Synergistic role of pipronyl butoxide in improving the Spodoptera littoralis nucleopolyhedrosis virus effectiveness against Spodoptera littoralis and studying its histopathological biosafety

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Introduction
The Egyptian cotton leaf worm, Spodoptera littoralis (Lepidoptera: Noctuidae) is the most destructive, serious and widespread pest not only in Egypt but also in subtropical and tropical regions (Elmenofy et al., 2020).It is known as polyphagous insect as it infests and attack various economically important crops over the year causing economical yield losses in these crops (Mohamed et al., 2019).This insect develops high level of resistance against variety of chemical insecticides (Aydin and Gürkan, 2006).In addition, the excessive use of chemical insecticides leads to harmful effect on natural enemies and all other non-target insect, also they cause detrimental problems to humans, plant and animals and induce negative impact to environment.To avoid these chemical insecticides problems, there is great interest to encourage searching for safely alternative to chemical insecticides.Many biological control agents have been used for controlling insects, such as the use of the entomopathogenic fungi, bacteria and virus.Baculoviruses are the largest and most widely insect specific entomopathogens belonging to family Baculoviridae, they are promising alternatives to decrease dependency on deleterious chemical insecticides.They have greatest potential for use as microbial insect pest control agents against several insect pest (Lacey et al., 2015).They are safe and environmental friendly bio agents (Cheng and Lynn, 2009), also they are very specific as they infect only closely related species.Although baculoviruses infect more than 600 insect species and used successfully in controlling different insect pests (El-Salamouny et al., 2003), its use as bio-control agent is limited compared to pest control agents (Wood, 1996).Limitations to the use of baculoviruses include the cost of production, refrigerated storage and a relatively slow speed of kill (Dáder et al., 2020).Great efforts have been made to overcome these disadvantages by the synergistic combination with low concentrations of synergistic agents and using high molecular technology.Several studies have proved that the use of synergists in addition to insecticides is considered the best option for enhancing the insecticides efficacy and for accelerating its toxic effect (Corbel et al., 2010).Synergists have a very fundamental role in increasing the efficacy of insecticide as they inhibit the natural defense of the insects by altering the insecticide detoxification enzymes (Moores et al., 2009).
Synergists also act as allosterically in which they cause changing in the shape and function of protein (enzyme) by altering the binding site and combine to point other than the chemical active site (Gunning et al., 1999).Piperonyl butoxide (PBO) was the first and most effective developed synergist.As a result of its limited toxicity to mammals, humans and birds, it is used in mixture with insecticides for household, medical and veterinary uses (Breathnach, 1998 andKeane, 1998).PBO is an organic compound used as a successful synergist and main component of pesticide formulations.Although it has no pesticide activity of its own, it enhances the effectiveness of several pesticides (Bingham et al., 2011).The prevailing situation of PBO among others synergists has been attributed to several factors.For example, it has been proved for its synergistic effect with a wide range of major classes of insecticides such as pyrethrins, pyrethroids, carbamates, organophosphates, and many insect growth regulators and insecticides formulation (Keane, 1998).Farmers and farm animals are exposed to environmental pollutants including agro pesticides through ingestion, inhalation or dermally, which are then distributed through the circulation to affect different organs' tissues and cells such as liver, kidneys, lungs, and heart.Experimental animals could be used as surrogate indicators of toxic exposures, and as a tool to evaluate risk to human health and to measure the toxicity degree of substances for humans and animals (Manfo et al., 2020 andOkail et al., 2020).Liver and kidneys are largely known as the most important organs involved in detoxification of different elements, via metabolism and excretion.Agro pesticides induced Hepatotoxicity and nephrotoxicity have been illustrated by many evaluations in laboratory animals (Manfo et al., 2020).Liver and kidney damage is often accompanied by oxidative stress or inflammation (Rapa et al., 2019 andRoghani et al., 2020).This study aims to improve and develop the effect of the nucleopolyhydrosis virus on the cotton leaf worm S. littoralis in combination with a biosafety synergistic agent (PBO).

Material and Methods
Test organism: Laboratory strain of the Egyptian cotton leaf worm S. littoralis was obtained from Insect Pathogen Unit-Plant Protection Research Institute-Agriculture Research Center.The larvae were fed on semisynthetic diet described by Shorey and Hale (1965) and reared at 25±2 o C and 65±5 RH with natural photoperiod (El-Defrawi et al., 1964) under highly controlled condition to avoid any contamination.

