DNA INSECTICIDES BASED ON IAP 3 GENE FRAGMENTS OF CABBAGE LOOPER AND GYPSY MOTH NUCLEAR POLYHEDROSIS VIRUSES SHOW SELECTIVITY FOR NON-TARGET INSECTS

DNA insecticides are novel preparations based on short single-stranded fragments of anti-apoptotic (IAP) genes of nuclear polyhedrosis viruses. In this article we report about the harmlessness of the DNA insecticides based on singlestranded fragments of the LdMNPV (Lymantria dispar multiple nucleocapsid polyhedrosis virus) IAP3 gene for tobacco hornworm and black cutworm, and present the first evidence of significant insect-specific insecticidal effect of singlestranded TnSNPV (Trichoplusia ni single nucleocapsid polyhedrosis virus) IAP3 gene fragments on the viability of cabbage looper and their harmlessness for black cutworm and wheat weevil. Our results show that DNA insecticides based on LdMNPV IAP3 and TnSNPV IAP3 gene fragments can be selective in action and have potential use in insect pest control.


INTRODUCTION
The selectivity and safety of insecticides are probably the main characteristics that have to be checked before the application of insecticides in order not to cause ecological imbalance.At the same time, approximately 40% of the crops grown in the world today are destroyed by pests, with insects contributing 13%, plant pathogens 12%, and weeds 12% (Sanchis, 2011;Pimentel, 2005).Farmers are faced with the dilemma of choosing whether to make a profit that harms the ecosystem with the use of effective but unselective insecticides, or by protecting the environment by using less effective but selective insecticides that cost more.
Each year many new insecticides appear in the market.An early report by the United Nations En-vironmental Program (Sanchis, 2011) suggested that insecticide resistance ranked as one of the top four environmental problems of the world.For example, with the introduction of every new insecticide class (cyclodienes, carbamates, formamidines, organophosphates, pyrethroids, even Bacillus thuringiensis), cases of resistance surfaced within two to twenty years (Daly et al., 1998).
Of note, the use of many insecticides led to substantial protection of the crop, many of them are selfsupporting and their application is justified economically.Usually there is a positive correlation between the use of insecticides and productivity of land and the increased mutagenic, carcinogenic and toxic impact on ecosystems.The challenge is to find a better group of insecticides that possess more selective and effective features than the existing preparations.

