Wild silk moths produce a variety of cocoons and silks. Given our interest in both the evolution and material properties of silk, we analyzed the silk genes of the saturniid moths Antheraea pernyi, Antheraea yamamai, Samia cynthia ricini, Samia cynthia pryeri, Rhodinia fugax, Saturnia japonica, and Cricula trifenestrata by sequencing clones of genomic DNA libraries and/or cDNA libraries from silk glands. We also analyzed the silk genes of a spider (Araneus ventricosus) and a hornet (Vespa simillima xanthoptera) using cDNA libraries. In saturniid species, the deduced amino acid sequences of fibroins, the major components of silk, consist primarily of repetitive units (motifs) containing polyalanine blocks. Each type of motif was conserved within species, but extreme sequence diversity was observed between species. Polyalanine-containing motifs were common in spider fibroins, although the fibroin of the domesticated silkworm Bombyx mori lacked polyalanine-containing motifs. Hornet silk was composed of four major fibroins with a complex of alanine-rich and serinerich sequences. We have been attempting to introduce fragments of fibroin genes from wild silk moths and spiders into B. mori using transgenic techniques. These studies will enable us to understand the dynamic evolution of silk genes and produce genetically engineered silks.

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Course of growth of eri silkmoth, Samia ricini Donovan was studied by rearing larvae on leaves of two food plants (castor, Ricinus communis; Tapioca, Manihot utilissima) with four combinations (TT, TC, CT and CC) at fixed abiotic conditions (26±2℃ temperature and 60–90% relative humidity). It was observed that peak of growth was reached on 22nd day in TT-fed larvae. It was one day sooner on TC. It was 22nd day on CT and 21st day on CC dietary regimen. It is observed that highest point of growth is reached in CC and lowest peak of growth was seen in TT diet. Peak in CT dietary regimen was little higher than that of TT. Value of angle theta, 89.515 was largest on CC diet followed by on TC, 89.500, CT, 89.069 and TT, 88.493. The above observations indicate that CC diet was best followed by other dietary regimens in the order of TC, CT and TT.

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The nucleopolyhedrovirus of Chinese oak silkmoth, Antheraea pernyi, (AnpeNPV) isolated in Liaoning Province, China, was plaque-purified by using the cell line NISES-AnPe-428 derived from A. pernyi embryos. We employed one of the purified virus, clone A, for constructing a baculovirus expression vector system and succeeded the efficient recombinant protein production in diapausing pupa which is an excellent natural bioreactor storable more than one year in refrigerator. To understand molecular mechanisms of the virus - host cell interaction and improve the vector system, we have sequenced the circular dsDNA genome of AnpeNPV clone A and revealed that the genome is 126,592 bp in size, relatively GC-rich (53.4% G+C), with 150 predicted open reading frames (ORFs), 95% of which (142 ORFs) are homologues in other baculoviruses. Based on the absence of AvrII restriction site in the genome, two AvrII sites have been introduced at 5’ and 3’ UTRs of the polyhedrin gene for linearizing the AnpeNPV DNA. In addition, attR1 and attR2 sites for site-specific cloning of the gene of interest from Gateway® (Invitrogen) entry clone have been inserted just upstream and downstream of the two AvrII sites, respectively. The resulting viral DNA has allowed us to obtain recombinant virus by direct gene transfer in vitro without any purification steps, demonstrating the virus vector applicable for high-throughput production of proteins.

