Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Šimo et al., 2009b, 2013). Among these, the neuropeptide SIFamide-along with its cognate receptor-were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.

• MeSH
• klíště * genetika   metabolismus   MeSH
• neurony metabolismus   MeSH
• neuropeptidy * genetika   metabolismus   MeSH
• slinné žlázy metabolismus   MeSH
• sliny MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. "Kissing bugs" are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

• MeSH
• hmyz metabolismus   fyziologie   MeSH
• hmyzí proteiny metabolismus   MeSH
• juvenilní hormony metabolismus   MeSH
• lipoproteiny metabolismus   MeSH
• metabolismus lipidů * MeSH
• oocyty metabolismus   MeSH
• oogeneze fyziologie   MeSH
• ovarium metabolismus   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• Triatominae * metabolismus   fyziologie   MeSH
• vitelogeneze fyziologie   MeSH
• vitelogeniny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Juvenile hormone (JH) controls insect reproduction and development through an intracellular receptor complex comprising two bHLH-PAS proteins, the JH-binding Methoprene-tolerant (Met) and its partner Taiman (Tai). Many hemimetabolous insects including cockroaches strictly depend on JH for stimulation of vitellogenesis. In termites, the eusocial hemimetabolans, JH also regulates the development of caste polyphenism. Studies addressing the agonist ligand binding to recombinant JH receptors currently include three species belonging to two holometabolous insect orders, but none that would represent any of the hemimetabolous orders. Here, we examined JH receptors in two representatives of Blattodea, the cockroach Blattella germanica and the termite Prorhinotermes simplex. To test the JH-binding capacity of Met proteins from these species, we performed chemical synthesis and tritium labeling of the natural blattodean JH homolog, JH III. Our improved protocol increased the yield and specific activity of [10-3H]JH III relative to formerly available preparations. Met proteins from both species specifically bound [3H]JH III with high affinity, whereas Met variants mutated at a critical position within the ligand-binding domain were incapable of such binding. Furthermore, JH III and the synthetic JH mimic fenoxycarb stimulated dimerization between Met and Tai components of the respective JH receptors of both species. These data present primary evidence for agonist binding by JH receptors in any hemimetabolous species and provide a molecular basis for JH action in cockroaches and termites.

• MeSH
• Ectobiidae metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• Isoptera metabolismus   MeSH
• seskviterpeny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.

• MeSH
• bourec * genetika   ultrastruktura   MeSH
• endoplazmatické retikulum metabolismus   ultrastruktura   MeSH
• fibroiny genetika   metabolismus   ultrastruktura   MeSH
• hedvábí * chemie   genetika   MeSH
• mutace MeSH
• mutageneze cílená metody   MeSH
• slinné žlázy * cytologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Many lepidopteran larvae produce silk secretions to build feeding tubes and cocoons that play important protective roles in their lives. Recent research on the silk of bombycoid and pyralid moths has shown that it contains several highly abundant silk components with remarkable mechanical properties. It was also found to contain a number of other proteins of which the functions have yet to be identified. To gain an overview of the silk composition in more primitive lepidopteran species and to identify the core silk components common to most species, we analyzed the cocoon proteins of Tineola bisselliella, which belongs to the basal ditrysian moth line. Using de novo transcriptome sequencing combined with mass spectrometry (MS)-based proteomics, we detected more than 100 secretory proteins in the silk cocoons. Fibroin, sericins, and protease inhibitors were found to be the most abundant proteins, along with several novel candidate silk components. We also verified the tissue and developmental stage specificity of the silk protein expression and characterized the morphology of both the silk glands and silk in T. bisselliella. Our study provides a detailed analysis of silk in the primitive moth, expands the known set of silk-specific genes in Lepidoptera, and helps to elucidate their evolutionary relationships.

• MeSH
• biologická evoluce * MeSH
• fibroiny metabolismus   MeSH
• hedvábí * chemie   genetika   metabolismus   MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• inhibitory proteas metabolismus   MeSH
• larva genetika   metabolismus   fyziologie   MeSH
• můry * genetika   metabolismus   fyziologie   MeSH
• proteomika metody   MeSH
• sericiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

• MeSH
• chromatografie kapalinová * MeSH
• Diptera chemie   MeSH
• Heteroptera chemie   MeSH
• Hymenoptera chemie   MeSH
• juvenilní hormony analýza   chemie   MeSH
• Lepidoptera chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