DNA INSECTICIDES BASED ON IAP3 GENE FRAGMENTS OF CABBAGE LOOPER AND GYPSY MOTH NUCLEAR POLYHEDROSIS VIRUSES SHOW SELECTIVITY FOR NON-TARGET INSECTS
There are three main groups of insecticides manufactured and applied today: non-organic compounds (compounds of sulfur, copper, fluorine, etc.), organic compounds (imidacloprid, carbaryl, deltamethrin, etc.) and biological preparations (baculoviruses, fungi, bacteria etc.).Biological preparations are the most selective in action and probably carry the lowest level of side effects on the surrounding environment.Viruses, bacteria and fungi usually have significant effect on 1-2 organisms and, as a rule, this effect may spread up to the narrow range of organisms that belong to the same genus or family, whereas chemical insecticides (non-organic or organic) can effectively kill phylogenetically distant organisms, for example mammals and insects.One of the principle reasons why biological preparations have not found wide practical application is because their production is based on the cultivation of a large number of host organisms (Moscardi, 1999;Inceoglu et al., 2006).This technology is time and labor intensive, making it expensive to produce the required quantity of viruses or bacteria to control pest populations.For comparison, just 1 g of pyrethroid deltamethrin that belongs to the group of chemical insecticides is able to kill 20 tons of cockroaches (Tkachev, 2004).No biological preparation can provide the same efficiency today.Beyond the economic cost of production, biological preparations take longer to kill their target than chemical insecticides (Rosell et al., 2008).
For the group of phytophagous insects, an alternative to the current insecticides may be DNA insecticides, a technique based on the application of singlestranded viral DNA fragments possessing insecticidal activity (Oberemok, 2009;Simchuk et al., 2012;Oberemok and Zaitsev, 2014;Oberemok and Skorokhod, 2014;Oberemok and Nyadar, 2014).Oberemok and Skorokhod (2014) showed the insecticidal potential of the fragments of BIR (baculovirus inhibitor of apoptosis repeat) and RING (really interesting new gene) domains of the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) IAP3 gene that can be used to create safe, relatively inexpensive and fast-acting DNA insecticides to control the quantity of gypsy moth populations, a serious pest of forests.LdMNPV is frequently found in gypsy moth populations (Oberemok, 2008b) and regulates their quantity but is rarely used as a means of pest control due to its high cost and slow speed of action.
All IAP genes isolated from different baculoviruses display two distinct structural features, BIR and RING domains.The BIR domain has been shown to be necessary for the interaction of IAP proteins with diverse pro-apoptotic factors, including invertebrate death inducers and vertebrate and invertebrate members of the caspase family of proteases (Ikeda et al., 2011).RING domains often function as modules that confer ubiquitin protein ligase (E3) activity and, in conjunction with an ubiquitin activity enzyme (E1) and an ubiquitin conjugating enzyme (E2), catalyze the transfer of ubiquitin to target proteins (Duckett et al., 1996).
The creation of effective biological preparations based on small fragments of DNA has a positive perspective due to the broad variability and specificity of DNA sequences and their relatively high chemical stability.Single-stranded DNA fragments of a virus that Means and SE are represented for 3 replicates; significance of difference between BIR+RING and control is marked as * when P <0.05 coincide with genes of a host cell should influence its biochemical activity in a manner similar to antisense molecules (weiss et al., 1999;Dias and Stein, 2002;Lu et al., 2004) and by mechanisms that resemble those of DNA interference (Kawai-Toyooka et al., 2004) and RNA interference (Fire et al., 1998;wang et al., 2011).
For example, if we block insect anti-apoptosis genes with viral DNA fragments, we initiate apoptosis and consequently death of the insect pest.Although we do not at present have complete genomic sequences of many insect pests in databases, many viruses have already been sequenced (Kuzio et al., 1999;willis et al., 2005) and found in IcTVdb (http://www.ictvonline.org).In this situation, viral DNA can provide valuable information on some vitally important genes that have cellular origin, such as anti-apoptosis genes (hughes, 2002) that can be used for insect pest control.Thus, it is possible to create fast-acting, safe and relatively inexpensive DNA insecticides to control insect pests and our recent studies with gypsy moth provide proof of this (Oberemok, 2009;Simchuk et al., 2012;Oberemok and Zaitsev, 2014;Oberemok and Skorokhod, 2014;Oberemok and Nyadar, 2014).
This research paper is devoted to an investigation of the influence of LdMNPV IAP3 gene fragments on the viability of tobacco hornworm and black cutworm and the impact of TnSNPV IAP3 gene fragments on the viability of cabbage looper, black cutworm and wheat weevil so as to evaluate the selectivity of the DNA insecticides for non-target insects.

MATERIALS AND METHODS
Two DNA fragments from BIR (sense chain) and two DNA fragments from RING (antisense chain) domains of the anti-apoptotic gene IAP3 of nuclear polyhedrosis viruses of gypsy moth (Lymantria dispar: Lepidoptera: Erebidae) -LdMNPV and cabbage looper (Trichoplusia ni: Lepidoptera: Noctuidae) -TnSNPV were chosen for the experiments.we designed DNA fragments according to the viral genome sequences found in IcTVdb and synthesized them by Eurofins MwG Operon (Germany) with hPSF grade.The sequences of the 2 single-stranded DNA fragments of LdMNPV IAP3 gene were 5ʹ-Gcc GGc GGA AcT GGc ccA -3ʹ (BIR domain), and 5ʹ-cGA cGT GGT GGc AcG GcG-3ʹ (RING domain) (Kuzio et al., 1999).The sequences of the 2 singlestranded DNA fragments of TnSNPV IAP3 gene were: 5ʹ-Acc cAT AGA GTT GGc AAT-3ʹ (BIR domain), and 5ʹ-cGA cAT GAc cGc AAG GTA-3ʹ (RING domain) (willis et al., 2005).The DNA fragments were diluted in distilled water to a concentration 100 pmol/µl.caterpillars were treated either with 0.3 μl of solution per caterpillar (tobacco hornworm and black cutworm) by putting a small drop in the middle of a caterpillar's body (approximately 3 rd -4 th setae of the thorax abdomen) or were immersed in a solution containing DNA fragments for 5 min (cabbage looper caterpillars and imago of wheat weevil).The immersion was used for cabbage looper and wheat weevil because it was very hard to treat them with drops of the solution due to their body peculiarities.Insects from the control groups were treated with distilled water and polyA oligonucleotide (5'-AAAAAAAAA AAAAAAAAA-3') at a concentration of 100 pmol/ μl.Lepidopteran larvae were grown on standard wheat germ-based forage and adults of wheat weevil were reared on wheat grains.Tobacco hornworm (Manduca sexta (Lepidoptera: Sphingidae)), black cutworm (Agrotis ipsilon, (Lepidoptera: Noctuidae)),

