Comparative in Vitro Evaluation of Three Geographically Different Isolates of Nucleopolyhedrovirus of Spodoptera littoralis in Egypt

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the interaction between insects and their environment. The goal of the journal is to advance the scientific understanding of mechanisms of toxicity. Emphasis will be placed on toxic effects observed at relevant exposures, which have direct impact on safety evaluation and risk assessment. The journal therefore welcomes papers on biology ranging from molecular and cell biology, biochemistry and physiology to ecology and environment, also systematics, microbiology, toxicology, hydrobiology, radiobiology and biotechnology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
The cotton leafworm, Spodoptera littoralis (Biosd.)(Lepidoptera: Noctuidae), is a serious polyphagous insect pest in Egypt.It attacks numerous economically important crops throughout the year.Conventional insecticides were successful in controlling insect pests.However, use of these chemical pesticides has led to several problems, including environmental pollution and endangered human health, such as cancer and several immune system disorders (Devine and Furlong, 2007).
Due to extensive using of insecticide groups, many populations of S. littoralis have acquired resistance towards most of them (Alford, 2000).The problems and hazards that have arisen as a result of using conventional insecticides were incentives for the search of alternative control agents.Microbial control agents are a primary means of biological control for insect pests.The use of microbial control agents is targeted for a particular pest species.The entomopathogens that have most been used in biological control include representatives of bacteria, fungi, viruses, nematodes, protozoa and insect growth regulator (Dent, 2000, Abdel-Aziz, 2012, Bakr et al., 2013and El-Sheikh, 2017) Baculoviruses are considered to be the largest and most broadly studied insect viruses.They are infectious for arthropods, particularly insects of the order Lepidoptera.Baculovirus infections have been reported in over 600 insect species of the orders Hymenoptera, Diptera, Coleoptera, Neuroptera, Trichoptera, and Thysanura, as well as in the Crustaceae order Decapoda (Murphy et al., 1995).Baculoviridae includes nuclearpolyhedrovirus (NPV) which has polyhedron-shaped occlusion bodies.The baculovirus isolates have a limited host range, and infect only closely related species as for insects mostly of order Lepidoptera (Moscardi, 1999).Aim of the present investigation is to evaluate the effect of three Egyptian isolates of baculovirus against S. littoralis (Boisd.), to finding for bioinsectsid nuclearpolyhedrovirus isolate (s) with better insecticidal characteristics.Fergani, (2015) andEl Sayed, et al., (2016).

Virus isolate, virus propagation and viral occlusion bodies (VOBs) purification:-
The original virus isolate was obtained from diseased S. littoralis larvae collected manually from cotton, tomato, and maize fields in three Governorates, El-Qalioubia, Al-Fayoum and El-Beheira.The larvae showed baculovirus infection symptoms were brought to our laboratory and examined to confirm the presence of virus by light microscope with Giemsa staining according to (Mustafa, et al, 2001), in which a thin smear of infected worm tissue was prepared on glass slide and dried in air.The smear was immersed for 1-2 min in Giemsa, rinsed under running tap water for 5-10 sec then the smear was stained for two hours in 10% Giemsa stain (10g of Giemsa dissolved in 100 ml.distilled water), the dye was rinsed off in running tap water for 5-10 sec and allowed to dry in air then examined under light microscope to detect the Occlusion Bodies (OBs).After the examination the diseases larvae kept at -80 o C until the purification of OBs (polyhedra).
The propagation of the virus isolate was performed by inoculation of the 3 rd instar larvae of S. littoralis with SlNPV isolate which collected from the field and tested by light microscopy by surface contamination of the artificial diet.The inoculated larvae were observed daily to identify the NPV infected ones based on the sign and symptoms of disease.The tissues of dead larvae were examined as soon as possible with the naked eye and tissue smears under light microscopy as mentioned above.
The method of OBs purification was done as (Sudhakar et al., 1997) with some modification.The individually dead larvae showing symptoms of NPV were transferred to a micro centrifuge tube and homogenized in 300 µl. of distilled water.The homogenates were filtered through a cheese cloth.The filtrate were subjected to sucrose layer (60% wt/vol) and centrifuged for 30 min at 10.000 rpm.The band was formed on top of the layer containing OBs was collected and again subjected to sucrose layer (40% wt/vol) and centrifuged at 10.000 rpm for 30 min.The band at the bottom of the gradient containing the OBs was collected and washed with distilled water.All the above steps were carried out at 4 o C. Pure OBs were suspended in distilled water and stored at -80 o C. For evaluation of OBs purification method, slide of OBs was stained with Giemsa stain as mentioned above and examined under light microscopy.The number of OBs was counted by using Neubaur Hemocytometer to determine the concentration of PIB /ml.Five concentrations were prepared from the viral OBs mother suspension by serial dilution to be used in bioassay experiment.

Rearing of the S. littoralis (Boisd):
Egg masses of a sensitive strain of the cotton leaf worm, S. littoralis (Boisd.)were incubated under laboratory condition at 27±2 O C, 60 ± 5% RH and 8:16 LD photoperiod (Smits, 1987).The original insect culture was obtained from the Research Division of the Cotton Leaf worm, Plant Protection Research Institute.Newly hatched larvae were transferred to clean glass jars covered with muslin cloth held in position with rubber bands.Larvae fed on artificial diet described by (Shorey and Hale, 1965).
The toxicity experiment was carried out using diet surface treatment procedure (Addy, 1969). 2 nd instars larvae of S. littoralis were starved for 8-10 h at 30 o C (Smits and Vlak, 1988) then transfer to cups with contaminated artificial diet with 20µl of (1x10 10 , 1x10 9 , 1x10 8 , 1x10 7 , and 1x10 6 PIB/ml) concentrations individually.After 2 days of feeding, the diet had become unpalatable, and the larvae were transferred to clean cups of diet and observed daily.Bioassays with 30 larvae per virus concentration plus 30 larvae as control were replicated 3 times.The experiment was conducted at a constant temperature 30 o C. Larval mortality was recorded at 2 day intervals during 10 days.The mortality percentages were corrected according to Abbott's formula (Abbott, 1925).
Toxicity was presented graphically as log/probit regression lines, and LC 25 ,LC 50 , and LC 90 values as well as the slope of the probit lines were calculated (Finney, 1971).

