Insect diversity on Calotropis gigantea (L.) in Sri Lanka

: Calotropis gigantea is a drought-resistant and salt-tolerant medicinal plant native to Sri Lanka. Although C. gigantea is widely distributed in Sri Lanka, information on insects associated with the plant is less understood. The objective of the study is to identify the diversity of insect fauna associated with C. gigantea . Surveys were conducted in 120 sites covering all provinces of Sri Lanka to document the insect fauna associated with C. gigantea and their biotic associations. The insects found in C. gigantea were cataloged as pests, pollinators, and occasional visitors. A total of thirteen morphospecies of phytophagous pests, six species of pollinators, and fourteen species of occasional visitors were documented. Dacus persicus and Paramecops farinosa were the highly damaging pests while Sphaeroderma sp. was more widespread. Xylocopa spp. were the most abundant insect pollinators. Dacus persicus and P. farinosa were identified as monophagous species of C. gigantea. Occasional visitors belonged to five orders and their diversity was very high. As the initial record from Sri Lanka, the findings of the study provide information on the identification of the insect fauna associated with Calotropis and their association with C. gigantea .


INTRODUCTION
Calotropis gigantea (L.) Dryand (Apocynaceae), commonly known as Arka  Madura, Yercum (Sethi, 2014), Crown flower, or Giant milkweed (Kadiyala et al., 2013;Saikia et al., 2015), is native to India, China, Bangladesh, Burma, Indonesia, Malaysia, Pakistan, Thailand, Philippines, and Sri Lanka (Kumar and Kumar, 2015). In many countries in Asia, C. gigantea is used as a medicinal plant to cure various ailments, including bronchial asthma, cholera, convulsions, pneumonia, ringworm infection, smallpox infection, toothache, epilepsy, fever, leprosy, rheumatism, catarrh, cold, cough, inflammation, tumors, mental disorders, snakebite infection, and tuberculosis Kumar and Kumar, 2015;Abeysinghe, 2018). In Sri Lanka, C. gigantea is widely used in Ayurvedic medicine, for the treatment of pain and inflammation (Shukla et al., 2018). Furthermore, in Sri Lankan traditional medicine, C. gigantea is used to treat scorpion poisoning (Ediriweera et al., 2018) and snake bites (Herath, 2017). Similarly, the root is used to treat dysentery (Gunaratna et al., 2015). Also, Sri Lankan farmers used latex of the plant as a sticky material in sticky traps. Alpha and Beta calotropeol and Beta amyrin in latex act as excellent compounds for crop pest control (Widanapathirana and Dassanayake, 2013). Especially sugarcane farmers use latex of the plant to control termite attacks on their crop fields (Wanasinghe et al., 2018). Furthermore, under laboratory conditions, extractions of C. gigantea are effective in controlling the cotton mealybugs (Prishanthini and Vinobaba, 2014). Sri Lankan Buddhists offer Calotropis flowers to Load Buddha and in Thailand, flowers are used to decorate temple ceremonies (Gaur, et al., 2013).
Insects associated with C. gigantea utilize the plant as a feeding substrate, a shelter, a hunting ground (Salau and Nasiru, 2015) as well as a breeding place. Phytophagous insects associated with C. gigantea play an important ecological role while they act as pests, predators as well as parasites (Saikia et al., 2015). The diversity of insect fauna associated with C. gigantea varies in different regions of the World. Although latex of C. procera is considered toxic to insects (Al dhafer et al., 2011) large numbers of insect pests cause considerable damage to the plant. Dhileepan (2014) explains that there are sixty-five phytophagous insect species associated with Calotropis spp. (including C. gigantea) in their native range. Among them, more than 50% of insects feed on leaves while others feed on flowers, stems, seeds, and fruits. Most of the insects associated with Calotropis species were recorded from India.
Aphids, grasshoppers, and caterpillars of Danaus spp. are the common plant feeders associated with C. procera (Al dhafer et al., 2011). Danaus spp. is a pest of Calotropis species in Australia, Hawaii, Fiji, Brazil, Jamaica, and Puerto Rico (Dhileepan, 2014). The gregarious feeding nature of Aphis nerii Boyer leads to defoliation and dieback of shoots and immature fruits of C. procera (Dhileepan, 2014). Most of the insects associated with C. procera are polyphagous except for Paramecops farinosa Schoenherr and Dacus persicus Hendel. They are highly destructive, monophagous pests (Dhileepan, 2014). Dacus persicus is distributed in India, Sri Lanka, Pakistan, Iran, and Iraq. Another fruit fly species, Dacus longistylus attacking C. procera fruits is widely distributed in Afrotropical regions. Paramecops farinosa is distributed in India and Pakistan with a less dispersal range than Dacus spp. (Dhileepan, 2014). Also, a study from India reveals that several other species of pests of C. gigantea cause considerable damage to the plant (Tara and Madhu, 2011). As the initial record, Platycorynus sp. was recorded on C. gigantea plants in West Bengal. Platycorynus sp. feeds on leaves and flowers of C. gigantea plants (Sudip et al., 2004).
Calotropis flower is a good nectar source for pollinators. The majority of insect pollinators of the plant belonged to the order Hymenoptera (Salau and Nasiru, 2015). A study in Israel found two carpenter bees, Xylocopa pubescens Spinola and X. sulcatipes Maa as major pollinators of C. procera. Carpenter bees are widely distributed in Asia and Africa (Eisikowitch, 1986;Zafar et al., 2018). In India, Apis dorsata Fabricius, Apis florae Fabricius, and Apis mellifera Linnaeus are active diurnal flower visitors of Calotropis spp. (Sudan, 2013). Dipterans of Family Muscidae, Sarcophagidae, and Syrphidae also visit C. procera flowers. Butterflies of the family Nymphalidae, Noctuidae, and Lycaenidae are occasional pollinators of C. procera (Sudan, 2013).
Diversity of predators associated with Calotropis spp. varies according to the different regions of the World. In the Central region of Saudi Arabia, 22 species of insects are reported as predators associated with C. procera (Al dhafer et al., 2011). Occasional visitors display a "neutral" relationship with the C. procera plant. They do not feed on the plant or prey on associated insects (Al dhafer et al., 2011). Further, the study of Al dhafer et al. (2011) explains that occasional visitors may feed around the plant without having a direct association with C. procera.
Although C. gigantea is widely distributed in Sri Lanka, no systematic surveys have been conducted so far. Therefore, the information on insects associate with C. gigantea in Sri Lanka is lacking and only a few records are available related to insect fauna of C. gigantea in Sri Lanka. Few species of bees including Amegilla comberi Cockerell, Amegilla fallax Smith, Amegilla violacea Lepeletie, Xylocopa fenestrata Fabricius and Xylocopa tenuiscapa Westwood were identified as pollinators of Calotropis in Sri Lanka (Karunaratne, et al., 2005). Also, butterfly larvae of Danaus chrysippus chrysippus were recorded as feeders on Calotropis leaves and flowers (Jayasinghe et al., 2013;Perera and Wickramasinghe, 2014).
There is no information on insects associated with C. gigantea in Sri Lanka except in the above studies. Therefore, the present study was conducted to fill the important gaps in the survey, catalog, and identification of insect fauna of C. gigantea in Sri Lanka.

