Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control

https://doi.org/10.1016/j.pestbp.2014.05.005Get rights and content

Highlights

  • Oligo-DNA insecticide is based on anti-apoptotic gene of Lymantria dispar virus LdMNPV.

  • Cutaneous application leads to significant mortality of L. dispar in 4–11 days.

  • Absence of insecticidal effect on Manduca sexta and Agrotis ipsilon.

  • Selective cloning can be used to predict site of DNA insecticide action.

  • Main features of proposed DNA insecticide: specificity and relatively rapid action.

Abstract

This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L. dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture.

Introduction

Today, during the rapid increase of human population, the regulation of pest insect quantity in agrobiocenoses and artificial plantations is very important. Regulation of pest quantity requires a safe, an effective and a cheap method. The history of insecticides evolution is very far from over, and the question of serious improvement in that field is relevant. One of the important problems is to reduce the insecticide resistance that obliges researchers to constantly search for new types of pesticides. For example, with the introduction of every new insecticide class – cyclodienes, carbamates, formamidines, organophosphates, pyrethroids, even Bacillus thuringiensis – cases of resistance surfaced within 2–20 years [1], [2].

There are two main kinds of insecticides, manufactured and applied today: chemical pesticides and bio-insecticides. Beyond resistance to insecticide developed in insects with the time, the main advantage of chemical pesticides such as DDT, lindane, etc. is that they are very fast in action while the main disadvantages are their unspecific action and the lengthy period of half-life that can last up to several decades. Nevertheless, many of them are still being produced and successfully used on vast territories against insects (mosquitoes, tsetse, etc.) unfortunately accompanied by the side effects on ecosystems [3]. Shortly, we may determine the action of chemical pesticides as “they save lives, paying a very high price”. The other modern insecticides, bio-insecticides, are not perfect as well and have different disadvantages according to the group they belong to. For example, viral bio-preparations based on baculoviruses have enormous potential in ecologically based microbiological control of the quantity of pests [4]. The basic problem in the use of baculoviruses is the slow infection process and delay in the death of the host [5], [6], [7]. A large field of research is devoted to improve baculoviruses by increasing the speed of their infection process through genetic modifications [8]. A possible way to improve insecticides is the development of a new preparation that will manifest the sum of their best characteristics: fastness and cheapness of chemical pesticides and safety from baculoviral preparations.

Biopreparations based on viral DNA fragments, termed DNA insecticides [9], [10], [11], [12], could be a safe, cheap and an effective alternative. The creation of effective biological preparations based on small DNA fragments is promising due to the important role viral DNA plays in host cells, the great variability on the one hand and specificity of sequences on the other hand, and the relatively high chemical stability. They may influence cells through mechanisms characteristic of antisense oligonucleotides [13], [14], mRNA-antisense DNA hybrids [15] and by mechanisms that resemble those both of DNA interference [16] and RNA interference [17], [18], [19]. We cannot also ignore the fact that DNA is able to interact with a large number of proteins and thus, can affect their functional activity [20].

One of the possible and practically convenient application ways of the DNA insecticides penetration into the body of a caterpillar is an external one. It is known that the presence of developed epicuticule limits the permeability of a caterpillar’s covers for most insecticides. Nevertheless, chlororganic, organophosphate and other contact insecticides get through the epicuticule easily into the organism of an insect through the most permeable areas of the covers [19]. Experiments on RNA interference have shown that negatively charged double-stranded RNA (dsRNA) fragments are able to penetrate through the cuticle of the round worms [21] and insects [22] which shows that despite the extra barriers, uptake of the dsRNA by whole insect bodies is possible [22], [23]. The induction of RNA interference (RNAi) by topical application of dsRNA could be explained by passage to interior tissues via the tracheal system [19].

Our idea was to investigate the influence of fragments of a single-stranded DNA of Lymantria dispar MNPV IAP-3 gene on the viability of gypsy moth, a serious insect pest and a destructive defoliator with a broad host range, and one of the most recognized pests of forests and ornamental trees in the world [24]. Baculoviruses have two classes of anti-apoptosis genes – p35 and IAP genes that can block apoptosis through various mechanisms in a phylogenetically wide range of organisms [25], [26]. Most of the baculoviral IAPs with anti-apoptotic functions belong to the IAP-3 group, with certain exceptions [27]. Of note, some recent studies suggest that Ld-IAP-3 induces apoptosis of insect cells through initiator caspase activation [28], although the authors state that “since analysis of pro-apoptotic functions of Ld-IAP-2 and Ld-IAP-3 in LdMNPV infected Ld652Y cells is lacking in the present study, further analyses are required to find a conclusion”.

