New field of insect science: Research on the use of insect properties

Authors


Correspondence: Satoshi Takeda, Tsukuba Research Laboratory, Hamamatsu Photonics K.K., Tsukuba, Ibaraki, Japan.

Email: stakeda@crl.hpk.co.jp

Abstract

In recent years, research on “insect properties” has been attracting much attention for industrial applications of insect technology. This is a new field of research that attempts to analyze specific physiological properties of insects to develop technology for helping humankind. The term “insect properties” has been used to refer to “specific biological functions of insects” since around 1986, and it is now widely accepted in Japan. From 1996, the National Institute of Sericultural and Entomological Science (NISES) promoted “Research for utilization of insect properties” as a “Center of Excellence (COE)” project funded by the Science and Technology Agency. At this point, a new research field called “Research for utilization of insect properties” was initiated, and this led to the recognition of this field by the academic community. In the 21st century, remarkable results, including the development of transgenic silkworms and the full decoding of the silkworm genome, have been achieved. It is expected that this advanced technology will be a powerful tool for progress of research on the use of insect properties. This review presents an overview of the current state of research on use of insect properties as a new technology.

Introduction

Insects are the most prosperous group of organisms on the earth. There are more than one million known species. Insects that are involved in human activities, such as useful insects and harmful pests, represent only a few of the many species of insects; most have little impact on our life.

Among the useful insects, silkworms and bees are well-known examples. Silk and honey are produced by silkworm and honeybees, respectively, and have been used by humans for a long time. Such use has been regarded as a kind of industrial applications of insects for human use.

Industrial use of insects can be divided into the following four categories: (i) utilization of individuals; (ii) utilization of their products; (iii) utilization of microorganisms related to insects; and (iv) utilization of their specific biological properties (Fig. 1).

Figure 1.

Categories of industrial utilization of insects.

Utilization of individuals

Well-known cases of utilization of individual insects are natural enemies and insect pollination. Natural enemies have been used since ancient times for pest management. In recent years, some companies produce large numbers of natural enemies to be sold for insect pest management. This can be referred to “natural enemy formulation”. Examples of taxa used for natural enemy formulation are spider mites and parasitoid wasps, which are mass-produced in the laboratory. In addition, by utilizing the pollen-borne behavior of insects, honey bees and bumble bees have been widely used for pollination of crops.

Utilization of their products

Since ancient times, humans have used polymeric substances produced by insects. Typical examples are silk and honey produced by silkworms and bees, respectively. Particularly, in Japan, since the opening of the country in the late 1800s, the sericulture industry had been a key factor in the economy of the country. For honeybee utilization, the recent trend is an increased demand for functional byproducts such as royal jelly and propolis in addition to honey. Also, in Southeast Asia, the production of white wax and rack, which is resin produced by scales, has been established as an industry.

Utilization of insect-related microorganisms

Parasitic insect-related microorganisms such as bacteria, viruses and fungi have been utilized as a microbial pesticide for pest control. As control agents of lepidopterous pests, Bacillus thuringiensis (Bt) has been studied since the late 1970s, and has been commercialized. Further, as a unique system using nuclear polyhedrosis virus (BmNPV), a virus pathogenic to silkworm, has been established for producing a useful substance in the silkworm and has been commercialized.

Utilization of insect-specific properties

The utilization of insect specific properties is attracting much attention in recent years as industrial applications of insect technology have expanded. It is a new field of research attempting to analyze the specific physiological properties of insects and establish a technology for helping humans by using the most advanced techniques such as molecular biology and genetic engineering. The target properties for utilization are certain insect-specific genes, metabolism, products and useful cells derived from insects.

This review provides an overview of the current state of research on utilization of insect properties as a new technology.

Novel utilization of insect materials and properties

Definition of insect properties

The term “insect properties” is unfamiliar to the general audience. In Japan, since about 1986, it has been used to mean “specific biological functions of insects”, and is now widely accepted even in the general society. As mentioned above, insects are the most abundant organisms in nature. More than one million known insect species account for about 70% of all the animal species. In addition, insects, which appeared on earth approximately 400 million years ago, have gained considerable diversity in the process of adaptation and evolution to the present day. The long process of evolution and their adaptation to various environments and expanding habitat were the driving forces for acquiring their unique physiological properties. We use the term “insect properties” to refer to those specific physiological properties.