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Eri silkworm (Philosamia ricini Bosid) is an economic insect which is brought to be reared and used for many beneficial aspects because of it strength, easy rearing and established rearing technique as well as domesticated silkworm (Bombyx mori Linn.). The main foods for eri silkworm are cassava (Manihot esculenta L.) and castor bean (Ricinus communis L.) leaves. The eri silkworm egg production and its rearing at the farmer practice project in northeastern Thailand has been conducted in 2008–2009 (for 2 years) at The Queen Sirikit of Sericulture Center (Nongkai and Chaiyaphum provinces) to produce eri silkworm eggs and distribute them to farmers for rearing. In this case farmers can earn money by selling fresh cocoons. The farmers who participated this project comprised persons from 4 provinces nearby area (Khonkaen, Chaiyaphum, Mahasarakham and Mukdaharn provinces). The total number of farmers was 20 persons, among whom were five farmers selected from each province to join the project. The workshop and training course of “Eri Silkworm Rearing” had been made for 4 days at The Queen Sirikit of Sericulture Center (Nongkai) to farmers who joined this project. Then, eri silkworm eggs were distributed to them for rearing. The results indicated that the entire farmer who had been trained could rear the eri silkworm very well which produced good quality of eri silkworm cocoons and high fresh cocoon weight in particular at the farmer from Chaiyaphum and Khonkaen provinces. Therefore, farmers could earn supplementary income and had more experiences to rear eri silkworm with high potential.

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The purpose of this study was to induce trimolters in four Thai Polyvoltine silkworm, Bombyx mori L; Nang Lai, Nang Tui, Hua Fai and Sumrong strains, at 48 hr of the fourth instar larvae by treatment with KK-42 (1-benzyl-5-[(E)-2, 6-dimethyl-1, 5- heptadienyl] imidazole with the doses of 25 and 30 ppm/larva. The result showed that the induced trimolters was the hightest as 84.2 ± 3.5 % in Nang Tui strain and the lowest as 30 ± 2.4 % in Sumrong strain. The cocoon weight, cocoon shell weight and percentage of cocoon shell in the treated groups were lower than the control groups. The cocoon filament in the treated groups was shorter than the control groups; however, the size of filament in the treated groups was thinner (0.6 ± 0.0 to 07 ± 0.3 d.) than the control group (1.5 ± 0.1 to 1.7 ± 0.2 d.). These results clearly showed that treatment of KK-42 to four polyvoltine silkworm strains at 48 hr of the fourth instar larvae remarkably induced trimolters and the thinner filament than untreated tetramolters.

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By-product from reeling eri cocoons is degumming waste water. Therefore, the objective of this research is to exploit the extracted protein from eri waste water as an ingredient of indigenous knowledge cosmetics for value added creation. In this study, liquid soap; shampoo and lotion were prepared using protein from degumming water as ingredient with different formulae (treatments). For liquid soap, 3 formulae were tested, Formula 1 (control); Formula 2 (plus protein from degumming water at the end concentration of 0.01%) and Formula 3 (plus protein from degumming water at the end concentration of 0.05%). The result showed that the soap of Formula 2 was clear brown color. Formula 3 had clear brown but slightly darker color than Formula 2 whereas Formula 1 was clear in color. The soap were evaluated for preference by users. The result revealed that there was no irritation and feel better (66.67% of the users) after application. Fifty percent of users preferred the soap Formula 2 with rating of the most preference score and feel ungreased, soft skin, much moistening feeling and easy to clean. Whereas, shampoo was developed according to 3 formulae, Formula 1 (control), Formula 2 (plus protein from degumming water at the end concentration of 0.01%) and Formula 3 (plus protein from degumming water at the end concentration of 0.05%). The shampoo Formula 3 exhibited clear brown color and slightly darker than Formula 2 while Formula 1 had no color. All Formulae were not irritated and feel better after use by 100% preference of the evaluators. The users preferred shampoo Formula 3 than others in the most preference score with following information: good out-look hair, lustrous, weighing, soft hair and good spring. In making lotions, 10 formulae (treatments) were developed. Most users provided information, good feeling, moisture and soft skin after use of the lotions. They (33.33%) preferred in the level of most preference score for Formula 1(B) with 0.05% protein and Formula 2(A) with 0.01% protein according to soft, moisture, smooth, slightly fragrant, and ungreased. In conclusion, all of these tested eri cosmetics are accepted better than commercial products based on protein concentration and preparation procedure. Moreover, degumming waste water of Thai mulberry silkworm varieties were also tested. This research contributes the potential of eri silk protein from degumming waste water, which can be applied as an ingredient in cosmetics towards the farmer’s income generating.

