The present study was carried out in the Bio-control Laboratory of the Department of Entomology, University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India. Experiments were conducted at 25 ± 0.5 °C, 70 ± 5% relative humidity and 12L:12D photoperiod.
A stock culture of P. xylostella was established at 25 ± 0.5 °C, 70 ± 5% RH and 12L:12D photoperiod in the laboratory from field collected larvae. The larvae were fed on cauliflower (Brassica oleracea var botrytis L.) leaves or mustard (Brassica campestris L.) seedlings in insect rearing cages (45 × 45 × 45 cm) fitted with glass on 3 sides and a nylon net on the front side. Larval food was changed daily or on alternate days (depending upon the requirement) until all the larvae pupated. Pupae were collected and placed in jars for adult emergence. Newly emerged adults were shifted to the insect rearing cages for mating and provided with 30% honey solution (in cotton swabs) as food and fresh leaves of cauliflower with their petioles inserted in a glass/plastic tube containing water as substrate for oviposition. After 24 h, the leaves with eggs were shifted to another cage for hatching. Neonate larvae were transferred carefully to the fresh cauliflower leaves for further rearing under laboratory conditions. P. xylostella was reared and multiplied for 2 generations before experiments were carried out.
A culture of C. vestalis (national accession number: NBAII-GN-BRA-04) was maintained from cocoons, obtained from the National Bureau of Agricultural Insect Resources, Bengaluru, Karnataka, India. The emerged adults were transferred to rearing cages (45 × 45 × 45 cm, fitted with glass on 3 sides and a nylon net on the front side) and provided with 30% honey solution (in cotton swabs) for feeding and the 2nd/3rd instar larvae of P. xylostella (on cauliflower leaves) for oviposition. After 24 h, the larvae were removed and a new batch of larvae was provided to the parasitoids. The process was repeated till all the parasitoids died. Parasitized larvae of P. xylostella were reared at 25 ± 0.5 °C, 70 ± 5% RH and 12L:12D photoperiod. The food of the adult parasitoid was renewed daily. C. vestalis was reared for 2 generations to obtain a sufficient number of parasitoids for the experiments.
Preference of C. vestalis for different larval stages of P. xylostella
To study the relative preference of C. vestalis for different larval ages of P. xylostella, parasitoid adults were provided by all the larval instars of P. xylostella simultaneously. Newly emerged parasitoid adults were sexed and each pair was confined in a glass tube (15 × 2.5 cm) containing honey streak on the side wall for 24 h to ensure mating. After 24 h, in a choice experiment, a pair of C. vestalis was provided by 20 larvae (replicated 5 times), 5 each of the 1st, 2nd, 3rd and 4th instars of the host on a cauliflower leaf simultaneously in a rearing cage. Larval instars of P. xylostella were differentiated on the bases of their head capsule width, body colour and size (Alizadeh et al., 2011). The parasitized host larvae of different ages were reared separately as described earlier until pupation and then adult emergence. The data on the number of parasitized larvae for each instar were recorded.
Developmental biology of C. vestalis parasitizing different host instars
Developmental biology of C. vestalis was studied by offering different larval instars of P. xylostella for parasitization. For this purpose, newly emerged parasitoid adults were sexed and each pair was offered by 10 larvae of the 2nd, 3rd or 4th instar larvae of the host insect separately in a no-choice experiment. After 24 h, the parasitized larvae were replaced with a new batch of ten larvae. The parasitized larvae were reared as per the procedure described above for the emergence of the parasitoids. Because the egg and larval stages of C. vestalis developed inside the body of the larva of P. xylostella, egg and larval instars were combined as egg-larval stage. Data on the duration of egg + larva (stinging to cocoon formation), prepupa + pupa (cocoon stage), adult longevity, sex ratio and fecundity (in terms of progeny developed) were recorded.
Population growth parameters of C. vestalis parasitizing different host instars
Population growth parameters of C. vestalis on P. xylostella were measured, using fertility tables. Fifty larvae of each instar parasitized by C. vestalis on a single day were separated and reared as described earlier. To obtain parasitized larvae, individual larva of P. xylostella was exposed to a 24-h-old mated female parasitoid in a glass tube and replaced with another one after being stung once by the parasitoid. In this way, nearly 20 parasitized larvae per hour were obtained. The female of C. vestalis was replaced after stinging 5 larvae. Host larva stung by the parasitoid was presumed to be parasitized. Adults thus obtained were sexed and each couple of parasitoid adults was offered by 20 host larvae of the desired age. After 24 h, the old batch of larvae was replaced by a new batch and the process was continued until all the parasitoids died. Daily survival and fecundity data were used to construct fertility-based life tables of the parasitoid on each host instar separately to calculate the population growth parameters (Birch 1948 and Carey 2001) as follows:
Net reproductive rate (Ro) = Σlxmx.
Intrinsic rate of increase (rm) was calculated by using the expression Σ (e−rmx lxmx) = 1.
Mean generation time (T) = Loge Ro/rm.
Finite rate of natural increase (λ) = Antiloge rm.
Weekly multiplication rate (WM) = e7rm
Doubling time (DT) = loge2/rm.
Where x = age of the individuals in days (pivotal age), lx = the proportion of females still alive at age x (survival rate) and mx = the number of female eggs (based on sex ratio) per female at the age x (fecundity rate).
The jack-knife method was used to generate pseudo-replication for obtaining standard errors of parameters with 5 replications in each case (Maia et al. 2000, Meyer et al. 1986).
Data on different parameters were subjected to one-way analysis of variance (ANOVA), using OP-STAT available at http://www.hu.ernet.in., and the significantly different means were separated by least significant difference (LSD).