Natural history of Platypria (Platypria) hystrix (Fabricius, 1798) on Fabaceae host plants, with notes on other Platypria species in India (Chrysomelidae, Cassidinae, Hispini)

Abstract The leaf-beetle genus Platypria Guérin-Méneville, 1840 comprises two subgenera and 34 species (Chrysomelidae: Cassidinae: Hispini). Host plants are documented for eight species and indicate mostly perennial species of Fabaceae and Rhamnaceae. Larvae and pupae have been documented for two Platypria species. This paper presents novel natural history data, based on a field study of populations of Platypria (Platypria) hystrix (Fabricius, 1798) on Erythrinastricta Roxb. and Puerariaphaseoloides (Roxb.) Benth. in Kerala, south India and on Erythrinavariegata L., Puerariamontanavar.lobata (Willd.) Maes. & S. Almeida and Mucunapruriens (L) DC in Assam, northeast India. Three new Fabaceae hosts are reported for P. (P.) hystrix. Brief notes and new host records, based on field observations, are also provided for the other three species of Platypria in India – P. (P.) chiroptera Gestro, 1899, P. (P.) echidna Guérin-Méneville, 1840 and P. (P.) erinaceus (Fabricius, 1801). Platypria females slit the leaf to lay a single egg which is covered with secretions that harden as an ootheca, the egg covering in Cassidinaes. l. There are five larval stages, each with the typical ‘hispine’ mining form and behaviour – a flattened cream-coloured body, chitinised head capsule and claws, and feeding on mesophyll and leaving irregular blotch mines on the host leaves. Pupation occurs in an independent pupal mine and lasts about a week. These observations suggest new potential phylogenetic character hypotheses that can stimulate better data collection on leaf-mining Cassidinae and help resolve evolutionary patterns amongst these basal mining genera.

The genus Platypria Guérin-Méneville, 1840 comprises two subgenera (Platypria, Dichirispa) and 34 species (Staines 2015). The two subgenera are separated by the elytra margins expanded both at the humeri and posteriorly, with long spines and with "windows" in the nominotypical subgenus (fenestrate; Würmli 1975Würmli , 1978. This paper focuses on four species found in India (Fig. 1). Adults of Indian Platypria are morphologically distinct : the body is oblong, but the margins of the pronotum and elytra are expanded into broad rounded lobes and have prominent spinose extensions (Maulik 1919;Uhmann 1954b;Würmli 1975). The antenna has nine antennomeres, as the last three are apparently fused (Maulik 1919). Platypria is distributed across the Afrotropical and Oriental Regions. Hosts are known for eight of the 34 species in the genus (Table 1). Kalshoven (1957) noted that Platypria is amongst a few Oriental hispine genera atypically associated with eudicotyledonous plants, often belonging to unrelated families; other such genera are Notosacantha Chevrolat, 1837 (Rane et al. 2000), Oncocephala Agassiz 1846 (Calcetas et al. 2020), Dactylispa, Dicladispa, Hispa and Monohispa (Staines 2015).
We present the first natural history notes on P. (P.) hystrix from two widely-separated localities in India. This species is widespread in southeast Asia and is documented from 16 States in India -Assam, Bihar, Chhattisgarh, Goa, Gujarat, Himachal Pradesh,  (Maulik 1919;Basu 1999;Borowiec and Świętojańska 2007;Borowiec and Sekerka 2010;Staines 2015). At present, 20 host plants in six families have been recorded for this species (Table 1). We report observations of the species on four host plants, three being new records for this species. We also present brief biological notes for the other three Indian Platypria species based on field observations and provide a key to identify these four species in India. This paper is the first step in an ongoing process; a detailed comparative morphology study is our next goal.

Materials and methods
The study is based on independent observations by SR and KDP of live populations of P. (P.) hystrix at two sites in India, 2,500 km apart (Fig. 1). Authors SR, KDP and HVG started observations independently and now are collaborating; we pool data here in this phase 1 of a long-term study.   (Faizal et al. 2006). In 2019 and 2020, the beetles were found only on P. phaseoloides at Vellayani (Figs 14-21).

