Generation of transgenic silkworms for production of erythropoietin in Bombyx mori

There have been many attempts to generate various essential proteins using transformed E. coli systems. However, prokaryote systems are not equipped with the protein maturation mechanisms necessary to generate eukaryotic proteins. In this sense, among the eukaryotes, silkworms have major merits in overcoming the difficulties. Such protein maturation mechanisms are available in silkworms. In this study, a transgenic silkworm producing rhEPO in the cocoon was generated and purified. Specifically, we constructed a transgenic silkworm using a vector system that could be controlled to the next generation. To accomplish this, we microinjected the system into eggs laid during the preblastoderm stage. The rhEPO was then purified from transgenic silkworm cocoons using a Con A affinity column. The biological activity of rhEPO isolated from the cocoon of transgenic silkworms was then assessed in a cell culture system using an EPO-dependent cell line, F-36E. Next, PCR analysis was used to demonstrate that stable gene expression can occur in the embryos of the silkworm, Bombyx. mori. Transgenic silkworms were then selected and observed to ensure that the transgenic silkworm was maintained and transmitted to their progeny. The rhEPO was subsequently purified from the transgenic silkworm cocoon and the electrophoretic pattern of the purified rhEPO revealed a protein band with a molecular weight of approximately 20 kDa. A total of 3 mg of rhEPO was eluted from 10 g of cocoons. The proliferation of F36E cells for 25 ng/ml rhEPO was 1.32, while the proliferation for 2.5 IU/ml hEPO was 1.32. The proliferation of these cells could be induced by commercial hEPO, as well as by rhEPO from transgenic silkworm cocoons. An in vivo test of mice treated with rhEPO revealed relatively high RBC values when compared to normal mice. These results indicated that purified glycosylated EPO from transgenic silkworms had biological activities. Overall, the transgenic silkworm technique will be very useful for the large scale production of proteins for diagnostic and therapeutic purposes.     

Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm,Bombyx mori

Bombyx mori cocoon has a multi-layer structure that provides optimal protection for silkworm pupa. Research on the mechanical properties of the multi-layer structure revealed structure-property relationships of the cocoon. Here, we investigated the protein components of the B. mori cocoon in terms of its multi-layer structure. Liquid chromatography-tandem mass spectrometry identified 286 proteins from the multiple cocoon layers. In addition to fibroins and sericins, we identified abundant protease inhibitors, seroins and proteins of unknown function. By comparing protein abundance across layers, we found that the outermost layer contained more sericin1 and protease inhibitors and the innermost layer had more seroin1. As many as 36 protease inhibitors were identified in cocoons, showing efficient inhibitory activities against a fungal protease. Thus, we propose that more abundant protease inhibitors in the outer cocoon layers may provide better protection for the cocoon. This study increases our understanding of the multi-layer mechanism of cocoons, and helps clarify the biological characteristics of cocoons. The data have been deposited to the ProteomeXchange with identifier PXD001469.     

Maintenance of a narrow host range by Oxyops vitiosa; a biological control agent of Melaleuca quinquenervia

Host range expansion in insect herbivores is often thought to be mediated by several factors, principal among them are secondary plant metabolites. In weed biological control, the host range of a prospective agent is one of the most important considerations in its implementation. Extensive host testing tests seek to determine the behavioral acceptance and nutritional value of different test plant species to the potential agent. A list of test plants is compiled that comprises species that are close taxonomic relatives of the target weed plus other species of economic or ecologic importance. The host testing of the Melaleuca quinquenervia biological control agent Oxyops vitiosa indicated that larvae would accept and complete development on the Australian target weed M. quinquenervia, two Australian ornamental species, Callistemon citrina, Callistemon viminalis (all Myrtaceae). However, the larvae did not complete development when fed a North American species Myrica cerifera (Myricaceae). The study reported here confirms these results and examines the nutritional and performance differences in O. vitiosa larvae fed leaves of these species. The leaf quality factors, percent moisture, percent nitrogen, toughness, and terpenoid content were related to larval survival, performance and digestive indices. The results indicate that plant quality among the Myrtaceae species was generally similar and correspondingly larval survival, performance and digestive indices differed little when larvae were fed leaves of these species. However, significant differences occurred in the plant quality of the North American M. cerifera compared with the Australian species which had leaves with the lowest percent moisture, lowest leaf toughness, highest percent nitrogen. This species, however, is not a physiological host as none of the neonates survived to pupate. When third instars were switched to M. cerifera from their normal host M. quinquenervia reductions were found in survival, biomass gain, digestive efficiency, and conversion of digested food to insect biomass. The marginal acceptance of this North American native plant in laboratory bioassays appears related to the terpenoid chemistry that has similarities to the taxonomically unrelated host M. quinquenervia. However, the high larval mortality corresponds to several novel terpenoids that are not present in the host. For weed biological control host testing these results indicate that M. cerifera is a poor host for O. vitiosa. Additionally, future test plant lists should include plants with secondary metabolites similar to the target weed as these compounds may constitute behavioral cues that are relevant to these specialized herbivores.     

