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Studies on important natural enemies associated with bark beetles were followed as per the standard methods adopted by Dahlsten and Stephen (1974) and Narendran et al. (2001) with little adjustments.
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For recording the seasonal incidence of hymenopteran parasitoids associated with bark beetles viz., I. stebbingi, P. major and P. scitus experiments were conducted during 2015–2016 in Nowpora village (33°61.078′ N, 075°18.700′ E, elevation 5920 ft.) in Anantnag District, Jammu & Kashmir. The samples were collected for a period of two years (from April to November 2015 and from April to November 2016). After every month ten branches (1–8 cm in diameter, 20–50 cm in length) by careful observation were cut from host trees (P. wallichiana) naturally infested with bark beetles. The sample branches were brought to the laboratory and kept in rearing boxes made up of glass fitted with white muslin cloth for the possible emergence of parasitoids(Figures 2–4).. After every ten days emerged parasitoids from infested branches were counted and some branches were also debarked to examine the activities of parasitoid stages associated with bark beetles. The same procedure was followed throughout the year and the parasitoids collected were listed and the proportion of a particular species out of the total (i.e., dominance coefficient) was determined. The coefficient of dominance of a parasitoid species was calculated as follows:
During year 2016, the same sampling procedure was followed and the dominance coefficient (%) was determined as per the above formula.
For recording the seasonal occurrence of predators associated with bark beetles viz., I. stebbingi, P. major and P. scitus, experiments were conducted in the same aforementioned study area. The samples were collected for a period of two years (from April to November 2015 and from April to November 2016) with the interval of 15 days between the successive sampling instances. Since three aforementioned bark beetle species occupy different parts of the host tree, three wooden frames (sampling units) were made available, one for each species. The dimensions of the wooden frames were 0.06 m2, 0.10 m2 and 0.16 m2 for P. scitus, P. major and I. stebbingi respectively. After every fifteen days, a total of thirty samples were taken (ten from each species) from severely infested logs, first by marking the bark surface by using wooden frames, then by carefully debarking the sample area occupied by each species (Figs. 3–4). Data of each predator species with its associated host beetle stage were recorded. The same procedure was followed throughout the year and the predators collected were listed and the proportion of a particular species out of the total (i.e., dominance coefficient) was determined as per the aforementioned formula used for parasitoids for both years.
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Studies on use of entomopathogenic fungi against bark beetles was followed as per the earlier standard methods adopted by Batta (2007) and Jakus and Blanzee (2011)
Naturally infested branches of P. wallichiana were collected during 2017(April to November 2017) from a severely infested pine stand located in Nowpora village (33°61.078′ N, 075°18.700′ E, elevation 5920 ft.) in Anantnag District, Jammu & Kashmir (Figure 1) and forest check point, Tangmarg (340 03.797′ N, 074024.948′ E, Elevation 7552 feet) in Baramulla District, Jammu & Kashmir (Figures 1¬¬¬¬–3). The infested branches were selected after observing bark beetle infestations (Figure 4–5). The sample branches were transported to the Animal House, Department of Zoology, University of Kashmir in plastic boxes for the evaluation of fungal treatments against I. stebbingi.
The commercial bioprepration of three entomopathogenic fungi viz., B. bassiana, M. anisopliae and L. lecanii were obtained from Green Life Biotech Laboratory, Somanur, Coimbatore, India. Experiment was performed from April to November 2017. A total of 90 branches naturally infested with bark beetles, categorized into five groups (G1–G5), were used in the experiment for each bark beetle species. Each replicate represented three infested branches and six replicates per experimental treatment were used for each bark beetle species (Table 1). The used insecticide was cyclone (active ingredient: Chlorpyriphos 50% + Cypermethrin 5%).
The fungal preparation was diluted in water: 1ml biopreparation/1000 ml water with four drops of a common detergent as a wetting agent. Each fungal suspension contained 1.0 × 109 spores of fungi in 1 ml. The fungal suspensions were applied with a hand sprayer at 500 ml per log (Table 1). High volumes of fungal suspensions were used for effective treatment so that suspensions would penetrate spontaneously after application. After 10 days nine branches from three treated replicates in each group were carefully debarked and the percentage mortality of each bark beetle species were calculated and compared (Table 1). The same procedure was applied for calculating percentage mortality of each bark beetle species after 20 days of treatment.
In this method a total of 15 petri dishes containing filter papers were used; three replicates were maintained for each treatment. The treatments were performed by applying two rapid jetting sprays standardized at 1.0 ml per replicate using a small calibrated hand sprayer (1 liter capacity) equipped with a nozzle suited to low-volume spray application (Batta, 2007). In each petri dish 40 adults of each bark beetle species were introduced before spraying. The same spray volumes (1 ml per replicate) were applied in the other treatments (Table 2). The mortality percentage from each treated group was evaluated after 2, 4 and 6 days after treatment. This mortality was shown either by the lack of movement of treated adults within five minute period of continuous observation or by the appearance of mycelial growth on the bodies of dead adults. The beetles were then incubated in petri dishes under humid conditions for one week to promote mycelial growth with the conidia and the conidiophores on their bodies.
Photographs during the field study were taken by using Canon PowerShot SX60 camera fitted with macro lens (Raynox MSN-505, 37mm). Analysis of digital images was done by using ImageJ analysis software (Version 2006. 02.01). For morphometric description of collected predators images were taken from haplotype with a Leica DFC295 camera attached to a Leica M205A Stereozoom binocular microscope. Multiple images with different focal levels were combined into a single image using Leica Automontage Software (V4.10). Measurements were also taken from the type specimens with Leica Automontage Software. All the specimens are deposited in the Museum, Department of Zoology, University of Kashmir, Srinagar.
The data obtained during the present study were tabulated and graphically presented as per the required statistical methods. Arithmetic mean, Variance, Range, Standard Error and SD (Standard deviation) were used to analyze the data. The correlation between maternal galleries and fecundity of females was analyzed by Karl Pearson’s correlation method. Head capsule width of different larval instars was used to calculate total larval instars by Dyar’s ratio (Dyar, 1890). Statistical analysis was done by using SPSS (Version 10.00).
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