Collection and identification of E. scitula
Predatory larvae of E. scitula feeding on colonies of C. cirripediformis infesting C. diurnum (Fig. 1) were collected from the CSIR-CIMAP Research Farm, Lucknow, Uttar Pradesh, India (26°53′38″ N; 80°58′50″ E, elevation:120 MSL) during June 2021. Five barnacle scale infested shoots (25 cm long) of C. diurnum were collected from 20 plants and placed in bags individually, brought to the laboratory and placed in the insect-rearing cages (30 × 30 × 30 cm3) and kept under the laboratory under controlled conditions (25 ± 5 °C; 65 ± 5% RH and 12L:12D photoperiod) for further observations. After adult eclosion, the adult moths were dissected and the predator was identified based on male genitalia at the National Pusa Collection, Division of Entomology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi), and the identity was also confirmed using sequences of COI gene of mitochondrial DNA.
For molecular identification, E. scitula last larval instar was collected from the field and reared in insect-rearing cages (dimension: 90 × 40 mm, shape: circular, mesh pore size: 0.053 µm, Hi-Media®, India) under above-described laboratory conditions until adult emergence which were utilized for DNA extraction and identification.
DNA extraction and amplification, sequencing and alignment
Insect DNA was extracted, following standard protocols of Dey et al. (2021). A fragment of the mitochondrial gene cytochrome oxidase subunit I (COI) was amplified by polymerase chain reaction (PCR), using 2 × PCR master mix (Thermo Fisher Scientific, USA), using primers, LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′ TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). The following thermal cycle parameters for 25μL amplification reaction: initial denaturation step of 94 °C for 1 min, followed by 4 cycles of 94 °C, 30 s; annealing at 45 °C for 90 s, 72 °C for 1 min; followed by 35 cycles of 94 °C, 30 s; 51 °C for 90 s, 72 °C for 1 min and the final extension at 72 °C for 5 min were used. PCR product was tested by electrophoresis on an agar gel, and if a single band was observed, it was purified, using a SureExtract PCR purification kit (Genetix Biotech Asia Pvt. Ltd.) and subjected for sequencing.
Sequence analysis and phylogenetic tree construction
PCR product was sequenced by the Sanger method; sequencing was performed bidirectionally by the automated sequencer (ABI Prism 3130 XL DNA Analyzer, USA) at the specific commercial facilities (Biokart Pvt. Ltd., Bangalore, India). Sequence was assembled and edited in Codon Code Aligner 10.0.1. Partial sequences for cytochrome oxidase I (COI) were compared in the GenBank database with the already available reference sequences. Multiple sequence alignment was performed employing present study sequence and database available sequences by using ClustalW and constructed phylogenetic tree by neighbor-joining (NJ) method with 1000 bootstrap iterations using MEGA X software (Kumar et al. 2018).
Occurrence of E. scitula
Experiment was conducted to observe the occurrence of predatory larvae of E. scitula on C. cirripediformis from June to September 2021. Field abundance of E. scitula was enumerated by investigating 15 plants of C. diurnum naturally infested with C. cirripediformis. Two infested shoots (25 cm in length) from each plant were observed for the occurrence of predatory larvae feeding in colonies of C. cirripediformis (Dean and Meyerdirk 1982) and recorded the number of larvae at 10-day intervals. The count of shield-like scale structures on the infested shoots was used to calculate the total larval population of E. scitula. No insecticides were applied to the plants during the study period.
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