The new microsporidia S80 isolated from, Bombyx mori L. in Korea showed ovoid in the morphology of the spores and the size were measured $2.9{\pm}0.28{\mu}$ in length and $1.7{\pm}0.29{\mu}$ width. No other microsporidian spore like this has not been so far isolated from Silkworm. The length of the polar filament extruded in hydrogen peroxide ($H_2O_2$) at $30^{\circ}C$ was $26{\mu}$ of a round cytoplasm on the top. The spores were partly stained with Giemsa, Safranin-O and Gram as the same staining properties as Nosema bombycis, Microsporidia K 79 and other microsporidian spores. The fine structures were observed under scanning eleceron microscope through ultrathin sectioning. The spore wall was composed of three layers ; the thin exospore of an electron dense rippled layer, the thick electron lucent endospore which was thinning considerably at the polar filament insertion point, and the inner limiting membrane. Polar cap present at the sporeapex, with a long polar filament of 12-13 coils, subtending angle of $60^{\circ}$ to spore axis, which is tubular made up of a multilayered and are a benes core, light ring structure enclosing the dance core, the dark ring structure enclosing the inner light ring structure and the other than and light ring structure bounded from cytoplasm. Lamellate polaroplast occupied the anterior part of the spore, and the two neclei with dense nucleoplasm bounded by a double nuclear envelope were cited in the slight downer middle portion of spore. From the characteristics of the shape, size and fine structures, it is certain to reason the Microsporidia S80 belong to the phylum Microspora, class Microspora, order Microsporida, order Microsporida. The shape of two nuclei cited seems to be genus Nosema, but in the classification for the suborder it should be defined wheather pansporoblasts be formed or not and for the genis especial attempts have been made to define the characters which distinguish the disporous genera in the life cycle. Survey through the infection of the bad cocoons during 1980 to 1982 in South Korea the areas contaminated with new microsporidia were revealed 5 provinces of Kyung-Gi, Kang-Won, Chung-Nam and Chun-Nam. Pathological effects inoculated per os at second instar larvae of silkworm, the LD 50 was $7.1{\times}10^7/ml$ as lower pathogenecity than that of Nosema bombycis Naegeli of $1.2{\times}10_7/ml$. While on the other hand the inoculation of the microsporidia at fourth instar larvae lowerd the whole cocoon weight and cocoon shell weight and significant at 1% level. The microsporidia S80 defined it can not be transmitted transovarially from the result of predictive and collective examination of 21 egg batches from the infected female moth.
Ha, Nam-Gyu;Kim, Seung-Yul;Kang, Jin-Ho;Kang, Pil-Don;Sung, Gyoo-Byung;Hong, In-Pyo
Journal of Sericultural and Entomological Science
/
v.47
no.1
/
pp.12-17
/
2005
To develop technique for the production of P. tenuipes stromata on a large scale, the infection of P. tenuipes and the growth of stroma were investigated by silkworm (Bombyx mori) variety. Also, studied about biological activites of fruiting body formed on silkworm. Infection rate of the 5th instar larvae of the silkworm with P. tenuipes was the highest in Yangwonjam, followed by Hachojam, Baegokjam and Chilbojam in that order. Also, as the inoculation times was increased, infection rate tended to be raised. The rate of fruiting body formation of the silkworm pupae infected with P. tenuipes was the highest in Baegokjam, followed by Yangwonjam and Chilbojam in the order. But, actually the fruiting body formation of the 5th instar larvae of the silkworm tested was good in Chilbojam, followed by Yangwonjam and Baegokjam in that order in 3 times spraying inoculation. The fruiting bodies of Yangwonjam and Chilbojam infected with P. tenuipes had high amount of Mannitol, but Baegokjam and Hachojam had high concentration of Glucose on a dry weight basis. The mean content of total amino acid in the fruiting bodies of P. tenuipes was 1.03 ${\mu}mole/g$. The distribution rate of amino acid components decreased in the order of Arginine (12.2%)>Glycine (10.5%)> Proline (9.6)>Tyrosine (8.9%)>Serine>Leucine>Threonine. The most abundant amino acid in the fruiting bodies of the Baegokjam, Chilbojam and Hachojam infected with P. tenuipes was arginine, while Yangwonjam was Glycine. The most abundant fatty acid in P.tenuipes was Oleic acid on a dry weight basis. The unsaturated fatty acids such as Oleic acid, Linoleic acid and Linolenic acid accounted for more than 78% of the total fatty acids.
