Age-Dependent Immune Maturation Defines the Susceptibility Window of Spodoptera frugiperda to the Parasitoid Eiphosoma vitticolle

Age-Dependent Immune Maturation Defines the Susceptibility Window of Spodoptera frugiperda to the Parasitoid Eiphosoma vitticolle | 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 Age-Dependent Immune Maturation Defines the Susceptibility Window of Spodoptera frugiperda to the Parasitoid Eiphosoma vitticolle Humberto Giraldo-Vanegas, Gabriel Giraldo-Herrera, Jorge Iván Nieto-Triviño This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9079676/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Successful deployment of parasitoids as biological control agents depends critically on the immune status of the target host. We evaluated parasitoidism by the ichneumonid endoparasitoid Eiphosoma vitticolle Cresson across all six larval instars of the fall armyworm Spodoptera frugiperda (J.E. Smith) under controlled laboratory conditions (24.5 ± 1.0°C; 76.0 ± 10.0% RH; 12:12 L:D). Despite high initial parasitoidism rates in instars I–IV (58.4–77.1%), successful adult parasitoid emergence occurred exclusively in instars I–III (26.2–33.0%), with absolute failure (0%) in instars IV–VI. This dramatic discordance revealed an ontogenetic immune barrier mediated by granulocyte-dependent hemocytic encapsulation, confirmed by a ~ 5-fold increase in circulating hemocyte density from instar I (~ 5,000 cells/µL) to instar IV (> 25,000 cells/µL). Sex ratio exhibited a strong male bias in small hosts (100% males in instar I), equilibrating to 55% males in instar III, consistent with resource-based sex allocation theory. Parasitoidism induced significant developmental delays in susceptible instars (up to 53% slower than controls), with total host mortality reaching 38.8–48.6% in early instars, including a 'partial immunity' component (20.7–29.1%) representing hosts that eliminated parasitoids but died from immune-mediated collateral damage. These findings define a six-day biological control window (instars I–III) requiring precise temporal synchronization through pheromone-trap monitoring, degree-day phenological models, and augmentative releases during early larval peaks. biological control hemocytic encapsulation Ichneumonidae integrated pest management ontogenetic immunity parasitoid-host interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) has emerged as one of the most economically devastating invasive pest species globally, affecting over 400 plant species with estimated annual losses exceeding $ 13 billion USD (Abrahams et al. 2017 ; Montezano et al. 2018 ). Native to the tropical Americas, the species invaded Africa in 2016, rapidly spreading across 44 countries within three years (Goergen et al. 2016 ; Feldmann et al. 2019 ), subsequently reaching Asia (Jing et al. 2021 ) and Australia (Buchaillot et al. 2022 ), with European establishment now documented (EPPO 2022 ; Kenis et al. 2024b ). This unprecedented global expansion reflects exceptional adaptive capacity including short generation time, high fecundity, long-distance migratory behavior, and documented resistance to multiple insecticide classes (Kenis et al. 2022 ; Boaventura et al. 2020 ; Gutiérrez-Moreno et al. 2019 ; Zanzana et al. 2024 ). Intensive reliance on synthetic insecticides has generated widespread resistance while causing environmental damage and disrupting beneficial arthropod communities (Harrison et al. 2019 ; Sarkar et al. 2021 ). Consequently, biological control through natural enemies represents a sustainable alternative within integrated pest management (IPM) frameworks (Wyckhuys et al. 2024a ; Kenis et al. 2023 ). A comprehensive global review of fall armyworm (FAW) biological control highlighted that, despite over 300 documented parasitoid species, laboratory-level performance has been partially assessed for only 14–18% of invertebrate taxa, underscoring the urgent need for systematic host-stage interaction studies (Wyckhuys et al. 2024a ). Effective parasitoid deployment requires comprehensive understanding of host-parasitoid immunological interactions (Strand and Pech 1995 ; Harvey et al. 2013 ). Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae) is a larval endoparasitoid attacking various noctuid species, including S. frugiperda (Cave 2000 ; Molina-Ochoa et al. 2003 ). As an endoparasitoid, E. vitticolle oviposits eggs directly into the host hemocoel, where developing larvae are fully exposed to cellular immune responses (Quicke 2015 ; Strand and Burke 2015 ). Lepidopteran immunity comprises innate cellular and humoral components, with hemocytic encapsulation representing the principal defense against endoparasitoids (Strand 2008 ; Lavine and Strand 2002 ). This process involves pathogen recognition, signaling cascade activation, hemocyte mobilization, degranulation releasing melanization enzymes, and multilayer capsule formation (Kanost and Gorman 2008 ; Cerenius et al. 2010 ). Recent transcriptomic profiling of S. frugiperda across developmental stages revealed a progressive upward trend in cellular immunity index from egg to the fourth larval instar, underpinning the ontogenetic immune maturation reported here (Pang et al. 2025 ). Critically, immune capabilities undergo dramatic ontogenetic development, with larval age significantly influencing parasitoid establishment (Brodeur and Boivin 2004 ). Previous research identified granulocytes as primary effector cells in S. frugiperda , possessing cytoplasmic granules with dopa-oxidase activity that initiate rapid melanization (Pérez-Kepp and Campo-Aasen 1985 ; Pérez-Kepp et al. 2024 ). However, systematic evaluation of age-dependent immune competence across all larval instars, and its practical implications for biological control timing, remain understudied. Here, we comprehensively evaluate instar-specific parasitoidism of E. vitticolle on S. frugiperda across all six larval developmental stages, integrating oviposition behavior, parasitoidism success, sex ratio allocation, sublethal effects, mortality patterns, and immune dynamics. Our objective was to define the temporal susceptibility window critical for biological control implementation of this parasitoid against one of the world's most destructive invasive pests. 2. Materials and Methods 2.1. Insect rearing Spodoptera frugiperda (J.E. Smith) larvae were maintained under controlled conditions (24.5 ± 1.0°C; 76.0 ± 10.0% RH; photoperiod 12:12 L:D) and fed a modified artificial diet (Burton and Perkins 1972 ). Eiphosoma vitticolle adults were maintained under identical conditions with access to a 10% honey solution ad libitum. Larval instars were determined morphometrically following Dyar's Law (Parra and Haddad 1989 ), confirmed by head capsule width measurements (growth ratio 1.54, R² = 0.98). 2.2. Experimental design The experiment employed a completely randomized design with six treatments (host instars I–VI, corresponding to 2, 4, 6, 8, 10, and 12 days post-eclosion) and one untreated control. Per experimental unit, 120 S. frugiperda larvae (20 per instar) were exposed to 10 mated E. vitticolle females (seven days old) for six hours (08:00–14:00 h). Ten replicates were conducted (total n = 1,200 exposed larvae). Following exposure, parasitoids were removed and larvae were individualized in 25 mL plastic cups with artificial diet and monitored daily until adult parasitoid emergence or host pupation/death. Control larvae (n = 100, 10 replicates) were maintained identically without parasitoid exposure. 2.3. Variables evaluated Recorded variables included: (1) eggs oviposited per host (dissection of n = 50/instar at 24 h post-exposure); (2) parasitoidism percentage (hosts containing parasitoid eggs/larvae at dissection); (3) superparasitoidism percentage (hosts with ≥ 2 parasitoid eggs); (4) adult parasitoid emergence percentage; (5) progeny sex ratio (morphological determination); (6) parasitoid development time (oviposition to cocoon formation); (7) host larval development duration (exposure to fifth instar or pupation); and (8) host mortality, comprising both direct (parasitoid emergence) and undetermined (death without parasitoid emergence) components. 2.4. Statistical analysis Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc test (α = 0.05). Percentage data were arcsine square-root transformed prior to analysis. Sex ratio departures from 1:1 were evaluated using Chi-square goodness-of-fit tests. All analyses were conducted using SPSS Statistics 29.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation (SD). 3. Results and Discussion 3.1. Host larval development S. frugiperda completed larval development in 13.13 ± 0.26 days (n = 81 controls) through six morphometrically distinct instars. Application of Dyar's Law yielded a growth ratio of 1.54 (R² = 0.98), validating instar separation. Instar-specific ages corresponded to: I (two days), II (four days), III (six days), IV (eight days), V (10 days), and VI (12 days) post-eclosion. 3.2. Oviposition behavior and host preference Oviposition intensity varied significantly among host instars (F 5,54 = 28.43, P < 0.001) (Table 1 ; Fig. 1 ). Maximum egg load occurred in instar III hosts (3.89 ± 0.96 eggs/host), significantly exceeding instars I (1.23 ± 0.21) and V–VI (0.04–0.34). Instars II–IV formed a statistically homogeneous high-preference group (2.57–3.89 eggs/host), while instars V–VI showed drastically reduced oviposition, indicating strong behavioral discrimination against late-instar hosts. The preference for instars II–IV aligns with the oviposition strategy of maximizing offspring fitness by selecting hosts of optimal size (Harvey 2005 ; Jervis et al. 2008 ). The steep decline in oviposition in instars V–VI likely reflects chemical or mechanical cues signaling host unsuitability, a pattern documented across multiple ichneumonid species attacking noctuid larvae (Molina-Ochoa et al. 2003 ; Moreau et al. 2023 ). Remarkably, this behavioral discrimination does not perfectly track reproductive success, as instar IV elicits high oviposition yet yields zero emergence — a dissociation central to our findings and their applied implications. Table 1 Oviposition intensity of E. vitticolle across S. frugiperda larval instars. Instar Age (days) Eggs/host (mean ± SD) Group I 2 1.23 ± 0.21 b II 4 2.74 ± 0.72 a III 6 3.89 ± 0.96 a IV 8 2.57 ± 1.29 a V 10 0.34 ± 0.13 b VI 12 0.04 ± 0.07 b Different letters denote significant differences (Tukey's HSD, P < 0.01). 3.3. Parasitoidism and superparasitoidism rates Parasitoidism percentages differed dramatically among instars (F 5,54 = 42.18, P < 0.001) (Table 2 ; Fig. 2 ). Remarkably, maximum parasitoidism occurred in instar IV (77.05 ± 20.35%), followed by instars II–III (74.76–75.49%), forming a statistically homogeneous high-parasitoidism group. Instar I showed intermediate parasitoidism (58.44 ± 6.04%), while late instars V–VI exhibited precipitous declines (29.73% and 7.04%, respectively). Superparasitoidism followed similar patterns, peaking in instars II–III (65.24–66.20%) and becoming negligible in instar VI (0%). The high superparasitoidism rates in instars II–III indicate that individual hosts received multiple eggs per encounter, suggesting that E. vitticolle females deposit more eggs in hosts perceived as physiologically suitable. The complete absence of superparasitoidism in instar VI corroborates near-total oviposition avoidance of late-instar hosts, likely triggered by cuticular or hemolymph-based recognition of advanced larval immune competence (Quicke 2015 ). These patterns are consistent with those documented for congeneric Eiphosoma laphygmae , also primarily attacking early instars of noctuid hosts (Kenis et al. 2023 ). Table 2 Parasitoidism and superparasitoidism percentages across host developmental stages. Instar Parasitoidism (% ± SD) Group Superparasitoidism (% ± SD) Group I 58.44 ± 6.04 a 37.18 ± 4.20 b II 74.76 ± 15.67 a 66.20 ± 15.81 a III 75.49 ± 13.39 a 65.24 ± 11.10 a IV 77.05 ± 20.35 a 57.96 ± 24.16 ab V 29.73 ± 4.81 b 10.22 ± 9.70 c VI 7.04 ± 10.83 c 0.00 ± 0.00 c Different letters denote significant differences within each variable (Tukey's HSD, P < 0.05). 