Morphological, Molecular Characterization, Life Cycle And Age Specic Life Table Parameters of Coconut Rugose Spiralling Whitey, Aleurodicus Rugioperculatus Martin: An Exotic Pest of India

The present study aimed at reporting the incidence of exotic pest on Coconut rugose spiralling whitey, Aleurodicus rugioperculatus Martin in Tamil Nadu, India. The taxonomical, morphometric, molecular characterization, life cycle and age specic life table parameters of the exotic pest were studied on coconut during 2017-19. The results revealed that rugose spiralling whitey (RSW) is an exotic and polyphagous in nature and had total life period of 56.33± 1.01 days which includes 6.9±0.88, 19.57±2.17, 10.9±0.78 and 26.00±2.34 days of egg, nymphal, pupal and adult period, respectively. The rugose spiralling whitey male adult was 2.6300± 0.0596 mm in length and 2.1220± 0.0576 mm width and female adult were 3.1460± 0.0329 mm in length and 2.1654± 0.0246 mm width. A. rugioperculatus was bigger than other previously reported whiteies in India. The operculum was wrinkled and having compound pores with dagger shaped axial pores. Ligula of A. rugioperculatus extended beyond vasiform orice. The identity of the species was further conrmed through amplication of mitochondrial COI gene 249 bp was obtained for all the isolates which were sequenced. The sequenced DNA product was submitted to NCBI- Gen Bank (Accession No. MT540964, MT679537, MT679538, MT679539, MT679540 and MT682638). Since, it is a rst study on the age specic life table parameters of A. rugioperculatus and the results revealed that the net reproductive rates (Ro) 24.08 females/female/generation, intrinsic rate of increase 0.0646/day, nite rate of Increase (λ) 1.066/ day, mean fecundity (F) 41.28 eggs/female and mean generation time (T) 49.25 days, respectively


Introduction
Coconut (Cocos nucifera L) is one of the most important crops in tropical areas and referred as 'tree of heaven' or 'kalpavriksha' because it provides more useful and diverse products to the people's utility. In India, Kerala, Tamil Nadu, Goa, Karnataka, Maharashtra, Orissa, West Bengal and Assam are the major coconut producing states. India consumes 50% of annual production for their culinary and religious purpose, 35% used as copra, 2% for manufacturing of value added products, 11% for tender uses and 2% for seed purpose (Nadanasabapathy and Kumar, 2013). In Tamil Nadu, the coconut is cultivated in various districts viz., Coimbatore, Tiruppur, Erode, Theni, Pudukkottai and Kanyakumari. Tamil Nadu stands third position in India having 461.06 thousand hectares of cultivable land with production of 6570.63 million nuts. More than 900 species of pests are associated with coconut palm. This includes both invertebrates and vertebrates (Kumara et al., 2015). Rugose spiralling white y (RSW) is an exotic pest of coconut and rst reported by Shanas et al. (2016) from Kerala, India. Further, it has been reported from peninsular India viz., Tamil Nadu, Karnataka and Andhra Pradesh (Sundararaj and Selvaraj, 2017). RSW is a polyphagous which is likely to expand the host range and mainly infests the coconut palms and other broad-leaved hosts in its native range . RSW feeding causes stress to the host plant by removing water and nutrients and secrete honeydew, which favors the growth of sooty mold on the lower leaves which reduces the photosynthesis (Taravati and Mannion, 2016). Presently it is spread all over Tamil Nadu and southern states of India and the farmers were facing hard ships due to this infestation of RSW in coconut ecosystem. Any change in population structure or unexpected dynamics may lead high damage and yield losses of coconut. As there are nil systematic studies and research on the life table parameters of A. rugioperculatus are scanty in India. .Therefore, the aim of this study on the morphology, molecular characters, life cycle and construct the life table of A. rugioperculatus fed on coconut to determine the survival rate and rate of increase of this exotic pest for effective pest management in India.