Flacherie, as one of the most prevalent silkworm diseases, causes severe economic damage to sericultural industry and its pathogens have been proved to be flacherie virus (FV) and densonucleosis virus (DNV). Multiplications of the viruses in the larvae of the silkworm, Bombyx mori, were studied by the sucrose density gradient centrifugation and electron microscopy. The quantitative and qualitative changes of nucleic acids and proteins were investigated from the midgut and hemolymph in the silkworm larvae infected separately with FV and DNV. The histopathological changes of epithelial cells of infected midgut also were examined by an electron microscope. 1. Purified fractions of FV or DNV in a sucrose density gradient centrifugation yielded one homogenous and sharp peak without a shoulder, suggesting no heterogenous materials in the preparation. Electron microscopy also revealed that FV and DNV were spherical particles, 27nm and 21nm in diameter, respectively. 2. Silkworm larvae showed a decrease in body weight on the 6th day and in midgut weight on the 3rd day after inoculation with FV or DNV. 3. DNA content was higher in the midgut when infected with FV or DNV, but the hemolymph of the infected larvae showed no difference during first 6 days after inoculation, after which DNA concentration declined rapidly. 4. RNA synthesis of silkworm larvae infected separately with FV and DNV was stimulated in the midgut, but RNA content was reduced in the hemolymph at the early stage of virus multiplication. At the late stage of virus multiplication, however, it was extremely reduced in both midgut and hemolymph. 5. The concentration of protein in the midgut and hemolymph of silkworm larvae infected separately with FV and DNV showed no difference from that of the healthy larvae at the early stage of virus multiplication, but it was significantly reduced at the late stage of virus multiplication. 6. There was no difference in the electrophoretic patterns of RNAs extracted from the midgut of healthy or virus-infected larvae. 7. The electrophoresis of proteins extracted from the midgut infected with FV or DNV, when carried out on the 1st and 5th day after virus inoculation, showed no difference from that of the healthy larvae. But, there was an additional band with medium motility in the proteins on the 8th day after virus inoculation, while a band with low mobility shown in the proteins of healthy larvae disappeared in the infected larvae. However, a band with high mobility in the healthy larvae was separated into two fractions in the infected larvae. 8. The electrophoretic pattern of hemolymph proteins of the silkworm larvae infected separately with FV and DNV was similar to that of the healthy larvae, but the concentration of hemolymph proteins in the infected larvae was lower than that of the healthy larvae at the late stage. 9. Two types of inclusion bodies were shown by the double staining of pyronin-methyl green in the columnar cell of the midgut on the 8th day after FV inoculation. 10. Electron microscopy of the infected midgut revealed that the 'cytoplasmic wall' of the goblet cell thickened on the 5th day after FV inoculation and several types of the cytopathogenic structures, such as virus$.$specific vesicles, virus particles, linear structures, tubular structures, and high electron-dense matrices were observed in the cytoplasm of the goblet cell. The virus particles were also observed in the microvilli and the structures similar to spherical virus particles were observed around the virus-specific vesicles, suggesting the virus assembly in the cytoplasm. 11. Fluorescence micrograph of the infected midgut stained with acridine orange showed that the nucleus, the site of DNV multiplication in the columnar cell, enlarged on the 5th day after virus inoculation. 12. Electron microscopic examination of DNV infected midgut revealed that the nucleolus of the columnar cell was broken into granules and those granules dispersed into apical region of the nucleus on the 5th day after virus inoculation. On the 8th day after inoculation, it was also observed that the nucleus of the columnar cell was full with the high electron-dense virogenic stroma which were similar to virus particles. These facts suggest that the virogenic stroma were the sites of virus assembly in the process of DNV multiplication.
