Identification and expression of short neuropeptide F and its receptors in the tick Ixodes ricinus.

In Europe, the tick Ixodes ricinus is the most important vector of numerous pathogens that are transmitted during blood feeding on their vertebrate hosts. To elucidate mechanisms controlling blood intake and associated transmission of pathogens we identified and described expression of short neuropeptide F (sNPF) and its receptors which are known to regulate feeding in insects. Using in situ hybridization (ISH) and immunohistochemistry (IHC) we stained numerous neurons producing sNPF in the central nervous system (CNS; synganglion), while a few peripheral neurons were detected anteriorly to the synganglion, and on the surface of the hindgut and leg muscles. Apparent sNPF expression was also found in enteroendocrine cells individually scattered in anterior lobes of the midgut. In silico analyses and BLAST search for sNPF receptors revealed two putative G protein-coupled receptors (sNPFR1 and sNPFR2) in the I. ricinus genome. Aequorin-based functional assay in CHO cells showed that both receptors were specific and sensitive to sNPF in nanomolar concentrations. Increased expression levels of these receptors in the gut during blood intake suggest that sNPF signaling may be involved in regulation of feeding and digestion processes of I. ricinus.

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.

Expression analysis of peptidergic enteroendocrine cells in the silkworm Bombyx mori.

Enteroendocrine cells (ECs) in the insect midgut respond to physiological changes in the intestine by releasing multiple peptides to control food intake, gastrointestinal activity and systemic metabolism. Here, we performed a comprehensive mapping of ECs producing different regulatory peptides in the larval midgut of Bombyx mori. In total, we identified 20 peptide genes expressed in different ECs in specific regions of the midgut. Transcript-specific in situ hybridisation combined with antibody staining revealed approximately 30 subsets of ECs, each producing a unique peptide or a combination of several different peptides. Functional significance of this diversity and specific roles of different enteroendocrine peptides are largely unknown. Results of this study highlight the importance of the midgut as a major endocrine/paracrine source of regulatory molecules in insects and provide important information to clarify functions of ECs during larval feeding and development.

Camptothecin, triptolide, and apoptosis inducer kit have differential effects on mitochondria in colorectal carcinoma cells.

Mitochondrial fission and fusion are required for cell survival, and several studies have shown an imbalance between fission and fusion in cancer. High levels of mitochondrial fusion are observed in drug-resistant tumor cells, whereas mitochondrial fission may be important in sensitizing tumor cells to chemotherapy drugs. Based on current knowledge, we hypothesized that different chemotherapeutics might differentially affect mitochondrial dynamics and energy production. Thus, we selected chemotherapeutics with different mechanisms of action (camptothecin, triptolide and apoptosis inducer kit) and investigated their effect on mitochondria in colorectal carcinoma cells. We report that these chemotherapeutics decreased the activity of complex I and reduced the mitochondrial membrane potential, and also decreased the size of mitochondria in the colorectal carcinoma cell lines DLD1 and HCT-116. Treatment with camptothecin, triptolide and/or apoptosis inducer kit results in differential effects of fission on apoptosis in these cells. Our results suggest that fission is an important process in apoptosis induced by chemotherapeutics.

Identification and function of ETH receptor networks in the silkworm Bombyx mori.

Insect ecdysis triggering hormones (ETHs) released from endocrine Inka cells act on specific neurons in the central nervous system (CNS) to activate the ecdysis sequence. These primary target neurons express distinct splicing variants of ETH receptor (ETHR-A or ETHR-B). Here, we characterized both ETHR subtypes in the moth Bombyx mori in vitro and mapped spatial and temporal distribution of their expression within the CNS and peripheral organs. In the CNS, we detected non-overlapping expression patterns of each receptor isoform which showed dramatic changes during metamorphosis. Most ETHR-A and a few ETHR-B neurons produce multiple neuropeptides which are downstream signals for the initiation or termination of various phases during the ecdysis sequence. We also described novel roles of different neuropeptides during these processes. Careful examination of peripheral organs revealed ETHRs expression in specific cells of the frontal ganglion (FG), corpora allata (CA), H-organ and Malpighian tubules prior to each ecdysis. These data indicate that PETH and ETH are multifunctional hormones that act via ETHR-A and ETHR-B to control various functions during the entire development-the ecdysis sequence and associated behaviors by the CNS and FG, JH synthesis by the CA, and possible activity of the H-organ and Malpighian tubules.

Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori.

Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination.

Multiple neuropeptides produced by sex-specific neurons control activity of the male accessory glands and gonoducts in the silkworm Bombyx mori.

The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.

Gene expression in reproductive organs of tsetse females - initial data in an approach to reduce fecundity.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, and their vectorial capacity results in a major public health emergency and vast economic losses in sub-Saharan Africa. Given the limited ability of trypanosome prevention and eradication, tsetse vectors remain major targets of control efforts. Larvae of all three instars are developed in mothers' uteri, nourished through milk, and 'larviposited' shortly before pupation. The past few years have witnessed the emergence of approaches based on knockdown of genes involved in milk production, resulting in a significant reduction of fecundity. RESULTS: In order to identify further genes applicable in the control of tsetse flies, we determined the expression of protein-coding genes in ovaries and uteri from both virgin and heavily pregnant Glossina morsitans morsitans females. Comparison of expression profiles allowed us to identify candidate genes with increased expression in pregnant individuals. Lists with the highest increases include genes involved in oocyte and embryonic development, or nourishment. Maximum ovarian fold change does not exceed 700, while the highest uterine fold change reaches to more than 4000. Relatively high fold changes of two neuropeptide receptors (for corazonin and myosuppressin) propose the corresponding genes alternative targets. CONCLUSIONS: Given the higher fold changes in the uterus, targeting gene expression in this tissue may result in a more evident reduction of fecundity. However, ovaries should not be neglected, as manifested by several genes with top fold changes involved in early developmental stages. Apart from focusing on the highest fold changes, neuropeptide receptors with moderate increases in expression should be also verified as targets, given their roles in mediating the tissue control. However, this data needs to be considered initial, and the potential of these genes in affecting female fecundity needs to be verified experimentally.

Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick, Amblyomma variegatum.

Tick saliva is a rich source of antihemostatic compounds. We amplified a cDNA from the salivary glands of the tropical bont tick (Amblyomma variegatum) using primers based on the variegin sequence, which we previously identified as a novel thrombin inhibitor from the same tick species. The transcript encodes a precursor protein comprising a signal peptide and 5 repeats of variegin-like sequences that could be processed into multiple short peptides. These peptides share 31 to 34% identity with variegin. Here, we structurally and functionally characterized one of these peptides named "avathrin." Avathrin is a fast, tight binding competitive inhibitor with an affinity of 545 pM for thrombin and is 4 orders of magnitude more selective towards thrombin than to the other serine proteases of the coagulation cascade. The crystal structure of thrombin-avathrin complex at 2.09 Å revealed that avathrin interacts with the thrombin active site and exosite-I. Although avathrin is cleaved by thrombin, the C-terminal cleavage product continues to exert prolonged inhibition. Avathrin is more potent than hirulog-1 in a murine carotid artery thrombosis model. Such precursor proteins that could be processed into multiple thrombin inhibiting peptides appear to be widespread among Amblyomminae, providing an enormous library of molecules for development as potent antithrombotics.-Iyer, J. K., Koh, C. Y., Kazimirova, M., Roller, L., Jobichen, C., Swaminathan, K., Mizuguchi, J., Iwanaga, S., Nuttall, P. A., Chan, M. Y., Kini, R. M. Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick, Amblyomma variegatum.

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.

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.

In vitro study of partially hydrolyzed poly(2-ethyl-2-oxazolines) as materials for biomedical applications.

Polymers based on 2-oxazoline, such as poly(2-ethyl-2-oxazolines) (PETOx), are considered to be a type of 'pseudopeptide' with the ability to form novel biomaterials. The hydrolysis of PETOx was carried out to evaluate its use in biomedical applications. In the present work, PETOx samples with a range of molar masses were prepared by living cationic polymerization. Hydrolysis was carried out at time intervals ranging from 15 to 180 min to prepare copolymers with different amounts of ethylene imine units. (1)H NMR spectroscopy was used to identify the structure of the hydrolyzed polymers. The dependence of in vitro cell viability on the degree of hydrolysis was determined using three different model cell lines, namely, mouse embryonic 3T3 fibroblasts, pancreatic ßTC3 cells, and mouse lymphoid macrophages P388.D1. It was demonstrated that increasing the degree of hydrolysis decreased cell viability for all cell types. Fibroblast cells displayed the highest tolerance; additionally, the effect of polymer size showed no observable significance. Macrophage cells, immune system representatives, displayed the highest sensitivity to contact with hydrolyzed PETOx. The effect of polymer hydrolysis, polymer concentration and the incubation time on cell viability was experimentally observed. Confocal laser-scanning microscopy provided evidence of cellular uptake of pyrene-labeled (co)polymers.

