RESEARCH ARTICLE

Seasonal and Microclimate-Driven Variation in Developmental Morphology of Papilio demoleus in Raipur, Chhattisgarh

Harshita Singh Bais¹*, Vibha Choubey ² and Kavita Das¹

¹Department of Zoology, Govt. Nagarjuna PG. College of Science, Raipur, Chhattisgarh, India.

²Department of Zoology, Govt. J.Y. Chhattisgarh College, Raipur, Chhattisgarh, India.

Authors Email- singhbaisharshita@gmail.com; vibhachoubey.bio@gmail.com; drkavitadas6@gmail.com

*Corresponding Author Email- singhbaisharshita@gmail.com

Graphical abstract

Figure 1. Graphical abstract illustrating the study design and life cycle of Papilio demoleus in Raipur district, Chhattisgarh. The diagram represents the study area, complete developmental stages (egg, larval instars, pupa, and adult), and key influencing factors such as host plant, temperature, and larval growth. It also highlights the methodology involving field-based data collection and subsequent data analysis to understand the temporal, spatial, and energetic dynamics of larval development. © Harshita Singh, 2026. Created using BioRender and Microsoft PowerPoint. Image credit: Available image source: Butterfly reference image

Introduction

Citrus (Family: Rutaceae) is believed to have originated in tropical and subtropical regions of Southeast Asia, particularly India and China (dos Santos Bezerra et al. 2022). The genus Citrus comprises a wide range of species and cultivars, among which mandarin, sweet orange (mosambi, malta or satgudi), lime, and grapefruit are economically and agriculturally important fruit crops (Devi et al. 2019).

Citrus plants and their associated species are highly susceptible to insect herbivores, among which the citrus butterfly Papilio demoleus (Lepidoptera: Papilionidae) is recognized as a major defoliator. The larvae are polyphagous in the family Rutaceae and can feed and breed on a variety of host plants like Citrus sinensis, Citrus aurantifolia, Citrus reticulata, Citrus hystrix, Murraya koenigii, and Aegle marmelos (Atwal 1964). This species is a serious pest of citrus in tropical and subtropical areas due to its wide host range and high feeding potential.

Its range extends from the Middle East, Iran and Saudi Arabia to India, Nepal, China, Taiwan and Japan. It is also reported from South East Asia including Malaysia, Indonesia, New Guinea and Australia. The species has expanded to the Caribbean islands in the recent decades including the Dominican Republic, Cuba, Puerto Rico and Jamaica which suggests a high invasive potential (Lewis 2009). Chatterjee et al. (2000) found P. demoleus as an extreme citrus pest in India, with infestation levels rising with maximum temperature and relative humidity.

The state of Chhattisgarh demonstrates significant butterfly variety, with over 170 species documented across diverse habitats such as hills, plateaus, plains, agroforests, and anthropogenic environments (Tandan et al. 2023). Some findings also shows that P. demoleus is more vulnerable to vehicle collisions in Gariaband District, Chhattisgarh (Tandan and Tandan 2025).

Population dynamics of Papilio demoleus shows distinct seasonal fluctuations, with peak incidence generally occurring between August and October (Maheswarababu 1988; Mathur and Upadhyay 1996; Rampratap et al. 2000). Seasonal outbreaks are highly dependent on environmental factors, mainly temperature and humidity, which regulate growth rate, survival and reproductive potential (Poorten 2004).

The life cycle of Papilio demoleus involves complete metamorphosis with egg, five larval instars, pupal and adult stages (Smith 1978). In Papilio demoleus, larval morphology varies throughout development. Early instars are dark with white markings like bird droppings to protect against predators, whereas later instars are green with saddle-like patterns to conceal amid host plant foliage. This ontogenetic change reflects adaptive survival strategies at different growth stages (El Khidir 1968).

Temperature is an important ecological factor controlling insect development, particularly in Lepidoptera. Papilio demoleus development, survival, and seasonal population dynamics depend on temperature. Lower temperatures prolong development, but higher temperatures accelerate growth and reproduction (Islam et al. 2019a). Additional experimental evidence supports the strong role of temperature in development, where thermal regimes between 23–32 °C significantly decreased incubation, larval and pupal durations with increasing temperature (Kumar et al. 2024). However, variations in laboratory conditions and host plant availability may extend the life cycle up to approximately 41 days, indicating the combined effect of environmental and nutritional factors (Devkar and Chati 2025).

