Rearing larvae of S. recurvalis on semi-synthetic diet
The colony started with larvae collected from sugar beet fields in Sakha at Kafr El Sheikh Governorate, Egypt and transferred to the laboratory at the Faculty of Agriculture, Cairo University, Giza Governorate. They were reared on daily fresh sugar beet leaves until pupation took place. Pupae were placed in egg-laying cages of cylindrical glass (19 cm in diameter and 20 cm in height), furnished with tissue paper, and bottomed and topped with plastic plates (20 cm in diameter and 3 cm in height). The bottom plate was provided with a small cap (3 cm in diameter), provided with a 1:15 solution of bee honey:water, as food for the emerged moths. Eggs were collected from the tissue paper by cutting and placed in Petri-dishes until hatching.
Newly hatched larvae were reared till pupation on a semi-synthetic diet formulated according to Shorey and Hall (1965). The diet was composed of 500 ml distilled water, 9.9 g agar, 12.5 g yeast, 3.7 mg vitamin B-complex, 1.9 g nepagin, 0.5 g sorbic acid, and 60 g matched cocked beans (Phaseolus vulgaris). The agar was added gradually to the distilled water under continuous stirring in boiling water bath for 40 min. With the exception of sorbic acid, all the components were then mixed with the agar in the hot water bath for another 30 min. Thereafter, the mixture was left to cool down to about 45 °C. Then, the sorbic acid was added, also under stirring. The prepared diet was poured into sterile plastic plates (15 cm in diameter and 5 cm in height) in a 2-cm-thick diet layer and left to cool. Diet plates were secured by polyethylene covers, fitted in place by rubber band to avoid dryness, and kept in the refrigerator until needed.
The rearing plates were covered by their top plates like Petri-dishes and incubated at 25 °C. The developing larvae produced a slight silky web cover on the diet under, which they feed on. As mining in the published literature, this could be the first trial to rear the beet web larvae on semi-synthetic diet (Fig. 1). Surplus diet was added to the developing larvae when needed till pupation took place. Pupae were collected and kept in similar egg laying containers till moth’s emergence, copulation, and egg laying.
For standardization of the L2 larvae for the bioassay test, reared larvae of the L1 that reached the moulting stage with the characteristic erected head capsule, swollen cervicum, and remaining motionless were gently transferred by a wetted fine camel-hair brush onto the treated diet. Within 12 h, they moult into the 2nd instar (L2) and after a short while, they started feeding on the treated or untreated (control) diet.
Tested Bacillus thuringiensis
The commercial formulation Dipel 2X-wettable powder based on Bacillus thuringiensis subsp. kurstaki (B.t.k.) (Abbott Laboratories. Illinois, Chicago, USA) was used to treat the larvae of S. recurvalis (L2) by mixing the B.t.k. formulation into the semi-synthetic diet. The formulation contains 32,000 international unit of potency per mg measured on Galleria mellonella L. and 1452 billion IU/pound representing 6.4% as active ingredient (spore-endotoxin-complex) and 83.6% inert ingredients.
Bioassay test
Five grams of Dipel 2X were mixed into 95 g of the semi-synthetic diet to prepare a diet containing 5% concentration of the commercial B.t.k. formulation. Successive concentrations (5.0, 2.5, 1.25, 0.6, and 0.3%) were prepared by mixing them with the diet. Then, 500 larvae (L2) were left to feed each of 100 ones on the tested diet (concentration) for 24 h, devided into four replicates, each of 25 larvae. Thereafter, larvae fed on each concentration were transferred onto untreated diet and mortality rate was recorded daily for 1 week post treatment. One hundred larvae (L2) in four replicates each of 25 ones were reared on untreated diet and served as control. The test was carried out in the laboratory under incubation at 25 °C.
Histopathology
The B.t.k.-treated diet (concentration 1.25%), on which the larval mortality reached 100% on the fifth day post treatment estimated through the bioassay test, was used for feeding 50 larvae (L2) of S. recurvalis to obtain a slow killing time facilitating the daily histopathological, follow the induced changes in the structure of the mid-gut (El Husseini 1976; Omar 2004). Five larvae from those fed on the treated diet and five others from the control (reared on untreated diet) were fixed daily in Bouin’s alcohol fixative during the next successive 5 days after treatment. Fixation process lasted 16 h; thereafter, larvae were processed in the common way for preparing paraffin serial sections of 6–8 μm with a double staining by eosin and hematoxylin; followed by mounting in Canada Balsam as permanent preparations (El Husseini 1980). Prepared cross sections in the mid-gut were examined with a light microscope in both treated larvae and those of the control. A Leica camera fixed to the microscope and connected to a PC served in shooting the needed photos.