Effect of insect growth regulators combined with nucleopolyhedrovirus on certain biological and histological aspects of Spodopera littoralis

Five insect growth regulators (IGR's) were tested for increasing the susceptibility of the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) to its homologous nucleopolyhedrovirus (SpliMNPV). S. littoralis MNPV was tested alone or in combination with IGR's at LC10 level against the 2 instar larvae of the pest. An increased viral infection rate was detected in the mixture treatment in the case of using Chlorfluazuron, Flufenoxuron, Triflumuron, Hexaflumuron or Teflubenzuron at 10%. The LC50 value of the virus alone treatment 1x10 PIB's was reduced to 4.3x10, 9.9x10, 4.9x10, 3.1x10 and 1.69x10 PIB's, with the tested five IGR's, respectively. It was observed that Flufenoxuron and Triflumuron mixtures slightly prolongated larval duration compared either untreated control or IGR's alone treatments. The highest rate of decrease in the pupation percentage (47%) was recorded in case of Triflumuron mixed with SpliMNPV followed by Flufenoxuron and Chlrofluazuron mixes. On the other hand there is no significance difference in pupal weight for all treatments. Changes Adult longevity was increased for all treatments compared to untreated control. Addition Flufenxuron (LC10) to SpliMNPV (LC50) showed histopathological effects to mid gut e.g. loss of the compact appearance of the muscularis layer, vaculation and exfoliation of the columnar cells.


INTRODUCTION
The extensive use of insecticides to control Spodoptera littoralis (Boisd.)larvae has led to its resistance to various classes of insecticides (Tabashink et al., 1987), residual toxicity and environmental pollution (Frank et al., 1990) and negative effects on non-target organisms (Franz, 1974).Numerous studies have been undertaken for unconventional control agents owing to the hazards of conventional pesticides.Among such agents are the baculoviruses.Several efforts have been made to enhance the baculoviruses efficiency by increasing insect host susceptibility to virus by using certain additives such as Fluorescent brighteners (El-Salamouny, 2004).Flufenoxuron (IGR) promoted infection of the silkworm Bombyx mori 5 th instar larvae by B. mori nucleopolyhedrovirus (BmNPV) which could be due to interference with chitin synthesis of peritrophic membrane (Arakawa, 2002).Therefore, the present study is undertaken to further investigate the combined effect of the mixture of IGR and NPV.Certain IGR's were evaluated as additives to increase the susceptibility of S. littoralis larvae to its homologous nucleopolyhedrovirus (SpliMNPV).

Methods:-1-Bioassay and follow up:
The cotton leafworm, Spodoptera littoralis (Boisd.)(Lepidoptera: Noctuidae) was maintained at the "Insect Virology Unit", Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, under laboratory conditions of 25±2°C and 60±5% RH.The standard test insect used in the bioassay of all experiments was three to four days old 2 nd instar larvae.Newly moulted 2 nd instar larvae were treated with a mixture of the LC 10 of the tested (IGR's) mixed with the LC 50 of SpliMNPV.Treated larvae were examined daily to determine the post treatment effects on those insects survived the treatments, (e.g., the larval duration, pupation %, pupal weight and adult emergence %).These parameters were compared with the untreated control larvae.

2-Histopathological study:
The Histopathological study was undertaken on the mid gut of late 6 th larval instars treatments as 2 nd instars with the mixture of IGR + SpliMNPV.The tested tissues were fixed in aqueous Bouin's solution for 24 hr.The normal paraffin wax embedding procedure was followed.The sections were cut 6µ thick and stained with heamatoxylin and eosin for microscopic examination.Control sections of non-treated larvae were also carried out.

3-Statistical analysis:
Statistical analysis (ANOVA) of the obtained data was performed using COSTAT program, which runs under WIN.Also, the difference between means was conducted by using Duncan's multiple range test (Duncan, 1955).Mortalityconcentration response was estimated according to Finney (1971).
The use of IGR s is based on its effect on the chitin synthesis process.The obtained result that the insect growth regulator "Flufenoxuron" proved to be a synergistic additive to viral pesticides of Spodoptera littoralis is similar to that found in case of the silkworm, Bombyx mori by Arakawa ( 2002).However, the rate of enhance-ment of 204.08 fold with IGR additive is much less compared to 2000 fold in case of the silkworm.This could be attributed to differences among insects in the rate of interference with chitin synthesis of peritrophic membrane (PM).This explanation agrees with the theory of enhancement of baculovirus by protease in the enhancing protein as indicated by Lepore et al. (1996).Increasing the rate of mortality which is reflected by decreasing the LC 50 value of SpliMNPV in present results could be due to facilitating the virus invasion through the midgut epithelial cells by IGR's.2. Effect of either single or combined treatments of tested IGR's and SpliMNPV (PIB's), on some biological aspects of the host.
The mean larval duration in Table (2) recorded highest value for both Flufenoxuron, Triflumuron alone (27.3 ± 0.17 and 27 ± 0.29 days) compared to (25 ± 0.58 and 25.5± 0.16) days for the mixture.The virus alone treatment recorded 25.5 ± 0.12 days compared to the untreated control (24.5± 0.29 days).The larval weight demonstrated in Table (2) recorded insignificant differences between treatments and the untreated control.Obtained results agree with Abd-El Wahed et al., (2010) as SpliMNPV reduced larval duration as well as adult emergence of S. littoralis .Results about the reduction in pupation due to viral infection go in line with those obtained by Dutton et al. (2003).Also, the data in  insignificant result for either SpliMNPV alone, IGR's alone or the mixture of them .The data presented in Table (2) demonstrate that, the highest reduction in the pupation percentage (47%) was recorded in case of Triflumuron mixed with SpliMNPV followed by Flufenoxuron & Chlrofluazuron (LC 10 ) mixed with SpliMNPV (LC 50 ) (80%) compared to 100% in case of both the untreated control and IGR's alone.The adult emergence (Table 3) was remarkably reduced in case of Triflumuron alone (47%) followed by Flufenoxuron alone (73%), Teflubenzuron alone (87%), Flufenoxuron + SpliMNPV (88%) and Teflubenzuron+ SpliMNPV (97%),while it recorded 100% for the other treatments.

Histpathological effect on mid gut of treated larvae:
The histological structure of mid gut in normal larvae (fig. 1) is well documented (Chapman, 1988).The light microscope examination of S. littoralis treated with LC 50 SpliMNPV alone in (Fig. 2) Shows vacuolization of the columnar cells.The peritrophic membrane was considerably deteriorated.On the other hand, the larvae treated with the IGR Flufenxuron alone (Fig 3) Show exfoliation and vacuolization of the midgut epithelium.The peritrophic membrane was completely disrupted.However, treatment with the mixture SpliMNPV (LC 50 ) + Flufenxuron (LC 10 ) (Fig. 4) Shows a loss of the compact appearance of the muscular layer, vacuolation and exfoliation of the columnar cells.Many of the histological alterations reported in the present study for the midgut of S. littoralis larvae treated with IGR are similar to those reported by Thabit et al. (2010). Also, Federici (1993) found that the ingestion of toxicant by the insects releases a toxic peptide which binds to sites on the microvillar membrane of the mid gut causing cytolysis, which leads to paralysis and subsequently death of the insect.
same letter are not significantly different (p<0.05).

Fig. 2 :
Fig. 2: Photomicrograph of longitudinal section in the mid gut of late 6 th larval instar of S. littoralis treated as 2 nd instar larvae with LC 50 of SpliMNPV(X400).A: Vaculization of columnar cells.B: Shrinking of the peritrophic membrane.D: longtuidinal muscle layer L.M.L. C: Loss of striated border of epithelial cell.E: circular muscle layer C.M.L.

Fig. 3 :
Fig. 3: Photomicrograph of longitudinal section in the mid gut of late 6 th larval instar of S. littoralis treated with LC 50 of Flufenoxuron as 2 nd instar larvae (X400).A: The colomnar cell lost its compact appearance.B: Completely disrupted peritrophic membrane.C: Vaculization of midgut epithelial.

Table 1 :
Viral mortality among Spodoptera littoralis 2 nd instar larvae treated with LC 10 of IGR's mixed with different concentrations of SpliMNPV.

Table 2 :
Rate of pupation, adult emergence and adult longevity of S. littoralis treatment as 2 nd instar larvae with either insect growth regulators; SpliMNPV or their mixture.
Table (2) demonstrate that the pupal weight recorded ♦ Total tested larvae.