Materials And Methods
Insect sample collection Surveys were conducted in different agro ecological zones of Tamil Nadu were coconut growing districts of Tamil Nadu viz., Thondamuthur (Coimbatore), Udumalpet (Tiruppur), Chennimalai (Erode), Periyakulam (Theni), Veppangudi (Pudukkottai) and Thatkalai Kanyakumari) during August 2017 to February 2019. As the occurrence of RSW was con rmed with the typical characteristic like eggs on the underside of leaves in a concentric circular or spiral pattern and cover it with white waxy matter on leaves and the different stages of white y viz., eggs, nymphs, pupa and adult were collected from coconut. Adult insects were collected using aspirator and other stages were collected with help of camel brush. The collected RSW samples was kept in 70% alcohol and brought to the bio control laboratory, Department of Entomology, Tamil Nadu Agricultural University, Coimbatore for detailed study about morphological, molecular characterization and age speci c life table parameters.

Taxonomic Characterization
Field collected samples of A .rugioperculatus (pupal stage) were mounted on slides as per the protocol of Martin (2004), maceration of body contents is carried out by warming to around 80°C in a 10 % Potassium Hydroxide solution for 5-10 minutes or longer, until visible contents have become translucent.
A small puncture may be made in the ventral surface of each specimen in order to speed up processes, and to help prevent osmotic collapse. De-waxing of the cuticle was carried out by gently warming the specimens in carbol-xylol (Xylene with 10% dissolved Phenol). Pale puparia staining was be carried out by adding an excess of Glacial Acetic Acid and a few drops of Acid fuchsin stain solution. Di staining was done using 95% Ethanol. Finally dehydration of specimens was carried out by soaking in absolute Ethanol for a few minutes. Clove Oil was dropped on the specimen and placed on slide for observed under microscope.

Morphometrics of Aleurodicus rugioperculatus
The morphometric studies were made on life stages of A. rugioperculatus. The sample drawn from the survey collections were used for the study. Measurements on eggs, nymphal instars (I to IV), pseudo puparium and adults were made using phase contrast research microscope at 40x for studying the essential characters. Adult characters (male and female) studied include wing span, Length and width of the antenna, measurement on left and right claspers of male. Measurements and photographs were taken in Leica image analyser (Leica M205C) at biosystematics laboratory, Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore.

Dna Isolation
To extract the DNA from a single white y, lysis method of DNA extraction was followed as detailed below (Zeidan and Czosnek, 1991). A petri dish lid was covered with aluminum foil and para lm one on another.
Then 5µl of lysis buffer (5µl of Tris Hcl, 1µl of EDTA, 5µl of (Triton X 100), 50µl of proteinase K 20 mg/ ml and 939µl of ddH 2 O) was spotted on the surface of the lid covered with para lm and single white y was placed on the buffer spot using brush and crushed with the edge of PCR tube. After crushing, the entire content was transferred into fresh PCR collection tube. In addition to this, edge of the PCR tube which was used to crush the white y was washed with 35µl of lysis buffer. The entire content was kept in ice for 5 minutes and incubated at 65ºC for 15 minutes followed by 95ºC for 10 minutes and placed immediately on ice. The contents were spun for ve seconds and supernatant was processed for PCR.

Mitochondrial Cytochrome Oxidase (Mtcoi) Subunit I Ampli cation:
The mtCOI gene sequence was analyzed for white y samples collected from six locations for genetic identi cation. Approximately 249 bp of the mtCOI gene fragment was ampli ed using forward primer (1852) 5' GGGGGAACTGGTTGAACAAT 3' and reverse primer (2100) 5' AAAAGTTCTATTAAAATTTCGATCT3' (Dickey et al.,2015). The 25µl reaction volume containing 12.5 µl of 2 X smART master mix, Cat. No. 280311 (readymade mix of taq polymerase, dNTPs and PCR buffer), 5µl of template DNA, (approximately 50 ng) 3.5µl of sterile distilled water and 2µl of each forward and reverse primer (15pg each). The PCR was performed with initial denaturation at 94 o C for 3 minutes, followed by 40 cycles each consists of denaturation for 30 seconds at 94 o C, annealing for 40 seconds at 53°C with nal extension for one minute at 72°C followed by nal extension for 20 minutes at 72°C. The PCR products were gel puri ed and sequenced availing the commercial facility.

