Habitat description and field treatment
Accidentally, during a visit to a large area planted with the Egyptian clover Trifolium alexandrinum L. at Al Badrashin village in Giza governorate in April 2019 to collect nymphs and adults of the earwig Labidura riparia Pallas for studies, the field was found highly infested with the cotton leafworm Spodoptera littoralis (Boisd.), S. exigua L., and few larvae of the semi-loopers Trichoplusia ni (Hübner) and Autographa circumflexa (L.). The semi-loopers were found on wild plants of Cruciferae in the clover field. Two trees of mulberry (Morus alba L.) were standing in the middle of the field on which a swarm of the house sparrow was resting and fly landing in the field feeding on the lepidopteran larvae visible on the clover plants. This behavior is repeated several times a day. Many trees of nearly 20-m-high white willows Salix tetrasperma Roxb. (Calicaceae) are growing along the irrigation canal adjacent to the field. A colony of the cattle egret Bubulcus ibis Bon. inhabited these Salix trees. When the clover field is irrigated after cutting, a large number of the B. ibis was found in the field feeding vigorously on the floating insects especially on the lepidopteran larvae. By evening, the house sparrows overnight on the mulberry trees and the cattle egret on the white willow trees. Under both tree species, a large number of droppings were excreted by these birds. Thus, the idea of the present study was born. The field under the mulberry trees and the area adjacent to the irrigation canal was sprayed at the evening by the nucleopolyhedrosis of S. littoralis at the concentration of 5 × 108 PIBs/ml, and after 6 days (1–2 days shorter than the virus infected larvae die), the clover was cut and the field was irrigated to attract more B. ibis for feeding on the floated and moribund larvae infected with the virus in order to collect and test the following droppings for presence of viable virus PIBs.
Collecting bird droppings
Plastic sheets were placed under the mulberry and white willows trees at the 6th day post application of the S. littoralis nucleopolyhedrosis virus (SlNPV). As previously mentioned, this period is required as not full incubation period (moribund larvae) enabling infection with the virus and its replication in cell nuclei of nearly most larval body tissues before death and rupture of the body cuticle. Six days post treatment with SlNPV, the clover foliage was cut as green fodder for farm animals, and the field was flooded by irrigation water, a routine practice after cutting the clover. This agricultural practice enforces any insects in soil cracks or under the remaining cut clover including the diseased larvae to float on the water surface, which attract a large number of the cattle egret B. ibis to visit the field and vigorously feed on them as well as did the house sparrow P. domesticus. The birds excrete most of their droppings at night; droppings were collected in the early morning from the plastic sheets placed under the trees at the 2nd, 3rd, and 4th days post irrigation, kept in paper bags, and transferred to the laboratory.
Extraction of PIBs from bird droppings
The collected droppings of the 3 days were mixed well together, and 50 g from droppings of each bird species were soaked in 100 ml distilled water and homogenized by vortexing for 5 min to disrupt any clumping in. The homogenized dropping suspensions were passed for primary filtration through 4 layers of muslin cloth. The filtrate was centrifuged at 900 rpm for 2 min to remove the undigested insect pats and debris. The supernatant was further centrifuged at 5000 rpm for 10 min. The pellet containing the polyhedral inclusion bodies (PIBs) was resuspended in 5 ml sterile distilled water as stock suspension, and the PIB concentrations were counted using a hemocytometer. The stock suspensions of PIBs were refrigerated at 4 °C until needed for the tests.
PIB extraction from feces of L. riparia and P. pallens
Both adults and nymphs (130 individuals) of L. riparia and 3rd instar larvae of P. pallens were offered S. littoralis NPV-diseased larvae, which were 6 days previously infected with SlNPV to enable multiplication of the virus and formation of PIBs in nuclei of all tissue cells of the body. The feces particles were separated from the butter paper furnished in the Petri dishes by means of a sculpt on the 1st day post feeding on diseased prey and shifted into sterilized test tube. Feces were collected for 3 successive days parallel to feeding on S. littoralis-diseased larvae. The daily excreted feces of each predatory species were mixed together and suspended in 1 ml sterilized distilled water. The suspension was homogenized by vortexing for 3 min followed by centrifugation at 900 rpm for 2 min to remove the debris, and the supernatant was further centrifuged at 5000 rpm for 10 min to extract the PIBs of the SlNPV, where the pellets here were resuspended each in 1 ml sterilized distilled water and kept at 4 °C.