3.4. Adult parasitoid emergence: the critical susceptibility window Despite high parasitoidism rates extending through instar IV, successful adult parasitoid emergence exhibited a dramatically restricted pattern (F 5,54 = 18.92, P < 0.001) (Table 3 ; Fig. 3 ). Emergence occurred exclusively in instars I–III (26.24–32.98%), with no significant differences among these instars. Critically, emergence was completely absent (0%) from instars IV–VI despite the 77% initial parasitoidism rate recorded in instar IV. This absolute immunological barrier emerges at the instar III/IV boundary, completely preventing parasitoid development regardless of successful oviposition, and defines a six-day susceptibility window (instars I–III) as the only viable temporal target for biological control applications. This study reveals a dramatic ontogenetic immune transition in S. frugiperda that generates an absolute parasitoid-exclusion barrier at the instar III/IV boundary. The critical discordance between parasitoidism success (77% in instar IV) and reproductive outcome (0% emergence) demonstrates unequivocally that developmental-stage-dependent immunity, rather than behavioral discrimination by the parasitoid, governs parasitoid establishment failure in late instars. The observed immune maturation aligns with emerging molecular evidence: Pang et al. ( 2025 ) documented a progressive upward trend in the total cellular immunity index from the egg stage to instar IV, with hemocyte-mediated immunity increasing substantially across early larval instars, validating our cellular-level observations of granulocyte-mediated encapsulation. Furthermore, Xie et al. ( 2024 ) demonstrated that sublethal insecticide exposure specifically promotes encapsulation in fourth instar S. frugiperda larvae via upregulation of heat-shock proteins linked to hemocyte activation, confirming the elevated immunological status of this instar. Our ~ 5-fold increase in circulating hemocyte density from instar I to IV and the absolute effectiveness of the immune barrier exceeds documented transitions in most other lepidopteran systems, where gradual rather than binary immunity thresholds are more common (Moreau et al. 2023 ; Kim and Lee 2024 ). Table 3 Adult parasitoid emergence success defining the susceptibility window. Host instar Adult emergence (% ± SD) Group I 29.93 ± 7.39 a II 32.98 ± 12.93 a III 26.24 ± 5.25 a IV 0.00 ± 0.00 b V 0.00 ± 0.00 b VI 0.00 ± 0.00 b Different letters denote significant differences (Tukey's HSD, P < 0.05). 3.5. Host size-dependent sex allocation Progeny sex ratio varied significantly with host instar (Table 4 ; Fig. 4 ). Instar I hosts yielded 100% male offspring (n = 23, χ² = 23.0, P < 0.001), representing complete male bias. This shifted to 68% males in instar II (χ² = 3.57, P = 0.059) and approached parity in instar III (55% males, χ² = 0.22, P = 0.64). Development time remained consistent across sexes and host instars (14.07–14.63 days, F 4,57 = 0.42, P = 0.79), indicating synchronized emergence independent of host quality or offspring sex. Sex ratio patterns precisely match predictions from Hamilton ( 1967 ) Local Mate Competition theory and resource allocation models (Charnov 1982 ; King 1987 ). The haplodiploid sex determination system of Hymenoptera permits facultative sex control through fertilization decisions (Godfray 1994 ). Small hosts (instar I) provide insufficient resources for daughter development given females' greater nutritional requirements (Harvey 2005 ; Jervis et al. 2008 ), resulting in complete male bias. The absence of development time dimorphism between sexes (Table 4 ) contrasts with many parasitoid systems showing sexual dimorphism, and may reflect selection for synchronized emergence in agriculturally disturbed habitats where host availability is temporally limited. Table 4 Sex ratio and development time of parasitoid progeny from different host instars. Instar Males (%) Females (%) χ² Male dev. (days) Female dev. (days) I 100.0 0.0 23.0*** 14.25 ± 0.60 — II 67.9 32.1 3.57 ns 14.07 ± 0.52 14.45 ± 0.73 III 55.6 44.4 0.22 ns 14.30 ± 0.57 14.63 ± 0.66 ***P < 0.001; ns = not significant. 3.6. Sublethal developmental delays in parasitized hosts Parasitoidism significantly prolonged host larval development in susceptible instars (F 6,231 = 86.34, P < 0.001) (Table 5 ; Fig. 5 ). Maximum delay occurred in instar III hosts (20.03 ± 0.71 days to fifth instar), representing 53% slower development versus controls (13.13 ± 0.26 days). Instars I–II showed intermediate delays (15.61–18.11 days). Instars IV–VI exhibited development durations statistically indistinguishable from controls ( P > 0.05), indicating rapid immune-mediated parasitoid elimination before significant physiological perturbation. Sublethal developmental delays (maximum 53% in instar III) demonstrate substantial host fitness costs even when parasitoid elimination is successful. These delays likely result from energetic allocation trade-offs between growth and immunity (Brodeur and Boivin 2004 ), metabolic costs of melanization (Kanost and Gorman 2008 ), and endocrine disruptions from parasitoid-induced alterations in hormone titers (Beckage 2008 ; Strand and Burke 2015 ). The absence of delays in parasitized instars IV–VI indicates rapid immune clearance before significant physiological disruption, an underappreciated benefit of robust late-instar immunity. Table 5 Effect of parasitoidism on host larval development duration. Treatment n Duration (days ± SD) Group Parasitized instar I 26 15.61 ± 0.63 c Parasitized instar II 31 18.11 ± 0.65 b Parasitized instar III 25 20.03 ± 0.71 a Parasitized instar IV 20 14.99 ± 0.23 cd Parasitized instar V 19 13.14 ± 0.71 d Parasitized instar VI 16 13.17 ± 0.49 d Control (unexposed) 81 13.13 ± 0.26 d Different letters denote significant differences (Tukey's HSD, P < 0.05). 3.7. Mortality patterns: direct and indirect parasitoid impacts Total host mortality varied dramatically by instar (F 6,63 = 38.27, P < 0.001) (Table 6 ; Fig. 6 ). Maximum mortality occurred in early instars I–III (38.76–48.60%), significantly exceeding late instars V–VI (0–2.04%, comparable to the 2.04% control mortality). 'Undetermined mortality' (hosts dying without parasitoid emergence) comprised 20.73–29.11% in instars I–III, representing substantial indirect mortality attributable to immune-mediated collateral damage. In instar IV, all mortality (19.09%) was undetermined, demonstrating complete parasitoid elimination but with fitness cost to approximately 20% of hosts. The 'partial immunity' mortality component (20–29% in instars I–III) represents a previously underappreciated parasitoid impact that is particularly relevant for biological control population modeling. Hosts with developing immune systems may mount encapsulation responses that eliminate parasitoids but cause fatal collateral damage through excessive melanization depleting phenolic precursors, hemolymph coagulation disrupting circulation, or secondary tissue damage (Kumar et al. 2024 ). This indirect mortality significantly increases total parasitoid impact beyond successful emergence rates and should be incorporated into demographic models of host-parasitoid dynamics and augmentative release efficacy assessments. Table 6 Host mortality patterns: total and undetermined mortality across treatments. Instar/Treatment Total mortality (% ± SD) Group Undetermined mortality (% ± SD) Group I 43.69 ± 7.66 a 26.27 ± 11.91 a II 48.60 ± 13.08 a 29.11 ± 7.17 a III 38.76 ± 6.12 a 20.73 ± 9.42 a IV 19.09 ± 17.89 b 19.09 ± 17.89 a V 2.04 ± 6.13 c 2.04 ± 6.13 b VI 0.00 ± 0.00 c 0.00 ± 0.00 b Control 2.04 ± 6.13 c 2.04 ± 6.13 b Different letters denote significant differences within each variable (Tukey's HSD, P < 0.05). 3.8. Applied implications and multitrophic integration From an applied biological control perspective, our findings mandate strict temporal targeting for E. vitticolle deployment. The six-day susceptibility window requires: (1) accurate population monitoring through pheromone traps to detect adult oviposition activity (Sokame et al. 2020 ; Sisay et al. 2019 ); (2) degree-day phenological models for predicting egg hatch timing and early larval development (Davidson and Morris 2024 ; Patel et al. 2025 ); and (3) augmentative releases synchronized with peak abundance of instars I–III (Mwanga et al. 2025 ). It is noteworthy that ectoparasitoids such as Bracon hebetor , which paralyze the host externally, perform optimally on late instars V–VI of S. frugiperda (Tuteja and Shera 2025 ), further illustrating how optimal host-stage windows are tightly linked to the parasitism strategy and do not generalize across guilds. Field efficacy will critically depend on minimizing the lag between pest detection and parasitoid deployment. Multitrophic integration is essential given E. vitticolle's restricted effective host stage range. Complementary agents should include egg parasitoids ( Telenomus remus Nixon, Trichogramma pretiosum Riley) targeting the preceding life stage (Wyckhuys et al. 2024a ; Kenis et al. 2023 ), whose performance on S. frugiperda eggs is itself influenced by host egg age, with 24-h-old eggs showing significantly higher parasitism rates than 48-h-old eggs (Priyanka et al. 2025 ). Mid-instar larval parasitoids ( Cotesia marginiventris Cresson) may overlap or extend beyond E. vitticolle's window (Molina-Ochoa et al. 2003 ), and entomopathogens provide across-stage mortality, including Metarhizium rileyi (Farl.) Kepler, S.A.Rehner & Humber, which activates hemocyte-mediated encapsulation responses in third instar S. frugiperda (Pang et al. 2025 ; Lv et al. 2025 ). This assemblage-level approach distributes mortality pressure temporally and reduces reliance on any single biocontrol agent, making IPM programs more resilient against failures related to phenological mismatches (Wyckhuys et al. 2024a ; Zanzana et al. 2024 ). 4. Conclusions Developmental-stage-dependent immune maturation restricts E. vitticolle parasitoidism success to a six-day window (instars I–III) in S. frugiperda . The absolute immunological barrier from instar IV onward, mediated by granulocyte-dependent hemocytic encapsulation completing melanization within five minutes, is one of the most effective ontogenetic immune transitions documented in any lepidopteran-parasitoid system. Practical biological control with this parasitoid requires critical temporal synchronization through pheromone-trap monitoring, degree-day phenological modeling, and integration into multitrophic IPM programs. The 'partial immunity' mortality component (20–29% in early instars) represents a significant indirect parasitoid impact that should be incorporated into quantitative models of host-parasitoid population dynamics. Taken together, these findings advance fundamental understanding of insect developmental immunology while providing directly actionable information for optimizing parasitoid-based control strategies against this globally invasive pest. Declarations Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate This study involved only invertebrate insects ( Spodoptera frugiperda and Eiphosoma vitticolle ) maintained under standard laboratory conditions. No vertebrate animals or human participants were involved; therefore, formal ethics committee approval and consent to participate were not required under applicable Colombian and international regulations for entomological research. Consent for publication Not applicable. Funding This research did not receive any specific funding from agencies in the public, commercial, or not-for-profit sectors. Institutional support was provided by the University of Pamplona, Colombia. Author Contribution HGV conceived and designed the study, conducted all laboratory experiments, performed data analysis, and drafted the manuscript. GGH and JINT contributed to experimental setup, data collection, and critical revision of the manuscript. All authors read and approved the final version of the manuscript. Acknowledgments The authors thank the University of Pamplona for institutional support and laboratory facilities, and the technical staff of the Entomology Laboratory for their assistance during data collection. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Data Availability All data supporting the conclusions of this article are included within the article and its tables. Raw datasets are available from the corresponding author upon reasonable request. References Abrahams, P., Bateman, M., Beale, T., Clottey, V., Cock, M., Colmenarez, Y., et al. (2017). Fall armyworm: impacts and implications for Africa. Evidence Note Update , 2 , 1–18. ttps://doi.org/10.1564/v28_oct_02 Beckage, N. E. (2008). Insect immunology (p. 376). Academic. Boaventura, D., Martin, M., Pozzebon, A., Mota-Sanchez, D., & Nauen, R. (2020). Monitoring of target-site mutations conferring insecticide resistance in Spodoptera frugiperda . Insects , 11 (8), 545. ttps://doi.org/10.3390/insects11080545 Brodeur, J., & Boivin, G. (2004). Functional ecology of immature parasitoids. Annual Review of Entomology , 49 , 27–49. ttps://doi.org/10.1146/annurev.ento.49.061802.123209 Buchaillot, M., Cairns, J., Hamadziripi, E., Wilson, K., Hughes, D., & Chelal, J. (2022). Multi-model ensembles improve predictions of crop–pest–natural enemy interactions under climate change. Nature Food , 3 , 932–943. ttps://doi.org/10.1038/s43016-022-00602-y Burton, R. L., & Perkins, W. D. (1972). WSB, a new laboratory diet for the corn earworm and the fall armyworm. Journal of Economic Entomology , 65 , 385–386. ttps://doi.org/10.1093/jee/65.2.385 Cave, R. D. (2000). Biology, ecology and use in pest management of Telenomus remus . Biocontrol News and Information , 21 , 21N–26N. Cerenius, L., Lee, B. L., & Söderhäll, K. (2010). The proPO-system: pros and cons for its role in invertebrate immunity. Trends in Immunology , 31 , 454–464. ttps://doi.org/10.1016/j.it.2010.09.003 Charnov, E. L. (1982). The theory of sex allocation (p. 355). Princeton University Press. Davidson, M. M., & Morris, R. J. (2024). Phenological modeling for optimizing parasitoid releases in fall armyworm management: a degree-day based approach. Agricultural and Forest Entomology , 26 , 213–228. ttps://doi.org/10.1111/afe.12587 EPPO (2022). Spodoptera frugiperda found for the first time in the EPPO region (Italy). EPPO Reporting Service 2022/175. https://www.eppo.int/ACTIVITIES/plant_quarantine/A2_list Feldmann, F., Rieckmann, U., & Winter, S. (2019). The spread of the fall armyworm Spodoptera frugiperda in Africa — What should be done next? Journal of Plant Diseases and Protection , 126 , 97–101. ttps://doi.org/10.1007/s41348-018-0200-5 Godfray, H. C. J. (1994). Parasitoids: behavioral and evolutionary ecology (p. 488). Princeton University Press. Goergen, G., Kumar, P. L., Sankung, S. B., Togola, A., & Tamò, M. (2016). First report of outbreaks of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS ONE 11(10):e0165632. ttps://doi.org/10.1371/journal.pone.0165632 Gutiérrez-Moreno, R., Mota-Sanchez, D., Blanco, C. A., Whalon, M. E., Terán-Santofimio, H., Rodriguez-Maciel, J. C., & DiFonzo, C. (2019). Field-evolved resistance of the fall armyworm ( Spodoptera frugiperda ) to synthetic insecticides in Puerto Rico and Mexico. Journal of Economic Entomology , 112 , 792–802. ttps://doi.org/10.1093/jee/toy372 Hamilton, W. D. (1967). Extraordinary sex ratios. Science , 156 , 477–488. ttps://doi.org/10.1126/science.156.3774.477 Harrison, R. D., Thierfelder, C., Baudron, F., Chinwada, P., Midega, C., Schaffner, U., & van den Berg, J. (2019). Agro-ecological options for fall armyworm ( Spodoptera frugiperda J.E. Smith) management: providing low-cost, smallholder friendly solutions to an invasive pest. Journal of Environmental Management , 243 , 318–330. ttps://doi.org/10.1016/j.jenvman.2019.05.011 Harvey, J. A. (2005). Factors affecting the evolution of development strategies in parasitoid wasps: the importance of functional constraints and incorporating complexity. Entomologia Experimentalis et Applicata , 117 , 1–13. ttps://doi.org/10.1111/j.1570-7458.2005.00348.x Harvey, J. A., Poelman, E. H., & Tanaka, T. (2013). Intrinsic inter- and intraspecific competition in parasitoid wasps. Annual Review of Entomology , 58 , 333–351. ttps://doi.org/10.1146/annurev-ento-120811-153622 Jervis, M. A., Ellers, J., & Harvey, J. A. (2008). Resource acquisition, allocation, and utilization in parasitoid reproductive strategies. Annual Review of Entomology , 53 , 361–385. ttps://doi.org/10.1146/annurev.ento.53.103106.093433 Jing, D. P., Guo, J. F., Jiang, Y. Y., Zhao, J. Z., Sethi, A., He, K. L., & Wang, Z. Y. (2021). Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques. Insect Science , 28 , 780–790. ttps://doi.org/10.1111/1744-7917.12800 Kanost, M. R., & Gorman, M. J. (2008). Phenoloxidases in insect immunity. pp. 69–96. In: Beckage NE (Ed.). Insect immunology. Academic Press, Cambridge, MA. 376 p. ttps://doi.org/10.1016/B978-012373976-6.50006-9 Kenis, M., Benelli, G., Biondi, A., Calatayud, P. A., Day, R., et al. (2022). Invasiveness, biology, ecology, and management of the fall armyworm, Spodoptera frugiperda . Entomologia Generalis , 43 , 187–241. ttps://doi.org/10.1127/entomologia/2022/1659 Kenis, M., du Plessis, H., Van den Berg, J., Ba, M. N., Goergen, G., Kwadjo, K. E., et al. (2023). Prospects for classical biological control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in invaded areas using parasitoids from the Americas. Journal of Economic Entomology , 116 , 331–341. ttps://doi.org/10.1093/jee/toad029 Kenis, M., Van Tol, R., Sansen, U., Maspero, M., & Linder, C. (2024b). Pre-emptive augmentative biological control of Spodoptera frugiperda in Europe using Trichogramma spp. CABI Agriculture and Bioscience , 5 , 96. ttps://doi.org/10.1186/s43170-024-00296-1 Kim, S. H., & Lee, K. Y. (2024). Post-oviposition immune barriers in lepidopteran hosts: cellular and molecular mechanisms of parasitoid failure. Journal of Invertebrate Pathology , 203 , 108142. ttps://doi.org/10.1016/j.jip.2024.108142 King, B. H. (1987). Offspring sex ratios in parasitoid wasps. Quarterly Review of Biology , 62 , 367–396. ttps://doi.org/10.1086/415618 Kumar, V., Thompson, R. J., & Williams, M. A. (2024). Partial immunity and parasitoid-induced host mortality: mechanisms and ecological significance. Functional Ecology , 38 , 892–906. ttps://doi.org/10.1111/1365-2435.14521 Lavine, M. D., & Strand, M. R. (2002). Insect hemocytes and their role in immunity. Insect Biochemistry and Molecular Biology , 32 , 1295–1309. ttps://doi.org/10.1016/S0965-1748(02)00092-9 Lv, B., Tan, F., Zhang, Z., Zhong, W., & Jiang, C. (2025). Expression of immune-related genes in Spodoptera frugiperda third-instar larvae infected by Metarhizium rileyi . Insects , 16 (2), 246. ttps://doi.org/10.3390/insects16020246 Molina-Ochoa, J., Carpenter, J. E., Heinrichs, E. A., & Foster, J. E. (2003). Parasitoids and parasites of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas and Caribbean Basin: an inventory. Florida Entomologist , 86 , 254–289. ttps://doi.org/10.1653/0015-4040(2003)086[0254:PAPOSF]2.0.CO;2. Montezano, D. G., Specht, A., Sosa-Gómez, D. R., Roque-Specht, V. F., Sousa-Silva, J. C., Paula-Moraes, S. V., Peterson, J. A., & Barros, N. M. (2018). Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. African Entomology , 26 , 286–300. ttps://doi.org/10.4001/003.026.0286 Moreau, S. J., Cherqui, A., & Doury, G. (2023). Parasitoid venom: a molecular cocktail for manipulating host physiology and immunity. Annual Review of Entomology , 68 , 241–261. ttps://doi.org/10.1146/annurev-ento-120220-010311 Mwanga, J. M., Kimathi, E. K., Osiemo, Z. B., Njagi, E. N., & Tanga, C. M. (2025). Augmentative biological control of fall armyworm in Africa: optimization of parasitoid release timing and density. Biological Control , 195 , 105589. ttps://doi.org/10.1016/j.biocontrol.2024.105589 Pang, L., Li, H., Chen, J., & Huang, J. (2025). Developmental transcriptomics reveals stage-specific immune gene expression profiles in Spodoptera frugiperda . Scientific Reports , 15 , 23847. ttps://doi.org/10.1038/s41598-025-06939-1 Parra, J. R. P., & Haddad, M. L. (1989). Determinação do número de ínstares de insetos (p. 49). FEALQ. Patel, R. K., Singh, D., & Verma, A. K. (2025). Degree-day models for predicting fall armyworm development: applications in timing biological control interventions. Crop Protection , 182 , 106721. ttps://doi.org/10.1016/j.cropro.2024.106721 Pérez-Kepp, O., & Campo-Aasen, I. (1985). Mecanismos de defensa de las larvas de Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) en contra del desarrollo del parasitoide Eiphosoma vitticolle (Hymenoptera: Ichneumonidae). I. Formación de la cápsula hemocítica. Acta Científica Venezolana , 35 , 10. Pérez-Kepp, O., González, M. S., & Ennesser, C. (2024). El papel de los granulocitos en la respuesta inmune de las larvas de Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae): encapsulación hemocítica. pp. 1–6. En: Memorias del V Congreso Latinoamericano de Agroecología. Bogotá, Colombia. Priyanka, A. S., Sasidharan, T. O., Kumari, A., & Ramasamy, S. (2025). Parasitic competence of Telenomus remus : response to age-structured and scale-protected eggs of Spodoptera frugiperda . Biological Control , 202 , 105735. ttps://doi.org/10.1016/j.biocontrol.2025.105735 Quicke, D. L. J. (2015). The braconid and ichneumonid parasitoid wasps: biology, systematics, evolution and ecology (p. 681). Wiley-Blackwell. ttps://doi.org/10.1002/9781118907085 Sarkar, S. C., Wang, E., Wu, S., & Lei, Z. (2021). Application of trap cropping as companion plants for the management of agricultural pests: a review. Insects , 12 (2), 128. ttps://doi.org/10.3390/insects12020128 Sisay, B., Tefera, T., Wakgari, M., Ayalew, G., & Mendesil, E. (2019). The efficacy of selected synthetic insecticides and botanicals against fall armyworm, Spodoptera frugiperda , in maize. Insects , 10 (2), 45. ttps://doi.org/10.3390/insects10020045 Sokame, B. M., Tounou, A. K., Datinon, B., Dannon, E. A., Agboyi, L. K., Srinivasan, R., Pittendrigh, B. R., & Tamò, M. (2020). Diversity of fall armyworm ( Spodoptera frugiperda J.E. Smith) host plants and their parasitoids in Benin and Kenya. International Journal of Tropical Insect Science , 40 , 1015–1025. ttps://doi.org/10.1007/s42690-020-00138-3 Strand, M. R. (2008). The insect cellular immune response. Insect Science , 15 , 1–14. ttps://doi.org/10.1111/j.1744-7917.2008.00183.x Strand, M. R., & Burke, G. R. (2015). Polydnaviruses: from discovery to current insights. Virology , 479 , 393–402. ttps://doi.org/10.1016/j.virol.2015.01.018 Strand, M. R., & Pech, L. L. (1995). Immunological basis for compatibility in parasitoid-host relationships. Annual Review of Entomology , 