Using the fluorescent differential display technique, a special band named Bm541 was identified by screening for differentially expressed genes in the resistant silkworm strain Qiufeng, the susceptible strain Huaba35, and the near isogenic line BC6, which carries the resistant gene to Bombyx mori densonucleosis virus (BmDNV). After applying the 5'RACE technique with specially designed primers, a 1148 bp cDNA clone containing a 387 bp open reading frame (ORF) was obtained. This gene was registered in GenBank under the accession number AY860950. The deduced amino acid sequence showed a 73.1% identity to the protein kinase C inhibitor (PKCI) of Drosophila pseudoobscura. In the deduced sequence of BmPKCI, the histidine triad (HIT) motif, which is essential for PKCI function, and the α-helix region, which is conserved among the PKCI family, were present. The data from quantitative real-time PCR (QRT-PCR) suggest that the expression levels of bmpkci in BC6 and Qiufeng both with BmDNV-Z are significantly higher than those in Huaba35, which indicate that BmPKCI plays a role in resistance to BmDNV-Z.
Cocoon weight and shell weight are the key economic traits ultimately determining silk yield. In order to detect the main quantitative trait loci (QTL) associated with the cocoon traits of the mulberry silkworm, Bombyx mori, the parents of larvae that produced cocoons that differed greatly in weight and shell weight were screened using 240 primer pairs of single nucleotide polymorphic markers (SNPs) representing all the 28 linkage groups in silkworm. Out of the 240 primer pairs, 48 (20%) revealed distinct polymorphism between the parents, which was confirmed by the co-dominant expression of both polymorphic PCR products in the F1 generation. The bulked segregant analysis (BSA) was used to compare the SNP profiles of the parents, F1 and F2 bulks using the 48 informative SNP primers. This revealed that out of 48 primer pairs, only one pair, i.e., No. 04124 of the linkage group 4 showed clear differences in the amplified products between the bulks corresponding to that of the parents with different cocoon traits suggesting that the DNA regions amplified by this primer pair are closely linked to the QTL controlling the cocoon traits. The results were also confirmed by screening the backcross (BC) progeny. This is the first report of the identification of a QTL using SNPs with BSA. The results of the present study indicate that it might be possible to use SNPs for marker assisted selection (MAS) in silkworm breeding programs aimed at improving cocoon traits. and Sivaramakurup Sreekumar, Southekal K. Ashwath, Monika Slathia, Sundaramurthy N. Kumar, Syed M.H. Qadri.
RFLP clones harbouring multi-copy DNA sequences were isolated from the Pst I sub-genomic library of the indigenous silkworm race, Nistari, and were used for DNA fingerprinting studies in 13 stocks of silkworm, Bombyx mori L. Six multilocus probes produced 180 RFLP markers that showed a high level (98%) of polymorphism and are highly useful in molecular mapping, genotype characterization and marker assisted selection (MAS). The dendrogram derived from UPGMA analysis clearly divides the 13 silkworm stocks into two major clusters: high- and low-yield stocks. Furthermore, adopting multiple regression analyses, the RFLP marker(s) associated with characters of economic importance were identified, a first of its kind for any species of insect of commercial importance. The results obtained create an opportunity of using germplasm stocks directly for isolating specific RFLP band(s) and use it for MAS in breeding programs.
The uzifly, Exorista sorbillans (Diptera: Tachinidae), a parasite of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), causes heavy losses to the silk industry. This parasitoid harbours a Wolbachia endosymbiont, which controls the fly's reproduction. In the present study a method for curtailing this notorious pest by administering Wolbachia-targeted tetracycline via its silkworm host's diet is investigated. Tetracycline not only influenced the larval growth of the silkworms' by decreasing larval duration, increased silk production and fecundity, without affecting hatchability, it also decreased the reproductive fitness of the uzifly endoparasite by killing the Wolbachia. The antibiotic exerts a beneficial influence by affecting the intestinal flora of silkworm larvae. On the other hand the reproductive fitness of uzifly was greatly reduced in terms of different reproductive abnormalities. When male and female flies that emerged from treated host silkworms were crossed and males from untreated hosts and females from treated hosts were crossed, approximately 72% and 97% of the eggs failed to hatch, respectively. However, of the eggs from crosses between male and female flies that emerged from untreated hosts and between males from treated hosts with females from untreated hosts, an average of 30% failed to hatch and the Wolbachia infection enhanced the fecundity of uziflies. These results demonstrate that the Wolbachia may be essential for uzifly reproduction and that Wolbachia-targeted antibiotics have a beneficial influence on silkworm growth while decreasing the reproductive fitness of the uzifly, E. sorbillans.
