Phenotypic plasticity of Spodoptera frugiperda (J. E. Smith) and Spodoptera litura (Hubner) raised on different hosts and seasons: A case study on age-dependent larval colour polymorphism

Introduction

The genus Spodoptera includes several economically important, destructive agricultural pests. Among them, fall armyworm, Spodoptera frugiperda (J. E. Smith), and tobacco cutworm, Spodoptera litura (Hubner), are two closely related major polyphagous pests belonging to the family Noctuidae under the order Lepidoptera (EPPO, 2015; Goergen et al., 2016; Pogue, 2002) [10,11,25] . Spodoptera frugiperda is an invasive pest in India and started infesting maize in 2018 (Sharanabasappa et al., 2018) [28] . It was also noticed to infest maize in West Bengal in the latter part of 2018 (Dhar et al., 2019) [9]. In contrast, Spodoptera litura is an indigenous pest to the Indian sub-continent (Herrero et al., 2016) [13] . However, both pests are distributed worldwide (Bragard et [2,3,10,11,16,25,31] . They have a wide host range of over 350 plant species, including cereals, vegetables, and cash crops, contributing to their rapid spread and establishment (Kenis et al., 2022; Montezano et al., 2018; Qin et al., 2004) [17,21,26] .They cause significant economic losses in a wide range of crops such as maize, soybean, cotton, and beans (Demis and Jemal, 2025 [8,15,20,22,25,33,36] . Both species undergo complete metamorphosis (holometabolous development) consisting of egg, larval, pupal, and adult stages (Chapman, 2013; Triplehorn and Johnson, 2005) [6,35] . Although they share similarities, distinct morphological traits allow developmental differentiation, especially in the larval and adult stages (EPPO, 2015; Pogue, 2002) [10,25] . Tobacco cutworm and fall armyworm exhibit phenotypic plasticity through distinct colour morphs in the larval stage.

Larval colouration shifts with age, population density, host species, and environmental factors such as temperature and seasons (Higo et al., 2022; Tojo, 1991; Wen et al., 2025) [14,34,37] . Such phenotypic plasticity through different colour morphs serves as more than just a morphological trait; it is a valuable indicator of pest population status and offers practical applications across multiple IPM components. Body colour can serve as an intuitive "indicator light" for assessing population density and outbreak risk, aiding early warning and decision-making.

Materials and Methods

Experimental site

The experiments were conducted at the laboratory of the Department of Agricultural Entomology, Uttar Banga Krishi Viswavidyalaya, Pundibari (26.404879ºN Latitude, 89.386219ºE Longitude and Altitude 18 meters mean sea level), Cooch Behar, West Bengal, India, to find out the phenotypic plasticity on colour variations into bacco caterpillar, Spodoptera litura, and fall armyworm, Spodoptera frugipardaat different larval stages. Both pest species were raisedon maize and castor as hosts during the winter and spring months.

Insect sample collection and rearing

Insect samples, including larvae of the fall armyworm, S. frugiperda and S. litura were collected from the maize and cabbage fields of the instructional farm of the University, respectively. The collected insect larvae were maintained on maize (Zea mays L.) and castor (Ricinus communis L.) leaves as a diet up to F-2 generations under laboratory conditions at normal ambient room temperature and relative humidity during both winter and spring months.The maize plants (Hybrid maize 91A21) were grown in both pots and fields without chemical application, and castor leaves were collected from the farm boundaries and used as hosts. The larvae were reared upto adult emergence. Adults were maintained using a 10% honey solution soaked on cotton pads in a small watch glass (7.5 cm diameter) placed inside the jar, which was replaced daily until the moths died. The adult moths were allowed to deposit eggs on maize and castor leaves by keeping them separately at a 1:1 ratio in the oviposition jar (25 cm× 15 cm, 15 cm diameter), which was wrapped in black art paper (A1 size, 59 cm × 84 cm, 220 GSM). Maize and castor leaves were used as oviposition substrates. The jar's lid was covered with muslin. Eggs were collected and placed in separate jars (size: 15cm × 10.5cm) for hatching. The egg masses were observed at 12-hr intervals until the larvae hatched. The newly emerged neonates (n = 28) were individually kept in small plastic jars (7.5cm × 5.5cm) filled with fresh maize leaves. The leaves were changed every 12-hr intervals, and the rearing jar was cleaned. Neonates emerged from the eggs and were transferred to a small rearing plastic jar in specific host plants. Larvae were fed artificially on a natural diet of maize and castor (leaves up to the sixth instars) till cessation of feeding. During an experiment, observations were taken using the digital microscope Zoomstar III (Observation head inclined at 45°, rotatable at 360°, Eye piece with wide-field 10X paired, Magnification range 8X-50X, with a working distance of 115mm).

Results and Discussion

Table 1 demonstrates that the larval colour of Spodoptera frugiperda exhibited pronounced phenotypic plasticity in response to both host plant and season, with the degree of pigmentation increasing progressively from the first to the sixth instar. Seasonal variation was evident throughout larval development, while host-induced differences became increasingly prominent in the middle and later instars. During the first and second instars (Table 1), larvae remained predominantly light green irrespective of host plant, indicating that pigmentation is weakly expressed during the early stages of development. However, subtle differences were already apparent between treatments. Winter-reared larvae on castor exhibited a faint brownish or yellowish-green tint compared with maize-fed larvae. In contrast, spring larvae, particularly those reared on maize, appeared almost transparent or pale green. The persistence of a black head capsule and pinacula in all treatments indicates that these diagnostic structures remain stable despite changes in body colour. The limited variation during the early instars is expected because newly hatched larvae possess a thin and weakly sclerotized cuticle with minimal melanin deposition (Chapman, 2013) [6] . From the third instar onwards, colour variation became increasingly pronounced. Larvae reared during winter gradually changed from pale brownish green on maize to dark brownish green on castor, while spring larvae retained a comparatively lighter green to olive-green colouration. Simultaneously, longitudinal stripes, pinacula, setae, and the characteristic dorsal and dorso-lateral lines became clearly visible. The earlier appearance and greater contrast of these markings in winter and castor-fed larvae suggest enhanced cuticular melanization and pigmentation under these conditions. A similar age-dependent development of colour patterns has been reported by Pogue (2002) [25] , who described progressive darkening and increasing prominence of longitudinal stripes in later instars of S. frugiperda. In the later stage of larval development (fourth to sixth instars), the hosts and seasons had more pronounced effects on larval colour development (Fig 1). The fourth instar represented a transitional stage where host-mediated differences became more evident. Larvae reared on maize during winter developed a light brownish green body with conspicuous white dorsal and dorso-lateral stripes and distinct black spots on the penultimate abdominal segment. In contrast, castor-fed larvae developed heavy pigmentation ranging from dark brown to blackish brown with intense striping, although the abdominal spots were relatively smaller. Spring larvae remained considerably lighter, retaining greenish or light brown colouration. These observations indicate that host plant quality and environmental temperature interact to regulate pigment deposition within the cuticle. Castor leaves contain defensive secondary metabolites, such as ricinine, which may stimulate physiological stress responses and, consequently, increase melanin synthesis. In contrast, maize provides a comparatively balanced nutritional environment, resulting in lighter pigmentation (Montezano et al., 2018). The greatest colour differentiation occurred during the fifth and sixth instars, when larvae attained maximum body size and cuticular development. Winter-reared larvae on maize became dark brown with a prominent inverted "Y"-shaped frontal suture, whereas castor-fed larvae developed an almost black body with highly contrasting longitudinal stripes. During spring, larvae remained olive-green to dark brown with comparatively less intense pigmentation. The sixth instar showed the highest degree of melanization, particularly in castor-fed winter larvae, which developed a deep blackish-brown body with very prominent white stripes. This progressive darkening reflects increasing cuticular sclerotization associated with larval maturation and environmental adaptation. Similar observations were reported by Sparks (1979) [32] , who noted that late instars of S. frugiperda exhibit considerable variation in body colour while retaining stable diagnostic characters, such as the inverted "Y" mark on the head capsule. Table 2 elucidated that Spodoptera litura exhibited pronounced phenotypic plasticity in larval colouration in response to host plant, season, and larval age. Progressive changes in body pigmentation and characteristic markings were observed from the first to the sixth instar, indicating that colour expression is developmentally regulated and strongly modified by environmental conditions. Overall, larvae reared during winter developed darker pigmentation than those reared during spring, while the host plant influenced the intensity and expression of characteristic colour patterns. During the first instar, larvae were generally pale or almost transparent, irrespective of host plant, possessing a characteristic black head capsule and prothoracic shield. Winter larvae reared on maize appeared pale white with a greenish tinge, whereas castor-fed larvae were light green. Spring larvae remained almost transparent with either a whitish or greenish tinge. These observations suggest that pigmentation is minimal immediately after hatching because the cuticle is thin, weakly sclerotized, and contains little melanin. Similar descriptions of newly hatched S. litura larvae have been reported by Ahmad et al. (2013) [1] and Capinera (2017) [5] , who noted that early instars are pale and gradually acquire pigmentation following successive molts. In the second instar, the body became pale green with an orange head capsule, and amber-brown lateral spots appeared below the first abdominal segment. These spots subsequently increased in size and prominence with successive molts. Although both host plants produced similar colour patterns, winter larvae exhibited slightly greater pigmentation than spring larvae. The appearance of these lateral spots represents the onset of species-specific colour pattern formation associated with cuticular differentiation. Previous studies have likewise reported that abdominal markings become increasingly distinct after the second instar and constitute important diagnostic features of S. litura larvae (Ravishankar et al., 2025) [27] . During the third instar, clear differences among treatments became evident. Winter larvae reared on maize developed a light blackish-green body, whereas castor-fed larvae appeared greyish green to yellowish green with brighter pigmentation than spring larvae. In all treatments, three characteristic longitudinal yellow lines (one dorsal and two lateral) became well developed. The emergence of these longitudinal stripes corresponds to increasing cuticular melanization and differentiation of epidermal pigments during larval growth. Similar ontogenetic development of longitudinal stripes has been described by CABI (2024) [4] and Capinera (2017) [5] , who reported that the dorsal and lateral lines become progressively more conspicuous from the third instar onward.

