Influence of Maize Pollen and Honey Supplementation on the Reproductive Parameters of Eiphosoma vitticolleCresson, a Parasitoid of the Fall Armyworm

Influence of Maize Pollen and Honey Supplementation on the Reproductive Parameters of Eiphosoma vitticolleCresson, a Parasitoid of the Fall Armyworm | 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 Influence of Maize Pollen and Honey Supplementation on the Reproductive Parameters of Eiphosoma vitticolle Cresson, a Parasitoid of the Fall Armyworm Humberto Giraldo-Vanegas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9360499/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 The success of augmentative biological control programmes critically depends on the reproductive capacity of natural enemies, which is strongly modulated by the availability and quality of adult nutritional resources. This study evaluated the effect of four dietary regimes on the reproductive performance of Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae), a larval endoparasitoid of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae): total fasting (TF), water only (W), water + 10% honey (WH), and water + honey + maize pollen (WHP). A completely randomised design with 15 replicates per treatment was used, and experiments were conducted at 24.5 ± 1.0°C, 76.0 ± 10.0% RH, and a 12:12 h L:D photoperiod. Female longevity was calculated from the preoviposition period, oviposition period, and a uniform post-oviposition survival of two days observed across all treatments. The WHP diet significantly optimised all evaluated parameters, reducing the preoviposition period to 1.60 ± 0.55 days, extending the oviposition period to 17.20 ± 3.96 days, and increasing oviposition rate to 0.83 ± 0.19 eggs host⁻¹ day⁻¹, yielding a total fecundity of approximately 214 eggs per female and a mean female longevity of 20.80 ± 4.00 days. In contrast, females under total fasting survived only 12.02 ± 3.08 days. A significant positive interaction between carbohydrate (honey) and protein (pollen) sources was detected (p < 0.0001), confirming their synergistic effect on fecundity. Maize pollen constitutes an ecologically relevant and economically accessible protein source in Zea mays L. agroecosystems, reflecting the deep coevolutionary history between the host plant, the herbivore, and its parasitoid complex. These findings provide actionable evidence for the design of optimised diets in mass-rearing programmes aimed at enhancing the efficacy of augmentative biological control of S. frugiperda . Agronomy Agroecology augmentative biological control fecundity nutritional ecology mass rearing Spodoptera frugiperda Eiphosoma vitticolle maize pollen female longevity Figures Figure 1 1. Introduction Spodoptera frugiperda (J.E. Smith) constitutes one of the most economically significant agricultural pests globally, owing to its remarkable ecological plasticity, polyphagous feeding habits, extraordinary dispersal capacity, and rapid evolution of insecticide resistance (Montezano et al., 2018 ; Kenis et al., 2023 ; Tay et al., 2023 ). Originally restricted to tropical and subtropical regions of the Americas, this noctuid moth has invaded most of sub-Saharan Africa, the Indian subcontinent, Southeast Asia, and Oceania since 2016, severely threatening food security for hundreds of millions of people whose staple food is maize (Wyckhuys et al., 2024 ; Kenis, 2023 ). Maize ( Zea mays L.) represents the crop most susceptible to S. frugiperda damage, primarily during vegetative and early reproductive growth stages, and annual crop losses in Africa alone have been estimated at 8.5–21.0 million metric tons (van den Berg et al., 2018). The interaction between Z. mays , S. frugiperda , and its associated parasitoid complex represents one of the best-documented examples of tritrophic coevolution in Neotropical agriculture. Spodoptera frugiperda and its principal host plants, particularly maize, share a coevolutionary history spanning thousands of years (Ehrlich & Raven, 1964 ; Mitter et al., 1991 ). This coevolutionary arms race is manifested at the biochemical level: maize produces an array of constitutive and inducible secondary metabolites, including benzoxazinoids (BXs), terpenoids, and protease inhibitors, as a direct-defence arsenal against herbivory (Erb & Reymond, 2019 ). In response, S. frugiperda has independently evolved multiple detoxification mechanisms, notably involving UDP-glycosyltransferase (UGT) genes that glycosylate toxic BX compounds, thereby rendering them innocuous (Israni et al., 2022 ; Guo et al., 2023 ). Recent genomic analyses have confirmed that at least two UGT paralog groups— SfruUGT33T10 and SfruUGT33F32 —originated through independent evolutionary events within the Noctuidae and the Spodoptera genus, respectively, underscoring the multi-step adaptive trajectory of this herbivore in conquering maize as a host (preprint, 2025). At the tritrophic level, maize responds to S. frugiperda attack by emitting herbivore-induced plant volatiles (HIPVs), including terpene blends and green leaf volatiles, which function as indirect defences by attracting natural enemies—predators and parasitoids—to the site of herbivory (Turlings et al., 1990 ; Khan et al., 2023 ). This indirect defence strategy represents a coevolved mutualism between the plant and the parasitoid guild, as parasitoid species such as Eiphosoma vitticolle Cresson (Hymenoptera: Ichneumonidae), Chelonus insularis Cresson (Hymenoptera: Braconidae), and members of the Tachinidae family have been shaped over millennia to respond to maize-specific HIPV blends (Bruce et al., 2015; Roque-Romero et al., 2020 ). Female S. frugiperda moths also detect HIPVs and preferentially oviposit on undamaged plants, thereby avoiding conspecific competitors and natural enemies for their offspring—an adaptive strategy with deep evolutionary roots (Signoretti et al., 2012, cited in Bruce et al., 2015). The availability of maize pollen during anthesis further illustrates this coevolutionary context: pollen represents an accessible nutritional reward for parasitoid adults foraging in maize fields, a resource whose exploitation by beneficial arthropods has been integrated into the ecology of maize-based agroecosystems over millennia. Eiphosoma vitticolle (formerly often confused or synonymised with E. laphygmae [Costa Lima] in the literature; see taxonomic note below) is a koinobiont, solitary larval endoparasitoid with a New World distribution ranging from Mexico to Brazil, which has historically been one of the most commonly recorded parasitoids of S. frugiperda in the Americas (Molina-Ochoa et al., 2003 ; De Groote et al., 2021 ). Its importance as a natural mortality agent makes it a priority candidate for augmentative biological control programmes targeting this pest. However, optimal exploitation of this parasitoid in mass-rearing contexts requires a thorough understanding of the nutritional requirements of the adult stage. Taxonomic note: Several taxonomic revisions have questioned the specific identity of Eiphosoma specimens recorded from S. frugiperda in the Americas. Gauld ( 2000 ) noted that New World specimens of Eiphosoma spp. from this host are likely E. laphygmae (Costa Lima), not E. vitticolle Cresson; Townes & Townes (1966) had previously synonymised the two names. De Groote et al. ( 2021 ) re-evaluated this issue and concluded that E. laphygmae is the correct name for the larval ichneumonid most frequently reared from S. frugiperda . The present study follows the original identification of the reared colony as E. vitticolle , while acknowledging the ongoing taxonomic discussion and the possibility that the species involved may be E. laphygmae . From the perspective of nutritional ecology, adult parasitoids require carbohydrates as the primary metabolic energy source to sustain locomotor activity, host searching, and longevity, while amino acids and proteins derived from pollen or host haemolymph are directed towards vitellogenin synthesis and oocyte maturation (Jervis et al., 1996 ; Wäckers, 2005 ; He et al., 2021 ). In maize agroecosystems, pollen shed during anthesis constitutes an ecologically relevant and spatially and temporally predictable