Rearing
We marked and numbered leaves with larval mines to observe their behaviour and development. In Assam, we followed 15 larvae and four successfully reached adulthood. In Kerala, about 20 larval and pupal mines were studied. Some specimens were taken to the lab to rear and collect certain life stages for vouchers, photography and measurements.
In addition to the detailed study of P. (P.) hystrix above, HVG, PKD and SR observed and collected the other three Indian Platypria species on Ziziphus and other hosts in India and provide these brief notes below.   Bhasin (1942) and Mathur and Singh (1961) recorded P. (P.) chiroptera (as Platypria garthwaitei Bhasin, 1942) on Ziziphus incurva (Rhamnaceae).

Taxonomic identifications
Erythrina stricta (Fabaceae) was identified by A. K. Pradeep, Calicut University Herbarium, previously for Faizal et al. (2006). No plant voucher was collected at that time as no flowers were produced under Vellayani conditions; now the plant has become locally extinct. Pueraria phaseoloides was identified by A. P. Balan, Malabar Botanical Garden. Erythrina variegata, P. montana var. lobata and M. pruriens (all Fabaceae) from Assam were identified by G. Krishna, Central National Herbarium (CAL), Botanical Survey of India. The beetles were identified independently by authors KDP and SR as P. (P.) hystrix using the species key by Maulik (1919) and compared with photos of type specimens deposited in Berlin Museum of Natural History and Kiel University, Germany. A key to identify the four Platypria species in India is developed.

Specimen collections and repository
Specimens collected by KDP and associates over years from various localities in India are deposited in the Travancore Insect Collection, Kerala Agricultural University, Vellayani (KAU). In addition to KAU, specimens of beetles will be deposited also at the National Bureau of Agricultural Insect Resources, Bengaluru, India (NBAIR). Specimens of P. (P.) echidna and P. (P.) erinaceus are deposited at the Modern College of Arts, Science and Commerce, Pune, India. Additionally, a specimen series of P. (P.) hystrix is on loan from KDP to CSC for further study. Vouchers of P. phaseoloides (Accession no. 7019), P. montana var. lobata (Accession no. 7030, 7031) and M. pruriens (Accession no. 7037, 7038) are deposited in the Calicut University Herbarium, Department of Botany, University of Calicut, Kerala.

Host plant ecology
The four Fabaceae hosts are native to southeast Asia. Each is used for multiple purposes in agro-ecosystems. Erythrina stricta is a spinose tree on which cultivated black pepper (Piper nigrum L.) is trailed. It is also grown as a hedge plant and shade tree. Leaves are used as fodder for sheep and rabbit (Prathapan, personal observations; Sastri 1952). Erythrina variegata (Fig. 6) is a tropical soft-wood tree, closely resembling E. stricta; however, its stem is usually unarmed. It is cultivated as an avenue tree and a live fence and it is used as a shade tree in plantations of tea and coffee and to trail betel vine and black pepper (Sastri 1952;Prathapan, personal observations). Pueraria phaseoloides (Figs 14-18; tropical kudzu) is a perennial climbing vine, trailing over trees, shrubs, bananas and grasses in and around the Instructional Farm of Kerala Agricultural University, Vellayani, India. It is grown as a cover crop in rubber plantations and for fodder (see Keung 2002). Pueraria montana var. lobata, known for rapid and competitive growth, is used as a pasture, fodder and hay crop in North America (Lindgren et al. 2013). Mucuna pruriens is used for its medicinal properties and as fodder (Choudhary et al. 2012;Patiri and Borah 2007). These five hosts have moderately large, trifoliate leaves.
The genus Ziziphus Mill. includes about 58 species of spiny shrubs and trees (El Maaiden et al. 2020). It is extensively used in folk and traditional medicine in arid and semi-arid regions for the treatment of diarrhoea, dysentery, cholera, diabetes, hypertension, inflammation, intestinal spasm, liver, malaria and other diseases (El Maaiden et al. 2020). Ziziphus mauritiana Lam., called Indian jujube or ber, is a tropical shrub or small tree, of considerable commercial importance and is widely cultivated for its fruits. Ziziphus rugosa Lam., called wild jujube, is a thorny tree or straggling shrub, common in foothills and low mountains in India (Chadha 1976). Fruits are collected from the wild for consumption. Ziziphus xylopyrus (Retz.) Willd., locally called 'kath ber', is an erect shrub or small tree, common in dry and deciduous forests (Chadha 1976). Ziziphus nummularia (Burm.f.) Wight & Arn., occurring in semi-arid areas from Iran to the Indian subcontinent, is a multipurpose branched thorny shrub reaching a height of 1-3 m, with medicinal, nutritional, industrial and economic values (Zandifar et al. 2020).

