Functional monandry in Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae): a single mating saturates the spermatheca and restores balanced offspring sex ratios

Functional monandry in Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae): a single mating saturates the spermatheca and restores balanced offspring sex ratios | 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 Functional monandry in Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae): a single mating saturates the spermatheca and restores balanced offspring sex ratios Humberto Giraldo-Vanegas, Gabriel Giraldo-Herrera This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9534338/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 Understanding mating systems in parasitoid Hymenoptera is fundamental to optimizing mass-rearing programs for biological control. Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae) is a solitary koinobiont endoparasitoid of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), a globally invasive pest of major economic importance. We investigated the effect of mating frequency on adult emergence rates and offspring sex ratios of E. vitticolle under controlled laboratory conditions using five treatments: virgin females (Hv), virgin females exposed to physically impeded males (Hv+Mi), singly mated females (Hc), multiply mated females (Hcc), and females in permanent cohabitation with males (HM). Second- to third-instar larvae of S. frugiperda served as hosts. Offspring from unmated treatments consisted exclusively of males (Hv: 45♂:0♀; Hv+Mi: 38♂:0♀; χ² ≥ 38.00, P 0.05 in all mated treatments). No significant differences in adult emergence rates were detected among treatments. These findings establish E. vitticolle as a functionally monandrous species and demonstrate that a single supervised mating per female is biologically sufficient for mass-rearing operations targeting augmentative biological control of S. frugiperda in the Neotropical region. arrhenotoky biological control fall armyworm haplodiploidy Ichneumonidae mass rearing monandry sex ratio Spodoptera frugiperda Figures Figure 1 Key Message Virgin females of Eiphosoma vitticolle produce exclusively male offspring (100%), confirming strict arrhenotokous reproduction and demonstrating that courtship without insemination does not trigger egg fertilization. A single mating event fully saturates the spermatheca, restoring offspring sex ratios to the theoretical 1:1 equilibrium throughout the female's reproductive lifespan; additional copulations confer no measurable reproductive benefit. These findings establish E. vitticolle as a functionally monandrous species and provide an empirical basis for streamlining mass-rearing protocols for augmentative biological control of Spodoptera frugiperda in Neotropical agroecosystems: a single supervised mating per female is biologically sufficient, reducing male-cohort maintenance costs without compromising female offspring production. Introduction The fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is one of the most economically devastating invasive arthropod pests globally, attacking 353 plant species and causing annual yield losses exceeding US $ 13 billion across sub-Saharan Africa (Day et al. 2017 ; Montezano et al. 2018 ). Native to the tropical Americas, the species was first detected in Africa in 2016 and spread across 44 countries within three years (Goergen et al. 2016 ; Feldmann et al. 2019 ), subsequently extending its range into Asia (Jing et al. 2021 ), Australia (Fagan-Jeffries et al. 2024 ), and most recently Europe (Kenis et al. 2024 ). This extraordinary adaptive capacity is underpinned by a short generation time, high fecundity, long-distance migratory behavior, and documented resistance to multiple insecticide classes (Kenis et al. 2022 ; Boaventura et al. 2020 ). Augmentative biological control using larval parasitoids has emerged as a sustainable, environmentally sound component of integrated pest management (IPM) strategies against this polyphagous noctuid (Wyckhuys et al. 2024 ; Kenis et al. 2023 ; Molina-Ochoa et al. 2003 ; Sisay et al. 2019 ). Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae) is a solitary koinobiont endoparasitoid whose primary host in the Neotropical region is S. frugiperda . As a member of the subfamily Cremastinae, this species attacks early-instar host larvae and completes development within the host body before killing it at the pupal stage (Molina-Ochoa et al. 2003 ). A taxonomic note is warranted: recent revisions indicate that most records of Eiphosoma spp. attacking S. frugiperda in the Americas may refer to Eiphosoma laphygmae Costa-Lima rather than E. vitticolle sensu stricto (Gauld 2000 ; Allen et al. 2021 ; Kenis et al. 2023 ). The specimens used in the present study were identified as E. vitticolle following morphological examination of the rearing colony maintained at the University of Pamplona, Colombia; molecular verification is recommended for future studies. Recent experimental work has established that successful adult parasitoid emergence from S. frugiperda is restricted to a six-day susceptibility window corresponding to larval instars I–III, owing to an ontogenetic immune transition mediated by granulocyte-dependent hemocytic encapsulation from instar IV onwards (Giraldo-Vanegas et al. 2025 ). This immunological constraint directly informs the choice of host larval age for laboratory experiments and augmentative release strategies. In the Hymenoptera, sex determination operates predominantly through arrhenotokous haplodiploidy: unfertilized, haploid eggs develop into males, whereas fertilized, diploid eggs develop into females (Flanders 1939 ; Heimpel and de Boer 2008 ). Females of most hymenopteran species possess a spermatheca that functions as a long-term sperm storage organ (Flanders 1939 , 1946 ). Species that require only a single mating event to saturate the spermatheca are termed monandrous, whereas those requiring multiple copulations are termed polyandrous (Ridley 1993 ; Godfray 1994 ). Monandry appears to be the predominant mating strategy in parasitoid wasps (≈ 70% of species; Ridley 1993 ). Sex ratio theory predicts that haplodiploid females should allocate offspring sex facultatively in response to host resource availability (Hamilton 1967 ; Charnov 1982 ; King 1987 ). Sperm supply constraints can substantially disrupt optimal sex allocation: sperm-depleted or unmated females produce exclusively male offspring, whereas sperm-replete females regulate offspring sex ratios according to host quality cues (Boivin 2013 ; Boulton et al. 2015 ). Empirical determination of whether a species is monandrous or polyandrous is therefore essential for designing efficient mass-rearing programs, because maintaining unnecessarily large male cohorts diverts resources and reduces overall production efficiency (Godfray 1994 ). This study aimed to: (i) characterize the reproductive mode of E. vitticolle ; (ii) determine the minimum number of copulations required to maintain balanced offspring sex ratios throughout the female's reproductive lifespan; and (iii) evaluate the effect of mating frequency on adult emergence rates from S. frugiperda host larvae under controlled laboratory conditions. Materials and Methods Parasitoid and host colonies Colonies of E. vitticolle and S. frugiperda were maintained at 26 ± 1°C, 70 ± 5% relative humidity, and a 12:12 h light:dark photoperiod. Spodoptera frugiperda larvae were reared on artificial diet following standard protocols (Burton and Perkins 1972 ). Adult parasitoids were provided with a 10% honey solution as a carbohydrate source and held in ventilated plexiglass cages (30 × 30 × 30 cm). Experimental design Bioassays were conducted using five seven-day-old females per treatment (five mating treatments × five replicates; n = 25 females in total). Host larvae