Research on insects, until recently, has been mainly focused on more efficient utilization of beneficial insects and the control of pest species. However, when one closely examines the so-called “ordinary insects” that have little impact on our lives, it is clear that they are likely to have good properties, which are completely unknown and can be expected to be of potential value. We can use these unknown properties for the development of new insect technologies.

Start of “research for utilization of insect properties”

The aim of “research for utilization of insect properties” is to discover new insect properties that can lead to their commercial use. This is a new and unique field in Japan. One of the factors that prompted the origin of this research was the decline of the silk industry in our country. Since the Meiji era, sericulture played a very important role as a key industry in the development of the Japanese economy. Since 1930, however, when cocoon production reached its peak, the sericulture industry has decreased continuously.

During this decline, national, public and private research institutions as well as universities supported the sericulture industry through cooperation. They have continued to study a wide range of topics from basic science to applied techniques. However, the decline of this industry forced a reduction in such studies, and in 1983, the national organization, Sericultural Experiment Station (“Sanshishikenjo”), the hub of sericultural research in Japan, reduced the size of its research by nearly half. Private and public research institutions related to sericulture were also either reduced in size or ceased to exist.

Five years later, in 1988, the Sericultural Experiment Station was reorganized as the “National Institute of Sericultural and Entomological Sciences (NISES)”, and technical knowledge on silk thread ranging from basic to applications was passed on as a new research field on the “effective utilization of insect properties”. At this point, the new research field called “research for utilization of insect properties” started, leading eventually to the recognition of this research field by the academic community.

Center of Excellence at NISES

In 1996, NISES proposed the “Research for utilization of insect properties” as a “Center of Excellence (COE)” project within the Science and Technology Promoting Program, and the proposal was adopted. The outline of this project proposed by NISES at that time is reviewed below (National Institute of Sericultural and Entomological Science 2001a,b) (Fig. 2).

Figure 2.

Cover of pamphlet of “Research for utilization of insect properties” as a Center of Excellence (COE) project of Science and Technology Agency, by National Institute of Sericultural and Entomological Sciences in 1996.

The project “Research for utilization of insect properties” consisted of the following two studies: (i) Creation of new materials based on insect properties; and (ii) Modeling of insect properties and their utilization.

The first study involved analyses of the regulatory mechanisms of metabolism of specific substances found in insects (e.g., fibroin, chitin and host-defense proteins) at the molecular level and chemical and other modification of these molecules, and development of a system for their large-scale production. From these steps, biomaterials with novel properties can be created.

The second study involved analyses of insect-specific sensory systems, stimulus perception, information processing, exoskeletal structure and motor functions (flying, walking, etc.) and their use in developing future technologies such as biochips, biosensors and micromachines. Although the above two studies were proposed, the principal focus of the research was on the former. The appropriation of the research funding focused to the deployment of the former studies. The study “Creation of new materials based on insect properties” was further divided into the following three sub-themes.

Search for new materials, analysis of their structure and investigation of their mechanisms of biosynthesis

Host-defense systems without involving antibodies, anticoagulant activity found in blood-sucking insects, and biosynthesis of macromolecules such as fibroin and chitin have attracted close attention as insect-specific properties. The proposal is to study these properties at the molecular levels (DNA, protein, etc.) and clarify their action in detail for the purpose of obtaining new antibacterial substances and drugs.

Modification of new materials, evaluation of their properties and development of technology for their utilization

Following clarification of the properties of insect-derived macromolecules such as silk fibroin and chitin, the proposal attempted to modify these molecules chemically and physically, evaluating the functions of the modified molecules and developing hybrid materials by combining them with macromolecules derived or synthesized from other organisms. It also attempted to develop techniques for attaching new properties to synthetic antibacterial proteins and anticoagulant factors and to evaluate them.