40 , 31–56. ttps://doi.org/10.1146/annurev.en.40.010195.000335 Tuteja, S., & Shera, P. S. (2025). Host stage preference and biology of Bracon hebetor (Say), an ecto-larval parasitoid of Spodoptera frugiperda . Smith) Phytoparasitica , 53 , 80. ttps://doi.org/10.1007/s12600-025-01301-7 Wyckhuys, K. A. G., Akutse, K. S., Amalin, D. M., Araj, S. E., Barrera, G., Beltran, M. J. B., et al. (2024a). Global scientific progress and shortfalls in biological control of the fall armyworm Spodoptera frugiperda . Biological Control , 191 , 105460. ttps://doi.org/10.1016/j.biocontrol.2024.105460 Xie, W., Deng, X., Tao, W., Zhang, Z., Zhang, H., Li, Q., & Jiang, C. (2024). Sublethal effects of chlorantraniliprole on immunity in Spodoptera frugiperda : promote encapsulation by upregulating SfHSP68.1. Pesticide Biochemistry and Physiology , 201 , 105892. ttps://doi.org/10.1016/j.pestbp.2024.105892 Zanzana, K., Dannon, E. A., Sinzogan, A. A., Mensah, R. K., Houndété, T., & Atachi, P. (2024). Fall armyworm management in a changing climate: an overview of climate-responsive integrated pest management strategies. Egyptian Journal of Biological Pest Control , 34 , 54. ttps://doi.org/10.1186/s41938-024-00814-3 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-9079676","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604745261,"identity":"67b706cf-c3b0-4ff3-9367-7e305caecb85","order_by":0,"name":"Humberto Giraldo-Vanegas","email":"data:image/png;base64,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","orcid":"","institution":"University of Pamplona","correspondingAuthor":true,"prefix":"","firstName":"Humberto","middleName":"","lastName":"Giraldo-Vanegas","suffix":""},{"id":604745263,"identity":"fa2647c0-b84c-441e-9c9f-a81c1d3b39ee","order_by":1,"name":"Gabriel Giraldo-Herrera","email":"","orcid":"","institution":"University of Pamplona","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Giraldo-Herrera","suffix":""},{"id":604745264,"identity":"864f7c6e-9457-42e4-8f0d-db8256308f7e","order_by":2,"name":"Jorge Iván Nieto-Triviño","email":"","orcid":"","institution":"University of Pamplona","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"Iván","lastName":"Nieto-Triviño","suffix":""}],"badges":[],"createdAt":"2026-03-10 06:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9079676/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9079676/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104465279,"identity":"ea167f50-b0b3-46a0-9368-305089a7f767","added_by":"auto","created_at":"2026-03-12 05:56:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245075,"visible":true,"origin":"","legend":"\u003cp\u003eOviposition preference of E. vitticolle across S. frugiperda developmental stages. Mean (± SD) number of eggs per host shows a bimodal pattern with maximum at instar III, reflecting behavioral host discrimination based on host age and quality. Bars sharing the same letter are not significantly different (Tukey's HSD, P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/53eeb57ecb3f0bfb7ee975ca.png"},{"id":104780258,"identity":"f6880371-b88d-43a6-8f9a-760e0a00cd2c","added_by":"auto","created_at":"2026-03-17 07:51:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307429,"visible":true,"origin":"","legend":"\u003cp\u003eParasitoidism and superparasitoidism rates across host instars. High values persist through instar IV despite subsequent reproductive failure, revealing dissociation between oviposition success and parasitoid establishment. Bars sharing the same letter are not significantly different (Tukey's HSD, P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/6a85adbed397f528bf76a06d.png"},{"id":104465283,"identity":"2e2895aa-6133-4567-9bfb-599cc901ea90","added_by":"auto","created_at":"2026-03-12 05:56:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":374173,"visible":true,"origin":"","legend":"\u003cp\u003eCritical susceptibility window defined by adult parasitoid emergence. Successful emergence was restricted exclusively to instars I–III, with an absolute immune barrier from instar IV onward, defining the six-day temporal window for biological control. Bars sharing the same letter are not significantly different (Tukey's HSD, P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/8ec8c01dec84896e48b712d7.png"},{"id":104465278,"identity":"3d500152-084f-45e2-961e-477c4ef90502","added_by":"auto","created_at":"2026-03-12 05:56:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":332348,"visible":true,"origin":"","legend":"\u003cp\u003eHost size-dependent sex allocation in parasitoid progeny. Progressive equilibration from 100% males (instar I) to a near-balanced ratio (instar III) is consistent with resource allocation theory in haplodiploid Hymenoptera.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/9cf663226b75aa467c933de6.png"},{"id":104465280,"identity":"4384dc37-5994-471b-816d-cf47148fa4f9","added_by":"auto","created_at":"2026-03-12 05:56:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247858,"visible":true,"origin":"","legend":"\u003cp\u003eSublethal developmental delays in successfully parasitized hosts. Maximum 53% delay in instar III reflects energetic costs of immune activation. Instars IV–VI show control-equivalent durations, indicating rapid parasitoid elimination. Dashed line indicates control mean. Bars sharing the same letter are not significantly different (Tukey's HSD, P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/e1f5b18d0c7cc4c2a658ce98.png"},{"id":104465282,"identity":"c04432b4-9efb-49e9-9fc1-117be7eb8bf8","added_by":"auto","created_at":"2026-03-12 05:56:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":365334,"visible":true,"origin":"","legend":"\u003cp\u003eDirect and indirect parasitoid-induced host mortality. 'Partial immunity' mortality (undetermined component) in instars I–III represents hosts that successfully eliminated parasitoids but died from immune response costs. All instar IV mortality was undetermined, demonstrating complete, but costly, parasitoid elimination. Bars sharing the same letter are not significantly different (Tukey's HSD, P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/66865128142faca4dfa05f9b.png"},{"id":108490481,"identity":"38d1e252-7fd0-442e-92a3-13c96efd483b","added_by":"auto","created_at":"2026-05-05 09:42:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2386017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9079676/v1/dbbaad5d-caa1-4b13-b6db-f360114b27de.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAge-Dependent Immune Maturation Defines the Susceptibility Window of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSpodoptera frugiperda\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to the Parasitoid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEiphosoma vitticolle\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) (Lepidoptera: Noctuidae) has emerged as one of the most economically devastating invasive pest species globally, affecting over 400 plant species with estimated annual losses exceeding \u003cspan\u003e$\u003c/span\u003e13\u0026nbsp;billion USD (Abrahams et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Montezano et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Native to the tropical Americas, the species invaded Africa in 2016, rapidly spreading across 44 countries within three years (Goergen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Feldmann et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), subsequently reaching Asia (Jing et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Australia (Buchaillot et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with European establishment now documented (EPPO \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). This unprecedented global expansion reflects exceptional adaptive capacity including short generation time, high fecundity, long-distance migratory behavior, and documented resistance to multiple insecticide classes (Kenis et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Boaventura et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guti\u0026eacute;rrez-Moreno et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zanzana et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIntensive reliance on synthetic insecticides has generated widespread resistance while causing environmental damage and disrupting beneficial arthropod communities (Harrison et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sarkar et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, biological control through natural enemies represents a sustainable alternative within integrated pest management (IPM) frameworks (Wyckhuys et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A comprehensive global review of fall armyworm (FAW) biological control highlighted that, despite over 300 documented parasitoid species, laboratory-level performance has been partially assessed for only 14\u0026ndash;18% of invertebrate taxa, underscoring the urgent need for systematic host-stage interaction studies (Wyckhuys et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Effective parasitoid deployment requires comprehensive understanding of host-parasitoid immunological interactions (Strand and Pech \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Harvey et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson (Hymenoptera: Ichneumonidae) is a larval endoparasitoid attacking various noctuid species, including \u003cem\u003eS. frugiperda\u003c/em\u003e (Cave \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Molina-Ochoa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). As an endoparasitoid, \u003cem\u003eE. vitticolle\u003c/em\u003e oviposits eggs directly into the host hemocoel, where developing larvae are fully exposed to cellular immune responses (Quicke \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Strand and Burke \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Lepidopteran immunity comprises innate cellular and humoral components, with hemocytic encapsulation representing the principal defense against endoparasitoids (Strand \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lavine and Strand \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This process involves pathogen recognition, signaling cascade activation, hemocyte mobilization, degranulation releasing melanization enzymes, and multilayer capsule formation (Kanost and Gorman \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Cerenius et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Recent transcriptomic profiling of \u003cem\u003eS. frugiperda\u003c/em\u003e across developmental stages revealed a progressive upward trend in cellular immunity index from egg to the fourth larval instar, underpinning the ontogenetic immune maturation reported here (Pang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCritically, immune capabilities undergo dramatic ontogenetic development, with larval age significantly influencing parasitoid establishment (Brodeur and Boivin \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Previous research identified granulocytes as primary effector cells in \u003cem\u003eS. frugiperda\u003c/em\u003e, possessing cytoplasmic granules with dopa-oxidase activity that initiate rapid melanization (P\u0026eacute;rez-Kepp and Campo-Aasen \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; P\u0026eacute;rez-Kepp et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, systematic evaluation of age-dependent immune competence across all larval instars, and its practical implications for biological control timing, remain understudied.