Rearing of test insects
Field-collected larvae of the 4 species, S. littoralis, S. exigua, T. ni, and A. circumflexa, were reared on the standard diet described by Shorey and Hale (1965). G. mellonella was reared on a simple diet according to Ibrahim et al. (1984). Adults and nymphs of the earwig L. riparia were collected from soil barrows at borders of the clover field, placed in a 2-L plastic container, filled with paper strips, to avoid cannibalism, and transported to the laboratory. Individuals were confined solitary in Petri dishes furnished with butter paper and supplied daily with young larvae of reared S. littoralis. This predator has only 3 generations/2 years (El Husseini 1969). Accordingly, adults and nymphs develop in a long period of many months. Thus, the field-collected specimens were used as wild individuals in the test. Adults of the lacewing P. pallens were collected by a light trap and reared as described by Abou-Bakr (1989). Larvae were fed first on aphids (Aphis craccivora Koch) till reaching the 3rd larval instar (L3), and then fed on young larvae of S. littoralis. All test insects were reared or maintained under laboratory conditions of 25–27 °C and 60–7% RH.
Bioassay of PIBs extracted from droppings
From the wide host range highly concentrated stock suspension of S. littoralis viral PIBs extracted from droppings of each B. ibis and P. domesticus, 5 successive concentrations of (1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 PIBs/ml) were prepared in sterilized distilled water. Fresh uncontaminated leaves of clover were hanged on a standing holder and sprayed with the tested PIB concentration using a fine perfume hand atomizer and left to evaporate. The treated clover leaves were placed in Petri dishes (5 cm in diameter) furnished with butter paper. Each dish separately received 10 neonate larvae (L1) from the targeted noctuid insect pests, i.e., S. littoralis, S. exigua, T. ni, and A. circumflexa that left feeding on for 48 h. Five replicates were set for each test. A control was set also in 5 replicates offering the larvae untreated clover leaves previously sprayed with sterilized distilled water. Then, the treated larvae and the control were provided daily with fresh untreated clover leaves for the next 5 days. Mortality among treatments and controls was recorded daily for 1 week post treatment. Concerning bioassay versus larvae of G. mellonella, 1 g of the diet was flat pressed on butter paper in thickness of 1 mm and sprayed with the tested PIBs desired concentration and then divided into 5 portions each on a disc of butter paper. The treated diet was shifted underlined with the butter paper into 2 oz metal cups with caps and provided each with 10 neonate larvae of G. mellonella. They were left feeding on the treated diet and provided with new untreated if needed. The test runs in 5 replicates for each concentration, beside an untreated control. Mortality was also recorded among 7 days post treatment.
Bioassay of PIBs extracted from insect predator’s feces
PIBs extracted from feces of L. riparia adults and nymphs were prepared in sterilized distilled water from the stock solution in concentrations of 1 × 101, 1 × 102, 1 × 103, 1 × 104, and 1 × 105 PIBs/ml. The 5 PIB concentrations were administrated to the 4 targeted noctuid larvae on contaminated clover leaves by the same technique described for bioassay of those extracted from avian droppings, beside an untreated control with clover leaves previously sprayed with sterilized distilled water. The pyralid G. mellonella was bioassayed as previously described. Larvae were left feeding on the treated clover leaves for 48 h and supplied daily with fresh untreated leaves. Meanwhile, the wax moth larvae were provided with untreated diet if needed. Mortality was also recorded among 7 days post treatment. Although microscopic examination of the extracted pellet suspension obtained from larval feces of the chrysopid predator P. pallens showed absence of the PIBs of the S. littoralis nucleopolyhedrosis virus, it was tested versus the targeted lepidopteran larvae on clover leaves sprayed with this extract without dilution to assure absence of the PIBs parallel to untreated controls. It was also tested versus neonates of G. mellonella by mixing in the diet as previously described. The treatments and control were inspected daily in the next 7 days for registering any mortality appearing among the larvae of the test.
Statistical analysis
Data were processed by analysis of variance using the Costat Statistical Software (1990).