Biotechnology and Insects (Insects)

Biotechnology can be broadly defined to include all practical uses of living organisms. As such, biotechnology has been practiced since beginning of the recorded history through endeavors such as fermentation of microorganisms for production of beer, selective breeding of crops, beekeeping for the production of honey, and maintenance of silkworms for the production of silk. Laboratory techniques developed within the last 20 years that enable transfer of genes from one organism to another have resulted
in tremendous scientific and commercial interest and investment in biotechnology. The word “biotechnology” is now commonly used to refer to manipulation of organisms at the molecular level. This article reviews insect-derived tools used for biotechnological research and the use of recombinant DNA technology for management of insect pests and insect-borne disease.

INSECT-DERIVED TOOLS USED FOR BIOTECHNOLOGICAL RESEARCH

Expression of Foreign Proteins in Insect Cells

Production of large amounts of a particular protein is extremely valuable for both research and industrial purposes. Baculoviruses, which are double-stranded DNA viruses that infect mainly insects, have been developed as baculovirus expression vectors (BEVs) by genetic modification to include a gene of interest. BEVs can replicate in lepidopteran cells and larvae, thereby efficiently transferring foreign genes into eukaryotic cells. The foreign gene is usually under transcriptional control of a viral promoter so that the gene is transcribed by the virus, but translated by the host cell biosynthetic machinery. The BEV system is one of the best tools for recombinant protein expression in a eukaryotic host and has been used for the production of many different proteins for research purposes. The BEV system also has potential industrial application for the production of proteins used in vaccines, therapeutic agents, and diagnostic reagents. Advantages of this protein production system include production of large quantities of foreign protein, and eukaryotic protein processing allowing production of more authentic eukaryotic proteins. The BEV expression system is only transient, however, because the baculovirus ultimately kills the host cells. Baculoviruses do not infect vertebrates and therefore provide relative safety for laboratory manipulation. The use of a baculovirus for production of a foreign protein was first demonstrated by expression of human (3-interferon and Escherichia coli (3-galactosidase.
Insect cells can also be engineered directly to express the recombinant protein, without the baculovirus expression vector intermediate. Such insect cells are stably transformed to constitu-tively express a foreign gene. Expression levels are usually lower than for the BEV system, but stably transformed cells produce recom-binant proteins continuously and process them more efficiently than infected cells.

Insect-Derived Genes Used in Biotechnology

Reporter enzymes allow monitoring of gene expression in living tissues and cells. The gene encoding the reporter enzyme is typically inserted under control of the promoter of the gene of interest, and production of the enzyme is monitored by means of an enzyme assay. Luciferases belong to a unique group of enzymes that produce light as an end product of catalysis. The luciferases derived from the North American firefly Photinus pyralis (Coleoptera) and the Jamaican click beetle Pyrophorus plagiophthalamus (Coleoptera) have been used as genetic reporter enzymes in virtually every experimental biological system, including prokaryotic and eukaryotic cell cultures, transgenic plants and animals, and cell-free expression systems. These luciferases, which evolved for the nocturnal mating behavior of the beetles, use ATP, oxygen, and d-luciferin as substrates in the catalysis of a light-producing reaction. The ease and reliability with which luciferase can be assayed, combined with the sensitivity of the technique, has made this enzyme a highly valuable research tool.

USE OF BIOTECHNOLOGY FOR MANAGEMENT OF INSECT PESTS IN AGRICULTURE

The ability to move genes from one organism to another and to silence genes has enabled scientists to develop insect-resistant trans-genic crops and insect pathogens with enhanced insecticidal properties. The technology also has the potential to protect beneficial insects from chemical pesticides.

Insect-Resistant Transgenic Plants

Despite the progress made in recent years, a significant proportion of the world’s food supply is lost to the activities of insect pests. The deleterious impact of chemical pesticides on the environment, combined with the emergence of technologies enabling plants to be transformed with foreign genes, has driven the seed industry to develop transgenic plants as novel, environmentally benign means of pest control. Insect-protected crops were among the first products of biotechnology to have a significant impact on crop protection, and at times their use has resulted in decreased application of classical chemical pesticides.
The bacterium Bacillus thuringiensis (Bt) kurstaki has served as a microbial insecticide for many years, but widespread use was limited by its instability when exposed to ultraviolet light and its poor retention on plant surfaces in wet weather. The high toxicity of the Bt toxins to a variety of insect pests, and the ease with which the gene could be isolated from bacterial plasmids, made it an obvious choice for development of the first insect-resistant transgenic plants. The active Bt toxin binds to a receptor in cells lining the insect gut and creates a channel allowing free passage of ions. The cells lining the gut die, and very soon, the insect dies, too. Different strains of Bt contain plas-mids encoding different toxins with different specificities of action against insects. A particular toxin is generally effective against only a limited range of closely related species. Bt toxins are used in a variety of transgenic crops, including cotton, for protection against various lepidopteran pests, corn (maize), for protection against the European corn borer Ostrinia nubilalis (Lepidoptera), and potatoes, for protection against the Colorado potato beetle Leptinotarsa decemlineata.
Industry has expended enormous effort to identify new isolates of Bt, with different specificities and increased virulence for development of insect-resistant crops. Other bacteria also provide a resource for identification of insect-specific toxin genes such as those derived from Bacillus cereus and the entomopathogenic nematode-associated bacterium Photorhabdus luminescens.
Plants have a variety of strategies to avoid or survive attack by insects, and genes encoding endogenous plant-defensive compounds are also candidates for enhancing the resistance of crops to insect pests. Such factors include inhibitors of digestive proteinases that disrupt digestion, and lectins that bind specifically to carbohydrate residues in the gut of phytophagous insects. The levels of protection conferred to plants by these agents have been variable.
Silencing of insect genes (RNA inhibition), which prevents translation of gene products, also has potential for crop protection. Insect resistance has been demonstrated for transgenic plants designed to silence essential insect genes or a gene that allows the insect to tolerate a plant-defensive compound.