Development of technology for large-scale production of new materials

This theme aimed to develop improved baculovirus expression vectors for utilization in insects or cultured insect cells, with the goal of establishing a technique of efficient and continuous production of novel functional substances, which can be evaluated in the research described above, as well as useful substances attached with new properties also described above.

Furthermore, it aimed to develop simulation models for insect growth, etc. and “insect factories”, which are fully automated systems for large-scale insect rearing and production of useful substances so that new materials and substances with new properties can be produced on a large scale at low cost. At the time of the proposal, although the abovementioned “Research for utilization of insect properties” had been proposed as a COE research project, the proposed contents were merely a part of the more general research for utilization of insect properties. Basically, the category of research for utilization of insect properties was rather vague, and its definition differed among researchers.

In the next section I introduce the main topics in recent years, according to research themes that were proposed and promoted by the COE project of NISES at the time.

Recent research topics in research for utilization of insect properties

Search for new materials, analysis of their structure and investigation of their mechanisms of biosynthesis

Useful functional materials and analysis of their function

To explore useful functional materials and substances produced by insects, selection of a target is important. Materials and substances that are useful for human life should be the first to be considered. In this sense, the potential for medical demand would be very high. Also, insect-derived materials with anti-cancer effects or inhibition of multiplication of pathogens resistant to conventional drugs would be attractive as a new category of substance. Many functional substances have been found in insects, and research continues to analyze their mechanism of action.

Antimicrobial protein

One of the most important elements of the defense reactions in insects is antimicrobial proteins. These are assumed to elicit the reactive secondary humoral immune response. Antimicrobial proteins, on invasion by bacteria or fungi, are produced mainly in the fat body and are secreted into the hemolymph. Hundreds of proteins and anti-microbial peptides have been isolated from 50 species of insects and mites. Ishibashi (2009) reviewed the structural characteristics of antimicrobial proteins found in the genomes of five species of insects including Drosophila melanogaster, Bombyx mori, Anopheles gambiae, Apis mellifera and Triborium castaneum, whose genome sequences had been analyzed by the end of 2009.

At the initial stages of the project, researchers tried to separate a variety of antibacterial proteins from various species of insects and mites, with the aim of commercializing the antibacterial proteins and peptides by modification of their structures. Although such proteins and peptides have not led to practical uses so far, study is now focused on the immune system and the basic mechanism of their actions. The evolution of the innate immune systems of lepidopteran insects has also been studied by using silkworm genomic information, after the silkworm genome was reported (Tanaka et al. 2008).

Anti-blood coagulant

An interesting substance was found in the blood-sucking insect Rhodnius prolixus (Yuda et al. 1996). This substance, designated as Prolixin-S, has possible applications in the medical field. Prolixin-S inhibits human blood clotting; its detailed mechanism of action has been investigated.

This substance, secreted from the salivary glands of R. prolixus, inhibits blood coagulation in combination with another factor in the human blood coagulation system. Furthermore, it was also found to act as a relaxing factor for vascular smooth muscle. This vasorelaxant effect is due to the biochemical characteristics of this substance; that is, Prolixin-S is a hemoprotein. Prolixin-S combines with nitrogen oxide (NO), which causes, after release, relaxation of vascular smooth muscle. Prolixin-S had potential application as a medicine for the prevention and treatment of various diseases related to the human circulatory system (Chinzei 2000). Unfortunately, this substance has pharmaceutical problems, such as high production costs, dosing methods and allergenicity; therefore its applications have not been realized so far.

Anti-cancer and cancer cell growth inhibitory substances
Pierisin

A substance that effectively kills cancer cells was found in the cabbage butterfly Pieris rapae, and has been studied for its practical applications. This substance was named “pierisin” from the butterfly genus Pieris. Pierisin was discovered by a group from the National Cancer Institute. Piericin, which exists in the hemolymph of cabbage butterfly larvae, pupae and adults, kills gastric cancer cells (TNK-1) in cell culture. Based on research on the mechanism of action of pierisin, it is thought that the very strong killing effect against cancer cells is due to apoptosis (Watanabe et al. 1999).

However, the problem for using it as an anti-cancer agent is that pierisin acts not only on cancer cells but also on normal cells. Further research on its applications is underway.