In addition to temperature, host plant quality and feeding behaviour play a crucial role in determining growth and developmental efficiency. Detailed biological studies have shown increased food consumption in successive larval instars and variation in adult longevity under different dietary conditions (Dharavath et al. 2021; Mangrio and Sahito 2022). Similarly, morphometric and developmental variations have been observed based on host plant species, which affects growth rate, sex ratio and overall life cycle duration (Patel et al. 2017). Laboratory studies also confirm its high reproductive potential, with high egg hatching success and considerable variation in survival and growth during larval stages under controlled conditions (Mangrio and Sahito 2021). Field-based studies further contribute to the ecological adaptability of Papilio demoleus by reporting multiple overlapped generations (up to 13 per year) and developmental periods from 22.5 to 46.5 days under different environmental conditions, especially temperature (Munir and Siddiqui 2017).

Owing to its high adaptability, rapid development, and strong association with citrus host plants, Papilio demoleus has emerged as a major pest species in the Asia–Pacific region. Its successful establishment and propagation are mainly attributed to its ecological plasticity and efficient utilization of host resources. Hence, integrated approach of chemical, biological and eco-friendly control methods is necessary for effective management strategies (Riaz et al. 2020). Green synthesis of nanoparticles using biological sources such as plants and microorganisms offers an eco-friendly alternative for pesticide delivery, pest control and pesticide remediation, thereby supporting safer and sustainable agricultural practices (Maravi and Nistala 2021).

Methodology

Study Area

We studied on the campus of Govt. Nagarjuna P.G. College of Science, Raipur, Chhattisgarh, India (21.25°N latitude, 81.63°E longitude; ~298 m above mean sea level). The region is situated in the central plains of India and experiences a tropical climate, characterized by hot summers, a monsoon season with heavy rainfall and mild winters. These environmental conditions support a diverse citrus vegetation and associated lepidopteran fauna including Papilio demoleus.

Study Duration and Design

The study was conducted over a period of one year from April 2025 to April 2026. A seasonal comparative experimental design was followed, and observations were categorized into three seasons: summer, monsoon, and winter. The objective was to evaluate seasonal and microclimatic effects on developmental morphology and life cycle duration of Papilio demoleus.

Microclimatic Observations

Microclimatic parameters including ambient temperature (°C), relative humidity (%) and light intensity (lux) were recorded during the entire study period.

·         Temperature measurements were taken from the AccuWeather application and validated with records from local meteorological stations.

·         Relative humidity was measured with a mobile app based on a digital hygrometer.

·         Light intensity was estimated using a Lux Light Meter application 

All environmental characteristics were recorded daily and averaged season-wise for analysis.

Experimental Design and Sample Collection

A preliminary field-based experimental study with limited replication was conducted. A total of 30 larvae (n = 10 per season) were collected from naturally infested citrus plants.

Larvae were collected manually using a soft brush to prevent physical damage. Selection was random but ensured representation of each seasonal condition (summer, monsoon, winter). The study focused on comparing developmental duration and morphological changes under natural seasonal variation.

Ten larvae were chosen for each season due to seasonal restrictions on field collecting and the requirement to raise each larva separately in a controlled laboratory setting. Despite the small sample size, detailed findings on seasonal fluctuations in morphology and developmental length were obtained through continuous monitoring during all developmental phases. As a result, the results should be regarded as initial baseline data for the Raipur area.

Host Plant

Larvae were predominantly gathered from citrus plants of the Rutaceae family, mainly Citrus aurantifolia and other locally available citrus species. Fresh leaves were used as larval food throughout the study.

Rearing Conditions

Collected larvae were reared under laboratory conditions in transparent, well-ventilated insect rearing cages. Fresh citrus leaves were provided daily as food. Containers were cleaned regularly to maintain hygienic conditions and prevent fungal or microbial contamination. Larvae were maintained under ambient laboratory temperature conditions corresponding to seasonal variation.

No artificial temperature or humidity control was applied, ensuring near-natural rearing conditions.

Observation and Data Collection

Observations of larvae were made every day until adult emergence. The following parameters were recorded:

·         Body length (mm)

·         Coloration pattern changes

·         Feeding behaviour

·         Moulting and instar transitions

·         Duration of each larval instar

·         Pupal duration

·         Total life cycle duration (egg to adult emergence where available)

Body length was measured using a standard millimetre scale. All observations were recorded systematically in a laboratory notebook and later compiled for statistical analysis.

Morphological Analysis

Morphological changes across five larval instars were documented visually and descriptively. Key features included:

·         Early instars showing bird-dropping mimicry (dark brown/black with white markings)

·         Intermediate instars showing increased body size and structural development

·         Late instars showing green coloration with saddle-like dorsal markings for camouflage

·         Late instars showing green coloration with saddle-like dorsal markings for camouflage

Morphological variation was analyzed in relation to seasonal conditions and environmental parameters.

Statistical Analysis

Data were analyzed using descriptive statistics (mean ± standard deviation). Seasonal variation in developmental duration was assessed using one-way ANOVA to determine statistical significance among summer, monsoon, and winter groups.

Table 1: Summary of experimental methods, observation schedule, and roles.