of S. frugiperda at six days of age (L2–L3 instar) were used in all treatments, corresponding to the validated susceptibility window for this parasitoid (Giraldo-Vanegas et al. 2025 ). The five mating treatments were: (i) Hv – virgin females maintained without male contact since emergence; (ii) Hv + Mi – virgin females exposed to physically impeded males (male genitalia immobilized with fine surgical thread to prevent intromission while permitting full courtship behavior); (iii) Hc – singly mated females that completed one copulation 24 h prior to the assay, after which the male was immediately removed; (iv) Hcc – multiply mated females that cohabited with males from emergence until the day prior to the assay; and (v) HM – females maintained in permanent cohabitation with males, including during the oviposition window. Parasitism bioassays In each experimental unit, one female was confined with 40 S. frugiperda L2–L3 larvae in a plexiglass arena (diameter 15 cm, height 20 cm) for a six-hour exposure period (08:00–14:00 h). Host larvae were subsequently individualized in 30-mL plastic cups with fresh artificial diet until either pupal formation of S. frugiperda or emergence of parasitoid cocoons. Emerged adult parasitoids were sexed under a stereomicroscope and counted. Statistical analysis Emergence proportion data were arcsine-square-root transformed [arcsin(√ p )] prior to analysis. Differences in emergence rates among treatments were evaluated by one-way ANOVA followed by Tukey's HSD post-hoc test (α = 0.05). Offspring sex ratios in each treatment were compared against the theoretical 1:1 null hypothesis using Pearson's chi-squared (χ²) goodness-of-fit test. All analyses were performed in R version 4.4.1 (R Core Team 2024 ). Results Reproductive mode Phenotypic analysis of progeny confirmed that E. vitticolle reproduces via strict arrhenotoky. In both treatments without effective sperm transfer (Hv and Hv + Mi), 100% of offspring were male (Table 1; Fig. 1 ). Treatment Hv yielded 45 males and 0 females (χ² = 45.00, P < 0.001); treatment Hv + Mi yielded 38 males and 0 females (χ² = 38.00, P < 0.001), demonstrating that courtship without insemination does not stimulate egg fertilization. Effect of mating frequency on offspring sex ratio All treatments with at least one completed copulation (Hc, Hcc, HM) produced mixed-sex offspring (Table 1; Fig. 1 ). In treatment Hc, 22 males and 18 females emerged (ratio 1.22:1; χ² = 0.40, P = 0.527). Treatment Hcc yielded 24 males and 18 females (ratio 1.33:1; χ² = 0.86, P = 0.354), and treatment HM yielded 22 males and 20 females (ratio 1.10:1; χ² = 0.09, P = 0.764). None of the mated treatments differed significantly from the theoretical 1:1 ratio, nor did they differ significantly from one another. Adult emergence rates No statistically significant differences in adult emergence rates were detected among the five mating treatments (one-way ANOVA: P > 0.05). Mating frequency had no significant effect on the total number of adult parasitoids that successfully emerged from S. frugiperda host larvae. Discussion The present study provides the first experimental characterization of the mating system and its reproductive consequences in E. vitticolle . The complete male bias of offspring from unmated females (Hv and Hv + Mi) unequivocally confirms that this species reproduces through strict arrhenotoky, the ancestral and numerically dominant mode of sex determination across the Hymenoptera (Flanders 1939 ; Heimpel and de Boer 2008 ). The inability of courtship behavior alone (treatment Hv + Mi) to influence offspring sex composition further demonstrates that genital coupling and physical sperm transfer — rather than any pre-copulatory signal — are the primary determinants of egg fertilization in E. vitticolle . The central finding — that a single copulation event is sufficient to restore offspring sex ratios to the theoretical 1:1 equilibrium — establishes E. vitticolle as a functionally monandrous species. This is consistent with the predominant mating strategy across the Ichneumonidae and parasitoid Hymenoptera more broadly, where approximately 70% of species exhibit monandry (Ridley 1993 ; Godfray 1994 ). The absence of significant differences among the Hc, Hcc, and HM treatments indicates that, once the spermatheca has been loaded by a single ejaculate, additional copulations confer no measurable reproductive benefit (Fig. 1 ). Similar patterns have been documented in other ichneumonid and braconid parasitoids, where the spermatheca effectively serves as a lifetime sperm reserve (Jackson 1958 ; Simmonds 1953 ; Boivin 2013 ). The sex ratio results reported here should be interpreted in the context of host-size-dependent sex allocation. A companion study demonstrated that host developmental stage critically modulates offspring sex ratio in E. vitticolle : instar I hosts yield exclusively male offspring, whereas instar III hosts produce near-parity ratios (Giraldo-Vanegas et al. 2025 ). This pattern is consistent with resource-based sex allocation theory (Hamilton 1967 ; Charnov 1982 ; King 1987 ): small hosts provide insufficient nutritional resources to support the greater developmental demands of female offspring, leading mothers to deposit unfertilized (male) eggs selectively. In the present experiments, L2–L3 hosts were used because they fall within the susceptibility window confirmed by Giraldo-Vanegas et al. ( 2025 ) and are large enough to sustain mixed-sex offspring production in sperm-replete females. The absence of significant differences in total adult emergence rates among treatments suggests that the mating history of E. vitticolle females does not measurably affect foraging efficiency or oviposition behavior during the six-hour exposure window. This contrasts with reports in some polyandrous parasitoid species, where additional matings increase fecundity through supplementary sperm transfer and seminal fluid proteins (Boulton et al. 2015 ). The absence of such an effect in E. vitticolle is entirely consistent with its monandrous strategy: if a single mating saturates the spermatheca, further copulations are functionally redundant. The monandrous nature of E. vitticolle has direct practical implications for mass-rearing operations targeting augmentative biological control of S. frugiperda . Our results demonstrate that a single controlled mating event per female is biologically sufficient to maintain female offspring production — the primary functional agents in augmentative biocontrol releases. Mass-rearing protocols can therefore be simplified by implementing brief, supervised mating periods (as in treatment Hc), followed by immediate male removal, with no penalty to reproductive efficiency. This approach would substantially reduce the labor and resources currently allocated to maintaining large male populations, thereby maximizing the productive capacity of rearing facilities. Furthermore, given that successful parasitoid establishment is restricted to the first three larval instars of S. frugiperda (Giraldo-Vanegas et al. 2025 ), precise synchronization of parasitoid releases with early host larval peaks — informed by pheromone-trap monitoring and degree-day phenological models — is essential for translating laboratory efficiency gains into field efficacy (Kenis et al. 2023 ; Wyckhuys et al. 2024 ). Future studies should examine the role of female age and progressive sperm depletion on sex ratio shifts over the full reproductive lifespan of E. vitticolle , as well as the interactive effects of host larval instar, female nutritional status, and ambient temperature on offspring sex allocation. Molecular verification of the taxonomic identity of the Colombian colony relative to E. laphygmae sensu Gauld ( 2000 ) is also recommended, given ongoing uncertainty in the taxonomy of Eiphosoma species attacking S. frugiperda in the