Yamamarin

“Yamamayu” silkworm Antheraea yamamai is a large moth native to Japan. Diapause in this moth is referred to as a “pre-larval diapause”. After the body of the “yamamayu” larvae is formed within the egg, the larvae remain there for about 8 months without hatching. A peptide consisting of five amino acids that induces the pre-larval diapause was found by a research group at Iwate University (Yan et al. 2004) and was named “yamamarin”. Surprisingly, yamamarin showed significant growth inhibitory effects against rat liver cancer cells. This effect was found to be reversible, from which control of cell synchronization has been inferred (Yan et al. 2004). In other words, it is thought that yamamarin causes a similar phenomenon against intact rat liver cancer cells as it does in maintaining diapause in A. yamamai.

Growth regulation in insects and mechanism of hormonal action

The endocrine system of insects, i.e. hormones, is involved in major physiological events such as development and diapause, which are regulated by hormones. Juvenile hormone and molting hormone, which are fat-soluble and have low molecular weights, are the most important in insects. Both are involved in various physiological phenomena such as embryogenesis, metamorphosis, molting and diapause. For this reason, it is very important for research for utilization of insect properties to clarify, in detail, the mechanism of action of these hormones and to elucidate the key elements in the endocrine system, which is expected to lead to pest control and production of useful substances.

These two hormones have been studied since 1940, and in recent years remarkable progress has been achieved in molecular studies based on genome sequencing analysis of various insects. An overview of the recent research in this field is summarized by Kamimura (2009); the present review will introduce some of the research topics that have been done by Japanese researchers using the silkworm Bombyx mori and were published in the past 2–3 years.

Molting hormone (ecdysteroids)
Molting hormone-degrading enzyme obtained from fungus

The most important action of insect molting hormone (ecdysteroids) is the induction of molting. Therefore, the amount of ecdysteroids in the blood is strictly controlled, so as to induce molting and metamorphosis accurately. The fungus Nomuraea rilleyi, which causes a silkworm disease, produces an enzyme that specifically degrades ecdysteroids, i.e. ecdysteroid-22-oxidase (E220), was isolated and characterized (Kamimura et al. 2012). Application of E220 to silkworm larvae is effective in reducing ecdysteroids concentrations in the blood. Furthermore, the degradation of ecdysteroids by E220 has been confirmed, not only in the silkworm, but in other insect orders as well. From these results, E220 appears to have the potential to be effectively utilized for controlling various pests.

Juvenile hormone
Three-dimensional structure of JH-binding protein

Juvenile hormone (JH) is very slightly soluble in water. Therefore, when JH is transferred to the blood from the secretory organ (corpora allata) to target organs, it is combined with a protein called JH binding protein (JHBP). However, the detailed mechanism of the molecular structure of JHBP was unknown. Recently, the mechanism of JH transfer to the blood and its protection from degrading enzyme were clarified by elucidation of the three-dimensional structure of JHBP (Suzuki et al. 2011). JHBP–JH complex was first prepared, and its three-dimensional structure was determined in solution and the crystalline state, by nuclear magnetic resonance (NMR) and X-ray crystallography, respectively. JH can be protected from enzymatic degradation in the blood and be transferred to the target cells by being stored in the portion of the pocket structure within JHBP, without binding to portions other than the target organ. This success in determining the three-dimensional structure of JH–JHBP complex is the first report of this type.

Identification of precocious metamorphosis

There are many silkworm mutant strains, which vary in the number of times of molting. One of these strains is “dimolting (mod)”. Whereas the normal silkworm metamorphoses to pupa after four larval molts, “dimolting” metamorphoses precociously after the third instar, having molted twice. Until recently, it was supposed that this dimolting strain had some abnormality in hormone secretion, but no molecular-level analysis had been performed. Now, based on the silkworm genome information, Daimon et al. (2012) revealed the mechanism of action of the genes involved in this phenomenon. Identified gene CYP15C1 is key in the synthesis of juvenile hormone in the corpora allata. It was found that dimolting does not have juvenile hormone in its body, due to the loss of function of this gene, thus leading to precocious metamorphosis.

