Toxicological and molecular Studies of newly compounds extracted from wastes against cotton leaf worm Spodoptera littoralis

An experiment was conducted under laboratory condition to evaluate the insecticidal activity of three newly compounds extracted from wastes from natural origin, Cyano acetyl urea (CAU), Benzimidazolyl acetyl urea (BAU) from urea and Cyano acetyl hydrolyzate (CAH) from rice straw, on cotton leaf worm (Spodoptera littoralis) through exposure of fourth instar larvae to castor bean leaves which immersed in different concentrations from tested compounds. In addition, the morphogenic abnormalities were recorded. The other objective of this study is to evaluate the potential of the random amplified polymorphic DNA (RAPD) assay for the detection of genetic polymorphism between control and treated S. littoralis larvae, which have been exposed to the tested compounds at both LC25 and LC70. Five primers namely: OP-01, OP-02, OP-03, OP-04, and OP-05 were used in this study. The present findings clear that, LC25 estimates of fourth instar larvae ranged from 0.092 to 0.154 % of (CAU), 0.191 to 0.225 % of (BAU) and 0.009 to 0.021 % of (CAH) while, LC70 estimates of fourth instar larvae ranged from 1.289 to 2.009 % of (CAU), 1.445 to 2.159 % of (BAU) and 0.339 to 0.627 % of (CAH). In addition, five primers OP-01, OP-02, OP-03, OP-04, and OP-05 generated a maximum of 26, 24, 20, 13 and 23 bands, respectively. RAPD profiles generated by these primers revealed differences between control and treated samples with visible changes in number and size of amplified DNA fragments. Polymorphism ranged from 44.4 to 100% as screened by the five primers among all samples. Taking all data together, higher polymorphism was recorded at LC70 (77.8, 84.4, and 86.4%) comparing corresponding values at LC25 (69.6, 80.3, and 79.4%) for larvae treated with CAU, BAU and CAH, respectively. Based on LC70, the highest polymorphism (86.4%) was observed in those treated with CAH comparing those either treated with CAU (77.8%) or with BAU (84.4%). Definitely, RAPD data confirmed the susceptibility test as well as the morphological study, and suggest that DNA damage and the possible occurred mutations may appear to be the main factor influencing the evident polymorphism between control and treated larvae.


INTRODUCTION
The cotton leafworm, Spodoptera littoralis (Boisd.) is considered the most serious pest for Egyptian cotton plant.Its larvae feed not only on cotton leaves, but also they attack other economically important crops.Control program of the insect in Egypt mostly depends on the use of various conventional insecticides which has a Reda F.A. Bakr et al. residual effect and increase the pest resistance.In order to avoid these hazards, there is a great need to develop alternative safe control agents with new modes of action.
Waste products from natural origin considered environmentally safe, less hazardous to non target biota, simple inexpensive and can be applied effectively by using techniques more suitable for developing countries ( El-Maghraby et al., 2012).
Sublethal insecticide exposure can lead to physiological and behavioral changes in the organism (Hyne and Maher 2003).These responses can be measured using specific biomarkers that provide a measure of sublethal effects (McCarthy and Shugart 1990).
The Polymerase Chain Reaction (PCR) initially described by Saiki et al., (1985) revolutionized the molecular biology and is still widely used for a variety of purposes.Classical PCR assay simply requires a target DNA sequence and two synthetic oligonucleotide primers complementary to opposite strands of the target DNA.Indeed, mutations may affect the annealing of the primers whereas DNA damage may interfere with the DNA polymerase activity, thus altering the number of newly synthesized amplicons.Hence, despite the advantages of using traditional PCR assays for detecting DNA alterations, there are a number of potential difficulties.First, the nucleotide sequence flanking upstream and downstream the target DNA needs to be determined (Newton and Graham, 1997).Second, the size of the PCR products play a crucial role in the detection of DNA damage because the amplification of short fragments (e.g. less than 300 bp) may be slightly reduced and possibly not inhibited at all even in case of extensive DNA damage.Third, it is conceivable that any reduction in the intensity of PCR reaction caused by factors other than DNA damage such as residual phenol after DNA extraction (Newton and Graham, 1997).Alternatively, if an amplicon entirely disappears, it could be argued that the PCR did not work at all, irrespective of the presence of DNA damage (Atienzar et al., 2002b).
Such disadvantages can be eliminated by using the random amplified polymorphic DNA (RAPD-PCR) (Williams et al., 1990).Generally, RAPD reactions are performed with a single 10 bp primer and multiple amplifiable fragments (from different loci) are usually present for each set of primers across each entire genome (Lynch and Milligan, 1994).The reactions generate a number of amplicons of variable lengths (e.g. between 100 and 4000 bp) (Atienzar et al., 2002b) and the number of loci that can be examined is essentially unlimited (Lynch and Milligan, 1994).RAPD-PCR is a versatile and inexpensive tool.Its main advantages lie in its rapidity and applicability to any organism (since no information on the nucleotide sequence, cell cycle, or chromosome complement is required) (Lushai et al., 2000;Atienzar et al., 2001).Furhtermore, Allozyme patterns may represent an environmentally-induced phenotype expression of the genome.In contrast, RAPD and related fingerprinting methods infer the genetic variability directly at the genome level; provide a less-biased genomic sample (Fritsch and Riesberg, 1996).
The objectives of this study are to estimate the sublethal concentration of some waste extracts from natural origin: Cyano acetyl urea(CAU), Benzimidazolyl acetyl urea (BAU) and Cyano acetyl hydrolyzate (CAH), to record the morphogenetic changes and to evaluate the potential of the RAPD-PCR assay for the detection of genetic polymorphism between control and treated S. littoralis larvae which have been exposed to tested compounds at both LC 25 and LC 70 .

