMPs exerts insecticidal activity against \u003cem\u003eA. pisum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePresence of \u003cem\u003eR. insecticola\u003c/em\u003e had no impact on \u003cem\u003eA. pisum\u003c/em\u003e survival, however, the symbiont aggravated the negative impact of BCR4 on aphid weight. Secondary symbionts have been reported to positively or negatively affect aphids\u0026rsquo; fitness, depending on the environment or the stress to which the insect is subjected, including xenobiotic exposure (Lemoine et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zytynska et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, infection by \u003cem\u003eSerriatia symbiotica\u003c/em\u003e in \u003cem\u003eA. pisum\u003c/em\u003e (Skaljac et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and \u003cem\u003eRickettsia\u003c/em\u003e in the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e (Kontsedalov et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), is associated with increased susceptibility to chemical insecticides, while the infection by \u003cem\u003eHamiltonella defensa\u003c/em\u003e is associated with reduced susceptibility in the aphid \u003cem\u003eSitobion miscanthi\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In \u003cem\u003eA. pisum\u003c/em\u003e, infection by \u003cem\u003eR. insecticola\u003c/em\u003e is associated with reduced aphid survival during heat stress (Russel and Moran, 2005) or bacterial infection (Luo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, the symbiont confers protection against fungal (Scarborough et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Parker et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Parker et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) or viral infections (Higashi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating that the bacterium could contribute to host defences in different ways. Although symbionts have been reported to increase their hosts\u0026rsquo; sensitivity to insecticides (Kontsedalov et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Skaljac et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the underlying mechanisms remain unknown. Hosting secondary symbionts is a finely-tuned cost-benefit trade-off for aphids, as symbionts are associated with a permanent physiological cost, acting as sinks for metabolites (Zytynska et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Harboring the \u003cem\u003eR. insecticola\u003c/em\u003e symbiont has been shown to negatively impact the host fitness (Sochard et al. 2020; Man et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Here, the cost of hosting \u003cem\u003eR. insecticola\u003c/em\u003e may be exacerbated when \u003cem\u003eA. pisum\u003c/em\u003e experiences stressful conditions such as exposure to BCR4. Similarly, specific symbiont combinations may aggravate the whitefly \u003cem\u003eB. tabaci\u003c/em\u003e performance on unfavourable host plants (Benhamou et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe negative impact of BCR4 on survival and weight of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003episum\u003c/em\u003e could be direct, by affecting aphid cells, or indirect, by targeting symbionts and disrupting the obligate nutritional relationship between the insect and its symbiotic bacteria. Concerning the direct cytotoxic effet of BCR4 on aphid cells, it is important to underline that this could not be excluded, as it have been shown that high concentration of certain AMPs could exhert a cytotoxic activity, \u003cem\u003ein vitro\u003c/em\u003e, against mammalian cells, by distrupting cell membranes (Vaucher et al. 2009; Greco et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, Greco et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e have shown that when inoculated in rats, AMPs lose their cytotoxic potential. Moreover, eukaryotic cells are generally resistant to AMPs. Contrary to prokaryotes, eukaryotic cell membranes contain sterol and mainly zwitteronic phospholipids that stabilize the membrane and confer a protection against AMPs (Mason et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Anderson et al. 2016; Almeida et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo gain an in-depth understanding of the effects of BCR4 ingestion on aphids and their symbionts, dissection and histological observations were performed. These analyses revealed a similar and significant negative effect on the embryos of each aphid line. Following treatment, embryonic chains were more fragile and contained smaller and fewer embryos, for which it was impossible to distinguish appendages or organs on histological sections. Histological and FISH observations revealed that the bacteriocytes and the \u003cem\u003eB. aphidicola\u003c/em\u003e they contain were mostly grouped at the posterior pole of the embryo, a localization that is usually limited to the early stages of embryo development (Miura et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Braendle et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This, combined with the lack of bacteriocytes organization in distinct cells, demonstrates at least a delay, if not a disruption, of embryonic development of bacteriocytes morphogenesis. Whereas previous studies using indolicidin (Le-Feuvre et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) or scorpion AMPs (Luna-Ramirez et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have shown an adverse impact of these peptides on aphid fecundity, our study presents for the first time a disruption of aphid embryonic development caused by the ingestion of an AMP.\u003c/p\u003e \u003cp\u003eMoreover, analyses of FISH and H\u0026amp;E staining revealed an alteration in maternal bacteriocytes. While bacteriocytes in the control group are numerous and round-shaped, most bacteriocytes from treated aphids appear fewer and distorted in the three aphid lines. The effect on aphid bacteriocytes is similar to the one observed by Le-Feuvre et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) in \u003cem\u003eM. persicae\u003c/em\u003e, who reported distortion of bacteriocyte morphology and reduction of bacteriocyte numbers after treatment with indolicidin. With FISH, we also show that the \u003cem\u003eR. insecticola\u003c/em\u003e signal appears to be less intense around the bacteriocytes than in the rest of the body.