Comparison of multilocus RFLPs and PCR-based marker systems for genetic analysis of the silkworm, Bombyx mori.

The utility of multilocus RFLPs and three PCR-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat-PCR (ISSR-PCR) and simple sequence repeats (SSRs) for genetic characterization was examined using 13 diverse silkworm strains. All four approaches successfully discriminated the 13 silkworm varieties but differed in the amount of polymorphism detected. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, EMR) and the amount of polymorphism detected (diversity index, DI). For example, the six multilocus RFLP probes produced 180 products of which 97% were polymorphic; 15 SSR loci gave rise to an average of 8 alleles each, of which 86% were polymorphic. The ISSR-PCR produced 39 fragments of which 76.98% were polymorphic. The highest diversity index was observed for ISSR-PCR (0.957) and the lowest for RAPDs (0.744). The RAPD, ISSR-PCR and RFLP assays clearly separated the diapausing and non-diapausing silkworm varieties. These results are discussed in terms of choice of appropriate marker technology for different aspects of silkworm genome analysis.

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR.

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3'-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

Influence of thyroxine on different ion-dependent ATPase activities in fat body of tasar silkworm, Antheraea mylitta D.

The activities of Na(+)-K(+)-, Ca(2+)-, and Mg(2+)-ATPase of tasar silkworm, Antheraea mylitta D. fat body were investigated from fifth larval stage to adult emergence after injection of various doses (0.5, 1.0, 2.0, and 5.0 micrograms/g) of mammalian thyroxine (T4) to 1-hr-old fifth instar larvae. In normal silkworms, both sexes exhibited maximum enzyme activity before spinning (Day 12). Na(+)-K(+)-, Ca(2+)-, and Mg(2+)-ATPase activities in fat body of silkworms markedly declined after pupation and more so on the 1-day-old adults. All doses of thyroxine treated on fifth instar larvae significantly altered ATPase activity in the larval, pupal, and adult stages in both sexes. ATPase activity was not altered by lower dose of T4 (0.5 micrograms/g) in 2-day-old fifth stage larvae, while the higher dose (5.0 micrograms/g) surprisingly caused a reduction in ATPase activity during the different developmental stages. The fat body ATPases were influenced by thyroxine in all the stages of silkworm in a dose-dependent manner. Our results thus indicate that thyroxine has a controlling influence on the ATPase system in silkworm fat body.

L-thyroxine (T4) elevates the free amino acid pool of haemolymph plasma of tasar silkworm, Antheraea mylitta drury (Lepidoptera: Saturniidae).

Concentration of free amino acids (FAA) in the haemolymph plasma of male and female tasar silkworm, A. mylitta, during fifth larval instar were determined by amino acid analyzer after administration of vertebrate thyroxine (T4). Twenty FAA have been identified in haemolymph plasma in both sexes, out of which ten were found to be predominating. Treatment with anabolic doses of thyroxine (0.5 and 1.0 micrograms/g) were able to enhance the titre of all the individual FAA which has been also reflected in the total concentrations. A specific pattern of variations in plasma FAA recorded during 5th larval instar, was not altered by the administered thyroxine. Our results thus indicate that T4 has a controlling influence on the FAA profiles of silkworm as observed in other biochemical parameters of the insect.

Complications from unrecognized defects in endotracheal tubes.

Considerable effort has been made to improve endotracheal tubes and make them safer than they have been in the past; yet complications continue to occur, due, in most instances, to inherent defects in the tube. It is not possible to identify these defects in a routine preoperative examination. Three cases are presented illustrating how defects in anode endotracheal tubes almost caused fatalities during anesthesia. Other possible complications and contributing factors causing obstruction of endotracheal tubes are also reviewed. Until some way is devised for the automatic compensation of the increases in volumes and pressures in endotracheal tube cuffs, hourly deflation is recommended to prevent complications.