Doleschallia bisaltide (Lepidoptera: Nymphalidae) profile and oviposition preference on Graptophyllum pictum (L.) Griff.

Doleschallia bisaltide (Lepidoptera: Nymphalidae) profile and oviposition preference on Graptophyllum pictum (L.) 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Griff. Peni Lestari, Tri Lestari Mardiningsih, Dewi Sartiami, Wage Ratna Rohaeni, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4820341/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 Doleschallia bisaltide Cramer (Lepidoptera: Nymphalidae) is a main pest on Graptophyllum pictum (L.) Griff. in the Lepidopteran order, the first-instar larval performance is determined by adult oviposition. An adult oviposition preference can be used to determine the resistance level of some G. pictum accessions in early-stage breeding programs. The study aimed to identify the profile of D. bisaltide and its oviposition preferences on G. pictum accessions. The herbivore-host interaction was utilized as a guide to select cultivars resistant to D. bisaltide and identify repellent characteristics for the insect. The research was conducted at the Indonesian Spice and Medicinal Crops Research Institute (ISMCRI), using nested Randomized Complete Block Design (RCBD). G. pictum accessions nested on flight periods. The study revealed that insect mating occurred from dusk to night, while oviposition occurred the following morning. The adult mating was dominantly in the humid area, suggesting a potential population outbreak during the rainy season. The highest oviposition preference was observed for accessions with higher anthocyanin-to-chlorophyll content. In contrast, the lowest oviposition was recorded for accession 12 content of higher chlorophyll-to-anthocyanins. D. bisaltide oviposition preferences were influenced by leaf pigment, saponins, and the C/N ratio. Phytochemicals such as flavonoids and glycosides in G.pictum leaves acted as oviposition stimulants for specialists such as D. bisaltide. anthocyanin herbivore-host interaction oviposition stimulant phytochemicals resistance level specialists Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The caricature plant, Graptophyllum pictum (L.) Griff. (Syn. Justicia picta Linn.), belonging to the family Acanthaceae is known in Indonesia as handeuleum (Sundanese), wungu leaf (Javanese) (Sartika & Indradi 2021 ), and purple pudding (Umami et al. 2020 ). This species is integral to Indonesian ethnobotany and is renowned for its medicinal properties, such as anti-hemorrhoid, antimicrobial, anti-inflammatory, analgesic, and wound-healing effects (Rahmawati et al. 2020 ). Additionally, G. pictum leaves are reported to alleviate coughs (Umami et al. 2020 ) and plaque reduction in teeth (Makkiyah et al. 2021 ). The health benefits of the G. pictum are attributed to various active compounds, including alkaloids, flavonoids, steroids, saponins, glycosides, and emollients, present in the leaves (Lestari et al. 2015 ), along with information on its pharmacological effects (Hutagalung 2019 ; Makkiyah et al. 2021 ). Doleschallia bisaltide Cramer (Lepidoptera: Nymphalidae) is a specialist (monophagous) insect known to feed leaves of the Acanthaceae family, particularly G. pictum and Asystasia gangetica (L.) Anders (Handayani et al. 2019). Yield losses of G. pictum leaves due to D. bisaltide larvae have been reported to range from 300.28 to 745.86 cm² per plant, equivalent to 70% of the leaves, based on (Lestari et al. 2015 ). G. pictum leaves are essential for harvesting for medicinal purposes; therefore, larvae infestation poses a risk of reducing the quantity and quality of its harvest. Understanding herbivore-plant interactions is crucial in developing plants resistant to herbivores (Wouters et al. 2016 ; Chabaane et al. 2022 ; Kamaliah et al. 2022 ), including the oviposition preferences of adult insects on host plants. Early instar larvae of Lepidopterans can only access food resources near their eggs (Wouters et al. 2016 ; Griese et al. 2020 ). Thus, oviposition preferences of adults on specific plants and the plant's antixenosis and antibiosis mechanisms are crucial in determining plant resistance levels against pests. Insect host preferences are influenced by plant morphology, which can serve as a selection factor in breeding initiatives for resistant varieties, this may serve as a selection factor (Lestari et al. 2015 ). Feeding preferences of D. bisaltide across Acanthaceae species and within G. pictum have been studied, along with population fluctuations in nature (Adi et al. 2021 ). However, information on oviposition preferences within Graptophyllum species still needs to be available. Based on literature studies, this is the first study to discuss the interaction of D. bisaltide and G. pictum regarding oviposition preference and plant phytochemical characteristics. Identifying the behavior of D. bisaltide adults and larvae is the first step in designing strategies for its management in G. pictum plantations, including developing resistant plant varieties. Insects naturally exhibit varied responses to plants with diverse characteristics. Plant chemistry, especially for female butterflies, plays a role in determining niches for oviposition and feeding activities (Griese et al. 2020 ). (Sokolinskaya et al. 2020 ) explained that Insects have created an impossible-to-miss and one of the foremost advanced chemoreception frameworks, which abuses at the slightest three receptor superfamilies giving discernment of scent and taste and chemical communication in these creatures. Badenes-Pérez ( 2023 ) said that there is an effect of host plant indolic glucosinolate content on oviposition preferences and the complexity index of glucosinolates and aliphatic glucosinolates without sulfur-containing side chains on total oviposition is smaller in P. rapae than in Plutella xylostella L. (Lepidoptera: Plutellidae ), another lepidopteran that specializes in containing plant glucosinolates. Variations in genetics, nutritional status, and environmental conditions cause significant differences in the phytochemical content among varieties under the same species. This diversity explains the oviposition preference and feeding preferences of some insects to some varieties over others. This study aims to explore the oviposition preferences and larvae development of D. bisaltide reared on 13 G. pictum accessions. The herbivore-host interaction in this study is used as a guide to develop G. pictum cultivars resistant to D. bisaltide. Material and Methods The research was conducted during three flight periods of D. bisaltide adults: the end of the rainy season (May-June), the beginning of the dry season (June-July), and the peak of the rainy season (November-December). The length of each period was adjusted until the last adult died. The research took place in the greenhouse of the Indonesian Spice and Medicinal Crops Research Institute (ISMCRI) (nowadays the Indonesian Agency for Agricultural Instrument Standardization/IAAIS), Bogor, Indonesia (240 m above sea level). The number of rainy days and total rainfall during the three flight periods are shown in Table 1 . Table 1 Number of rainy days and total precipitation during the study Flight period Number of rainy days (day) Total precipitation during a month (mm) May-June 33 771.0 June-July 23 501.4 November-December 37 693.4 Data was obtained from the weather station at the ISMCRI Research Station Preparation of Test Plants Thirteen G. pictum accessions were obtained from the germplasm collection of the Department of Agronomy and Horticulture, IPB University. Their tolerance level to D.bisaltide was described in Table 2 . Cuttings that were 15 cm long were planted in sandy soil. Healthy and uniform seedlings, 30 days old, were transplanted into a planting medium mixed with soil, manure, and rice husks charcoal (1:1:1). Seedlings were maintained under 55% light intensity using artificial shade. Plants were watered twice a day. The 250 ml of balanced NPK (16-16-16) liquid fertilizer (concentration 2 g/L water) was applied to the plants every two weeks. Plants were in the vegetative phase during the testing period. Table 2 Tolerance level of G. pictum accessions to D. bisaltide larvae Accessions Resistance level 1 Medium resistant 2 Susceptible 3 Medium resistant 4 Medium resistant 5 Susceptible 6 Medium resistant 7 Resistant 8 Resistant 9 Susceptible 10 Medium resistant 11 Medium resistant 12 Medium resistant 13 Resistant Tolerance level on every G. pictum accession to D. bisaltide based on feeding preference (non choice test) (Lestari et al. 2015 ) Preparation of Test Insects The adult insect population in this experiment from a field sample first breed generations, originated from eggs collected from the G. pictum population in the ISMCRI collection garden. Eggs were kept in a rearing box. After hatching, the first instar larvae were moved to another box until they reached the third instar stage, then transplanted into a 0.25 m³ cylindrical mesh cage until they became pupae (Schäpers et al. 2015 ). During rearing, larvae were fed a mixture of leaves from the 13 tested G. pictum accessions every morning and evening. Pupae were transferred to the rearing box. The hatched butterflies were reared in another mesh cage and maintained until testing. Food sources for D. bisaltide butterflies were nectars from flowers of Ixora sp. and Hydrangea sp., as well as cotton moistened with a 10% concentration of multiflora honey solution. Experimental Design The experiment was carried out in three flight periods: May-June (end of the rainy season), June-July (transition from rainy to dry season), and November-December (peak of the rainy season). In each experimental set, 13 G. pictum accessions were arranged based on a Randomized Complete Block Design with three replications and five plants per group. The morphological characteristics and mating activity of D. bisaltide were observed in the adult population during the experiment. Oviposition preference testing was carried out based on the choice method. Fifty pairs of butterflies that came out from pupae on the same day were flown in a greenhouse containing the G. pictum accessions tested. Dead butterflies were replaced until the seventh day of flight, then started to count the adults. The profile of D. bisaltide was observed daily until the last adult died. The lifespan of the butterflies was calculated based on the reduction in the number of male or female butterflies compared to the initial number flown. The frequency of visits of D. bisaltide adults was calculated based on the number of egg batches on a particular accession. The oviposition preference of D. bisaltide was estimated based on the total number of eggs laid on each plant accession compared to the total number of eggs laid on all plants during one flight period (%). The larger the egg batches and the more number eggs laid on accession, the higher the D. bisaltide adult preference for that accession (Lestari et al., 2015 ; Griese et al., 2020 ). The eggs counted were those laid on that day. First instar larvae were selected and maintained on each accession (non-choice) to observe their development. The frequency of adult visits, oviposition preference, number of eggs, and larval growth and development were counted daily during the experiment. Environmental variables, such as temperature and