Neotropical region (Allen et al. 2021 ; Kenis et al. 2023 ). In conclusion, E. vitticolle is a strictly arrhenotokous, functionally monandrous parasitoid. A single copulation saturates the spermatheca and maintains balanced offspring sex ratios at approximately 1:1 throughout the female's reproductive period. Neither additional copulations nor courtship without insemination improved reproductive outcomes over a single controlled mating (Fig. 1 ; Table 1). These findings provide an empirical basis for streamlining mass-rearing protocols by adopting single-mating strategies, thereby optimizing resource allocation in biological control programs targeting S. frugiperda in Neotropical agricultural systems. Declarations Competing interests The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper. Funding This research did not receive specific funding from any public, commercial, or not-for-profit funding agency. Institutional support was provided by the University of Pamplona, Norte de Santander, Colombia. Author contributions HGV: Conceptualization, Methodology, Investigation, Formal Analysis, Data Curation, Writing – Original Draft. GGH: Investigation, Data Curation, Writing – Review & Editing. Both authors read and approved the final version. Data availability The dataset supporting the conclusions of this article is available from the corresponding author upon reasonable request and will be deposited in a public repository upon acceptance. Ethics approval This study involved only invertebrate insects ( Spodoptera frugiperda and Eiphosoma vitticolle ). No vertebrate animals or human participants were involved; consequently, formal ethics committee approval was not required under applicable Colombian regulations (Ley 84 de 1989) or international guidelines for entomological research. Acknowledgements The authors thank the technical staff of the Entomology Laboratory, Faculty of Agricultural Sciences, University of Pamplona, for assistance with insect colony maintenance, and two anonymous reviewers for critical comments that improved an earlier version of the manuscript. AI use disclosure AI-assisted language tools were used exclusively for grammar and style revision of an English-language manuscript prepared by non-native English speakers. All scientific content, data, interpretations, and conclusions are solely the intellectual product of the authors. References Allen T, Kenis M, Norgrove L (2021) Eiphosoma laphygmae , a classical solution for the biocontrol of the fall armyworm, Spodoptera frugiperda ? J Plant Dis Prot 128:1141–1156. https://doi.org/10.1007/s41348-021-00480-9 Boaventura D, Martin M, Pozzebon A, Mota-Sanchez D, Nauen R (2020) Monitoring of target-site mutations conferring insecticide resistance in Spodoptera frugiperda . Insects 11:545. https://doi.org/10.3390/insects11080545 Boivin G (2013) Sperm as a limiting factor in mating success in Hymenoptera parasitoids. Entomol Exp Appl 146:149–155. https://doi.org/10.1111/eea.12003 Boulton RA, Collins LA, Shuker DM (2015) Beyond sex allocation: the role of mating systems in sexual selection in parasitoid wasps. Biol Rev 90:599–627. https://doi.org/10.1111/brv.12126 Burton RL, Perkins WD (1972) WSB, a new laboratory diet for the corn earworm and the fall armyworm. J Econ Entomol 65:385–386. https://doi.org/10.1093/jee/65.2.385 Charnov EL (1982) The Theory of Sex Allocation. Princeton University Press, Princeton, NJ Day R, Abrahams P, Bateman M, Beale T, Clottey V, Cock M, Colmenarez Y, Corniani N, Early R, Godwin J, Gomez J, Moreno PG, Murphy ST, Oppong-Mensah B, Phiri N, Pratt C, Silvestri S, Witt A (2017) Fall armyworm: impacts and implications for Africa. Outlooks Pest Manag 28:196–201. https://doi.org/10.1564/v28_oct_02 Fagan-Jeffries EP, Broad GR, Atkin-Zaldivar CM, Dennis TE, Flett BJ, Gibbs G, Mally R, Peterson GM, Riegler M, Spafford H, Thistleton B (2024) Hymenopteran parasitoids of fall armyworm ( Spodoptera frugiperda ) in Australia, with the description of five new species in the families Braconidae and Eulophidae. Austral Entomol 63:136–174. https://doi.org/10.1111/aen.12682 Feldmann F, Rieckmann U, Winter S (2019) The spread of the fall armyworm Spodoptera frugiperda in Africa — what should be done next? J Plant Dis Prot 126:97–101. https://doi.org/10.1007/s41348-018-0200-5 Flanders SE (1939) Environmental control of sex in hymenopterous insects. Ann Entomol Soc Am 32:11–26. https://doi.org/10.1093/aesa/32.1.11 Flanders SE (1946) Control of sex and sex-limited polymorphism in the Hymenoptera. Q Rev Biol 21:135–143. https://doi.org/10.1086/395009 Gauld ID (2000) The Ichneumonidae of Costa Rica. Mem Am Entomol Inst 66:1–4534 Giraldo-Vanegas H, Giraldo-Herrera G, Nieto-Triviño JI (2025) Age-dependent immune maturation defines the susceptibility window of Spodoptera frugiperda to the parasitoid Eiphosoma vitticolle . Preprint Res Square. https://doi.org/10.21203/rs.3.rs-9079676/v1 Godfray HCJ (1994) Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton, NJ Goergen G, Kumar PL, Sankung SB, Togola A, Tamò M (2016) First report of outbreaks of the fall armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS ONE 11:e0165632. https://doi.org/10.1371/journal.pone.0165632 Hamilton WD (1967) Extraordinary sex ratios. Science 156:477–488. https://doi.org/10.1126/science.156.3774.477 Heimpel GE, de Boer JG (2008) Sex determination in the Hymenoptera. Annu Rev Entomol 53:209–230. https://doi.org/10.1146/annurev.ento.53.103106.093441 Jackson DJ (1958) Observations on the biology of Caraphractus cinctus Walker (Hymenoptera: Mymaridae), a parasitoid of the eggs of Dytiscidae (Coleoptera). Trans R Entomol Soc Lond 110:533–554. https://doi.org/10.1111/j.1365-2311.1958.tb01304.x Jing DP, Guo JF, Jiang YY, Zhao JZ, Sethi A, He KL, Wang ZY (2021) Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques. Insect Sci 28:780–790. https://doi.org/10.1111/1744-7917.12800 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 . Entomol Gen 43:187–241. https://doi.org/10.1127/entomologia/2022/1659 Kenis M, du Plessis H, Van den Berg J, Ba MN, Goergen G, Kwadjo KE et al (2023) Prospects for classical biological control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in invaded areas using parasitoids from the Americas. J Econ Entomol 116:331–341. https://doi.org/10.1093/jee/toad029 Kenis M, Van Tol R, Sansen U, Maspero M, Linder C (2024) Pre-emptive augmentative biological control of Spodoptera frugiperda in Europe using Trichogramma spp. CABI Agric Biosci 5:96. https://doi.org/10.1186/s43170-024-00296-1 King BH (1987) Offspring sex ratios in parasitoid wasps. Q Rev Biol 62:367–396. https://doi.org/10.1086/415618 Molina-Ochoa J, Carpenter JE, Heinrichs EA, Foster JE (2003) Parasitoids and parasites of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas and the Caribbean Basin: an inventory. Fla Entomol 86:254–289. https://doi.org/10.1653/0015-4040 (2003)086[0254:PAPOSF]2.0.CO;2 Montezano DG, Specht A, Sosa-Gómez DR, Roque-Specht VF, Sousa-Silva JC, Paula-Moraes SV, Peterson JA, Hunt TE (2018) Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. Afr Entomol 26:286–300. https://doi.org/10.4001/003.026.0286 R Core Team (2024) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/ Ridley M (1993) Clutch size and mating frequency in parasitic Hymenoptera. Am Nat 142:893–911. https://doi.org/10.1086/285579 Simmonds FJ (1953) Observations on the biology and mass-breeding of Spalangia drosophilae Ashmead (Hymenoptera: Pteromalidae), a parasite of the frit-fly, Oscinella frit . Bull Entomol Res 44:773–778. https://doi.org/10.1017/S0007485300025591 Sisay B, Simiyu J, Malusi P, Likhayo P, Mendesil E, Elibariki N, Wakgari M, Ayalew G, Tefera T (2019) First report of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), natural enemies from Africa. J Appl Entomol 143:110–114. https://doi.org/10.1111/jen.12534 Wyckhuys KAG, Akutse KS, Amalin DM, Araj S-E, Barrera G, Beltran MJB et al (2024) Global scientific progress and shortfalls in biological control of the fall armyworm Spodoptera frugiperda . Biol Control 191:105460. https://doi.org/10.1016/j.biocontrol.2024.105460 Tables Table 1 Offspring sex ratio of Eiphosoma vitticolle Cresson under five mating frequency treatments and comparison with the theoretical 1:1 ratio (Pearson's χ² goodness-of-fit test; α = 0.05). Treatment Males (n) Females (n) Sex ratio (♂:♀) χ ² (df = 1) Hv (virgin) 45 0 45:0 45.00** Hv+Mi (impeded mating) 38 0 38:0 38.00** Hc (single mating) 22 18 1.22:1 0.40 ns Hcc (multiple matings) 24 18 1.33:1 0.86 ns HM (permanent cohabitation) 22 20 1.10:1 0.09 ns ** P 0.05). For Hv and Hv+Mi, the χ² goodness-of-fit test was applied against the theoretical 1:1 expectation. Additional Declarations The authors declare no competing interests. 