Over the last several years, animal welfare concerns have forced scientists to reduce the number of vertebrates, especially mammals, used as experimental animal models, and alternative animal models that do not require approval by the ethics committee have been sought. Given that silkworms have been used in the silk industry for centuries and their application for research does not require ethical clearance, their use as experimental animal models is both less complicated and less costly compared to vertebrates.

The utilization of silkworms in the silk industry has facilitated its domestication. The proper methods of feeding and maintaining silkworms are well-established. The development of artificial diet in the 1960s replaced the seasonally available mulberry leaves and allowed year-round utilization of silkworms. The feeding of artificial diet not only facilitated breeding and rearing but also contributed to a uniform quality of silkworms. Uniformity in quality is important for robust and reproducible results in studies performed using model animals. The widespread distribution of the silk industry makes it easy to obtain fertilized eggs, thus dramatically reducing the time and labor required to care for silkworms. As fifth-instar day 2 larvae are utilized for infection assays, the total silkworm rearing time required from hatching of the eggs to using the larvae for infection assays is <3 weeks, which is far shorter than the time required for mammals. Utilization of aseptic procedures during rearing allows the generation of germ-free larvae. Furthermore, larvae molt four times; thus making it easy to distinguish each instar stage and reducing individual genetic differences.

A silkworm rearing room generally includes an incubator and a safety cabinet. Thus, silkworm rearing does not require large and expensive equipment, making it much less expensive than other animal models. Furthermore, in contrast to mammalian models, large numbers of larvae can be reared in a single cage, which significantly reduces the cost of maintaining silkworms. Based on our experience, the cost of using silkworms is 1% that of the cost of utilizing mice.

In general, experiments utilizing silkworms require less time than those utilizing mammalian models. Further, silkworm larvae have a large enough body size for easy handling by human hands, and samples can be injected with the same syringe type used for medical purposes in humans (Figure 1A). Samples can be injected into the hemolymph, which corresponds to intravenous injection in mammals, and the midgut, corresponding to oral administration. Moreover, silkworm organs such as the midgut can be isolated for experimental use. Silkworms are easy to work with; they do not have sharp horns/hair, teeth, or claws that can sting, and do not bite. Moreover, the adult moths cannot fly and the movement of silkworm larvae is slow and weak, making them easy to handle for injecting samples. This weak and slow movement also makes it difficult for silkworms to escape from their cages, allowing us to perform infection assays for level 2 pathogens. As silkworms have been very highly domesticated through the long history of the silk industry, they cannot survive or reproduce in a natural environment. This further adds to the safety of using silkworms for injection of pathogenic organisms and reduces the biohazard potential.

Attempts to decode the B. mori genome were performed in 2004 and 2008 (Xia et al., 2004; The International Silkworm Genome Consortium, 2008). The availability of the genome sequences facilitated the development of basic genetic and molecular genetic tools and markers. Over the last decade, the development of genetic technologies for B. mori has greatly advanced. Genetic modification may be achieved by transposon-based technology, such as transgene integration or expression; RNA interference-based gene silencing; gene- and enhancer-trap methods and genome editing technology utilizing zinc finger nucleases; transcription activator-like effector nucleases (TALENs); and CRISPR/Cas9 (Xu and O'Brochta, 2015). Furthermore, Dr. Sezutsu's group in the National Agriculture and Food Research Organization, Japan, recently succeeded in utilizing these various techniques to edit the silkworm genome (Inagaki et al., 2015; Sakurai et al., 2015; Takasu et al., 2016). With these recent developments, the silkworm now has a sophisticated genetic modification system and can thus be used to establish disease models and humanized models to screen candidate compounds and analyze physiologic processes.

Silkworm organs, such as the gut, fat body, and malpighian tubule, correspond to the intestine, liver, and kidney, respectively, in mammals, and are involved in the metabolism and excretion of external compounds (Figure 1B). Pharmacokinetic parameters are very important from the view of therapeutic activity. Even compounds that exhibit good activity in vitro will not be effective in vivo if they have poor pharmacokinetic parameters. We demonstrated that the half-lives of model compounds in silkworms are similar to those in mammals (Hamamoto et al., 2009). We also found that a model compound is metabolized in silkworms by the cytochrome P450 enzyme, follows the metabolic pathway via the conjugation reaction, and exhibits the similar pharmacokinetics as in mammals (Hamamoto et al., 2009). We also demonstrated that the general non-specific transport of molecules through paracellular routes is comparable between the mammalian intestine and the silkworm midgut (Hamamoto et al., 2004, 2005). In addition, the doses of cytotoxic chemicals that are lethal in 50% of the animals (LD₅₀) are similar between silkworms and mammals, indicating that the toxicity of compounds listed in the OECD guidelines can be evaluated using silkworms (Usui et al., 2016). Based on this, we have used silkworms to optimize antimicrobial agents for less acute toxicity and succeeded to increase the median lethal dose (LD₅₀) from 100 to 230 μg/g larvae (Paudel et al., 2013).