Maintenance of culture
Spodoptera littoralis larvae obtained from the laboratory culture of plant protection Research Institute, Agricultural Research Center (Cairo, Egypt).This strain was reared under constant laboratory conditions of 27±2 o C and 65±5 % RH (EL-Defrawy et al., 1964).

Insecticidal activity:
To assess the insecticidal activity of the tested compounds, different concentrations were prepared ranged from 2 to 0.025%.The larvae used in the experiments were freshly molted 4 th instar larvae within 6 hrs after ecdysis.The leaf dipping technique was adopted, where freshly castor oil beans leaves were dipped for 30 second in one of the prepared concentrations.The treated leaves were left to dry for approximately 5 minutes at room temperature before being offer to S. littoralis larvae.Three replicates contained 20 larvae / jar were used for each treatment and also for the control experiment.The mortality percentages of treated larvae were corrected against those of the control by using Abbott ̕ ̕ s formula, (Abbott, 1925).Probit analysis was determined to calculate LC 25s ,LC 50s , LC70s and slope values of the tested compounds (Finney, 1971), through software computer program.
Amplification was performed using DNA thermal cycler (Progeny 30, Techno, Cambridge Ltd.Dux ford Cambridge, UK).The thermal conditions were as follows: 94 ºC for 5 min (initial denaturation), 40 cycles: 1 min denaturation at 94 ºC, 1min annealing at 40 ºC, 2 min extension at 72 ºC, and 7 min for final extension at 72 ºC Then the PCR reaction was kept at 4 ºC.The PCR products were subjected to electrophoresis in 1% agarose gel, stained with ethidium bromide and visualized under UV light.

Insecticidal activity:
Insecticidal activity of three newly compounds extracted from waste products, Cyano acetyl urea (CAU), Benzimidazolyl acetyl urea (BAU) from urea and Cyano acetyl hydrolyzate (CAH) from rice straw against 4 th larval instars of Spodoptera littoralis was recorded as shown in Table (2).Data revealed that CAH was the most effective compound followed by CAU and finally BAU.The LC 25 values of CAH, CAU and BAU were 0.015, 0.123 and 0.191 % respectively, while The LC 50 values of CAH, CAU and BAU were 0.101, 0.515 and 0.660 %.On the other hand, The LC 70 values of CAH, CAU and BAU were 0.442, 1.569 and 1.730 %.

