BACKGROUND: Leishmaniasis is a group of neglected vector-borne diseases transmitted by phlebotomine sand flies. Leishmania parasites must overcome various defenses in the sand fly midgut, including the insects's immune response. Insect immunity is regulated by the ecdysone hormone, which binds to its nuclear receptor (EcR) and activates the transcription of genes involved in insect immunity. However, the role of ecdysone in sand fly immunity has never been studied. Phlebotomus perniciosus is a natural vector of Leishmania infantum; here, we manipulated its neuroendocrine system using azadirachtin (Aza), a natural compound known to affect ecdysone synthesis. METHODS: Phlebotomus perniciosus larvae and adult females were fed on food containing either Aza alone or Aza plus ecdysone, and the effects on mortality and ecdysis were evaluated. Genes related to ecdysone signaling and immunity were identified in P. perniciosus, and the expression of antimicrobial peptides (AMPs), EcR, the ecdysone-induced genes Eip74EF and Eip75B, and the transcription factor serpent were analyzed using quantitative polymerase chain reaction (PCR). RESULTS: Aza treatment inhibited molting of first-instar (L1) larvae to L2, with only 10% of larvae molting compared to 95% in the control group. Serpent and Eip74EF, attacin, defensin 1, and defensin 2 genes were downregulated by Aza treatment in larvae. Similarly, Aza-treated adult females also presented suppression of ecdysone signaling-related genes and the AMPs attacin and defensin 2. Notably, all gene repression caused by Aza was reversed by adding ecdysone concomitantly with Aza to the larval or female food, indicating that these genes are effective markers for ecdysone repression. CONCLUSIONS: These results highlight the critical role of ecdysone in regulating the development and immunity of P. perniciosus, which potentially could interfere with Leishmania infection.
- Klíčová slova
- Phlebotomus perniciosus, Antimicrobial peptides, Azadirachtin, Ecdysone,
- MeSH
- antimikrobiální peptidy genetika farmakologie MeSH
- ekdyson * MeSH
- hmyz - vektory účinky léků genetika parazitologie imunologie MeSH
- hmyzí proteiny genetika metabolismus MeSH
- larva * účinky léků imunologie genetika MeSH
- limoniny * farmakologie MeSH
- Phlebotomus * účinky léků genetika parazitologie imunologie MeSH
- shazování tělního pokryvu účinky léků MeSH
- signální transdukce * účinky léků MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antimikrobiální peptidy MeSH
- azadirachtin MeSH Prohlížeč
- ekdyson * MeSH
- hmyzí proteiny MeSH
- limoniny * MeSH
We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.
- Klíčová slova
- Lutzomyia, Phlebotomus, Endogenous control gene, Gene expression, Gene stability, Reference gene,
- MeSH
- esenciální geny * MeSH
- hmyz - vektory genetika MeSH
- hmyzí geny MeSH
- larva genetika MeSH
- Leishmania genetika MeSH
- Phlebotomus * genetika MeSH
- Psychodidae genetika MeSH
- stanovení celkové genové exprese metody MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.
- MeSH
- dánio pruhované * genetika růst a vývoj metabolismus MeSH
- larva * účinky léků genetika metabolismus růst a vývoj MeSH
- metylace DNA * účinky léků MeSH
- organofosfáty * toxicita MeSH
- retardanty hoření toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- organofosfáty * MeSH
- retardanty hoření MeSH
- triphenyl phosphate MeSH Prohlížeč
The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.
- Klíčová slova
- Fibrohexamerin-like protein, Lepidoptera, Silk, Silk antibacterial proteins, Silk kinase, Silk laccase,
- MeSH
- hedvábí * metabolismus genetika MeSH
- hmyzí proteiny * metabolismus genetika MeSH
- larva metabolismus genetika růst a vývoj MeSH
- můry * genetika metabolismus MeSH
- proteomika MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hedvábí * MeSH
- hmyzí proteiny * MeSH
Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.
- Klíčová slova
- Tenebrio molitor, digestion, expression patterns, phylogenetic analysis, polypeptidases, serine peptidase homologs, serine peptidases,
- MeSH
- fylogeneze MeSH
- hmyzí proteiny * genetika metabolismus MeSH
- larva genetika růst a vývoj MeSH
- sekvence aminokyselin MeSH
- serinové proteasy genetika metabolismus MeSH
- stanovení celkové genové exprese * MeSH
- Tenebrio * genetika enzymologie MeSH
- transkriptom * MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hmyzí proteiny * MeSH
- serinové proteasy MeSH
One of the fundamental questions in insect evolution is the origin of their wings and primary function of ancestral wing precursors. Recent phylogenomic and comparative morphological studies broadly support a terrestrial ancestor of pterygotes, but an aquatic or semiaquatic ancestor cannot be ruled out. Here new features of the branchial system of palaeodictyopteran larvae of several different instars of Katosaxoniapteron brauneri gen. et sp. nov. (Eugereonoidea) from the late Carboniferous collected at Piesberg (Germany) are described, which consist of delicate dorsolateral and lamellate caudal abdominal gills that support an aquatic or at least semiaquatic lifestyle for these insects. Moreover, the similar form and surface microstructures on the lateral abdominal outgrowths and thoracic wing pads indicate that paired serial outgrowths on segments of both tagmata presumably functioned as ancestral type of gills resembling a protopterygote model. This is consistent with the hypothesis that the wing sheaths of later stage damselfly larvae in hypoxic conditions have a respiratory role similar to abdominal tracheal gills. Hence, the primary function and driving force for the evolution of the precursors of wing pads and their abdominal homologues could be respiration.