\u003c/p\u003e \u003cp\u003eTaken together, the disturbance in the anatomical organization of bacteriocytes and the reduction of \u003cem\u003eR. insecticola\u003c/em\u003e signal suggests an effect of BCR4 on both symbiotic populations. To test this hypothesis, we used flow cytometry to get insights in symbiotic bacterial numbers. This showed a massive reduction (72\u0026ndash;84%) of \u003cem\u003eB. aphidicola\u003c/em\u003e numbers in BCR4-treated aphids. This method also showed that BCR4 has a strong, but lower effect (58%) on \u003cem\u003eR. insecticola\u003c/em\u003e numbers. Comparable results have been obtained by Luna-Ramirez et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), who showed that ingestion of scorpion AMPs reduces the symbiotic load (\u003cem\u003eB. aphidicola\u003c/em\u003e and \u003cem\u003eSerratia symbiotica)\u003c/em\u003e in \u003cem\u003eA. pisum\u003c/em\u003e, and argue that insecticidal effects of those AMPs are due to a direct effect on symbionts. Importantly, previous studies have shown that BCR4 is bactericidal \u003cem\u003ein vitro\u003c/em\u003e against \u003cem\u003eE. coli\u003c/em\u003e, a Gram-negative bacterium closely related to \u003cem\u003eB. aphidicola\u003c/em\u003e (Uchi et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Loth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Taken together, these results suggest that BCR4 could similarly have a \u003cem\u003ein vivo\u003c/em\u003e bactericidal effect on \u003cem\u003eA. pisum\u003c/em\u003e symbiont, thereby having a negative, indirect impact on the growth and survival of the aphid.\u003c/p\u003e \u003cp\u003eInterestingly, \u003cem\u003eB. aphidicola\u003c/em\u003e symbionts appear to be more sensitive to BCR4 than \u003cem\u003eR. insecticola\u003c/em\u003e. Defensins typically target bacterial membranes, often leading to membrane disruption or pore formation (Cociancich et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Shai \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wimley, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Agadi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and it has been shown that BCR4 alter \u003cem\u003eE. coli\u003c/em\u003e morphology and membrane permeability (Uchi et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been demonstrated that the \u003cem\u003eB. aphidicola\u003c/em\u003e outer membrane lacks lipopolysaccharides (LPS) (Shigenobu et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Charles et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which may explain its greater vulnerability compared to \u003cem\u003eR. insecticola\u003c/em\u003e. As for other AMPs, one could propose that BCR4 exhibits a selective activity, being more specific against one symbiont (here \u003cem\u003eB. aphidicola\u003c/em\u003e) than another (here \u003cem\u003eR. insecticola\u003c/em\u003e). For instance, in the bean bug \u003cem\u003eRiptortus pedestris\u003c/em\u003e, several AMPs called Crypt-specific Cysteine-Rich peptides (CCRs) are expressed in the specialized posterior midgut region M4, prior to the acquisition of orally transmitted microbiota. This results in a selective barrier against unwanted bacteria, that facilitates the colonization by its beneficial symbiont \u003cem\u003eCaballeronia insecticola\u003c/em\u003e (formerly known as \u003cem\u003eBurkholderia insecticola\u003c/em\u003e) that is resistant to host CCRs (Lachat et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Once established, beneficial C. \u003cem\u003einsecticola\u003c/em\u003e becomes vulnerable to host AMPs due to an alteration in its LPS, which allows the host to regulate its gut symbionts (Kim et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, in \u003cem\u003eDrosophila\u003c/em\u003e, AMPs expressed in the gut have been shown to exhibit high selectivity depending on the invading pathogen (Hanson et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As an example, the commensal, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, is resistant to host AMPs during infection phases, allowing it to stably colonize the fly gut (Arias-Rojas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther investigations are needed to explore the potential of orally administered BCRs in selectively targeting different facultative symbionts in \u003cem\u003eA. pisum\u003c/em\u003e, and more generally in aphids.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study highlights the detrimental effect of BCR4 on aphids when administered orally. Further research is needed to understand the functions of the BCR-family in aphids and their potential use as bioinsecticides. Targeting insect symbionts in the context of pest management is increasingly being discussed (Arora \u0026amp; Douglas \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Gonella et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Noman et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sinno et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gonella \u0026amp; Alma \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rupawate et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Combined with advances in plant bioengineering (Bisht et al. 2020; Suhag et al. 2020; Mateos Fern\u0026aacute;ndez et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Komal et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and because they are restricted to the aphid lineage (Loth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), BCR delivery could be a powerful tool to specifically control aphid pest through their interference with their symbionts\u0026rsquo; populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eM\u0026eacute;lanie Ribeiro-Lopes, Federica Calevro and Pedro Da Silva conceived and designed research. Chryst\u0026egrave;le Jouve and Gabrielle Duport prepared the AP3 medium. Catherine Sivignon and Vincent Auchard produces the BCR4 peptide. Hugo Terrasson, Isabelle Rahioui and Chryst\u0026egrave;le Jouve conducted bioassays experiments. Hugo Terrasson and Sylvain Benhamou performed the survival and mass monitoring and analyses the data. Hugo Terrasson, Karen Gaget and Garance Lapetoule conducted histological preparations and observations. Hugo Terrasson, Karen Gaget and Garance Lapetoule conducted dissections and macroscopical observations. Karen Gaget and Garance Lapetoule performed flow cytometry experiments and Karen Gaget analyses the data. Jean-Christophe Simon provide both YR2 \u003cem\u003eA. pisum\u003c/em\u003e lines. Hugo Terrasson prepared the figures and wrote the first draft. Fran\u0026ccedil;ois Renoz, Sylvain Benhamou, Jean-Christophe Simon M\u0026eacute;lanie Ribeiro-Lopes Federica Calvero and Pedro Da Silva reviewed and corrected the draft. All authors read and approved the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eNo international, national and institutional guidelines for the care and use of animals were needed and followed.