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The effects of neem extract was studied on economically important yield of the Thai silkworm race, Nang Noi. The experiment was focused on 3 sets of concentrations of the extract. Each concentration set was composed of 5 levels: the first concentration set, 0.0002, 0.0004, 0.0006, 0.0008 and 0.0010 mg l ^-1, the second concentration set, 0.002, 0.004, 0.006, 0.008 and 0.010 mg l ^-1 and the third concentration set, 0.02, 0.04, 0.06, 0.08 and 0.10 mg l ^-1. The neem extract was applied to the 1st to 5th instar of Nang Noi silkworm larvae by mulberry leaf dipping method which was replicated three times. The control was fed with mulberry leaf without the extract. The results showed that the significantly longest larval period at-tained was 430 hrs (3.9% longer than the control) when treated with 0.008 mg l ^-1 of the substance of the second concentration set on the 5th instar larvae. The cocoon weight, pupal weight, cocoon shell weight, shell ratio and filament length were significantly increased by 17.18, 16.29, 36.78, 14.60 and 20.12%, respectively when treated with 0.006 mg l ^-1 of the second concentration set on the 5th instar larvae. This result clearly shows the effect that neem extract had by enhancing the economically important yield of the Nang Noi silkworm race at 0.006 mg l^-1.

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The toxicity of carbosulfan and esterase enzyme activity after carbosulfan exposure in Nang Noi silkworm larvae (Bombyx mori L.) was investigated by the leaf dipping method. Commercial grade carbosulfan was diluted into 5 concentrations to test the toxicity value in terms of the LC₅₀ on 1st-5th silkworm instar larvae. The results showed that the toxicity dramatically increased when the concentration and the time exposure were increased in each instar larvae. Carbosulfan had the highest toxicity in the 1st instar larvae, and the lowest toxicity in the 5th instar larvae. The In vivo study by spectophotometer absorbance at 400 nm revealed that carbosulfan decreased esterase enzyme activities. The expression of esterase enzyme activity was decreased but not significantly different from the untreated in the early instar larvae, 1st-2nd instar, but significantly decreased in the later instar larvae, 3rd-5th instar. Moreover, the results showed that the esterase enzyme activity in each instar decreased when the concentration was increased. In each instar larvae, the esterase enzyme activity was high when treated with the lowest of the carbosulfan concentrations, and the activity was low when they were treated with the highest concentration. The results indicated that carbosulfan was toxic to Nang Noi silkworm larvae. Therefore, carbosulfan insecticides should be carefully used for mulberry insect pest control, for silkworm safety.

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The feeding behavior of Bombyx mori larvae deprived of food after the fourth ecdysis was analyzed by recording temporal feeding patterns over a 3-h period to determine the effects of different foods (mulberry leaves and artificial diets) and food experiences up until the fourth instar. Log-survivorship analyses of the gap length between feedings, in which the data were pooled for each experimental group, revealed that bout criteria classifying between intra- and inter-meal gaps were longer in larvae with different food experiences. Although the change in slope in plots of feeding length was not particularly prominent, a minor change was observed in larvae with different food experiences, suggesting a period of food evaluation prior to eating. The total time spent feeding per meal and inter-meal interval were longer in larvae tested with mulberry leaves than in those tested with an artificial diet. Larvae having different feeding experiences had fewer meals, longer feeding times per meal, and a longer first inter-meal interval. These differences are likely due to nutrient feedback, which was monitored under each experimental condition. The results suggest that food sources and experiences can affect the temporal patterns of feeding behavior and meal characteristics. These characteristics may be useful in investigating the adaptive mechanisms of feeding behavior, even in a monophagous species such as B. mori.

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The knobbed mutant of the silkworm, Bombyx mori, is a dermal mutant that is characterized as having paired knobs at specific regions of larval segments. In order to clarify the details of knob formation mechanisms, cellular morphology of the knobbed region during the fourth instar was investigated by light and electron microscopy. Cell proliferation which induces knob formation of larval epidermis was also detected by whole mount immunohistochemistry using an antibody specific for M-phase nuclei. The epidermal mitotic activities were compared between the knobbed and the surrounding non-knobbed regions during the fourth larval instar.
Mitosis in epidermis of the non-knobbed region was mainly observed to occur at 48 h (mid feeding stage) after ecdysis. On the other hand, numerous mitotic figures were observed in the knobbed region at 24 and 48 h after ecdysis. Clear mitotic figures were also observed at the stage just preceding the molting period (72 h). In the observation using the light and electron microscopy, the epidermal cells at the knobbed region were considerably longer and more slender than those of the non-knobbed region, as clearly seen at the end of intermolt stage. The irregular shape of epidermal cells in the knobbed region appeared to be due to such abnormally prolonged mitotic activity. These results suggest that mitosis occurs at the knobbed regions during most of the intermolt/feeding stage, and that the prolonged cell proliferation observed in the epidermal cells results in an increase of cell number, causing irregular epidermal cell shape and conspicuous outgrowths of integument which takes the form of swollen knobs. We will discuss the sequential processes of knob formation mechanism.

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Sericin has been utilized in various products and commercialized with high value added. Silk degumming waste water of intense yellow color from silk factory in Bangkok, Thailand, which contains sericin, gives two choices to corporate managers; isolation for production of commercial goods or disposal after digestion by protease. In this study, sericin isolation by pH adjustment and salting out with (NH₄)₂SO₄ and organic solvents, and sericin digestion with proteases were investigated. Isolation of sericin by adjustment of pH of waste water to 1-9 and by salting out yielded 93% w/v sericin. In contrast, ethanol and methanol treatments could not precipitate sericin from the waste water. Sericin isolated at pH 7 had the highest antioxidant activity compared to those obtained from other methods. Its activity was much lower than that of vitamin C. Digestion by a commercial protease or by crude papain for 24 h resulted in reduction of sericin concentration in the waste water as much as 66.04±3.06% and 55.66±2.31%, respectively. When low antioxidant activity of isolated sericin and the cost for sericin isolation were considered, digestion of sericin with papain is the most appropriate way to deal with sericin in silk degumming waste water.

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Photoperiodic system consists of several functional units: (I) photoreceptor, (II) circadian/photoperiodic clock, (III) photoperiodic counter and (IV) endocrine switch. To tweeze out these subunits in Antheraea pernyi, we used the probes for the following targets: (1) neurotransmitters, (2) neurotransmitter receptors, (3) neurotransmitter enzymes and (4) circadian clock gene products. We also followed dynamics of these transmitters, the receptor transcripts, clock gene products, cyclic nucleotides, ecdysteroid titer and neurotransmitter enzyme activities.
The results showed; (1) PER- and DBT-expressing cells were identical. These seem to be the circadian clock cells in Antheraea. (2) These cells express melatonin synthesizing penultimate enzyme, arylalkylamine N-acetyltransferase (NAT). (3) These cells also express ultimate melatonin synthesizing enzyme, hydroxyindol O-methytransferase (HIOMP) and melatonin itself. (4) Melatonin content and NAT activity in the brain fluctuated in a circadian manner. These data suggest that melatonin is the output signal mediator of the photoperiodic/circadian clock in Antheraea. (5) Dynamics of neurotransmitter monoamines showed parallel change to the fate of development. Catecolamines decreased, while 5HT increased as diapause was being terminated either by chilling or long days. NAT activity increased when the brain was activated both by long days and low temperature. This means that regulation of NAT activity could function as the photoperiodic counter. (6) PER-expressing cells and PTTH-producing cells juxtapose to each other and the cell expressing 5HT receptor (5HTR)-like IHC reactivity also is located in this vicinity and IHC reactivity against human melatonin receptor, MT2 is also shown in the PTTH cells, which suggests that melatonin could function as a neuroendocrine switch mechanism, binding to this 5HT receptor. Thus NAT-indolamine pathway constitutes the core mechanism for photoperiodism in Antheraea pupae. A tentative synthesis for current knowledge was proposed.

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