Biological insecticide: Spodoptera littoralis nucleopolyhedrosis virus (SlNPV)
Preparation of virus isolate Virus isolate propagation Propagation of virus was carried out by inoculation of S. littoralis 3 rd instar larvae (Li and Skinner, 2005) with SlNPV isolate provided by Ahmed et al. (2016) by surface contamination of artificial diet.The inoculated larvae were distinguished every day to observe the contaminated ones based on viral disease manifestations.Infected larvae were examined preliminary by naked eye then by light microscope with Giemsa staining method according to Mustafa et al. (2001) for detection of viral occlusion bodies (VOBs) .After the observation, the contaminated larvae were collected and preserved in -20 o C till purification of VOBs.

Viral occlusion bodies (VOBs) purification
VOBs were purified by using Sudhakar et al. (1997) method with slight modification.Infected larvae showing virus infection symptoms were transported to micro centrifuge tube and homogenized in 300µl distilled water.The homogenate was filtered through chess cloth and subjected to 60% wt/vol sucrose layer then centrifuged at 10000 rpm for 30min.VOBs formed band on the top of the layer, this band was collected and subjected to 40%wt/vol sucrose layer and centrifuged at 10000 rpm for 30min.VOBs band which again formed near the bottom of the filtrate was collected and washed with distilled water.All previous steps were done at 4 o C. Purified VOBs were suspended in distilled water and stored at -20 o C. Then the confirmation of purification effectiveness method caused by making OBs slide and examined under light microscope by Giemsa staining method according to Mustafa et al. (2001).The concentration of VOBs/ml was determined by counting their number using Neubaur Hemocytometer (Alves and Moraes, 1998).Five concentrations were prepared from VOBs mother suspension by serial dilution to be used in toxicity experiment.

Toxicity test
Five SlNPV concentrations (10 10 , 10 9 , 10 8 , 10 7 and 10 6 POB/ml) were tested against 2 nd and 4 th instar larvae.The experiment was conducted in plastic cups containing semisynthetic diet by applying diet surface treatment procedure (Addy, 1969), in which 250µl of the virus suspension was pipetted on the semisynthetic diet surface, distributed then left to dry.The virus suspension was replaced by distilled water in the control treatment.Larvae of 2 nd and 4 th instar were allowed to feed on treated diet for 48 hour then transfer to clean cup contain untreated diet.Three replicates each of ten larvae per virus concentration plus three replicates for control were used.All treatments were incubated at 25°C, 60-70% RH and the larval mortality was recorded at two days for a period of ten days.The mortality percentage and lethal concentration (LC 50 ) were calculated according to Finney (1971) using Ldp line software (Bakr, 2000).

Synergistic material: Pipronyl butoxide (PBO)
Piperonyl butoxide (99%) was purchased from Sigma-Aldrich Company as oil and solved in analytical grade acetone to prepare its tested concentrations (Huang et al., 2010).

Pipronyl butoxide toxicity test
Concentrations of 0.1, 0.2 and 0.3% were prepared from PBO by using analytical grade acetone for testing their toxicity against S. littoralis 2 nd and 4 th instar larvae according to Diab (2011) with slight modification.The two tested instar were placed in cups containing treated artificial diet with PBO and allowed to feed for 48 hour, then transferred to clean cups containing untreated diet and observed daily.Thirty larvae for PBO concentration plus 30 larvae treated with acetone only as a control were replicated 3 times.Larval mortality was listed after 24 hours till 96 hours (Radova, 2010).

Histopathological evaluations of SlNPV and PBO biosafety Animals
Fifty-six adult male Sprague-Dawley rats weighing 162±13 g were used in the present experiment.Rats were brought from the National Research Center (NRC), Giza, Egypt, and then housed in plastic cages (7 rats/cage) at normal atmospheric temperature (26 ± 3 °C) with normal 12-h light/dark cycle.Diet and water were provided ad libitum.Rats were allowed to be acclimatized for one week prior to experimental use to exclude any infection.All experimental animal studies were approved by

Suez university research ethics committee
Experimental design: Animals were divided into 8 groups as following Group I: The normal control group.Group II: Rats were given a single oral dose of SlNPV 1 x 10 5 PIB (1ml/100gm body weight).Group III: Piperonyl butoxide (PBO) group, this group was subdivided into 3 subgroups, in each of which rats were given a single oral dose of PBO as 0.1.0.2, or 0.3% mg/kg b.w (1 ml/100 gm b.w).Group IV: PBO+ SlNPV group, this group was subdivided into 3 subgroups, in each of which rats were given PBO as 0.1, 0.2, or 0.3% mg/kg b.w + a single oral dose of SlNPV 1x 10 5 PIB.All animals were observed for 21 days after all treatments, and then were sacrificed by cervical dislocation, dissected out and then their livers and kidneys were removed and were cut into small pieces.Liver and kidneys specimens were fixed in 10% neutral formalin, dehydrated, cleared in xylene and embedded in paraffin wax for histological and immunohistochemical examinations.Moreover, sera were obtained for biochemical analyses.