Sequence Analysis
All mtCOI sequences of different group of white ies were downloaded from the National Center for Life table of A. rugioperculatus was studied on dwarf coconut trees (Chowghat orange variety) at 25 to 30°C with relative humidity of 70 to 85 per cent at coconut farm, Tamil Nadu Agricultural University(11.01° N, 76.93° E). The leaves with egg spirals were collected and kept in a plastic container for the emergence of nymphs from the eggs. The leaves were examined every 24 hours interval for the nymphal emergence and assessing the incubation period of eggs. After the emergence, the nymphs were examined under hand magni er (15x) on every day to observe the period of each instar from rst to fourth instar nymphs and the total developmental time was recorded. The fourth instar nymph of RSW was considering as pseudo puparial stage and it was covered with small leaf clip-cage (10 X 10 X 5 cm) to trap the emerging RSW adults from pseudo puparium and ve freshly emerged males and females were collected with the help of aspirator and con ned within in a small leaf clip-cage. The caged adults were observed daily for survival rate, mortality and fecundity. The period between the release of adults and the adult mortality was recorded as the adult longevity. Life stage parameters of A. rugioperculatus from egg to adults were collected from thirty individuals. The data of all thirty individuals of A. rugioperculatus were pooled and analyzed through age speci c-two sex life table software of TWOSEX-MS chart programme described by Chi (2018).

Results
Life cycle and morphometrics of A. rugioperculatus Eggs are smooth, elliptical, whitish to yellow, translucent and laid mainly on under surface of the leaves of coconut in characteristic spiral manner covered with waxy coating. Eggs were 0.3426 ± 0.0028 mm length and 0.1720 ± 0.0034 mm width in characteristic spiral manner covered with waxy coating. There were four distinct nymphal instars. The rst instar nymphs were translucent, yellowish green, elliptical with a convex dorsum, functional walking legs and antennae. The rst instar nymphs were 0.4098 ± 0.0068 mm in length and 0.2556 ± 0.0450 mm in width. First instar nymphal period was 5.80 ± 0.78 days (Fig. 2). The second instar nymphs were oval, translucent and had many marginal fringes of wax covering the body of dorsum. The second instar nymphs were 0.9540 ± 0.0340 mm in length and 0.7300 ± 0.029 mm in width. Puparium is oval shaped, size 1.5940 ± 0.0385 mm. Operculum was wrinkled and having compound pores with dagger shaped axial pores. Ligula extended beyond vasiform ori ce and elongate, triangular at proximal end almost sharply pointed (Fig. 1). Pupa period was 10.9 ± 0.78 days (Table.2). Adults emerged through a 'T' shape exit slit on the dorsal surface of the pupae. The wings of newly emerged adults were clear after unfurling, later was covered with a coat of white waxy powder.
Eyes dark reddish brown, forewings with three irregular brown bands on the upper side near costal and hind margin and one towards ternal margin. Male adult with 2.6300 ± 0.0596 mm length and 2.1220 ± 0.0576 mm width and having longevity 23.00 ± 3.48 days. Female adult was 3.1460 ± 0.0329 mm length and 2.1654 ± 0.0246 mm width and having longevity 26.00 ± 2.34 days, respectively (Table.1).
Molecular con rmation and phylogenetic tree analysis of rugose spiralling white y Genomic DNA was isolated from individual adults of A. rugioperculatus, which were subjected to PCR analysis for con rmation of rugose spiralling white y using speci c primers (Dickey et al., 2015). The expected amplicon size was found to be 249 bp in all the samples were collected from six locations in Tamil Nadu (Fig. 3). The nucleotides were aligned and edited using Bioedit software. After aligning the sequences, these were subjected to BLAST analysis and compared with previously deposited sequences of A. rugioperculatus in the NCBI database. Based on the nucleotide sequence analysis of A. rugioperculatus isolates (CO1, TI1, ER1, PK1 and TH1) shared 99 per cent similarity with previously reported isolate (MK883218). Whereas, KK1 isolate of A. rugioperculatus exhibit 97 per cent similarity with A. rugioperculatus (MK883215). The nucleotides sequences were deposited in the NCBI database (Accession No. MT540964, MT679537, MT679538, MT679539, MT679540 and MT682638) ( Table.3). Our study isolates also share 99 per cent identity with KY209909 and KP032219 isolates of A. rugioperculatus. A phylogenetic tree was constructed on the basis of mtCOI nucleotide sequence (Fig. 4.) clearly showed that the populations CO1, TI1, ER1, PK1, and TH1 were clustered with A. rugioperculatus group. Though the population KK1 belonged to A. rugioperculatus group, it branched off from A. rugioperculatus due to some substitutions in nucleotide sequence. The phylogenetic tree was divided three main branches; in that rst main branch was sub divided into two clades and each clade contain single group of white y population from different countries were placed together viz., Paraleyrodes bondari and Aleurothrixus occosus, whereas the second main branch contain Paraleyrodes minei population. Third main branch was divided into two sub clades which contain A. dispersus and A. rugioperculatus populations were well separated and clustered together.

Life table parameters of A. rugioperculatus
Age-speci c survival rate (lx) and fecundity (mx) of A. rugioperculatus shown in Fig. 5. Survivorship curves shown in the gure were based on the mortality rate from egg to adult stage of A. rugioperculatus. The survivorship study of A. rugioperculatus revealed that most of the mortality happened at later instar of life cycle and less mortality in the earlier life stages of an organism. The life table parameters of A. rugioperculatus revealed that the net reproductive rates (Ro) 24.08 females/female/generation, intrinsic rate of increase ® 0.0646/day, nite rate of Increase (λ) 1.066/ day, mean fecundity (F) 41.28 eggs/female and mean generation time (T) 49.25 days, respectively (Table.4).

Discussion
Rugose spiralling white y, A. rugioperculatus had total life period of 56.33 ± 1.01 days which includes 6.9 ± 0.88, 19.57 ± 2.17, 10.9 ± 0.78 and 26.00 ± 2.34 days of egg, nymphal, pupal and adult period, respectively. Similarly, Kumar et al. (2013) also documented that RSW females lay eggs on underside of the leaves in concentric circular or spiral pattern covered with waxy matter and also reported eggs are elliptical and creamy yellow in colour. Egg period of A. rugioperculatus was 6.90 ± 0.88 days. Boughton et al. (2015) also reported that the egg period of RSW was 8.3 ± 0.1 days on bird of paradise plants. Mannion (2010) documented that A. rugioperculatus crawlers molt into immature stages that are immobile, oval and at initially but become more convex with progression of its life cycle. Stocks and Hodges (2012) reported that the nymphs are light to golden yellow in colour and nymphs will produce a dense cottony wax as well as long, thin waxy laments reported by Boughton et al. (2015) stated that life durations of rst instar, second instar ,third instar and fourth instar nymphs of A. rugioperculatus were 6.7 ± 1.2 days, 3.8 ± 0.6 days, 5.5 ± 0.8 days and 8.2 ± 0.2 days respectively. The same observations were also reported by Stocks and Hodges (2012)       Phylogenetic dendrogram based on mtCOI partial nucleotide sequences of Aleurodicus rugiosperculatus Phylogenetic tree generated from aligned partial mtCOI nucleotide sequences of Aleurodicus rugiosperculatus genotypes with other selected white ies. Tree was generated by neighbour joining method by aligning the sequences in MEGA 7 using ClustalW. Vertical branches are arbitrary; horizontal branches are proportional to calculated mutation distances; values at nodes indicate percentage boot straps values (1000 replicates).