Pathogens respond and adapt to the signals appearing in the host environment to successfully infect the host and in most cases, the host's response is critical to determine the degree of pathogenicity. An ideal animal model should provide a response similar to that of humans, during infections. Although insects like silkworm lack acquired immunity, innate immunity is widely conserved among mammals and insects (Hoffmann and Reichhart, 2002). Immune response by silkworms includes humoral response such as: production of various proteins like phenoloxidase, antimicrobial peptides, lysozymes, lectins, serine proteases, etc.; and cellular response such as hemocyte-mediated phagocytosis, encapsulation, nodule formation, etc. We previously reported the activation of innate immunity in silkworm during pathogen invasion and found that a cytokine-like paralytic peptide induced humoral and cellular responses and played an important role in silkworm immunity. Further, we found that the paralytic peptide promoted the engulfment of bacteria and induced nitric oxide (NO) production, that is required for both p38 and MAPK activation (Ishii et al., 2013, 2015a).

All the features of silkworms mentioned above suggest that they are a suitable alternative evaluation system for determining the therapeutic effectiveness of candidate drugs. In addition, the availability of a large number of silkworms with low cost and no ethical issues allows us to screen novel antibiotics for their therapeutic effectiveness.

We collected soil samples from various places within Japan and isolated bacteria from these samples. We screened the supernatants of the soil bacteria for in vitro antimicrobial activity against methicillin-resistant Staphylococcus aureus as a primary screening. Of the 14,651 supernatants, 2,794 (19%) had in vitro inhibitory activity against S. aureus. Surprisingly, only 23 of the 2,794 culture supernatants had therapeutic activity in the silkworm infection model. This low hit rate for the therapeutic effectiveness of the culture supernatants indicates that the silkworm infection model is highly effective for excluding antibiotics that do not have therapeutic effectiveness. We purified antibiotics from the culture supernatant of a Lysobacter, which has recently attracted high interest due to their capacity to produce antibiotics and other natural products (Panthee et al., 2016). Purification based on the therapeutic activity observed in silkworms infected with S. aureus from the culture broth of Lysobacter sp. RH2180-5 led to the identification of a therapeutically active novel antibiotic, lysocin E (Hamamoto et al., 2015) (Table 3, Figure 3A). The detailed purification process showed that the in vitro activity was higher for the partially purified butanol extract than purified lysocin E. This indicated that the culture broth contained both therapeutically active and inactive antimicrobial components and our success in finding a therapeutically active antibiotic relied on utilization of the silkworm screening system.

Lysocin E is a cyclic peptide that exhibits antimicrobial activity against various Staphylococci, Bacillus subtilis, Bacillus cereus, and Listeria monocytogenes with MICs ranging from 1 to 4 μg/ml (Table 4). Based on the genetic and biochemical analysis, we identified menaquinone, a membrane molecule important for the bacterial electron transport chain, as the lysocin E target (Figure 3B). Lysocin E is the first antibiotic discovered that targets menaquinone (Hamamoto et al., 2015; Paudel et al., 2016). Lysocin E also has potent therapeutic activity in a mouse infection model of methicillin-resistant S. aureus (ED₅₀: 0.5 mg/kg). Lysocin E did not exhibit acute toxicity in mice at a dose up to 400 mg/kg. The chemical structure of lysocin E comprises 12 amino acids arranged linearly by two large multimodular non-ribosomal peptide synthetases named lysocin E synthetase (LesA and LesB) that have a total of 12 modules and 43 domains in a 1.7-MDa core peptide (Figure 3C) (Panthee et al., 2017). The potent therapeutic activity and low toxicity of lysocin E in animal infection models suggest its high potential for clinical application and the identification of its gene cluster opened avenues for further derivatization of the structure with enhanced activity.

To identify antifungal compounds, the clinical fungal isolate A. fumigatus FP1305 was used as a test strain. Astellas group screened culture broths of 310 fungal strains for in vitro activity against A. fumigatus FP1305, followed by testing the ability of the broths to cure silkworms infected with FP1305. The culture broth of Acremonium persicinum MF-347833 had therapeutic activity in the silkworm infection model. To purify the antifungal compound, they first attempted to purify the activity based on the in-vitro antifungal activity, which failed to identify some of the therapeutically active fractions. This finding led to the speculation that the culture broth of A. persicinum MF-347833 contained a mixture of antifungal compounds with therapeutic activity or without therapeutic activity. They next utilized the silkworm infection assay-guided purification method to identify therapeutically active antifungal compounds and successfully identified ASP2397 (Nakamura et al., 2017a,b) (Figure 4). ASP2397 has an MIC of 0.2 μg/ml against A. fumigatus FP1305 and is also effective against azole-resistant A. fumigatus (Arendrup et al., 2016), displays therapeutic activity in mice infected with A. fumigatus, and has no cytotoxicity toward mammalian cells at a concentration up to 50 μg/ml, indicating its potential as a therapeutic agent. The planar structure of ASP2397 indicates that it is a metal ion chelator that harbors aluminum, indicating the importance of metal chelation for its therapeutic activity. The fact that ASP2397 was not identified by the conventional approach highlights the utility of the silkworm model for identifying therapeutically active drug molecules.

KS is a consultant for Genome Pharmaceutical Institute, Co. Ltd. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.