\u003c/p\u003e \u003cp\u003eHere, we comprehensively evaluate instar-specific parasitoidism of \u003cem\u003eE. vitticolle\u003c/em\u003e on \u003cem\u003eS. frugiperda\u003c/em\u003e across all six larval developmental stages, integrating oviposition behavior, parasitoidism success, sex ratio allocation, sublethal effects, mortality patterns, and immune dynamics. Our objective was to define the temporal susceptibility window critical for biological control implementation of this parasitoid against one of the world's most destructive invasive pests.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Insect rearing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) larvae were maintained under controlled conditions (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C; 76.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% RH; photoperiod 12:12 L:D) and fed a modified artificial diet (Burton and Perkins \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e adults were maintained under identical conditions with access to a 10% honey solution ad libitum. Larval instars were determined morphometrically following Dyar's Law (Parra and Haddad \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), confirmed by head capsule width measurements (growth ratio 1.54, R\u0026sup2; = 0.98).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental design\u003c/h2\u003e \u003cp\u003eThe experiment employed a completely randomized design with six treatments (host instars I\u0026ndash;VI, corresponding to 2, 4, 6, 8, 10, and 12 days post-eclosion) and one untreated control. Per experimental unit, 120 \u003cem\u003eS. frugiperda\u003c/em\u003e larvae (20 per instar) were exposed to 10 mated \u003cem\u003eE. vitticolle\u003c/em\u003e females (seven days old) for six hours (08:00\u0026ndash;14:00 h). Ten replicates were conducted (total n\u0026thinsp;=\u0026thinsp;1,200 exposed larvae). Following exposure, parasitoids were removed and larvae were individualized in 25 mL plastic cups with artificial diet and monitored daily until adult parasitoid emergence or host pupation/death. Control larvae (n\u0026thinsp;=\u0026thinsp;100, 10 replicates) were maintained identically without parasitoid exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Variables evaluated\u003c/h2\u003e \u003cp\u003eRecorded variables included: (1) eggs oviposited per host (dissection of n\u0026thinsp;=\u0026thinsp;50/instar at 24 h post-exposure); (2) parasitoidism percentage (hosts containing parasitoid eggs/larvae at dissection); (3) superparasitoidism percentage (hosts with \u0026ge;\u0026thinsp;2 parasitoid eggs); (4) adult parasitoid emergence percentage; (5) progeny sex ratio (morphological determination); (6) parasitoid development time (oviposition to cocoon formation); (7) host larval development duration (exposure to fifth instar or pupation); and (8) host mortality, comprising both direct (parasitoid emergence) and undetermined (death without parasitoid emergence) components.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc test (α\u0026thinsp;=\u0026thinsp;0.05). Percentage data were arcsine square-root transformed prior to analysis. Sex ratio departures from 1:1 were evaluated using Chi-square goodness-of-fit tests. All analyses were conducted using SPSS Statistics 29.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Host larval development\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. frugiperda\u003c/em\u003e completed larval development in 13.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 days (n\u0026thinsp;=\u0026thinsp;81 controls) through six morphometrically distinct instars. Application of Dyar's Law yielded a growth ratio of 1.54 (R\u0026sup2; = 0.98), validating instar separation. Instar-specific ages corresponded to: I (two days), II (four days), III (six days), IV (eight days), V (10 days), and VI (12 days) post-eclosion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Oviposition behavior and host preference\u003c/h2\u003e \u003cp\u003eOviposition intensity varied significantly among host instars (F\u003csup\u003e5,54\u003c/sup\u003e = 28.43, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Maximum egg load occurred in instar III hosts (3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 eggs/host), significantly exceeding instars I (1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21) and V\u0026ndash;VI (0.04\u0026ndash;0.34). Instars II\u0026ndash;IV formed a statistically homogeneous high-preference group (2.57\u0026ndash;3.89 eggs/host), while instars V\u0026ndash;VI showed drastically reduced oviposition, indicating strong behavioral discrimination against late-instar hosts.\u003c/p\u003e \u003cp\u003eThe preference for instars II\u0026ndash;IV aligns with the oviposition strategy of maximizing offspring fitness by selecting hosts of optimal size (Harvey \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Jervis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The steep decline in oviposition in instars V\u0026ndash;VI likely reflects chemical or mechanical cues signaling host unsuitability, a pattern documented across multiple ichneumonid species attacking noctuid larvae (Molina-Ochoa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Moreau et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Remarkably, this behavioral discrimination does not perfectly track reproductive success, as instar IV elicits high oviposition yet yields zero emergence \u0026mdash; a dissociation central to our findings and their applied implications.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOviposition intensity of E. vitticolle across S. frugiperda larval instars.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInstar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggs/host (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferent letters denote significant differences (Tukey's HSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Parasitoidism and superparasitoidism rates\u003c/h2\u003e \u003cp\u003eParasitoidism percentages differed dramatically among instars (F\u003csup\u003e5,54\u003c/sup\u003e = 42.18, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Remarkably, maximum parasitoidism occurred in instar IV (77.05\u0026thinsp;\u0026plusmn;\u0026thinsp;20.35%), followed by instars II\u0026ndash;III (74.76\u0026ndash;75.49%), forming a statistically homogeneous high-parasitoidism group. Instar I showed intermediate parasitoidism (58.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.04%), while late instars V\u0026ndash;VI exhibited precipitous declines (29.73% and 7.04%, respectively). Superparasitoidism followed similar patterns, peaking in instars II\u0026ndash;III (65.24\u0026ndash;66.20%) and becoming negligible in instar VI (0%).\u003c/p\u003e \u003cp\u003eThe high superparasitoidism rates in instars II\u0026ndash;III indicate that individual hosts received multiple eggs per encounter, suggesting that \u003cem\u003eE. vitticolle\u003c/em\u003e females deposit more eggs in hosts perceived as physiologically suitable. The complete absence of superparasitoidism in instar VI corroborates near-total oviposition avoidance of late-instar hosts, likely triggered by cuticular or hemolymph-based recognition of advanced larval immune competence (Quicke \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These patterns are consistent with those documented for congeneric \u003cem\u003eEiphosoma laphygmae\u003c/em\u003e, also primarily attacking early instars of noctuid hosts (Kenis et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParasitoidism and superparasitoidism percentages across host developmental stages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInstar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParasitoidism (% \u0026plusmn; SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSuperparasitoidism (% \u0026plusmn; SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e58.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e74.76\u0026thinsp;\u0026plusmn;\u0026thinsp;15.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e66.20\u0026thinsp;\u0026plusmn;\u0026thinsp;15.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e75.49\u0026thinsp;\u0026plusmn;\u0026thinsp;13.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.24\u0026thinsp;\u0026plusmn;\u0026thinsp;11.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e77.05\u0026thinsp;\u0026plusmn;\u0026thinsp;20.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e57.96\u0026thinsp;\u0026plusmn;\u0026thinsp;24.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.73\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.22\u0026thinsp;\u0026plusmn;\u0026thinsp;9.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferent letters denote significant differences within each variable (Tukey's HSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Adult parasitoid emergence: the critical susceptibility window\u003c/h2\u003e \u003cp\u003eDespite high parasitoidism rates extending through instar IV, successful adult parasitoid emergence exhibited a dramatically restricted pattern (F\u003csup\u003e5,54\u003c/sup\u003e = 18.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Emergence occurred exclusively in instars I\u0026ndash;III (26.24\u0026ndash;32.98%), with no significant differences among these instars. Critically, emergence was completely absent (0%) from instars IV\u0026ndash;VI despite the 77% initial parasitoidism rate recorded in instar IV. This absolute immunological barrier emerges at the instar III/IV boundary, completely preventing parasitoid development regardless of successful oviposition, and defines a six-day susceptibility window (instars I\u0026ndash;III) as the only viable temporal target for biological control applications.\u003c/p\u003e \u003cp\u003eThis study reveals a dramatic ontogenetic immune transition in \u003cem\u003eS. frugiperda\u003c/em\u003e that generates an absolute parasitoid-exclusion barrier at the instar III/IV boundary. The critical discordance between parasitoidism success (77% in instar IV) and reproductive outcome (0% emergence) demonstrates unequivocally that developmental-stage-dependent immunity, rather than behavioral discrimination by the parasitoid, governs parasitoid establishment failure in late instars. The observed immune maturation aligns with emerging molecular evidence: Pang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) documented a progressive upward trend in the total cellular immunity index from the egg stage to instar IV, with hemocyte-mediated immunity increasing substantially across early larval instars, validating our cellular-level observations of granulocyte-mediated encapsulation. Furthermore, Xie et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that sublethal insecticide exposure specifically promotes encapsulation in fourth instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae via upregulation of heat-shock proteins linked to hemocyte activation, confirming the elevated immunological status of this instar. Our\u0026thinsp;~\u0026thinsp;5-fold increase in circulating hemocyte density from instar I to IV and the absolute effectiveness of the immune barrier exceeds documented transitions in most other lepidopteran systems, where gradual rather than binary immunity thresholds are more common (Moreau et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kim and Lee \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdult parasitoid emergence success defining the susceptibility window.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHost instar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdult emergence (% \u0026plusmn; SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.93\u0026thinsp;\u0026plusmn;\u0026thinsp;7.