Antiviral proteins in midgut of the silkworm, Bombyx mori, fed on different food sources

Midgut proteins from the silkworms, Bombyx mori, fed on an artificial diet containing dried powder of the spiral blue-green alga, Spirulina platensis, had a very low fluorescence intensity. This indicated that chlorophyll in this alga is not utilizable for synthesizing the red fluorescent protein (RFP) which has been considered as an effective antiviral protein in silkworm midgut. Many electrophoretic bands of the midgut proteins from the mulberry-fed silkworms emitted red fluorescence under UV light. It is thus concluded that the RFP as a whole is more than one midgut protein. Enzymatic analyses showed that silkworms fed on mulberry leaves and artificial diets had similar protease activities, but they had different phospholipase C activities. Therefore, it is suggested that the antiviral activity of purified RFP in the silkworm gut juice is attributable to phospholipase C.

---

Résumé Les protéines de l'intestin moyen de Bombyx mori, nourri avec un aliment artificiel contenant de la poudre séchée de l'algue bleue spiralée, Spirulina pratensis, avaient une très faible fluorescence. Cesi indique que la chlorophylle a de cette algue n'est pas utilisable pour la synthèse de la protéine fluorescente rouge (RFP), considérée comme une protéine antivirale efficace de l'intestin moyen. Après séparation par électrophorèse sur gel de polyacrylamine DISC plusieurs bandes de la protéine de l'intestin moyen de B. mori élevé sur mûrier émettaient une fluorescence rouge en lumière U.V. On peut conclure que RFP est un complexe plutôt qu'une protéine particulière de l'intestin moyen. Les analyses enzymatiques ont montré que la phospholipase C et non les protéases ont une activité antivirale dans la RFP purifiée provenant de vers à soie nourris à partir de mûrier ou sur régime artificiel.

     

Studies on the utilization of single cell protein by the silkworm Bombyx mori

Final-instar larvae of Bombyx mori fed mulberry leaves, supplemented with Spirulina fusiformis (Woronichin) as a source of single cell protein (SCP), required 6 days to attain a maximum larval weight of 2090 mg; control group larvae needed 9 days to attain a final larval weight of 1470 mg. Quantity of feeding, assimilation and conversion efficiencies increased substantially in the SCP-fed group. Significant improvements in the economic characters such as cocoon, pupal, and shell weights were obtained in the SCP supplemented larvae in comparison to the normal leaf fed larvae. About 15% of the labelled S. fusiformis was directly incorporated into larval tissue. Presence of SCP in the gut facilitated better conversion of consumed leaf protein.

---

Etudes sur l'utilisation des protéines de cellules isolées par le ver à soie, Bombyx mori

Résumé Des chenilles du dernier stade de Bombyx mori, alimentées sur mûrier additionné de Spirulina fusiformis comme source de protéine de cellule isolée (SCP), atteignent en 6 jours le poids larvaire maximum de 2090 mg; les chenilles témoins consommaient pendant 9 jours pour obtenir leur poids larvaire final de 1470 mg. Les quantités consommées, les coefficients d'assimilation et de conversion ont augmenté substantiellement chez les chenilles avec SCP. Des augmentations significatives de critères économiques, comme les poids de cocon, de nymphe et de cogul, ont été observées avec l'addition de SCP par rapport aux témoins. Environ 15% du S. fusiformis marqué a été incorporé directement dans les tissus larvaires. La présence de SCP dans l'intestin a permis une meilleure conversion des protéines foliaires consommées.

     

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.     

The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p ^Sa + ^p W + ^od )Fem, derived from the translocation-carrying W chromosome (p ^Sa + ^p W + ^od ), is inert as femaleness determinant. Moreover, this Df(p ^Sa + ^p W + ^od )Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p ^Sa + ^p W + ^od )Fem chromosome are produced. Initially, to investigate whether the Df(p ^Sa + ^p W + ^od )Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(p ^Sa + ^p W + ^od )Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(p ^Sa + ^p W + ^od )Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(p ^Sa + ^p W + ^od )Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(p ^Sa + ^p W + ^od )Fem chromosome is viable. Therefore, we concluded that the Df(p ^Sa + ^p W + ^od )Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(p ^Sa + ^p W + ^od )Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(p ^Sa + ^p W + ^od )Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, p ^Sa + ^p W + ^od .     

Morphological and electrophysiological characteristics of the epipharyngeal sensilla of the silkworm, Bombyx mori

One pair of gustatory sensilla was found on the epipharynx ofBombyx mori larvae, and some morphological and electrophysiological characteristics of the epipharyngeal sensilla were investigated. They are sensilla coeloconica composed of a small papilla with a pore at the tip and a swelling of cuticle encircling the papilla. Three bipolar neurons innervate each sensillum. One neuron is an inositol receptor which responds to inositol only. Another cell responds with action potentials of relatively large amplitude to some feeding deterrent substances, such as strychnine nitrate. The thresholds of these cells for inositol and strychnine nitrate are approximately 10⁻⁴ M and 10⁻⁷ M, respectively. At least two kinds of spikes can be observed when these sensilla are stimulated with some salts and acids. Dose-response relationships and time courses of responses to inositol and strychnine nitrate were also examined in this study.     