Morphogenic abnormalities:
Treatment of 4 th larval instar of S. littorais with CAU, BAU and CAH resulted in different morphogenic effects.
CAU treatment, induced 6 th instar larva surrounded by old exuvium of 5 th instar larva in the posterior part of the larva In all cases the rate of malformations occurrence was directly proportional to the concentration used, the sequences of events were identical.

Molecular studies:
RAPD profiles generated by the different 5 primers revealed differences between control and treated S. littoralis 6 th instar larvae, with visible changes in the number and size of amplified DNA fragments as the following: Primer OP-01 produced 26 scorable polymorphic bands (Fig. 2.1, Table 3).The control sample was identified by the presence of 3 unique bands (236, 306, and 433 bp).Samples treated with Cyano acetyl urea (CAU) were recognized by the 2 unique bands (774 and 1065 bp) for LC 25 and any of unique bands for LC 70 .On the other hand, one unique band (1694 bp) distinguished samples treated with benzimidazolyl acetyl urea (BAU) for LC 25 .For samples treated with Cyano acetyl hydrolyzate (CAH) one unique band (959 bp) was detected for LC 25 and 2 unique ones (266 and 318 bp) were detected for LC 70.Absolutely, no bands were obtained by RAPD profiles for samples treated with BAU (for LC 70 ) using this primer.4) illustrate the RAPD profile generated by the primer OP-02.A sum of 24 polymorphic bands was generated by this primer from which, 15 were shared ones.The control sample was identified by the presence of two unique bands at about 879 and 648 bp.Samples treated with CAU were recognized by the presence of the three unique bands; at about 1882 bp (for LC 25 ) ; at 1288 and 1111 bp (for LC 70 ).On the other hand, three unique bands were distinguished samples treated with BAU; two bands at 1135 and 363 bp (for LC 25 ) and one at 560 bp (for LC 70 ).Only, one unique band at about 283 bp distinguished samples treated with CAH (for LC 70 ).
The primer OP-03 generated a total of 20 polymorphic bands (Fig. 2.3, Table 5) and two unique bands at about 983 and 322 bp were detected in the control sample.Two other unique ones were scored for those treated with CAU at about 1301 bp (for LC 25 ) and 1817 bp (for LC 70 ).No unique band characterized samples treated with BAU.Samples treated with CAH were recognized by the presence of the three unique bands at about 1635, 900, 770 bp (for LC 25 ) and one at 800 bp (for LC 70 ).Primer OP-04 generated a total of 13 polymorphic bands.The lowest number (3) of unique bands was recorded by this primer (Fig. 2.4, Table 6).The first band was recorded at about 453 bp in the control sample (lane 1) while the second one was identified in the samples treated with BAU at about 400 bp.The third band (at 346bp) distinguished samples treated with CAH.No unique band was detected in samples treated with CAU.Primer OP-05 generated 23 polymorphic (Fig. 2.5, Table 7).One unique band was detected for the control (at 366 bp).Samples treated with CAU were distinguished by one unique band (for LC 25 ) at 1430.8 bp and 3 unique bands for LC 70 (at 1804, 1590, and 1343 bp).On the other hand, 1 unique band (1766 bp) was determined for samples treated with BAU (for LC 25 ).No unique bands were determined for the same compound at LC 70.As to individuals treated with CAH, 2 unique bands distinguished treated samples at LC 25 (428, 719 bp) while 3 unique ones were detected for LC 70 (at 400, 542, 1088 bp).
From the above results, The assay disclosed that a maximum of 26, 24, 20, 13 and 23 DNA bands were generated by the primers OP-01, OP-02, OP-03, OP-4 and OP-05, respectively.The size of the amplified fragments ranged from about 1922 bp (OP-02) to 236 bp (OP-01) across the profiles generated by the five primers.The polymorphic ratios between control and treated larvae were also calculated (Table 8).RAPD data confirmed the insecticidal activity as well as the morphological study.Indeed, polymorphism ranged from 44.4 to 100% (Table 8) among all treated samples.Taking all data together, higher polymorphism was recorded at LC 70 (77.8, 84.4, and 86.4%) comparing corresponding values at LC 25 (69.6, 80.3, and 79.4) for larvae treated with CAU, BAU and CAH, respectively.