Study sites
The study was conducted from December 2014 to June 2015, at a monthly interval, to identify the associated insect fauna of C. gigantea in Sri Lanka. Field visits were conducted covering 120 sites in nine provinces ( Figure 1). Sampling was done only once for each site. Roadside sampling sites were selected randomly at thirty minutes intervals while traveling on a vehicle with a speed of fifty kilometers per hour. If a new site with C. gigantea plants was not observed after thirty minutes, traveling was continued until a site with C. gigantea was found (Wijeweera et al., 2021). In all sampling sites, the distribution of C. gigantea (GPS coordinates) was recorded. At each site, associated insect fauna of C. gigantea was observed for thirty minutes. During the survey, insects associated with the plant were observed and photographed. The insects were collected directly from various parts of the plant i.e. leaves, flowers, flower buds, stems, and fruits by hand-picking. Two or three individuals of the same species in each site were collected into small plastic vials for morphological identification.

Identification of insects
The specimens were preserved, pinned, and lodged in the laboratory of the Department of Zoology at the University of Ruhuna, Sri Lanka. Specimens were identified up to genus/species level under the guidance of Entomologists of Entomology Division, National Plant Quarantine Service, Department of Agriculture, Gannoruwa. Unidentified specimens were sent to Mr. Justin Bartlett, Technical Officer (Taxonomy), Biosecurity Queensland, Department of Agriculture and Fisheries (DAF), Australia for identification.