humidity, were recorded daily using a thermohygrometer (data not shown). Observation of G. pictum Leaf Pigments The leaves chlorophyll, anthocyanins, and carotenoids were measured using spectrophotometric methods based on the Sims & Gamon ( 2002 ) method. The other phytochemical substances were examined in the ISMCRI laboratory utilizing these techniques: nitrogen was measured using the Kjeldahl method; calcium was identified via the Atomic Absorption Spectroscopy (AAS) method; organic carbon was determined by spectrophotometry; and fiber was assessed based on the gravimetric method (SNI 01-2891-1992). Data Analysis Data were tabulated and prepared using Microsoft Excel 2019 ( www.microsoft.com , USA). The normality of data distribution was analyzed using the Shapiro-Wilk test. Data were analyzed using analysis of variance followed by the Tukey post hoc test (α = 5%). The relationship between observed variables was estimated using Pearson correlation in Minitab v.18 software ( www.minitab.com ). Mattjik & Sumertajaya ( 2013 ) suggest that correlation analysis describes the nature of the relationship between one character and another. The closer it is to 1 or -1, the stronger the correlation between the two characters. Conversely, the closer it is to 0, the more unrelated the two characters are. Results Profile of D. bisaltide on G. pictum The adult of D. bisaltide has brown wings, ranging from yellowish-brown to grayish-brown. Male D. bisaltide butterflies have characteristically darker wings with white patches on the lower-part wings, while female butterflies do not have these spots (Fig. 1 ). The average lifespan of butterflies in this study was 9.00 ± 7.00 days across the three flight periods. Characteristics of D. bisaltide are shown in Fig. 2 . Insect of D. bisaltide has four life stages: egg, larva, pupa, and adult (butterfly). Generally, D. bisaltide butterflies take flight around 8 o'clock when the weather is clear and end at sunset. In contrast, D. bisaltide mating activity starts in the afternoon, before sunset, and ends at night. The mating process begins with flying together as a calling stage, continuing with chasing (mating stage) (Ma et al. 2019 ). The butterflies who have found their mate then separate themselves and perch in pairs on the ceiling of the screen house. The male and female abdomens unite until the mating process is complete (Fig. 1 ). The mating process lasts six to eight hours, as (Ma et al. 2019 ) report on other Lepidopterans ( Scopula subpunctaria Herrich-Schaeffer). As many as 40 of 50 pairs of butterflies copulate near water sources. The following day, the female will lay eggs on the underside of the leaves. During the test period, mating occurred within the first 3 weeks. The eggs of D. bisaltide were pearly white when laid (Fig. 2 a), turned cream, and finally became clear with black spots at the poles (Fig. 2 b) when ready to hatch. If they turned black (Fig. 2 f), they failed to hatch. The black spot was the head of the larva. The eggs were spherical (round) with a diameter of ± 1 mm. They bent inward at one pole (Fig. 2 c). D. bisaltide adults laid all eggs, or divided into several egg batches, under the leaf surface (Fig. 2 d). Sometimes, eggs were laid on the stem (Fig. 2 e). The egg numbers reach 50 eggs. The eggs would hatch within 4 days (Fig. 1 ). The number of eggs laid per female differed between flight periods (n = 3, p < 0.001) as well as between replicates per period (n = 6, p = 0.002). Females laid more eggs during the flight period in November-December (125.78 eggs), followed by June-July (96.57 eggs) and May-June (51.20 eggs) (Table 3 ). However, the hatching percentage was highest in May-June and differed from June-July (Table 4 ). More eggs were laid in plant populations near water sources. Table 3 Egg numbers in 13 G. pictum accessions during the three flight periods Accession Number of eggs (eggs)* Average May-June June-July November - December 1 67.70d 143.18a 114.57a 108.48a 2 54.33d 130.14c 135.44c 106.64a 3 67.16b 82.64h 143.73h 97.84a 4 54.45c 102.19e 164.61e 107.08a 5 56.21e 117.11b 149.56b 107.63a 6 40.84i 61.08j 129.25j 77.06a 7 66.69g 100.10d 100.21d 89.00a 8 21.92k 119.21d 132.35d 91.16a 9 76.88a 102.85f 125.73f 101.82a 10 52.78h 86.94i 152.35i 97.36a 11 43.27j 97.43g 148.90g 96.53a 12 7.64l 21.48k 19.05k 16.06b 13 55.76f 91.03f 119.43f 88.74a Average 51.20c 96.57b 125.78a Columns with the same letter are not significantly different based on the Tukey post hoc at α = 5%. Each period is tested using Kruskal-Wallis The more significant number of egg batches found on one plant indicates that the more frequently adults visit that plant, the higher the oviposition preference of D. bisaltide females on that accession. The lowest visiting frequency was obtained for accession 12, followed by accessions 6 and 13, whereas accessions 1, 5, and 9 were most regularly visited (Fig. 3 ). The number of egg batches laid in other accessions varies in each period. The number of eggs laid in accession 12 was lowest in each flight period (May-June (H = 2.94, p = 0.003), June-July (H = 3.37, p = 0.001), November-December (H = 2.85, p = 0.005)). This value was only 1.33% of the eggs in 13 plant accessions, followed by accessions 6 and 13. In contrast, most of the D. bisaltide adult individuals laid eggs in accessions 1, 5, and 9 with a percentage of eggs ≥ 10% of the total eggs. The larvae development stage lasts around 14–16 days, followed by a pupation period of 14 days (Fig. 4 ). Table 4 Hatched egg presentation in 13 G. pictum accessions during the three flight periods Accession Hatched Egg (%) Average May-June June-July November - December 1 76.38ab 63.14ab 63.29a 67.60ab 2 67.50ab 31.34b 44.00a 47.61b 3 57.59ab 55.63ab 70.28a 61.17ab 4 38.15b 67.72ab 76.50a 60.79ab 5 62.84ab 69.68ab 70.24a 67.58ab 6 73.20ab 57.34ab 63.13a 64.56ab 7 73.05ab 61.82ab 57.60a 64.16ab 8 50.11b 47.09ab 47.22a 48.14b 9 59.68ab 34.72b 45.23a 46.55b 10 60.55ab 71.15ab 73.34a 68.34ab 11 77.12ab 39.00ab 37.58a 51.24b 12 97.62a 55.51ab 58.40a 70.51ab 13 80.50ab 80.42a 76.99a 79.30a The average season 67.25a 56.50b 60.29ab They live on the lower surface of leaves. No cannibalism was found, except for fifth instar larvae, which ate young pupae. When hatched, D. bisaltide larvae had pale yellow bodies with a shiny black head region and soft hair on each body segment (Fig. 4 a). After they ate the leaves, the bodies of the first instar larvae were blackish, like jelly (Fig. 4 b). In the second instar larvae, bristly hairs were developed on all the body segments (Fig. 4 c). The third instar larvae began to display white lines on their dorsal and lateral sides (Fig. 4 d). These lines become more prominent as they develop to the fourth and 5th instars. From the fourth instar larvae, orange sockets appeared on the lateral sides and metallic blue sockets on the dorsal sides (Fig. 4 e and 4 f). The fifth instar larvae had reached maximum body size, the most extended lifespan, and the highest eating ability (Fig. 4 f). The larvae need 2.04 ± 0.09 days for the first instar, 2.14 ± 0.22 days for the second instar, 2.42 ± 0.28 days for the third instar, 2.70 ± 0.30 days for the fourth instar, and 5.85 ± 0.64 days for fifth instar. At the end of the fifth instar, the larvae ceased feeding and underwent a morphological change in which their bodies shortened and hung down, forming the shape of a letter J with their heads at the bottom. The pupae of D. bisaltide had an obtecta shape, characterized by prospective antennae, wings, and legs that appear faint and are tightly attached to the body. The pupa was wrapped in cremaster thread. The color of the pupa was initially pink (Fig. 4 h), gradually becoming light brown to dark brown, with a yellow tinge in some parts (Fig. 4 i). Butterflies would emerge in the next 7–9 days (Fig. 4 j). There were neatly arranged black dots on the dorsal side of the pupa. The highest mortality occurred during the initial phase of pupa formation, likely due to the consumption of soft pupal tissues by fifth instar larvae. The extent of damage to the pupa can significantly impact the success of metamorphosis into a butterfly as an adult form of D. bisaltide . The emerge-adult can take one of three forms: perfect, deformed, or deceased. Characteristics of G. pictum and Its Relation to D. bisaltide Oviposition The results revealed significant variations in the percentage of anthocyanin, chlorophyll, and carotenoid pigments, suggesting differences in the composition of these pigments among the accessions. The highest concentration of anthocyanin pigments was in accession 1, and the lowest was in accession 12 (Table 5 ). The proportion of anthocyanin to chlorophyll to carotenoid pigments varied among accessions, with accession 1 having a ratio of 5:3:2 and accession 12 of 1:6:3. In addition, eleven other accessions exhibited a similar proportion of anthocyanin to chlorophyll to carotenoid pigments, with a ratio of 4:4:2. Visually, the 12 G. pictum accessions have similar leaf colors, viz. reddish purple to blackish purple. Only accession 12 has a green-white variegation color (Fig. 5 ). Table 5 Proportion of pigment content on the leaves of G. pictum Accession Leaf pigment proportion (%)* Anthocyanin Total chlorophyll Carotenoid 1 52.23 28.81 18.96 2 - - - 3 43.67 35.87 20.46 4 38.87 38.87 22.27 5 39.54 36.95 23.51 6 36.86 40.91 22.23 7 36.11 41.09 22.80 8 38.36 37.62 24.02 9 42.77 35.78 21.45 10 36.44 41.19 22.37 11 35.51 41.38 23.11 12 13.80 60.08 26.11 13 36.21 41.20 22.58 *) Measured on the third leaf of a fully opened shoot using the (Sims dan Gamon 2002) method. Pigment measurements were not carried out on accession two due to insufficient availability The relationship between the number of eggs and the pigment content and secondary metabolites of 13 G. pictum accessions showed that the number of eggs was significantly positively correlated with the anthocyanin content, C/N ratio, and saponin content of G. pictum leaves and negatively correlated with chlorophyll content (Table 6 ). In contrast, the frequency of D. bisltide visits was uncorrelated with any plant traits. However, the correlation value is relatively high in leaves with high saponin content or low calcium content. Table 6 Correlation values between plant characteristics and egg number and frequency of adult arrival G. pictum characters Correlation value with Egg number Visiting frequency Anthocyanin 0.71** 0.03 Chlorophyll -0.74** -0.11 Carotenoid -0.51 -0.14 C organic 0.03 0.10 Nitrogen -0.52 -0.15 C/N ratio 0.63* 0.13 Calcium 0.37 -0.59 Fiber 0.16 0.21 Alkaloids -0.38 -0.10 Saponin 0.62* 0.51 Flavonoids -0.02 0.32 Triterpenoids 0.31 0.22 Steroids -0.26 -0.44 Note: * means significant (P value < 0.05) and ** means very significant (P value < 0.01) based on Pearson correlation analysis Discussions Like autumn leaves, the brown wings are characteristic of D. bisaltide , making it known as the autumn-leaf butterfly (Fig. 1 ). Several publications link the variation in wing color of D. bisaltide to its functions, such as mimicry, mating preferences, defense from predators, and response to pollutant concentrations in the environment (Sekimura & Nijhout 2017 ; Penz & Williams 2020 ). Sekimura dan Nijhout (2017) concluded that the evolution and diversity of Nymphalidae wings are influenced by genetics and changes in environmental temperature. Both