Table 2: Duration of different developmental stages of Papilio demoleus under laboratory conditions. The data include minimum, maximum, and mean duration (± standard deviation) for each life stage.

The duration of development in different life stages of Papilio demoleus is affected by environmental factors such as temperature, humidity and host plant quality. The early instars are of a relatively short duration and the later instars are of a longer duration. This indicates that the advanced developmental stages require more nutrition and a greater amount of biomass. Especially the length of the fifth instar indicates a significant period of energy storage before pupation. The total life cycle length observed (23–41 days) is consistent with previous studies, but with a larger variability likely due to differences in experimental conditions and temperature regimes. Development is known to be accelerated by higher temperatures, while lower temperatures prolong the larval and pupal periods. In addition, the longer adult longevity for females than males may be due to reproductive needs such as oviposition.

These results show the biological plasticity of Papilio demoleus that allows it to adapt to different environmental conditions, which is a key to its success as a wide-spread agricultural pest. Knowledge of such trends is essential if population changes are to be anticipated and appropriate pest control practices devised.

Table 3 presents the variation in body length of Papilio demoleus larvae across different instars, based on observations of three individuals under varying seasonal conditions. A progressive increase in larval body length is evident from the first to the fifth instar, reflecting continuous growth and development.

The first instar shows the smallest size range (2–4 mm; mean 3 mm), indicating the initial post-hatching stage with limited feeding capacity. A gradual increase in length is observed in the second and third instars (mean lengths of 6.5 mm and 11 mm, respectively), corresponding to enhanced feeding activity and metabolic growth.

A significant rise in body length occurs during the fourth instar (mean 22 mm), suggesting accelerated growth and increased resource utilization. The fifth instar exhibits the maximum body length (25–35 mm; mean 30 mm), representing the peak growth phase, where larvae accumulate maximum biomass and energy reserves prior to pupation.

Overall, the data demonstrate a consistent and stage-wise increase in larval size, highlighting the relationship between developmental stage, feeding intensity, and energy accumulation in Papilio demoleus.

The seasonal developmental data clearly demonstrate temperature-dependent variation in the developmental cycle of Papilio demoleus. The shortest developmental duration was recorded in summer, while the longest was in winter.

Monsoon conditions showed intermediate values. This pattern confirms that higher ambient temperatures accelerate metabolic activity and reduce developmental time, whereas lower temperatures prolong larval and pupal stages.

Table 3: Summary table of larval body length across different instars of Papilio demoleus.

Table 4: Seasonal variation in developmental duration of Papilio demoleus under laboratory conditions.

Figure 2. Representative larval stages of Papilio demoleus illustrating morphological and functional changes during development. (a) exhibit camouflage adaptations, while mid-instars (b) show increased feeding and growth. The late instar (c) represents peak biomass accumulation with distinct eye spots for predator deterrence. The pre-pupal stage (d) shows reduced mobility and color transition, marking the onset of metamorphosis.

The developmental pattern of Papilio demoleus larvae in five instars indicates a clear increase in size. The larvae are relatively small (2-13 mm) and dark in colour with white patches, closely resembling bird droppings in the early stages (1st to 3rd instar). Mimicry is a good defence against predators. As the larvae enter the 4th instar, a dramatic change in coloration occurs, with the body becoming green and exhibiting a conspicuous white saddle-like marking. This suggests a shift from mimicry-based defence to camouflage in the host plant environment. The larvae in the 5th instar reach their maximal size (25–35 mm) and are totally green, plump, and well developed. At this time, feeding activity is decreased as the larva prepares for pupation.

Overall, the table shows a gradual increase in body size and corresponding adaptive changes in morphology that enhance survival at different life stages. The graph of larval growth and temperature-dependent development clearly shows different patterns in Papilio demoleus.

Figure 3. Scientific line diagram of the larva of Papilio demoleus illustrating detailed external morphology. The figure shows a lateral view of the caterpillar with clearly labelled body regions, including the head capsule, thoracic segments (T1–T3), and abdominal segments (A1–A10). Key structures such as antennae, ocelli (simple eyes), mandibles, prothoracic shield, spiracles, thoracic legs, abdominal prolegs with crochets, anal prolegs, and anal claspers are distinctly marked. The presence of eye-like spots (false eyes) is also indicated as a defensive adaptation. © Harshita Singh, 2026. Original figure based on author’s sketch and digitally prepared with assistance from ChatGPT.

Figure 4. Growth pattern of Papilio demoleus larvae showing progressive increase in body length across five instars. The graph represents minimum, maximum, and approximate mean larval length (mm), indicating continuous growth and variation during development.

Table 5: Duration of different developmental stages of Papilio demoleus reared at two different temperatures.

Figure 5. Effect of temperature on larval developmental duration of Papilio demoleus, showing faster development at higher temperature compared to lower temperature.