For the prevention of worldwide prevalent disease of nuclear polyhedrosis virus (NPV), environmental conditions and their incidence of grasserie was investigated through 57 cases of silkworm rearing from the year of 1979 to 1993 in the countries of Korea, Japan, and Philippines. Relationship between the occurrence of NPV and environmental factors were also analysed from the aspect of causal pathogenesis. Unfavorable foactors related to the prevalence of NPV disease was reconfirmed by the assay of experimental rearing. Silkworms reared on mulberry leaves or artificial diet appeared similar result on the occurrence of grasserie. Disinfection by formalin and simple sweeping or washing was not significantly different on the occurrence of NPV disease. Following insufficient ventilation on the younger larvae. from the 1st to 3rd instar, the disease by NPV at the later stage was remarkably emphasized those insidence. An experimental rearing from 1993 to 1996 demonstrated the prevention of NPV disease by simple cleaning of sweeping under the condition of air forced ventilation, the customal practice of disinfection with formalin or any other chemical agents could be omissible.
The Electrophoretic separation in agarose gel on the esterase and acid phosphatase of blood, midgut and silk gland was carried out with 2 original variginal varieties and 7 F$_1$ hybrids. 1. The midgut of larvae fed on mulberry leaves showed one or two more esterase bands than that of larvae fed on artificial diet. 2. The midgut of C 15 larvae being excellently respondent to artificial diet showed one or two more esterase bands than that of larvae being bad respondent to artificial diet. 3. Electrophoretic separation of esterase bands appeared to be greatly different among newly hatched larvae, 1st and 2nd install larvae of F$_1$hybrids. However the difference among the silkworm varieties was not recognized. 4. According to the change in rearing temperature, the number of the active band of midgut esterase was varied. At the temperature of 28$^{\circ}C$ 5 active esterase bands were found. At temperature of 35$^{\circ}C$ 4 bands were noted at 3rd install and 6 or 7 bands at 4th instar. 5. No similar esterase bands conld be found among midgut, blood and silkgland. There are five esterase bands in the midgut, one in blood and three in silkgland. 6. There was rather small difference in acid phosphatase types of midgut and blood according to varieties and rearing temperature. No active band was shown in silkgland. In midgut, there was one acid phosphatase band at 3rd install, two at 4th instar and three at 5th instar. In blood, One active band at 3rd or 4th instar and three bands at 5th inatar were detected.
For fine-denier cocoon filament production, the precocious trimolting silkworms were induced by the treatments with both the imidazole compound "KK-42" hating anti-juvenile hormone activity and high temperature, and their growth, dietary efficiency, cocoon and cocoon filament qualities were compared with those of normal tetramolters as control. 1. The percentage of precocious trimolters was higher in the application of KK-42 than the treatment of high temperature. The effective concentration of KK-42 was 10$\mu\textrm{g}$/1arva. The high temperature treatment for 48 hour was more effective than 36 hour treatment, and silkworm larvae fed on mulberry leaves were more sensitive than artificial diet to induce trimolters. 2. The larval duration of the trimolters induced by KK-42 was 5.17 day shorter than that of normal tetramolters in mulberry leaves rearing, and the increasing pattern of body weight in the 4th inster larvae of trimotlers was similar to the 5th instar larvae of normal tetramolters. 3. The qualities such as cocoon weight, cocoon layer weight and cocoon layer ratio of precocious trimolter induced by KK-42 were much lower those of normal tetramolters: the cocoon weight and cocoon layer ratio were 0.78g, 14.2cg and 18.4% in mulberry leaves rearing, and 0.86 cg, 10.3cg and 12.3% in artificial diet rearing, respectively. 4. The size of cocoon and cocoon filament was smaller in the precocious trimolter, both KK-42 and high temperature, as compared with that of the normal tetramolters. 5. The efficiency of cocoon layer production of the precocious trimolters by the KK-42 was lower than that of the normal tetramolters: amount of the cocoon layer production per 1g of dry mulberry leaves ingested was 7.97cg in the precocious trimotors, while 9.20cg in the normal tetramolters.
Forty-nine plant species as additives to silkworm artificial diet and 5 species as cellulose sources for artificial diet were screened for their economic values as feed-resources for the silkworm. Feeding response to artificial diet was tested on 82 silkworm strains. The effect of rearing conditions on feeding response and enzyme activities in the silkworm was investigated. The results were summarized as follows. 1. Seven species out of 49, Vigna sinensis ENDL, Ipomoea vatatas Lamarck, Cyperus anuricus Var. Laxus, Alnus japonica Stendel, Trifolium repens L, Prunus serrulata Lindley. Var, Glycine max L increased feeding response, compared with the basic formula of artificial diet. 2. The economic values of Vigna sinensis ENDL, Ipomoea vatatas Lamarck, Cyperus anuricus Var. Laxus, Ainus japonica Stendel, Cassia tera L, Erigeron canedensis L as feed-resources for artificiale diet were recognized, through feeding experiment during the entire larval stage. 3. Mulberry cellulose showed the best results in rearing and cocoon characteristics. 4. The extent of feeding response varied according to strains and varieties. Varieties in japanese strains showed higher feeding response than those in chinese and european varieties, with considerable variations among a varieties in strains. 5. The begining of 4th instar seems to be a proper time to convert from mulberry to artificial diet, or artificial diet to mulberry, however the middle of 3rd instar seems acceptable. 6. The optimum temperature for artificial diet rearing is 30$^{\circ}C$ during the period of 1st-3rd instar and 28$^{\circ}C$ for 4th-5th instar. 7. Electrophoretic isozyme patterns of esterase and acid phosphatase on agarose gel, as affected by strain. rearing temperature and feed-resources, were observed as follow. (1) Isozyme patterns of mid-gut esterase varied, depending on instar. One or two more isozyme bands were observed in the larvae than feed on the mulberry fed for the artificial diet. (2) A strain, chinese-15 with a higher feeding response, had 1∼2 more bands than chinese-60 with a lower feeding response. (3) Five bands of mid-gut esterase in 3rd and 4th instar larvae reared at 28$^{\circ}C$. and 4 for 3rd instar and 6∼7 for 4th instar larvae at 35$^{\circ}C$ were observed. (4) No similar esterase bands could be found among mid-gut, blood and silkgland. There are five esterase bands in the midgut, one in blood and three in silkgland. (5) There was rather small digerence in acid phosphatase types of mid-gut and blood according to varieties and rearing temperature. No active band was shown in silkgland. In midgut, there was one acid phosphatase band at 3rd instar, two at 4th instar and three at 5th instar. In blood, one active band at 3rd or 4th instar and three bands at 5th inster wire detected.
Since 1966, practical use of active heavy analogs of the hormones has been also worked out as an insecticide and brought the features of it to the light as cocoon producer. On the other hand, it is expected to afford the increase of silk productivity resulted from control of the fifth larval period by delaying normal development. With these regards, some of analogs have been tried to apply practically to the silkworm for an increase of cocoon productivity. One of the synthesized juvenile hormone available is "Manina". And it is presently used for the increase of silk productivity in Korea. For the practical use, it is very essential the varietal differences in the increase of silk productivity by topical application was tested and the obtained results are summarized as follows. 1. It was evident that the fifty larval period was extended by topical application after 48 hrs. of the last ecdysis, ranging from 8 hrs. to one day, as compared to the control. 2. In pupal rates, there is no significance between control and treatments. It proved that there was no toxicity to silkworm by topical application in general, except jam 120. With regards to an increase of cocoon yield in Japanese, it was resulted from 17∼24% of cocoon yield from 10,000 larvae, as compared to that of control. In case of Chinese, the incrasing rates were varied from 15∼26% of cocoon yield, 17.8kg of it with 26% increase for Jam 122 and 16.7kg of it with 25% increase for Jam 118. In case of all hybrids, an increase of the cocoon yield took places from 20% to 31% and the weight of cocoon layer for the Japanese increased by 6 to 14%, those for the Chinese by 4 to 7% and those for the hybrids ranged from 21 to 29% increase. 3. It was recognized that the hybrid vigor rate took places with the hybrids between high responsing parents to juvenile hormones.
The pathogenicity of hexagonal and tetragonal cytoplasmic polyhedron virus to one of present leading silkworm varieties, Jam 103${\times}$Jam 104, and its parents, Jam 103 and Jam 104, was investigated. The activation of occult virus by oral inoculation of foreign virus as well as the interference phenomena between the activated and inoculated cytoplasmic polyhedron viruses was observed and the results obtained are as follows. 1. The pathogenicity of cytoplasmic polyhedrosis viruses. 1) There was a high significant difference in the pathogenicity of hexagonal polyhedron virus between the hybrid and its parents showing the lowest infection rate in the hybrid (Jam 103${\times}$Jam 104), medium infection rate in the Japanese line (Jam 103) and the highest infection rate in the Chinese line (Jam 104). In the pathogenicityof tetragonal polyhedron virus, a significant difference was observed only between the hybrid (Jam 103${\times}$Jam 104) and the Chinese line (Jam 104) by showing a higher infection rate in the Chinese line than in the hybrid. 2) The pathogenicity of both hexagonal and tetragonal polyhedron viruses showed a high significant difference in different silkworm instars inoculated by showing a higher infection rate at the second instar than at the fourth instar. 3) The pathogenicity of both hexagonal and tetragonal polyhedron viruses was increased as the concentration of viruses inoculated increased. 2. The phenomena of induction and interference by oral inoculation of foreign virus. 1) The induction rate of cytoplasmic polyhedrosis virus was higher in the parents than in the hybrid. In the parents. a higher rate in the Chinese line than in the Japanese line was observed. 2) The effect of inoculation at different instars on the induction was studied and the induction rate was higher at the second instar inoculated than at the fourth instar inoculated. 3) The degree of activation of hexagonal polyhedron virus with inoculation of tetragonal polyhedron virus was very high when a lower concentration of virus was inoculated and it was very low when a higher concentration of virus was inoculated 4) The degree of activation of tetragonal polyhedron virus with inoculation of hexagonal polyhedron virus was very low when a lower concentration of virus was inoculated and it was not observed when a higher concentration of virus was inoculated. 5) The mixed infection rate with hexagonal and tetragonal polyhedron viruses was higher at the second instar inoculated than at the fourth instar inoculated. 6) It was observed that the secondary hexagonal pc]yhedron virus activated interfered with the primary tetragonal polyhedron virus inoculated when the inoculated concentration of the primary virus is low and the primary virus inoculated interfered with the secondary virus activated when the inoculated concentration of the primary virus is high.
In general, the mean silkmoth lifespan is around 8 days for female and 5 days for male. But, the duration of J037 strain's lifespan is remarkably long in both sexes. On the contrary, the Daizo(sdi) strain has a remarkably short lifetime. The differences in adult lifetime among various silkworm strains has been suggested that the adult lifetime may be genetically controlled. In this experiment, using J037 and Daizo strains we investigated genetic factors related to the adult lifetime of silkworm. We constructed the full-length cDNA library from the adult male of the J037 strain. A total of 2,688 clones were randomly selected, and we performed a differential display hybridization with cDNA probes generated from J037 and Daizo adult males. In conclusion, 193 clones were identified as differential expressed genes, and 154 unique genes were generated after the assembly of 193 clones. Of the 154 unique genes, the most abundant genes were cytochrome oxidase subunit-1 gene(9 times) and unknown(clone ID; 1-50) gene(5 times). The functional groups of these unique genes with matches in the AmiGo database were constructed according to their putative molecular functions. Among thirteen functional categories, the largest group was unclassified protein(24%). In addition, we analyzed the nucleotide and deduced amino acid sequences of the most highly occurred gene(1-50, EF434397), which consisted of 240 amino acids. However, it is confirmed yet that these genes really have an affected on the silkworms longevity. Further studies on these molecules biological roles will give us well-fined information about mechanisms of insect aging and/or scenesence.
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