Histological and morphometric examinations
Sections of 5 μm thickness were stained with hematoxylin and eosin (Drury and Wallington, 1980).The criteria for scoring liver injury depended on vacuolization and pyknosis of hepatocytes, activation of Kupffer cells and sinusoids enlargement.Injury of kidney tissue was evaluated depending on degeneration of Bowman capsules, glomeruli, proximal and distal tubules, in addition to, vascular congestion and interstitial edema.Five nonoverlapped fields from each specimen (X400) was examined to score liver and kidney histomorphometric alterations.Histopathological scoring of each specimen was done via a scale ranging from 0 -3 (0: none, 1: mild, 2: moderate, and 3: severe) for each criterion.The total score was 9 for both liver and kidney tissues (Ibrahim et al., 2010).

Immunohistochemical examination
For immunohistochemical localization, 4 μm thickness paraffin sections were stained by using the avidin-biotin peroxidase method.Sections were incubated with primary antibody for 60 min for each of: 1-Caspase-3 according to Jackson et al. (2008).2-Proliferating cell nuclear antigen (PCNA) immunoreactivity was performed according to Eldridge and Goldsworthy (1996).
Then slides were incubated with a biotinylated secondary antibody, then incubated with horseradish peroxidase conjugated with streptavidin for further 15 min, followed by adding 3-amino-9ethylcarbasole (Dako Cytomation, USA).

Image analyses
Semi-quantitative scoring system (Image J software, Java based application for analyzing images) was used to analyze digital images.The immunohistochemical-stained sections were analyzed in 10 microscopic fields under high-power field (X400) microscope to calculate each of the following: a.The degree of reaction of Capase3 and TNF-α area were recorded.Percentage of positive stained area (%) was calculated as mean of 10 fields/slide.b.PCNA-Labeling Index (PCNA-LI): Randomly, five microscopic fields per slide were chosen.The PCNA-LI for each field was evaluated as a percentage of immuno-labeled cells to all basal cells.For each section, the mean ±SD was calculated (Abdel-Dayem, 2009).c.Calculation of Apoptotic index (AI)(%): AI = (number of apoptotic cells/the total number of counted cells) × 100%.

Biochemical studies
Sera were obtained through blood samples centrifugation, and then stored at -20°C until using to estimate the following parameters: 1-The lipid peroxidation marker, malondialdhyde (MDA) according to Ohkawa et al. (1979).2-The total anti-oxidant capacity (TAC) according to Sedlak and Lindsay (1968).

Cell culture Sulforhodamine B (SRB) cytotoxicity assay
For more confirmation of cytotoxic effects of PBO different concentrations used in the presented study, the effect of PBO on cell viability by SRB assay to evaluate the growth inhibition colorimetrically, as the cell numbers is evaluated indirectly by staining total cellular protein with SRB dye according to Skehan et al. (1990).SRB assay was applied on two different cell lines OEC (Oral epithelial cell) and Vero (Green monkey kidney).Cell lines were obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt).Cells were maintained in DMEM media supplemented with 100mg/mL of streptomycin, 100 units/mL of penicillin and 10% of heat-inactivated fetal bovine serum in humidified, 5% (v/v) CO 2 atmosphere at 37 °C.

Synergistic effect of PBO
According to PBO toxicity test and histopathological evaluation of SlNPV and PBO biosafety studies, 0.1% PBO concentration was used for examination its effect on S. littoralis detoxification enzymes and synergistic potential toward SlNPV in vitro.

Estimation of enzymes activities
The 4 th instar larvae were allowed to feed on treated artificial diet with PBO for 1h for detection the disruption the detoxification enzymes (Dadzie et al., 2017), then collected and frozen in -20 o C till enzymes estimation test.

Sample preparation
Treated 4 th instar larvae were homogenized in distilled water (50mg/1ml).Homogenates were centrifuged at 8000 rpm for 15 min at 5 o C in a refrigerated centrifuge.The deposits were discarded and the supernatants were kept in a deep freezer till use.

Estimated enzymes Acetylcholinesterase (AchE)
AchE activity was measured according to the method described by Simpson et al. (1964) using acetylcholine bromide (AchBr) as substrate.

Lactate dehydrogenase (LDH)
LDH activity was determined according to the method derived from the formulation recommended by the German Society for clinical chemistry (Deutsche Gesellschaft Fur Klinische Chemie) (DGKC, 1972).
Oxidase Oxidase activity was determined according to Hansen and Hodgson, (1971) with slight modification.