Transgenic Arthropod Natural Enemies

Recombinant DNA methods may be applied to produce improved strains of natural enemies such as predatory arthropods and parasitoids, but techniques are in the early stages of development. For example, the western predatory mite, Metaseiulus occidentalis (Acari), is among a group of mites that are mass-reared for the control of spider mites. However, pesticides applied for control of other pest species often wipe out the predatory mites. Engineering beneficial insects such as the western predatory mite with insecticide resistance genes would in theory provide protection from chemical sprays applied for control of insect pest species.

Engineered Insect Pathogens for Pest Control

Insect pathogenic bacteria, viruses, fungi, and nematodes have been used for the management of insect pests in various niche markets. However, each agent suffers from at least one major limitation, such as susceptibility to environmental stresses, temperature extremes, desiccation, or solar radiation. Most work has been done on the genetic enhancement of bacteria, viruses, and fungi. Genetic engineering to enhance the insecticidal properties of entomopatho-genic nematodes is in its infancy.
Genetic engineering has been used to enhance the insecticidal efficacy of various strains of Bt by increasing virulence, extending host range, and increasing field stability, and by introducing alternative toxins to facilitate resistance management. Techniques have been developed for production by genetic means of new strains of Bt with new combinations of toxin genes.
Considerable progress has been made toward optimization of entomopathogenic viruses at the genetic level. The baculoviruses are arthropod-specific viruses that have been studied extensively both as protein expression vectors and as insect pest control agents. These viruses have been genetically engineered with genes encoding insec-ticidal peptides or insect-specific toxins that are active within the hemocoel of the insect. Upon infection of the insect host, the toxin is produced as the virus replicates, and the infected insect dies from the effects of the toxin delivered by the virus. Recombinant baculovi-rus insecticides have been developed that now approach the efficacy of the classical chemical insecticides.
A similar strategy has been used for genetic enhancement of the entomopathogenic fungus Metarhizium anisopliae, with the fungus delivering a scorpion venom-derived, insect-specific toxin into the insect hemocoel.

USE OF BIOTECHNOLOGY FOR MANAGEMENT OF INSECT PESTS AND INSECT-BORNE DISEASE

Transgenic Insects

Transposable elements are mobile segments of DNA that can move from site to site within a genome and can be used for delivery of foreign DNA into the genomes of insects. Although the vinegar fly Drosophila melanogaster (Diptera) was, in 1982, the first organism to be transformed, leading to tremendous advancements in genetics research, the application of this technology to other insects has been slow. Recent successes, however, indicate that stable transformation of insects may become more routine in the foreseeable future. Transformation using transposable elements has been achieved for relatively few species, mostly within the Lepidoptera and Diptera (Table I) . Other gene transfer systems using viruses or gene expression from transformed bacterial endosymbionts (so-called paratransgenesis) have been used for some species that are not amenable to direct transformation. The genomes of bacteria and viruses are also significantly easier to engineer than eukary-otic genomes. Bacteria and viruses have been used as vectors for both transient and stable foreign gene expression in insects. For example, the bacterial symbionts of the kissing bug, Rhodnius prolixus, were successfully engineered to reduce the quantity of Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease and is carried by this vector. The bacterial endosymbionts were engineered to express an antimicrobial peptide or antibodies that specifically target the parasite. Similar methods are being developed to prevent transmission of the malaria parasite Plasmodium by its mosquito vectors.

GENETIC APPROACHES FOR MANAGEMENT OF INSECT PEST POPULATIONS

The sterile insect technique (SIT) relies on release of large numbers of sterile male insects that mate with wild females, thereby reducing reproductive potential or, if sufficient numbers of males are released over time, resulting in eradication of the pest population in

a given area. Successful SIT programs have been conducted against the screwworm, Cochliomyia hominivorax. the Mediterranean fruit fly, Ceratitis capitata. and the tsetse fly, Glossina spp. One of the problems associated with SIT is that laboratory rearing and sterilization of males result in reduced fitness of the insects.
Alternative genetic control systems include use of natural sterility such as cytoplasmic incompatibility induced by infection with the bacterium Wolbachia, and conditional lethal traits. For a conditional lethal release, insects are engineered to carry a lethal trait that is active only under certain conditions, such as certain temperatures, or at diapause. Because the trait is not lethal immediately, it can spread in a population. Genetic techniques have also been developed that allow induction of female-specific lethality. These autocidal control strategies have been demonstrated only in the model organism Drosophila thus far. The ability to insert the desired genes into insect genomes will be critical to the success of these genetic approaches for management of insect pests in the future.