Recent Advances in Biological Control of Pest Insects by Using Viruses in China

Insect viruses are attractive as biological control agents and could be a feasible alternative to chemical insecticides in the management of insect infestations. This review describes recent advances in the development of wild-type and genetically modified viruses as insecticides. A new strategy of application of insect viruses in China is reviewed. Also, the assessment of biosafety of genetically modified Helicoverpa armigera Nucleopolyhedovirus (HearNPV) is emphasized as a case-study.     

重组病毒杀虫剂应用研究进展

应用分子生物学技术可以将昆虫特异性的毒素基因、某些酶基因等外源基因插入昆虫病毒基因组,或通过改造昆虫病毒基因组等方法构建重组病毒杀虫剂,提高杀虫效果。温室及田间释放实验证实,重组病毒杀虫剂可以显著提高现场防治效果。连续多代抗性筛选实验表明,宿主被诱导产生对重组病毒杀虫剂抗性的速度低于野生型病毒杀虫剂。采用在剂型中添加光增白剂等保护剂、在基因组中插入具有增效作用的基因、应用病毒增强蛋白等技术可以提高重组病毒杀虫效果。随着基因工程技术的发展和安全性研究的深入,以重组杆状病毒为主的重组昆虫病毒杀虫剂的应用研究正面临着突破。     

Not all GMOs are crop plants: non-plant GMO applications in agriculture

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.     

Behavior of a recombinant baculovirus in lepidopteran hosts with different susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

Persistence of viruses and DNA in soil

With growing environmental concerns over the use of chemical pesticides for insect control in both agriculture and forestry, increased emphasis is being placed on the development of alternative, biological pesticides such as genetically modified baculoviruses. Before the large-scale use of genetically modified viruses (GMV) can be realized, fate of GMV and their DNA in soil should be investigated. There are a number of factors that have the potential to affect persistence of both wild-type and genetically modified viruses and their DNA in soil. In this mini-review, the persistence of viral particles and DNA in soil is examined with particular emphasis on baculoviruses.     

Persistence of viruses and DNA in soil

With growing environmental concerns over the use of chemical pesticides for insect control in both agriculture and forestry, increased emphasis is being placed on the development of alternative, biological pesticides such as genetically modified baculoviruses. Before the large-scale use of genetically modified viruses (GMV) can be realized, fate of GMV and their DNA in soil should be investigated. There are a number of factors that have the potential to affect persistence of both wild-type and genetically modified viruses and their DNA in soil. In this mini-review, the persistence of viral particles and DNA in soil is examined with particular emphasis on baculoviruses.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus

Spodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD₅₀) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT₅₀) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.     

The impact of secondary pests on Bacillus thuringiensis (Bt) crops

The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to insecticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered insect‐resistant crops could mitigate many of the negative side effects of insecticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro‐ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here, we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if nonsusceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible long‐term ecological trophic interactions of employing this technology.     

Genetically improved potatoes: protection from damage by Colorado potato beetles

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.     

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.     

Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus

Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.     

Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae

Production of molecules with toxic activity by genetically transformed symbiotic bacteria of pest insects may serve as a powerful approach to biological control. The symbiont, Enterobacter gergoviae, isolated from the gut of the pink bollworm (PBW), has been transformed to express Cyt1A, a cytolytic protein toxin lethal to mosquito and black fly larvae, as a model system. These transgenic bacteria might be used to spread genes encoding insecticidal proteins to populations of agricultural insects or as replacement for chemical insecticides such as malathion used in bait formulation to control specific insect pests, because of extreme public pressure against organophosphate pesticide spraying. Received: 27 November 2000 / Accepted: 29 December 2000     

昆虫碱性磷酸酶的研究进展

碱性磷酸酶存在于昆虫的头、唾液腺（唾液）、肠道、马氏管、表皮、血淋巴、脂肪体、生殖系统、附肢等部位,广泛参与了昆虫的发育、神经传导、激素合成、物质代谢、滞育、社会型昆虫亚种形成等过程。同时碱性磷酸酶与昆虫抗性有关,特别涉及到对Bt制剂的阻滞作用,其本身也是某些农药的靶标酶,某些生物源化合物及病毒、真菌也可以影响其活性。昆虫碱性磷酸酶的研究,将有助于提高对昆虫生化机制及代谢过程的认识,并为害虫治理和资源昆虫饲养提供新的思路。本文综述了国内外对昆虫碱性磷酸酶的研究状况,并描述了昆虫碱性磷酸酶的生化性质及其与生理功能的关系。     

Integrating chemical control with sterile insect releases in an integrated pest management programme for Thaumatotibia leucotreta

False codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is an important indigenous pest of citrus in southern Africa. Successful control is dependent upon integration of area‐wide sterile insect releases and other suppression methods. The aim of this work was to test pyrethroid and organophosphate‐based insecticides (tau‐fluvalinate and chlorpyrifos) for their residual effect on mortality of released irradiated T. leucotreta male moths. Both of these insecticides were effective in killing irradiated T. leucotreta for 7 days after application on leaves, after which degradation of the active ingredient resulted in a marked reduction in efficacy after 14 days and rendering them harmless. Mortality was found to be similar for irradiated and non‐irradiated male T. leucotreta after treatment. Consequently, even though these insecticides might have an effect on moths in the field, ratios of sterile:wild moths should not be altered. Supporting field data from six sites in the Sundays River Valley over a season of sterile insect releases showed the conventional chemical crop protection programme to be as effective as an integrated pest management programme in facilitating effective control of T. leucotreta through sterile insect releases. The study also confirmed that the ratios of sterile:wild male moths in the commercial citrus orchards were not affected by the application of insecticides. These findings confirm the high potential of sterile insect releases for control of T. leucotreta in citrus.     

Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

Purpose

Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.

Method

Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.

Result

Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.

Conclusion

This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

     

Genetically modified microbial symbionts as arthropod pest controllers: risk assessment through the European legislations

The genetic modification and applied use of microbial symbionts have been identified as novel tools to protect beneficial insects such as pollinators or parasitoids or to fight insects that constitute pests or are vectors of infectious diseases. The deliberate release of insect pest or disease vector control products containing genetically modified micro‐organisms (GMMs) can raise questions about health and environmental safety. Different national and international authorities have established legal requirements to ensure the safe use of conventional pesticides and insecticides as well as GMMs. A key requirement is to conduct a scientific risk assessment to determine whether the product is safe to be placed in the market. In this study, we address the legal framework, the regulatory requirements, and the criteria for the environmental risk assessment of GM symbionts that currently apply within the European Union.     

A review on biological interactions and management of the cotton bollworm,Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is one of the most damaging insect pests globally, causing estimated global economic losses of over 3 billion US dollars annually. Crops most affected include cotton, tomato, soybean, grain crops such as corn and sorghum, chickpea and other pulses. Adults of this species possess strong migratory abilities (>2000 km), high fecundity and rapid reproductive rates; completing 4–6 generations per year in most cropping regions. Furthermore, the larvae are polyphagous, with a wide and diverse host range and possess the ability to enter diapause in order to survive adverse climatic conditions. At present, it is distributed across most of Oceania, Asia, Africa and southern Europe and has recently spread to South America. Various control measures have been trialled or proposed for the treatment of this pest, including synthetic insecticides, phytopesticides, microbial pesticides, macro-biocontrol agents (both parasitoids and predators) and the development of genetically modified crops (e.g. Bt cotton). Successful control necessitates the use of an integrated pest management (IPM) approach, wherein biological, chemical and physical control measures are combined for the greatest control efficacy.