Silk production quantity is the most economically important characteristic of the domesticated silkworm moth, Bombyx mori. It is controlled by multiple loci. The cocoon and silk production quantity of silkworm strains Jingsong and Lan10 have significantly diverged. A backcross population (BC1) was bred using Jingsong and Lan10 as parents to identify quantitative trait loci (QTLs) for silk quality. In this research, a genetic linkage map of the silkworm was constructed using the BC1 mapping population. The map contained 85 sequence-tagged site markers, 80 simple sequence repeat markers, and 16 single nucleotide polymorphisms. A linkage map was constructed from the data, which consisted of 181 markers distributed over 28 expected linkage groups and spans 2147.1 cM in total length. Fourteen QTLs were detected for cocoon filament length, whole cocoon weight, pupae weight, filament weight, and cocoon shell weight. The 14 QTLs were distributed in 5 linkage groups (linkage groups 1, 14, 18, 23 and 25) based on the constructed linkage map. In addition, five QTLs, which had the highest logarithm (base 10) of odds (LOD) values, were located on the first chromosome, three of which located at the same region in linkage group 1. These results represent an important foundation for the map-based cloning of QTLs and marker-assisted selection for improving the silk quality of economically important silkworm strains., Bing Li ... [et al.]., and Obsahuje seznam literatury
Two glutathione S-transferase (GST) cDNAs, GSTD2 and GSTS2, were cloned from the silkworm Bombyx mori. The B. mori GSTD2 (BmGSTD2) gene spans 4371 bp and consists of four introns and five exons that encode 222 amino acid residues. The deduced amino acid sequence of BmGSTD2 showed 58% protein sequence identity to the Delta-class GST of Maduca sexta. The B. mori GSTS2 (BmGSTS2) gene spans 3470 bp and consists of three introns and four exons that encode 206 amino acid residues. The deduced amino acid sequence of BmGSTS2 revealed 67%, 63%, and 61% protein sequence identities to the Sigma-class GSTs from B. mori, Platynota idaeusalis, and M. sexta, respectively. The BmGSTD2 and BmGSTS2 cDNAs were expressed as 25 kDa and 23 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot and Western blot analyses showed that BmGSTD2 and BmGSTS2 were specifically expressed in three gut regions, indicating that the gut is the prime site for BmGSTD2 and BmGSTS2 synthesis in B. mori larvae.
The brain and subesophageal ganglion (BR-SG) of the commercial silk worm, Bombyx mori, were stained immunohistochemically at the larval stage for circadian clock neurons with antibodies against Doubletime (DBT) of B. mori and Period (PER) of Periplaneta americana. The BR-SGs were also stained with antisera against [Arg7]-corazonin, which has been known to be present in B. mori and co-localized with PER in Manduca sexta, and against [His7]-corazonin, a homolog identified in other species. From co-localization of [Arg7]-corazonin and PER-like reactivities in the pars lateralis, [Arg7]-corazonin is suspected to be a downstream regulator of the circadian clock in M. sexta. DBT- and corazonin-like immunohistochemical reactivities were found in both the neurosecretory cells of the pars intercerebralis (PIC) and pars lateralis (PL) in B. mori. Small numbers of neurons shared both reactivities against anti-DBT and anti-corazonin. The majority of the immunopositive cells were common to both corazonins, but some cells were unique in expressing either reactivity against [His7]-corazonin or [Arg7]-corazonin only. The results suggest that there is a diversity in the clock output pathway among lepidopterans and that [His7]-corazonin may be present in B. mori, as well as [Arg7]-corazonin, although the former has not been chemically identified in this species. Corazonin may be a downstream regulator of circadian clocks in B. mori because of the co-localization of [His7]-corazonin at PIC and [Arg7]-corazonin at PL with anti-DBT.
