Differential expression of ITP and ITPL indicate multiple functions in the silkworm Bombyx mori.

Ion transport peptide (ITP) and a longer ITP-like (ITPL) are alternatively spliced insect neuropeptides involved in the regulation of development and water homeostasis. Using in situ hybridisation and immunohistochemistry, we determined site- and stage-specific expression of each peptide in Bombyx mori. Each peptide was differentially expressed, except for the prominent overlapping expression of both peptides in six pairs of the brain neurosecretory cells Ia2. After metamorphosis, ITP appeared in the male-specific neurons of the abdominal neuromere 9 (MAN9) that innervate the reproductive organs. ITPL was detected in a pair of dorsolateral interneurons (IN-DL) in each thoracic and abdominal ganglion, and in the thoracic neurosecretory cells (NS-VTL2) which terminate in the vicinity of the prothoracic gland. Feeding larvae showed ITPL expression in the abdominal neurosecretory cells M5. ITPL was also expressed in the peripheral L1 neurons that project axons into the thoracic and abdominal transverse nerves. Our results suggest that ITP and ITPL exhibit different sex- and stage-specific functions that may include regulation of reproduction and steroid production. For future functional studies, we identified an upstream regulatory region controlling ITP/ITPL expression in the brain and L1 neurons, and prepared stable transgenic line pITP-Gal4.2 using the piggyBac system.

Molecular cloning, expression and identification of the promoter regulatory region for the neuropeptide trissin in the nervous system of the silkmoth Bombyx mori.

Trissin has recently been identified as a conserved insect neuropeptide, but its cellular expression and function is unknown. We detected the presence of this neuropeptide in the silkworm Bombyx mori using in silico search and molecular cloning. In situ hybridisation was used to examine trissin expression in the entire central nervous system (CNS) and gut of larvae, pupae and adults. Surprisingly, its expression is restricted to only two pairs of small protocerebral interneurons and four to five large neurons in the frontal ganglion (FG). These neurons were further characterised by subsequent multiple staining with selected antibodies against insect neuropeptides. The brain interneurons innervate edges of the mushroom bodies and co-express trissin with myoinhibitory peptides (MIP) and CRF-like diuretic hormones (CRF-DH). In the FG, one pair of neurons co-express trissin with calcitonin-like diuretic hormone (CT-DH), short neuropeptide F (sNPF) and MIP. These neurons innervate the brain tritocerebrum and musculature of the anterior midgut. The other pair of trissin neurons in the FG co-express sNPF and project axons to the tritocerebrum and midgut. We also used the baculovirus expression system to identify the promoter regulatory region of the trissin gene for targeted expression of various molecular markers in these neurons. Dominant expression of trissin in the FG indicates its possible role in the regulation of foregut-midgut contractions and food intake.