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by INSA Lyon (Institut National des Sciences Appliqu\u0026eacute;es de Lyon), INRAE (Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement) and the French ANR BIOFAMILY project (ANR-19-CE11-0004).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.R.L., F.C. and P.D.S. conceived and designed research. C.J. and G.D. prepared the AP3 medium. C.S. and V.A. produces the BCR4 peptide. H.T., I.R., and C.J. conducted bioassays experiments. H.T. and S.B. performed the survival and mass monitoring and analyses the data. H.T., K.G. and G.L. conducted histological preparations and observations. H.T., K.G. and G.L. conducted dissections and macroscopical observations. K.G. and G.L. performed flow cytometry experiments and K.G. analyses the data. J.C.S. provide both YR2 A. pisum lines. H.T. prepared the figures and wrote the first draft F.R., S.B., J.C.S., M.R.L., F.C. and P.D.S. reviewed and corrected the draft. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors would like to thank Hubert Charles for his suggestions about statistical analysis, and Aur\u0026eacute;lie Herbomez for secretarial assistance. This work was supported by INSA Lyon (Institut National des Sciences Appliqu\u0026eacute;es de Lyon), INRAE (Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement) and the French ANR BIOFAMILY (ANR-19-CE11-0004) program grant.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available in the supplementary materials\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgadi N, Maity A, Jha AK, et al (2022) Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides. Biochimica et Biophysica Acta (BBA) - Biomembranes 1864:184047. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbamem.2022.184047\u003c/span\u003e\u003cspan address=\"10.1016/j.bbamem.2022.184047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmeida CV, de Oliveira CFR, dos Santos EL, et al (2021) Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes. Biochimica et Biophysica Acta (BBA) - General Subjects 1865:129937. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbagen.2021.129937\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagen.2021.129937\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndersson DI, Hughes D, Kubicek-Sutherland JZ (2016) Mechanisms and consequences of bacterial resistance to antimicrobial peptides. Drug Resistance Updates 26:43\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.drup.2016.04.002\u003c/span\u003e\u003cspan address=\"10.1016/j.drup.2016.04.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArias-Rojas A, Frahm D, Hurwitz R, et al (2023) Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in \u003cem\u003eDrosophila\u003c/em\u003e. Proceedings of the National Academy of Sciences 120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.2305649120\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2305649120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArora AK, Douglas AE (2017) Hype or opportunity? Using microbial symbionts in novel strategies for insect pest control. Journal of Insect Physiology 103:10\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2017.09.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2017.09.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarass\u0026eacute; V, Jouvensal L, Boy G, et al (2023) Discovery of an insect neuroactive helix ring peptide from ant venom. Toxins 15:600. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins15100600\u003c/span\u003e\u003cspan address=\"10.3390/toxins15100600\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaumann P (2005) Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annual Review of Microbiology 59:155\u0026ndash;189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.micro.59.030804.121041\u003c/span\u003e\u003cspan address=\"10.1146/annurev.micro.59.030804.121041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenhamou S, Rahioui I, Henri H, et al (2021) Cytotype affects the capability of the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e MED species to feed and oviposit on an unfavorable host plant. mBio 12:e0073021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mBio.00730-21\u003c/span\u003e\u003cspan address=\"10.1128/mBio.00730-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisht DS, Bhatia V, Bhattacharya R (2019) Improving plant-resistance to insect-pests and pathogens: the new opportunities through targeted genome editing. Seminars in Cell \u0026amp; Developmental Biology 96:65\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.semcdb.2019.04.008\u003c/span\u003e\u003cspan address=\"10.1016/j.semcdb.2019.04.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraendle C, Miura T, Bickel R, et al (2003) Developmental origin and evolution of bacteriocytes in the aphid\u0026ndash;\u003cem\u003eBuchnera\u003c/em\u003e symbiosis. PLOS Biology 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.0000021\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.0000021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalatayud P-A (2000) Influence of linamarin and rutin on biological performances of \u003cem\u003ePhenacoccus manihoti\u003c/em\u003e in artificial diets. Entomologia Experimentalis et Applicata 96:81\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1570-7458.2000.00681.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1570-7458.2000.00681.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalevro F, Tagu D, Callaerts P (2019) \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Trends in Genetics 35:781\u0026ndash;782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tig.2019.07.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tig.2019.07.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharles H, Balmand S, Lamelas A, et al (2011) A genomic reappraisal of symbiotic function in the aphid/\u003cem\u003eBuchnera\u003c/em\u003e symbiosis: reduced transporter sets and variable membrane organisations. PLOS ONE 6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0029096\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0029096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCociancich S, Ghazi A, Hetru C, et al (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in \u003cem\u003eMicrococcus luteus\u003c/em\u003e. Journal of Biological Chemistry 268:19239\u0026ndash;19245. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0021-9258(19)36505-6\u003c/span\u003e\u003cspan address=\"10.1016/S0021-9258(19)36505-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarby AC, Douglas AE (2003) Elucidation of the transmission patterns of an insect-borne bacterium. Applied and Environmental Microbiology 69:4403\u0026ndash;4407. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.69.8.4403-4407.2003\u003c/span\u003e\u003cspan address=\"10.1128/AEM.69.8.4403-4407.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDedryver C-A, Le Ralec A, Fabre F (2010) The conflicting relationships between aphids and men: a review of aphid damage and control strategies. Comptes Rendus Biologies 333:539\u0026ndash;553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.crvi.2010.03.009\u003c/span\u003e\u003cspan address=\"10.1016/j.crvi.2010.03.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFebvay G, Delobel B, Rahb\u0026eacute; Y (1988) Influence of the amino acid balance on the improvement of an artificial diet for a biotype of \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e (Homoptera: Aphididae). Can J Zool 66:2449\u0026ndash;2453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/z88-362\u003c/span\u003e\u003cspan address=\"10.1139/z88-362\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrari J, Scarborough CL, Godfray HCJ (2007) Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids. Oecologia 153:323\u0026ndash;329. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00442-007-0730-2\u003c/span\u003e\u003cspan address=\"10.1007/s00442-007-0730-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrago E, Mala M, Weldegergis BT, et al (2017) Symbionts protect aphids from parasitic wasps by attenuating herbivore-induced plant volatiles. Nat Commun 8:1860. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-017-01935-0\u003c/span\u003e\u003cspan address=\"10.1038/s41467-017-01935-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonella E, Alma A (2023) The role of symbiont-targeted strategies in the management of Pentatomidae and Tephritidae pests under an integrated vision. Agronomy 13:868. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy13030868\u003c/span\u003e\u003cspan address=\"10.3390/agronomy13030868\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonella E, Orr\u0026ugrave; B, Marasco R, et al (2020) Disruption of host-symbiont associations for the symbiotic control and management of pentatomid agricultural pests\u0026mdash;a review. Front Microbiol 11:547031. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2020.547031\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.547031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoulson D, 232 signatories (2018) Call to restrict neonicotinoids. Science 360:973\u0026ndash;973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.aau0432\u003c/span\u003e\u003cspan address=\"10.1126/science.aau0432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreco I, Molchanova N, Holmedal E, et al (2020) Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides. Sci Rep 10:13206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-69995-9\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-69995-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGula LT (2023) Researchers helping protect crops from pests. NIFA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nifa.usda.gov/about-nifa/blogs/researchers-helping-protect-crops-pests\u003c/span\u003e\u003cspan address=\"https://www.nifa.usda.gov/about-nifa/blogs/researchers-helping-protect-crops-pests\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 26 Jun 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansen AK, Moran NA (2014) The impact of microbial symbionts on host plant utilization by herbivorous insects. Molecular Ecology 23:1473\u0026ndash;1496. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mec.12421\u003c/span\u003e\u003cspan address=\"10.1111/mec.12421\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanson MA, Dost\u0026aacute;lov\u0026aacute; A, Ceroni C, et al (2019) Synergy and remarkable specificity of antimicrobial peptides \u003cem\u003ein vivo\u003c/em\u003e using a systematic knockout approach. eLife 8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.44341\u003c/span\u003e\u003cspan address=\"10.7554/eLife.44341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigashi CHV, Nichols WL, Chevignon G, et al (2023) An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen. Molecular Ecology 32:936\u0026ndash;950. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mec.16801\u003c/span\u003e\u003cspan address=\"10.1111/mec.16801\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHlina BL, Birceanu O, Robinson CS, et al (2021) The relationship between thermal physiology and lampricide sensitivity in larval sea lamprey (\u003cem\u003ePetromyzon marinus\u003c/em\u003e). Journal of Great Lakes Research 47\u0026ndash;S284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jglr.2021.10.002\u003c/span\u003e\u003cspan address=\"10.1016/j.jglr.2021.10.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHo TNT, Turner A, Pham SH, et al (2023) Cysteine-rich peptides: from bioactivity to bioinsecticide applications. Toxicon 230:107173. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.toxicon.2023.107173\u003c/span\u003e\u003cspan address=\"10.1016/j.toxicon.2023.107173\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHogenhout SA, Ammar E-D, Whitfield AE, Redinbaugh MG (2008) Insect vector interactions with persistently transmitted viruses. Annual Review of Phytopathology 46:327\u0026ndash;359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.phyto.022508.092135\u003c/span\u003e\u003cspan address=\"10.1146/annurev.phyto.022508.092135\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJahan H, Khudr MS, Arafeh A, Hager R (2023) Exposure to heat stress leads to striking clone-specific nymph deformity in pea aphid. PLOS ONE 18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0282449\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0282449\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JK, Son DW, Kim C-H, et al (2015) Insect gut symbiont susceptibility to host antimicrobial peptides caused by alteration of the bacterial cell envelope. Journal of Biological Chemistry 290:21042\u0026ndash;21053. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M115.651158\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M115.651158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoga R, Tsuchida T, Sakurai M, Fukatsu T (2007) Selective elimination of aphid endosymbionts: effects of antibiotic dose and host genotype, and fitness consequences. FEMS Microbiology Ecology 60:229\u0026ndash;239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6941.2007.00284.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6941.2007.00284.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoga R, Meng X-Y, Tsuchida T, Fukatsu T (2012) Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte\u0026ndash;embryo interface. Proceedings of the National Academy of Sciences 109:E1230\u0026ndash;E1237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1119212109\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1119212109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomal J, Desai HR, Samal I, et al (2023) Unveiling the genetic symphony: harnessing CRISPR-Cas genome editing for effective insect pest management. Plants 12:3961. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants12233961\u003c/span\u003e\u003cspan address=\"10.3390/plants12233961\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKontsedalov S, Zchori-Fein E, Chiel E, et al (2008) The presence of \u003cem\u003eRickettsia\u003c/em\u003e is associated with increased susceptibility of \u003cem\u003eBemisia tabaci\u003c/em\u003e (Homoptera: Aleyrodidae) to insecticides. Pest Management Science 64:789\u0026ndash;792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.1595\u003c/span\u003e\u003cspan address=\"10.1002/ps.1595\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLachat J, Lextrait G, Jouan R, et al (2024) Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts. Proceedings of the National Academy of Sciences 121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.2401802121\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2401802121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe-Feuvre RR, Ram\u0026iacute;rez CC, Olea N, Meza-Basso L (2007) Effect of the antimicrobial peptide indolicidin on the green peach aphid \u003cem\u003eMyzus persicae\u003c/em\u003e (Sulzer). Journal of Applied Entomology 131:71\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1439-0418.2006.01117.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0418.2006.01117.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLemoine MM, Engl T, Kaltenpoth M (2020) Microbial symbionts expanding or constraining abiotic niche space in insects. Current Opinion in Insect Science 39:14\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cois.2020.01.003\u003c/span\u003e\u003cspan address=\"10.1016/j.cois.2020.01.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Fan J, Sun J, et al (2018) Plant-mediated horizontal transmission of \u003cem\u003eHamiltonella defensa\u003c/em\u003e in the wheat aphid \u003cem\u003eSitobion miscanthi\u003c/em\u003e. J Agric Food Chem 66:13367\u0026ndash;13377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.8b04828\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.8b04828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Sun J, Qin Y, et al (2021) Reduced insecticide susceptibility of the wheat aphid after infection by the secondary bacterial symbiont \u003cem\u003eHamiltonella defensa\u003c/em\u003e. Pest Management Science 77:1936\u0026ndash;1944. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.6221\u003c/span\u003e\u003cspan address=\"10.1002/ps.6221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLibbrecht R, Gwynn DM, Fellowes MDE (2007) \u003cem\u003eAphidius ervi\u003c/em\u003e preferentially attacks the green morph of the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. J Insect Behav 20:25\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10905-006-9055-y\u003c/span\u003e\u003cspan address=\"10.1007/s10905-006-9055-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoth K, Parisot N, Paquet F, et al (2022) Aphid BCR4 structure and activity uncover a new defensin peptide superfamily. International Journal of Molecular Sciences 23:12480. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms232012480\u003c/span\u003e\u003cspan address=\"10.3390/ijms232012480\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuna-Ramirez K, Skaljac M, Grotmann J, et al (2017) Orally delivered scorpion antimicrobial peptides exhibit activity against pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e) and its bacterial symbionts. Toxins 9:261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins9090261\u003c/span\u003e\u003cspan address=\"10.3390/toxins9090261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo C, Belghazi M, Schmitz A, et al (2021) Hosting certain facultative symbionts modulates the phenoloxidase activity and immune response of the pea aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Insect Science 28:1780\u0026ndash;1799. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1744-7917.12888\u003c/span\u003e\u003cspan address=\"10.1111/1744-7917.12888\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMan Y, Li D, Wang M, et al (2023) Indirect and direct interactions between grain aphid and parasitoid in the presence of symbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e. CABI Agriculture and Bioscience 4:59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43170-023-00202-1\u003c/span\u003e\u003cspan address=\"10.1186/s43170-023-00202-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMason AJ, Marquette A, Bechinger B (2007) Zwitterionic phospholipids and sterols modulate antimicrobial peptide-induced membrane destabilization. Biophysical Journal 93:4289\u0026ndash;4299. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1529/biophysj.107.116681\u003c/span\u003e\u003cspan address=\"10.1529/biophysj.107.116681\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez AJ, Doremus MR, Kraft LJ, et al (2018) Multi-modal defences in aphids offer redundant protection and increased costs likely impeding a protective mutualism. Journal of Animal Ecology 87:464\u0026ndash;477. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1365-2656.12675\u003c/span\u003e\u003cspan address=\"10.1111/1365-2656.12675\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMateos Fern\u0026aacute;ndez R, Petek M, Gerasymenko I, et al (2022) Insect pest management in the age of synthetic biology. Plant Biotechnology Journal 20:25\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13685\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13685\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiura T, Braendle C, Shingleton A, et al (2003) A comparison of parthenogenetic and sexual embryogenesis of the pea aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e (Hemiptera: Aphidoidea). Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 295B:59\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jez.b.3\u003c/span\u003e\u003cspan address=\"10.1002/jez.b.3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontllor CB, Maxmen A, Purcell AH (2002) Facultative bacterial endosymbionts benefit pea aphids \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e under heat stress. Ecological Entomology 27:189\u0026ndash;195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-2311.2002.00393.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2311.2002.00393.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoran NA, Russell JA, Koga R, Fukatsu T (2005) Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Applied and Environmental Microbiology 71:3302\u0026ndash;3310. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.71.6.3302-3310.2005\u003c/span\u003e\u003cspan address=\"10.1128/AEM.71.6.3302-3310.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoman MS, Liu L, Bai Z, Li Z (2020) Tephritidae bacterial symbionts: potentials for pest management. Bulletin of Entomological Research 110:1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0007485319000403\u003c/span\u003e\u003cspan address=\"10.1017/S0007485319000403\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliver KM, Russell JA, Moran NA, Hunter MS (2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proceedings of the National Academy of Sciences 100:1803\u0026ndash;1807. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0335320100\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0335320100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParker BJ, Hrček J, McLean AHC, Godfray HCJ (2017) Genotype specificity among hosts, pathogens, and beneficial microbes influences the strength of symbiont-mediated protection. Evolution 71:1222\u0026ndash;1231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/evo.13216\u003c/span\u003e\u003cspan address=\"10.1111/evo.13216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParker BJ, Spragg CJ, Altincicek B, Gerardo NM (2013) Symbiont-mediated protection against fungal pathogens in pea aphids: a role for pathogen specificity? Applied and Environmental Microbiology 79:2455\u0026ndash;2458. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.03193-12\u003c/span\u003e\u003cspan address=\"10.1128/AEM.03193-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeccoud J, Bonhomme J, Mah\u0026eacute;o F, et al (2014) Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes. Insect Science 21:291\u0026ndash;300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1744-7917.12083\u003c/span\u003e\u003cspan address=\"10.1111/1744-7917.12083\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePons I, Renoz F, No\u0026euml;l C, Hance T (2019a) Circulation of the cultivable symbiont \u003cem\u003eSerratia symbiotica\u003c/em\u003e in aphids is mediated by plants. Front Microbiol 10:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2019.00764\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.00764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePons I, Renoz F, No\u0026euml;l C, Hance T (2019b) New insights into the nature of symbiotic associations in aphids: infection process, biological effects, and transmission mode of cultivable \u003cem\u003eSerratia symbiotica\u003c/em\u003e bacteria. Applied and Environmental Microbiology 85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.02445-18\u003c/span\u003e\u003cspan address=\"10.1128/AEM.02445-18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahb\u0026eacute; Y, Febvay G (1993) Protein toxicity to aphids: an in vitro test on \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Entomologia Experimentalis et Applicata 67:149\u0026ndash;160. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1570-7458.1993.tb01663.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1570-7458.1993.tb01663.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahb\u0026eacute; Y, Febvay G, Delobel B, Bournoville R (1988) \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e performance in response to the sugar and amino acid composition of artificial diets, and its relation to lucerne varietal resistance. Entomologia Experimentalis et Applicata 48:283\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1570-7458.1988.tb01175.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1570-7458.1988.tb01175.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRenoz F (2024) The nutritional dimension of facultative bacterial symbiosis in aphids: current status and methodological considerations for future research. Current Research in Insect Science 5:100070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cris.2023.100070\u003c/span\u003e\u003cspan address=\"10.1016/j.cris.2023.100070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRenoz F, Pons I, Vanderpoorten A, et al (2019) Evidence for gut-associated \u003cem\u003eSerratia symbiotica\u003c/em\u003e in wild aphids and ants provides new perspectives on the evolution of bacterial mutualism in insects. Microb Ecol 78:159\u0026ndash;169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-018-1265-2\u003c/span\u003e\u003cspan address=\"10.1007/s00248-018-1265-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibeiro Lopes M, Gaget K, Renoz F, et al (2022) Bacteriocyte plasticity in pea aphids facing amino acid stress or starvation during development. Front Physiol 13:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2022.982920\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2022.982920\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibeiro Lopes M, Simonet P, Duport G, et al (2021) Isolation of insect bacteriocytes as a platform for transcriptomic analyses. Methods Mol Biol 2170:185\u0026ndash;198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-0716-0743-5_13\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-0743-5_13\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitz C, Baty F, Streibig JC, Gerhard D (2015) Dose-response analysis using R. PLOS ONE 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0146021\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0146021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRupawate PS, Roylawar P, Khandagale K, et al (2023) Role of gut symbionts of insect pests: a novel target for insect-pest control. Front Microbiol 14:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1146390\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1146390\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRussell JA, Moran NA (2005) Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures. Proceedings of the Royal Society B: Biological Sciences 273:603\u0026ndash;610. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2005.3348\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2005.3348\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSapountzis P, Duport G, Balmand S, et al (2014) New insight into the RNA interference response against cathepsin-L gene in the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e: molting or gut phenotypes specifically induced by injection or feeding treatments. Insect Biochemistry and Molecular Biology 51:20\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ibmb.2014.05.005\u003c/span\u003e\u003cspan address=\"10.1016/j.ibmb.2014.05.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScarborough CL, Ferrari J, Godfray HCJ (2005) Aphid protected from pathogen by endosymbiont. Science 310:1781\u0026ndash;1781. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1120180\u003c/span\u003e\u003cspan address=\"10.1126/science.1120180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShai Y (2002) Mode of action of membrane active antimicrobial peptides. Peptide Science 66:236\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/bip.10260\u003c/span\u003e\u003cspan address=\"10.1002/bip.10260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShigenobu S, Stern DL (2013) Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont. Proceedings of the Royal Society B: Biological Sciences 280:20121952. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2012.1952\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2012.1952\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShigenobu S, Watanabe H, Hattori M, et al (2000) Genome sequence of the endocellular bacterial symbiont of aphids \u003cem\u003eBuchnera\u003c/em\u003e sp. APS. Nature 407:81\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/35024074\u003c/span\u003e\u003cspan address=\"10.1038/35024074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon J-C, Boutin S, Tsuchida T, et al (2011) Facultative symbiont infections affect aphid reproduction. PLOS ONE 6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0021831\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0021831\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimonet P, Duport G, Gaget K, et al (2016) Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis. Sci Rep 6:19967. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep19967\u003c/span\u003e\u003cspan address=\"10.1038/srep19967\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimonet P, Gaget K, Balmand S, et al (2018) Bacteriocyte cell death in the pea aphid/\u003cem\u003eBuchnera\u003c/em\u003e symbiotic system. Proceedings of the National Academy of Sciences 115:E1819\u0026ndash;E1828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1720237115\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1720237115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinno M, B\u0026eacute;zier A, Vinale F, et al (2020) Symbiosis disruption in the olive fruit fly, \u003cem\u003eBactrocera oleae\u003c/em\u003e (Rossi), as a potential tool for sustainable control. Pest Management Science 76:3199\u0026ndash;3207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.5875\u003c/span\u003e\u003cspan address=\"10.1002/ps.5875\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkaljac M, Kirfel P, Grotmann J, Vilcinskas A (2018) Fitness costs of infection with \u003cem\u003eSerratia symbiotica\u003c/em\u003e are associated with greater susceptibility to insecticides in the pea aphid. Pest Management Science 74:1829\u0026ndash;1836. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.4881\u003c/span\u003e\u003cspan address=\"10.1002/ps.4881\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSochard C, Le Floch M, Anton S, et al (2021) Limited influence of gain and loss of symbionts on host plant selection in specialized pea aphid genotypes. Entomologia Generalis 39\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1127/entomologia/2020/1076\u003c/span\u003e\u003cspan address=\"10.1127/entomologia/2020/1076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith TE, Moran NA (2020) Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and \u003cem\u003eBuchnera\u003c/em\u003e. Proceedings of the National Academy of Sciences 117:2113\u0026ndash;2121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1916748117\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1916748117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuhag A, Yadav H, Chaudhary D, et al (2021) Biotechnological interventions for the sustainable management of a global pest, whitefly (\u003cem\u003eBemisia tabaci\u003c/em\u003e). Insect Science 28:1228\u0026ndash;1252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1744-7917.12853\u003c/span\u003e\u003cspan address=\"10.1111/1744-7917.12853\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTherneau T (2024). \u003cem\u003eA Package for Survival Analysis in R.\u003c/em\u003e R package version 3.7-0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://CRAN.R-project.org/package=survival\u003c/span\u003e\u003cspan address=\"https://CRAN.R-project.org/package=survival\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuchida T, Koga R, Fukatsu T (2004) Host plant specialization governed by facultative symbiont. Science 303:1989\u0026ndash;1989. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1094611\u003c/span\u003e\u003cspan address=\"10.1126/science.1094611\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuchida T, Koga R, Meng XY, et al (2005) Characterization of a facultative endosymbiotic bacterium of the pea aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Microb Ecol 49:126\u0026ndash;133. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-004-0216-2\u003c/span\u003e\u003cspan address=\"10.1007/s00248-004-0216-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuchida T, Koga R, Fujiwara A, Fukatsu T (2014) Phenotypic effect of \u0026ldquo;\u003cem\u003eCandidatus Rickettsiella viridis\u003c/em\u003e,\u0026rdquo; a facultative symbiont of the pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e), and its interaction with a coexisting symbiont. Applied and Environmental Microbiology 80:525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.03049-13\u003c/span\u003e\u003cspan address=\"10.1128/AEM.03049-13\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUchi N, Fukudome M, Nozaki N, et al (2019) Antimicrobial activities of cysteine-rich peptides specific to bacteriocytes of the pea aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Microbes and Environments 34:155\u0026ndash;160. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1264/jsme2.ME18148\u003c/span\u003e\u003cspan address=\"10.1264/jsme2.ME18148\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaucher RA, Teixeira ML, Brandelli A (2010) Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells. Curr Microbiol 60:1\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00284-009-9490-z\u003c/span\u003e\u003cspan address=\"10.1007/s00284-009-9490-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWernegreen JJ (2012) Endosymbiosis. Current Biology 22:R555\u0026ndash;R561. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2012.06.010\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2012.06.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWimley WC (2010) Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol 5:905\u0026ndash;917. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/cb1001558\u003c/span\u003e\u003cspan address=\"10.1021/cb1001558\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Q, Patočka J, Kuča K (2018) Insect antimicrobial peptides, a mini review. Toxins 10:461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins10110461\u003c/span\u003e\u003cspan address=\"10.3390/toxins10110461\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZytynska SE, Tighiouart K, Frago E (2021) Benefits and costs of hosting facultative symbionts in plant-sucking insects: A meta-analysis. Molecular Ecology 30:2483\u0026ndash;2494. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mec.15897\u003c/span\u003e\u003cspan address=\"10.1111/mec.15897\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aphididae, bacteriocyte-specific cysteine rich peptide, bio-insecticidal peptide, symbiont, pest management","lastPublishedDoi":"10.21203/rs.3.rs-5386556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5386556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAphids are major crop pests capable of colonizing the main plants grown for human consumption. They have specialized cells, the bacteriocytes, which house the obligatory symbionts \u003cem\u003eBuchnera aphidicola \u003c/em\u003ethat provide them with essential nutrients missing from their diet. Bacteriocyte-specific cysteine-rich peptides (BCRs) are encoded by a defensin gene family exclusively present in aphids and specifically expressed in bacteriocyte. One BCR family member, BCR4, has been shown to have insecticidal properties against the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e(Hemiptera: Aphididae). In the present study, we exposed the pea aphid to different doses of BCR4 and examined the impact on aphid survival, mass, anatomy, fecundity, as well as on bacterial symbiosis. As different pea aphid lines with various symbiotic status may be differently affected by stress, we investigated the effect of BCR4 ingestion on three different \u003cem\u003eA. pisum\u003c/em\u003e lines: LL01 and YR2-amp, that are mono-infected with \u003cem\u003eB. aphidicola\u003c/em\u003e, and YR2-\u003cem\u003eRi\u003c/em\u003e, that is genetically identical to YR2-amp but also contains the extracellular facultative symbionts \u003cem\u003eRegiella insecticola\u003c/em\u003e. Our results show a strong dose-response effect of BCR4 on LL01 survival and a more moderate effect on both YR2 lines, while an impact on the mass was observed in the three lines. Histological analyses revealed severe embryonic developmental defects due to the treatment. Finally, BCR4 treatment \u0026nbsp;reduced symbiont quantity, with \u003cem\u003eB. aphidicola\u003c/em\u003e being more affected than \u003cem\u003eR. insecticola\u003c/em\u003e. This study supports the idea that BCR4 could act as a key regulator of aphid symbiosis and development, and highlights its potential as a candidate bioinsecticide for pest control.\u003c/p\u003e","manuscriptTitle":"Strong impact of orally administered BCR4 defensin on aphid survival, embryo development and symbiotic cells in three Acyrthosiphon pisum parthenogenetic lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 19:09:04","doi":"10.21203/rs.3.rs-5386556/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-11T18:31:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-11T17:27:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177351112797268984498765705630459546220","date":"2025-02-10T01:14:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206538592264233261516263635281965427692","date":"2025-02-05T12:55:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-16T09:09:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171728674720364668854650292801249570219","date":"2025-01-10T10:19:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63753639022533406085408033561454299350","date":"2024-12-11T19:50:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36609906769263642012379451359867829643","date":"2024-12-05T19:44:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-18T10:57:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-09T07:42:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-09T07:41:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Pest Science","date":"2024-11-04T09:01:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"173f4853-5318-42b3-86d2-c580aeff364d","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:05:23+00:00","versionOfRecord":{"articleIdentity":"rs-5386556","link":"https://doi.org/10.1007/s10340-025-01923-0","journal":{"identity":"journal-of-pest-science","isVorOnly":false,"title":"Journal of Pest Science"},"publishedOn":"2025-06-18 15:57:53","publishedOnDateReadable":"June 18th, 2025"},"versionCreatedAt":"2024-11-27 19:09:04","video":"","vorDoi":"10.1007/s10340-025-01923-0","vorDoiUrl":"https://doi.org/10.1007/s10340-025-01923-0","workflowStages":[]},"version":"v1","identity":"rs-5386556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5386556","identity":"rs-5386556","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}