Ladislav Roller

Bombyx prothoracicostatic peptides activate the sex peptide receptor to regulate ecdysteroid biosynthesis

Insect molting and metamorphosis are induced by steroid hormones named ecdysteroids, whose production is regulated by various neuropeptides. We cloned the gene and analyzed the expression of the prothoracicostatic peptide, a unique neuropeptide shown to suppress the production of ecdysteroids in the prothoracic gland of the silkworm, Bombyx mori. We also characterized a Bombyx G protein-coupled receptor, which has previously been identified as an ortholog of the Drosophila sex peptide receptor, as a functional prothoracicostatic peptide receptor. This receptor responded specifically to the prothoracicostatic peptides when examined using a heterologous expression system. The receptor was highly expressed in the prothoracic gland on the day before each larval and pupal ecdysis, when prothoracicostatic peptides are synthesized at a high level in the epiproctodeal glands. These results suggest that the sex peptide receptor functions as a prothoracicostatic peptide receptor in Bombyx and that the peripheral neurosecretory cells as well as the central neuroendocrine system play stage-specific roles in regulating ecdysteroidogenesis.

Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects

Antisera against corazonin were used to investigate distribution of immunoreactive cells in the central nervous system (CNS) of representatives of six insect orders: Ctenolepisma lineata (Zygentoma), Locusta migratoria (Orthoptera), Oxya yezoensis (Orthoptera), Gryllus bimaculatus (Orthoptera), Pyrrhocoris apterus (Hemiptera), Arge nigrinodosa (Hymenoptera), Athalia rosae (Hymenoptera), Bombyx mori (Lepidoptera) and Anomala cuprea (Coleoptera). Corazonin-like immunoreactive (CLI) cells were detected in the brain and ventral ganglia of all insects studied except for the albino strain of L. migratoria and the beetle A. cuprea. Implantation of the brain or different ganglia from insects with detected immunoreactivity induced dark coloration in the albino locust, providing further evidence for the presence of authentic corazonins [His7- and Arg7-isoforms] in these insects. The protocerebral lateral neurosecretory cells projecting into the ipsilateral retrocerebral neurohemal organs and bilateral longitudinal tracts extending and branching throughout the entire CNS seem to be a well-conserved part of the corazonin system in insects. The bilateral longitudinal tracts were formed by species-specific numbers of bilateral interneurons segmentally distributed in the ventral ganglia. Additional immunoreactive somata, mostly interneurons, were detected in the CNS of various insects. The distribution of corazonin in the cephalic neurosecretory system and in the bilateral interneurons suggests that corazonin acts as a hormone as well as a neurotransmitter or a neuromodulator. An ancient origin of corazonin is suggested by the presence of a corazonin-like substance in the primitive insect, C. lineata. These results support previous findings on the common occurrence of corazonin among insects, except for the albino strain of L. migratoria and the Coleoptera.

Corazonin reduces the spinning rate in the silkworm, Bombyx mori

The insect neuropeptide, [Arg(7)]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.

Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis.

Biosynthesis of ecdysteroids, the insect steroid hormones controlling gene expression during molting and metamorphosis, takes place primarily in the prothoracic gland (PG). The activity of the PG is regulated by various neuropeptides. In the silkworm Bombyx mori, these neuropeptides utilize both hormonal and neuronal pathways to regulate the activity of the PG, making the insect an excellent model system to investigate the complex signaling network controlling ecdysteroid biosynthesis. Here we report another group of neuropeptides, orcokinins, as neuronal prothoracicotropic factors. Using direct mass spectrometric profiling of the axons associated with the PG, we detected several peptide peaks which correspond to orcokinin gene products in addition to the previously described Bommo‐FMRFamides (BRFas). In situ hybridization and immunohistochemistry revealed that orcokinins are produced in the prominent neurosecretory cells in the ventral ganglia, as well as in numerous small neurons throughout the central nervous system and in midgut endocrine cells. One of the two pairs of BRFa‐expressing neurosecretory cells in the prothoracic ganglion coexpresses orcokinin, and these neurons project axons through the transverse nerve and terminate on the surface of the PG. Using an in vitro PG bioassay, we show that orcokinins have a clear prothoracicotropic activity and are able to cancel the static effect of BRFas on ecdysteroid biosynthesis, whereas the suppressive effect of BRFas on cAMP production remained unchanged in the presence of orcokinins. The discovery of a second regulator of PG activity in these neurons further illustrates the potential importance of the PG innervation in the regulation of insect development. J. Comp. Neurol. 519:238‐246, 2011.

