Entomopathogenic fungi culture
The EPF, L. lecanii Vl-8 strain, was grown and maintained in Sabouraud Dextrose Yeast agar (SDYA) Medium at 25 ± 1 °C for 8–10 days. After the growth of pure culture in the plates, the fungal spore culture was inoculated into Sabouraud Dextrose Broth media (SDYB) liquid media. It was then placed in shaker at 25 ± 1 °C and 150 rpm for 8 days. The 8-day-old culture was then mixed with the pre-autoclaved talc in the ratio 1:2 (500 ml fungi grown broth: 1 kg talc). Then, the mixed talc was covered with a black cloth and kept for drying for 3 days under aseptic conditions. Quality analysis of EPF was done by checking the spore count through hemocytometer and serial dilution method. Briefly, 1 g of talc was mixed to 9 ml of water and was vortexed and 1 ml from it was transferred to another vial to make it 10–2. The same method was repeated up to 10–12 dilution. Further 1 ml taken from 10–6, 10–8, 10–10, 10–12 dilutions and transferred to a Petri plate containing SDYA medium. The colony count was observed in 10–8 plates to confirm that the talc contains 10–8 spores. After the spore count was quantified, the suspensions were finally prepared by serial dilution in distilled water by adding 0.1% of Tween-80 and stored in the refrigerator at 4 °C until further use.
Insect culture
Frankliniella schultzei
Adults of F. schultzei were reared on chemical free beans in a plastic container (1 L capacity) with a lid. The lid of the container was cut open and stuck with a fine mesh for aeration. Daily bean pods were collected from the adult container and kept for hatching to get desirable stage.
Blaptostethus pallescens
Adult of B. pallescens was obtained from the stock culture reared at ICAR-NBAIR laboratory, Bengaluru, and subcultured in plastic pearl pet containers of 500 ml capacity. Corcyra cephalonica eggs ad libitum were given as food, and 4–5 pieces of bean pods each of size 5–6 cm were provided as ovipositional substrate in each container which can hold up to 20 adult B. pallescens. Collection of bean pods was done on daily basis to collect the eggs of B. pallescens and kept for hatching. The newly hatched nymphs were reared in plastic containers using the same procedure as followed for adults. Corcyra eggs were replenished on daily basis as food. A small cotton piece soaked in water was introduced into the containers to maintain the moisture.
Biology and feeding potential on Vl-8 treated and untreated thrips
Laboratory studies were carried out at 26 ± 2 °C and 65 ± 2% R.H. Second instar larvae of thrips were taken and sprayed with 1 × 108 spores/ml of Vl-8 fungal strain such that the entire body surface is covered. After 24 h. of spraying, the treated thrips were then counted and provided as feed for the predator. The predator B. pallescens was acclimatized to laboratory conditions for 2 generations on untreated F. schultzei thrips before commencing the experiment. Biology was also studied on untreated thrips.
Thirty newly hatched nymphs of the predator were taken for the experiment. Each nymph was placed individually in plastic containers (90 mm diameter) with tissue paper in the bottom and Vl-8-treated thrips as feed. Two bean pods were introduced in each plastic container for feeding for thrips. Ten number of treated or untreated second instar thrips larvae were provided per nymph initially up to first two instars of predatory bug and were increased to about 30 for late instars and adults because of increase in requirement by the advance stages of predatory bug. Observations on the number of thrips consumed and the time of molting were recorded on daily basis in both the treatments. Thrips were replenished on daily basis to all the containers. The experimental setup was maintained till emergence of adults. The nymphal duration for each individual was recorded. After the adult emergence, the sex ratios were recorded. Further, the adults were left for mating in order to observe the fecundity and per cent hatching. Longevity of male and female predator was also recorded. Morphometrics of each nymphal instars and adults were recorded using LEICA M205C microscope.
F1 generation
The eggs laid by the adult bugs reared on Vl-8 treated or untreated thrips were collected and kept for hatching. Twenty nymphs were taken and reared on UV-treated C. cephalonica eggs to check if there was any adverse or sublethal effect of Vl-8 on nymphal duration of F1.
Prey preference of different instars of predatory bugs
Second, third, fourth and fifth nymphal instars of B. pallescens along with female adults were taken and provided with Vl-8 treated and untreated second instar thrips larvae at the same time. Fifteen treated and fifteen untreated thrips were provided for second and third instar, and the number of thrips was increased to 20 each for fourth instar to adult stage. Experiment setup of each bug was maintained in a large Petri plate and kept for 24 h. The thrips consumed by the bugs were recorded.
Adult preference at different time intervals of thrips infection
Three-day-old adult bugs were offered 20 untreated and 20 Vl-8 treated second instar thrips larvae at different intervals, i.e., 24, 48, 72, 96 h. post-inoculation, and were checked for their preference. The thrips consumed by the bugs were recorded after 24 h.
Statistical analyses
Prey preference of different instars of B. pallescens was analyzed using an independent t test comparing the means of Vl-8 treated and untreated thrips. Manly’s preference index for prey type was calculated using the formula given below:
$$\beta 1 = \frac{{\log \left( {\frac{{e{1}}}{A1}} \right)}}{{\log \left( {\frac{e1}{{A1}}} \right) + {\text{log}}\left( {\frac{e2}{{A2}}} \right)}}$$
where β1 is the prey preference toward prey type 1, e1 is the remaining prey after the experiment, and A1 is the number of prey provided initially. The value near to 0.5 reveals that there is no preference toward provided prey. If the value is nearing to 1, it shows the preference toward prey type 1 and value nearing to 0 shows the preference toward prey type 2 (Varshney and Ballal 2018). In this study, untreated thrips were chosen as prey type 1. One-sample t test was used to compare the Manly’s preference index for untreated thrips setting a value 0.5 to test the null hypothesis that predator prefers prey at random.