However, it still remains a mystery why precocious metamorphosis does not occur at the first or second instar and happens for the first time in the third stage (Fig. 3).

Figure 3.

Cocoons and moths of the precocious metamorphosed dimolting silkworm Bombyx mori. Left: Cocoon (upper) and moth (lower) of normal silkworms that molted 4 times. Right: Those of dimolting mutant that metamorphosed precociously before the 4th instar. Photograph by T Daimon of NIAS.

Metamorphosis-suppressor gene

JH is a hormone known to inhibit metamorphosis from larva to pupa. The kruppel homolog 1 gene (Kr-h1) plays a key role in the repression of metamorphosis by JH based on experiments in the beetle T. castaneum. However, the detailed molecular mechanism of JH-mediated induction of the action of Kr-h1 was not known. Research groups from the NIAS investigated its mechanism using a silkworm cell line (Kayukawa et al. 2012). As a result, they succeeded in identifying a region as the JH response element in the Kr-h1 gene and found two key proteins involved in JH-dependent activation of Kr-h1. These two proteins combine to form a complex with JH to activate Kr-h1 by interacting with the JH response element.

It is expected that this system can be used for the effective screening of JH antagonists and JH agonists for the development of new pesticides.

Analysis of silkworm genome

Because one of the goals of research for utilization of insect properties is to create an industry based on insect properties, the functional analysis of specific genes of insects is a very important point. Silkworm is one of the most important insects, not only limited to research for utilization of insect properties, but for more general industrial utilization. In 2004, a draft analysis of the silkworm genome was finished by both Japanese and Chinese research groups at the same time, independently.

In addition, in 2006, Japanese and Chinese researchers signed a joint research agreement to integrate the draft data of the two projects. As a result, in January 2009, the full silkworm genome was decoded (The International Silkworm Genome Consortium 2008). Currently, decoding of the sequence exceeds 90%, which covers 88% of the silkworm chromosomes.

It is estimated that the number of genes in the silkworm is about 17 000, 90% of which are sequenced and aligned to their positions on the chromosomes. The integrated database “KAIKObase” was built and published for open access. This database includes the total genetic information, including expressed genes and genetic map of the genes.

In advancing research for utilization of insect properties, the comprehensive public database KAIKObase will be of important significance. As described above, the main outputs of research for utilization of insect properties are: (i) production of useful substances derived from insects; and (ii) pest control based on gene function analysis. In the former, the silkworm is mainly used and basic information in KAIKObase is a useful tool for identification of useful genes and information mining from gene expression analysis. Further, since about 40% of crop pests are lepidopterans, in the latter case we will be able to identify target genes based on the silkworm genome information for advancing the development of pesticides.

One of the interesting outcomes obtained by using silkworm genome information is discussed below.

A group from NIAS, in collaboration with French researchers, revealed that the silkworm and some lepidopterous pest insects were similar in their genomic structure (d'Alençon et al. 2010). After sequencing of the genomes of two important noctuid pests, Helicoverpa amigera and Spodoptera frugiperda, genome structures were compared with the silkworm. The arrangement of genes on their genomes was found to be almost the same. However, in detail, there were considerable differences in the distances between genes, gene orientations and the numbers of amplification. These findings were achieved effectively by using the silkworm genome information, and it is shown that analysis of lepidopterous gene dynamics can lead to the development of new pesticides.

Development and utilization of transgenic silkworm

In 2000, a research group from NIAS developed a transgenic silkworm (Tamura et al. 2000). The jellyfish green fluorescent protein gene (GFP) was successfully introduced to the silkworm for the first time. Since then, transgenic silkworms have become a powerful tool for industrial use, in particular, producing useful substances and modifying the properties of silk protein. Moreover, they have also become an important technique in the study of fundamental analysis of gene function.

Production of useful substances

The advantages for utilizing transgenic silkworms are as follows.