Transgenesis approaches for functional analysis of peptidergic cells in the silkworm Bombyx mori

The domestic silkworm, Bombyx mori represents an insect model of great scientific and economic importance. Besides the establishment of a stable germline transformation using the PiggyBac vector, technically feasible methods for in vivo gene delivery and transient gene expression were developed using viral based vectors, especially Sindbis viruses and baculoviruses. The recombinant baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), commonly used for large-scale protein production in permissive cell lines or insects, has been used for foreign gene transfer into specific peptidergic cells of B. mori in vivo. Since targeted gene expression is essential for functional analysis of neuropeptide genes and their receptors, the baculovirus-mediated gene transfer can serve as a reliable approach in reverse genetic studies in the silkworm. We review various strategies employing the baculovirus vector system for transient expression of molecular markers and transcription factors in specific peptidergic cells to investigate their roles in B. mori. We also use this system for functional analysis of neuropeptide signaling in the ecdysis behavioral sequence. Our data indicate that the AcMNPV vector is suitable for efficient delivery of foreign genes and their expression directed into specific peptidergic neurons and endocrine cells of B. mori larvae and pupae. However, some modifications of the vector and steps for optimization are necessary to minimize negative effects of viral infection on the host development. The transient gene expression using the AcMNPV and other virus vectors are promising tools for analysis of molecular mechanisms underlying various neuroendocrine processes during development of B. mori.

Immunization with recombinant subolesin does not reduce tick infection with tick-borne encephalitis virus nor protect mice against disease

Tick-borne encephalitis (TBE) is a growing zoonotic disease caused by tick-borne encephalitis virus (TBEV) infection. Although effective vaccines for TBEV are available, on-going vaccination efforts are insufficient to prevent increase in TBE cases annually. Vaccination with arthropod vector antigens to reduce vector infestations and vector capacity allows control of several vector-borne diseases by targeting their common vector. Subolesin (SUB) is a tick protective antigen that has a role in tick innate immunity and other molecular pathways and has been shown to protect against tick infestations and infection by vector-borne pathogens. However, SUB expression and the effect of SUB immunization have not been evaluated for tick-borne viruses. Herein, we showed that SUB expression is downregulated during Ixodes ricinus tick feeding but induced in ticks infected with TBEV, thus supporting a role for this molecule in tick innate immune response to virus infection. Immunization with recombinant SUB reduced SUB mRNA levels in nymphs co-feeding with infected females and suggested and effect on tick infestations in mice. However, SUB immunization did not reduce tick infection with TBEV nor protect mice against TBE. These results suggested that SUB is not a good candidate antigen for vaccination against TBEV and support the characterization of tick-pathogen interactions to identify mechanisms that could be targeted to reduce TBEV infection and transmission by ticks.

Expression of RYamide in the nervous and endocrine system of Bombyx mori

RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion.

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.

Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori

Allatotropin (AT) and related neuropeptides are widespread bioactive molecules that regulate development, food intake and muscle contractions in insects and other invertebrates. In moths, alternative splicing of the at gene generates three mRNA precursors encoding AT with different combinations of three structurally similar AT-like peptides (ATLI-III). We used in situ hybridization and immunohistochemistry to map the differential expression of these transcripts during the postembryonic development of Bombyx mori. Transcript encoding AT alone was expressed in numerous neurons of the central nervous system and frontal ganglion, whereas transcripts encoding AT with ATLs were produced by smaller specific subgroups of neurons in larval stages. Metamorphosis was associated with considerable developmental changes and sex-specific differences in the expression of all transcripts. The most notable was the appearance of AT/ATL transcripts (1) in the brain lateral neurosecretory cells producing prothoracicotropic hormone; (2) in the male-specific cluster of about 20 neurons in the posterior region of the terminal abdominal ganglion; (3) in the female-specific medial neurons in the abdominal ganglia AG2-7. Immunohistochemical staining showed that these neurons produced a mixture of various neuropeptides and innervated diverse peripheral organs. Our data suggest that AT/ATL neuropeptides are involved in multiple stage- and sex-specific functions during the development of B. mori.