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.

Analysis of lipolysis underlying lactation in the tsetse fly, Glossina morsitans.

Female tsetse flies undergo viviparous reproduction, generating one larva each gonotrophic cycle. Larval nourishment is provided by the mother in the form of milk secretions. The milk consists mostly of lipids during early larval development and shifts to a balanced combination of protein and lipids in the late larval instars. Provisioning of adequate lipids to the accessory gland is an indispensable process for tsetse fecundity. This work investigates the roles of Brummer lipase (Bmm) and the adipokinetic hormone (AKH)/adipokinetic hormone receptor (AKHR) systems on lipid metabolism and mobilization during lactation in tsetse. The contributions of each system were investigated by a knockdown approach utilizing siRNA injections. Starvation experiments revealed that silencing of either system results in prolonged female lifespan. Simultaneous suppression of bmm and akhr prolonged survival further than either individual knockdown. Knockdown of akhr and bmm transcript levels resulted in high levels of whole body lipids at death, indicating an inability to utilize lipid reserves during starvation. Silencing of bmm resulted in delayed oocyte development. Respective reductions in fecundity of 20 and 50% were observed upon knockdown of akhr and bmm, while simultaneous knockdown of both genes resulted in 80% reduction of larval production. Omission of one bloodmeal during larvigenesis (nutritional stress) after simultaneous knockdown led to almost complete suppression of larval production. This phenotype likely results from tsetse's inability to utilize lipid reserves as loss of both lipolysis systems leads to accumulation and retention of stored lipids during pregnancy. This shows that both Bmm lipolysis and AKH/AKHR signaling are critical for lipolysis required for milk production during tsetse pregnancy, and identifies the underlying mechanisms of lipid metabolism critical to tsetse lactation. The similarities in the lipid metabolic pathways and other aspects of milk production between tsetse and mammals indicate that this fly could be used as a novel model for lactation research.

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.

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.

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.

Functional role of 64P, the candidate transmission-blocking vaccine antigen from the tick, Rhipicephalus appendiculatus.

Anti-ectoparasite vaccines offer attractive alternatives to the use of chemical pesticides, especially if they also control the pathogens that ectoparasites transmit. However, selection of suitable antigens is a major constraint on vaccine development. The recombinant tick cement protein, 64TRP, derived from the African brown ear tick, Rhipicephalus appendiculatus, acts as a transmission-blocking vaccine in a mouse model of tick-borne encephalitis virus (TBEV) transmission, protecting immunised mice against lethal challenge with TBEV after exposure to infected ticks. 64TRP acts as a dual action vaccine, targeting both 'exposed' antigens in tick saliva and 'concealed' antigenic epitopes in the tick midgut. To assess further the suitability of 64TRP as a vaccine antigen, we examined the function (including localisation) of the protein, and its sequence variability. Histological profiles of normal hamster skin showed similarities between normal skin proteins in the epidermis (keratin) and dermis (collagen/reticulin) and the tick cement cone. Immuno-reactivity of anti-64TRP sera with hamster skin suggests a potential sequence similarity of 64P with host skin proteins and may reflect previously reported sequence similarities of 64P with skin keratin and collagen proteins. Variability in the N-terminal signal peptide and in the C-terminal glycine-rich amino acid repeats of 64P protein was detected; previous studies showed the C-terminal region to be immunologically non-protective. Using in situ hybridisation and quantitative reverse transcriptase-PCR, 64P mRNA was detected in the types II and III salivary gland acini. The highest levels of 64P mRNA were observed in 1-day fed females, and 1- and 7-day fed males. Salivary glands of longer feeding females and unfed ticks as well as midguts of both sexes were negative. Early expression in tick salivary glands is consistent with previously published data that 64P is a cement protein, and contributes to its candidacy as a vaccine antigen. However, further studies are required to assess whether cross-reactivity with skin proteins may induce autoimmunity.

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.