Glutamic pyruvic transaminase (GPT) and Glutamic oxaloacetic transaminase (GOT)
GPT and GOT were determined according to the method of Reitman and Frankel (1957).

Synergistic assay of PBO with SlNPV LC 50
Viral LC 50 calculated previously was tested individually against two tested instar larvae and the mortality rate recorded daily and also combined to 0.1% PBO with volume ratio 1:1 then pipetted and distributed on the artificial diet.The treated artificial diet was introduced to 2 nd and 4 th instar larvae for 48 hours then replaced by untreated diet.Acetone was applied as a control.All treatments were kept at a constant temperature 30°C and larval mortality was recorded daily.

Statistical analysis
Statistical analysis was performed using SPSS v.16.Results were calculated as mean ± standard deviation (SD) and all statistical comparisons were made by means of one-way ANOVA test followed by Duncan's multiple range test post hoc analysis.A P value (P > 0.01) considered significant.

External symptoms of insect infestation with SlNPV
Infected larvae were examined by naked eye showing that they were smaller than normal one, slow motion, pale and milky color cuticle, swollen larval body and Liquefied larvae releasing millions of VOBs to the environment (Fig .1)

VOBs Light microscope observation
After virus propagation and purification, the presence of VOBs as polyhedral and negative stain particles was confirmed under light microscope (Fig. 2)

Toxicity test of SlNPV
The results obtained from toxicity test of SlNPV against 2 nd and 4 th instar larvae of S. littoralis (Table 1&2 and Fig. 3&4) demonstrated that larval mortality was directly proportional to viral concentration and time elapsed post treatment.The high viral concentration used 10 10 PIB/ml caused 93% which is considered the maximum mortality rate for 2 nd instar larvae, and 83% for 4 th instar larvae after 10 days post treatment.The remaining concentrations produced mortality percentage ranged from 60 -83 % and 56 -77 % for 2 nd and 4 th instar larvae respectively.It is clear from the data that 2 nd instar larvae were more susceptible to all tested concentrations than 4 th instar ones.

Estimation of enzymes activities
The activities of AchE, GST, LDH, GOT, GPT, MFO, αesterase and βesterase of S. littoralis treated with 0.1% of PBO were assayed and differences in the metabolic enzyme activities between the control and treated larvae were illustrated in Table 5, 6 &7.The mean level of all enzymes were significantly lower than control except GPT, MFO and LDH.The activity ratio for these enzymes were 0.41, 0.71, 1.40, 0.99, 1.63, 1.26, 0.84 and 0.85 respectively.Histological examination of liver parenchyma of both control group and SlNPV treated group revealed a normal liver histoarchitecture with arranged hepatocytes in hepatic cords surrounding the central vein and separated by free sinusoids.The sinusoids were lined with endothelium containing phagocytic Kupffer cells.Each individual hepatocyte was polyhedral in shape with central rounded nucleus, where some hepatocytes were binucleated.kidney sections of both control group and SlNPV treated group showed the typical structure of the renal corpuscles and tubules as SlNPV treated rats did not show any histological changes by comparing with control group (Figs 6a : 6d).Treating rats with the PBO in three different doses led to the progression of histopathological changes in a direct relation between the high concentration of the PBO and the severity of histopathological changes in both kidney and liver tissues.Rats treated with PBO at dose levels 0.3% induced marked histopathological alterations and significant increase in injury score in liver and kidney when compared with control group and 0.2%, 0.1% treated groups.liver tissues revealed leucocytic infiltration, blood venues congestion and cytoplasmic vaculation.Hemorrhagic foci, necrotic cells with pyknotic nuclei were also observed.Fatty degeneration was observed markedly.Moreover, it was also noted that, animals co-treated with 0.3% PBO plus SlNPV didn't reveal any difference when compared with rats treated with 0.3% PBO alone.Examination of renal tissues of 0.3% PBO treated rats showed periglomerular and interstitial necrosis associated with inflammatory cells infiltration.Congested blood vessels and interstitial hemorrhage, tubular degeneration and vaculation were abundant, glomerular atrophy, widening in Bowman's capsule and tubular dilatation.There was the same degree of histopathological changes in the parallel group that dual treated with 0.3% PBO plus SlNPV, without much difference (Figs 6e: 6h).liver and kidney tissues of rats treated with 0.2% PBO, and 0.2% PBO plus SlNPV showed marked improvement in both liver and kidney tissues architecture, in addition to, a marked decrease of injury score in liver and kidney when compared with 0.3% PBO, and 0.3% PBO plus SlNPV respectively, as the degenerative changes were markedly declined, but the liver tissues still displaying vaculation and fatty degeneration, while a mild leucocyte infiltration and vaculation in kidney tissue (Figs 6i : 6L).Continuing the dose dependent gradual improvement, microscopic examinations of 0.1% PBO, and 0.1% PBO plus SlNPV(showed advanced degree of improvement of liver and kidney tissues, and injury score as hepatocytes rearranged in radiating strands, showing normal nuclei and cytoplasm which didn't include any water vacuoles or fat droplets .Moreover, kidney tissues returned to the classical appearance as corpuscles and tubules appeared more or less normal like when compared with control animals (Figs.6m: 6P) .The histopathological alterations in different animal groups were summarized in table (8).

