A study was undertaken to identify and ascertain the cellular encapsulation response in
Spodoptera litura (Fabricius) to the entomopathogens was carried out during 2022-23 in Insect Pathology Laboratory, Department of Entomology, College of Agriculture, Rajendranagar, Hyderabad. The materials used and procedures followed for the study were as under.
Insect rearing
The
Spodoptera litura larvae were collected from the college farm of college of agriculture, professor jayashankar telangana state agricultural univesrity (PJTSAU) and farmer fields in rajendranagar, Hyderabad. The collected larvae were maintained on the artificial diet
(Gupta et al., 2005) with relative humidity 70-75% and temperature 20±2Úc under laboratory conditions. The larvae which were in the stage to pupate were transferred into the tray containing soil. After pupation, the pupae were collected in a tray and covered with a muslin cloth. The emerged adults with a male and female ratio of 2:3, were transferred into the cages containing 15% sucrose solution (
Santharam, 1985). The egg masses were collected and maintained in the Insect Pathology Laboratory, Department of Entomology, College of Agriculture, PJTSAU, Rajendranagar, Hyderabad.
Entomopathogens and inoculation procedure
Commercial formulations of different test entomopathogens
i.
e.,
Beauveria bassiana,
Metarhizium anisopliae,
Bacillus thuringiensis and Nuclear Polyhedrosis virus (
Sl NPV) (Table 1) used in the present study were sourced from a noted agri biotech company, AgriLife (India) Private Limited, Hyderabad. Leaf dip method for
Bt and
Sl NPV and topical application for entomopathogenic fungi (Table 1) were used for inoculating the larvae. Once
S.
litura larvae moulted to 3rd instar, they were starved for 24 hours before inoculating with the leaf dip method. For inoculating the larvae with
Bt and
Sl NPV, the castor leaves were dipped in suspensions of
Bt for treatment three and in viral suspensions for treatment four as described in Table 1. The treated castor leaves were then dried for 30 seconds and the dried leaves were placed on the filter paper within a petri dish (one leaf per petri dish). Two larvae per petri dish were released and four such replications for each treatment were maintained. Then the larvae were allowed to feed on the infested leaves. Since oral infection is not the primary infection route for entomopathogenic fungi, for treatment with
B.
bassiana and
M.
anisopliae the third instar larvae were topically exposed by adding 10ìl of the solution with a sampler device, following which they were placed on filter paper for 30 seconds and transferred two larvae per petri dish and four such replications were maintained for the treatment. For control, castor leaves were dipped in 10ìl of Tween 80 (0.05%) solution, then dried for 30 seconds and placed in the petri dish. Two larvae per petri dish were released.
Collection of haemolymph from infected larvae
Haemolymph was collected by cutting the tip of a proleg of the infected larvae from each treatment, previously anesthetized on ice using fine scissors and needle (
Rosenberger and Jones, 1960). Gentle pressure was applied on the insect abdomen for getting more quantity of haemolymph
(Barakat et al., 2002). The collected haemolymph from the infected larvae was used to prepare blood smears, by spreading a drop (20µl) of haemolymph on a glass slide and smeared by drawing a second slide across the first at an angle of 45° and then dried at room temperature. The air-dried haemolymph smear slides were then dipped into methanol two times and air dried. Slides were then stained with Giemsa stain (diluted five times with phosphate-buffer saline (PBS) and filtered before use) for 20 min and then rinsed in distilled water. The smear was washed in 0.02% acetic acid followed by rinsing in distilled water. After drying, permanent microscopy slides were prepared using permount or canada balsam.
Cellular encapsulation
Immediately after the haemolymph extraction, a 3 mm long piece of nylon monofilament was fully placed into the punctured wound of each larva to reduce the risk of rupturing the midgut. Larvae that survived were fed for 24 hours. The remaining larvae were then frozen and after the death of the larvae, the nylon monofilament was removed and placed on a slide and captured in a photograph. ImageJ, GIMP, photoshop and other software were used to quantify the degree of melaninization and the area of cell cover. The larval samples with gut ruptures and that were failed to recover the nylon filament during the dissection were discarded
(Black et al., 2022).
Data analysis
Each treatment was replicated four times and the data was analyzed by completely randomized design (CRD) using Web Agri Stat Package (WASP 2.0) software. The data was subjected to the standard statistical analysis using techniques of analysis of variance. Differences between samplings were considered statistically significant at a probability more than 5% (
p≤0.05).