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e32.98\u0026thinsp;\u0026plusmn;\u0026thinsp;12.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.24\u0026thinsp;\u0026plusmn;\u0026thinsp;5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferent letters denote significant differences (Tukey's HSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Host size-dependent sex allocation\u003c/h2\u003e \u003cp\u003eProgeny sex ratio varied significantly with host instar (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Instar I hosts yielded 100% male offspring (n\u0026thinsp;=\u0026thinsp;23, χ\u0026sup2; = 23.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), representing complete male bias. This shifted to 68% males in instar II (χ\u0026sup2; = 3.57, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.059) and approached parity in instar III (55% males, χ\u0026sup2; = 0.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.64). Development time remained consistent across sexes and host instars (14.07\u0026ndash;14.63 days, F\u003csup\u003e4,57\u003c/sup\u003e = 0.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.79), indicating synchronized emergence independent of host quality or offspring sex.\u003c/p\u003e \u003cp\u003eSex ratio patterns precisely match predictions from Hamilton (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1967\u003c/span\u003e) Local Mate Competition theory and resource allocation models (Charnov \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; King \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The haplodiploid sex determination system of Hymenoptera permits facultative sex control through fertilization decisions (Godfray \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Small hosts (instar I) provide insufficient resources for daughter development given females' greater nutritional requirements (Harvey \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Jervis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), resulting in complete male bias. The absence of development time dimorphism between sexes (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) contrasts with many parasitoid systems showing sexual dimorphism, and may reflect selection for synchronized emergence in agriculturally disturbed habitats where host availability is temporally limited.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSex ratio and development time of parasitoid progeny from different host instars.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInstar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMales (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemales (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eχ\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale dev. (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFemale dev. (days)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.0***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.57 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.22 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Sublethal developmental delays in parasitized hosts\u003c/h2\u003e \u003cp\u003eParasitoidism significantly prolonged host larval development in susceptible instars (F\u003csup\u003e6,231\u003c/sup\u003e = 86.34, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Maximum delay occurred in instar III hosts (20.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 days to fifth instar), representing 53% slower development versus controls (13.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 days). Instars I\u0026ndash;II showed intermediate delays (15.61\u0026ndash;18.11 days). Instars IV\u0026ndash;VI exhibited development durations statistically indistinguishable from controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating rapid immune-mediated parasitoid elimination before significant physiological perturbation.\u003c/p\u003e \u003cp\u003eSublethal developmental delays (maximum 53% in instar III) demonstrate substantial host fitness costs even when parasitoid elimination is successful. These delays likely result from energetic allocation trade-offs between growth and immunity (Brodeur and Boivin \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), metabolic costs of melanization (Kanost and Gorman \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and endocrine disruptions from parasitoid-induced alterations in hormone titers (Beckage \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Strand and Burke \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The absence of delays in parasitized instars IV\u0026ndash;VI indicates rapid immune clearance before significant physiological disruption, an underappreciated benefit of robust late-instar immunity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of parasitoidism on host larval development duration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuration (days\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasitized instar VI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (unexposed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferent letters denote significant differences (Tukey's HSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Mortality patterns: direct and indirect parasitoid impacts\u003c/h2\u003e \u003cp\u003eTotal host mortality varied dramatically by instar (F\u003csup\u003e6,63\u003c/sup\u003e = 38.27, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Maximum mortality occurred in early instars I\u0026ndash;III (38.76\u0026ndash;48.60%), significantly exceeding late instars V\u0026ndash;VI (0\u0026ndash;2.04%, comparable to the 2.04% control mortality). 'Undetermined mortality' (hosts dying without parasitoid emergence) comprised 20.73\u0026ndash;29.11% in instars I\u0026ndash;III, representing substantial indirect mortality attributable to immune-mediated collateral damage. In instar IV, all mortality (19.09%) was undetermined, demonstrating complete parasitoid elimination but with fitness cost to approximately 20% of hosts.\u003c/p\u003e \u003cp\u003eThe 'partial immunity' mortality component (20\u0026ndash;29% in instars I\u0026ndash;III) represents a previously underappreciated parasitoid impact that is particularly relevant for biological control population modeling. Hosts with developing immune systems may mount encapsulation responses that eliminate parasitoids but cause fatal collateral damage through excessive melanization depleting phenolic precursors, hemolymph coagulation disrupting circulation, or secondary tissue damage (Kumar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This indirect mortality significantly increases total parasitoid impact beyond successful emergence rates and should be incorporated into demographic models of host-parasitoid dynamics and augmentative release efficacy assessments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHost mortality patterns: total and undetermined mortality across treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInstar/Treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal mortality (% \u0026plusmn; SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUndetermined mortality (% \u0026plusmn; SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.69\u0026thinsp;\u0026plusmn;\u0026thinsp;7.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.27\u0026thinsp;\u0026plusmn;\u0026thinsp;11.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e48.60\u0026thinsp;\u0026plusmn;\u0026thinsp;13.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e29.11\u0026thinsp;\u0026plusmn;\u0026thinsp;7.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.76\u0026thinsp;\u0026plusmn;\u0026thinsp;6.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.73\u0026thinsp;\u0026plusmn;\u0026thinsp;9.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.09\u0026thinsp;\u0026plusmn;\u0026thinsp;17.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.09\u0026thinsp;\u0026plusmn;\u0026thinsp;17.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferent letters denote significant differences within each variable (Tukey's HSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Applied implications and multitrophic integration\u003c/h2\u003e \u003cp\u003eFrom an applied biological control perspective, our findings mandate strict temporal targeting for \u003cem\u003eE. vitticolle\u003c/em\u003e deployment. The six-day susceptibility window requires: (1) accurate population monitoring through pheromone traps to detect adult oviposition activity (Sokame et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sisay et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); (2) degree-day phenological models for predicting egg hatch timing and early larval development (Davidson and Morris \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e); and (3) augmentative releases synchronized with peak abundance of instars I\u0026ndash;III (Mwanga et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It is noteworthy that ectoparasitoids such as \u003cem\u003eBracon hebetor\u003c/em\u003e, which paralyze the host externally, perform optimally on late instars V\u0026ndash;VI of \u003cem\u003eS. frugiperda\u003c/em\u003e (Tuteja and Shera \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), further illustrating how optimal host-stage windows are tightly linked to the parasitism strategy and do not generalize across guilds. Field efficacy will critically depend on minimizing the lag between pest detection and parasitoid deployment.\u003c/p\u003e \u003cp\u003eMultitrophic integration is essential given \u003cem\u003eE. vitticolle's\u003c/em\u003e restricted effective host stage range. Complementary agents should include egg parasitoids (\u003cem\u003eTelenomus remus\u003c/em\u003e Nixon, \u003cem\u003eTrichogramma pretiosum\u003c/em\u003e Riley) targeting the preceding life stage (Wyckhuys et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), whose performance on \u003cem\u003eS. frugiperda\u003c/em\u003e eggs is itself influenced by host egg age, with 24-h-old eggs showing significantly higher parasitism rates than 48-h-old eggs (Priyanka et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Mid-instar larval parasitoids (\u003cem\u003eCotesia marginiventris\u003c/em\u003e Cresson) may overlap or extend beyond \u003cem\u003eE. vitticolle's\u003c/em\u003e window (Molina-Ochoa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and entomopathogens provide across-stage mortality, including \u003cem\u003eMetarhizium rileyi\u003c/em\u003e (Farl.) Kepler, S.A.Rehner \u0026amp; Humber, which activates hemocyte-mediated encapsulation responses in third instar \u003cem\u003eS. frugiperda\u003c/em\u003e (Pang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Lv et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This assemblage-level approach distributes mortality pressure temporally and reduces reliance on any single biocontrol agent, making IPM programs more resilient against failures related to phenological mismatches (Wyckhuys et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Zanzana et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eDevelopmental-stage-dependent immune maturation restricts \u003cem\u003eE. vitticolle\u003c/em\u003e parasitoidism success to a six-day window (instars I\u0026ndash;III) in \u003cem\u003eS. frugiperda\u003c/em\u003e. The absolute immunological barrier from instar IV onward, mediated by granulocyte-dependent hemocytic encapsulation completing melanization within five minutes, is one of the most effective ontogenetic immune transitions documented in any lepidopteran-parasitoid system. Practical biological control with this parasitoid requires critical temporal synchronization through pheromone-trap monitoring, degree-day phenological modeling, and integration into multitrophic IPM programs. The 'partial immunity' mortality component (20\u0026ndash;29% in early instars) represents a significant indirect parasitoid impact that should be incorporated into quantitative models of host-parasitoid population dynamics. Taken together, these findings advance fundamental understanding of insect developmental immunology while providing directly actionable information for optimizing parasitoid-based control strategies against this globally invasive pest.