larvae and adults of D. bisaltide are active from morning to evening, but mating activity generally occurs in the afternoon to evening, with ovipositioning the following day. The long duration of mating causes damage to the hind wings of D. bisaltide adults (Fig. 1 ). Most Nymphalidae butterflies (depending on the species) are reported to actively mate and lay eggs at dawn, dusk, or both (Penz & Williams 2020 ). Forty of the fifty pairs of D. bisaltide chose water sources for copulation, indicating that the insect population increases in the rainy season or in areas with high humidity. Observations showed that most eggs were laid during the rainy season (November-December) (Table 3 ), as well as the hatching percentage (Table 4 ) when the number of rainy days and precipitation was higher than in the dry season (Table 1 ). Air humidity in November-December reaches 90%, decreasing to 48–53% from June to July and around 60% from May to June. This study supports Adi et al. ( 2021 ), who stated that humidity affects the fecundity of adult D. bisaltide . Based on the average total number of eggs (Table 3 ) and the frequency of adult visits in three flight periods (Fig. 3 ), it was concluded that adult D. bisaltide females had the lowest oviposition preference in accession 12 and the highest in accessions 1, 5, and 9. The number of eggs laid is unrelated to the number of eggs that hatch but is in line with feeding activity. The lowest percentage of hatched eggs was in accession 9 (46.55%), and the highest was in accession 13 (79.30%). Meanwhile, our previous study on feeding activity showed that accessions 5 and 9 were susceptible to D. bisaltide larvae, while accessions 7 and 12 were resistant (Table 2 ) (Lestari et al., 2015 ). Both accessions 7 and 12 were rich in fiber content. Apart from insect factors, such as food consumed and the genetics of adults, plant factors also influence egg hatching. The presence of herbivore eggs acts as an inducer of plant defense mechanisms, and then they respond physiologically (Hilker & Fatouros 2016 ) by changes in secondary metabolic contents or structure. The presence of insect eggs is an essential form of early-stage plant defense evolution. The ability to detect and create unsuitable conditions for egg development potentially inhibits eggs from hatching, resulting in fewer larvae consuming the plant (Griese et al. 2020 ). Plant characteristics influence the interaction of herbivore insects with their hosts through antixenosis and antibiosis mechanisms. There is a role for plant chemicals in differential preferences between host races related to distantly related plant taxa (Katte et al. 2022 ). Each adult was not labeled in this study, so whether individual butterflies return to the same plant for subsequent oviposition cannot be tracked. However, Nataraj et al. ( 2024 ) explained that insects return to similar flowers or plants based on previous experience and limit their visits to other potential oviposition niches. It has been proven that larvae-infested and uninfested maize plants affect the oviposition preference of three different Lepidoptera species (Ntiri et al. 2018 ). The decision of adult female insects to oviposit on a plant is made after landing on the leaves influenced by the physical and chemical characteristics of the plant. Females were found to prefer plants with high stomatal density, a large stomatal area, and fewer trichomes as oviposition hosts, and the depth of egg placement was determined by leaf thickness (Zhang et al. 2021 ). During the host search phase, flying insects like Lepidoptera rely on sight and smell to identify their host plants. This research showed that plant color influences insect landing and ovipositioning activities. Accession 12 is the least choice, while accessions 1, 5, and 9 are the most preference. The proportion of anthocyanins to chlorophyll and carotenoids is thought to help D. bisaltide find its host (Table 5 ). Apart from leaf pigment, the egg-laying preference of D. bisaltide is also related to high saponin content and C/N ratio (Table 6 ). Anthocyanins (flavonoids) and saponins, glycosides, are reported as deterrents and antifeedants in many generalist insects, such as Spodoptera littoralis Boisduval. and Helicoverpa armigera Hubner (Boate & Abalis 2020). In constrast, we suspect that plants with high anthocyanin concentrations, namely accessions 1, 5, and 9, tend to be more frequently selected by D. bisaltide adults for oviposition. In contrast, accession 12, with the lowest anthocyanin content, is the least preferred by D. bisaltide to oviposit, as Cepero et al. ( 2015 ) concluded that Monarch butterflies prefer purple color to green ones. The behavior of monophagous insects (specialist insects), such as D. bisaltide revealed that toxic secondary metabolites in plants are stimulants for specialist insects, which generalist insects avoid (Wouters et al. 2016 ). Generalist insects have a range of multiple hosts from different families, while specialist insects are monophagous or oligophagous, eating plants in the same family that release defense compounds. Adult females generally lay eggs on the lower surface of young leaves for several pairs of leaves later. Consistent with the theory that the phytochemical content of plants is distributed with varying concentrations between plant parts Idris et al. ( 2023 ) explained that secondary metabolites are not synthesized in young leaves but are translocated from fully developed leaves to young leaves. Thus, the first instar larvae can easily feed on and digest the soft tissues of the plant. The choice of young leaves as a place to lay eggs is thought to be an insect's response to its host's defense. The diversity of characters between G. pictum genotypes influences egg-laying activity but only slightly influences larval development. This pattern is also observed in other specialist insects, such as Spodoptera frugiperda J.E. Smith on corn (Wouters et al. 2016 ) and Euphydryas editha Bayansis on three different host plants (Haan et al. 2018 ). All larvae that feed on different G. pictum accessions can complete the larval cycle within 14–16 days, two days faster than larvae fed on A. gangetica , based on research by (Handayani et al. 2019). However, the duration of each D. bisaltide larval instar obtained in this study was shorter than the observations of Sartiami et al. ( 2009 ) in the field. This suggests that an unlimited food source enables the larvae to reach their maximum size earlier. Conclusion Flying activities of D. bisaltide and its larvae-feeding activities occur during the day. Their mating activities take place in the dusk until evening. Meanwhile, oviposition activities were held the following morning. The insects prefer the leaf with higher anthocyanin-to-chlorophyll content, high saponin content, and a higher carbon-to-nitrogen ratio. They tend to avoid leaves with high calcium content. The variation in leaf characteristics among G. pictum genotypes affects oviposition preference, though it has only a minor impact on larval development. Accession 12 was suspected to be resistant to D. bisaltide based on oviposition preference. Therefore, it is necessary to conduct genetic studies on accessions 12 to use it as donor parents in a breeding program of G. pictum for resistance trait to D. bisaltide attack. Declarations Consent for publication Not applicable. Conflict of Interest: The authors have no competing interests to declare relevant to this article's content. Funding This research was supported in collaboration between ISMCRI and IPB University Author Contribution PL, TLM, DS – conceptualization, ideas, and research design formulation. PL, WRR, RR, BOO - observation and analysis data, writing the manuscript. PL, TLM, WRR, BOO, IM – insect collecting and mass rearing. PL, DS, RR, IM – morphology identification. PL, TLM, DS, WRR, RR, BOO, IM – writing, editing, managing all communication, and approving the manuscript. All authors listed have significantly contributed to the article’s development and writing. Acknowledgement We greatly appreciate our esteemed lecturers, Dr. Ir. Nurul Khumaida and Dra. Natalini Nova Kristina, thank you for all their suggestions. Thanks to Mr. Cucu Sukmana, who assisted with this experiment. Data availability The authors confirm that the data supporting the findings of this study are available within the article. References Adi MBS, Susanti D, Wijaya NR. 2021. Fluctuation of Doleschallia bisaltide larvae (Lepidoptera: Nymphalidae) on Caricature Plant (Graptophyllum pictum). J Hama dan Penyakit Tumbuh Trop . 21(1):1–7. doi:10.23960/j.hptt.1211-7. Badenes-Pérez FR. 2023. Plant Glucosinolate Content and Host-Plant Preference and Suitability in the Small White Butterfly (Lepidoptera: Pieridae) and Comparison with Another Specialist Lepidopteran. Plants . 12(11). doi:10.3390/plants12112148. Boate, Ukoroije R, Abalis, Otayor R. 2020. Review on the Bio-insecticidal Properties of Some Plant Secondary Metabolites: Types, Formulations, Modes of Action, Advantages and Limitations. Asian J Res Zool ., siap terbit. Cepero LC, Rosenwald LC, Weiss MR. 2015. The Relative Importance of Flower Color and Shape for the Foraging Monarch Butterfly (Lepidoptera: Nymphalidae). J Insect Behav . 28(4):499–511. doi:10.1007/s10905-015-9519-z. Chabaane Y, Marques Arce C, Glauser G, Benrey B. 2022. Altered capsaicin levels in domesticated chili pepper varieties affect the interaction between a generalist herbivore and its ectoparasitoid. J Pest Sci (2004) . 95(2):735–747. doi:10.1007/s10340-021-01399-8. Griese E, Pineda A, Pashalidou FG, Iradi EP, Hilker M, Dicke M, Fatouros NE. 2020. Plant responses to butterfly oviposition partly explain preference–performance relationships on different brassicaceous species. Oecologia . 192(2):463–475. doi:10.1007/s00442-019-04590-y. Haan NL, Bakker JD, Dunwiddie PW, Linders MJ. 2018. Instar-specific effects of host plants on survival of endangered butterfly larvae. Ecol Entomol . 43(6):742–753. doi:10.1111/een.12656. Handayani V, Dahelmi, Herwina H. 2019. Life cycle of the butterfly Doleschallia bisaltide (Lepidoptera: Nymphalide). J Educ Dev . 7(3):301–303. [in Indonesian]. https://doi.org/10.37081/ed.v7i3.1277. Hilker M, Fatouros NE. 2016. Resisting the onset of herbivore attack: Plants perceive and respond to insect eggs. Curr Opin Plant Biol . 32:9–16. doi:10.1016/j.pbi.2016.05.003. Hutagalung MSB. 2019. Phlebotrophic Effect of Graptophyllum Pictum (L.) Griff on Experimental Wistar Hemorrhoids. J Biomed Transl Res . 5(1):1. doi:10.14710/jbtr.v5i1.3704. Idris NS, Khandaker MM, Rashid ZM, Majrashi A, Alenazi MM, Adnan AFM, Mahmud K, Mat N. 2023. Discrimination of Syzygium samarangense cv. ‘Giant Green’ Leaves at Different Maturity Stages by FTIR and GCMS Fingerprinting. Horticulturae . 9(5). doi:10.3390/horticulturae9050609. Kamaliah TL, Hidayat P, Maharijaya A, Syukur M. 2022. Preference Bemisia tabaci Genn . and Its Relation to Leaf Anatomical and Morphological Characters of Chili ( Capsicum annuum L .). J Agron Indones . 50(3):291–298. [in Indonesia]. https://doi.org/10.24831/jai.v50i3.40312. Katte T, Shimoda S, Kobayashi T, Wada-Katsumata A, Nishida R, Ohshima I, Ono H. 2022. Oviposition stimulants underlying different preferences between host races in the leaf-mining moth Acrocercops transecta (Lepidoptera: Gracillariidae). Sci Rep . 12(1):1–12. doi:10.1038/s41598-022-18238-0. Lestari P, Khumaida N, Sartiami D, Mardiningsih TL. 2015. Selection criteria of Graptophyllum pictum resistance to Doleschallia bisaltide cramer (Lep: Nymphalidae) attack based on insect feeding preference. SABRAO J Breed Genet . 47(2):172–184. https://www.researchgate.net/publication/282279312. Ma T, Shi X, Lin N, Wang Z, Xiao Q, Sun Z, Wen X. 2019. Temporal pattern of adult emergence and sexual behavior of Scopula subpunctaria (Lepidoptera: Geometridae). Phytoparasitica . 47(1):17–29. doi:10.1007/s12600-019-00713-6. Makkiyah F, Rahmi EP, Revina R, Susantiningsih T, Setyaningsih Y. 2021. Graptophyllum pictum (l.) griff. (syn: Justicia picta linn.) and its effectiveness: A well-known indonesian plant. Pharmacogn J . 13(3):835–838. doi:10.5530/pj.2021.13.106. Mattjik A, Sumertajaya I. 2013. Experiment design by application SAS and Minitab . Bogor, Indonesia: IPB Press. [in Indonesia]. Nataraj N, Hansson BS, Knaden M. 2024. Learning-based oviposition constancy in insects. Front Ecol Evol . 12(1351400):1–8. doi:10.3389/fevo.2024.1351400. Ntiri ES, Calatayud PA, Musyoka B, Van den Berg J, Le Ru BP. 2018. Influence of feeding-damaged plants on the oviposition responses within a community of female moths. Phytoparasitica . 46(5):607–615. doi:10.1007/s12600-018-0695-1. Penz CM, Williams SF. 2020. Wing morphology and body design in opsiphanes and caligo butterflies match the demands of male mating displays (lepidoptera: nymphalidae). Ann Entomol Soc Am . 113(3):207–215. doi:10.1093/AESA/SAZ073. Rahmawati N, Widiyastuti Y, Purwanto R, Lestari SS, Amir Sene IH, Bakari Y. 2020. Medicinal Plants Used by Traditional Healers for the Treatment of Various Diseases in Ondae Sub-ethnic of Poso District in Indonesia. Di dalam: 4th International Symposium on Health Research . Antlantis Press. hlm 460–468. Sartiami D, Mardiningsih T, Khumaida N, Kristina N. 2009. Sartiami D, Mardiningsih TL, Khumaida N, Kristina NN. Di dalam: Proceedings of the National seminar: the role of biosystematics. Faculty of Biology, University of Jenderal Soedirman, Purwokerto . hlm 563–568. Sartika S, Indradi RB. 2021. Indonesian Journal of Biological Pharmacy Pharmacological Activities of Daun Ungu Plants (Graptophyllum pictum L. Griff). Indones J Biol Pharm . 1(2):88–96. Schäpers A, Carlsson MA, Gamberale-Stille G, Janz N. 2015. The Role of Olfactory Cues for the Search Behavior of a Specialist and Generalist Butterfly. J Insect Behav . 28(1):77–87. doi:10.1007/s10905-014-9482-0. Sekimura T, Nijhout HF. 2017. Diversity and Evolution of Butterfly Wing Patterns. An Integrative Approach . Sekimura T, Nijhout F, editor. Singapore: Springer Nature. Sims DA, Gamon JA. 2002. Relationships between leaf pigment content and spectral reflectance across a wide range of species , leaf structures and developmental stages. Remote Sens Environ . 81:337–354. Sokolinskaya EL, Kolesov D V., Lukyanov KA, Bogdanov AM. 2020. Molecular Principles of Insect Chemoreception. Acta Naturae . 12(3):81–91. doi:10.32607/actanaturae.11038. Umami Z, Mutiah R, Annisa R. 2020. Antitussive activity of the combination formula of ethanol extracts of red ginger (Zingiber officinale var. rubrum) and caricature plant (Graptophyllum pictum) in guinea pigs (Cavia porcellus). Majalan Kesehat . 7(4):212–219. Wouters FC, Blanchette B, Gershenzon J, Vassão DG. 2016. Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores. Phytochem Rev . 15(6):1127–1151. doi:10.1007/s11101-016-9481-1. Zhang L, Qin Z, Liu P, Yin Y, Felton GW, Shi W. 2021. Influence of plant physical and anatomical characteristics on the ovipositional preference of Orius sauteri (hemiptera: Anthocoridae). Insects . 12(4). doi:10.3390/insects12040326. Additional Declarations No competing interests reported. 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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-4820341","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342966483,"identity":"d6135c41-e253-45ee-9b1f-71b833f62d00","order_by":0,"name":"Peni Lestari","email":"","orcid":"","institution":"National Research and Innovation Agency","correspondingAuthor":false,"prefix":"","firstName":"Peni","middleName":"","lastName":"Lestari","suffix":""},{"id":342966485,"identity":"92bf64a6-4f34-4239-9418-e45a745ba249","order_by":1,"name":"Tri Lestari Mardiningsih","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFACxsYDCQY1cgYMjA1gPhtDAn4NPECVBz5UHDMmRQsDw8EZZ5gTNyDECGixlz7ccJi3jS19u/ThtgcMv2wY+NgJ2cKXCNIik7uzL7HdgLEvjYGN5wEBLTyMYFtyN5xhbJNg7DnMwCZByBaIFuZ0A5K0gLyfANbC8IMYLWcggWy4sweoJbEhjYegX9h72B8+AEalvDkP+zOJD39s5OTbCdiCChLbwBFFEvhDqoZRMApGwSgYCQAAay9AwIkjjoQAAAAASUVORK5CYII=","orcid":"","institution":"National Research and Innovation Agency","correspondingAuthor":true,"prefix":"","firstName":"Tri","middleName":"Lestari","lastName":"Mardiningsih","suffix":""},{"id":342966487,"identity":"a56dea18-8410-489f-9cfe-43fb34bf5742","order_by":2,"name":"Dewi Sartiami","email":"","orcid":"","institution":"IPB University","correspondingAuthor":false,"prefix":"","firstName":"Dewi","middleName":"","lastName":"Sartiami","suffix":""},{"id":342966490,"identity":"d6c17f11-dc98-429f-86f5-c93123dcbd43","order_by":3,"name":"Wage Ratna Rohaeni","email":"","orcid":"","institution":"Indonesian Agency for Agricultural Instrument Standardization","correspondingAuthor":false,"prefix":"","firstName":"Wage","middleName":"Ratna","lastName":"Rohaeni","suffix":""},{"id":342966492,"identity":"695a6d87-0724-4660-8dd8-ba65efe6c6b0","order_by":4,"name":"Rohimatun Rohimatun","email":"","orcid":"","institution":"National Research and Innovation Agency","correspondingAuthor":false,"prefix":"","firstName":"Rohimatun","middleName":"","lastName":"Rohimatun","suffix":""},{"id":342966494,"identity":"bf68fee3-b386-4996-a07e-4fd53dea9814","order_by":5,"name":"Bruce Ochieng Obura","email":"","orcid":"","institution":"International Potato Center","correspondingAuthor":false,"prefix":"","firstName":"Bruce","middleName":"Ochieng","lastName":"Obura","suffix":""},{"id":342966495,"identity":"256e8f37-cd53-4cff-b54c-f440c6a8d052","order_by":6,"name":"Ifa Manzila","email":"","orcid":"","institution":"National Research and Innovation Agency","correspondingAuthor":false,"prefix":"","firstName":"Ifa","middleName":"","lastName":"Manzila","suffix":""}],"badges":[],"createdAt":"2024-07-29 08:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4820341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4820341/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64814816,"identity":"ef52c3a0-fdc5-43c7-bd93-19eaecb74a08","added_by":"auto","created_at":"2024-09-19 06:34:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":384174,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of the adult \u003cem\u003eD. bisaltide\u003c/em\u003e. Female (B) and male (B) adult of \u003cem\u003eD. bisaltide\u003c/em\u003e. The arrow indicates the white spots, a characteristic of males that differentiates them from females\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/b3afcb7151614be737ef91fc.png"},{"id":64814815,"identity":"94be2b48-6d03-4352-ae2b-bb3450089ff4","added_by":"auto","created_at":"2024-09-19 06:34:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":637039,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of \u003cem\u003eD. bisaltide\u003c/em\u003e eggs. Newly laid eggs (A), microscopic characteristics of eggs (B), eggs about to hatch (C), a female adult was laying eggs on the undersurface of leaves (D), a cluster of eggs laid on stems (E), and eggs that failed to hatch (F)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/bc4d51c9752f1e30e6114f5f.png"},{"id":64815481,"identity":"24559d48-ab87-4d7e-b344-b22b5689f553","added_by":"auto","created_at":"2024-09-19 06:42:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37028,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of\u003cem\u003e D. bisaltide\u003c/em\u003e visits in 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/d3ac56c17789b22fac569189.png"},{"id":64816054,"identity":"39910d85-969a-4675-9def-390737dd877f","added_by":"auto","created_at":"2024-09-19 06:50:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":656921,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of \u003cem\u003eD. bisaltide\u003c/em\u003e larvae and pupae. Neonate (A), first instar larvae (B), second instar larvae (C), third instar larvae (D), fourth instar larvae (E), fifth instar larvae (F), prepupa (G), early pupa (H), pupa (I), and newly adult came out from pupae (J) of \u003cem\u003eD. bisaltide\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/ee9278785aa264557220e117.png"},{"id":64814812,"identity":"119f8b9b-77fd-43e9-bbc4-4c6bff9298b9","added_by":"auto","created_at":"2024-09-19 06:34:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":849626,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf morphology of \u003cem\u003eG. pictum\u003c/em\u003e. Numbers 1-13 mean \u003cem\u003eG. pictum \u003c/em\u003eaccession à angka pada gambar diganti fonnta dg Helvetica/Arial\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/8f6946dbd381668d4c00621f.png"},{"id":65028213,"identity":"eeae91c9-b5b5-48da-9102-f6a3b7059a08","added_by":"auto","created_at":"2024-09-22 18:59:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3998776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4820341/v1/7a87586a-595c-47ba-8c5d-6aaa4994cc72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Doleschallia bisaltide (Lepidoptera: Nymphalidae) profile and oviposition preference on Graptophyllum pictum (L.) Griff.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe caricature plant, \u003cem\u003eGraptophyllum pictum\u003c/em\u003e (L.) Griff. (Syn. \u003cem\u003eJusticia picta\u003c/em\u003e Linn.), belonging to the family Acanthaceae is known in Indonesia as \u003cem\u003ehandeuleum\u003c/em\u003e (Sundanese), \u003cem\u003ewungu\u003c/em\u003e leaf (Javanese) (Sartika \u0026amp; Indradi \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and purple pudding (Umami et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This species is integral to Indonesian ethnobotany and is renowned for its medicinal properties, such as anti-hemorrhoid, antimicrobial, anti-inflammatory, analgesic, and wound-healing effects (Rahmawati et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, \u003cem\u003eG. pictum\u003c/em\u003e leaves are reported to alleviate coughs (Umami et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and plaque reduction in teeth (Makkiyah et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The health benefits of the \u003cem\u003eG. pictum\u003c/em\u003e are attributed to various active compounds, including alkaloids, flavonoids, steroids, saponins, glycosides, and emollients, present in the leaves (Lestari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), along with information on its pharmacological effects (Hutagalung \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Makkiyah et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDoleschallia bisaltide\u003c/em\u003e Cramer (Lepidoptera: Nymphalidae) is a specialist (monophagous) insect known to feed leaves of the Acanthaceae family, particularly \u003cem\u003eG. pictum\u003c/em\u003e and \u003cem\u003eAsystasia gangetica\u003c/em\u003e (L.) Anders (Handayani \u003cem\u003eet al.\u003c/em\u003e 2019). Yield losses of \u003cem\u003eG. pictum\u003c/em\u003e leaves due to \u003cem\u003eD. bisaltide\u003c/em\u003e larvae have been reported to range from 300.28 to 745.86 cm\u0026sup2; per plant, equivalent to 70% of the leaves, based on (Lestari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eG. pictum\u003c/em\u003e leaves are essential for harvesting for medicinal purposes; therefore, larvae infestation poses a risk of reducing the quantity and quality of its harvest.\u003c/p\u003e \u003cp\u003eUnderstanding herbivore-plant interactions is crucial in developing plants resistant to herbivores (Wouters et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chabaane et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kamaliah et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), including the oviposition preferences of adult insects on host plants. Early instar larvae of Lepidopterans can only access food resources near their eggs (Wouters et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Griese et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, oviposition preferences of adults on specific plants and the plant's antixenosis and antibiosis mechanisms are crucial in determining plant resistance levels against pests. Insect host preferences are influenced by plant morphology, which can serve as a selection factor in breeding initiatives for resistant varieties, this may serve as a selection factor (Lestari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Feeding preferences of \u003cem\u003eD. bisaltide\u003c/em\u003e across Acanthaceae species and within \u003cem\u003eG. pictum\u003c/em\u003e have been studied, along with population fluctuations in nature (Adi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, information on oviposition preferences within \u003cem\u003eGraptophyllum\u003c/em\u003e species still needs to be available. Based on literature studies, this is the first study to discuss the interaction of \u003cem\u003eD. bisaltide\u003c/em\u003e and \u003cem\u003eG. pictum\u003c/em\u003e regarding oviposition preference and plant phytochemical characteristics. Identifying the behavior of \u003cem\u003eD. bisaltide\u003c/em\u003e adults and larvae is the first step in designing strategies for its management in \u003cem\u003eG. pictum\u003c/em\u003e plantations, including developing resistant plant varieties.\u003c/p\u003e \u003cp\u003eInsects naturally exhibit varied responses to plants with diverse characteristics. Plant chemistry, especially for female butterflies, plays a role in determining niches for oviposition and feeding activities (Griese et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). (Sokolinskaya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) explained that Insects have created an impossible-to-miss and one of the foremost advanced chemoreception frameworks, which abuses at the slightest three receptor superfamilies giving discernment of scent and taste and chemical communication in these creatures. Badenes-P\u0026eacute;rez (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) said that there is an effect of host plant indolic glucosinolate content on oviposition preferences and the complexity index of glucosinolates and aliphatic glucosinolates without sulfur-containing side chains on total oviposition is smaller in \u003cem\u003eP. rapae\u003c/em\u003e than in \u003cem\u003ePlutella xylostella\u003c/em\u003e L. (Lepidoptera: \u003cem\u003ePlutellidae\u003c/em\u003e), another lepidopteran that specializes in containing plant glucosinolates. Variations in genetics, nutritional status, and environmental conditions cause significant differences in the phytochemical content among varieties under the same species. This diversity explains the oviposition preference and feeding preferences of some insects to some varieties over others.\u003c/p\u003e \u003cp\u003eThis study aims to explore the oviposition preferences and larvae development of \u003cem\u003eD. bisaltide\u003c/em\u003e reared on 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions. The herbivore-host interaction in this study is used as a guide to develop \u003cem\u003eG. pictum\u003c/em\u003e cultivars resistant to \u003cem\u003eD. bisaltide.\u003c/em\u003e\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eThe research was conducted during three flight periods of \u003cem\u003eD. bisaltide\u003c/em\u003e adults: the end of the rainy season (May-June), the beginning of the dry season (June-July), and the peak of the rainy season (November-December). The length of each period was adjusted until the last adult died. The research took place in the greenhouse of the Indonesian Spice and Medicinal Crops Research Institute (ISMCRI) (nowadays the Indonesian Agency for Agricultural Instrument Standardization/IAAIS), Bogor, Indonesia (240 m above sea level). The number of rainy days and total rainfall during the three flight periods are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eNumber of rainy days and total precipitation during the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlight period\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of rainy days (day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal precipitation during a month (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMay-June\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e771.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJune-July\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e501.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovember-December\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e693.4\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\u003eData was obtained from the weather station at the ISMCRI Research Station\u003c/p\u003e \u003cp\u003ePreparation of Test Plants\u003c/p\u003e \u003cp\u003eThirteen \u003cem\u003eG. pictum\u003c/em\u003e accessions were obtained from the germplasm collection of the Department of Agronomy and Horticulture, IPB University. Their tolerance level to \u003cem\u003eD.bisaltide\u003c/em\u003e was described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Cuttings that were 15 cm long were planted in sandy soil. Healthy and uniform seedlings, 30 days old, were transplanted into a planting medium mixed with soil, manure, and rice husks charcoal (1:1:1). Seedlings were maintained under 55% light intensity using artificial shade. Plants were watered twice a day. The 250 ml of balanced NPK (16-16-16) liquid fertilizer (concentration 2 g/L water) was applied to the plants every two weeks. Plants were in the vegetative phase during the testing period.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTolerance level of \u003cem\u003eG. pictum\u003c/em\u003e accessions to \u003cem\u003eD. bisaltide\u003c/em\u003e larvae\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccessions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResistance level\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResistant\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\u003eTolerance level on every \u003cem\u003eG. pictum\u003c/em\u003e accession to \u003cem\u003eD. bisaltide\u003c/em\u003e based on feeding preference (non choice test) (Lestari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ePreparation of Test Insects\u003c/p\u003e \u003cp\u003eThe adult insect population in this experiment from a field sample first breed generations, originated from eggs collected from the \u003cem\u003eG. pictum\u003c/em\u003e population in the ISMCRI collection garden. Eggs were kept in a rearing box. After hatching, the first instar larvae were moved to another box until they reached the third instar stage, then transplanted into a 0.25 m\u0026sup3; cylindrical mesh cage until they became pupae (Sch\u0026auml;pers et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). During rearing, larvae were fed a mixture of leaves from the 13 tested \u003cem\u003eG. pictum\u003c/em\u003e accessions every morning and evening. Pupae were transferred to the rearing box. The hatched butterflies were reared in another mesh cage and maintained until testing. Food sources for \u003cem\u003eD. bisaltide\u003c/em\u003e butterflies were nectars from flowers of \u003cem\u003eIxora\u003c/em\u003e sp. and \u003cem\u003eHydrangea\u003c/em\u003e sp., as well as cotton moistened with a 10% concentration of multiflora honey solution.\u003c/p\u003e \u003cp\u003eExperimental Design\u003c/p\u003e \u003cp\u003eThe experiment was carried out in three flight periods: May-June (end of the rainy season), June-July (transition from rainy to dry season), and November-December (peak of the rainy season). In each experimental set, 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions were arranged based on a Randomized Complete Block Design with three replications and five plants per group.\u003c/p\u003e \u003cp\u003eThe morphological characteristics and mating activity of \u003cem\u003eD. bisaltide\u003c/em\u003e were observed in the adult population during the experiment. Oviposition preference testing was carried out based on the choice method. Fifty pairs of butterflies that came out from pupae on the same day were flown in a greenhouse containing the \u003cem\u003eG. pictum\u003c/em\u003e accessions tested. Dead butterflies were replaced until the seventh day of flight, then started to count the adults. The profile of \u003cem\u003eD. bisaltide\u003c/em\u003e was observed daily until the last adult died. The lifespan of the butterflies was calculated based on the reduction in the number of male or female butterflies compared to the initial number flown.\u003c/p\u003e \u003cp\u003eThe frequency of visits of \u003cem\u003eD. bisaltide\u003c/em\u003e adults was calculated based on the number of egg batches on a particular accession. The oviposition preference of \u003cem\u003eD. bisaltide\u003c/em\u003e was estimated based on the total number of eggs laid on each plant accession compared to the total number of eggs laid on all plants during one flight period (%). The larger the egg batches and the more number eggs laid on accession, the higher the \u003cem\u003eD. bisaltide\u003c/em\u003e adult preference for that accession (Lestari et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Griese et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The eggs counted were those laid on that day. First instar larvae were selected and maintained on each accession (non-choice) to observe their development. The frequency of adult visits, oviposition preference, number of eggs, and larval growth and development were counted daily during the experiment. Environmental variables, such as temperature and humidity, were recorded daily using a thermohygrometer (data not shown).\u003c/p\u003e \u003cp\u003eObservation of \u003cem\u003eG. pictum\u003c/em\u003e Leaf Pigments\u003c/p\u003e \u003cp\u003eThe leaves chlorophyll, anthocyanins, and carotenoids were measured using spectrophotometric methods based on the Sims \u0026amp; Gamon (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) method. The other phytochemical substances were examined in the ISMCRI laboratory utilizing these techniques: nitrogen was measured using the Kjeldahl method; calcium was identified via the Atomic Absorption Spectroscopy (AAS) method; organic carbon was determined by spectrophotometry; and fiber was assessed based on the gravimetric method (SNI 01-2891-1992).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eData were tabulated and prepared using Microsoft Excel 2019 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.microsoft.com\u003c/span\u003e\u003cspan address=\"http://www.microsoft.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, USA). The normality of data distribution was analyzed using the Shapiro-Wilk test. Data were analyzed using analysis of variance followed by the Tukey post hoc test (α\u0026thinsp;=\u0026thinsp;5%). The relationship between observed variables was estimated using Pearson correlation in Minitab v.18 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.minitab.com\u003c/span\u003e\u003cspan address=\"http://www.minitab.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Mattjik \u0026amp; Sumertajaya (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) suggest that correlation analysis describes the nature of the relationship between one character and another. The closer it is to 1 or -1, the stronger the correlation between the two characters. Conversely, the closer it is to 0, the more unrelated the two characters are.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eProfile of \u003cem\u003eD. bisaltide\u003c/em\u003e on \u003cem\u003eG. pictum\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe adult of \u003cem\u003eD. bisaltide\u003c/em\u003e has brown wings, ranging from yellowish-brown to grayish-brown. Male \u003cem\u003eD. bisaltide\u003c/em\u003e butterflies have characteristically darker wings with white patches on the lower-part wings, while female butterflies do not have these spots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average lifespan of butterflies in this study was 9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00 days across the three flight periods. Characteristics of \u003cem\u003eD. bisaltide\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eInsect of \u003cem\u003eD. bisaltide\u003c/em\u003e has four life stages: egg, larva, pupa, and adult (butterfly). Generally, \u003cem\u003eD. bisaltide\u003c/em\u003e butterflies take flight around 8 o'clock when the weather is clear and end at sunset. In contrast, \u003cem\u003eD. bisaltide\u003c/em\u003e mating activity starts in the afternoon, before sunset, and ends at night. The mating process begins with flying together as a calling stage, continuing with chasing (mating stage) (Ma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The butterflies who have found their mate then separate themselves and perch in pairs on the ceiling of the screen house. The male and female abdomens unite until the mating process is complete (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mating process lasts six to eight hours, as (Ma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) report on other Lepidopterans (\u003cem\u003eScopula subpunctaria\u003c/em\u003e Herrich-Schaeffer). As many as 40 of 50 pairs of butterflies copulate near water sources. The following day, the female will lay eggs on the underside of the leaves. During the test period, mating occurred within the first 3 weeks.\u003c/p\u003e \u003cp\u003eThe eggs of \u003cem\u003eD. bisaltide\u003c/em\u003e were pearly white when laid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), turned cream, and finally became clear with black spots at the poles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) when ready to hatch. If they turned black (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), they failed to hatch. The black spot was the head of the larva. The eggs were spherical (round) with a diameter of \u0026plusmn;\u0026thinsp;1 mm. They bent inward at one pole (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). \u003cem\u003eD. bisaltide\u003c/em\u003e adults laid all eggs, or divided into several egg batches, under the leaf surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Sometimes, eggs were laid on the stem (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The egg numbers reach 50 eggs. The eggs would hatch within 4 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of eggs laid per female differed between flight periods (n\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as well as between replicates per period (n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;=\u0026thinsp;0.002). Females laid more eggs during the flight period in November-December (125.78 eggs), followed by June-July (96.57 eggs) and May-June (51.20 eggs) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the hatching percentage was highest in May-June and differed from June-July (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). More eggs were laid in plant populations near water sources.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEgg numbers in 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions during the three flight periods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNumber of eggs (eggs)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay-June\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJune-July\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNovember - December\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.70d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e143.18a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114.57a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.48a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.33d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130.14c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e135.44c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106.64a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.16b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.64h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e143.73h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.84a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.45c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102.19e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164.61e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e107.08a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.21e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e117.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e149.56b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e107.63a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.84i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.08j\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e129.25j\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.06a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.69g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.10d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.21d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.92k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e119.21d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e132.35d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.16a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.88a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102.85f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125.73f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.82a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.78h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.94i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e152.35i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.36a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.27j\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.43g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.90g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.53a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7.64l\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e21.48k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e19.05k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e16.06b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.76f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.03f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119.43f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.74a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.20c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.57b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125.78a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eColumns with the same letter are not significantly different based on the Tukey post hoc at α\u0026thinsp;=\u0026thinsp;5%. Each period is tested using Kruskal-Wallis\u003c/p\u003e \u003cp\u003eThe more significant number of egg batches found on one plant indicates that the more frequently adults visit that plant, the higher the oviposition preference of \u003cem\u003eD. bisaltide\u003c/em\u003e females on that accession. The lowest visiting frequency was obtained for accession 12, followed by accessions 6 and 13, whereas accessions 1, 5, and 9 were most regularly visited (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The number of egg batches laid in other accessions varies in each period. The number of eggs laid in accession 12 was lowest in each flight period (May-June (H\u0026thinsp;=\u0026thinsp;2.94, p\u0026thinsp;=\u0026thinsp;0.003), June-July (H\u0026thinsp;=\u0026thinsp;3.37, p\u0026thinsp;=\u0026thinsp;0.001), November-December (H\u0026thinsp;=\u0026thinsp;2.85, p\u0026thinsp;=\u0026thinsp;0.005)). This value was only 1.33% of the eggs in 13 plant accessions, followed by accessions 6 and 13. In contrast, most of the \u003cem\u003eD. bisaltide\u003c/em\u003e adult individuals laid eggs in accessions 1, 5, and 9 with a percentage of eggs\u0026thinsp;\u0026ge;\u0026thinsp;10% of the total eggs. The larvae development stage lasts around 14\u0026ndash;16 days, followed by a pupation period of 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHatched egg presentation in 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions during the three flight periods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eHatched Egg (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay-June\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJune-July\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNovember - December\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.38ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.14ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.29a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.60ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.50ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.34b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.61b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.59ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.63ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.28a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61.17ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.15b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.72ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.50a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.79ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.84ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.68ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.24a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.58ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.20ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.34ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.13a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.56ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.05ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.82ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.60a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.16ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.09ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.22a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.14b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.68ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.72b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.23a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.55b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.55ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.15ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.34a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68.34ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.12ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.00ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.24b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.62a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.51ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.40a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70.51ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.50ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80.42a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.99a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.30a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe average season\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.25a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.50b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.29ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThey live on the lower surface of leaves. No cannibalism was found, except for fifth instar larvae, which ate young pupae. When hatched, \u003cem\u003eD. bisaltide\u003c/em\u003e larvae had pale yellow bodies with a shiny black head region and soft hair on each body segment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After they ate the leaves, the bodies of the first instar larvae were blackish, like jelly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In the second instar larvae, bristly hairs were developed on all the body segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The third instar larvae began to display white lines on their dorsal and lateral sides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These lines become more prominent as they develop to the fourth and 5th instars. From the fourth instar larvae, orange sockets appeared on the lateral sides and metallic blue sockets on the dorsal sides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The fifth instar larvae had reached maximum body size, the most extended lifespan, and the highest eating ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The larvae need 2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 days for the first instar, 2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 days for the second instar, 2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 days for the third instar, 2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 days for the fourth instar, and 5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 days for fifth instar. At the end of the fifth instar, the larvae ceased feeding and underwent a morphological change in which their bodies shortened and hung down, forming the shape of a letter J with their heads at the bottom.\u003c/p\u003e \u003cp\u003eThe pupae of \u003cem\u003eD. bisaltide\u003c/em\u003e had an obtecta shape, characterized by prospective antennae, wings, and legs that appear faint and are tightly attached to the body. The pupa was wrapped in cremaster thread. The color of the pupa was initially pink (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), gradually becoming light brown to dark brown, with a yellow tinge in some parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Butterflies would emerge in the next 7\u0026ndash;9 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). There were neatly arranged black dots on the dorsal side of the pupa. The highest mortality occurred during the initial phase of pupa formation, likely due to the consumption of soft pupal tissues by fifth instar larvae. The extent of damage to the pupa can significantly impact the success of metamorphosis into a butterfly as an adult form of \u003cem\u003eD. bisaltide\u003c/em\u003e. The emerge-adult can take one of three forms: perfect, deformed, or deceased.\u003c/p\u003e \u003cp\u003eCharacteristics of \u003cem\u003eG. pictum\u003c/em\u003e and Its Relation to \u003cem\u003eD. bisaltide\u003c/em\u003e Oviposition\u003c/p\u003e \u003cp\u003eThe results revealed significant variations in the percentage of anthocyanin, chlorophyll, and carotenoid pigments, suggesting differences in the composition of these pigments among the accessions. The highest concentration of anthocyanin pigments was in accession 1, and the lowest was in accession 12 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The proportion of anthocyanin to chlorophyll to carotenoid pigments varied among accessions, with accession 1 having a ratio of 5:3:2 and accession 12 of 1:6:3. In addition, eleven other accessions exhibited a similar proportion of anthocyanin to chlorophyll to carotenoid pigments, with a ratio of 4:4:2. Visually, the 12 \u003cem\u003eG. pictum\u003c/em\u003e accessions have similar leaf colors, viz. reddish purple to blackish purple. Only accession 12 has a green-white variegation color (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProportion of pigment content on the leaves of \u003cem\u003eG. pictum\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLeaf pigment proportion (%)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthocyanin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal chlorophyll\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarotenoid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*) Measured on the third leaf of a fully opened shoot using the (Sims dan Gamon 2002) method. Pigment measurements were not carried out on accession two due to insufficient availability\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe relationship between the number of eggs and the pigment content and secondary metabolites of 13 \u003cem\u003eG. pictum\u003c/em\u003e accessions showed that the number of eggs was significantly positively correlated with the anthocyanin content, C/N ratio, and saponin content of \u003cem\u003eG. pictum\u003c/em\u003e leaves and negatively correlated with chlorophyll content (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, the frequency of \u003cem\u003eD. bisltide\u003c/em\u003e visits was uncorrelated with any plant traits. However, the correlation value is relatively high in leaves with high saponin content or low calcium content.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation values between plant characteristics and egg number and frequency of adult arrival\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eG. pictum\u003c/em\u003e characters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCorrelation value with\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEgg number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVisiting frequency\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnthocyanin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.71**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.74**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarotenoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC organic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC/N ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.63*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFiber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaloids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaponin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriterpenoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteroids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: * means significant (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and ** means very significant (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) based on Pearson correlation analysis\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eLike autumn leaves, the brown wings are characteristic of \u003cem\u003eD. bisaltide\u003c/em\u003e, making it known as the autumn-leaf butterfly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Several publications link the variation in wing color of \u003cem\u003eD. bisaltide\u003c/em\u003e to its functions, such as mimicry, mating preferences, defense from predators, and response to pollutant concentrations in the environment (Sekimura \u0026amp; Nijhout \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Penz \u0026amp; Williams \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sekimura dan Nijhout (2017) concluded that the evolution and diversity of Nymphalidae wings are influenced by genetics and changes in environmental temperature. Both larvae and adults of \u003cem\u003eD. bisaltide\u003c/em\u003e are active from morning to evening, but mating activity generally occurs in the afternoon to evening, with ovipositioning the following day. The long duration of mating causes damage to the hind wings of \u003cem\u003eD. bisaltide\u003c/em\u003e adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Most Nymphalidae butterflies (depending on the species) are reported to actively mate and lay eggs at dawn, dusk, or both (Penz \u0026amp; Williams \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eForty of the fifty pairs of \u003cem\u003eD. bisaltide\u003c/em\u003e chose water sources for copulation, indicating that the insect population increases in the rainy season or in areas with high humidity. Observations showed that most eggs were laid during the rainy season (November-December) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), as well as the hatching percentage (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) when the number of rainy days and precipitation was higher than in the dry season (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Air humidity in November-December reaches 90%, decreasing to 48\u0026ndash;53% from June to July and around 60% from May to June. This study supports Adi et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who stated that humidity affects the fecundity of adult \u003cem\u003eD. bisaltide\u003c/em\u003e. Based on the average total number of eggs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and the frequency of adult visits in three flight periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it was concluded that adult \u003cem\u003eD. bisaltide\u003c/em\u003e females had the lowest oviposition preference in accession 12 and the highest in accessions 1, 5, and 9.