Rows
Based on LC 70, the highest polymorphism (86.4%) was observed in those treated with CAH comparing those either treated with CAU (77.8%) or with BAU (84.4%).Thus, these results had confirmed the susceptibility test as well as the morphological study; a considerable percentage of larvae treated with CAH completely failed to transform to normal pupae, and moreover, the most malformed structures and morphological abnormalities were produced after its application.
Hundred percent polymorphism (100%) was noted for 4 samples.Out of them, 2 samples were amplified by primer OP-02: in individuals treated with CAU (for LC 70 ) and also for those treated with BAU (for LC 25 ).The third sample was amplified by primer OP-03 in larvae treated with CAH (for LC 70 ).The forth sample was treated with BAU and revealed no bands after screening by primer OP-01.

DISCUSSION
The principal criterion in this study is conversion of wastes to economic products as insecticides which uses on large scale in controlling pests in the entire world.Three newly compounds extracted from wastes from natural origin [Cyano acetyl urea (CAU), Benzimidazolyl acetyl urea (BAU) from urea and Cyano acetyl hydrolyzate (CAH) from rice straw] were evaluated as insecticide against 4 th larval instars of Spodoptera littorallis.The tested compounds revealed differences in LC 25 , LC 50, LC 70 values and the slope functions of the regression lines.In addition, remarkable variations in the potency of tested compounds which possess parallel regression lines of nearly equal slope values.This may suggest that these compounds have the same mode of action against the tested larvae.These results are in agreement with the findings of Mesbah et al., (1996) who found that chlorfluazuron (urea derivatives) causes toxic effect, decreasing the pupation and adult emergence of S. littoralis.EL-Nemaky (2004) found that benzoylphenyl ureas were effective against one day old larvae of the sping bollworms.Similary, El-Maghraby et al., (2012) observed the toxicity of rice straw derivatives on mosquito larvae.
In the present study, the application of either urea derivatives (CAU & BAU) or rice straw waste extract (CAH) induced molting disturbances, e.g.hanging larval exuvium, larval -pupal intermediates and pupal-adult intermediates.All morphogenetic effects indicate that these compounds may act as an insect growth regulator where (wright 1970) indicated that a pupal-adult intermediate is a true juvenile hormone effect.( Emam & Degheele, 1993) and (Hassanein & Zidan, 1996) tested benzoylphenyl ureas on fourth-instar larvae of S. littoralis and of Plodia interpunctella respectively,with sublethal doses.Ecdysis to the next instar was disrupted, with an extra moult being induced in some cases.Treatment resulted in malformations in pupae and adults.In addition Baker et al., (2006) showed sever malformations when 4 th larval instar of S. littoralis treated with rice bran extract.
The RAPD technique was very sensitive to detect genetic differences between the control and the treated S. littoralis 6 th instar larvae.This conclusion was noted also by other authors who tested different toxicants (Atienzar et al., 2001;2002 a&b;Abd-Allah et al., 2003;Atienzar and Jha, 2004;Barreto et al., 2005).
The present data suggest that DNA damage, and the possible occurred mutations may appear to be the main factor influencing RAPD patterns; the different polymorphism between control and treated larvae.Definitely, RAPD data confirmed the results about insecticidal activity as well as the morphological abnormalities.Indeed, resulting DNA profiles, gain and loss of bands among individuals depending on 1-presence/absence of priming sites 2-priming complementary completeness/incompleteness or 3-the distance between priming sites (Fritch and Riesberg, 1996).Hence, some important DNA effects must have occurred due to CAU, BAU and CAH treatments.These DNA effects (DNA adducts, DNA breaks, point mutations, genomic rearrangements, insertions and deletions) affect the presence/absence of primer sites, their complementarities to primers and /or the distance between priming sites.RAPD bands of different molecular weights are interpreted as separate loci which are scored on a present (amplification) or absent (non-amplification) basis ((Fritch and Riesberg, 1996).Thus, the new PCR products could be amplified because some sites become accessible to the primers after structural changes or because some mutations have occurred in the