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 confirmed 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 whitefly 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 specific life table parameters.
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.
To extract the DNA from a single whitefly, lysis method of DNA extraction was followed as detailed below (Zeidan and Czosnek, 1991). A petri dish lid was covered with aluminum foil and parafilm 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 ddH2O) was spotted on the surface of the lid covered with parafilm and single whitefly 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 whitefly 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 five seconds and supernatant was processed for PCR.
Mitochondrial Cytochrome Oxidase (Mtcoi) Subunit I Amplification:
The mtCOI gene sequence was analyzed for whitefly samples collected from six locations for genetic identification. Approximately 249 bp of the mtCOI gene fragment was amplified 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 94oC for 3 minutes, followed by 40 cycles each consists of denaturation for 30 seconds at 94oC, annealing for 40 seconds at 53°C with final extension for one minute at 72°C followed by final extension for 20 minutes at 72°C. The PCR products were gel purified and sequenced availing the commercial facility.
All mtCOI sequences of different group of whiteflies were downloaded from the National Center for Biotechnology Information (NCBI) GenBank. [https://www.ncbi.nlm.nih.gov/ Blast.cgi]. Sequence alignment was performed employing MUSCLE implemented in Seaview (Thomp-son et al. 1994). Genetic divergence was calculated employing MEGA 7 using ClustalW (Tamura et al. 2011). The mtCOI DNA sequences generated in this study were submitted to the NCBI database.
Life table parameters of A. rugioperculatus
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 magnifier (15x) on every day to observe the period of each instar from first 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 five freshly emerged males and females were collected with the help of aspirator and confined 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 specific-two sex life table software of TWOSEX-MS chart programme described by Chi (2018).