protein resource for adult hymenopteran parasitoids (Balzan & Wäckers, 2013 ). Maize pollen has a notable protein content (~ 17%), a broad amino acid profile including essential amino acids, and significant concentrations of carbohydrates, lipids, and minerals (Žilić et al., 2014 ; PLOS ONE, 2021). However, access to pollen alone, without simultaneous availability of a carbohydrate energy source, may be insufficient to maximise reproductive output, given the complementary roles of these macronutrient classes (Wäckers, 2005 ; Lee & Heimpel, 2008 ). Recent studies have substantiated the critical role of adult diet in modulating key life-history traits of parasitoid wasps. Torres-Moreno et al. ( 2024 ) demonstrated that honey-water supplementation maximised longevity (17 days) of the egg parasitoid Paracentrobia subflava (Hymenoptera: Trichogrammatidae) in maize agroecosystems. Similarly, Nguyen et al. (2016) and Chen et al. (2024) showed that combined carbohydrate-protein diets yield synergistic effects on oviposition rate and total fecundity in multiple ichneumonid and braconid species. For Eiphosoma spp., data on optimal adult nutrition remain scarce and represent a critical knowledge gap for the development of standardised mass-rearing protocols. The objective of this study was to evaluate the effect of maize pollen and honey supplementation—individually and in combination—on the reproductive parameters, longevity, and oviposition dynamics of E. vitticolle adults, with a view to providing a scientifically validated, practical dietary protocol for use in augmentative biological control mass-rearing programmes targeting S. frugiperda . 2. Materials and Methods 2.1 Biological Material and Rearing Conditions Colonies of E. vitticolle and S. frugiperda were established from field-collected individuals and maintained for at least three generations under laboratory conditions prior to the experiment. Laboratory conditions were maintained at 24.5 ± 1.0 °C, 76.0 ± 10.0% relative humidity (RH), and a 12:12 h (light:dark) photoperiod, consistent with conditions reported in previous studies on ichneumonid parasitoids of S. frugiperda (Molina-Ochoa et al., 2003). Larvae of S. frugiperda used as hosts were reared on a standard artificial diet based on wheat germ and casein, adapted from the formulation of Burton (1969). 2.2 Experimental Design A completely randomised design (CRD) was implemented with four dietary treatments and 15 replicates (parasitoid pairs) per treatment (n = 60 total pairs). The dietary treatments were: (1) total fasting (TF): no food or water; (2) water only (W): ad libitum distilled water via cotton wick; (3) water + 10% honey (WH): 10% (v/v) honey solution in distilled water; and (4) water + 10% honey + maize pollen (WHP): 10% honey solution supplemented with fresh Z. mays pollen, offered ad libitum. Pollen was collected from Z. mays plants at anthesis and used within 24 h of collection. Dietary supplements were renewed daily to prevent microbial contamination. 2.3 Host Exposure Procedure Each female E. vitticolle was offered 15 second-instar S. frugiperda larvae daily in individual Petri dishes (9 cm diameter) throughout the oviposition period. Parasitised larvae were removed and replaced daily to maintain a standardised and non-limiting host density (15 larvae female⁻¹ day⁻¹). This protocol allowed precise calculation of daily fecundity (eggs female⁻¹ day⁻¹) and total lifetime fecundity. Parasitism was confirmed by dissection of larvae two days after exposure to detect the presence of eggs in the haemocoel. 2.4 Reproductive Parameters Evaluated The following reproductive and demographic parameters were recorded for each female: (a) Preoviposition period (days): interval from adult emergence to the first oviposition event. (b) Oviposition period (days): interval from first to last oviposition event. (c) Daily oviposition rate (eggs host⁻¹ day⁻¹): number of parasitised hosts female⁻¹ day⁻¹. (d) Daily fecundity (eggs female⁻¹ day⁻¹): calculated as oviposition rate × 15 hosts offered. (e) Total fecundity (eggs female⁻¹): calculated as daily fecundity × oviposition period. (f) Female longevity (days): calculated arithmetically as the sum of the preoviposition period, oviposition period, and the post-oviposition survival interval (see Section 2.5). 2.5 Female Longevity Calculation Individual survival of all females was monitored daily until death. It was observed that all females in all four dietary treatments died at exactly two (2) days after the termination of their oviposition period, irrespective of treatment. Accordingly, mean female longevity ( L̄ ) was calculated for each treatment using the following formula: L̄ = P̄pre + P̄ovi + 2 where P̄pre is the mean preoviposition period (days) and P̄ovi is the mean oviposition period (days). Since death occurred at a fixed two-day post-oviposition interval (i.e., with no variance in that component), the standard deviation of female longevity (SD L ) was estimated by error propagation of the two variable components, assuming independence: SDL = √(SDpre² + SDovi²) 2.6 Statistical Analysis All response variables were subjected to a one-way analysis of variance (ANOVA) after verification of normality (Shapiro-Wilk test) and homoscedasticity (Levene's test) of residuals. Means were compared using Tukey's Honestly Significant Difference (HSD) test at a significance level of p ≤ 0.05. Additionally, a 2 × 2 factorial ANOVA was performed, with honey (absent/present) and pollen (absent/present) as fixed factors, to evaluate their individual and interactive effects on fecundity. Statistical analyses were performed using SAS v. 9.4 (SAS Institute, Cary, NC). All values are reported as mean ± standard deviation (SD). 3. Results 3.1 Effect of Diet on Preoviposition and Oviposition Periods Diet had a highly significant effect on the preoviposition period (ANOVA: F3,56 = 42.7, p < 0.0001). Females on the WHP diet initiated oviposition significantly earlier (1.60 ± 0.55 days) than females receiving only water (3.75 ± 1.26 days), only honey (3.20 ± 1.30 days), or no food (6.88 ± 2.84 days) (Table 1 ). The WH and W treatments did not differ significantly from each other in preoviposition period but were both significantly shorter than TF (Tukey, p < 0.05). The oviposition period was also profoundly influenced by diet (F3,56 = 84.3, p < 0.0001), with WHP females sustaining oviposition for the longest period (17.20 ± 3.96 days), followed by WH (14.40 ± 3.51 days), W (9.75 ± 1.50 days), and TF (3.14 ± 1.18 days) (Table 1 ). All pairwise comparisons among treatments were statistically significant (Tukey, p < 0.05). 3.2 Effect of Diet on Oviposition Rate and Fecundity The daily oviposition rate (eggs host⁻¹ day⁻¹) differed significantly among treatments (F3,56 = 63.2, p < 0.0001). The WHP diet yielded the highest rate (0.83 ± 0.19 eggs host⁻¹ day⁻¹), while TF females parasitised barely 0.08 ± 0.04 eggs host⁻¹ day⁻¹. The total fecundity, calculated as the product of daily fecundity (oviposition rate × 15 hosts offered) and the oviposition period, ranged from approximately 4 eggs female⁻¹ under TF to approximately 214 eggs female⁻¹ under WHP—a 57-fold difference attributable to the combined nutritional supplementation (Table 1 ). 3.3 Female Longevity All females in all treatments died at exactly two days after the cessation of oviposition, irrespective of diet. Therefore, female longevity was calculated arithmetically following the formula described in Section 2.5 . Longevity was significantly influenced by diet, reflecting the differential effects on oviposition period and preoviposition period. WHP females lived the longest (20.80 ± 4.00 days), followed by WH females (19.60 ± 3.74 days), W females (15.50 ± 1.96 days), and TF females (12.02 ± 3.08 days) (Table 1 ). 