Isolation and reconstitution of the RNA replicase of the cytoplasmic polyhedrosis virus of silkworm,Bombyx mori

Summary After irradiation of the virus particles of CPV, the RNA replicase associated with the virion was isolated in the form of a genome-replicase complex with DEAE-Sephadex A-25 chromatography. This complex was then treated with Triton X-100 and purified by phosphocellulose column chromatography. The RNA replicase reconstituted with the doublestranded RNA of CPV showed both the enzyme activity of RNA polymerase and methyltransferase. The single-stranded RNA could not serve as the template for the RNA replicase. The role of the RNA replicase of CPV is discussed.     

Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2^v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome 11. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the S-2^v variant polypeptide is lower by approximately 62,500 than that of the S-2 polypeptide. Amino acid analysis indicates that the two sericin polypeptides have similar compositions. These results are consistent with the idea that the variant allele arose by deletion within the S-2 coding sequence in the Src-2 gene locus as the result of unequal recombination.     

Induction of dauer pupae by fenoxycarb in the silkworm,Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.     

Molecular phylogeny of the domesticated silkworm,Bombyx mori, based on the sequences of mitochondrialcytochrome b genes

Pupae from the Chinese wild mulberry silkworm,Bombyx mandarina, and 11 representative strains of the domesticated silkworm,Bombyx mori were selected for preparation of mitochondrial DNA. The 5′-end fragments ofcytochrome b genes (Cytb) were generated by polymerase chain reaction products and sequenced directly. The homologous sequences of the JapaneseB. mandarina and three strains ofB. mori were from the GenBank database. The sequences of the 16 silkworm strains were analysed with DNASTAR software and a phylogenic tree was constructed using PHYLIP software. The result showed that: (i) The sequence divergence between the strains ofB. mori and the JapaneseB. mandarina was larger (5.4% ≈ 5.8%) compared with that between strains ofB. mori and the ChineseB. mandarina (0.8% ≈ 1.9%). Analysis of clustering also showed that the sequences ofB. mori strains and ChineseB. mandarina clustered into group (B group), while that of JapaneseB. mandarina (A group) was outside this cluster. This may be evidence for the hypothesis thatB. mori originated from ChineseB. mandarina. (ii) Among 14 strains ofB. mori, sequence divergence was small and the most divergence was seen between strains Yanhe-1 and Chuxiong, whose sequences branched off from those of the otherB. mori strains on the phylogenetic tree. From this and from historical records, we infer that the strains Yanhe-1 and Chuxiong originated independently from southwest China.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.     

Hierarchical clustering of 54 races and strains of the mulberry silkworm,Bombyx mori L: Significance of biochemical parameters

Summary A detailed analysis was undertaken to test the efficacy of hierarchical agglomerative clustering (UPGMA method) in grouping the races and strains of the mulberry silkworm, Bombyx moti L., and to ascertain the importance of biochemical parameters in the clustering process. The analysis was based on data from two rearing seasons with 54 selected races/strains of different geographic origin and varying yield potentials. The results indicate that seven clusters can be realised with yield parameters alone, whereas the inclusion of biochemical parameters in clustering resulted into two broad groups: one having all the breeds with high cocoon weight and shell weight, the other having all the low-yielding silkworm strains both from India and from other countries. Further sub-grouping under these two groups highlights genetical differences associated with the differentiation of various groups of races in temperate and tropical areas as well as their significance for silkworm breeding. Estimates of all ten variables were further subjected to quick clustering and the results showed that cluster 5, constituted by 38 lowyielding strains of India, China and Europe, had the highest values of the final cluster centre for amylase and the effective rate of rearing (ERR), while clusters 1 and 4 had the highest values for invertase and alkaline phosphatase. The evolutionary aspect of the genetic channelisation of silkworm races from various countries is discussed against the background of differences in the biochemical parameters and yield variables.     

Role of Sodium Ions and Their Uptake by Cells of Cultured Blue-Green Algae, Spirulina platensis and Spirulina maxima

The growth of the blue-green algae Spirulina platensis and Spirulina maxima, cultured in complete mineral Zarouk medium containing Na⁺ or Na⁺-deficient medium, was studied over a period of 24 h. The optical densities of S. platensis and S. maxima cells, determined during the last hour of exposure to sodium deficiency, amounted to 55.6 and 32.6%, respectively, of the optical densities of the same cells grown in complete Zarouk medium. Moreover, the cultures grown in Na⁺-deficient medium exhibited increased ability to take up sodium (which was low in S. platensis and S. maxima cells cultured in complete mineral medium). It is concluded that the two species studied are characterized by periodic, on the order of minutes, changes in the cellular uptake and release of sodium.     

Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L

Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.     

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase