Mass culturing of Aleurodicus rugioperculatus
Aleurodicus rugioperculatus infested coconut leaflets were collected at the Tamil Nadu Agricultural University (TNAU) orchard (11.0123° N, 76.9355° E), Coimbatore, Tamil Nadu, India and released onto potted (41 cm diameter) plants of coconut, banana, sapota, and guava, which were kept in a separate mini net house (270 × 150 × 210 cm with a nylon net mesh size of 120 microns). Aleurodicus rugioperculatus rearings were maintained in the Insectary, Department of Agricultural Entomology at 31 ± 2 °C, 60 to 75℅ RH under natural light (Fig. 1).
Mass culturing of Encarsia guadeloupae
The initial stock culture of E. guadeloupae was established by collecting the adults using an aspirator at the TNAU orchard. Encarsia guadeloupae adults were released onto A. rugioperculatus infested potted banana plants maintained in a mini net house as described previously.
Molecular identification of test insects
Aleurodicus rugioperculatus and E. guadeloupae DNA was extracted using the HotSHOT method (Montero‐Pau et al., 2008). First, a single A. rugioperculatus and E. guadeloupae adult was washed in Milli-Q water, transferred to a 2 ml microcentrifuge tube containing 50 µl of alkaline lysis buffer (pH 12; Sigma-Aldrich), and crushed using a sterile pipette tip. The lysate was incubated at 95 °C for 30 min and subsequently incubated on ice for 3 to 4 min; 50 µl of neutralizing buffer (pH 5; Sigma-Aldrich) was added to each tube, and the samples were vortexed and spun down. DNA was quantified using the Nanodrop Spectrophotometer (ND-1000). Based on the nanodrop readings, DNA was diluted in TE buffer to make a final concentration of 50 to 60 ng µl–1 and stored at 40 °C for further use (Sambrook et al., 1989). The quality of genomic DNA was examined in a 0.8℅ agarose gel. Electrophoresis was performed at 65 V for 1 h. Amplified genomic DNA was visualized in a UV transilluminator (Bio-Rad, USA) and documented using the gel documentation system (GELSTAN 1312).
After DNA quality and quantity check, a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI) was amplified using LCO 1490 and HCO 2198 primers (Folmer et al., 1994). PCR was performed using Master Cycler Gradient-Eppendorf (Pro S model, Germany) with a 25 µl reaction mixture containing 5 µl of aliquots of DNA lysate (50 ng/µl), 2.5 µl of 1X PCR buffer and 0.5 µl Taq DNA polymerase, 1.0 µl MgCl2 (25 mM), 2.5 µl dNTPs (2.5 mM each), 1.5 µl of primers (10 µM each), 9.5 µl of nuclease-free water, and 1.0 µl of dimethylsulfoxide (DMSO). The thermocycling program was as follows: initial denaturing step, at 94 °C for 2 min followed by 35 cycles of amplification (94 °C for 1 min, 52 °C for 1 min, 72 °C 1 min), and a final extension at 72 °C for 10 min. A total of 5 µl of PCR product (mixed with 2 µl of loading dye) was loaded on the agarose gel (1.5℅) and electrophoresed at 65 V for 1 h. Finally, the products were visualized in a UV transilluminator, and the gel was documented using a gel documentation system (GELSTAN, 1312).
The PCR product was purified using the Pure Link PCR purification Kit, and sequencing PCR was set up using the Bigdye Terminator V3.1 Cycle Sequencing Kit. The resulting sequencing information was retrieved from the client database of the SciGenom Labs online portal. Finally, the COI fragments of insect samples were sequenced bi-directionally at SciGenom, Kerala, India. Next, the sequences were aligned, edited, and trimmed using the program Geneious and identified through the BLASTn algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained nucleotide sequence was submitted to NCBI under accession number: ON619568 (A. rugioperculatus) and ON166641 (E. guadeloupae). The neighbour-joining phylogenetic tree was constructed with the bootstrap values of 1000 replicates by using the MEGA version 11.0.
Antibiotic treatments through parafilm feeding chambers
Aleurodicus rugioperculatus adults were fed with the antibiotic added sugar solution using parafilm feeding chamber method adopted by Ruan et al. (2006) given in the Fig. 2. Newly emerged pair of adult whiteflies were taken into small plastic containers (3.5 cm height, 2.0 cm width). Top portion of container was stretched with parafilm and feeding solution was placed above parafilm layer. Another layer of stretched parafilm covered with feeding solution to avoid air bubbles. Minute hole was made on the sides of the container for aeration of the adults. Adults were allowed to feed the antibiotic solution for 24 - 48 h and transferred to clip cages. Then, clip cages were placed on four host plants for further development. Antibiotic (Carbenicillin 100 μg ml-1 + Ciprofloxazin 5 μg ml-1) feeding solution (0.2 ml) consisted of 5 mmoll-1 phosphate buffers (pH 7.0), 25% sucrose (w/v) along with antibiotics and control feeding solution without antibiotics were used for the above experiment.
Antibiotic solutions were fed to the subsequent progeny generations until complete elimination of cultivable gut bacteria which was confirmed through culture-dependent bacterial isolation method after each antibiotic feeding experiment. The resulting antibiotic treated A. rugioperculatus populations were used for parasitization experiments.
Culture-dependent bacterial isolation
For the gut bacterial isolation, fifty numbers of second nymphal stage (Preferred stage for
E. guadeloupae parasitization) from antibiotic treated A. rugioperculatus adults were collected on coconut, banana, sapota and guava plants using a hair-brush under the microscope and starved for 24 h to eliminate the transient gut bacteria. Then, the nymphs were surface sterilized with 70% ethanol followed by 5% sodium hypochlorite for 1 min and washed with sterile water 3–5 times to remove adhering contaminants. Then, 0.1 ml of the final wash was added to 5 ml of Tryptic soy broth (TSB) and incubated for 48 h at 28 ± 2 °C to ensure surface sterilization. No growth was noticed in TSB. The second nymphal stage of A. rugioperculatus is very small, and it is difficult to dissect the gut. Hence, the whole A. rugioperculatus nymph was taken for gut bacteria isolation after surface disinfection (Malathi et al., 2017). The nymphs were homogenized with 0.1 M phosphate buffer (pH 7.0). The nymphal homogenates were serially diluted in sterile distilled water and 0.1 mL was spreaded on nutrient agar media (M/s. HiMedia Laboratories, Mumbai, India). Petri plates containing gut homogenates were incubated for 48 h at 28 ± 2 °C and monitored for bacterial growth at an interval of 24 h (Saranya et al., 2022).
Parasitoid infectivity on symbiotic and antibiotic treated population of Aleurodicus rugioperculatus
This experiment was performed to study the adaptation behaviour of parasitoids against symbiont mediated defenses in host insects. Genetically identical whitefly population with symbiotic (untreated), antibiotic treated and its parasitoid, E. guadeloupae were collected from A. rugioperculatus infested host plants kept under mini net house condition were used for this study. Experiments consisted of eight insect lines, which includes four symbiotic populations (untreated) of A. rugioperculatus reared on coconut, banana, sapota, guava and four antibiotic treated population of A. rugioperculatus reared on coconut, banana, sapota, guava. Each insect lines were maintained in separate mini net house condition.
Symbiotic (untreated) and antibiotic treated A. rugioperculatus lines infested in potted plants of coconut, banana, sapota and guava were placed in separate mini net house condition to maintain respective
A. rugioperculatus cultures. Thirty individuals of E. guadeloupae were released onto each insect line maintained in the mini net house. Subsequent parasitoid progenies (30 individuals) emerging from the above experiments were released onto respective symbiotic (untreated) and antibiotic treated (F4, F5 and F6 progenies)
A. rugioperculatus lines maintained under mini net house condition to observe its adaptation behaviour against symbiont mediated defenses in host.
Statistical analysis
Means of untreated and antibiotic treated populationns were compared using Paired t test after arcsine and square root transformations. All the data analyses were performed by using IBM SPSS Statistics 22 (Spss, 2013).