Experiments with TnSNPV IAP3 gene fragments on cabbage looper, wheat weevil and black cutworm
Experiments with single-stranded fragments of BIR and RING domains of TnSNPV IAP3 gene revealed a susceptibility of cabbage looper (χ 2 =5.625;P<0.05) and resistance of black cutworm and wheat weevil to the applied DNA insecticides after 7 days of treatment (Table 1).Further rearing up to 14 days did not show additional differences in mortality compared to the control groups.In our opinion, adult wheat weevils showed the highest resistance to the applied DNA fragments partially because elytra provided some protection from the contact insecticide.This is the first evidence of the insecticidal effect of Tn-SNPV IAP3 gene fragments and together with our previous results with the LdMNPV IAP3 gene (Oberemok, 2009;Oberemok and Zaitsev, 2014;Oberemok and Skorokhod, 2014;Oberemok and Nyadar, 2014).These results point to the good potential for the use of DNA insecticides on the basis of anti-apoptosis genes of insect-specific nuclear polyhedrosis viruses.Interestingly, the effect of DNA insecticides can be faster when compared with the direct effect of the virus because during experimental infection of insects with nucleopolyhedroviruses, the latent period of illness lasts from 3-5 up to 10-12 days (Van Beek and Davis, 2007).
Generally, cabbage looper caterpillars from the BIR+RING-treated group stopped eating, they moved slower and appeared more dehydrated and smaller than caterpillars from the control group.Such alterations have been shown to correspond to apoptotic patterns at the cell (Schliess and haussinger, 2005) and organism (hamshou et al., 2013) levels.These signs point to apoptosis processes in the caterpillars that lead to insect death, and correspond to our concept of the use of viral anti-apoptosis gene fragments for insect pest control (Oberemok and Skorokhod, 2014).
Fragments of the RING domain might have an insecticidal role in the BIR+RING pair, probably by acting as a small antisense molecule.Since the fragment of BIR belongs to a sense strand, its role is not clear for the moment and may be an auxiliary one.
Our recent experiments show that separate use of the antisense fragment of the RING domain of the LdMNPV IAP3 gene leads to a significantly smaller accumulation of biomass by gypsy moth caterpillars in comparison with the control, whereas use of the sense fragment of the BIR domain of the LdMNPV IAP3 gene in the same concentration does not (unpublished data).
PolyA fragments at a concentration of 100 pmol/ μl did not have a significant effect on cabbage looper in comparison with the control.This result suggests that only specific DNA fragments can cause significant elevated mortality of the insect.DNA insecticides based on TnSNPV IAP3 fragments proposed here could be used in the future against cabbage looper that feeds on a diverse range of plants and is an important pest on Brassicaceae plants (chow et al., 2005).The 10 pmol/μl concentration of TnSNPV IAP3 gene fragments turned out to be harmless for cabbage looper caterpillars, indicating the importance of the appropriate concentrations of DNA insecticides that could be used for insect pest control.

Experiments with LdMNPV IAP3 gene fragments on tobacco hornworm and black cutworm
Our results show that single-stranded fragments of the LdMNPV IAP3 gene did not have a significant effect on the viability of tobacco hornworm and black cutworm second-instar caterpillars after 7 days, when compared with the control and polyA samples, which emphasizes the selectivity of DNA insecticides (Table 2).Further rearing up to 14 days also did not show a difference in mortality compared to the control groups.
It is worth noting that first-and second-instar gypsy moth caterpillars were significantly affected during the same period of time by the smaller dose of DNA insecticides based on the same fragments of BIR and RING domains of the LdMNPV IAP3 gene (Oberemok, 2008a;Oberemok, 2009;Simchuk et al., 2012;Oberemok and Zaitsev, 2014;Oberemok and Skorokhod, 2014;Oberemok and Nyadar, 2014).Gypsy moth caterpillars have similar or comparatively bigger average body mass than tobacco hornworm has (8-6 mg, respectively) and approximately a 4-times bigger body mass in comparison with black cutworm (8-2 mg, respectively) at this stage of development.Thus, a higher dose of DNA insecticides per 1 mg of insect tissue was used for non-target insects than for gypsy moth, but they were not significantly affected.This implies that DNA insecticides based on the fragments of BIR and RING domains of the Ld-MNPV IAP3 gene have a reliable margin of safety in action and harmlessness for non-target insects.The results obtained here with DNA insecticides based on fragments of BIR and RING domains of the Ld-MNPV IAP3 gene correspond to the data provided by Oberemok and Skorokhod (2014) who reared insects for 11 days after the treatment.These findings provide additional evidence of the reproducibility of the observed harmlessness of the DNA insecticides for the non-target insects.
we also evaluated the reduction of biomass of caterpillars as one of the apoptotic patterns (Schliess and häussinger, 2005).In our recent experiments with gypsy moth using DNA insecticides we observed a decrease in body size of caterpillars, which corresponds to apoptotic patterns on the cell level (Oberemok and Skorokhod, 2014).we decided to evaluate biomass accumulation of tobacco hornworm and black cutworm after treatment with the DNA insecticides and did not find significant differences in biomass accumulation in the BIR+RING and polyA groups compared to the control (Table 3).Of note, for both tobacco hornworm and black cutworm we found that in the BIR+RING group, caterpillars had a lower average body mass by 5.1% and 11.8 %, respectively, in comparison with the control, and this may indicate a non-specific response of cells on fragments of BIR and RING domains of the LdMNPV IAP3 gene similar to that shown for wheat sprouts 24 h after the treatment (Oberemok et al., 2013).