Immunohistochemical Results
i. Capase3 Immunoexpression Immunohistochemical examinations of livers and kidneys sections of control and SlNPV-treated rats show negative immunoreactivity of Caspase-3.Rats treated with 0.3% PBO, or 0.3% PBO plus SlNPV revealed a significant increase in Caspase-3 immunoexpression in the cytoplasm of both hepatic and renal cells as indicated by dark brown color when compared with all other groups.
Considerable reduction in Caspase-3 immunostaining was noted in livers and kidneys of 0.2% PBO, or 0.2 % PBO plus SlNPV.Microscopic observation of hepatic and renal tissues of rats treated with 0.1% PBO, or 0.1 % PBO plus SlNPV displayed weak reaction for caspase3 in the liver and kidney (Fig 7 and table 8).
ii. PCNA Immunoexpression Liver and kidney tissues stained for PCNA antibodies of both normal control group and SlNPV treated groups showed limited immune reaction in hepatocytes, while PCNA-positive cells were not found in the kidney sections.In 0.3% PBO, and 0.3% PBO plus SlNPV treated rats, both liver and kidney cells showed abundant nuclear PCNA-positive immunostaining with a significant increase in PCNA-LI, as proliferating cells in hepatic parenchyma, while in renal tissues PCNA-positive cells were demonstrated in interstitial tissue and within tubular cells.Examination of the 0.2% PBO, or 0.2 % PBO plus SlNPV treated groups revealed a significant decrease in PCNA-LI in both liver and kidney tissues when compared with 0.3% PBO, or 0.3% PBO plus SlNPV treated rats, where kidney cells demonstrated a marked reduction of positive stained nuclei, but some hepatocytes were still positively stained.Examination of the liver and kidney sections of 0.1% PBO, or 0.1 % PBO plus SlNPV groups showed a limited PCNA-positive nuclear immune reaction when compared with other treated groups (Figure 8 and table 8).
iii-TNF-α immunoexpression Regarding TNF-α immunoexpression, it was not surprising to observe the significant increase of TNFα cytoplasmic expression in hepatic and renal cells of rats that were treated with 0.3% PBO, or 0.3% PBO plus SlNPV when compared with other groups.However, such strong positive TNF-α immunoreactivity was declined in animals subjected to the treatment of 0.2% PBO, as well, in rats subjected to the dual treatment 0.2% PBO plus SlNPV .The minimal immunoexpression of TNF-α was observed in each of 0.1% PBO, or 0.1% PBO plus SlNPV which revealed insignificant difference in TNF-α reactivity with both control and SlNPV treated group neither in liver nor in kidney (Fig. 9 and table 8).    9 Sera of both control and SlNPV treated groups showed normal values of MDA and TAC with insignificant difference between control group and SlNPV group neither in MDA nor in TAC values.Treating animals with 0.3% PBO, or with 0.3% PBO + SlNPV induced a significant increase of MDA and a significant reduction of TAC when compared with other groups, however, no statistical differences were detected between 0.3% PBO, and 0.3% PBO + SlNPV treated groups.Improvement of oxidative statues were achieved by reducing PBO treatment dose, as at rats group treated with 0.2% PBO alone or in combination SlNPV resulted in reducing MDA and increase in TAC, and then ideal oxidative status was detected in animals treated with 0.1% PBO, or with 0.1% PBO + SlNPV as MDA and TAC values were nearly similar to those of control and SlNPV groups (table 9).

In-vitro SRB cytotoxicity assay
Data in table 10 showed OEC and Vero cell lines treated with different concentrations of PBO (0.3%, 0.2%, 0.1%) compared with a control of untreated cells.Data recorded revealed that viability percentage and PBO dose concentration were related.OEC and Vero cells that were treated with 0.3% PBO revealed the highest cytotoxic activities and the lowest viability percentage, when compared with cells treated with 0.2% PBO, and 0.1% PBO, while 0.2% PBO treated OEC and Vero caused higher viability percentage.The highest cell viability percentage in both OEC and Vero cells was achieved in 0.1% PBO treatment that increased significantly when compared with both 0.2% PBO, and 0.3% PBO.Insignificant difference when compared with control untreated group, significant differences at (p < 0.01).