Photographs
Specimens were colour-photographed using a AF Micro Nikkor 60 mm macrolens, mounted on a Nikon D3000 SLR camera. The camera was mounted on a Wemacro stack rail, positioned vertically. Three Ikea 201.696.58 Jansjo Desk Work LED Lamps, with suitable diffusers, were used to uniformly illuminate the specimen. A Wemacro rail android Bluetooth control app, installed on a smartphone, was used to remotely control the imaging system. Multiple images at different depths of plane were taken and were stacked together using Helicon focus software. The high-resolution images, thus obtained, were edited with Adobe Photoshop 2020. Field photographs were taken using a Canon EFS 55-250 mm lens mounted on a Canon EOS 1300D SLR camera or Micro Nikkor 60 mm macrolens mounted on a Nikon D3000 SLR camera.

Measurements
Life stages of P. (P.) hystrix were measured using a standardised ocular micrometer placed in one eyepiece of a stereoscopic microscope. Measurements of host plant leaves and leaf mines were taken using a Vernier caliper. In our Assam lab, we measured three adults, one instar I, one instar III, one instar V, one pupa and one pupal mine. In our Kerala lab, we measured 10 adults, 20 pupal mines, 10 pupae, and seven oothecae.

Taxonomy
We use the current plant names according to the online catalogue (Tropicos 2020) and current beetle names according to the catalogue of Staines (2015).

1
Antenna thick, hardly extending beyond scutellum over pronotum; third antennomere not longer than 2.5 times width ..... P. (P.) erinaceus (F.) (Fig. 4 (Fig. 9). Eggs were observed on E. variegata in Assam as well as on P. phaseoloides in Kerala. They were laid singly on the adaxial side of leaves. Up to four eggs were observed on a single leaflet. Individual eggs were inserted into a depression made on the mesophyll and were covered with a creamy-brown secretion to form the oblong-oval ootheca, that measured 1.03-1.32 mm (1.10 mm -mean of 7 observations) long and 1.07-1.48 times (1.24 times -mean of 7 observations) longer than wide. A characteristic, long, thread-like process, arising from the middle of the ootheca, enabled easy identification of the ootheca under low power of the microscope. From the abaxial side, the ootheca appeared like a minute, brown speck. The egg appeared soft and was easily ruptured when we attempted to separate it from the oothecal covering. The thread-like process and the outer wall of the ootheca remained intact even after hatching and formation of the leaf mine. In Assam, a female was observed on the host plant for ten days; oviposition and egg hatch were noted. Twenty-one leaflets were observed, each with about 3-4 beetle eggs. These eggs hatched in about 4-7 days. Many eggs remained unhatched or the larvae died prematurely.
The larva hatched out of the egg mines into the adjacent mesophyll without breaking the oothecal covering. It feeds and moves within the leaf creating mines by consuming mesophyll tissue. The first instar larva grew up to 1.8 mm. The larva has chitinous brown head and translucent-greenish body. The alimentary canal appears dark green due to the presence of food. While observing it against sunlight, the mines appeared occupied and small larvae were apparent through the epidermis. The larval mine in P. phaseoloides appeared less apparent in the abaxial view (Fig. 17), but clear and rather transparent from the adaxial side of the leaf lamina (Fig. 18). The larval mines are irregular blotch mines. Six leaf mines were observed on a 65 mm wide leaflet of P. phaseoloides (Figs 17-18) at Vellayani. The mines contained excreta, exuviae and often remnants of dead larvae. The leaf mines of the late instars were noticeable as some of them were approximately 1 cm wide and 10 cm long and irregularly shaped. The final instar was about 5 mm long.

Pupation
The pupation takes place in a separate pupal mine. Emergence from the larval mine and construction of the pupal mine were observed in Assam. The mature larva (Fig. 13) exits the larval mine, moves towards the other leaf end and initiates the pupal mine. Construction of the pupal mine by a single larva that was observed took 23 minutes to conceal itself. Four pupal mines were observed in Assam on E. variegata. The average size of the pupal mine was 9 × 4 mm (n = 4). Excreta was present next to every pupal mine's single opening. The pupal period in Assam lasted for about seven days. On four occasions, the pupa was observed moving out of the mine and adults emerged in early morning.
About 20 pupal mines were observed on P. phaseoloides at Vellayani. The length of pupal mines ranged from 7.5-10.1 mm (9.98 mm; mean of 10 observations) and width 3.5-4.5 mm (4.01; mean of 10 observations). All, except two, were formed along a leaf vein. Two were formed between the veins on the leaf lamina. The pupal mines are U-shaped, resembling a pocket, with its distal end closed and the proximal end, from where the larva initiated the mine, remaining open. The resident pupa has the head orientated to the closed end and its rear end towards the mine opening. In Assam, we observed that a pupating larva spent one day in the pupal mine, then cast the last larval skin and pupated. This individual took 9 days from formation of the pupal mine to adult emergence. Generally, 1-2 pupal mines were observed on a single leaflet on both E. variegata in Assam and P. phaseoloides in south India (Vellayani, Kerala). The fresh pupa is yellow in colour that turns coffee-brown in a few days. Prior to the emergence of the adult, the pupa exited the mine and shed the exuviae. In the case of the single individual observed by PKD in the laboratory, the exuviae of the pupa remained about 3 cm away from the pupal mine. Thus, the pupa can move out of the mine to eclose.
The adults (n = 10; length 4.29-5.24 mm) were observed feeding mainly by scraping on adaxial surface of leaves. Sexual dimorphism was not distinct to the naked eye. Copulation was recorded in the morning as well as in the evening. Pairs were in copula for more than an hour.

Dormancy and aestivation
In Assam, the adults were seen until the first week of December 2019, after which they were not found anymore. They appeared on the same plant in the first week of March 2020. Further south, at Vellayani in Kerala, the population of P. (P.) hystrix on P. phaseoloides was active throughout the year, as adults and leaf mines were observed even during the summer months of March and April. Apparently, no dormant stage of the insect occurs in Kerala as extremes of climate are absent in this part of the country.

Longevity
Although our observations are still in progress, we noticed that adults emerging in September 2019 in Assam were active, with mating and egg laying observed during March 2020. We suspect that the adults survive for at least one year.

Mating behaviour
Copulation was observed in the third week of March after several thunder showers in Assam. On 23 March, we noted four pairs on E. variegata. In the case of two pairs, a single female was pursued by two males. The male mounted the female, keeping fore-and middle legs on the elytra of the female, the hind legs being on the substratum. The pair remained coupled for more than one hour per observation. On a few occasions, coupled pairs were observed for 4-6 hours. The female moved around, carrying the male and even fed while in copula. During a single sighting, we found a maximum of eight beetles on a single sapling of Erythrina at Assam, indicating that it is not a major pest.

Natural enemies
At the Vellayani site, we observed a Braconidae wasp (Hymenoptera) parasitising a mature larva of P. (P.) hystrix (Fig. 11) and ant (Hymenoptera: Formicidae) predation of a pupa. Both the wasp and ant specimens are deposited at KAU. In the Pune locality, we observed a chalcid wasp (Hymenoptera) laying its egg on a late larval instar on 27 May 1999 and subsequently, we detected a chalcid infestation of the larval and pupal stages of P. (P.) erinaceus. Bernon and Graves (1979) is the only other report of Hymenoptera parasites of Platypria; they noted that Platypria was an alternative host of the Hymenoptera parasites of the Coelaenomenodera pest. (2004) has discussed many aspects of leaf mining by Chrysomelidae and Chaboo (2007: 46-47) provides an overview of Cassidinae pupation. We discuss here aspects of the biology and behaviour of Platypria species and compare with the other members of the tribe and Cassidinae s. l. generally. We discuss refinements for the current morphology and behaviour-based phylogenetic characters of Chaboo (2007).

Plant relations
Platypria is associated mainly with two plant families, Fabaceae and Rhamnaceae (Table 1). We found several citations in Indian literature about the genus that should be added to the online catalogue of Staines (2015). Records on other plants -Fagaceae [Nair (1986), Euphorbiaceae (Hua 2002) and Poaceae (Anand 1989;Maulik 1919Maulik , 1937] -need confirmation as there is little information on immatures from these observations. We can call only those plants as 'hosts' where larval development occurs successfully. In that sense, P. (P.) echidna may sometimes feed on Z. nummularia, but we have never observed larvae or pupa of this species on Z. nummularia. Similarly, we never observed larvae/pupae of any Platypria species on Z. xylopyrus (Retz.) Willd. which we [HVG and SR] regularly visited to study bionomics of another cassidine. Further, although Z. oenoplia (L.) Mill. is reported as a host of one Platypria species (see Table 1), HVG never observed Z. oenoplia in Pune harbouring any Platypria. Kalshoven (1957) noted that Platypria is one amongst a few unusual Oriental hispine genera associated with dicotyledonous plants, often belonging to different families. He also commented that Platypria is unusual as it is one of the few hispine taxa specific to dicots and exhibits trophic selections between unrelated host plant families.

Life cycle
All life stages of P. (P.) hystrix (egg to adults) were observed in both south and northeast India. The natural history of the populations observed in Assam, northeast India and in Kerala, south India were rather identical, irrespective of the host species, though the populations are separated by a distance of > 2,500 km and climates are distinct. The south Indian population at Vellayani was active throughout the year as harsh winter or summer is absent here, while the northeast Indian population vanished as the winter peaked and re-appeared only after receipt of rains in summer, thus disappearing for at least three months from December to March.
Information is limited on the eggs and associated maternal behaviour for leafmining hispines. In P. (P.) hystrix, we observed females excavating a depression on the abaxial surface of leaves and laying a single egg there. Then she covered the egg with a yellow secretion that turned red brown on drying and formed a crusty oothecal covering. Thrusting single eggs into the leaf lamina is known in some leaf-mining hispines (Chen 1982;Chaboo et al. 2010;Shameem et al. 2016;Liao et al. 2018b), although Taylor (1937) noted that females of Promecotheca species may oviposit on the leaf surface or sink the egg into the leaf and the natal larva starts the mine. In Prionispa champaka Maulik, 1919 (Oncocephalini), the female oviposits 5-6 eggs into a channel she cuts on the leaf (Liao et al. 2018a). Chaboo (2007: 244) proposed two egg features (egg stalk and faecal cover) for phylogeny reconstruction; our data here suggest at least three new potential character hypotheses about the oviposition site (externally on leaf surface or thrust into the leaf tissue), egg grouping (single or massed) and maternal covering (naked with no covering, oothecal secretion, faecal/plant covering or oothecal secretion + faecal/plant covering). Verma and Kalaichelvan (2004) reported observations on oothecal structures in Indian Cassidinae; however, our observations of such secretions in Platypria indicate the behaviour of maternal coverings is more widespread across the cassidine tree of life. It is very important to document such information in fine detail to achieve better resolved phylogenies of Cassidinae s. l.
We observed all larvae of the four Indian species of Platypria making a blotch mine, as in some other mining Cassidinae (Bernon and Graves 1979;Chen 1982;Lee et al. 2009;Liao et al. 2014Liao et al. , 2018a. Figs 17-18 show six mines in one leaf; however, we are uncertain how many larvae can be sustained by the single leaflet to reach pupation and adulthood. We observed a single larva per mine, agreeing with observations in Javeta pallida Baly, 1858(Shameem et al. 2016 and Chaeridiona thailandica Kimoto, 1998 (Świętojańska andKovac 2007). This contrasts with those mining species whose larvae live gregariously in a common mine (e.g. Pr. champaka, Liao et al. 2018a).
The structure of the pupal mine appeared very similar in our observed Platypria species. In C. thailandica (Oncocephalini), the mature larva exits the larval mine, bores into the mid-rib forming a pupal chamber and then pupates with the head orientated towards the stem of the plant (Świętojańska and Kovac 2007). Members of Hispini, Notosacanthini and Oncocephalini, that live on eudicots, create more or less similar pupal mines. Chaboo (2007: 244) proposed Character 18 with four states for different pupation sites across Cassidinae s. l. Our new observations here suggest that the origin of the pupation mine can provide an additional character hypothesis with two states -within a larval mine or a separate mine.
The pupal mines of P. (P.) hystrix are U-shaped and the resident pupa is positioned such that its rear end is orientated to the single opening at the wider end. This facilitates respiration with the erect, tubular spiracles. Even in rains when the pupal mine may become flooded, the pupa can be seen projecting spiracles out of the opening; the pupa is motile and not glued like other Cassidinae. Similar pupal mines have been reported for P. (P.) echidna and P. (P.) erinaceus and some other basal Cassidinae, such as Chaeridiona picea Baly (personal observations SR; Oncocephalini), Notoscantha (Rane et al. 2000;Notosacanthini), and Oncocephala tuberculata Olivier,1792 (Oncocephalini). Notosacanthini is one of the historic transitional tribes between crown-clade Cassidinae, based on adult morphology and basal "hispines" (Chaboo 2007). The similarity of its pupal chamber to that of Platypria and Oncocephalini underscores the need for re-assessment of its taxonomic placement. Chatterjee and Bhasin (1936) and Kalshoven (1957) reported Platypria adults as exhibiting swarming behaviour on Rubus ellipticus Sm. (Rosaceae) in India. We did not observe such behaviour. Swarming has been reported for only one other Cassidinae, Caelaenomenodera elaeidis Maulik (Bernon and Graves 1979), where this behaviour appears to be cyclical. It could provide another set of phylogenetically informative characters.
Platypria females attract many males in a mating frenzy. Once a male is chosen, copulation lasts several hours. (See our supplementary movie file on the life cycle of Platypria in India).

Conclusions
This paper provides a first step in ongoing fieldwork and study of the four Indian species of Platypria. We discovered new hosts and note the specialisation of these species on Fabaceae and Rhamnaceae. We characterise aspects of the oviposition behaviour, egg, larvae, pupae, mining behaviour and adult courtship. A detailed morphological study is our next goal. and Ruie Nie, Chinese Academy of Sciences, Beijing, China for translating Chinese texts, Michael Schmitt, University of Greifswald for translating sections of Uhmann (1957Uhmann ( , 1958 and Hugh Rowell for a complete translation of Würmli (1975). We thank Rashmi Kant Shukla and Subodh Kr. Bajpai, the National Forest Library and Information Centre, Dehra Dun for Chatterjee and Bhasin (1936) and Singh (1959, 1961). SR acknowledges Vibhu Prakash for his encouragement and the Bombay Natural History Society for support. We also thank Charles Staines for sending several references. KDP thanks Koormath Mohammed Shameem for collaboration and camaraderie during fieldwork; SR and HVG also thank Neelesh Sunil Rane for collaboration during fieldwork. We thank Michael Geiser and Dmitry Telnov, British Museum of Natural History, London, United Kingdom, for a loan of specimens and Bernd Jaeger, Museum für Naturkunde, Berlin, Germany, for photographs of a lectotype and specimen information. Sangamesh Hiremath prepared the map of India. Chaboo thanks the U.S. National Science Foundation for Eager Grant #1663680 that supported her effort. HVG thanks the authorities of Modern College for access to facilities. We thank the three anonymous reviewers and editor Michael Schmitt for comments that improved the final manuscript. Finally, we all thank our respective institutions for support during this work.