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Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms.

To investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae produce reactive oxygen species (ROS) as part of the oxidative killing of invading pathogens, the production of ROS was measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or Xenorhabdus nematophila) hemolymph. No detectable ROS production was found. The spontaneous and activated ROS production measured with hemocytes, i.e. under the conditions when the antioxidative potential of hemolymph plasma was eliminated, was again undetectable. Likewise, ROS production by isolated hemocytes was observed by spectrophotometric (NBT test, cytochrome c assay) and fluorimetric (using dihydrorhodamine and hydroethidine probes) methods. Hence none of the experimental approaches used indicated the production of ROS by hemocytes of B. mori larvae as part of their immune response.

Animals↗

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages.

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus ( BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

Animals↗

Expression profiling of homeobox genes in silk gland development in the mulberry silkworm Bombyx mori.

The homeobox (Hox) genes constitute an evolutionarily conserved family encoding transcription factors which play major roles in segmental identity and organ specification, across species. The expression patterns of three Hox genes, Antennapedia (Antp), Ultrabithorax (Ubx) and even-skipped (eve) were analyzed during silk gland development in Bombyx mori. Antp followed a middle silk gland (MSG) restricted pattern of expression, whereas Ubx was specifically expressed in the posterior silk gland (PSG) during embryonic and larval developmental stages. Eve protein, on the other hand, was expressed in both MSG and PSG. We have also identified and characterized a novel Pax-like mRNA that is expressed exclusively in the PSGs during embryonic and larval development. The expression of Antp, Ubx and Pax-like reached maximum levels in the fifth larval intermoult and no expression was detected during the intervening moults. The region-specific expression of certain Hox genes appears to be responsible for the specification of silk gland compartments, whereas other Hox genes may play a global role in controlling the expression of genes encoding silk proteins.

Animals↗

Programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior silk gland of the silkworm, Bombyx mori.

Silk gland is a larval specific tissue of lepidopteran insects and begins to degenerate shortly before pupation. Programmed cell death (PCD) of the anterior silk gland of Bombyx mori last instar larvae was studied in vivo and in vitro, focusing on the effects of 20- hydroxyecdysone (20E). The glands began to exhibit signs of PCD in vivo 2 days after gut purge and completed PCD by 48 h. In vitro, 20E prematurely induced PCD, and its completion took 144 h (6 days). An oligo-nucleosomal ladder pattern was observed in DNA extracted at the end of PCD. Caspase 3 inhibitor inhibited attainment of full PCD, but it did not block chromatin condensation as revealed by acridine orange staining. alpha-Amanitin inhibited the PCD induced by 20E in vitro if added to the culture in the first 8 h. Similarly, cycloheximide and emetine completely blocked PCD when applied in the first 18 h of culture with 20E. These results indicate that 20E-stimulated transcription and protein synthesis for PCD are completed in 8 h and 18 h, respectively. Nevertheless, withdrawal of 20E from the medium at different times showed that 20E must be present in vitro for 42 h to elicit full PCD. Current results indicate that the effects of 20E on the progression of PCD are mediated by two distinct processes - one through nuclear hormone receptors, and the other independent from de novo gene expression.

Animals↗

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 ).

Animals↗

Genetic variability and genetic structure of wild and semi-domestic populations of tasar silkworm (Antheraea mylitta ) ecorace Daba as revealed through ISSR markers.

The genetic diversity in the wild and semi-domestic populations of Daba ecorace of Antheraea mylitta was studied to ascertain the distribution of variability within and among populations of semi-domestic bivoltine (DB), trivoltine (DT) and nature grown wild populations (DN) with inter-simple sequence repeat (ISSR) markers. A total of 138 markers were produced among 56 individuals of the three populations, of which 98% were polymorphic. For the individual populations, the percentage polymorphism was 58.69, 52.9 and 77.54 for DB, DT and DN, respectively. Average number of observed (1.791+/- 0.408) and effective alleles (1.389+/-0.348) was also high in the wild populations in comparison to the bivoltine and trivoltine semi-domestic populations. Genetic diversity (H(t)) in DB, DT and DN was 0.180+/- 0.033, 0.153+/- 0.032 and 0.235+/- 0.033, respectively and within-population genetic diversity (H(s)) ranged from 0.166 to 0.259 with a mean of 0.189. Mean gene differentiation (G(ST)) was found to be 0.25. Shanon's diversity index was 0.278, 0.237 and 0.361 for DB, DT and DN and overall it was 0.391. Gene flow (N(m)) among the populations was 1.509. The dendrogram produced by UPGMA with Dice's genetic distance matrices resulted in the formation of three major clusters separating the three populations. Considerable intra- and inter-population variability is found in all three populations. The population structure analysis further suggests that the semi-domestic populations of Daba ecorace are at the threshold of differentiating themselves. The high genetic variability present within wild Daba population of A. mylitta is of much importance for conservation as well as utilization in systematic breeding program.

Alleles↗

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori.

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W( Ze )). We succeeded in obtaining a fragment of the W( Ze ) chromosome designated as Ze (W), having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze (W) fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze (W) fragment, the Z chromosome was also broken into a large fragment (Z(1)) having the +( sch ) (1-21.5) and a small fragment (Z(2)) having the +( od ) (1-49.6). Moreover, a new chromosomal fragment (Ze (W)Z(2)) was generated by a fusion event between the Ze (W) and the Z(2) fragments. We analyzed the genetic behavior of the Z(1) fragment and the Ze (W)Z(2) fragment during male (Z/Z(1) Ze (W)Z(2)) and female (Z(1) Ze (W)Z(2)/W) meiosis using phenotypic markers. It was observed that the Z(1) fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze (W)Z(2) fragment and the Z chromosome and also between the Ze (W)Z(2) fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze (W)Z(2)/W) and males (2A: Z/Z(1)) resulting from non-disjunction between the Ze (W)Z(2) fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z(2) region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.

Animals↗

Comparative study of biological and technological characters in three generations of silkworm Bombyx mori L. ameiotic, parthenogenetically cloned lines.

PURPOSE: Detect any deviation in biologic and technologic characters of eight ameiotic-parthenogenetically cloned lines of Bombyx mori L. from different origins from a normal sexually reproduced control line in three generations. METHODS: Comparative study of the three generations was conducted in SES, Vratza, unit of the National Center for Agrarian Sciences of Bulgaria after fixing all environmental rearing conditions. RESULTS: The ameiotic-parthen-clones displayed good parthenogenetic development, although total hatchability was significantly less than the sexually reproducing control populations. Survival rates between clones and control were not significantly different. All clones displayed significantly longer larval periods. Slight decline in second generation, and a steeper one in the third generation were observed for all eight cloned lines in cocoon weight, shell weight, and shell ratio and these differences were statistically significant. Cocoon yield was significantly lower than the control throughout the three generations. CONCLUSIONS: Our parthen-cloning method has a high rate of success in comparison to other cloning methods, the cloned progeny populations although were weaker technologically (cocoon weight, shell weight, and shell ratio), the biological characters (parthenogenetic development and survival rate) were not compromised. Further study is needed to determine the thermal needs of the cloned embryos and metabolic rate of all stages.

Animals↗