DISCUSSION
Our research showed that DNA insecticides based on the LdMNPV IAP3 and TnSNPV IAP3 genes can be selective in action.Since every species has its own unique DNA sequence, it would appear to be possible to create the most effective and selective DNA insecticides that would manifest the highest effect on a target organism and provide safety assurance to other members of an ecosystem.we also recently evaluated the possible side effects of DNA insecticides on wheat (Triticum aestivum).we did not find negative effects on plants after prolonged exposure to DNA insecticides, as judged by the investigated biochemical parameters (glucose concentration, alkaline phosphatase activity), as well as the biomass of wheat sprouts (Oberemok et al., 2013).These results support the potential safety of the DNA insecticides described here.
Preservation of land not contaminated with harmful insecticides is vitally important.Therefore, the use of safe insecticides, such as effective biological preparations, as well as DNA insecticides, can assist in safe agriculture practice.with the use of molecular mechanisms such as DNA interference, RNA interference and antisense oligonucleotides techniques, we could create efficient insecticides, which will be an effective and safe method of pest control because they originate from nature.The scientific community is gradually coming round to this idea as well.For example, Belles (2010) says that RNA interference itself could be envisaged as an insect control tool through targeting vital genes, although efficient systems of double-stranded RNA formulation and delivery must be developed.In addition, the approach of administering double-stranded RNAs by feeding, as seen in Epiphyas postvittana (Turner et al., 2006) and Rhodnius prolixus (Araujo et al., 2006), paves the way in this field, and the possibility of delivering the dsRNA by soaking, as in Caenorhabditis elegans (Tabara et al., 1998), should also be considered, for example, in particularly permeable stages of aquatic insects.Of note, studies on gypsy moth Lymantria dispar (Oberemok, 2008a) and on the mosquito Aedes aegypti (Pridgeon et al., 2008) were the first to show that the mortality of insects can be induced by topical application of both short single-stranded DNA (Oberemok, 2008a) and relatively long double-stranded RNA (Pridgeon et al., 2008).while DNA insecticides may resemble mechanisms characteristic of antisense oligonucleotides (weiss et al., 1999), DNA interference (Kawai-Toyooka et al., 2004) and RNA interference (Fire et al., 1998;wang et al., 2011), it has its own peculiar features, such as external application, the use of small oligonucleotides and single-stranded DNA molecules, and the concept of using viral anti-apoptosis genes.This approach provides the same effect with less effort; i.e. instead of a baculovirus preparation, we could use small parts of a viral genome in high concentrations and obtain the same effect.The application of single-stranded DNA fragments as insecticides is a novel approach, without analogy in the literature.we predict that this approach could lead to the creation of selective and lower risk food webs in agroecosystems, and affordable and relatively fast-acting DNA insecticides for lepidopteran pests at the caterpillar stage.
The main disadvantages of DNA insecticides we see are the necessity of contacting the insect cuticle, and the relatively high cost of oligonucleotide production technology.An important expansion of DNA insecticides is in the reduction of the concentration of DNA fragments in the end-product through the addition of DNA carrier molecules to the preparation.
DNA insecticides based on LdMNPV IAP3 and TnSNPV IAP3 gene fragments can be selective in action, and the short single-stranded DNA fragments investigated here could be used for the creation of safe insecticides for crop protection.

Table 1 .
Percentages of different groups of black cutworm, wheat weevil and cabbage looper caterpillars that survived 7-days.

Table 2 .
Percentages of different groups of tobacco hornworm and black cutworm caterpillars that survived 7 days.Mann-Whitney test to evaluate the significance of insecticidal effect of the viral IAP3 gene fragments on the insects (Sofa Statistics 1.3.3software).

Table 3 .
Average body mass (in mg) of different groups of tobacco hornworm and black cutworm caterpillars that survived 11 days.