We sequenced nine introns of 25 silkworm (Bombyx mori L.) strains, assuming that the introns are particularly prone to mutation. Mean sequence divergence and maximum sequence divergence in each intronic sequence among 25 silkworm strains ranged from 0.81% (3.8 nucleotides) ~ 9.15% (85.2 nucleotides) and 1.2% (seven nucleotides) ~ 39.3% (366 nucleotides), respectively. The degree of sequence divergence in some introns is very variable, suggesting the potential of using intronic sequences for strain identification. In particular, some introns were highly promising and convenient strain markers due to the presence of a large indels (e.g., 403 bp and 329 bp) in only a limited number of strains. Phylogenetic analysis using the individual or the nine concatenated intronic sequences showed no clustering on the basis of known strain characteristics. This may further indicate the potential of the intronic sequences for the identification of silkworm strains.
Steroid hormone 20-hydroxyecdysone and the sesquiterpenoid juvenile hormone are the main regulators of insect development; however, it is unclear how they interact in the regulation of metamorphic events. Using the silkworm, Bombyx mori, we show that the juvenile hormone analogue fenoxycarb affects the cascade of ecdysone regulated genes that control the programmed cell death in the larval midgut. Morphological changes that occur during cell death were investigated by studying cross-sections of the midgut stained with hematoxylin and eosin. Apoptosis-specific DNA fragmentation was detected using TUNEL assay. Expression patterns of genes ATG8 and ATG12, which were used as indicators of autophagy, and genes of the ecdysone-regulated gene cascade were examined using real-time quantitative polymerase chain reaction. Fenoxycarb application on day 0 of the 5th larval instar extended the feeding period and postponed programmed cell death in mature larval midgut. This effect was probably due to a delay in ecdysone secretion and associated changes in gene expression were mostly not a direct response to the fenoxycarb. However, differences in the gene expression patterns in the control and fenoxycarb treated insects during the prepupal and early pupal stages indicated that fenoxycarb may also exert a more direct effect on some genes of the ecdysone regulated gene cascade., Ebru Goncu, Ramazan Uranli, Osman Parlak., and Obsahuje bibliografii
Wild type silkworm larvae have opaque white skin, whereas the mutants Sel (Sepialumazine) and Xan (Xanthous) are yellow-skinned. Previous genetic analysis indicated that Sel and Xan are on established linkage groups 24 (0.0) and 27 (0.0), respectively. However, in constructing a molecular linkage map using simple sequence repeat (SSR) loci, we found that the two mutations were linked. To confirm this finding, we developed a set of SSR markers and used them to score reciprocal backcross populations. Taking advantage of the lack of crossing-over in female silkworms, we found that the progeny of backcrosses between F1 females and males of the parental strains (BC1F) of the two visible mutations had the same inheritance patterns linked to the same SSR markers. This indicated that the two visible mutations belonged to the same chromosome. To confirm this finding, we tested for independent assortment by crossing Sel and Xan marker strains with each other to obtain F1 and F2 populations. Absence of the expected wild type class among 5000 F2 progeny indicated that the two visible mutations were located on the same linkage group. We carried out recombination analysis for each mutation by scoring 190 progeny of backcrosses between F1 males and parental females (BC1M) and constructed a linkage map for each strain. The results indicated that the Sel gene was 12 cM from SSR marker S2404, and the Xan gene was 7.03 cM from SSR marker S2407. To construct a combined SSR map and to avoid having to discriminate the two similar dominant mutations in heterozygotes, we carried out recombination analysis by scoring recessive wild type segregants of F2 populations for each mutation. The results showed that the Sel and Xan genes were 13 cM and 13.7 cM from the S2404 marker, respectively, consistent with the possibility that they are alleles of the same locus, which we provisionally assigned to SSR linkage group 24. We also used the F2 recessive populations to construct two linkage groups for the Sel and Xan genes.