  1. The silkworm can be stably supplied as a material due to the accumulated technology for breeding and knowledge of its biological nature.
  2. Once an interesting gene is introduced to the silkworm, by maintaining the strain, it is possible to produce useful substances for a long period.
  3. Transgenic silkworms can synthesize proteins having more complicated structures than those produced by gene-manipulated microorganisms.
  4. Transgenic silkworm can produce a lot of proteins derived from mammalian cells.

Currently, one private company has partnered with Gunma Prefecture, and is trying to produce drugs using transgenic silkworms. In 2012, they commissioned the “Cooperative society for breeding transgenic silkworms” for rearing 24 000 individuals of a transgenic silkworm. They have already started to supply human collagen for cosmetic materials and are now pursuing the development of human fibrinogen as hemostatic agents.

Functional modifications of silk

In the industrial utilization of transgenic silkworms, another important application is the biochemical modifications of silk that can alter its functional properties. By expressing a foreign gene in fibroin, which is the main component of the silk fiber, silk can acquire new functional properties. It is possible to introduce into fibroin several genes of fluorescent proteins such as green and red fluorescent proteins. NIAS has developed fluorescent clothing, such as jackets and scarves from fluorescent silk and silk cloth in cooperation with university laboratories, Gunma Prefecture and private companies (Fig. 4).

Figure 4.

Womenswear made by recombinant silk with fluorescent colors. Photographed under UV in the dark. Photograph by H Sezutsu of NIAS.

Analysis of gene function

The transgenic silkworm is a powerful tool for functional analysis of specific genes. Researchers often use mutant strains in order to determine the genes involved in specific characteristics. This is possible because many mutations are caused by a deficiency or dysfunction of specific genes. Most mutations are caused by unknown genes, and it is necessary to confirm that the isolated and identified gene is really involved in the transformation and functional expression of the mutation. For this purpose, one of the powerful techniques for analysis is transgenic silkworms. The gene responsible for the mutation can be confirmed if the gene function or the original trait before mutation is recovered by forced expression of the isolated gene introduced into the mutant.

However, this system utilizing transgenic silkworms still has some points that have to be improved. These are: (i) improvement in the amount of proteins produced; (ii) analysis and regulation of the factors that will increase the expression and the translation efficiency of the proteins; and (iii) development of new genetic markers. Also in industrial utilization, some problems will still remain as future challenges. These are systematization of technology in transgenic silkworm rearing and compliance with the law on production of useful substances in transgenic silkworm.

One example of novel gene identification by using transgenic silkworm is discussed below.

“Red egg” (Bm-re) is related to the metabolism on ommochrome pigment. Various mutants of the color of silkworm eggs are known. The trait of egg color (brown, purple or red pigment) is due to ommochrome metabolism. The “Red egg” mutant that has a distinctive dark red color is caused by abnormality in the synthesis of pigments in the ommochrome system. Effectively using the silkworm genome information, a research group from NISES clarified that the causative gene of the red-egg mutant was Bm-re (Osanai-Futahashi et al. 2012). They also found the Bm-re gene in several insects other than silkworm, except Drosophila. In these insects it is involved in metabolism of the pigment. It should be noted that Bm-re is used as a marker useful for gene recombination in the silkworm. It is because Bm-re has an advantage as a marker, i.e., one can detect the color with the naked eye whether or not an individual is transgenic. Bm-re may be used for various experiments on transgenic silkworms as an alternative to fluorescent markers that are currently used.

Another paper typical of this research field is introduced below.

It is the achievement, in which the causative gene of silkworm resistant to B. thuringiensis (Bt) toxin was identified (Atsumi et al. 2012). This proteinous toxin produced by the entomopathogenic bacteria Bt has strong and specific insecticidal activity to lepidopteran insects, and has been used as a pesticide. Genetically modified insect-resistant crops were also developed, in which Bt toxin gene was introduced.