Biological studies:
Newly hatched larvae from the maintained insect colony were collected and offered daily with artificial diet with 20µl of (1x10 10 , 1x10 9 , 1x10 8 , 1x10 7 , and 1x10 6 PIB/ml) Treated instars larvae were examined daily in order to study the following parameters: larval and pupal duration of each instar and percentage of pupation.Pupae were sexed and then placed in 3 pairs in the glass jars of the following combinations: treated male x treated female and for a control untreated male x untreated female.Subsequently, percentage of adult emergence, longevity of moths and the fecundity and fertility of eggs/female, were determined.

RESULTS AND DISCUSSION
Efficacy of the nuclearpolyhedrovirus isolates against 2 nd instars larvae of S. littoralis larvae was performed by testing five concentrations of occlusion bodies from each of the three nuclearpolyhedrovirus isolates (1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 and 1×10 10 PIB/ml).The data in Table (1 and 2) and fig.(1) indicate that the percentage mortality of the larvae was increased with increasing concentrations of tested isolates.Furthermore, NPV Al-Fayoum exhibited higher toxicity to 2 nd inster larvae followed by NPV El-Qalioubia , and NPV El-Beheira .The results of our study showed that, high concentrations of NPVs caused a high mortality rate; this conclusion is parallel to that found by (Duan and Otvos 2001) who reported that mortality was higher when younger larvae of Choristonura fumiferana were used.Similar findings were recorded with Abd-El Wahed et al.
(2011) who mentioned that viruset was more effective on 2 nd instar larvae than profect.LC 90 and LC 50 for viruset were 1x10 6 and 1x10 3 PIB/ml, respectively, corresponding values were 5x10 8 + 1.6x10 7 and 5x10 4 + 1.6x10 3 PIB/ml + IU/ml when profect was tested.The bioassay test revealed that the S. littoralis larvae showed symptoms during the first three days post-inoculation, these observations agree with (Federici 1997 The obtained results in Table (3) clarified the effect of the tested isolates virus on the mean larval duration, pupation%, and pupal duration.The treatment of the 2 nd larval instar with LC 50 of the three isolates decreased the mean larval duration about 0.6-1.6 day than untreated larvae (Table 2) this was in agreement with El Sayed, (2015) who found that the treatment of 2 nd instars larvae of S. littoralis with Littovir (NPV) has reduced the mean larval duration about 72 hr.than untreated larvae.The three isolates affect the percentages of larvae entering pupation which was decreased in 2 nd instar larvae treated than untreated larvae (47.6, 45.6 and 47%) for NPV El-Qalioubia , NPV Al-Fayoum and NPV El-Beheira respectively.Meanwhile, the pupal stage were decrease in all isolates of 2 nd instars larvae treated with NPV El-Qalioubia , NPV Al-Fayoum and NPV El-Beheira were decreased (14, 13, and 14.3 days) respectively, than untreated larvae (15.3 days).These results were agreed with those of Abd El-Kareem, (2012) who treated S. littoralis with Protecto, Viruset, and Profect has shorted the pupal duration of the treated instars larvae.
(Table 4) showed that the three isolates affect the percentage of adult emergence was only slightly decreased than the control to 91, 90.3 and 94.6% for the three isolates NPV El-Qalioubia , NPV Al-Fayoum and NPV El-Beheira respectively.Also, all tested isolates showed significantly shortening in the mean adult longevity for both males and females (Table 4).That was in coincides with Abd El-Kareem (2012) and El Sayed, (2015) who noticed decrease in the percentage of adult emergence and mean adult longevity of treated larvae of S. littoralis with Viruset.Table (5) showed the latent effect of the treated S. littoralis with LC 50 of tested isolates NPV on the mean number of laid and hatched eggs/female.All tested isolates NPV significantly decreased the mean number of eggs laid/female.NPV Al-Fayoum was the most effective virous, followed by NPV El- Qalioubia , and NPV El-Beheira .On the other hand, significantly reduction in the mean number of hatched eggs/female was observed when treated instar larvae with recommended dose of all tested (Table 5).That was in agreement with Abdel-Aziz, ( 2007), Abd El-Kareem, (2012) andEl Sayed, (2015).
The results of this study showed that the three isolates could be used in the pests control.The best isolates were the isolation of El-Fayoum Governorate (NPV Al-Fayoum ) which gave the highest percentage of mortality from other isolates as well as the largest reduction rate in all biological experiments, Followed by El-Qalioubia Governorate NPV El-Qalioubia and then the El-Beheira Governorate NPV El-Beheira .-Means with the same letter are not significantly different (p<0.05).

Table ( 3
): Effect of three of nuclearpolyhedrovirus isolates on larval duration, pupation rate and pupal duration of 2 nd instars larvae of S. littoralis.Means with the same letter are not significantly different (p<0.05).Table (4): Effect of three of nuclearpolyhedrovirus isolates on adult emergence percentage and adult longevity of 2 nd instars larvae of S. littoralis.Means with the same letter are not significantly different (p<0.05).Table (5): Effect of three of nuclearpolyhedrovirus isolates on fecundity of S. littoralis treated as new hatch instars.