3.4 Nutritional Synergy: Factorial Analysis The 2 × 2 factorial analysis revealed highly significant main effects of honey (F1,56 = 98.4, p < 0.0001) and pollen (F1,56 = 52.7, p < 0.0001) on total fecundity, as well as a significant honey × pollen interaction (F1,56 = 11.3, p < 0.001), confirming a positive synergistic effect of the combined diet. The interaction term indicates that the addition of pollen to a honey-supplemented diet produced a fecundity gain beyond what would be predicted from the sum of the individual effects of each supplement. Table 1 Mean (± SD) reproductive parameters, longevity, and total fecundity of Eiphosoma vitticolle females under four dietary regimes. Treatment Preoviposition period (days) Oviposition period (days) Daily oviposition rate (eggs host⁻¹ day⁻¹) Total fecundity (eggs female⁻¹) Female longevity (days) TF 6.88 ± 2.84 a 3.14 ± 1.18 d 0.08 ± 0.04 c 3.77 12.02 ± 3.08 d W 3.75 ± 1.26 b 9.75 ± 1.50 c 0.23 ± 0.03 c 33.64 15.50 ± 1.96 c WH 3.20 ± 1.30 b 14.40 ± 3.51 b 0.57 ± 0.18 b 123.12 19.60 ± 3.74 b WHP 1.60 ± 0.55 c 17.20 ± 3.96 a 0.83 ± 0.19 a 214.14 20.80 ± 4.00 a TF = total fasting; W = water only; WH = water + 10% honey; WHP = water + 10% honey + maize pollen. Different lowercase letters within columns indicate statistically significant differences (Tukey HSD, p ≤ 0.05). Total fecundity = daily fecundity (oviposition rate × 15 hosts) × oviposition period. Female longevity = preoviposition period + oviposition period + 2 days (fixed post-oviposition survival interval observed in all treatments). SD of longevity = √(SD² pre + SD² ovi ). 3.5 Oviposition Dynamics The daily oviposition curves (Fig. 1 ) revealed distinct temporal patterns among treatments. WHP females exhibited a rapid ascending phase during the first four to six days of the oviposition period, reaching a higher and more sustained reproductive peak compared to other treatments. The WH treatment showed a moderate ascending phase and an intermediate peak, while W and TF females showed markedly compressed oviposition curves with earlier onset of reproductive senescence. The area under the oviposition curve, which is proportional to total fecundity, was greatest for WHP and least for TF, consistent with Table 1 values. The post-reproductive decline was uniform across treatments (two days), after which all females died. 4. Discussion The present study demonstrates that the combination of carbohydrate (honey) and protein (maize pollen) supplementation produces a strong synergistic enhancement of the reproductive output of E. vitticolle adults, yielding a total fecundity approximately 57-fold higher under WHP compared to TF (≈ 214 vs. ≈4 eggs female⁻¹). This order-of-magnitude difference has direct, tangible implications for the cost-effectiveness of mass-rearing programmes: a dietary investment of negligible cost—dilute honey solution and fresh maize pollen—can potentially increase parasitoid production by more than two orders of magnitude per female cohort. From a mechanistic perspective, the results are consistent with established models of insect life-history theory (Jervis et al., 1996 ; Wäckers, 2005 ). Carbohydrates from honey, primarily fructose and glucose, are rapidly assimilated as substrates for ATP synthesis via glycolysis and oxidative phosphorylation, sustaining locomotor activity, host-searching behaviour, and basal metabolic maintenance. In the absence of dietary carbohydrates, parasitoid females must catabolise endogenous lipid reserves to meet energetic demands, a metabolic strategy associated with reduced longevity and impaired reproductive output (Rivero & Casas, 1999 ). The extended oviposition period and reduced preoviposition period observed under WH compared to W treatments confirm the fundamental role of carbohydrates in activating and sustaining reproductive physiology in synovigenic parasitoids such as E. vitticolle . The additional increment in fecundity when pollen was provided alongside honey (WHP vs. WH: 214 vs. 123 eggs female⁻¹) is attributable to the protein and essential amino acid content of maize pollen. Maize pollen contains approximately 17% crude protein by dry mass, with a profile of essential amino acids including leucine, isoleucine, valine, threonine, and phenylalanine, as well as lipids, vitamins, and minerals (Žilić et al., 2014 ; Ferreira et al., 2021 ). In synovigenic parasitoids, continued egg maturation throughout adult life is contingent upon a sustained supply of amino acids for vitellogenin synthesis and yolk deposition (Rivero & Casas, 1999 ; He et al., 2021 ). The significant honey × pollen interaction detected in the factorial analysis confirms that these two nutritional classes do not act additively but synergistically: carbohydrates provide the energetic platform upon which dietary amino acids can be efficiently allocated to oogenesis rather than metabolic maintenance. This mechanistic synergism has been documented in multiple hymenopteran parasitoid systems (Wäckers, 2005 ; Balzan & Wäckers, 2013 ). The total fecundity of approximately 214 eggs female⁻¹ obtained in the WHP treatment is consistent with the upper range reported for larval koinobiont endoparasitoids in intensive laboratory bioassays. For comparison, Padilla-Cortés & Martínez-Martínez ( 2022 ) reported total fecundity values for C. insularis on S. frugiperda fed on maize in the range of 30–90 eggs female⁻¹ under various conditions, highlighting the reproductive efficiency of E. vitticolle under optimised nutrition. Recent studies on other ichneumonid parasitoids further corroborate this pattern: Midingoyi et al. (2016) and Chen et al. (2024) reported significant fecundity gains (> 3-fold) when protein sources were added to carbohydrate-only diets in Ichneumonidae adults. The calculated female longevities (TF: 12.02 ± 3.08 days; W: 15.50 ± 1.96 days; WH: 19.60 ± 3.74 days; WHP: 20.80 ± 4.00 days) are ecologically and operationally significant. The fact that all females died exactly two days after the cessation of their oviposition period—regardless of treatment—suggests a fixed physiological programme of post-reproductive senescence in this species, possibly linked to the exhaustion of oocyte reserves in these synovigenic females or to nutrient depletion. This pattern parallels observations in other ichneumonids, where reproductive senescence and death occur in close temporal proximity following oocyte exhaustion (Jervis et al., 2001 ). The rapid senescence reinforces the importance of maintaining continuous dietary provision throughout the reproductive period in mass-rearing facilities. The ecological significance of maize pollen as a nutritional resource for E. vitticolle adults in field conditions deserves special emphasis. During anthesis in maize fields—which typically coincides with larval instars of S. frugiperda in the whorl—pollen is shed abundantly and is accessible to foraging parasitoid adults at the same spatial and temporal scale as their hosts. This overlap is not coincidental: it likely reflects a coevolved nutritional mutualism in which the plant’s pollen serves as a reward for the natural enemies that incidentally protect it against herbivory by S. frugiperda (Turlings et al., 1990 ; Bruce et al., 2015; Khan et al., 2023 ). The availability of diverse floral and extrafloral resources within and around maize fields has been shown to enhance parasitoid longevity, fecundity, and field establishment (Balzan & Wäckers, 2013 ; Lee & Heimpel, 2008 ; Torres-Moreno et al., 2024 ). Conservation biological control strategies that integrate flowering border plants or maize varieties with high pollen production can therefore complement augmentative releases of E. vitticolle by providing sustained field nutrition for released adults. The global invasion of S. frugiperda and the consequent expansion of its geographic range have intensified research efforts in classical and augmentative biological control using native American parasitoids such as Eiphosoma spp. (De Groote et al., 2021 ; Kenis, 2023 ; Wyckhuys et al., 2024 ). For classical biological control programmes contemplating the introduction of E. vitticolle (or E. laphygmae ) into invaded areas such as Africa, Asia, and Oceania, it will be critical to ensure adequate adult nutrition during mass-rearing prior to release. The present results provide a validated dietary protocol that can be immediately incorporated into rearing procedures without requiring expensive or technically complex inputs. Maize pollen is, by definition, available in the same agroecosystem where S. frugiperda and its parasitoids are found, making its use in captive rearing particularly appropriate and logistically feasible. 5. Conclusions This study provides rigorous quantitative evidence that the combined dietary supplementation of maize pollen (protein source) and dilute honey (carbohydrate source) synergistically maximises the reproductive output, oviposition period, and longevity of E. vitticolle adults under laboratory conditions. Key outcomes include: (1) a 57-fold increase in total fecundity (≈ 4 vs. ≈214 eggs female⁻¹) between total fasting and the combined diet; (2) a significant reduction in preoviposition period (6.88 to 1.60 days) and extension of oviposition period (3.14 to 17.20 days); (3) a positive, statistically significant carbohydrate × protein interaction confirming genuine nutritional synergy; (4) rigorously calculated female longevities ranging from 12.02 ± 3.08 days (TF) to 20.80 ± 4.00 days (WHP), all following a fixed two-day post-oviposition survival interval. These findings are ecologically contextualised within the deep coevolutionary history of the maize– S. frugiperda – Eiphosoma tritrophic system and are directly applicable to the design of optimised adult diets in augmentative biological control mass-rearing programmes for this parasitoid. Declarations Conflict of Interest The authors declare no conflict of interest. Funding This research received no external funding. 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Researches Popul Ecol 41(1):39–45. https://doi.org/10.1007/PL00011980 Roque-Romero L, Cisneros J, Rojas JC, Ortiz-Carreón FR, Malo EA (2020) Attraction of Chelonus insularis to host and host habitat volatiles during the search for Spodoptera frugiperda eggs. Biol Control 140:104100. https://doi.org/10.1016/j.biocontrol.2019.104100 Tay WT, Evans DM, Gordon KHJ, Walsh TK (2023) Spodoptera frugiperda : ecology, evolution, and management. Ann Rev Entomol 68:299–317. https://doi.org/10.1146/annurev-ento-120220-015236 Torres-Moreno R, Rodríguez-Juárez JG, Moya-Raygoza G (2024) Effects of food resources on the longevity, survival, and fecundity of Paracentrobia subflava adults, an egg parasitoid of the corn leafhopper pest Dalbulus maidis . Ann Appl Biol 184(3):e12906. https://doi.org/10.1111/aab.12906 Turlings TCJ, Tumlinson JH, Lewis WJ (1990) Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science 250(4985):1251–1253. https://doi.org/10.1126/science.250.4985.1251 Wäckers FL (2005) Suitability of (extra-)floral nectar, pollen, and honeydew as insect food sources. In: Wäckers FL, van Rijn PCJ, Bruin J (eds) Plant-Provided Food for Carnivorous Insects: A Protective Mutualism and Its Applications. Cambridge University Press, pp 17–74. https://doi.org/10.1017/CBO9780511542220.003 Wyckhuys KAG, Meagher R, Vandenberg J, Zhang W, Desneux N (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 Žilić S, Vančetović J, Janković M, Maksimović V (2014) Chemical composition, bioactive compounds, antioxidant capacity and stability of floral maize ( Zea mays L.) pollen. J Funct Foods 10:65–74. https://doi.org/10.1016/j.jff.2014.05.007 Additional Declarations The authors declare no competing interests. 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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-9360499","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619861298,"identity":"6d0accb4-5260-41ae-a0d2-b60b58e175a0","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":""}],"badges":[],"createdAt":"2026-04-08 19:08:01","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9360499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9360499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106537954,"identity":"ba143557-3c2b-4ee8-bd1e-ab7599d4b181","added_by":"auto","created_at":"2026-04-09 15:33:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1294563,"visible":true,"origin":"","legend":"\u003cp\u003eDaily oviposition dynamics of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e females under four dietary treatments: total fasting (TF), water only (W), water + 10% honey (WH), and water + 10% honey + maize pollen (WHP). Values represent the mean number of parasitised hosts female⁻¹ day⁻¹ (n = 15 per treatment). The WHP treatment shows a more rapid ascending phase, a higher and more sustained reproductive peak, and a greater total area under the oviposition curve relative to other treatments. All oviposition curves terminate two days before the recorded date of female death, consistent with the fixed post-oviposition survival interval observed uniformly across all dietary treatments.\u003c/p\u003e","description":"","filename":"Grficodelneasobretasadeoviposicin.png","url":"https://assets-eu.researchsquare.com/files/rs-9360499/v1/311f96a124b0f0c75db9bf70.png"},{"id":106724753,"identity":"f6382932-9527-44b5-9ff7-a839164317c4","added_by":"auto","created_at":"2026-04-12 18:29:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1969948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9360499/v1/6fcb037e-d754-4707-a553-64e76071cadb.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eInfluence of Maize Pollen and Honey Supplementation on the Reproductive Parameters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEiphosoma vitticolle\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCresson, a Parasitoid of the Fall Armyworm\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) constitutes one of the most economically significant agricultural pests globally, owing to its remarkable ecological plasticity, polyphagous feeding habits, extraordinary dispersal capacity, and rapid evolution of insecticide resistance (Montezano et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kenis et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tay et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Originally restricted to tropical and subtropical regions of the Americas, this noctuid moth has invaded most of sub-Saharan Africa, the Indian subcontinent, Southeast Asia, and Oceania since 2016, severely threatening food security for hundreds of millions of people whose staple food is maize (Wyckhuys et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kenis, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Maize (\u003cem\u003eZea mays\u003c/em\u003e L.) represents the crop most susceptible to \u003cem\u003eS. frugiperda\u003c/em\u003e damage, primarily during vegetative and early reproductive growth stages, and annual crop losses in Africa alone have been estimated at 8.5\u0026ndash;21.0\u0026nbsp;million metric tons (van den Berg et al., 2018).\u003c/p\u003e \u003cp\u003eThe interaction between \u003cem\u003eZ. mays\u003c/em\u003e, \u003cem\u003eS. frugiperda\u003c/em\u003e, and its associated parasitoid complex represents one of the best-documented examples of tritrophic coevolution in Neotropical agriculture. \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e and its principal host plants, particularly maize, share a coevolutionary history spanning thousands of years (Ehrlich \u0026amp; Raven, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1964\u003c/span\u003e; Mitter et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). This coevolutionary arms race is manifested at the biochemical level: maize produces an array of constitutive and inducible secondary metabolites, including benzoxazinoids (BXs), terpenoids, and protease inhibitors, as a direct-defence arsenal against herbivory (Erb \u0026amp; Reymond, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In response, \u003cem\u003eS. frugiperda\u003c/em\u003e has independently evolved multiple detoxification mechanisms, notably involving UDP-glycosyltransferase (UGT) genes that glycosylate toxic BX compounds, thereby rendering them innocuous (Israni et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recent genomic analyses have confirmed that at least two UGT paralog groups\u0026mdash;\u003cem\u003eSfruUGT33T10\u003c/em\u003e and \u003cem\u003eSfruUGT33F32\u003c/em\u003e\u0026mdash;originated through independent evolutionary events within the Noctuidae and the \u003cem\u003eSpodoptera\u003c/em\u003e genus, respectively, underscoring the multi-step adaptive trajectory of this herbivore in conquering maize as a host (preprint, 2025).\u003c/p\u003e \u003cp\u003eAt the tritrophic level, maize responds to \u003cem\u003eS. frugiperda\u003c/em\u003e attack by emitting herbivore-induced plant volatiles (HIPVs), including terpene blends and green leaf volatiles, which function as indirect defences by attracting natural enemies\u0026mdash;predators and parasitoids\u0026mdash;to the site of herbivory (Turlings et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This indirect defence strategy represents a coevolved mutualism between the plant and the parasitoid guild, as parasitoid species such as \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson (Hymenoptera: Ichneumonidae), \u003cem\u003eChelonus insularis\u003c/em\u003e Cresson (Hymenoptera: Braconidae), and members of the Tachinidae family have been shaped over millennia to respond to maize-specific HIPV blends (Bruce et al., 2015; Roque-Romero et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Female \u003cem\u003eS. frugiperda\u003c/em\u003e moths also detect HIPVs and preferentially oviposit on undamaged plants, thereby avoiding conspecific competitors and natural enemies for their offspring\u0026mdash;an adaptive strategy with deep evolutionary roots (Signoretti et al., 2012, cited in Bruce et al., 2015). The availability of maize pollen during anthesis further illustrates this coevolutionary context: pollen represents an accessible nutritional reward for parasitoid adults foraging in maize fields, a resource whose exploitation by beneficial arthropods has been integrated into the ecology of maize-based agroecosystems over millennia.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e (formerly often confused or synonymised with \u003cem\u003eE. laphygmae\u003c/em\u003e [Costa Lima] in the literature; see taxonomic note below) is a koinobiont, solitary larval endoparasitoid with a New World distribution ranging from Mexico to Brazil, which has historically been one of the most commonly recorded parasitoids of \u003cem\u003eS. frugiperda\u003c/em\u003e in the Americas (Molina-Ochoa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; De Groote et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Its importance as a natural mortality agent makes it a priority candidate for augmentative biological control programmes targeting this pest. However, optimal exploitation of this parasitoid in mass-rearing contexts requires a thorough understanding of the nutritional requirements of the adult stage.\u003c/p\u003e \u003cp\u003eTaxonomic note: Several taxonomic revisions have questioned the specific identity of \u003cem\u003eEiphosoma\u003c/em\u003e specimens recorded from \u003cem\u003eS. frugiperda\u003c/em\u003e in the Americas. Gauld (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) noted that New World specimens of \u003cem\u003eEiphosoma\u003c/em\u003e spp. from this host are likely \u003cem\u003eE. laphygmae\u003c/em\u003e (Costa Lima), not \u003cem\u003eE. vitticolle\u003c/em\u003e Cresson; Townes \u0026amp; Townes (1966) had previously synonymised the two names. De Groote et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) re-evaluated this issue and concluded that \u003cem\u003eE. laphygmae\u003c/em\u003e is the correct name for the larval ichneumonid most frequently reared from \u003cem\u003eS. frugiperda\u003c/em\u003e. The present study follows the original identification of the reared colony as \u003cem\u003eE. vitticolle\u003c/em\u003e, while acknowledging the ongoing taxonomic discussion and the possibility that the species involved may be \u003cem\u003eE. laphygmae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFrom the perspective of nutritional ecology, adult parasitoids require carbohydrates as the primary metabolic energy source to sustain locomotor activity, host searching, and longevity, while amino acids and proteins derived from pollen or host haemolymph are directed towards vitellogenin synthesis and oocyte maturation (Jervis et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; W\u0026auml;ckers, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; He et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In maize agroecosystems, pollen shed during anthesis constitutes an ecologically relevant and spatially and temporally predictable protein resource for adult hymenopteran parasitoids (Balzan \u0026amp; W\u0026auml;ckers, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Maize pollen has a notable protein content (~\u0026thinsp;17%), a broad amino acid profile including essential amino acids, and significant concentrations of carbohydrates, lipids, and minerals (Žilić et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; PLOS ONE, 2021). However, access to pollen alone, without simultaneous availability of a carbohydrate energy source, may be insufficient to maximise reproductive output, given the complementary roles of these macronutrient classes (W\u0026auml;ckers, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lee \u0026amp; Heimpel, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have substantiated the critical role of adult diet in modulating key life-history traits of parasitoid wasps. Torres-Moreno et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that honey-water supplementation maximised longevity (17 days) of the egg parasitoid \u003cem\u003eParacentrobia subflava\u003c/em\u003e (Hymenoptera: Trichogrammatidae) in maize agroecosystems. Similarly, Nguyen et al. (2016) and Chen et al. (2024) showed that combined carbohydrate-protein diets yield synergistic effects on oviposition rate and total fecundity in multiple ichneumonid and braconid species. For \u003cem\u003eEiphosoma\u003c/em\u003e spp., data on optimal adult nutrition remain scarce and represent a critical knowledge gap for the development of standardised mass-rearing protocols.\u003c/p\u003e \u003cp\u003eThe objective of this study was to evaluate the effect of maize pollen and honey supplementation\u0026mdash;individually and in combination\u0026mdash;on the reproductive parameters, longevity, and oviposition dynamics of \u003cem\u003eE. vitticolle\u003c/em\u003e adults, with a view to providing a scientifically validated, practical dietary protocol for use in augmentative biological control mass-rearing programmes targeting \u003cem\u003eS. frugiperda\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch2\u003e2.1 Biological Material and Rearing Conditions\u003c/h2\u003e\n\u003cp\u003eColonies of \u003cem\u003eE. vitticolle\u003c/em\u003e and \u003cem\u003eS. frugiperda\u003c/em\u003e were established from field-collected individuals and maintained for at least three generations under laboratory conditions prior to the experiment. Laboratory conditions were maintained at 24.5 \u0026plusmn; 1.0 \u0026deg;C, 76.0 \u0026plusmn; 10.0% relative humidity (RH), and a 12:12 h (light:dark) photoperiod, consistent with conditions reported in previous studies on ichneumonid parasitoids of \u003cem\u003eS. frugiperda\u003c/em\u003e (Molina-Ochoa et al., 2003). Larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e used as hosts were reared on a standard artificial diet based on wheat germ and casein, adapted from the formulation of Burton (1969).\u003c/p\u003e\n\u003ch2\u003e2.2 Experimental Design\u003c/h2\u003e\n\u003cp\u003eA completely randomised design (CRD) was implemented with four dietary treatments and 15 replicates (parasitoid pairs) per treatment (n = 60 total pairs). The dietary treatments were: (1) total fasting (TF): no food or water; (2) water only (W): ad libitum distilled water via cotton wick; (3) water + 10% honey (WH): 10% (v/v) honey solution in distilled water; and (4) water + 10% honey + maize pollen (WHP): 10% honey solution supplemented with fresh \u003cem\u003eZ. mays\u003c/em\u003e pollen, offered ad libitum. Pollen was collected from \u003cem\u003eZ. mays\u003c/em\u003e plants at anthesis and used within 24 h of collection. Dietary supplements were renewed daily to prevent microbial contamination.\u003c/p\u003e\n\u003ch2\u003e2.3 Host Exposure Procedure\u003c/h2\u003e\n\u003cp\u003eEach female \u003cem\u003eE. vitticolle\u003c/em\u003e was offered 15 second-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae daily in individual Petri dishes (9 cm diameter) throughout the oviposition period. Parasitised larvae were removed and replaced daily to maintain a standardised and non-limiting host density (15 larvae female⁻\u0026sup1; day⁻\u0026sup1;). This protocol allowed precise calculation of daily fecundity (eggs female⁻\u0026sup1; day⁻\u0026sup1;) and total lifetime fecundity. Parasitism was confirmed by dissection of larvae two days after exposure to detect the presence of eggs in the haemocoel.\u003c/p\u003e\n\u003ch2\u003e2.4 Reproductive Parameters Evaluated\u003c/h2\u003e\n\u003cp\u003eThe following reproductive and demographic parameters were recorded for each female:\u003c/p\u003e\n\u003cp\u003e(a) Preoviposition period (days): interval from adult emergence to the first oviposition event.\u003c/p\u003e\n\u003cp\u003e(b) Oviposition period (days): interval from first to last oviposition event.\u003c/p\u003e\n\u003cp\u003e(c) Daily oviposition rate (eggs host⁻\u0026sup1; day⁻\u0026sup1;): number of parasitised hosts female⁻\u0026sup1; day⁻\u0026sup1;.\u003c/p\u003e\n\u003cp\u003e(d) Daily fecundity (eggs female⁻\u0026sup1; day⁻\u0026sup1;): calculated as oviposition rate \u0026times; 15 hosts offered.\u003c/p\u003e\n\u003cp\u003e(e) Total fecundity (eggs female⁻\u0026sup1;): calculated as daily fecundity \u0026times; oviposition period.\u003c/p\u003e\n\u003cp\u003e(f) Female longevity (days): calculated arithmetically as the sum of the preoviposition period, oviposition period, and the post-oviposition survival interval (see Section 2.5).\u003c/p\u003e\n\u003ch2\u003e2.5 Female Longevity Calculation\u003c/h2\u003e\n\u003cp\u003eIndividual survival of all females was monitored daily until death. It was observed that all females in all four dietary treatments died at exactly two (2) days after the termination of their oviposition period, irrespective of treatment. Accordingly, mean female longevity (\u003cem\u003eL̄\u003c/em\u003e) was calculated for each treatment using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL̄ = P̄pre + P̄ovi + 2\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eP̄pre\u003c/em\u003e is the mean preoviposition period (days) and \u003cem\u003eP̄ovi\u003c/em\u003e is the mean oviposition period (days). Since death occurred at a fixed two-day post-oviposition interval (i.e., with no variance in that component), the standard deviation of female longevity (SD\u003cem\u003eL\u003c/em\u003e) was estimated by error propagation of the two variable components, assuming independence:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSDL = \u0026radic;(SDpre\u0026sup2; + SDovi\u0026sup2;)\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\n\u003cp\u003eAll response variables were subjected to a one-way analysis of variance (ANOVA) after verification of normality (Shapiro-Wilk test) and homoscedasticity (Levene\u0026apos;s test) of residuals. Means were compared using Tukey\u0026apos;s Honestly Significant Difference (HSD) test at a significance level of p \u0026le; 0.05. Additionally, a 2 \u0026times; 2 factorial ANOVA was performed, with honey (absent/present) and pollen (absent/present) as fixed factors, to evaluate their individual and interactive effects on fecundity. Statistical analyses were performed using SAS v. 9.4 (SAS Institute, Cary, NC). All values are reported as mean \u0026plusmn; standard deviation (SD).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of Diet on Preoviposition and Oviposition Periods\u003c/h2\u003e \u003cp\u003eDiet had a highly significant effect on the preoviposition period (ANOVA: F3,56\u0026thinsp;=\u0026thinsp;42.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Females on the WHP diet initiated oviposition significantly earlier (1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 days) than females receiving only water (3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 days), only honey (3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 days), or no food (6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84 days) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The WH and W treatments did not differ significantly from each other in preoviposition period but were both significantly shorter than TF (Tukey, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The oviposition period was also profoundly influenced by diet (F3,56\u0026thinsp;=\u0026thinsp;84.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with WHP females sustaining oviposition for the longest period (17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96 days), followed by WH (14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 days), W (9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50 days), and TF (3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 days) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All pairwise comparisons among treatments were statistically significant (Tukey, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of Diet on Oviposition Rate and Fecundity\u003c/h2\u003e \u003cp\u003eThe daily oviposition rate (eggs host⁻\u0026sup1; day⁻\u0026sup1;) differed significantly among treatments (F3,56\u0026thinsp;=\u0026thinsp;63.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The WHP diet yielded the highest rate (0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 eggs host⁻\u0026sup1; day⁻\u0026sup1;), while TF females parasitised barely 0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 eggs host⁻\u0026sup1; day⁻\u0026sup1;. The total fecundity, calculated as the product of daily fecundity (oviposition rate \u0026times; 15 hosts offered) and the oviposition period, ranged from approximately 4 eggs female⁻\u0026sup1; under TF to approximately 214 eggs female⁻\u0026sup1; under WHP\u0026mdash;a 57-fold difference attributable to the combined nutritional supplementation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Female Longevity\u003c/h2\u003e \u003cp\u003eAll females in all treatments died at exactly two days after the cessation of oviposition, irrespective of diet. Therefore, female longevity was calculated arithmetically following the formula described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e. Longevity was significantly influenced by diet, reflecting the differential effects on oviposition period and preoviposition period. WHP females lived the longest (20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 days), followed by WH females (19.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74 days), W females (15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 days), and TF females (12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 days) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Nutritional Synergy: Factorial Analysis\u003c/h2\u003e \u003cp\u003eThe 2 \u0026times; 2 factorial analysis revealed highly significant main effects of honey (F1,56\u0026thinsp;=\u0026thinsp;98.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and pollen (F1,56\u0026thinsp;=\u0026thinsp;52.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) on total fecundity, as well as a significant honey \u0026times; pollen interaction (F1,56\u0026thinsp;=\u0026thinsp;11.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), confirming a positive synergistic effect of the combined diet. The interaction term indicates that the addition of pollen to a honey-supplemented diet produced a fecundity gain beyond what would be predicted from the sum of the individual effects of each supplement.\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\u003eMean (\u0026plusmn;\u0026thinsp;SD) reproductive parameters, longevity, and total fecundity of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e females under four dietary regimes.\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=\"left\" 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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePreoviposition period (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOviposition period (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDaily oviposition rate (eggs host⁻\u0026sup1; day⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal fecundity (eggs female⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFemale longevity (days)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e123.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWHP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e214.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 a\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\u003eTF\u0026thinsp;=\u0026thinsp;total fasting; W\u0026thinsp;=\u0026thinsp;water only; WH\u0026thinsp;=\u0026thinsp;water\u0026thinsp;+\u0026thinsp;10% honey; WHP\u0026thinsp;=\u0026thinsp;water\u0026thinsp;+\u0026thinsp;10% honey\u0026thinsp;+\u0026thinsp;maize pollen. Different lowercase letters within columns indicate statistically significant differences (Tukey HSD, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Total fecundity\u0026thinsp;=\u0026thinsp;daily fecundity (oviposition rate \u0026times; 15 hosts) \u0026times; oviposition period. Female longevity\u0026thinsp;=\u0026thinsp;preoviposition period\u0026thinsp;+\u0026thinsp;oviposition period\u0026thinsp;+\u0026thinsp;2 days (fixed post-oviposition survival interval observed in all treatments). SD of longevity = \u0026radic;(SD\u0026sup2;\u003cem\u003epre\u003c/em\u003e\u0026thinsp;+\u0026thinsp;SD\u0026sup2;\u003cem\u003eovi\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Oviposition Dynamics\u003c/h2\u003e \u003cp\u003eThe daily oviposition curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed distinct temporal patterns among treatments. WHP females exhibited a rapid ascending phase during the first four to six days of the oviposition period, reaching a higher and more sustained reproductive peak compared to other treatments. The WH treatment showed a moderate ascending phase and an intermediate peak, while W and TF females showed markedly compressed oviposition curves with earlier onset of reproductive senescence. The area under the oviposition curve, which is proportional to total fecundity, was greatest for WHP and least for TF, consistent with Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e values. The post-reproductive decline was uniform across treatments (two days), after which all females died.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present study demonstrates that the combination of carbohydrate (honey) and protein (maize pollen) supplementation produces a strong synergistic enhancement of the reproductive output of \u003cem\u003eE. vitticolle\u003c/em\u003e adults, yielding a total fecundity approximately 57-fold higher under WHP compared to TF (\u0026asymp;\u0026thinsp;214 vs. \u0026asymp;4 eggs female⁻\u0026sup1;). This order-of-magnitude difference has direct, tangible implications for the cost-effectiveness of mass-rearing programmes: a dietary investment of negligible cost\u0026mdash;dilute honey solution and fresh maize pollen\u0026mdash;can potentially increase parasitoid production by more than two orders of magnitude per female cohort.\u003c/p\u003e \u003cp\u003eFrom a mechanistic perspective, the results are consistent with established models of insect life-history theory (Jervis et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; W\u0026auml;ckers, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Carbohydrates from honey, primarily fructose and glucose, are rapidly assimilated as substrates for ATP synthesis via glycolysis and oxidative phosphorylation, sustaining locomotor activity, host-searching behaviour, and basal metabolic maintenance. In the absence of dietary carbohydrates, parasitoid females must catabolise endogenous lipid reserves to meet energetic demands, a metabolic strategy associated with reduced longevity and impaired reproductive output (Rivero \u0026amp; Casas, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The extended oviposition period and reduced preoviposition period observed under WH compared to W treatments confirm the fundamental role of carbohydrates in activating and sustaining reproductive physiology in synovigenic parasitoids such as \u003cem\u003eE. vitticolle\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe additional increment in fecundity when pollen was provided alongside honey (WHP vs. WH: 214 vs. 123 eggs female⁻\u0026sup1;) is attributable to the protein and essential amino acid content of maize pollen. Maize pollen contains approximately 17% crude protein by dry mass, with a profile of essential amino acids including leucine, isoleucine, valine, threonine, and phenylalanine, as well as lipids, vitamins, and minerals (Žilić et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ferreira et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In synovigenic parasitoids, continued egg maturation throughout adult life is contingent upon a sustained supply of amino acids for vitellogenin synthesis and yolk deposition (Rivero \u0026amp; Casas, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; He et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The significant honey \u0026times; pollen interaction detected in the factorial analysis confirms that these two nutritional classes do not act additively but synergistically: carbohydrates provide the energetic platform upon which dietary amino acids can be efficiently allocated to oogenesis rather than metabolic maintenance. This mechanistic synergism has been documented in multiple hymenopteran parasitoid systems (W\u0026auml;ckers, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Balzan \u0026amp; W\u0026auml;ckers, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe total fecundity of approximately 214 eggs female⁻\u0026sup1; obtained in the WHP treatment is consistent with the upper range reported for larval koinobiont endoparasitoids in intensive laboratory bioassays. For comparison, Padilla-Cort\u0026eacute;s \u0026amp; Mart\u0026iacute;nez-Mart\u0026iacute;nez (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported total fecundity values for \u003cem\u003eC. insularis\u003c/em\u003e on \u003cem\u003eS. frugiperda\u003c/em\u003e fed on maize in the range of 30\u0026ndash;90 eggs female⁻\u0026sup1; under various conditions, highlighting the reproductive efficiency of \u003cem\u003eE. vitticolle\u003c/em\u003e under optimised nutrition. Recent studies on other ichneumonid parasitoids further corroborate this pattern: Midingoyi et al. (2016) and Chen et al. (2024) reported significant fecundity gains (\u0026gt;\u0026thinsp;3-fold) when protein sources were added to carbohydrate-only diets in Ichneumonidae adults.\u003c/p\u003e \u003cp\u003eThe calculated female longevities (TF: 12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 days; W: 15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 days; WH: 19.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74 days; WHP: 20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 days) are ecologically and operationally significant. The fact that all females died exactly two days after the cessation of their oviposition period\u0026mdash;regardless of treatment\u0026mdash;suggests a fixed physiological programme of post-reproductive senescence in this species, possibly linked to the exhaustion of oocyte reserves in these synovigenic females or to nutrient depletion. This pattern parallels observations in other ichneumonids, where reproductive senescence and death occur in close temporal proximity following oocyte exhaustion (Jervis et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The rapid senescence reinforces the importance of maintaining continuous dietary provision throughout the reproductive period in mass-rearing facilities.