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-9534338","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629845715,"identity":"7d8bc55c-efd8-47f5-84ed-e2fec81dec38","order_by":0,"name":"Humberto Giraldo-Vanegas","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0801-2714","institution":"Universidad de Pamplona","correspondingAuthor":true,"prefix":"","firstName":"Humberto","middleName":"","lastName":"Giraldo-Vanegas","suffix":""},{"id":629845716,"identity":"8fc712e7-b06e-4b5c-af8f-c23b36b0cf18","order_by":1,"name":"Gabriel Giraldo-Herrera","email":"","orcid":"","institution":"Universidad de Pamplona","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Giraldo-Herrera","suffix":""}],"badges":[],"createdAt":"2026-04-26 20:19:32","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9534338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9534338/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107990087,"identity":"8474e6a0-6de5-42a1-9119-32ffeeb785cd","added_by":"auto","created_at":"2026-04-28 10:00:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193676,"visible":true,"origin":"","legend":"\u003cp\u003eOffspring sex ratio (proportion of males) of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson under five mating treatments. Hv: virgin females; Hv+Mi: virgin females exposed to physically impeded males; Hc: singly mated females; Hcc: multiply mated females; HM: females in permanent cohabitation with males. The dashed horizontal line at 0.5 indicates the theoretical 1:1 sex ratio. Double asterisks (**) above bars indicate significant departure from 1:1 (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001); bars labeled ns are not significantly different from 0.5 (\u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05). Error bars represent ± standard error of the mean.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9534338/v1/69c7cd0e0d3ca7a3bd5a5cd4.png"},{"id":109067389,"identity":"63a01e32-26b2-4852-a5e6-e563eab94666","added_by":"auto","created_at":"2026-05-12 09:40:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":477133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9534338/v1/55588c15-e94b-4e46-95ab-89fb28035346.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eFunctional monandry in \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson (Hymenoptera: Ichneumonidae): a single mating saturates the spermatheca and restores balanced offspring sex ratios\u003c/p\u003e","fulltext":[{"header":"Key Message","content":"\u003cul\u003e\n \u003cli\u003eVirgin females of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e produce exclusively male offspring (100%), confirming strict arrhenotokous reproduction and demonstrating that courtship without insemination does not trigger egg fertilization.\u003c/li\u003e\n \u003cli\u003eA single mating event fully saturates the spermatheca, restoring offspring sex ratios to the theoretical 1:1 equilibrium throughout the female\u0026apos;s reproductive lifespan; additional copulations confer no measurable reproductive benefit.\u003c/li\u003e\n \u003cli\u003eThese findings establish \u003cem\u003eE. vitticolle\u003c/em\u003e as a functionally monandrous species and provide an empirical basis for streamlining mass-rearing protocols for augmentative biological control of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in Neotropical agroecosystems: a single supervised mating per female is biologically sufficient, reducing male-cohort maintenance costs without compromising female offspring production.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) (Lepidoptera: Noctuidae) is one of the most economically devastating invasive arthropod pests globally, attacking 353 plant species and causing annual yield losses exceeding US \u003cspan\u003e$\u003c/span\u003e13\u0026nbsp;billion across sub-Saharan Africa (Day et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Montezano et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Native to the tropical Americas, the species was first detected in Africa in 2016 and spread across 44 countries within three years (Goergen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Feldmann et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), subsequently extending its range into Asia (Jing et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Australia (Fagan-Jeffries et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and most recently Europe (Kenis et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This extraordinary adaptive capacity is underpinned by a short generation time, high fecundity, long-distance migratory behavior, and documented resistance to multiple insecticide classes (Kenis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Boaventura et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Augmentative biological control using larval parasitoids has emerged as a sustainable, environmentally sound component of integrated pest management (IPM) strategies against this polyphagous noctuid (Wyckhuys et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Molina-Ochoa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sisay et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson (Hymenoptera: Ichneumonidae) is a solitary koinobiont endoparasitoid whose primary host in the Neotropical region is \u003cem\u003eS. frugiperda\u003c/em\u003e. As a member of the subfamily Cremastinae, this species attacks early-instar host larvae and completes development within the host body before killing it at the pupal stage (Molina-Ochoa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A taxonomic note is warranted: recent revisions indicate that most records of \u003cem\u003eEiphosoma\u003c/em\u003e spp. attacking \u003cem\u003eS. frugiperda\u003c/em\u003e in the Americas may refer to \u003cem\u003eEiphosoma laphygmae\u003c/em\u003e Costa-Lima rather than \u003cem\u003eE. vitticolle\u003c/em\u003e sensu stricto (Gauld \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Allen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The specimens used in the present study were identified as \u003cem\u003eE. vitticolle\u003c/em\u003e following morphological examination of the rearing colony maintained at the University of Pamplona, Colombia; molecular verification is recommended for future studies. Recent experimental work has established that successful adult parasitoid emergence from \u003cem\u003eS. frugiperda\u003c/em\u003e is restricted to a six-day susceptibility window corresponding to larval instars I\u0026ndash;III, owing to an ontogenetic immune transition mediated by granulocyte-dependent hemocytic encapsulation from instar IV onwards (Giraldo-Vanegas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This immunological constraint directly informs the choice of host larval age for laboratory experiments and augmentative release strategies.