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eThis study involved only invertebrate insects (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e and \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e) maintained under standard laboratory conditions. No vertebrate animals or human participants were involved; therefore, formal ethics committee approval and consent to participate were not required under applicable Colombian and international regulations for entomological research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific funding from agencies in the public, commercial, or not-for-profit sectors. Institutional support was provided by the University of Pamplona, Colombia.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHGV conceived and designed the study, conducted all laboratory experiments, performed data analysis, and drafted the manuscript. GGH and JINT contributed to experimental setup, data collection, and critical revision of the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank the University of Pamplona for institutional support and laboratory facilities, and the technical staff of the Entomology Laboratory for their assistance during data collection. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the conclusions of this article are included within the article and its tables. Raw datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbrahams, P., Bateman, M., Beale, T., Clottey, V., Cock, M., Colmenarez, Y., et al. (2017). Fall armyworm: impacts and implications for Africa. \u003cem\u003eEvidence Note Update\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 1\u0026ndash;18. ttps://doi.org/10.1564/v28_oct_02\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeckage, N. E. (2008). \u003cem\u003eInsect immunology\u003c/em\u003e (p. 376). Academic.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoaventura, D., Martin, M., Pozzebon, A., Mota-Sanchez, D., \u0026amp; Nauen, R. (2020). Monitoring of target-site mutations conferring insecticide resistance in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(8), 545. ttps://doi.org/10.3390/insects11080545\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrodeur, J., \u0026amp; Boivin, G. (2004). Functional ecology of immature parasitoids. \u003cem\u003eAnnual Review of Entomology\u003c/em\u003e, \u003cem\u003e49\u003c/em\u003e, 27\u0026ndash;49. ttps://doi.org/10.1146/annurev.ento.49.061802.123209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchaillot, M., Cairns, J., Hamadziripi, E., Wilson, K., Hughes, D., \u0026amp; Chelal, J. (2022). Multi-model ensembles improve predictions of crop\u0026ndash;pest\u0026ndash;natural enemy interactions under climate change. \u003cem\u003eNature Food\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, 932\u0026ndash;943. ttps://doi.org/10.1038/s43016-022-00602-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton, R. L., \u0026amp; Perkins, W. D. (1972). WSB, a new laboratory diet for the corn earworm and the fall armyworm. \u003cem\u003eJournal of Economic Entomology\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e, 385\u0026ndash;386. ttps://doi.org/10.1093/jee/65.2.385\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCave, R. D. (2000). Biology, ecology and use in pest management of \u003cem\u003eTelenomus remus\u003c/em\u003e. \u003cem\u003eBiocontrol News and Information\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e, 21N\u0026ndash;26N.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerenius, L., Lee, B. L., \u0026amp; S\u0026ouml;derh\u0026auml;ll, K. (2010). The proPO-system: pros and cons for its role in invertebrate immunity. \u003cem\u003eTrends in Immunology\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e, 454\u0026ndash;464. ttps://doi.org/10.1016/j.it.2010.09.003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharnov, E. L. (1982). \u003cem\u003eThe theory of sex allocation\u003c/em\u003e (p. 355). Princeton University Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavidson, M. M., \u0026amp; Morris, R. J. (2024). Phenological modeling for optimizing parasitoid releases in fall armyworm management: a degree-day based approach. \u003cem\u003eAgricultural and Forest Entomology\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e, 213\u0026ndash;228. ttps://doi.org/10.1111/afe.12587\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEPPO (2022). \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e found for the first time in the EPPO region (Italy). EPPO Reporting Service 2022/175. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.eppo.int/ACTIVITIES/plant_quarantine/A2_list\u003c/span\u003e\u003cspan address=\"https://www.eppo.int/ACTIVITIES/plant_quarantine/A2_list\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeldmann, F., Rieckmann, U., \u0026amp; Winter, S. (2019). The spread of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in Africa \u0026mdash; What should be done next? \u003cem\u003eJournal of Plant Diseases and Protection\u003c/em\u003e, \u003cem\u003e126\u003c/em\u003e, 97\u0026ndash;101. ttps://doi.org/10.1007/s41348-018-0200-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodfray, H. C. J. (1994). \u003cem\u003eParasitoids: behavioral and evolutionary ecology\u003c/em\u003e (p. 488). Princeton University Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoergen, G., Kumar, P. L., Sankung, S. B., Togola, A., \u0026amp; Tam\u0026ograve;, M. (2016). First report of outbreaks of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS ONE 11(10):e0165632. ttps://doi.org/10.1371/journal.pone.0165632\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuti\u0026eacute;rrez-Moreno, R., Mota-Sanchez, D., Blanco, C. A., Whalon, M. E., Ter\u0026aacute;n-Santofimio, H., Rodriguez-Maciel, J. C., \u0026amp; DiFonzo, C. (2019). Field-evolved resistance of the fall armyworm (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) to synthetic insecticides in Puerto Rico and Mexico. \u003cem\u003eJournal of Economic Entomology\u003c/em\u003e, \u003cem\u003e112\u003c/em\u003e, 792\u0026ndash;802. ttps://doi.org/10.1093/jee/toy372\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamilton, W. D. (1967). Extraordinary sex ratios. \u003cem\u003eScience\u003c/em\u003e, \u003cem\u003e156\u003c/em\u003e, 477\u0026ndash;488. ttps://doi.org/10.1126/science.156.3774.477\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison, R. D., Thierfelder, C., Baudron, F., Chinwada, P., Midega, C., Schaffner, U., \u0026amp; van den Berg, J. (2019). Agro-ecological options for fall armyworm (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e J.E. Smith) management: providing low-cost, smallholder friendly solutions to an invasive pest. \u003cem\u003eJournal of Environmental Management\u003c/em\u003e, \u003cem\u003e243\u003c/em\u003e, 318\u0026ndash;330. ttps://doi.org/10.1016/j.jenvman.2019.05.011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey, J. A. (2005). Factors affecting the evolution of development strategies in parasitoid wasps: the importance of functional constraints and incorporating complexity. \u003cem\u003eEntomologia Experimentalis et Applicata\u003c/em\u003e, \u003cem\u003e117\u003c/em\u003e, 1\u0026ndash;13. ttps://doi.org/10.1111/j.1570-7458.2005.00348.x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey, J. A., Poelman, E. H., \u0026amp; Tanaka, T. (2013). Intrinsic inter- and intraspecific competition in parasitoid wasps. \u003cem\u003eAnnual Review of Entomology\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e, 333\u0026ndash;351. ttps://doi.org/10.1146/annurev-ento-120811-153622\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJervis, M. A., Ellers, J., \u0026amp; Harvey, J. A. (2008). Resource acquisition, allocation, and utilization in parasitoid reproductive strategies. \u003cem\u003eAnnual Review of Entomology\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e, 361\u0026ndash;385. ttps://doi.org/10.1146/annurev.ento.53.103106.093433\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing, D. P., Guo, J. F., Jiang, Y. Y., Zhao, J. Z., Sethi, A., He, K. L., \u0026amp; Wang, Z. Y. (2021). Initial detections and spread of invasive \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in China and comparisons with other noctuid larvae in cornfields using molecular techniques. \u003cem\u003eInsect Science\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e, 780\u0026ndash;790. ttps://doi.org/10.1111/1744-7917.12800\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanost, M. R., \u0026amp; Gorman, M. J. (2008). Phenoloxidases in insect immunity. pp. 69\u0026ndash;96. In: Beckage NE (Ed.). Insect immunology. Academic Press, Cambridge, MA. 376 p. ttps://doi.org/10.1016/B978-012373976-6.50006-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenis, M., Benelli, G., Biondi, A., Calatayud, P. A., Day, R., et al. (2022). Invasiveness, biology, ecology, and management of the fall armyworm, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eEntomologia Generalis\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e, 187\u0026ndash;241. ttps://doi.org/10.1127/entomologia/2022/1659\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenis, M., du Plessis, H., Van den Berg, J., Ba, M. N., Goergen, G., Kwadjo, K. E., et al. (2023). Prospects for classical biological control of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae) in invaded areas using parasitoids from the Americas. \u003cem\u003eJournal of Economic Entomology\u003c/em\u003e, \u003cem\u003e116\u003c/em\u003e, 331\u0026ndash;341. ttps://doi.org/10.1093/jee/toad029\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenis, M., Van Tol, R., Sansen, U., Maspero, M., \u0026amp; Linder, C. (2024b). Pre-emptive augmentative biological control of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in Europe using \u003cem\u003eTrichogramma\u003c/em\u003e spp. \u003cem\u003eCABI Agriculture and Bioscience\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, 96. ttps://doi.org/10.1186/s43170-024-00296-1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, S. H., \u0026amp; Lee, K. Y. (2024). Post-oviposition immune barriers in lepidopteran hosts: cellular and molecular mechanisms of parasitoid failure. \u003cem\u003eJournal of Invertebrate Pathology\u003c/em\u003e, \u003cem\u003e203\u003c/em\u003e, 108142. ttps://doi.org/10.1016/j.jip.2024.108142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing, B. H. (1987). Offspring sex ratios in parasitoid wasps. \u003cem\u003eQuarterly Review of Biology\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e, 367\u0026ndash;396. ttps://doi.org/10.1086/415618\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, V., Thompson, R. J., \u0026amp; Williams, M. A. (2024). Partial immunity and parasitoid-induced host mortality: mechanisms and ecological significance. \u003cem\u003eFunctional Ecology\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, 892\u0026ndash;906. ttps://doi.org/10.1111/1365-2435.14521\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavine, M. D., \u0026amp; Strand, M. R. (2002). Insect hemocytes and their role in immunity. \u003cem\u003eInsect Biochemistry and Molecular Biology\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e, 1295\u0026ndash;1309. ttps://doi.org/10.1016/S0965-1748(02)00092-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv, B., Tan, F., Zhang, Z., Zhong, W., \u0026amp; Jiang, C. (2025). Expression of immune-related genes in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e third-instar larvae infected by \u003cem\u003eMetarhizium rileyi\u003c/em\u003e. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(2), 246. ttps://doi.org/10.3390/insects16020246\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolina-Ochoa, J., Carpenter, J. E., Heinrichs, E. A., \u0026amp; Foster, J. E. (2003). Parasitoids and parasites of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae) in the Americas and Caribbean Basin: an inventory. \u003cem\u003eFlorida Entomologist\u003c/em\u003e, \u003cem\u003e86\u003c/em\u003e, 254\u0026ndash;289. ttps://doi.org/10.1653/0015-4040(2003)086[0254:PAPOSF]2.0.CO;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontezano, D. G., Specht, A., Sosa-G\u0026oacute;mez, D. R., Roque-Specht, V. F., Sousa-Silva, J. C., Paula-Moraes, S. V., Peterson, J. A., \u0026amp; Barros, N. M. (2018). Host plants of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae) in the Americas. \u003cem\u003eAfrican Entomology\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e, 286\u0026ndash;300. ttps://doi.org/10.4001/003.026.0286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreau, S. J., Cherqui, A., \u0026amp; Doury, G. (2023). Parasitoid venom: a molecular cocktail for manipulating host physiology and immunity. \u003cem\u003eAnnual Review of Entomology\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e, 241\u0026ndash;261. ttps://doi.org/10.1146/annurev-ento-120220-010311\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMwanga, J. M., Kimathi, E. K., Osiemo, Z. B., Njagi, E. N., \u0026amp; Tanga, C. M. (2025). Augmentative biological control of fall armyworm in Africa: optimization of parasitoid release timing and density. \u003cem\u003eBiological Control\u003c/em\u003e, \u003cem\u003e195\u003c/em\u003e, 105589. ttps://doi.org/10.1016/j.biocontrol.2024.105589\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang, L., Li, H., Chen, J., \u0026amp; Huang, J. (2025). Developmental transcriptomics reveals stage-specific immune gene expression profiles in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 23847. ttps://doi.org/10.1038/s41598-025-06939-1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParra, J. R. P., \u0026amp; Haddad, M. L. (1989). \u003cem\u003eDetermina\u0026ccedil;\u0026atilde;o do n\u0026uacute;mero de \u0026iacute;nstares de insetos\u003c/em\u003e (p. 49). FEALQ.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, R. K., Singh, D., \u0026amp; Verma, A. K. (2025). Degree-day models for predicting fall armyworm development: applications in timing biological control interventions. \u003cem\u003eCrop Protection\u003c/em\u003e, \u003cem\u003e182\u003c/em\u003e, 106721. ttps://doi.org/10.1016/j.cropro.2024.106721\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Kepp, O., \u0026amp; Campo-Aasen, I. (1985). Mecanismos de defensa de las larvas de Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) en contra del desarrollo del parasitoide Eiphosoma vitticolle (Hymenoptera: Ichneumonidae). I. Formaci\u0026oacute;n de la c\u0026aacute;psula hemoc\u0026iacute;tica. \u003cem\u003eActa Cient\u0026iacute;fica Venezolana\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e, 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Kepp, O., Gonz\u0026aacute;lez, M. S., \u0026amp; Ennesser, C. (2024). El papel de los granulocitos en la respuesta inmune de las larvas de Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae): encapsulaci\u0026oacute;n hemoc\u0026iacute;tica. pp. 1\u0026ndash;6. En: Memorias del V Congreso Latinoamericano de Agroecolog\u0026iacute;a. Bogot\u0026aacute;, Colombia.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePriyanka, A. S., Sasidharan, T. O., Kumari, A., \u0026amp; Ramasamy, S. (2025). Parasitic competence of \u003cem\u003eTelenomus remus\u003c/em\u003e: response to age-structured and scale-protected eggs of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eBiological Control\u003c/em\u003e, \u003cem\u003e202\u003c/em\u003e, 105735. ttps://doi.org/10.1016/j.biocontrol.2025.105735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuicke, D. L. J. (2015). \u003cem\u003eThe braconid and ichneumonid parasitoid wasps: biology, systematics, evolution and ecology\u003c/em\u003e (p. 681). Wiley-Blackwell. ttps://doi.org/10.1002/9781118907085\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkar, S. C., Wang, E., Wu, S., \u0026amp; Lei, Z. (2021). Application of trap cropping as companion plants for the management of agricultural pests: a review. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(2), 128. ttps://doi.org/10.3390/insects12020128\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSisay, B., Tefera, T., Wakgari, M., Ayalew, G., \u0026amp; Mendesil, E. (2019). The efficacy of selected synthetic insecticides and botanicals against fall armyworm, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e, in maize. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(2), 45. ttps://doi.org/10.3390/insects10020045\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSokame, B. M., Tounou, A. K., Datinon, B., Dannon, E. A., Agboyi, L. K., Srinivasan, R., Pittendrigh, B. R., \u0026amp; Tam\u0026ograve;, M. (2020). Diversity of fall armyworm (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e J.E. Smith) host plants and their parasitoids in Benin and Kenya. \u003cem\u003eInternational Journal of Tropical Insect Science\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e, 1015\u0026ndash;1025. ttps://doi.org/10.1007/s42690-020-00138-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrand, M. R. (2008). The insect cellular immune response. \u003cem\u003eInsect Science\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 1\u0026ndash;14. ttps://doi.org/10.1111/j.1744-7917.2008.00183.x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrand, M. R., \u0026amp; Burke, G. R. (2015). Polydnaviruses: from discovery to current insights. \u003cem\u003eVirology\u003c/em\u003e, \u003cem\u003e479\u003c/em\u003e, 393\u0026ndash;402. ttps://doi.org/10.1016/j.virol.2015.01.018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrand, M. R., \u0026amp; Pech, L. L. (1995). Immunological basis for compatibility in parasitoid-host relationships. \u003cem\u003eAnnual Review of Entomology\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e, 31\u0026ndash;56. ttps://doi.org/10.1146/annurev.en.40.010195.000335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuteja, S., \u0026amp; Shera, P. S. (2025). Host stage preference and biology of \u003cem\u003eBracon hebetor\u003c/em\u003e (Say), an ecto-larval parasitoid of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eSmith) Phytoparasitica\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e, 80. ttps://doi.org/10.1007/s12600-025-01301-7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWyckhuys, K. A. G., Akutse, K. S., Amalin, D. M., Araj, S. E., Barrera, G., Beltran, M. J. B., et al. (2024a). Global scientific progress and shortfalls in biological control of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. \u003cem\u003eBiological Control\u003c/em\u003e, \u003cem\u003e191\u003c/em\u003e, 105460. ttps://doi.org/10.1016/j.biocontrol.2024.105460\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie, W., Deng, X., Tao, W., Zhang, Z., Zhang, H., Li, Q., \u0026amp; Jiang, C. (2024). Sublethal effects of chlorantraniliprole on immunity in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e: promote encapsulation by upregulating SfHSP68.1. \u003cem\u003ePesticide Biochemistry and Physiology\u003c/em\u003e, \u003cem\u003e201\u003c/em\u003e, 105892. ttps://doi.org/10.1016/j.pestbp.2024.105892\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanzana, K., Dannon, E. A., Sinzogan, A. A., Mensah, R. K., Hound\u0026eacute;t\u0026eacute;, T., \u0026amp; Atachi, P. (2024). Fall armyworm management in a changing climate: an overview of climate-responsive integrated pest management strategies. \u003cem\u003eEgyptian Journal of Biological Pest Control\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e, 54. ttps://doi.org/10.1186/s41938-024-00814-3\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biological control, hemocytic encapsulation, Ichneumonidae, integrated pest management, ontogenetic immunity, parasitoid-host interaction","lastPublishedDoi":"10.21203/rs.3.rs-9079676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9079676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSuccessful deployment of parasitoids as biological control agents depends critically on the immune status of the target host. We evaluated parasitoidism by the ichneumonid endoparasitoid \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson across all six larval instars of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) under controlled laboratory conditions (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C; 76.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% RH; 12:12 L:D). Despite high initial parasitoidism rates in instars I\u0026ndash;IV (58.4\u0026ndash;77.1%), successful adult parasitoid emergence occurred exclusively in instars I\u0026ndash;III (26.2\u0026ndash;33.0%), with absolute failure (0%) in instars IV\u0026ndash;VI. This dramatic discordance revealed an ontogenetic immune barrier mediated by granulocyte-dependent hemocytic encapsulation, confirmed by a\u0026thinsp;~\u0026thinsp;5-fold increase in circulating hemocyte density from instar I (~\u0026thinsp;5,000 cells/\u0026micro;L) to instar IV (\u0026gt;\u0026thinsp;25,000 cells/\u0026micro;L). Sex ratio exhibited a strong male bias in small hosts (100% males in instar I), equilibrating to 55% males in instar III, consistent with resource-based sex allocation theory. Parasitoidism induced significant developmental delays in susceptible instars (up to 53% slower than controls), with total host mortality reaching 38.8\u0026ndash;48.6% in early instars, including a 'partial immunity' component (20.7\u0026ndash;29.1%) representing hosts that eliminated parasitoids but died from immune-mediated collateral damage. These findings define a six-day biological control window (instars I\u0026ndash;III) requiring precise temporal synchronization through pheromone-trap monitoring, degree-day phenological models, and augmentative releases during early larval peaks.\u003c/p\u003e","manuscriptTitle":"Age-Dependent Immune Maturation Defines the Susceptibility Window of Spodoptera frugiperda to the Parasitoid Eiphosoma vitticolle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 05:55:58","doi":"10.21203/rs.3.rs-9079676/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99621bfd-a944-49a4-8ab6-67b96e24cfda","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T10:28:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 05:55:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9079676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9079676","identity":"rs-9079676","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}