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F₂ progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16_{905 bp} showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F₂ progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16_{826 bp}). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk.     

Mulberry improvements via plastid transformation and tissue culture engineering

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in “molecular farming” is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.Key words: molecular farming, Morus spp., plastid transformation, protoplast electrofusion, sericultureThe importance of silk production is well recognized in sericulture industry that involves cultivation of host plants for silkworm rearing. India is one of the countries where sericulture is an important agro-based cottage industry involved in production of five different silk types—mori, muga, eri, tasar and oak types. This classification comes from type of host plant that act as feed for silkworm, and thus sericulture industry largely depends on the availability of host plant species. Bombyx mori (mulberry silkworm) feeds on mulberry leaves, Philosomia ricini (eri silkworm) on castor leaves, Anthraea assama (muga silkworm) on som and soalu leaves, Anthraea proylei (temperate/oak tasar silkworm) on oak leaves, and Anthraea mylitta (tropical tasar silkworm) on Terminalia leaves. A systematic and proper cultivation of novel primary and/or secondary host plants showing high yield, suitability to silkworm rearing, and resistance to different abiotic stress conditions i.e., tolerance to water stress, alkalinity and salinity are recommended for sericulture improvement.The genus Morus (commonly known as mulberry) belongs to the family Moraceae, is a group of dioecious woody trees/shrubs. Many varieties of these species are cultivated on a commercial scale in India, China, Japan and Korea for the sericulture industry.¹ In India, six species are found, namely, M. alba L., M. indica L., M. nigra L., M. atropurpurea Roxb., M. serrata Roxb. and M. laevigata Wall.² Due to higher economic return and greater employment potential, attempts are been made to increase productivity by developing high yielding mulberry varieties. At present, Mysore local, Bomaypiasbari, Kanva-2 (K2), Bilidevalaya, Kajli, Sujanpur-1 (S1), BC (2) 59, C776, RFS-175, S36 and Victory-1 are being cultivated extensively in different parts of India.     

Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana

Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon.     

Role of the cocoon ofAcrolepiopsis assectella (Lep., Hyponomeutoidae) in host recognition by the parasitoidDiadromus pulchellus (Hym., Ichneumonidae)

The study of various behavioural criteria of femaleDiadromus pulchellus parasitoids in the presence of theirAcrolepiopsis assectella hosts has shown the essentially chemical nature of the stimulant determining host recognition. The physical stimuli of the cocoon seem not to be implicated. Thus cocoons whose original texture has been completely altered, either mechanically or chemically, as well as the silk excreted by the host caterpillars significantly stimulates the female parasitoids. The cocoon contact kairomones are detected in testing the aqueous extracts ofA. assectella which provoke a positive behavioural response from the females in a threshold concentration of 1 cocoon-equivalent. The comparison of aqueous extracts ofA. assectella host cocoons, and of non-host species:Bombyx mori, Ephestia kuehniella andCacoecimorpha pronubana demonstrate the kairomone specificity of the silk, the extracts of non-host cocoons being ignored by the parasitoid, as were the silk threads left byE. kuehniella caterpillars. Finally the contact kairomones linked to the silk seem to be independent of the host plant and of the nutrient diet of the host caterpillars. The cocoons spun by the caterpillars reared on leeks or on artificial diets with or without powdered leek provoke similar responses in the parasitoids.     

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.     

Green cocoons in silkworm Bombyx mori resulting from the quercetin 5-O-glucosyltransferase of UGT86, is an evolved response to dietary toxins

The glycosylation of UDP-glucosyltransferases (UGTs) is of great importance in the control and elimination of both endogenous and exogenous toxins. Bm-UGT10286 (UGT86) is the sole provider of UGT activity against the 5-O position of quercetin and directly influences the formation of green pigment in the Bombyx cocoon. To evaluate whether cocoon coloration evolved for mimetic purposes, we concentrated on the expression pattern of Ugt86 and the activities of the enzyme substrates. The expression of Ugt86 was not only detected in the cocoon absorbing and accumulating tissues such as the digestive tube and silk glands, but also in quantity in the detoxification tissues of the malpighian tubes and fat body, as well as in the gonads. As in the green cocoon strains, Ugt86 was clearly expressed in the yellow and white cocoon strains. In vitro, the fusion protein of UGT86 showed quercetin metabolic activity. Nevertheless, Ugt86 expression of 5th instar larvae was not up-regulated in the silk gland by exogenous quercetin. However, it was significantly up-regulated in the digestive tube and gonads (P < 0.05). A similar result was observed in experiments where larvae were exposed to rutin, an insect resistance inducer and growth inhibitor typically found in plants, and to 20-hydroxylecdysone (20E), an insect endocrine and plant source hormone. On the contrary, up-regulated Ugt86 expression was almost nil in larvae exposed to juvenile hormone III (P > 0.05). The results of HPLC revealed that a new substance was formed by mixing 20E with the recombinant UGT86 protein in vitro, indicating that the effect of Ugt86 on 20E was similar to that on exogenous quercetin derived from plant food, and that the effect probably initiated the detoxification reaction against rutin. The conclusion is that the reaction of Ugt86 on the silkworm cocoon pigment quercetin is not the result of active mimetic ecogenesis, but derives from the detoxification of UGTs.     