- MeSH
- biologická evoluce * MeSH
- fylogeneze MeSH
- hmyz genetika MeSH
- křídla zvířecí * anatomie a histologie MeSH
- larva genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A panel of X-linked microsatellite markers was newly designed using the data from a previous sequencing project available in NCBI and used for a study of the Colorado potato beetle (CPB, Leptinotarsa decemlineata) X-haplotype variability. The analysis of scaffolds 49 and 61 (newly identified as fragments of CPB chromosome X) found ten high-quality markers, which were arranged in two PCR multiplexes and evaluated in both 420 CPB adults, collected from 14 localities of Czechia and Slovakia, and 866 larvae from five single-female families from two more Czech localities. Length polymorphisms found in 6 loci have predicted 192 potential X-haplotypes, however, only 36 combinations were detected in the adult males (N = 189), and seven additional ones in the larvae. The X-haplotypes were also generally unevenly distributed; five of the most frequent haplotypes were detected in 55% of males, 19 repeating up to ten-times in 38.7% of males and the remained 12 occurred uniquely in 6.3% of males. Bulk analysis of X-haplotypes dissimilarity indicated seven haplotype groups diversified by mutations and recombinations. Two haplotypes showed a distinctive regional distribution, which indicates an east-west disruption of CPB migration probably caused by different environments of localities in the South Bohemia region and Vysocina region. On the contrary, the results indicate a south-north migration corridor alongside the Vltava River. In the single-female families, from 6 to 13 distinct paternal haplotypes were detected, which proved and quantified a frequented polyandry in CPB.
The morphology of the larva, pupa, male, and female of Rhyacophila loeffleri Valladolid & Waringer, n. sp., from Austria, Czech Republic, France, and Italy is described. Data from molecular analysis are also provided, based on mitochondrial cytochrome c oxidase subunit I (mtCOI), including sequences from German and Swiss specimens, and compared with those of samples of the nominate species R. fasciata Hagen 1859, as well as with other species and subspecies in the Rhyacophila fasciata Species Complex: R. septentrionis McLachlan 1865, R. denticulata McLachlan 1879, R. sociata Navs 1916, R. kykladica Malicky & Sipahiler 1993, R. delici Kuini & Valladolid 2020, R. fasciata viteceki Valladolid & Kuini 2020 and R. macedonica Karaouzas, Valladolid & Ibrahimi 2022. Our study revealed morphological differences between R. fasciata and R. loeffleri, as well as genetic differences among the other taxa of the R. fasciata Complex, supporting the recognition of R. loeffleri as a new species. Also, Rhyacophila fasciata viteceki is recognized as a distinct species, R. viteceki (status promotus), based on results of the molecular analysis.
- MeSH
- hmyz s proměnou dokonalou * MeSH
- hmyz * MeSH
- larva genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa MeSH
Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.
- Klíčová slova
- AFB spread, American foulbrood, Epidemiology, Genotyping, Honeybee,
- MeSH
- genomika MeSH
- genotyp MeSH
- larva genetika mikrobiologie MeSH
- lidé MeSH
- multilokusová sekvenční typizace veterinární MeSH
- Paenibacillus larvae * genetika MeSH
- včely MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Slovenská republika epidemiologie MeSH
- Spojené státy americké MeSH
Genetically engineered (GE) maize expressing Bacillus thuringiensis Cry3Bb1 protein was developed to control Diabrotica spp. (Coleoptera: Chrysomelidae). However, Cry proteins have been reported to have effects on non-target arthropods. We therefore investigated whether the non-target pest Tetranychus urticae (Acari: Tetranychidae) was negatively affected by GE maize expressing the insecticidal Cry3Bb1 protein. Five treatments were used in the laboratory tests to determine the life history parameters of T. urticae on leaves of field-grown maize varieties: (1) GE maize MON 88017, (2) isogenic maize, (3) isogenic maize protected with the soil applied insecticide chlorpyrifos (Dursban 10G), and two unrelated varieties (4) Kipous and (5) PR38N86. Newly emerged T. urticae larvae were individually released on the upper surface of leaf discs placed on water saturated cotton wool. Immatures and adults survival, duration of developmental stages and female fecundity were recorded daily until T. urticae died. Age-stage, two-sex life table method and test for trends, revealed no significant differences in 13 of 18 studied parameters. The unrelated varieties Kipous and PR38N86 on one side and maize with the same genetic background, namely GE maize and isogenic maize with or without insecticide protection, on the other side, showed significant variations in male longevity, larval survival rate, preoviposition period, and fecundity. In addition to the differences between varieties, GE maize and insecticide-protected isogenic maize showed a substantial difference in age-specific fecundity, but not in the mean number of eggs laid by females. The obtained results do not indicate that consumption of Cry3Bb1 has negative effect on T. urticae and suggest that GE maize does not pose a risk with respect to the non-target mite pest T. urticae. The results may have implications for the approval and renewal of import and cultivation for GE crop in the European Union.
- Klíčová slova
- Cry3Bb1, GMO, Non-target effect, Risk assessment, Two-sex life-table, Two-spotted spider mite,
- MeSH
- brouci * genetika MeSH
- genetické inženýrství MeSH
- insekticidy * farmakologie MeSH
- kukuřice setá genetika metabolismus MeSH
- larva genetika MeSH
- ovum MeSH
- Tetranychidae * genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- insekticidy * MeSH