\u003c/p\u003e \u003cp\u003eThe number of eggs laid is unrelated to the number of eggs that hatch but is in line with feeding activity. The lowest percentage of hatched eggs was in accession 9 (46.55%), and the highest was in accession 13 (79.30%). Meanwhile, our previous study on feeding activity showed that accessions 5 and 9 were susceptible to \u003cem\u003eD. bisaltide\u003c/em\u003e larvae, while accessions 7 and 12 were resistant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Lestari et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Both accessions 7 and 12 were rich in fiber content. Apart from insect factors, such as food consumed and the genetics of adults, plant factors also influence egg hatching. The presence of herbivore eggs acts as an inducer of plant defense mechanisms, and then they respond physiologically (Hilker \u0026amp; Fatouros \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) by changes in secondary metabolic contents or structure. The presence of insect eggs is an essential form of early-stage plant defense evolution. The ability to detect and create unsuitable conditions for egg development potentially inhibits eggs from hatching, resulting in fewer larvae consuming the plant (Griese et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant characteristics influence the interaction of herbivore insects with their hosts through antixenosis and antibiosis mechanisms. There is a role for plant chemicals in differential preferences between host races related to distantly related plant taxa (Katte et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Each adult was not labeled in this study, so whether individual butterflies return to the same plant for subsequent oviposition cannot be tracked. However, Nataraj et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) explained that insects return to similar flowers or plants based on previous experience and limit their visits to other potential oviposition niches. It has been proven that larvae-infested and uninfested maize plants affect the oviposition preference of three different Lepidoptera species (Ntiri et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The decision of adult female insects to oviposit on a plant is made after landing on the leaves influenced by the physical and chemical characteristics of the plant. Females were found to prefer plants with high stomatal density, a large stomatal area, and fewer trichomes as oviposition hosts, and the depth of egg placement was determined by leaf thickness (Zhang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the host search phase, flying insects like Lepidoptera rely on sight and smell to identify their host plants. This research showed that plant color influences insect landing and ovipositioning activities. Accession 12 is the least choice, while accessions 1, 5, and 9 are the most preference. The proportion of anthocyanins to chlorophyll and carotenoids is thought to help \u003cem\u003eD. bisaltide\u003c/em\u003e find its host (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Apart from leaf pigment, the egg-laying preference of \u003cem\u003eD. bisaltide\u003c/em\u003e is also related to high saponin content and C/N ratio (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Anthocyanins (flavonoids) and saponins, glycosides, are reported as deterrents and antifeedants in many generalist insects, such as \u003cem\u003eSpodoptera littoralis\u003c/em\u003e Boisduval. and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e Hubner (Boate \u0026amp; Abalis 2020). In constrast, we suspect that plants with high anthocyanin concentrations, namely accessions 1, 5, and 9, tend to be more frequently selected by \u003cem\u003eD. bisaltide\u003c/em\u003e adults for oviposition. In contrast, accession 12, with the lowest anthocyanin content, is the least preferred by \u003cem\u003eD. bisaltide\u003c/em\u003e to oviposit, as Cepero et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) concluded that Monarch butterflies prefer purple color to green ones.\u003c/p\u003e \u003cp\u003eThe behavior of monophagous insects (specialist insects), such as \u003cem\u003eD. bisaltide\u003c/em\u003e revealed that toxic secondary metabolites in plants are stimulants for specialist insects, which generalist insects avoid (Wouters et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Generalist insects have a range of multiple hosts from different families, while specialist insects are monophagous or oligophagous, eating plants in the same family that release defense compounds.\u003c/p\u003e \u003cp\u003eAdult females generally lay eggs on the lower surface of young leaves for several pairs of leaves later. Consistent with the theory that the phytochemical content of plants is distributed with varying concentrations between plant parts Idris et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) explained that secondary metabolites are not synthesized in young leaves but are translocated from fully developed leaves to young leaves. Thus, the first instar larvae can easily feed on and digest the soft tissues of the plant. The choice of young leaves as a place to lay eggs is thought to be an insect's response to its host's defense.\u003c/p\u003e \u003cp\u003eThe diversity of characters between \u003cem\u003eG. pictum\u003c/em\u003e genotypes influences egg-laying activity but only slightly influences larval development. This pattern is also observed in other specialist insects, such as \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e J.E. Smith on corn (Wouters et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and \u003cem\u003eEuphydryas editha\u003c/em\u003e Bayansis on three different host plants (Haan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). All larvae that feed on different \u003cem\u003eG. pictum\u003c/em\u003e accessions can complete the larval cycle within 14\u0026ndash;16 days, two days faster than larvae fed on \u003cem\u003eA. gangetica\u003c/em\u003e, based on research by (Handayani \u003cem\u003eet al.\u003c/em\u003e 2019). However, the duration of each \u003cem\u003eD. bisaltide\u003c/em\u003e larval instar obtained in this study was shorter than the observations of Sartiami et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) in the field. This suggests that an unlimited food source enables the larvae to reach their maximum size earlier.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFlying activities of \u003cem\u003eD. bisaltide\u003c/em\u003e and its larvae-feeding activities occur during the day. Their mating activities take place in the dusk until evening. Meanwhile, oviposition activities were held the following morning. The insects prefer the leaf with higher anthocyanin-to-chlorophyll content, high saponin content, and a higher carbon-to-nitrogen ratio. They tend to avoid leaves with high calcium content. The variation in leaf characteristics among \u003cem\u003eG. pictum\u003c/em\u003e genotypes affects oviposition preference, though it has only a minor impact on larval development. Accession 12 was suspected to be resistant to \u003cem\u003eD. bisaltide\u003c/em\u003e based on oviposition preference. Therefore, it is necessary to conduct genetic studies on accessions 12 to use it as donor parents in a breeding program of \u003cem\u003eG. pictum\u003c/em\u003e for resistance trait to \u003cem\u003eD. bisaltide\u003c/em\u003e attack.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest:\u003c/strong\u003e \u003cp\u003eThe authors have no competing interests to declare relevant to this article's content.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported in collaboration between ISMCRI and IPB University\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePL, TLM, DS \u0026ndash; conceptualization, ideas, and research design formulation. PL, WRR, RR, BOO - observation and analysis data, writing the manuscript. PL, TLM, WRR, BOO, IM \u0026ndash; insect collecting and mass rearing. PL, DS, RR, IM \u0026ndash; morphology identification. PL, TLM, DS, WRR, RR, BOO, IM \u0026ndash; writing, editing, managing all communication, and approving the manuscript. All authors listed have significantly contributed to the article\u0026rsquo;s development and writing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe greatly appreciate our esteemed lecturers, Dr. Ir. Nurul Khumaida and Dra. Natalini Nova Kristina, thank you for all their suggestions. Thanks to Mr. Cucu Sukmana, who assisted with this experiment.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdi MBS, Susanti D, Wijaya NR. 2021. 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Influence of plant physical and anatomical characteristics on the ovipositional preference of Orius sauteri (hemiptera: Anthocoridae). \u003cem\u003eInsects\u003c/em\u003e. 12(4). doi:10.3390/insects12040326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"anthocyanin, herbivore-host interaction, oviposition stimulant, phytochemicals, resistance level, specialists","lastPublishedDoi":"10.21203/rs.3.rs-4820341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4820341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eDoleschallia bisaltide\u003c/em\u003e Cramer (Lepidoptera: Nymphalidae) is a main pest on \u003cem\u003eGraptophyllum pictum\u003c/em\u003e (L.) Griff. in the Lepidopteran order, the first-instar larval performance is determined by adult oviposition. An adult oviposition preference can be used to determine the resistance level of some \u003cem\u003eG. pictum\u003c/em\u003e accessions in early-stage breeding programs. The study aimed to identify the profile of \u003cem\u003eD. bisaltide\u003c/em\u003e and its oviposition preferences on \u003cem\u003eG. pictum\u003c/em\u003e accessions. The herbivore-host interaction was utilized as a guide to select cultivars resistant to \u003cem\u003eD. bisaltide\u003c/em\u003e and identify repellent characteristics for the insect. The research was conducted at the Indonesian Spice and Medicinal Crops Research Institute (ISMCRI), using nested Randomized Complete Block Design (RCBD). \u003cem\u003eG. pictum\u003c/em\u003e accessions nested on flight periods. The study revealed that insect mating occurred from dusk to night, while oviposition occurred the following morning. The adult mating was dominantly in the humid area, suggesting a potential population outbreak during the rainy season. The highest oviposition preference was observed for accessions with higher anthocyanin-to-chlorophyll content. In contrast, the lowest oviposition was recorded for accession 12 content of higher chlorophyll-to-anthocyanins. \u003cem\u003eD. bisaltide\u003c/em\u003e oviposition preferences were influenced by leaf pigment, saponins, and the C/N ratio. Phytochemicals such as flavonoids and glycosides in \u003cem\u003eG.pictum\u003c/em\u003e leaves acted as oviposition stimulants for specialists such as \u003cem\u003eD. bisaltide.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Doleschallia bisaltide (Lepidoptera: Nymphalidae) profile and oviposition preference on Graptophyllum pictum (L.) Griff.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-19 06:34:12","doi":"10.21203/rs.3.rs-4820341/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":"c3032ca0-8ee5-41cc-972a-781b1cbdebdc","owner":[],"postedDate":"September 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-22T18:51:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-19 06:34:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4820341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4820341","identity":"rs-4820341","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}