The fourth instar (Fig 2 ) exhibited a marked increase in pigmentation. Winter larvae on maize became blackish green with a distinct reticulation pattern along the lateral body surface, whereas castor-fed larvae were greyish green but darker than those reared during spring. Spring larvae remained comparatively lighter, ranging from light brown on maize to greyish green on castor. The development of the reticulation pattern indicates progressive cuticular maturation and increased melanin deposition. This pigmentation enhancement during intermediate larval stages has been associated with increased cuticle thickness and improved protection against environmental stress (Nation, 2022) [23] . The greatest colour variation occurred during the fifth and sixth instars (Fig 2). Fifth-instar winter larvae on maize changed from blackish green to brownish black with a highly conspicuous reticulation pattern, while castor-fed larvae developed an even darker blackish brown body. Spring larvae remained comparatively lighter, although the reticulation pattern was still evident. In the sixth instar, winter larvae reared on maize attained a deep black body with characteristic black dorsal spots on the first and eighth abdominal segments. Castor-fed larvae remained greyish black with similar dorsal spots, whereas spring larvae developed blackish grey or dark brown colouration. These findings indicate that maximum melanization occurs during the final larval instar, coinciding with completion of larval growth and cuticular sclerotization. Similar progressive darkening in mature S. litura larvae has been reported by Ahmad et al. (2013) [1] and Capinera (2017) [5] , who described substantial colour polymorphism among late instars under different environmental conditions. The consistently darker pigmentation observed during winter suggests that seasonal temperature strongly influences melanin biosynthesis in S. fruigiperda and S. litura. It also indicates that temperature-dependent melanization is an important adaptive mechanism in both S. frugiperda and S. litura. Melanin enhances absorption of solar radiation, enabling larvae to maintain favourable body temperatures under cooler environmental conditions. Increased melanization is also associated with enhanced immune defense, greater cuticular hardness, and improved tolerance to environmental stress. The phenomenon of thermal melanism has been extensively documented in insects, where individuals exposed to lower temperatures generally exhibit darker body pigmentation than those developing under warmer conditions (Clusella-Trullas et al., 2007; Gunn, 1998; Solensky and Larkin, 2003) [7,12,30] . Therefore, the darker winter phenotype observed in the present study represents an adaptive response that improves physiological performance during cold conditions. Host plants also played a significant role in regulating colour expression in many lepidopteran pests (Kesztheyli et al., 2024; Liao et al., 2025; Yamasaki et al., 2009; Yoshida et al., 2023) [18,19,38,39] . Larvae reared on castor consistently exhibited greater melanization than those reared on maize, particularly during the later instars. This finding supports the hypothesis that host plant chemistry influences insect phenotype through nutritional and biochemical pathways. Castor contains ricinine, phenolic compounds, and other allelochemicals that may alter digestive physiology and induce oxidative stress, thereby increasing melanin synthesis as part of the insect's defense mechanism.

Comparable host-induced phenotypic plasticity has been reported in several polyphagous noctuid species, in which changes in host quality modify larval colour, growth rate, and developmental characteristics (Montezano et al., 2018; Nylin and Gotthard, 1998; Simpson and Sword, 2008) [21,24,29] . The interaction between the host plant and season further enhanced phenotypic plasticity. Larvae reared on castor during winter generally developed the darkest phenotypes, whereas maize-fed larvae during spring remained relatively light. This phenomenon was more evident in S. frugiperda. This interaction indicates that it is a highly plastic trait rather than a genetically fixed character. Environmental cues act synergistically to regulate pigment production via endocrine and physiological pathways. Such plasticity provides adaptive advantages by improving camouflage, thermoregulation, and resistance to environmental stress, thereby increasing ecological fitness under diverse environmental conditions. Darker larvae may absorb heat more efficiently and survive low-temperature stress, whereas lighter larvae reduce excessive heat absorption during spring and blend more effectively with green foliage, thereby improving crypsis against natural enemies. Importantly, despite the extensive variation in body colour, diagnostic morphological characters remained unchanged. The inverted "Y"-shaped frontal suture, dorsal stripes, and arrangement of abdominal spots in S. frugiperda and longitudinal yellow stripes, reticulation pattern, and black dorsal spots on the first and eighth abdominal segments in S. litura were consistently present in all treatments, confirming that the observed variation represents environmentally induced phenotypic plasticity rather than genetic differentiation. This finding agrees with the comprehensive taxonomic revision of Spodoptera by Pogue (2002) [25] , who emphasized that colour is highly variable and should not be used alone for species identification.

Conclusion

Overall, the findings clearly demonstrate that larval colouration in S. frugiperda and S. litura is a highly plastic trait regulated by developmental stage, host plant quality, and seasonal environment. The progressive increase in melanization from the first to the sixth instar, coupled with darker pigmentation during winter and on castor, reflects adaptive physiological responses that enhance survival under varying ecological conditions. These results further emphasize the remarkable ecological adaptability of S. frugiperda and S. litura, which contribute to their success as wide spread polyphagous agricultural pests.

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