\u003c/p\u003e \u003cp\u003eIn the Hymenoptera, sex determination operates predominantly through arrhenotokous haplodiploidy: unfertilized, haploid eggs develop into males, whereas fertilized, diploid eggs develop into females (Flanders \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1939\u003c/span\u003e; Heimpel and de Boer \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Females of most hymenopteran species possess a spermatheca that functions as a long-term sperm storage organ (Flanders \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1939\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1946\u003c/span\u003e). Species that require only a single mating event to saturate the spermatheca are termed monandrous, whereas those requiring multiple copulations are termed polyandrous (Ridley \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Godfray \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Monandry appears to be the predominant mating strategy in parasitoid wasps (\u0026asymp;\u0026thinsp;70% of species; Ridley \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Sex ratio theory predicts that haplodiploid females should allocate offspring sex facultatively in response to host resource availability (Hamilton \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Charnov \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; King \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Sperm supply constraints can substantially disrupt optimal sex allocation: sperm-depleted or unmated females produce exclusively male offspring, whereas sperm-replete females regulate offspring sex ratios according to host quality cues (Boivin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Boulton et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Empirical determination of whether a species is monandrous or polyandrous is therefore essential for designing efficient mass-rearing programs, because maintaining unnecessarily large male cohorts diverts resources and reduces overall production efficiency (Godfray \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aimed to: (i) characterize the reproductive mode of \u003cem\u003eE. vitticolle\u003c/em\u003e; (ii) determine the minimum number of copulations required to maintain balanced offspring sex ratios throughout the female's reproductive lifespan; and (iii) evaluate the effect of mating frequency on adult emergence rates from \u003cem\u003eS. frugiperda\u003c/em\u003e host larvae under controlled laboratory conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParasitoid and host colonies\u003c/h2\u003e \u003cp\u003eColonies of \u003cem\u003eE. vitticolle\u003c/em\u003e and \u003cem\u003eS. frugiperda\u003c/em\u003e were maintained at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 12:12 h light:dark photoperiod. \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e larvae were reared on artificial diet following standard protocols (Burton and Perkins \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Adult parasitoids were provided with a 10% honey solution as a carbohydrate source and held in ventilated plexiglass cages (30 \u0026times; 30 \u0026times; 30 cm).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eBioassays were conducted using five seven-day-old females per treatment (five mating treatments \u0026times; five replicates; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25 females in total). Host larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e at six days of age (L2\u0026ndash;L3 instar) were used in all treatments, corresponding to the validated susceptibility window for this parasitoid (Giraldo-Vanegas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The five mating treatments were: (i) \u003cb\u003eHv\u003c/b\u003e \u0026ndash; virgin females maintained without male contact since emergence; (ii) \u003cb\u003eHv\u0026thinsp;+\u0026thinsp;Mi\u003c/b\u003e \u0026ndash; virgin females exposed to physically impeded males (male genitalia immobilized with fine surgical thread to prevent intromission while permitting full courtship behavior); (iii) \u003cb\u003eHc\u003c/b\u003e \u0026ndash; singly mated females that completed one copulation 24 h prior to the assay, after which the male was immediately removed; (iv) \u003cb\u003eHcc\u003c/b\u003e \u0026ndash; multiply mated females that cohabited with males from emergence until the day prior to the assay; and (v) \u003cb\u003eHM\u003c/b\u003e \u0026ndash; females maintained in permanent cohabitation with males, including during the oviposition window.\u003c/p\u003e\n\u003ch3\u003eParasitism bioassays\u003c/h3\u003e\n\u003cp\u003eIn each experimental unit, one female was confined with 40 \u003cem\u003eS. frugiperda\u003c/em\u003e L2\u0026ndash;L3 larvae in a plexiglass arena (diameter 15 cm, height 20 cm) for a six-hour exposure period (08:00\u0026ndash;14:00 h). Host larvae were subsequently individualized in 30-mL plastic cups with fresh artificial diet until either pupal formation of \u003cem\u003eS. frugiperda\u003c/em\u003e or emergence of parasitoid cocoons. Emerged adult parasitoids were sexed under a stereomicroscope and counted.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEmergence proportion data were arcsine-square-root transformed [arcsin(\u0026radic;\u003cem\u003ep\u003c/em\u003e)] prior to analysis. Differences in emergence rates among treatments were evaluated by one-way ANOVA followed by Tukey's HSD post-hoc test (α\u0026thinsp;=\u0026thinsp;0.05). Offspring sex ratios in each treatment were compared against the theoretical 1:1 null hypothesis using Pearson's chi-squared (χ\u0026sup2;) goodness-of-fit test. All analyses were performed in R version 4.4.1 (R Core Team \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReproductive mode\u003c/h2\u003e \u003cp\u003ePhenotypic analysis of progeny confirmed that \u003cem\u003eE. vitticolle\u003c/em\u003e reproduces via strict arrhenotoky. In both treatments without effective sperm transfer (Hv and Hv\u0026thinsp;+\u0026thinsp;Mi), 100% of offspring were male (Table\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Treatment Hv yielded 45 males and 0 females (χ\u0026sup2; = 45.00, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); treatment Hv\u0026thinsp;+\u0026thinsp;Mi yielded 38 males and 0 females (χ\u0026sup2; = 38.00, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), demonstrating that courtship without insemination does not stimulate egg fertilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of mating frequency on offspring sex ratio\u003c/h3\u003e\n\u003cp\u003eAll treatments with at least one completed copulation (Hc, Hcc, HM) produced mixed-sex offspring (Table\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In treatment Hc, 22 males and 18 females emerged (ratio 1.22:1; χ\u0026sup2; = 0.40, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.527). Treatment Hcc yielded 24 males and 18 females (ratio 1.33:1; χ\u0026sup2; = 0.86, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.354), and treatment HM yielded 22 males and 20 females (ratio 1.10:1; χ\u0026sup2; = 0.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.764). None of the mated treatments differed significantly from the theoretical 1:1 ratio, nor did they differ significantly from one another.\u003c/p\u003e\n\u003ch3\u003eAdult emergence rates\u003c/h3\u003e\n\u003cp\u003eNo statistically significant differences in adult emergence rates were detected among the five mating treatments (one-way ANOVA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Mating frequency had no significant effect on the total number of adult parasitoids that successfully emerged from \u003cem\u003eS. frugiperda\u003c/em\u003e host larvae.