Behavioral Attraction of Two Parasitoids,Venturia canescens and Bracon hebetor,to Silk Extracts of a Host Plodia interpunctella

Kairomonal activities of silk extracts of host Plodia interpunctella were determined by measuring the rates of behavioral responses of two parasitic wasps, Venturia canescens and Bracon hebetor. Silk of P. interpunctella larvae attracted both parasitic wasps but the cocoon silk of silkworm, Bombyx mori and the web silk of two-spotted spider mite, Tetranychus urticae did not. Silk components of P. interpunctella were extracted by using either hexane or methanol, and tested the rates of three serial responses of wasps; host location, antennal drumming and ovipositor probing behaviors. The patterns of each behavioral response were similar in two wasps. The rates of each response were increased at the higher concentrations of both extracts. Antennal drumming behavior was much more responsive to lower concentrations of both extracts than ovipositor probing behavior was. Furthermore, the rate of antennal drumming response was higher in hexane-extracts rather than methanol-extract in both wasps; V. canescens and B. hebetor for 20 and 17 times, respectively. However, ovipositor probing response was similar in two different extracts. Both extracts elicited 100% of antennal drumming response but ovipositor probing response was only 60 to 80% of all tested individuals. Our results were shown that silk extracts of host larvae elicited strong behavioral responses of two parasitic wasps and could be applied for practical application of parasitoids attraction in the biological control of agricultural pests.     

Agonosoma trilineatum (Heteroptera: Scutelleridae) a biological control agent of the weed bellyache bush,Jatropha gossypiifolia (Euphorbiaceae)

Bellyache bush, Jatropha gossypiifolia L., is a serious weed of northern Australia. Agonosoma trilineatum (F.) is an insect from tropical America released in Australia in 2003 as a biological control agent against bellyache bush. It feeds on seeds and has the potential to reduce seed production, thereby potentially reducing the rate of spread and recruitment. To test the host specificity of A. trilineatum, four biological responses to host plant species were determined: development of nymphs, oviposition preferences, adult feeding and frequency of mating. Development of nymphs to adults and adult feeding only occurred on three Jatropha spp. These species also supported mating and oogenesis but only J. gossypiifolia was accepted for oviposition. Mating did not occur in the presence of other plant species. The evidence indicates that there is little risk associated with the release of this insect species in Australia and probably other countries where this weed is a problem. The probability of this insect expanding its host range is low because multiple aspects of the biology would need to change simultaneously. A. trilineatum was released in Australia between 2003 and 2007. A Climex model indicated that coastal areas of Queensland and the Northern Territory would be climatically most suitable for this insect.     

Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases.     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.     

C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori

Two flavonoids containing the l-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

Application of Multi-vitamins as Supplementary Nutrients on Biological and Economical Characteristics of Silkworm Bombyx mori L

In order to investigate the effects of supplementary nutrients on silkworm, Bombyx mori, an experiment was conducted with multi-vitamins treatments. Leaves enriched with multi-vitamins (1, 2.5 and 5%) were fed once a day to 4th and 5th instar silkworm larva. The supplementation of the leaves was done by spraying the treatments on them. These treatments resulted in a significant increase in biological and economical parameters such as larval weight, female and male cocoon weight, pupal weight and egg productivity when compared with normal control. Multi-vitamins of 2.5% treatment increased the larval weight by 10.2%, which had the most weight enhancement in the fifth day of 5th instar larvae. However, 5% concentration of multi-vitamins decreased some biological characteristics. These treatments resulted in 4.7% increase of cocoon's weight. In most of the moths the number and weight of eggs had significant difference compared to control, but these supplementary materials caused relatively less egg hatching.     

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant.     

Preventive effects of N‐acetyl‐l‐cysteine against imidacloprid intoxication on Bombyx mori larvae

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N‐acetyl‐l ‐cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth‐instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth‐instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.     

Safety evaluation of four entomopathogenic nematode species against two silkworm species

Chinese oak silkworm (Antheraea pernyi) and mulberry silkworm (Bombyx mori) are economically important insects used for silk production and food resource. Entomopathogenic nematodes (EPNs) from the families of Steinernematidae and Heterorhabditidae are beneficial organisms currently considered in biological control. In this paper, we evaluated survival of two silkworm species exposed to four Steinernema species which are widely applied in pest control. The results showed that among four Steinernema species, S. bicornutum and S. feltiae did not have an effect on the larval survival to the two silkworm species, whereas S. carpocapsae and S. glaseri did have an effect. Each Steinernema species poses no threat to hatchability of eggs, pupation rate, larval durations and cocoon shell ratio.