genome (Atienzar et al., 2001).Even a single point mutation within the primer site can generate significant changes in RAPD patterns (Williams et al., 1990).It is also well known that DNA repair, and replication of damaged DNA can lead to point mutations (Livneh et al., 1993).Disappearance of amplified products could be a result of extensive DNA damage (Atienzar et al., 2000), since DNA lesions are expected to have detrimental effects on RAPD profiles, they not only can induced structural change, but also can reduce the polymerization of DNA and / or block the Taq DNA polymerase (Nelson et al., 1996).These reasons may explain that, in this study, absolutely no bands were obtained at in RAPD profiles at LC 70 for larvae treated by BAU using primer OP-01.Again, DNA damage and mutations are the main factors that influenced RAPD patterns and gain or loss of bands (Atienzar et al., 2000;2002 a&b).
RAPD data revealed that the highest polymorphism was observed in larvae treated with Cyano acetyl hydrolyzate and screened by the five mentioned primers, which in turn confirms the susceptibility test.Furthermore, a considerable percentage of larvae treated with this compound completely failed to transform to normal pupae.Moreover, the most malformed structures and morphological abnormalities were produced after its application.
Several Cyano-derivatives used as pesticides, known to induce DNA damage (Villarini et al., 1998;Undeger and Basaran, 2005;Patel et al., 2006).This may reflect the observed polymorphism between control and larvae treated with either CAU or CAH.On the other hand, CAU and BAU are urea derivatives.Many ureaderived insecticides are in wide use also in controlling insect pests.Whatever the mode of action, the inhibition of chitin synthesis (Hajjar and Cassida, 1979;Hassal, 1990) is considered as the obvious ultimate effect of urea-derived compounds (Abd-Allah et al, 2003).Urea derivatives have the property of DNA adduct formation (Lutz, 1979;Miller and Miller, 1983).In the present study, changes in genomic DNA may lead to the inhibition of chitin synthesis, and thus the observed malformations and mortality, which was also reported by Abd-Allah et al., (2003).Merzendorfer et al., (2012) examined the effect of diflubenzuron (urea-derivative) on Tribolium castenatum.They revealed that unexpectedly, genes encoding enzymes involved in chitin metabolism were unaffected, but many genes encoding cuticle proteins were affected.However, RAPD data must be considered as preliminary, semi-quantitative, and give an overview of DNA effects (Atienzar et al., 2001;De Wolf et al., 2004).Hence, a more specific method may be needed in further research.
In conclusion, newly compounds extracted from wastes, Cyano acetyl urea(CAU), Benzimidazolyl acetyl urea (BAU) and Cyano acetyl hydrolyzate (CAH), are effective chemicals to control cotton leaf worm, however, they must be improve the insecticidal characters to increase its toxicity and its stability to can use them in IPM program.In addition, RAPD assay could be considered as a relatively cheap but quick method to preliminary screen the polymorphism induced in S. littoralis 6 th instar larvae after treatment with tested compounds.However, the RAPD-PCR assay is a semi-quantitative tool until preliminary data further documented by a more specific method.
Figure (1.1).Beside, Larval pupal inter-mediate with larval head but the thorax has pupal structure.In addition shrinked pupa failed in shedding off the larval exuvium Figure (1.2).Pupal-adult intermediate was showed as head and thorax enclosed by old cuticle of pupa. Figure (1.3) revealed moth with poorly developed wings and distended abdomen.In the case of BAU, it caused intermediate instar between 4 th and 5 th larval instars Figure (1.4) and pupal cuticle remained adhered to the moth's body at different sites Figure (1.5).The wing failed to spread Figure (1.6).Also, CAH showed 6 th instar larva surrounded by old exuvium of 5 th instar larva in the middle parts of the larva Figure (1.7), shrinkage of the pupae Figure (1.8) and Pupal-adult intermediate, head and thorax enclosed by old cuticle of pupa Figure (1.9).In addition, Figure (1.10) showed moth with poorly developed wings.

Table ( 1
): The code and sequences of five RAPD primers.

Table ( 3): RAPD-PCR profile for Spodoptera littoralis 6 th instar larvae treated by newly-extracted compounds from waste and amplified by primer OP-01
* No bands revealed upon RAPD-PCR Figure 2.2 and Table (