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides the first experimental characterization of the mating system and its reproductive consequences in \u003cem\u003eE. vitticolle\u003c/em\u003e. The complete male bias of offspring from unmated females (Hv and Hv\u0026thinsp;+\u0026thinsp;Mi) unequivocally confirms that this species reproduces through strict arrhenotoky, the ancestral and numerically dominant mode of sex determination across the Hymenoptera (Flanders \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1939\u003c/span\u003e; Heimpel and de Boer \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The inability of courtship behavior alone (treatment Hv\u0026thinsp;+\u0026thinsp;Mi) to influence offspring sex composition further demonstrates that genital coupling and physical sperm transfer \u0026mdash; rather than any pre-copulatory signal \u0026mdash; are the primary determinants of egg fertilization in \u003cem\u003eE. vitticolle\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe central finding \u0026mdash; that a single copulation event is sufficient to restore offspring sex ratios to the theoretical 1:1 equilibrium \u0026mdash; establishes \u003cem\u003eE. vitticolle\u003c/em\u003e as a functionally monandrous species. This is consistent with the predominant mating strategy across the Ichneumonidae and parasitoid Hymenoptera more broadly, where approximately 70% of species exhibit monandry (Ridley \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Godfray \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The absence of significant differences among the Hc, Hcc, and HM treatments indicates that, once the spermatheca has been loaded by a single ejaculate, additional copulations confer no measurable reproductive benefit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar patterns have been documented in other ichneumonid and braconid parasitoids, where the spermatheca effectively serves as a lifetime sperm reserve (Jackson \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Simmonds \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1953\u003c/span\u003e; Boivin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe sex ratio results reported here should be interpreted in the context of host-size-dependent sex allocation. A companion study demonstrated that host developmental stage critically modulates offspring sex ratio in \u003cem\u003eE. vitticolle\u003c/em\u003e: instar I hosts yield exclusively male offspring, whereas instar III hosts produce near-parity ratios (Giraldo-Vanegas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This pattern is consistent with resource-based sex allocation theory (Hamilton \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Charnov \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; King \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1987\u003c/span\u003e): small hosts provide insufficient nutritional resources to support the greater developmental demands of female offspring, leading mothers to deposit unfertilized (male) eggs selectively. In the present experiments, L2\u0026ndash;L3 hosts were used because they fall within the susceptibility window confirmed by Giraldo-Vanegas et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and are large enough to sustain mixed-sex offspring production in sperm-replete females.\u003c/p\u003e \u003cp\u003eThe absence of significant differences in total adult emergence rates among treatments suggests that the mating history of \u003cem\u003eE. vitticolle\u003c/em\u003e females does not measurably affect foraging efficiency or oviposition behavior during the six-hour exposure window. This contrasts with reports in some polyandrous parasitoid species, where additional matings increase fecundity through supplementary sperm transfer and seminal fluid proteins (Boulton et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The absence of such an effect in \u003cem\u003eE. vitticolle\u003c/em\u003e is entirely consistent with its monandrous strategy: if a single mating saturates the spermatheca, further copulations are functionally redundant.\u003c/p\u003e \u003cp\u003eThe monandrous nature of \u003cem\u003eE. vitticolle\u003c/em\u003e has direct practical implications for mass-rearing operations targeting augmentative biological control of \u003cem\u003eS. frugiperda\u003c/em\u003e. Our results demonstrate that a single controlled mating event per female is biologically sufficient to maintain female offspring production \u0026mdash; the primary functional agents in augmentative biocontrol releases. Mass-rearing protocols can therefore be simplified by implementing brief, supervised mating periods (as in treatment Hc), followed by immediate male removal, with no penalty to reproductive efficiency. This approach would substantially reduce the labor and resources currently allocated to maintaining large male populations, thereby maximizing the productive capacity of rearing facilities. Furthermore, given that successful parasitoid establishment is restricted to the first three larval instars of \u003cem\u003eS. frugiperda\u003c/em\u003e (Giraldo-Vanegas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), precise synchronization of parasitoid releases with early host larval peaks \u0026mdash; informed by pheromone-trap monitoring and degree-day phenological models \u0026mdash; is essential for translating laboratory efficiency gains into field efficacy (Kenis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wyckhuys et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFuture studies should examine the role of female age and progressive sperm depletion on sex ratio shifts over the full reproductive lifespan of \u003cem\u003eE. vitticolle\u003c/em\u003e, as well as the interactive effects of host larval instar, female nutritional status, and ambient temperature on offspring sex allocation. Molecular verification of the taxonomic identity of the Colombian colony relative to \u003cem\u003eE. laphygmae\u003c/em\u003e sensu Gauld (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) is also recommended, given ongoing uncertainty in the taxonomy of \u003cem\u003eEiphosoma\u003c/em\u003e species attacking \u003cem\u003eS. frugiperda\u003c/em\u003e in the Neotropical region (Allen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kenis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, \u003cem\u003eE. vitticolle\u003c/em\u003e is a strictly arrhenotokous, functionally monandrous parasitoid. A single copulation saturates the spermatheca and maintains balanced offspring sex ratios at approximately 1:1 throughout the female's reproductive period. Neither additional copulations nor courtship without insemination improved reproductive outcomes over a single controlled mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;1). These findings provide an empirical basis for streamlining mass-rearing protocols by adopting single-mating strategies, thereby optimizing resource allocation in biological control programs targeting \u003cem\u003eS. frugiperda\u003c/em\u003e in Neotropical agricultural systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive specific funding from any public, commercial, or not-for-profit funding agency. Institutional support was provided by the University of Pamplona, Norte de Santander, Colombia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHGV: Conceptualization, Methodology, Investigation, Formal Analysis, Data Curation, Writing – Original Draft. GGH: Investigation, Data Curation, Writing – Review \u0026amp; Editing. Both authors read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is available from the corresponding author upon reasonable request and will be deposited in a public repository upon acceptance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study involved only invertebrate insects (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e and \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e). No vertebrate animals or human participants were involved; consequently, formal ethics committee approval was not required under applicable Colombian