Engineering silkworms for resistance to baculovirus through multigene RNA interference.

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus.

Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms.

RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L.

Polycalin (chlorophyllid A binding protein): a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L.

We studied a protein from the midgut of the silkworm Bombyx mori characterized by its ability to bind the prosthetic group of chlorophyll, that confers fluorescent properties to this protein. Several techniques, 2D electrophoresis purification, MS-MS and Maldi-TOF peptide sequencing, RT-PCR and nucleotide sequencing were used to obtain the nucleotide sequence and the deduced amino acid sequence. The coding sequence was compared to the gene sequence to define the number and size of introns and exons.

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms.

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines.

A helium burst biolistic device adapted to penetrate fragile insect tissues.

To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast.

Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori.

In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother.

3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards.

Transgenesis was recently achieved in Bombyx mori L., but it has proved difficult and time-consuming to screen the numerous progeny to identify the transgenic individuals. As the 3xP3-EGFP marker has been shown to be a suitable universal marker for transgenic insects (Nature 402 (1999) 370), we evaluated its use for embryonic-stage screening for B.

The biolistic method as a tool for testing the differential activity of putative silkmoth chorion gene promoters.

Bombyx mori unpaired early chorion gene copies 6F6.1,.2 and.

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B.

Hydroxy juvenile hormones: new putative juvenile hormones biosynthesized by locust corpora allata in vitro.

The in vitro production of sesquiterpenoids was investigated by using corpora allata (CA) of the African locust Locusta migratoria migratorioides. Labeled products from unstimulated biosynthesis were extracted, purified by normal phase HPLC, and derivatized to determine the functional groups present. An extra hydroxyl group was detected in each of two juvenile hormone (JH) biosynthetic products.

Gas chromatography-mass spectroscopy analysis of juvenile hormone released by insect corpora allata.

Corpora allata incubated in appropriate medium release several compounds including juvenile hormones. Juvenile hormones (14C labeled or unlabeled) were extracted with hexane and directly analyzed by gas chromatography-mass spectroscopy. This method allowed the qualitative and quantitative analysis of total released juvenile hormone.

Farnesol and farnesal dehydrogenase(s) in corpora allata of the tobacco hornworm moth, Manduca sexta.

The metabolism of [3H]farnesol was studied in cell-free preparations of corpora allata from the tobacco hornworm, Manduca sexta, to assess the role of this presumed biosynthetic precursor of juvenile hormone (JH) III. A reversed-phase ion-pair liquid chromatographic (RP-IPC) procedure was devised to separate farnesol from several potential intermediates in its presumed metabolism to JH III: farnesal, farnesoic acid, 10,11-epoxyfarnesoic acid, and methyl farnesoate. Following incubation of (2E,6E)-[1,5,9-3H]farnesol with homogenates of corpora allata from fifth instar larvae or adult female M.

Purification of an N-acetyl-D-glucosamine specific lectin (P.B.A.) from epidermal cell membranes of Pieris brassicae L.

We report the isolation and the purification of an N-acetyl-D-glucosamine specific lectin capable of agglutinating either fixed trypsinized rabbit erythrocytes or chitin particles. An agglutinin assay based on the affinity of this lectin for the chitin was devised with fluorescent particles of scorpion cuticle to measure lectin activity during purification steps. Lectin was isolated from epidermal cell membranes; its molecular weight was determined by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate.

[Variations in the level of molting hormone during the second cycle of embryonic life in cockroaches].

It was shown by gas chromatography/mass fragmentography and radio-immunology that moulting hormone is present in great quantity during the second cycle of the embryonic life of Blabera and that the hormone concentration changes during this cycle. The loss of the regenerative capacity corresponds to a beta-ecdysone peak.