A research group mainly composed of NIAS scientists has analyzed the genomes of individual silkworm strains resistant to Bt. They found that the gene responsible for resistance was recessive. From positional cloning based on the silkworm genome information, they could estimate that this gene corresponded to an adenosine triphosphate (ATP)-binding cassette (ABC) gene. Finally, using the transgenic silkworm, they confirmed that the gene itself is the ABC gene. From the following points, I would like to evaluate the topic as a typical achievement in “Research for utilization of insect properties”. The NIAS researchers:

  1. effectively used both resistant and susceptible strains found in the genetic resources of the silkworm in NIAS
  2. isolated resistant individuals by classical breeding experiments
  3. determined the position of the gene in the chromosome based on the already completed silkworm genome information
  4. estimated the corresponding gene to be the ABC gene by effectively using the silkworm genome information
  5. confirmed that the gene was in fact the ABC gene by using a transgenic silkworm.

In other words, this excellent result was achieved by an effective use of all the following assets: genetic resources, classical genetic techniques, and most advanced techniques such as genome information and transgenic silkworms. Furthermore, since the identified genes have high value in the industry as well, it is the desired type of outcome of “Research for utilization of insect properties”.

Modification of new materials, evaluation of their properties and development of technology for their utilization

Until now, silk has mostly been used as a textile fiber. However, recently silk has become a target for a new utilization beyond the textile field by regarding it as a material having characteristics of proteins.

Silk protein is secreted from silk glands in the silkworm. The glands have the ability to synthesize a very high amount of protein, reaching as much as about 0.4 g of protein per individual.

Silk consists of two proteins, fibroin and sericin; fibroin accounts for about 75% of the total protein. As a biomaterial, fibroin is more appropriate for utilization. The reason is that fibroin has the following advantages: (i) it is biocompatible and does not become as allergen; (ii) it can be supplied in a large amount easily; (iii) it is easily prepared as material; and (iv) chemically, it is highly reactive.

One method to modify the properties of silk protein is alteration of the structure of silk. Ultra-micronized silk protein, powdered by physically grinding, still retains excellent characteristics of silk, which are that it is hygroscopic and releases moisture. From this ultra fine silk powder, with an average particle size of 3-nm diameter, cosmetics composed of 100% silk have been developed (Tsubouchi & Fujiwara 2001). It is also possible to prepare silk film from silk protein by dissolving it in water and drying it on a glass plate. Since silk film has the effect of accelerating cell proliferation, development of a wound curing material and a cosmetic pack product has recently been attempted.

Further, much interest is focused on the 3D structure of fibroin (silk sponge) (Fig. 5). Until recently, there had been no effective method for preparing a sponge-like structure from silk protein. Therefore, development of a safe, simple and inexpensive method for preparing a silk sponge was a goal. In 2005, a NIAS research group succeeded in finding a simple way to prepare silk sponge by adopting the process of freezing and thawing of an aqueous solution of gelled fibroin (Tamada 2005). The silk sponge has excellent properties. It is strong, and has good biodegradability. In addition, active cell proliferation takes place in small pores of the sponge. Therefore, research for medical applications such as wound dressing material or cartilage regeneration continues. Since 2009, NIAS, medical laboratories and private companies have jointly participated in a 3-year project to promote the commercialization of silk sponge products. In this project, a cosmetic pack has been developed as a practical product, which has reached a stage close to commercialization.

Figure 5.

Scanning electron micrograph of fibroin sponge formed dimethyl sulfoxide (DMSO) at 1.0 vol.% as the solvent. Fibroin concentration was 4%. Photograph by Y Tamada of NIAS.

Development of technology for large-scale production of new materials

In the late 1990s, a system that produced useful substances using insects was referred to as an “insect factory”, with the bodies of insects regarded as the “factory”. Now, however, this term is not used as much, and the term “Useful substance production system using insects” has become more popular. Silkworms have mainly been used for those production systems in two major ways. One is the method of using B. mori nuclear polyhedrosis virus (BmNPV), which belongs to the baculovirus family and infects silkworms, causing disease. Another is that using the abovementioned transgenic silkworms.

Method using baculovirus (BmNPV)

A useful protein production system using BmNPV and silkworm was developed by Maeda et al. (1985). In this system, a useful gene is first introduced into BmNPV by gene recombination. Then, the silkworm is infected with the recombinant BmNPV. The gene of a useful protein in the recombinant BmNVP is multiplied in the body of the silkworm, and the multiplied BmNPVs then produce useful proteins. Because this useful protein is secreted into the hemolymph of silkworm larvae, it can be commercialized after removal of the viruses from the hemolymph and its purification.

Maeda et al. (1985) adopted BmNPV as the vector for production because of the following advantages of BmNPV: (i) it produces large amounts of the polyhedral protein; (ii) the polyhedral protein production is independent of the growth of BmNPV itself; and (iii) analysis of the genomic structure of BmNPV was progressing.

In 1992 the private company “Toray” developed a system producing medicine for cat calicivirus disease using the above system (Yanai et al. 2002). This pet medicine has been commercialized under the brand name “Intercat” since 1993. This substance withstands acidic conditions very well; thus efficient purification was achieved by taking advantage of this feature. In addition, the drug formulation IFN-y produced by the same system for dermatitis of dogs was marketed in Europe under the brand name “Interdog” in 2005.

In 1996, Katakura Industries Co. Ltd. started an on-demand protein production business called “KaikoExpress”, which uses the BmNPV–silkworm system. This service is intended to produce and provide active protein reagents for research use on request, and has a proven track record of more than 1500 orders. This sector was acquired by Sysmex Corporation in April 2011, and the service “KaikoExpress” continues under the new name “ProCube”.

Production of useful substances using transgenic silkworm

This system has been described above in the section about the production of useful material by the transgenic silkworm. Antibodies, enzymes for diagnostic medicine and vaccines for pets are proteins suitable for the production system using transgenic silkworms. However, in the case of pharmaceutical products targeting humans, development is still in the future because of the high authorization standards for genetically modified organisms and other requirements.

Advantages and disadvantages of the two useful substance production systems

The two useful substance production systems, i.e. BmNPV–silkworm system and the transgenic silkworm, have both advantages and disadvantages.

The advantages of the BmNPV–silkworm system are: (i) since the technique was established in 1992, it can easily be applied to new materials; and (ii) because of the short life cycle of the silkworm, it is suitable for producing a wide variety of proteins in small quantities simultaneously. On the other hand, the disadvantages are: (i) silkworms infected with recombinant BmNPV are not sustainable because the insects die in one generation; and (ii) purification of useful substances from silkworm blood may not be easy in some cases.

On the other hand, the advantages of the transgenic silkworm are: (i) the production of useful substances can be continued for a long period by maintaining silkworm strains with a successful gene recombination; and (ii) purification of a substance will be relatively easy by confining the gene expression within a specific tissue. However, there will remain some disadvantages, such as: (i) low efficiency in producing individuals having genetic recombination, and difficulty in obtaining the desired recombination of genes; and (ii) systematic maintenance of transgenic silkworms consumes much time and money. Therefore, when producing a useful substance in the silkworm, it is important to carefully consider the characteristics of the two methods as well as other necessary methods to be employed.

Future prospects for research into utilization of insect properties

The research field of utilization of insect properties is a new and unique and originated in Japan. The ultimate goal of this research is to create a new insect industry. It is also a new field of biotechnological applications in science and technology of insects. Suitable materials are desired for the development of new industries, as well as for basic research. From this perspective, the silkworm is one of the most appropriate insects for this project.

In fact, silkworm has a number of advantages not only as an industrial insect but also in insect biology research. These include: (i) the body size of silkworm is relatively big compared to other insects; (ii) silkworm can be reared throughout the year by technologies of artificial hatching and artificial diet; and (iii) numerous strains and mutants are conserved. In addition, only recently new biological and technological tools have become available for such a research field: transgenic silkworms and the complete silkworm genome information. These will be important tools for genetic engineering and gene functional analysis.

I hope that these advanced technologies will further contribute to the progress of the research project by combining the already accumulated knowledge and technologies of the silkworm as well as the derived materials, which may lead to creation of new insect industries. Furthermore, it is also expected that future advancement of the project will be applied to the more general field of pest and insect management and utilization.

Acknowledgments

I thank Dr Shigeru Hosoi (Tsukuba Research Laboratory, Hamamatsu Photonics) for his critical reading of the manuscript and I also thank some of researchers of National Institute of Agrobiological Science for their help in writing this manuscript.

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