EFLamide (EFLa) is a neuropeptide known for a long time from crustaceans, chelicerates and myriapods. Recently, EFLa-encoding genes were identified in the genomes of apterygote hexapods including basal insect species. In pterygote insects, however, evidence of EFLa was limited to partial sequences in the bed bug (Cimex), migratory locust and a few phasmid species. Here we present identification of a full length EFLa-encoding transcript in the linden bug, Pyrrhocoris apterus (Heteroptera). We created complete null mutants allowing unambiguous anatomical location of this peptide in the central nervous system. Only 2-3 EFLa-expressing cells are located very close to each other near to the surface of the lateral protocerebrum with dense neuronal arborization. Homozygous null EFLa mutants are fully viable and do not have any visible defect in development, reproduction, lifespan, diapause induction or circadian rhythmicity. Phylogenetic analysis revealed that EFLa-encoding transcripts are produced by alternative splicing of a gene that also produces Prohormone-4. However, this Proh-4/EFLa connection is found only in Hemiptera and Locusta, whereas EFLa-encoding transcripts in apterygote hexapods, chelicerates and crustaceans are clearly distinct from Proh-4 genes. The exact mechanism leading to the fused Proh-4/EFLa transcript is not yet determined, and might be a result of canonical cis-splicing, cis-splicing of adjacent genes (cis-SAG), or trans-splicing.

• MeSH
• fylogeneze MeSH
• Heteroptera genetika   metabolismus   MeSH
• hmyzí proteiny chemie   genetika   metabolismus   MeSH
• hormon uvolňující thyreotropin genetika   metabolismus   MeSH
• neuropeptidy chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera.

• MeSH
• fylogeneze MeSH
• glykoproteiny chemie   genetika   metabolismus   MeSH
• hedvábí genetika   metabolismus   MeSH
• hmyzí proteiny chemie   genetika   metabolismus   MeSH
• larva genetika   růst a vývoj   metabolismus   MeSH
• muciny chemie   genetika   metabolismus   MeSH
• můry genetika   růst a vývoj   metabolismus   MeSH
• proteom * MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• sericiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phlebotomus perniciosus and P. orientalis, the Old-World vectors of visceral leishmaniases causative agents. Expressed and purified YRPs from three different sand fly species were tested for their interactions with various biogenic amines, including serotonin, histamine and catecholamines. Using the L. longipalpis YRP LJM11 as a control, we have demonstrated the comparability of the microscale thermophoresis method with conventional isothermal titration calorimetry described previously. By homology in silico modeling, we predicted the surface charge and both amino acids and hydrogen bonds of the amine-binding motifs to influence the binding affinities between closely related YRPs. All YRPs tested bound at least two biogenic amines, while the affinities differ both among and within species. Low affinity was observed for histamine. The salivary recombinant proteins rSP03B (P. perniciosus) and rPorASP4 (P. orientalis) showed high-affinity binding of serotonin, suggesting their capability to facilitate inhibition of the blood vessel contraction and platelet aggregation.

• MeSH
• aminy metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• konformace proteinů MeSH
• Phlebotomus metabolismus   MeSH
• slinné proteiny a peptidy metabolismus   MeSH
• slinné žlázy metabolismus   MeSH
• statická elektřina MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

The attrition of telomeres, the ends of eukaryote chromosomes, and activity of telomerase, the enzyme that restores telomere length, play a role in the ageing process and act as indicators of biological age. A notable feature of advanced eusocial insects is the longevity of reproductive individuals (queens and kings) compared to those from non-reproductive castes (workers and soldiers) within a given species, with a proposed link towards upregulation of telomerase activity in the somatic tissues of reproductive individuals. Given this, eusocial insects provide excellent model systems for research into ageing. We tested telomerase activity and measured telomere length in Bombus terrestris, which is a primitively eusocial insect species with several distinct features compared to advanced social insects. In somatic tissues, telomerase activity was upregulated only in the fat bodies of pre-diapause queens, and this upregulation was linked to heightened DNA synthesis. Telomere length was shorter in old queens compared to that in younger queens or workers. We speculate that (1) the upregulation of telomerase activity, together with DNA synthesis, is the essential step for intensifying metabolic activity in the fat body to build up a sufficient energy reserve prior to diapause, and that (2) the lifespan differences between B. terrestris workers and queens are related to the long diapause period of the queen. A possible relationship between telomere length regulation and TOR, FOXO, and InR as cell signaling components, was tested.

• MeSH
• DNA biosyntéza   MeSH
• telomerasa metabolismus   MeSH
• tukové těleso enzymologie   MeSH
• včely enzymologie   MeSH
• zkracování telomer MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH