Influence of altitude on larval instar determination of Spodoptera frugiperda (Lepidoptera: Noctuidae) under high-mountain laboratory conditions

Influence of altitude on larval instar determination of Spodoptera frugiperda (Lepidoptera: Noctuidae) under high-mountain laboratory conditions | 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 altitude on larval instar determination of Spodoptera frugiperda (Lepidoptera: Noctuidae) under high-mountain laboratory conditions Humberto Giraldo-Vanegas¹, Sanly Natalia Núñez-García, Selena Marily Velásquez-López This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8876098/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a globally invasive and highly polyphagous pest whose developmental biology has been predominantly characterized under lowland, warm-temperature conditions. However, knowledge of its larval development under tropical high-mountain environments remains limited. This study evaluated the effects of altitude-associated low temperature on larval development, instar number variability, and growth dynamics of S. frugiperda under controlled laboratory conditions representative of high-mountain agroecosystems. Larvae were reared at 17°C and 65% relative humidity, and head capsule widths were measured to determine larval instars and assess developmental plasticity. Larval development was markedly prolonged, instar overlap increased, and deviations from classical growth ratios were observed relative to low-altitude reports. A proportion of individuals exhibited supernumerary instars, indicating strong thermal plasticity. These findings demonstrate that altitude-related thermal constraints significantly modify larval development of S. frugiperda, with direct implications for instar determination, phenological modeling, and integrated pest management strategies in high-altitude tropical cropping systems. fall armyworm thermal plasticity larval instars altitude head capsule width phenology integrated pest management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Spodoptera frugiperda is one of the most economically important lepidopteran pests worldwide, affecting maize and more than 80 host plant species. Originally native to the Americas, it has rapidly expanded its geographic range to Africa, Asia, and Oceania, posing a serious threat to global food security. Most biological and ecological studies on S. frugiperda have been conducted under lowland tropical or subtropical conditions, where temperatures favor rapid development and high population growth rates. Altitude is a key ecological factor influencing insect development, primarily through its effects on temperature, oxygen availability, and host plant physiology. In tropical regions, high-mountain agroecosystems present markedly cooler conditions compared with lowland areas, potentially altering insect life-history traits. For S. frugiperda, whose development is strongly temperature-dependent, such environments may induce developmental delays, changes in instar number, and increased phenotypic plasticity. Larval instar determination based on head capsule width remains a fundamental tool in insect developmental studies and pest management. Accurate instar identification supports population modeling, timing of control measures, and interpretation of laboratory and field bioassays. Nevertheless, environmental stressors such as low temperature may disrupt classical growth rules, including Dyar’s rule, leading to overlap among instars and variability in developmental trajectories. The objective of this study was to evaluate larval development and instar determination of S. frugiperda under laboratory conditions representative of tropical high-mountain environments. By quantifying head capsule widths and developmental duration at low temperature, we aimed to elucidate the extent of thermal plasticity in this species and discuss its implications for integrated pest management in high-altitude agricultural systems. 2. Materials and Methods 2.1. Insect Colony Larvae of Spodoptera frugiperda were obtained from a laboratory colony maintained at the Universidad de Pamplona, Colombia. The colony originated from field-collected individuals and was reared for several generations under controlled conditions prior to experimentation. 2.2. Experimental Conditions The bioassay was conducted under controlled laboratory conditions simulating a tropical high-mountain environment: temperature of 17 ± 1°C, relative humidity of 65 ± 5%, and a photoperiod of 12:12 h (L:D). 2.3. Larval Rearing and Diet Neonates were individually placed in rearing containers and fed fresh maize (Zea mays L.) leaf tissue. Larvae were supplied daily with leaf sections cut into approximately 2 × 2 cm pieces, obtained from healthy maize plants free of pesticide exposure. Leaf material was replaced regularly to maintain freshness and nutritional quality, and rearing containers were cleaned as needed to prevent microbial contamination. 2.4. Instar Determination Larvae were observed daily. After each molt, head capsules were collected and measured using a stereomicroscope equipped with an ocular micrometer. Head capsule width was recorded to the nearest 0.01 mm. Instars were determined based on frequency distributions of head capsule widths and growth ratios between successive instars. 2.5. Data Analysis Descriptive statistics were calculated for head capsule width and developmental duration of each instar. Growth ratios were compared with classical expectations for lepidopteran larvae. Variability in instar number and developmental time was assessed to evaluate thermal plasticity. 3. Results 3.1. Larval Development Duration Larval development patterns are illustrated in Figs. 1 and 3–5, which summarize head capsule growth patterns and instar-specific developmental trajectories under low-temperature conditions, and are quantitatively detailed in Tables 1–3. Larval development at 17°C was markedly prolonged compared with reports from low-altitude conditions (Table 1; Figs. 2–4). Total larval duration increased substantially, with extended duration observed in all instars. Table 1 Mean duration (days) ± standard deviation of each of the six larval instars and pupal stage of Spodoptera frugiperda reared under laboratory conditions. Instar N Mean duration (days) SD L1 70 5.19 0.39 L2 70 3.67 0.53 L3 70 4.36 0.54 L4 70 5.93 0.46 L5 70 6.39 1.22 L6 70 13.63 1.75 Total larval duration – 39.17 – Pupa 58 21.05 1.66 Table 2 Mean duration (days) ± standard deviation of each of the seven larval instars and pupal stage of Spodoptera frugiperda reared under laboratory conditions. Instar N Mean duration (days) SD L1 119 5.30 0.46 L2 119 3.52 0.53 L3 119 4.09 0.34 L4 119 5.41 0.56 L5 119 4.97 0.75 L6 119 6.17 0.90 L7 119 11.59 0.77 Total larval duration – 41.05 – Pupa 96 21.91 1.48 Table 3 Mean head capsule width (mm), standard deviation, and geometric growth progression of Spodoptera frugiperda larvae reared under laboratory conditions. Instar N Range (mm) Mean (mm) SD Geometric growth ratio* L1 29 0.28–0.36 0.32 0.01 1.44 L2 24 0.40–0.66 0.46 0.02 1.82 L3 22 0.80–0.98 0.84 0.01 1.53 L4 19 1.13–1.50 1.29 0.03 1.44 L5 17 1.80–2.00 1.87 0.01 1.19 L6 16 2.10–2.50 2.22 0.02 1.52 L7 14 2.65–2.98 2.74 0.03 – *Growth ratios calculated between successive instars. 3.2. Head Capsule Width and Instar Differentiation Head capsule width measurements allowed differentiation of larval instars; however, greater overlap among successive instars was observed compared with lowland studies (Table 3; Figs. 1 and 5). Variability in head capsule size increased in later instars. 3.3. Instar Number Variability Most individuals completed development through six instars, but a proportion exhibited an additional instar, indicating developmental plasticity under low-temperature conditions (Tables 1 and 2; Fig. 4). 3.4. Growth Ratios Growth ratios between successive instars deviated from classical Dyar’s rule, particularly in later instars, reflecting the influence of thermal stress on larval growth dynamics (Table 3; Fig. 1). 4. Discussion Low-temperature conditions associated with tropical high-mountain environments significantly influenced larval development of Spodoptera frugiperda . Prolonged development times observed in this study are consistent with the strong thermal dependence of metabolic and physiological processes in Lepidoptera, as widely documented for noctuid species exposed to suboptimal temperatures (Barfield and Ashley 1987; Esperk et al. 2007 ; Régnière et al. 2012 ). Most published studies on S. frugiperda report larval development under temperatures ranging from 25 to 30°C, conditions that promote rapid growth and typically result in six larval instars (Sparks 1979; Montezano et al. 2019 ; Du Plessis et al. 2018). In contrast, the low temperature evaluated in the present study (17°C) led to substantial developmental delays and increased variability in instar number. Similar patterns of prolonged development and instar number variability under cool conditions have been reported for S. frugiperda and other Lepidoptera in recent studies focusing on thermal stress and developmental plasticity (Pashley et al. 2020; Chen et al. 2021). The occurrence of supernumerary instars under suboptimal thermal conditions is generally interpreted as a compensatory mechanism allowing larvae to reach a critical size threshold before pupation (Esperk et al. 2007 ; Davidowitz et al. 2016). Such responses highlight the plastic nature of larval development in S. frugiperda and underscore the influence of environmental constraints on growth regulation. Deviations from Dyar’s rule observed in this study further illustrate the limitations of applying fixed growth ratios for instar determination under environmental stress. Although Dyar’s rule remains a useful heuristic under optimal conditions, its reliability decreases when larvae are exposed to low temperatures, poor host quality, or other stressors (Dyar 1890 ; Esperk et al. 2007 ; Yang et al. 2020). Therefore, instar determination protocols developed under lowland conditions may not be directly applicable to high-altitude agroecosystems. From an applied perspective, extended larval development at high altitude may have contrasting implications for pest management. Prolonged larval stages may increase exposure to natural enemies and enhance the effectiveness of biological control agents, as suggested for highland agroecosystems (Harrison et al. 2019; Hogg et al. 2022). Conversely, longer developmental periods may complicate phenological predictions and disrupt the timing of chemical or cultural control measures if degree-day models derived from lowland populations are applied without adjustment (Régnière et al. 2012 ; Tonnang et al. 2022). Overall, the results of this study emphasize the need to incorporate altitude- and temperature-specific biological parameters into integrated pest management programs targeting S. frugiperda in tropical high-mountain regions. Failure to account for developmental plasticity may lead to inaccurate monitoring, suboptimal intervention timing, and reduced control efficacy, particularly in newly colonized or marginal environments (Du Plessis et al. 2018; Tonnang et al. 2022). 5. Conclusions Altitude-associated low temperatures strongly affect larval development, instar number, and growth patterns of Spodoptera frugiperda . The species exhibits notable thermal plasticity, which must be considered when developing phenological models and integrated pest management strategies for high-altitude agricultural systems. Accurate instar determination under these conditions requires locally validated criteria rather than reliance on lowland-based developmental rules. Declarations Funding This research received no external funding. Ethical Approval This study did not involve human participants or vertebrate animals. The research was conducted using an insect species ( Spodoptera frugiperda ) under laboratory conditions and did not require approval from an ethics committee. Therefore, ethical approval is not applicable. Clinical trial number Not applicable. Author Contributions Conceptualization: H.G.-V.; Methodology: H.G.-V., S.N.N.-G., S.M.V.-L.; Data Collection: S.N.N.-G., S.M.V.-L.; Analysis: H.G.-V.; Writing—Original Draft: H.G.-V.; Writing—Review & Editing: All authors. All authors reviewed, edited, and approved the final version of the manuscript. Conflicts of Interest / Competing Interests The authors declare that they have no conflicts of interest. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. 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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-8876098","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594171069,"identity":"46816c37-5572-4349-b280-c95e7bc32c60","order_by":0,"name":"Humberto Giraldo-Vanegas¹","email":"data:image/png;base64,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","orcid":"","institution":"University of Pamplona","correspondingAuthor":true,"prefix":"","firstName":"Humberto","middleName":"","lastName":"Giraldo-Vanegas¹","suffix":""},{"id":594171070,"identity":"c5153f73-3ff5-473a-9940-b99832080e13","order_by":1,"name":"Sanly Natalia Núñez-García","email":"","orcid":"","institution":"University of Pamplona","correspondingAuthor":false,"prefix":"","firstName":"Sanly","middleName":"Natalia","lastName":"Núñez-García","suffix":""},{"id":594171071,"identity":"127543f3-3573-4f46-8b71-fe69dec217b6","order_by":2,"name":"Selena Marily Velásquez-López","email":"","orcid":"","institution":"University of Pamplona","correspondingAuthor":false,"prefix":"","firstName":"Selena","middleName":"Marily","lastName":"Velásquez-López","suffix":""}],"badges":[],"createdAt":"2026-02-14 01:23:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8876098/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8876098/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103506281,"identity":"1c1451af-15d0-4414-bec9-ae6931844f71","added_by":"auto","created_at":"2026-02-26 13:34:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":600969,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between larval instar and head capsule width of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e reared at 17 °C, showing the linear regression used to evaluate compliance with Dyar’s rule, including growth ratio and coefficient of determination (R²).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eFigure generated by Python 3.12\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/fe0698360a3c27e53cdc38d2.png"},{"id":103507170,"identity":"b3105ba6-d600-4e97-8caf-b226549d9e34","added_by":"auto","created_at":"2026-02-26 13:40:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17578,"visible":true,"origin":"","legend":"\u003cp\u003eMean duration (days ± SD) of each larval instar in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e individuals developing through six instars under laboratory conditions at 17 °C (N = 70).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eFigure generated by Python 3.12\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/1cbb73dad9083c89cbee3dec.png"},{"id":103362963,"identity":"e328f0f0-9d44-45f3-be27-95ad2bdd54fd","added_by":"auto","created_at":"2026-02-24 21:36:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17107,"visible":true,"origin":"","legend":"\u003cp\u003eMean duration (days ± SD) of each larval instar in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e individuals developing through seven instars under laboratory conditions at 17 °C (N = 119).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eFigure generated by Python 3.12\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/fc99aafefb12c6c36bd99963.png"},{"id":103362961,"identity":"8943ba8e-7d8e-41a9-82f7-ed6ba37d9ab1","added_by":"auto","created_at":"2026-02-24 21:36:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87246,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of developmental strategies in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e reared at 17 °C, showing instar-specific mean duration (days ± SD) for individuals developing through six or seven instars and highlighting compensatory responses in late larval stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eFigure generated by Python 3.12\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/fdbd5ddb4ab1c315e6909183.png"},{"id":103362959,"identity":"db85b836-3f3e-4061-9722-ee205529ef6c","added_by":"auto","created_at":"2026-02-24 21:36:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36099,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency distribution (histograms) of head capsule width measurements for successive larval instars of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003ereared at 17 °C, including mean values and standard deviations, illustrating overlap among instars under high-mountain thermal conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eFigure generated by Python 3.12\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/ece649de7d47f9c23fe8bb36.png"},{"id":103509808,"identity":"77c97d7e-9562-4a7d-a1d4-0eddd05e4714","added_by":"auto","created_at":"2026-02-26 14:01:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1145204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8876098/v1/eb235db3-7d57-44dd-9854-7c6931766c7b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of altitude on larval instar determination of Spodoptera frugiperda (Lepidoptera: Noctuidae) under high-mountain laboratory conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpodoptera frugiperda is one of the most economically important lepidopteran pests worldwide, affecting maize and more than 80 host plant species. Originally native to the Americas, it has rapidly expanded its geographic range to Africa, Asia, and Oceania, posing a serious threat to global food security. Most biological and ecological studies on S. frugiperda have been conducted under lowland tropical or subtropical conditions, where temperatures favor rapid development and high population growth rates.\u003c/p\u003e \u003cp\u003eAltitude is a key ecological factor influencing insect development, primarily through its effects on temperature, oxygen availability, and host plant physiology. In tropical regions, high-mountain agroecosystems present markedly cooler conditions compared with lowland areas, potentially altering insect life-history traits. For S. frugiperda, whose development is strongly temperature-dependent, such environments may induce developmental delays, changes in instar number, and increased phenotypic plasticity.\u003c/p\u003e \u003cp\u003eLarval instar determination based on head capsule width remains a fundamental tool in insect developmental studies and pest management. Accurate instar identification supports population modeling, timing of control measures, and interpretation of laboratory and field bioassays. Nevertheless, environmental stressors such as low temperature may disrupt classical growth rules, including Dyar\u0026rsquo;s rule, leading to overlap among instars and variability in developmental trajectories.\u003c/p\u003e \u003cp\u003eThe objective of this study was to evaluate larval development and instar determination of S. frugiperda under laboratory conditions representative of tropical high-mountain environments. By quantifying head capsule widths and developmental duration at low temperature, we aimed to elucidate the extent of thermal plasticity in this species and discuss its implications for integrated pest management in high-altitude agricultural systems.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Insect Colony\u003c/h2\u003e \u003cp\u003eLarvae of Spodoptera frugiperda were obtained from a laboratory colony maintained at the Universidad de Pamplona, Colombia. The colony originated from field-collected individuals and was reared for several generations under controlled conditions prior to experimentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Conditions\u003c/h2\u003e \u003cp\u003eThe bioassay was conducted under controlled laboratory conditions simulating a tropical high-mountain environment: temperature of 17\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and a photoperiod of 12:12 h (L:D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Larval Rearing and Diet\u003c/h2\u003e \u003cp\u003eNeonates were individually placed in rearing containers and fed fresh maize (Zea mays L.) leaf tissue. Larvae were supplied daily with leaf sections cut into approximately 2 \u0026times; 2 cm pieces, obtained from healthy maize plants free of pesticide exposure. Leaf material was replaced regularly to maintain freshness and nutritional quality, and rearing containers were cleaned as needed to prevent microbial contamination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Instar Determination\u003c/h2\u003e \u003cp\u003eLarvae were observed daily. After each molt, head capsules were collected and measured using a stereomicroscope equipped with an ocular micrometer. Head capsule width was recorded to the nearest 0.01 mm. Instars were determined based on frequency distributions of head capsule widths and growth ratios between successive instars.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data Analysis\u003c/h2\u003e \u003cp\u003eDescriptive statistics were calculated for head capsule width and developmental duration of each instar. Growth ratios were compared with classical expectations for lepidopteran larvae. Variability in instar number and developmental time was assessed to evaluate thermal plasticity.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e3.1.\u003c/h2\u003e\n \u003cp\u003eLarval Development Duration\u003c/p\u003e\n \u003cp\u003eLarval development patterns are illustrated in Figs.\u0026nbsp;1 and 3\u0026ndash;5, which summarize head capsule growth patterns and instar-specific developmental trajectories under low-temperature conditions, and are quantitatively detailed in Tables\u0026nbsp;1\u0026ndash;3. Larval development at 17\u0026deg;C was markedly prolonged compared with reports from low-altitude conditions (Table\u0026nbsp;1; Figs.\u0026nbsp;2\u0026ndash;4). Total larval duration increased substantially, with extended duration observed in all instars.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMean duration (days) \u0026plusmn; standard deviation of each of the six larval instars and pupal stage of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e reared under laboratory conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstar\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean duration\u003c/p\u003e\n \u003cp\u003e(days)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal larval duration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e39.17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePupa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMean duration (days) \u0026plusmn; standard deviation of each of the seven larval instars and pupal stage of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e reared under laboratory conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstar\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean duration\u003c/p\u003e\n \u003cp\u003e(days)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal larval duration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e41.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePupa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMean head capsule width (mm), standard deviation, and geometric growth progression of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e larvae reared under laboratory conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstar\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRange\u003c/p\u003e\n \u003cp\u003e(mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003cp\u003e(mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGeometric\u003c/p\u003e\n \u003cp\u003egrowth ratio*\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.28\u0026ndash;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.40\u0026ndash;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.80\u0026ndash;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.13\u0026ndash;1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.80\u0026ndash;2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.10\u0026ndash;2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.65\u0026ndash;2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*Growth ratios calculated between successive instars.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e3.2.\u003c/h2\u003e\n \u003cp\u003eHead Capsule Width and Instar Differentiation\u003c/p\u003e\n \u003cp\u003eHead capsule width measurements allowed differentiation of larval instars; however, greater overlap among successive instars was observed compared with lowland studies (Table 3; Figs. 1 and 5). Variability in head capsule size increased in later instars.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.3.\u003c/h2\u003e\n \u003cp\u003eInstar Number Variability\u003c/p\u003e\n \u003cp\u003eMost individuals completed development through six instars, but a proportion exhibited an additional instar, indicating developmental plasticity under low-temperature conditions (Tables\u0026nbsp;1 and 2; Fig.\u0026nbsp;4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.4.\u003c/h2\u003e\n \u003cp\u003eGrowth Ratios\u003c/p\u003e\n \u003cp\u003eGrowth ratios between successive instars deviated from classical Dyar\u0026rsquo;s rule, particularly in later instars, reflecting the influence of thermal stress on larval growth dynamics (Table\u0026nbsp;3; Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLow-temperature conditions associated with tropical high-mountain environments significantly influenced larval development of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. Prolonged development times observed in this study are consistent with the strong thermal dependence of metabolic and physiological processes in Lepidoptera, as widely documented for noctuid species exposed to suboptimal temperatures (Barfield and Ashley 1987; Esperk et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; R\u0026eacute;gni\u0026egrave;re et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Most published studies on \u003cem\u003eS. frugiperda\u003c/em\u003e report larval development under temperatures ranging from 25 to 30\u0026deg;C, conditions that promote rapid growth and typically result in six larval instars (Sparks 1979; Montezano et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Du Plessis et al. 2018). In contrast, the low temperature evaluated in the present study (17\u0026deg;C) led to substantial developmental delays and increased variability in instar number.\u003c/p\u003e \u003cp\u003eSimilar patterns of prolonged development and instar number variability under cool conditions have been reported for \u003cem\u003eS. frugiperda\u003c/em\u003e and other Lepidoptera in recent studies focusing on thermal stress and developmental plasticity (Pashley et al. 2020; Chen et al. 2021). The occurrence of supernumerary instars under suboptimal thermal conditions is generally interpreted as a compensatory mechanism allowing larvae to reach a critical size threshold before pupation (Esperk et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Davidowitz et al. 2016). Such responses highlight the plastic nature of larval development in \u003cem\u003eS. frugiperda\u003c/em\u003e and underscore the influence of environmental constraints on growth regulation.\u003c/p\u003e \u003cp\u003eDeviations from Dyar\u0026rsquo;s rule observed in this study further illustrate the limitations of applying fixed growth ratios for instar determination under environmental stress. Although Dyar\u0026rsquo;s rule remains a useful heuristic under optimal conditions, its reliability decreases when larvae are exposed to low temperatures, poor host quality, or other stressors (Dyar \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1890\u003c/span\u003e; Esperk et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yang et al. 2020). Therefore, instar determination protocols developed under lowland conditions may not be directly applicable to high-altitude agroecosystems.\u003c/p\u003e \u003cp\u003eFrom an applied perspective, extended larval development at high altitude may have contrasting implications for pest management. Prolonged larval stages may increase exposure to natural enemies and enhance the effectiveness of biological control agents, as suggested for highland agroecosystems (Harrison et al. 2019; Hogg et al. 2022). Conversely, longer developmental periods may complicate phenological predictions and disrupt the timing of chemical or cultural control measures if degree-day models derived from lowland populations are applied without adjustment (R\u0026eacute;gni\u0026egrave;re et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tonnang et al. 2022). Overall, the results of this study emphasize the need to incorporate altitude- and temperature-specific biological parameters into integrated pest management programs targeting \u003cem\u003eS. frugiperda\u003c/em\u003e in tropical high-mountain regions. Failure to account for developmental plasticity may lead to inaccurate monitoring, suboptimal intervention timing, and reduced control efficacy, particularly in newly colonized or marginal environments (Du Plessis et al. 2018; Tonnang et al. 2022).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAltitude-associated low temperatures strongly affect larval development, instar number, and growth patterns of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. The species exhibits notable thermal plasticity, which must be considered when developing phenological models and integrated pest management strategies for high-altitude agricultural systems. Accurate instar determination under these conditions requires locally validated criteria rather than reliance on lowland-based developmental rules.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or vertebrate animals. The research was conducted using an insect species (\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) under laboratory conditions and did not require approval from an ethics committee. Therefore, ethical approval is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: H.G.-V.; Methodology: H.G.-V., S.N.N.-G., S.M.V.-L.; Data Collection: S.N.N.-G., S.M.V.-L.; Analysis: H.G.-V.; Writing\u0026mdash;Original Draft: H.G.-V.; Writing\u0026mdash;Review \u0026amp; Editing: All authors. \u0026nbsp;All authors reviewed, edited, and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest / Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbrahams P, Bateman M, Beale T et al (2017) Fall armyworm: Impacts and implications for Africa. CABI Evidence Note Update (October 2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/CABICOMM-25-484\u003c/span\u003e\u003cspan address=\"10.1079/CABICOMM-25-484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. CABI\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcosta-Est\u0026eacute;vez A (2021) Biolog\u0026iacute;a y morfolog\u0026iacute;a externa de los estadios inmaduros de \u003cem\u003eSpodoptera dolichos\u003c/em\u003e (Lepidoptera: Noctuidae). 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Mol Ecol Resour 20(6):1682\u0026ndash;1696. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1755-0998.13219\u003c/span\u003e\u003cspan address=\"10.1111/1755-0998.13219\" 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":false,"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":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fall armyworm, thermal plasticity, larval instars, altitude, head capsule width, phenology, integrated pest management","lastPublishedDoi":"10.21203/rs.3.rs-8876098/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8876098/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe fall armyworm, Spodoptera frugiperda (J.E. Smith), is a globally invasive and highly polyphagous pest whose developmental biology has been predominantly characterized under lowland, warm-temperature conditions. However, knowledge of its larval development under tropical high-mountain environments remains limited. This study evaluated the effects of altitude-associated low temperature on larval development, instar number variability, and growth dynamics of S. frugiperda under controlled laboratory conditions representative of high-mountain agroecosystems. Larvae were reared at 17\u0026deg;C and 65% relative humidity, and head capsule widths were measured to determine larval instars and assess developmental plasticity. Larval development was markedly prolonged, instar overlap increased, and deviations from classical growth ratios were observed relative to low-altitude reports. A proportion of individuals exhibited supernumerary instars, indicating strong thermal plasticity. These findings demonstrate that altitude-related thermal constraints significantly modify larval development of S. frugiperda, with direct implications for instar determination, phenological modeling, and integrated pest management strategies in high-altitude tropical cropping systems.\u003c/p\u003e","manuscriptTitle":"Influence of altitude on larval instar determination of Spodoptera frugiperda (Lepidoptera: Noctuidae) under high-mountain laboratory conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 21:35:53","doi":"10.21203/rs.3.rs-8876098/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"182680944379863966653790969886948712513","date":"2026-05-10T23:04:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-10T15:24:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T01:12:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T01:11:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2026-02-14T01:19:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99621bfd-a944-49a4-8ab6-67b96e24cfda","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"182680944379863966653790969886948712513","date":"2026-05-10T23:04:41+00:00","index":29,"fulltext":""},{"type":"reviewersInvited","content":"20","date":"2026-05-10T15:24:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T15:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 21:35:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8876098","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8876098","identity":"rs-8876098","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}