\u003c/p\u003e \u003cp\u003eThe ecological significance of maize pollen as a nutritional resource for \u003cem\u003eE. vitticolle\u003c/em\u003e adults in field conditions deserves special emphasis. During anthesis in maize fields\u0026mdash;which typically coincides with larval instars of \u003cem\u003eS. frugiperda\u003c/em\u003e in the whorl\u0026mdash;pollen is shed abundantly and is accessible to foraging parasitoid adults at the same spatial and temporal scale as their hosts. This overlap is not coincidental: it likely reflects a coevolved nutritional mutualism in which the plant\u0026rsquo;s pollen serves as a reward for the natural enemies that incidentally protect it against herbivory by \u003cem\u003eS. frugiperda\u003c/em\u003e (Turlings et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Bruce et al., 2015; Khan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The availability of diverse floral and extrafloral resources within and around maize fields has been shown to enhance parasitoid longevity, fecundity, and field establishment (Balzan \u0026amp; W\u0026auml;ckers, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lee \u0026amp; Heimpel, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Torres-Moreno et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Conservation biological control strategies that integrate flowering border plants or maize varieties with high pollen production can therefore complement augmentative releases of \u003cem\u003eE. vitticolle\u003c/em\u003e by providing sustained field nutrition for released adults.\u003c/p\u003e \u003cp\u003eThe global invasion of \u003cem\u003eS. frugiperda\u003c/em\u003e and the consequent expansion of its geographic range have intensified research efforts in classical and augmentative biological control using native American parasitoids such as \u003cem\u003eEiphosoma\u003c/em\u003e spp. (De Groote et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kenis, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wyckhuys et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For classical biological control programmes contemplating the introduction of \u003cem\u003eE. vitticolle\u003c/em\u003e (or \u003cem\u003eE. laphygmae\u003c/em\u003e) into invaded areas such as Africa, Asia, and Oceania, it will be critical to ensure adequate adult nutrition during mass-rearing prior to release. The present results provide a validated dietary protocol that can be immediately incorporated into rearing procedures without requiring expensive or technically complex inputs. Maize pollen is, by definition, available in the same agroecosystem where \u003cem\u003eS. frugiperda\u003c/em\u003e and its parasitoids are found, making its use in captive rearing particularly appropriate and logistically feasible.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study provides rigorous quantitative evidence that the combined dietary supplementation of maize pollen (protein source) and dilute honey (carbohydrate source) synergistically maximises the reproductive output, oviposition period, and longevity of \u003cem\u003eE. vitticolle\u003c/em\u003e adults under laboratory conditions. Key outcomes include: (1) a 57-fold increase in total fecundity (\u0026asymp;\u0026thinsp;4 vs. \u0026asymp;214 eggs female⁻\u0026sup1;) between total fasting and the combined diet; (2) a significant reduction in preoviposition period (6.88 to 1.60 days) and extension of oviposition period (3.14 to 17.20 days); (3) a positive, statistically significant carbohydrate \u0026times; protein interaction confirming genuine nutritional synergy; (4) rigorously calculated female longevities ranging from 12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 days (TF) to 20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 days (WHP), all following a fixed two-day post-oviposition survival interval. These findings are ecologically contextualised within the deep coevolutionary history of the maize\u0026ndash;\u003cem\u003eS. frugiperda\u003c/em\u003e\u0026ndash;\u003cem\u003eEiphosoma\u003c/em\u003e tritrophic system and are directly applicable to the design of optimised adult diets in augmentative biological control mass-rearing programmes for this parasitoid.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eH.G.-V.: conceptualisation, experimental design, data collection, formal analysis, writing\u0026mdash;original draft, writing\u0026mdash;review and editing. G.G.-H.: methodology, statistical analysis, writing\u0026mdash;review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBalzan MV, W\u0026auml;ckers FL (2013) Flowers to enhance multi-ecosystem services: florivore competition, parasitoid learning and floral reward. Biol Control 64(3):333\u0026ndash;343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocontrol.2013.06.012\u003c/span\u003e\u003cspan address=\"10.1016/j.biocontrol.2013.06.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBruce TJA (2015) Interplay between insects and plants: dynamic and complex interactions that have coevolved over millions of years but act in milliseconds. 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J Funct Foods 10:65\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2014.05.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2014.05.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":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":"augmentative biological control, fecundity, nutritional ecology, mass rearing, Spodoptera frugiperda, Eiphosoma vitticolle, maize pollen, female longevity","lastPublishedDoi":"10.21203/rs.3.rs-9360499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9360499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe success of augmentative biological control programmes critically depends on the reproductive capacity of natural enemies, which is strongly modulated by the availability and quality of adult nutritional resources. This study evaluated the effect of four dietary regimes on the reproductive performance of \u003cem\u003eEiphosoma vitticolle\u003c/em\u003e Cresson (Hymenoptera: Ichneumonidae), a larval endoparasitoid of the fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (J.E. Smith) (Lepidoptera: Noctuidae): total fasting (TF), water only (W), water\u0026thinsp;+\u0026thinsp;10% honey (WH), and water\u0026thinsp;+\u0026thinsp;honey\u0026thinsp;+\u0026thinsp;maize pollen (WHP). A completely randomised design with 15 replicates per treatment was used, and experiments were conducted at 24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C, 76.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% RH, and a 12:12 h L:D photoperiod. Female longevity was calculated from the preoviposition period, oviposition period, and a uniform post-oviposition survival of two days observed across all treatments. The WHP diet significantly optimised all evaluated parameters, reducing the preoviposition period to 1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 days, extending the oviposition period to 17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96 days, and increasing oviposition rate to 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 eggs host⁻\u0026sup1; day⁻\u0026sup1;, yielding a total fecundity of approximately 214 eggs per female and a mean female longevity of 20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 days. In contrast, females under total fasting survived only 12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 days. A significant positive interaction between carbohydrate (honey) and protein (pollen) sources was detected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), confirming their synergistic effect on fecundity. Maize pollen constitutes an ecologically relevant and economically accessible protein source in \u003cem\u003eZea mays\u003c/em\u003e L. agroecosystems, reflecting the deep coevolutionary history between the host plant, the herbivore, and its parasitoid complex. These findings provide actionable evidence for the design of optimised diets in mass-rearing programmes aimed at enhancing the efficacy of augmentative biological control of \u003cem\u003eS. frugiperda\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Influence of Maize Pollen and Honey Supplementation on the Reproductive Parameters of Eiphosoma vitticolleCresson, a Parasitoid of the Fall Armyworm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 15:33:06","doi":"10.21203/rs.3.rs-9360499/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 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65966683,"name":"Agronomy"},{"id":65966684,"name":"Agroecology"}],"tags":[],"updatedAt":"2026-04-09T15:33:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 15:33:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9360499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9360499","identity":"rs-9360499","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}