Many investigations suggest an eukaryotic cellular origin for the viral IAPs [29]. Relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts [30], and the ability to affect their biochemical reactions through expression of homologous anti-apoptotic genes. All IAP genes isolated from different baculoviruses display two distinct structural features. The first of these is the presence of amino-terminal repeats of amino acid sequence termed a baculovirus IAP repeat (BIR). The second highly conserved feature of baculovirus IAP proteins is a zinc binding domain known as a RING (really interesting new gene) finger [31]. 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 [27]. RING domains are characterized by the presence of six to seven cysteins and one or two histidines that form cross brace architecture and coordinate two zinc ions. 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 [31]. For this study we chose two DNA fragments from relatively conserved BIR (sense chain) and highly conserved RING (antisense chain) domains of LdMNPV IAP-3 gene. Thus, if anti-apoptosis genes of a virus are homologous to the host anti-apoptosis genes – as it is known homologous sequences usually have the same, or very similar functions [32], then our hypothesis is the application of such fragments of viral anti-apoptosis genes is supposed to interfere with pro- and antiapoptotic pathways in the gypsy moth cells (for example, via mechanisms characteristic for the action of antisense oligonucleotides). The consequences of such application of viral IAP-3 gene fragments may be the blocking of anti-apoptosis proteins synthesis, the high level of apoptosis of affected cells and as a result, the death of an organism. Thus, viral IAP-3 gene fragments might have insecticidal effect on gypsy moth caterpillars. Besides this, every species has its own unique sequence of anti-apoptosis genes and it seems possible to create the most effective and selective DNA insecticides for certain species that will manifest the highest effect on a target insect and provide harmlessness to other members of an ecosystem.

Section snippets

Materials and methods

For the experiments three egg masses of gypsy moth (L. dispar (Lepidoptera: Erebidae) were identified and collected in the forest (Tours, France), after that they were randomized and reared under standard conditions. In average, 17 1st instar caterpillars were used per each control and experimental group for DNA insecticides treatment. The experiment was performed in 4 replicates. A water solution with single-stranded DNA fragments with a concentration of 100 pmol/μl was applied outwardly as a

DNA fragments from BIR and RING domains of LdMNPV IAP-3 gene induces mortality of gypsy moth caterpillars

After the treatment with the fragments of viral IAP-3 BIR and RING domains (BIR + RING treatment) significant increase in mortality was observed already on the 4th day in comparison with both control (water treated) and polyA groups (Fig. 1). The percentage of perished caterpillars continued to rise up to 11th day of the experiment, reaching in average 24.2% of caterpillar individuals perished in the BIR + RING experimental group, 11.1% of individuals perished in the experimental group treated with

Discussion

The found effect of the viral DNA fragments on its host can be used for the creation of selective fast-acting insecticides to protect plants from the gypsy moth and other phyllophagous insects [9]. Such phenomenon seems to be adaptively important for a system host-virus and may be one of natural pathways of interaction between the gypsy moth and L. dispar multicapsid nuclear polyhedrosis virus and requires further investigation.

Viral DNA fragments acted as inducers (for death) in the host cells

Conclusions

In this article we show the insecticidal potential of the viral DNA fragments 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 agriculture and forestry. For the used egg masses we found that effect of DNA insecticides does not depend on the infection of caterpillars with LdMNPV. PCR and DNA sequencing techniques helped us to find that proposed here DNA insecticides might act through the

Author contributions

Volodymyr V. Oberemok designed and performed experiments, analyzed data, and wrote the paper. Oleksii A. Skorokhod analyzed data and wrote the paper.

Conflict of interest disclosure

The authors declare no conflict of interest and no competing financial interests.

Acknowledgments

Dr. Volodymyr V. Oberemok is very much indebted to Dr. Elisabeth Herniou for inviting him to the Institut de Recherche sur la Biologie de l’Insecte, Université François-Rabelais (Tours, France) for a postdoctoral internship (supported by Grant of Cabinet of Ministers of Ukraine) where he could make a new step in a direction of deeper understanding of DNA insecticides’ phenomenon. We are also grateful to Dr. A. Bezier from the Institut de Recherche sur la Biologie de l’Insecte, Université

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