Double-Stranded RNAs High-Efficiently Protect Transgenic Potato from Leptinotarsa decemlineata by Disrupting Juvenile Hormone Biosynthesis.

RNA interference (RNAi) has been developed for plant pest control. In this study, hairpin-type double-stranded RNA (dsRNA) targeting the juvenile hormone (JH) acid methyltransferase ( JHAMT) gene ( dsJHAMT) was introduced in potato plants via Agrobacterium-mediated transformation. The results indicated that the transcriptional RNA of dsJHAMT accumulated in the transgenic plants. The transcripts and proteins of the L. decemlineata JHAMT gene were significantly reduced in larvae feeding on dsJHAMT transgenic foliage. The dsJHAMT had a significant negative effect on the growth and development of L. decemlineata, especially resulting in less oviposition. Importantly, in the field trials, transgenic plants are high-efficiently protected from insect damage mainly because surviving insects laid fewer or no eggs. Even full protection from beetle damage can be acquired by continuously lowering insect population size at large scale in the field over the years. Therefore, the transgenic plants expressing dsJHAMT successfully provided an additional option for plant pest control.

Identification and functional characterization of FGLamide-related allatostatin receptor in Rhodnius prolixus.

FGLamide-related ASTs (FGLa/ASTs) are a family of brain/gut peptides with numerous physiological roles, including inhibition of juvenile hormone (JH) biosynthesis by the corpora allata and inhibition of visceral muscle contraction. FGLa/ASTs mediate their effects by binding to a rhodopsin-like G-protein coupled receptor that is evolutionarily related to the vertebrate galanin receptor. Here we determine the cDNA sequence encoding FGLa/AST receptor (FGLa/AST-R) from the Chagas disease vector, Rhodnius prolixus (Rhopr-FGLa/AST-R), determine its spatial expression pattern using quantitative PCR and functionally characterize the receptor using a heterologous assay. Our expression analysis indicates that Rhopr-FGLa/AST-R is highly expressed in the central nervous system. The receptor is also expressed in various peripheral tissues including the dorsal vessel, midgut, hindgut and reproductive tissues of both males and females, suggesting a role in processes associated with feeding and reproduction. The possible involvement of Rhopr-FGLa/ASTs in the inhibition of JH biosynthesis is also implicated due to presence of the receptor transcript in the R. prolixus corpora cardiaca/corpora allata complex. The functional assay showed that various Rhopr-FGLa/ASTs activate the receptor, with EC50 values for the response in the nanomolar range. Moreover, Rhopr-FGLa/AST-R can couple with Gq alpha subunits and cause an increase in intracellular calcium concentration. Lastly, we tested various FGLa/AST analogs in our heterologous assay. These compounds also activated the receptor and thus have the potential to serve as insect growth regulators and aid in pest control.

Insect NMDA receptors mediate juvenile hormone biosynthesis.

In vertebrates, the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. We have now demonstrated immunohistochemically glutamatergic nerve terminals in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibited elevated cytosolic [Ca(2+)], but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg(2+). Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 microM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg(2+). Having obtained substantive evidence of functioning NMDAR in insect corpora allata, we used reverse transcription PCR to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, we used the same primers for DNMDAR1 to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. Our results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution.

Molecular characterization of a cDNA from Pseudaletia unipuncta encoding the Manduca sexta allatostatin peptide (Mas-AST).

A 15-residue neuropeptide, Manduca sexta allatostatin (Mas-AST), strongly inhibits juvenile hormone (JH) biosynthesis in vitro by corpora allata (CA) from Manduca fifth-stadium larvae and adult females as well as Helicoverpa zea adult females (Kramer et al., 1991 Proc. Natl. Acad. Sci (USA) 88, 9458-9462). In contrast, this study found that 1.0 microM Mas-AST has no JH biosynthesis inhibitory activity in Pseudaletia unipuncta sixth instar larvae or newly-emerged (day 0) adults but inhibited CA of 5-day-old adult females by 60%. From a P. unipuncta brain cDNA library, was isolated a cDNA that encodes a 125 amino acid polypeptide containing the Mas-AST sequence. Within the precursor, Mas-AST is situated at the carboxy terminus and is flanked by different dibasic proteolytic cleavage signals. The Pseudaletia gene specifying the Mas-AST peptide is present as a single copy per haploid genome. Expression of this gene was low in Pseudaletia sixth instar larvae, prepupae and early pupae but was relatively high in late pupae, and day 1 and 3 adults of both sexes. In day 5 adults, the relative transcript level appears to be maintained in females but declines in males. This pattern of Mas-AST expression does not correlate well with the profile of JH biosynthesis in Pseudaletia, which increases during the first 5 days of adult life, suggesting additional or alternative functions for this peptide.