The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Summary: The whitefly (Bemisia tabaci) causes tremendous damage to cotton production worldwide. However, very limited information is available about how plants perceive and defend themselves from this destructive pest. In this study, the transcriptomic differences between two cotton cultivars that exhibit either strong resistance (HR) or sensitivity (ZS) to whitefly were compared at different time points (0, 12, 24 and 48 h after infection) using RNA‐Seq. Approximately one billion paired‐end reads were obtained by Illumina sequencing technology. Gene ontology and KEGG pathway analysis indicated that the cotton transcriptional response to whitefly infestation involves genes encoding protein kinases, transcription factors, metabolite synthesis, and phytohormone signalling. Furthermore, a weighted gene co‐expression network constructed from RNA‐Seq datasets showed that WRKY40 and copper transport protein are hub genes that may regulate cotton defenses to whitefly infestation. Silencing GhMPK3 by virus‐induced gene silencing (VIGS) resulted in suppression of the MPK‐WRKY‐JA and ET pathways and lead to enhanced whitefly susceptibility, suggesting that the candidate insect resistant genes identified in this RNA‐Seq analysis are credible and offer significant utility. Taken together, this study provides comprehensive insights into the cotton defense system to whitefly infestation and has identified several candidate genes for control of phloem‐feeding pests.

The sex pheromone biosynthetic pathways of lepidopterans require the concerted actions of multiple gene products. A number of pheromone gland (PG)-specific genes have been cloned in recent years and, whereas in vitro characterizations have indicated functions consistent with roles in pheromone production, there have been no clear demonstrations in vivo . Using an RNA interference-mediated loss-of-function approach, we injected newly formed Bombyx mori pupae with dsRNAs corresponding to genes of interest [i.e., PG fatty acyl reductase ( pgFAR ), B. mori PG Z11/Δ10,12 desaturase ( Bmpgdesat1 ), PG acyl-CoA-binding protein ( pgACBP ), midgut ACBP, and pheromone biosynthesis activating neuropeptide receptor ( PBANR )] to assess their specific roles during pheromonogenesis. In all cases, the introduced dsRNAs induced a dose-dependent reduction in sex pheromone production with the corresponding decrease in transcript levels. No effects on pupal development or adult emergence were observed. Disrupting the PBANR gene resulted in a loss of the lipase activity that liberates pheromone precursors, whereas knockout of the pgACBP gene prevented the daily accumulation and fluctuation of the triacylglycerols that function as the cellular deposits for the pheromone precursors. Taken together, our results provide unequivocal evidence that the pgACBP, Bmpgdesat1, pgFAR, and PBANR gene products are essential during pheromonogenesis and demonstrate the power of this methodology for dissecting the molecular interactions that comprise biosynthetic pathways.

The straight-chain C ₁₀ to C ₁₈ unsaturated aliphatic compounds containing an oxygenated functional group (aldehyde, alcohol, or acetate ester) derived from saturated C ₁₆ or C ₁₈ fatty acids are a major class of sex pheromone components produced by female moths. In the biosynthesis of these pheromone components, various combinations of limited chain-shortening and regio- and stereospecific desaturation reactions significantly contribute to the production of a vast number of the species-specific pheromone components in Lepidoptera. Biosynthesis of the silkmoth sex pheromone bombykol, ( E,Z )-10,12-hexadecadien-1-ol, involves two consecutive desaturation steps, the second of which is unique in that it generates a conjugated diene system from the Δ11-monoene C ₁₆ intermediate. In experiments designed to characterize the acyl-CoA desaturases responsible for bombykol biosynthesis, we have cloned three cDNAs encoding desaturase family members from the pheromone gland of the inbred strain of the silkmoth, Bombyx mori . Transcript analyses by RT-PCR and subsequent functional assays using a Bac-to-Bac baculovirus expression system revealed that desat1 is the only desaturase gene prominently expressed during pheromonogenesis and that its gene product, B. mori Desat1, possesses both Z11 desaturation and Δ10,12-desaturation activities. Consequently, we have concluded that B. mori Desat1 is not only a bifunctional desaturase involved in bombykol biosynthesis but that it is also the enzyme responsible for both desaturation steps.

In most Lepidoptera, pheromone biosynthesis is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN). Although much is known about the cellular targets of PBAN, identification and functional characterization of the PBAN receptor (PBANR) has proven to be elusive. Given the sequence similarity between the active C-terminal regions of PBAN and neuromedin U, it was hypothesized that their respective receptors might also be similar in structure (Park, Y., Kim, Y. J., and Adams, M. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11423-11428). Consequently, utilizing primers constructed from the conserved regions of insect neuromedin U receptor homologues, a full-length 2780-nucleotide clone encoding a 46-kDa G protein-coupled receptor was amplified from a Bombyx mori pheromone gland cDNA library. Tissue distribution analyses revealed that the receptor transcript is specific to the pheromone gland where it undergoes significant up-regulation in the day preceding eclosion. When transiently expressed in Sf9 cells, the B. mori PBANR responds to PBAN by mobilizing extracellular calcium in a dose-dependent manner. Confocal microscopic studies demonstrated the specificity of enhanced green fluorescent protein-tagged B. mori PBANR for PBAN and showed that PBAN induces internalization of the PBANR.PBAN complex. The rapid onset of internalization is mediated by a 67-amino acid C-terminal extension absent in the cloned Helicoverpa zea PBANR, which suggests that receptor internalization in that species likely utilizes a different mechanism. From these results, we have concluded that the cloned receptor gene encodes the B. mori PBANR and that it is both structurally and functionally distinct from the H. zea PBANR.