Insect fauna of C. gigantea and their distribution in Sri Lanka
A total of 32 insect fauna (morphospecies) belonging to twenty-three families was observed on C. gigantea in Sri Lanka. Insects were categorized as phytophagous insects/ pests, pollinators/ flower visitors, and occasional visitors. High insect diversity was recorded in C. gigantea associated with the coastal belt of Sri Lanka. Also, well-established insect populations were observed in inland areas of the Southern, Northern, Eastern, and North-Central provinces of Sri Lanka.

Insect pests of C. gigantea in Sri Lanka
The majority of the insect fauna associated with C. gigantea in Sri Lanka were phytophagous species (Table 1).
The phytophagous insects associated with C. gigantea belonged to nine families, Chrysomelidae, Lygaeidae, Cercopidae, Membracidae, Tephritidae, Aphididae, Papilionidae, Curculionidae, and Cerambycidae, and feed on different plant parts including leaves, fruits, flowers, flower buds, fruits, seeds, and plant sap (Table 1). Spilostethuspanduru Militaris, Sphaeroderma sp., Graptostethus servus Fabricius, Spilostethus hospes Scopoli, and Aphis nerri Boyer de Fonscolombe were observed as gregarious feeders. Field observations revealed that all the pests caused minor damage to C. gigantea plants, except Phelipara moringae Aurivillius, Dacuspersicus Hendel, and Paramecops farinosa Schoenherr which are the major pests. Even though P. moringae is a destructive pest, they were less abundant within the country with respect to D. persicus and P. farinosa. Dacus persicus and P. farinosa were highly destructive pests as they damage fruits and seeds of C. gigantea plants influencing the reproductive output of the plant (Figure 2).

Pollinators and occasional visitors associated with Calotropis gigantea in Sri Lanka
The most common pollinators were Xylocopa fenestrate Fabricius, Xylocopa caerulea Fabricius and Apis cerana Fabricius. They consumed nectar as well as pollen of C. gigantea flowers while Spindasis lohita Horsfield, Danaus chrysippus Linnaeus, and Xylocopa spp. only feed on nectar (Figure 3).
Occasional visitors utilized C. gigantea plants as a shelter and hiding place. Coccinella sp. fed on aphids on C. gigantea plants. Different insect pollinators/ flower visitors and occasional visitors associated with C. gigantea are given in Table 2.

DISCUSSION
The insects collected from C. gigantea during this study in Sri Lanka are very similar to the insects reported on C. procera and C. gigantea from India (Pugalenthi and Livingstone, 1997;Chandra et al., 2011;Jana et al., 2012;Dhileepan 2014). A similar study had been conducted in Saudi Arabia related to C. procera and 99 insect species belonging to 43 families were identified (Al dhafer et al., 2011). According to a study in the Jabalpur district in India,  (Chandra et al., 2011), 8 species of insects from 6 families have been documented on C. procera. Similarly, a study in West Bengal in India reported 19 insect species from ten families of C. procera (Jana et al., 2012).
Thirteen phytophagous pest species belonging to nine families were recorded in Sri Lanka. In the study of Saudi Arabia (Al dhafer et al., 2011), three species; the carpenter moth, Semitocossus Johannes (Staudinger), scale insect Contigaspis zilla (Hall), and milkweed aphid Aphis nerii (Boyer de Fons-colombe) were pests of C. procera. Similarly, a study in India revealed eight species of pests associated with C. procera including A. chrysippus, S. pandurus, S. hospes, L. acuta, A. nerii, A. foveicollis, C. peregrinus, and C. sexmaculata (Chandra et al., 2011). Concerning both studies, the highest pest species richness was recorded in Calotropis in Sri Lanka.
Most pest species recorded in the present study were polyphagous feeders. Only P. farinosa (Aak weevil) and D. persicus (Aak fruit fly) were monophagous feeders (Dhileepan, 2014;Wijeweera et al., 2021). A similar observation was recorded in India (Dhileepan, 2014) and Pakistan (Sudan, 2013;Shabbir et al, 2019;Ali et al 2020; as well. Dacus persicus was found in 26 sites including coastal and inland regions of the country. Gravid females lay eggs inside C. gigantea fruits by penetrating the skin of the fruit with its ovipositor (Kumar and Kumar, 2015). The larval stage of D. persicus is a major destructive seed predator in Calotropis species (Sharma and Amriphale, 2008). Nourishment and development of larval stages of D. persicus are taken place within the fruit. Infected fruits rot and often drop prematurely. Pupation of this species occurs in the soil after the detachment of the fruit from the tree (Dhileepan, 2014). The damage is directly focused on the reproductive output of the plant which severely reduces the propagation and dispersal of the Calotropis species. Pakistan (Sudan, 2013), and Sri Lanka. The slow-moving nature of P. farinosa might limit their distribution into twenty inland sites and their relative frequency of occurrences was recorded as 16.67% (Table 1). Larval stages of P. farinosa feed and destroy fruits, and seeds while the adult weevil depends on the C. procera plant for feeding, sheltering, and oviposition. Paramecops farinosa feeds on leaves, flowers, and fruits (Sharma and Amriphale, 2007;Dhileepan, 2014). Field observations revealed that the adults prefer to feed on flower buds and tender leaves.

Paramecops farinosa naturally occurs in India,
Sphaeroderma sp. mostly occurred (67.5%) in many of the sampling sites (Table 1). It is considered a polyphagous pest feeding on leaves (Dhileepan, 2014). According to observations of the present study, it tends to aggregate as groups to feed on tender leaves. Damaged leaves appeared as a perforated mesh with emerging whitish latex. Later on, damaged leaves become brownish in color and dried out. According to a study in India (Saikia et al., 2015) both adult and larvae of Corynodes sp. are pests of C. gigantea plants. Corynodes beetles appear metallic-blue in color and they feed on leaves while larval stages act as stem borers.
Lygaeid bugs S. hospes, S. pandurus, and G. servus were the second most common (66.67%) polyphagous insect pests associated with C. gigantea plants (Table 1). Graptostethus servus Fabricius feeds on the seeds of Calotropis sp. Also, it is recorded in India, China, Pakistan, Indonesia, Turkey, Syria, and Australia (Hussain et al., 2014). The three species are seed predators of Calotropis plants (Dhileepan, 2014). Additionally, adult and nymphal stages of S. hospes feed on leaf sap of Calotropis sp. Nymphal stages inflict severe damage to the tender leaves by actively sucking leaf sap. Attacked leaves appear yellowish in color and dried (Saikia et al., 2015). These damages were observed in the present study too. Spilostethus hospes has also been recorded in Australia, China, Malaysia Archipelago, Pakistan, India, and New Caledonia while S. pandurus has been reported from Australia, India, and Pakistan (Chandra et al., 2015).
Abidma refula (Spittlebug) was recorded as the third most common insect pest of C. gigantea. It fed on plant sap and was recorded in 62 sites (51%) in Sri Lanka (Table 1). One species of the same genus was recorded in India which feeds on C. gigantea as well as C. procera (Dhileepan, 2014). Oxyrachis sp. (cow bug) was observed as a pest of C. gigantea in 57 sites. Oxyrachis sp. is widely distributed in South Asia, including in India, Sri Lanka, and Nepal. They occasionally feed on C. procera. Both nymphs and adults feed gregariously on the sap of immature stems, leaves, and flower buds (Sudan, 2013). The gregarious feeding nature of Oxyrachis sp. leads to the weakening of plants. They produce honeydew which leads to the attraction of ants to C. procera plants (Sudan, 2013) and a similar observation was recorded in the present study also.
Apart from pests and pollinators in C. gigantea, there was a great species richness in occasional visitors. Drosophila sp. was commonly observed under the shade of C. gigantea leaves in 33 sampling sites. Coccinella sp. visits C. gigantea plants to hunt small insects. They are predators of aphids and other small insects. Similarly, Brumoides suturalis Fabricius is a predatory beetle recorded in Pakistan, Bangladesh, China, Taiwan, and India that feeds on aphids and mealybugs associated with C. gigantea (Sudan, 2013).

CONCLUSIONS
The findings of the present study provide detailed records of insect fauna associated with the C. gigantea plant in Sri Lanka. Insects associated with Calotropis were cataloged as pests, pollinators, and occasional visitors. A total of thirtytwo morphospecies of insects belonging to twenty-three families were identified. Thirteen pests associated with C. gigantea were identified up to the genus/species level. Six species of pollinators and high diversity of occasional visitors belonged to five orders also documented.