regulations (Ley 84 de 1989) or international guidelines for entomological research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the technical staff of the Entomology Laboratory, Faculty of Agricultural Sciences, University of Pamplona, for assistance with insect colony maintenance, and two anonymous reviewers for critical comments that improved an earlier version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAI use disclosure\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAI-assisted language tools were used exclusively for grammar and style revision of an English-language manuscript prepared by non-native English speakers. All scientific content, data, interpretations, and conclusions are solely the intellectual product of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllen T, Kenis M, Norgrove L (2021) \u003cem\u003eEiphosoma laphygmae\u003c/em\u003e, a classical solution for the biocontrol of the fall armyworm, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e? J Plant Dis Prot 128:1141\u0026ndash;1156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s41348-021-00480-9\u003c/span\u003e\u003cspan address=\"10.1007/s41348-021-00480-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoaventura D, Martin M, Pozzebon A, Mota-Sanchez D, Nauen R (2020) Monitoring of target-site mutations conferring insecticide resistance in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. Insects 11:545. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects11080545\u003c/span\u003e\u003cspan address=\"10.3390/insects11080545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoivin G (2013) Sperm as a limiting factor in mating success in Hymenoptera parasitoids. Entomol Exp Appl 146:149\u0026ndash;155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/eea.12003\u003c/span\u003e\u003cspan address=\"10.1111/eea.12003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoulton RA, Collins LA, Shuker DM (2015) Beyond sex allocation: the role of mating systems in sexual selection in parasitoid wasps. Biol Rev 90:599\u0026ndash;627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/brv.12126\u003c/span\u003e\u003cspan address=\"10.1111/brv.12126\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton RL, Perkins WD (1972) WSB, a new laboratory diet for the corn earworm and the fall armyworm. J Econ Entomol 65:385\u0026ndash;386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jee/65.2.385\u003c/span\u003e\u003cspan address=\"10.1093/jee/65.2.385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharnov EL (1982) The Theory of Sex Allocation. Princeton University Press, Princeton, NJ\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDay R, Abrahams P, Bateman M, Beale T, Clottey V, Cock M, Colmenarez Y, Corniani N, Early R, Godwin J, Gomez J, Moreno PG, Murphy ST, Oppong-Mensah B, Phiri N, Pratt C, Silvestri S, Witt A (2017) Fall armyworm: impacts and implications for Africa. Outlooks Pest Manag 28:196\u0026ndash;201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1564/v28_oct_02\u003c/span\u003e\u003cspan address=\"10.1564/v28_oct_02\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFagan-Jeffries EP, Broad GR, Atkin-Zaldivar CM, Dennis TE, Flett BJ, Gibbs G, Mally R, Peterson GM, Riegler M, Spafford H, Thistleton B (2024) Hymenopteran parasitoids of fall armyworm (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) in Australia, with the description of five new species in the families Braconidae and Eulophidae. Austral Entomol 63:136\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/aen.12682\u003c/span\u003e\u003cspan address=\"10.1111/aen.12682\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeldmann F, Rieckmann U, Winter S (2019) The spread of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in Africa \u0026mdash; what should be done next? J Plant Dis Prot 126:97\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s41348-018-0200-5\u003c/span\u003e\u003cspan address=\"10.1007/s41348-018-0200-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlanders SE (1939) Environmental control of sex in hymenopterous insects. Ann Entomol Soc Am 32:11\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aesa/32.1.11\u003c/span\u003e\u003cspan address=\"10.1093/aesa/32.1.11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlanders SE (1946) Control of sex and sex-limited polymorphism in the Hymenoptera. Q Rev Biol 21:135\u0026ndash;143. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/395009\u003c/span\u003e\u003cspan address=\"10.1086/395009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGauld ID (2000) The Ichneumonidae of Costa Rica. Mem Am Entomol Inst 66:1\u0026ndash;4534\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiraldo-Vanegas H, Giraldo-Herrera G, Nieto-Trivi\u0026ntilde;o JI (2025) Age-dependent immune maturation defines the susceptibility window of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e to the parasitoid \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e. Preprint Res Square. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21203/rs.3.rs-9079676/v1\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-9079676/v1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodfray HCJ (1994) Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton, NJ\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoergen G, Kumar PL, Sankung SB, Togola A, Tam\u0026ograve; M (2016) First report of outbreaks of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS ONE 11:e0165632. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0165632\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0165632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamilton WD (1967) Extraordinary sex ratios. Science 156:477\u0026ndash;488. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.156.3774.477\u003c/span\u003e\u003cspan address=\"10.1126/science.156.3774.477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeimpel GE, de Boer JG (2008) Sex determination in the Hymenoptera. Annu Rev Entomol 53:209\u0026ndash;230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.ento.53.103106.093441\u003c/span\u003e\u003cspan address=\"10.1146/annurev.ento.53.103106.093441\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson DJ (1958) Observations on the biology of \u003cem\u003eCaraphractus cinctus\u003c/em\u003e Walker (Hymenoptera: Mymaridae), a parasitoid of the eggs of Dytiscidae (Coleoptera). Trans R Entomol Soc Lond 110:533\u0026ndash;554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2311.1958.tb01304.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2311.1958.tb01304.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing DP, Guo JF, Jiang YY, Zhao JZ, Sethi A, He KL, Wang ZY (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. Insect Sci 28:780\u0026ndash;790. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1744-7917.12800\u003c/span\u003e\u003cspan address=\"10.1111/1744-7917.12800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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. Entomol Gen 43:187\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1127/entomologia/2022/1659\u003c/span\u003e\u003cspan address=\"10.1127/entomologia/2022/1659\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenis M, du Plessis H, Van den Berg J, Ba MN, Goergen G, Kwadjo KE 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. J Econ Entomol 116:331\u0026ndash;341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jee/toad029\u003c/span\u003e\u003cspan address=\"10.1093/jee/toad029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenis M, Van Tol R, Sansen U, Maspero M, Linder C (2024) Pre-emptive augmentative biological control of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e in Europe using \u003cem\u003eTrichogramma\u003c/em\u003e spp. CABI Agric Biosci 5:96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43170-024-00296-1\u003c/span\u003e\u003cspan address=\"10.1186/s43170-024-00296-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing BH (1987) Offspring sex ratios in parasitoid wasps. Q Rev Biol 62:367\u0026ndash;396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/415618\u003c/span\u003e\u003cspan address=\"10.1086/415618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolina-Ochoa J, Carpenter JE, Heinrichs EA, Foster JE (2003) Parasitoids and parasites of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae) in the Americas and the Caribbean Basin: an inventory. Fla Entomol 86:254\u0026ndash;289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1653/0015-4040\u003c/span\u003e\u003cspan address=\"10.1653/0015-4040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e(2003)086[0254:PAPOSF]2.0.CO;2\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontezano DG, Specht A, Sosa-G\u0026oacute;mez DR, Roque-Specht VF, Sousa-Silva JC, Paula-Moraes SV, Peterson JA, Hunt TE (2018) Host plants of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae) in the Americas. Afr Entomol 26:286\u0026ndash;300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4001/003.026.0286\u003c/span\u003e\u003cspan address=\"10.4001/003.026.0286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team (2024) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRidley M (1993) Clutch size and mating frequency in parasitic Hymenoptera. Am Nat 142:893\u0026ndash;911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/285579\u003c/span\u003e\u003cspan address=\"10.1086/285579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimmonds FJ (1953) Observations on the biology and mass-breeding of \u003cem\u003eSpalangia drosophilae\u003c/em\u003e Ashmead (Hymenoptera: Pteromalidae), a parasite of the frit-fly, \u003cem\u003eOscinella frit\u003c/em\u003e. Bull Entomol Res 44:773\u0026ndash;778. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0007485300025591\u003c/span\u003e\u003cspan address=\"10.1017/S0007485300025591\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSisay B, Simiyu J, Malusi P, Likhayo P, Mendesil E, Elibariki N, Wakgari M, Ayalew G, Tefera T (2019) First report of the fall armyworm, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Lepidoptera: Noctuidae), natural enemies from Africa. J Appl Entomol 143:110\u0026ndash;114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jen.12534\u003c/span\u003e\u003cspan address=\"10.1111/jen.12534\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWyckhuys KAG, Akutse KS, Amalin DM, Araj S-E, Barrera G, Beltran MJB et al (2024) Global scientific progress and shortfalls in biological control of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. Biol Control 191:105460. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocontrol.2024.105460\u003c/span\u003e\u003cspan address=\"10.1016/j.biocontrol.2024.105460\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOffspring sex ratio of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson under five mating frequency treatments and comparison with the theoretical 1:1 ratio (Pearson\u0026apos;s \u0026chi;\u0026sup2; goodness-of-fit test; \u0026alpha; = 0.05).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Males (n)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Females (n)\u0026nbsp; \u0026nbsp;\u0026nbsp;Sex ratio (♂:♀)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026chi;\u003c/strong\u003e\u003cstrong\u003e\u0026sup2; (df = 1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHv (virgin)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;45\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;0\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;45:0\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;45.00**\u003c/p\u003e\n\u003cp\u003eHv+Mi (impeded mating)\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;38\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;0\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;38:0\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;38.00**\u003c/p\u003e\n\u003cp\u003eHc (single mating)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;22\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;18\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;1.22:1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;0.40 ns\u003c/p\u003e\n\u003cp\u003eHcc (multiple matings) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 24 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;18 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1.33:1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.86 ns\u003c/p\u003e\n\u003cp\u003eHM (permanent cohabitation) 22 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;20 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1.10:1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.09 ns\u003c/p\u003e\n\u003cp\u003e** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). For Hv and Hv+Mi, the \u0026chi;\u0026sup2; goodness-of-fit test was applied against the theoretical 1:1 expectation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\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":"arrhenotoky, biological control, fall armyworm, haplodiploidy, Ichneumonidae, mass rearing, monandry, sex ratio, Spodoptera frugiperda","lastPublishedDoi":"10.21203/rs.3.rs-9534338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9534338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding mating systems in parasitoid Hymenoptera is fundamental to optimizing mass-rearing programs for biological control. \u003cem\u003eEiphosoma vitticolle\u003c/em\u003eCresson (Hymenoptera: Ichneumonidae) is a solitary koinobiont endoparasitoid of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) (Lepidoptera: Noctuidae), a globally invasive pest of major economic importance. We investigated the effect of mating frequency on adult emergence rates and offspring sex ratios of \u003cem\u003eE. vitticolle\u003c/em\u003e under controlled laboratory conditions using five treatments: virgin females (Hv), virgin females exposed to physically impeded males (Hv+Mi), singly mated females (Hc), multiply mated females (Hcc), and females in permanent cohabitation with males (HM). Second- to third-instar larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e served as hosts. Offspring from unmated treatments consisted exclusively of males (Hv: 45♂:0♀; Hv+Mi: 38♂:0♀; χ² ≥ 38.00, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001), confirming strict arrhenotokous reproduction. A single mating event was sufficient to restore mixed-sex offspring production at ratios not significantly different from 1:1 (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05 in all mated treatments). No significant differences in adult emergence rates were detected among treatments. These findings establish \u003cem\u003eE. vitticolle\u003c/em\u003e as a functionally monandrous species and demonstrate that a single supervised mating per female is biologically sufficient for mass-rearing operations targeting augmentative biological control of \u003cem\u003eS. frugiperda\u003c/em\u003e in the Neotropical region.\u003c/p\u003e","manuscriptTitle":"Functional monandry in Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae): a single mating saturates the spermatheca and restores balanced offspring sex ratios","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 10:00:15","doi":"10.21203/rs.3.rs-9534338/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":"April 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T10:00:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 10:00:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9534338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9534338","identity":"rs-9534338","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}