Discussion
Viral symptoms obtained in this study was in agreement with Tanada and Kaya (1993), in which the larval cuticle appear pale and milky color and this symptom can be associated with infection of the hypodermal cells.Symptom appeared in the intermediate stage of infection was swollen larval bodies which could be due to hypertrophy that had occurred in the cells and increasing of the nucleus (the main organ of virus reproduction) size as a result of viral infection.Liquefaction is the final and most characteristic symptom of viral infection in which the larval body appeared full of millions of VOBs causing complete damage and destruction of all larval tissues and organs, then release to the environment to spread the infection and infect other hosts (El Salamouny, 2007).The viral infection begins when the larvae ingested food contaminated with occlusion bodies (OBs), which reached the larval midgut and dissolved by the alkaline digestive juice of the midgut lumen, (Moore and Manousis, 1986).SlNPV virions release and spread within the midgut to cause larval infection (Petrik et al., 2003).
Our SlNPV toxicity results confirm those of Abd El-Aziz (1999) and Mahmoud et al. (2009) who proved that the 2 nd instar larvae of S. littoralis was highly susceptible to NPV than the 4 th instar larvae.Sensitivity of the S. littoralis younger instar to NPV was reported by several authors (Pawar and Ramakrishnan, 1975;Klein and Podoler, 1978;Seufi andOsman, 2005 andSeufi, 2008).Stairs (1965) found that the susceptibility to NPV decreased as larvae of Malacosoma disstria grew older.In parallel Duan and Otvos (2001) reported that mortality was higher as younger instar larvae of Choristonura fumiferana were used, Wilson et al. (2000) also mentioned that 2 nd instar larvae were more susceptible to AcMNPV than 4 th instar larvae of cabbage looper, Trichoplusia ni.Also, Cherry et al. (2000) proved that the susceptibility of Helicoverpha armigera to HearNPV depends on the larval age, and Bourner et al. (1992) emphasized that earlier instar has speed of kill by AsNPV than older instar larvae of Agrotis segetum Our results showed that high NPVs concentrations caused high mortality rate and this conclusion is in line with Sutanto et al. (2014) who studied the infectivity of SlNPV against 1 st and 2 nd instar larvae of S. littoralis and found that mortality percentage of the larvae was greater at the highest concentration of the virus than at lowest concentrations.Baculovirus is potential microbial insecticide play a major role in the suppression of a variety of different types of insects.There are several reviews in using baculovirus for controlling different insect family (Szewczyk et al., 2006).The most serious disadvantage of using baculovirus as effective insecticide is slow speed of kill which give chance for the pests to infest the crops and cause complete damage to them.The long time required by baculovirus to kill insects is due to insect metabolic resistance which is considered common defense mechanism based on the enzymatic system that protect insects.Synergists are chemical compounds which significantly improve the efficacy of insecticides by increasing lethality and effectiveness of them.Using of the synergists in addition to insecticides is considered the best way to overcome resistance to insecticides by damaging binding site of the detoxification enzymes and make the insecticides more toxic to pests (Corbel et al., 2010 andPennetier et al., 2013).The most common and effective insecticide synergist used is PBO.Our results proved that when S. littoralis larvae were pre-treated with PBO before being treated with the viral isolate, there was significant enhancement of the larval susceptibility to the virus and increasing in the larval death in a short time .These results are in agreement with Cakir et al. (2008) who investigated the effect of PBO on the biological activity of three insecticides and showed that PBO extensively promoted their efficacy against different housefly population.In parallel, Dáder et al. (2019) examined a wide range of insecticides with different modes of action against Philaenus spumarius and indicated that, some of these insecticides are not effective when applied alone and their efficacy was enhanced by using PBO which caused a 3-fold increase in the mortality of P. spumarius nymphs.Dadzie et al. (2017) investigated the effect of PBO on the susceptibility of Anopheles gambiae mosquitoes treated with pyrethroid insecticides and found that when the mosquitoes were pre-exposed to PBO before exposure to deltamethrin or permethrin, there was a significant enhancement of their susceptibility to these insecticides.Ahmed and Vogel (2016) evaluated the synergistic effects of PBO on the toxic activity of 16 insecticides against Aedes aegypti adults and confirmed the strongly synergistic effect of PBO with all insecticides types used.AchE is key enzyme in detoxification of neurotoxic insecticides (their target is nervous system) and overproduction of AchE catalyzes the breakdown of acetylcholine and some other choline esters which acts as neurotransmitters (Byrne and Toscano, 2001).Nonspecific esterase (αesterase and βesterase) is another important enzyme, detoxify several pesticides by two main ways, hydrolysis of the ester bond and binding of the pesticide to the active site of esterase (Crow et al., 2007).GST plays critical role in detoxification of insecticides by conjugating reduced glutathione to the electrophilic centers of insecticides.(Enayati et al., 2005).Multi-function oxidases is one of the most important enzyme of insect resistance mechanism (Qi et al., 2016), it introduces one or more polar groups into the substrate in Phase I metabolism and make them suitable for conjugation at Phase II reaction of detoxification (Kranthi, 2005).LDH is an important glycolytic enzyme that is present approximately in all tissues (Kaplan and Pesce, 1996).It is involved in carbohydrate metabolism and has been used as an indicator for exposure to chemical stress (Diamantino et al., 2001).LDH is also a parameter used widely in toxicology and clinical chemistry to diagnose cell, tissue, and organ damage.GOT and GPT are also named aspartic transeferase (AST) and (ALT).They are key enzymes in the formation of amino acids, gluconeogenesis, metabolism of the nitrogen compound, and associated with protein synthesis (Mordue and Goldworthy, 1973).All these enzymes are involved in insect's defense process due to their genetic diversity, broad substrate specificity and catalytic versatility.It is clear from the results that the concentration of PBO used in this study enhance the effectiveness of viral isolate by making disturbance of the tested insect enzyme either with increase or decrease, and this indicates that toxic effect of PBO affects the synthesis or function of these enzymes by altering cytomorphology of the cells (Nath, 2000).Our findings are in line with Todeschini et al. ( 2017) who measure enzymes activities in Italian and Carniolan honey bees treated with PBO and some of its derivatives and found that only esterase enzyme activity was inhibited while other enzymes like MFO, GST and AchE were not affected and seem not to be targeted by the tested synergists.Pereira et al. (2014) studied the effect of PBO on the toxicity of organophosphate temephos against Aedes aegypti and showed that PBO cause inhibition of the total esterase activity while some concentration caused activation of MFO enzyme.Matowo et al. (2010) investigated the combined effect of permethrin and PBO on Anopheles arabiensis and their action on enzymes activity and noticed inhibition of the activity of both and B-esterase and concluded that PBO increase the efficacy of permethrin.In the same line Joffe et al. (2011) determined the efficacy of seven natural compounds compared with PBO in synergising pyrethrum in houseflies and proved that PBO was the most effective synergist and this was associated with inhibition of esterase and oxidase enzyme activity.Enhancement and inhibition in the enzyme activity are indication of dysfunction and alternation of the metabolic enzyme system.Increase the activity of some enzyme like GPT is related to increase of lipid peroxidase which mean generation of free radical in cell membrane resulting in cell damage (Icen et al., 2005).GOT and GPT enzyme are biomarker for determination of tissue injury of vertebrates (Patil et al., 2003), increase of the activity of them or one of them mean impairment in amino acid metabolism.Increasing and decreasing of enzyme activity recorded in our study may be due to depression or mutations of the regulating genes responsible for biosynthesis of polypeptide chains constructing this enzyme (Hassanien et al., 1996).The presented histopathological results showed that no statistical differences were recorded between 0.3% PBO + SlNPV, 0.2% PBO + SlNPV or 0.1% PBO + SlNPV, with 0.3% PBO, 0.2% PBO or 0.1% PBO treated groups respectively within all histological, morphometric, immunohistochemical, biochemical and cytological studies.These findings suggested that SlNPV with our suggested dose is safe and has not any pathological effects on rat tissues and cells.In addition, the ineffective impact of PBO on the nature and activity of SlNPV.The safety of SlNPV on experimental animals was previously discussed in several studies.Döller et al. (1983) reported that NPV didn't induce any pathological alterations in neither lymph nodes nor 125I-labeled polyhedrin binding capacity in NPV-fed pigs when compared with control animals.Ashour et al. (2007) studied the histopathological effects of SlNPV on stomach, intestine, liver, kidney, brain, spleen and lung tissues in SlNPV-orally treated male and female rats.The findings reported by Ashour et al. (2007) revealed that no histopathological changes induced by SlNPV oral or intraperitoneally injection.Moreover, no mortality over a 21 day-long observation period, no indications of toxicity were observed by hematological and clinical biochemical analyses.Infectivity of HaNPV in intravenous infected rats has been reported by Zhao et al. (2019) who found that no obvious toxic symptoms or mortality were observed during the experimental period.HaNPV didn't induce any amplification bands by PCR test, histopathological changes or specific lesion in brain, blood, lymph nodes, liver, spleen, lung, kidney, and cecum contents by PCR test.(Zhao et al., 2019).Current histological and cytotoxicity results showed the pathogenicity of 0.3% and 0.2% PBO as there were moderate degrees of liver and kidney tissues damages, which were clarified by immunohistochemical observations as apoptosis and inflammation through the significant increase of immunoreactivity of Caspase-3, PCNA-Li and TNFα respectively, then with serum biochemical analyses as oxidative stress.Pathological effects of increased doses of PBO on animals were reported by several authors.Fukuyama et al. (2013) suggested that PBO cause T-lymphocyte apoptosis via initiation of Caspase-3 activities in different cells.Phillips et al. (1997) reported that histological examinations of liver tissues of PBOtreated rats showed necrosis in hepatocytes.In addition, they observed that the necrosis severity was dose-dependent.PBO can damage DNA via oxidative stress in rats (Muguruma et al., 2007 andArslan et al., 2016).Therefore, the histopathological impacts of PBO in our study could be related to oxidative stress induced by PBO.Oxidative stress contributes to the increase in caspase-3 immunoreactivity, and proteolytic activation and subsequent DNA fragmentation, which eventually results in apoptotic cell death (Kim et al., 2000).Role of oxidative stress in disturbances of TNF-α, one of the most important inflammatory cytokines, has been evidenced.TNF-α is increased in the early stage of inflammation and initiate the ROS production (Zhu et al., 2020).Ibrahim et al. (2019) found that, the increased ROS and proinflammatory cytokines induce renal dysfunction and damage the increased TNF-α is associated with the increase of oxidative stress.TNFalpha activates different intracellular pathways to control inflammation, proliferation and apoptosis in both liver and kidney (Schwabe andBrenner, 2006 andVielhauer andMayadas, 2007).Regarding PCNA overexpression in liver and kidney tissues, it has been reported that PCNA formation takes place after oxidative stress in order to repair and attenuate the oxidative DNA damage (Balajee et al., 1999 andTsai et al., 2017).Dose concentrationdependent decreased histopathological changes were observed by comparing 0.3% and 0.2% PBO treated rats, which may also explain the significant depletion in pathogenicity of 0.1% PBO treated rats, where the liver and kidney tissues seems to normal control tissues.

Conclusion
In this study, the ultimate goal was to overcome on the viral slow speed of action which limits its application despite its great effectiveness in plant pests control and use as a safe and environmentally friendly biocontrol agent by using PBO as a synergistic agent with SlNPV.The results showed that 0.1% of PBO had a high degree of tissue biosafety and proved its synergistic effect, so that, it could be used as a synergistic agent with SlNPV for enhancing the viral activity to be used as a potential biocontrol agent

Fig.( 6 )
The histopathological changes in liver and kidney sections among different experimental groups (H and E, X400).(6a) Liver section from a control rat showing normal hepatic histoarchitecture with normal hepatocytes (h), sinusoid (s) and vascular channels as central vein (cv).(6b) Liver section of SlNPV treated rat showing normal hepatic tissue with normal portal area with normal appearance of portal vein (pv), and bile duct (bd).(6c) Kidney tissue of control rat showing the classic histological structure of renal tissue with normal glomeruli (G) and tubules (t).(6d) Kidney section of rat from SlNPV group showing the normal histological appearance of renal parenchyma.showing congestion of renal blood vessels (arrows).

Fig. 6 :
Fig. 6: (6i) Liver section of a rat treated with 0.2% PBO showing fatty degenerative changes (arrows) in some hepatocytes and limited hemorrhage (h).(6j) Liver section from 0.2% PBO + SlNPV treated rat showing severe vacuolated hepatocytes (arrows) with pyknotic nuclei.(6k) Kidney section of 0.2% PBO treated rat showing slight vaculation of epithelial lining some renal tubules and intertubular few inflammatory cells infiltration (arrows).(6L) Kidney section of 0.2% PBO + SlNPV treated rat showing vaculation of epithelial lining renal tubules (arrow).(6m) Liver section taken from 0.1% PBO treated rat showing normal architecture with normal appearance of hepatocytes (h), sinusoid (s) and vascular channels as central vein (cv).(6n) liver section taken from 0.1% PBO + SlNPV showing normal architecture of portal area with normal appearance of portal vein (pv), and bile duct (bd).(6o) Kidney section of rat from 0.1% PBO treated group showing normal renal tissue with no histopathological changes.(6p) Kidney section of a rat from 0.1% PBO + SlNPV treated group showing no histopathological changes in renal tissue.

Table - 7: Effect of PBO on α-estearase and β-esterase activities of 4 th instar larvae of S. littoralis.
SlNPV treated rats, which indicates the infectivity of SlNPV on liver and kidney.