Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for... more Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers and are produced in the middle part of the silk gland. So far, five genes encoding sericin proteins have been found in Bombyx mori. Sericins 1 and 3 are responsible for silk adhesion in the cocoon, while sericins 2, 4, and 5 are present in non-cocoon spun silk of younger larvae (including the early last instar). We found a new gene, which we named P150/sericin 6, which appears to be an ortholog of the sericin-like protein previously found in Galleria mellonella. The B. mori sequence of the P150/sericin 6 ORF was previously incorrectly predicted and assigned to two smaller, uncharacterized genes. We present a new P150/sericin 6 gene model and show that it encodes a large protein of 467 kDa. It is characterized by repeats with a high proportion of threonine residues and a short conserved region with a cysteine knot motif (CXCXCX) at the C-terminus. Expression analyzes have sho...
Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidop... more Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.
bioRxiv (Cold Spring Harbor Laboratory), Jan 6, 2020
Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mamma... more Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. In the present study, we observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). We altered Ado signaling using genetic tools and found that the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs), were able to minimize mHTT-induced mortality. We also identified the downstream targets of the AdoR pathway, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, we showed that a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. Our study provides important insights into how Ado regulates stress responses in Drosophila.
Developmental profiles of total RNA and four silk protein mRNAs exhibit different correlations wi... more Developmental profiles of total RNA and four silk protein mRNAs exhibit different correlations with ecdysteroid titre changes in course of the 180 hr-long last larval instar. Total RNA and heavy-chain fibroin mRNA increase gradually until 168 hr and 156 hr, respectively, dropping sharply afterwards. The content of sericin1 mRNA rises for 120 hr, remains high until 168 hr and then drops. The amounts of ligh-chain fibroin mRNA and sericin2 mRNA increase sharply at the start of the instar; the former rises until 168 hr, whereas the latter begins to decline already at 120 hr. The pupation-inducing ecdysteroid surge at 144-168 hr causes a drop in all examined RNA classes. Similar drop is induced precociously with exogenous ecdysterone, while a slow decrease of RNA contents to similar bottom levels occurs when the larvae are ligated behind prothorax.
Many Drosophila genes exist as members of multigene families and within each family the members c... more Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.
Entomopathogenic nematodes and their associated bacteria comprise together a highly pathogenic co... more Entomopathogenic nematodes and their associated bacteria comprise together a highly pathogenic complex which is able to invade and kill insect host within two days. Both bacteria and nematodes produce a variety of factors interacting with the insect immune system and help to overcome host defences. The tripartite model (Drosophila, nematodes, bacteria) was established and used to show an immune function for candidate genes using different Drosophila mutants or RNAi lines with defects in clotting or other branches of the immune system. Microarray analysis was used to compare gene expression of Drosophila larvae infected by the entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens with non-infected larvae. The role of candidate genes, selected based on genomic comparison, in response towards nematobacterial complex was further evaluated by in vivo infection assays. We demonstrated an immune function during nematode infection for known clotting enzymes and substrates, recognition molecules and eicosanoids. In conclusion, we show that the Heterorhabditis/Photorhabdus infection model is suitable to identify regulators of innate immunity in insects. Our research is supported by research grants from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), by grant from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047) and The Technology Agency of the Czech Republic (TA04020318).
Entomopathogenic nematodes and their associated bacteria comprise together highly pathogenic comp... more Entomopathogenic nematodes and their associated bacteria comprise together highly pathogenic complex able to invade and kill insect host within two days. Both bacteria and nematodes produce variety of factors interacting with insect immune system that help to overcome host defences. The tripartite model (Drosophila, nematodes, bacteria) was established and used to show an immune function for candidate genes using different Drosophila mutants or RNAi lines with defects in clotting or other branches of the immune system. We demonstrated an immune function during nematode infection for known clotting enzymes and substrates, recognition molecules, eicosanoids etc. In conclusion, we show that the Heterorhabditis/Photorhabdus infection model is suitable to identify novel regulators of innate immunity and we bring evidence of coagulation immune function in insects against nematode infection.
Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are obligate and lethal insect par... more Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are obligate and lethal insect parasites. These EPN are symbiotically associated with entomopathogenic bacteria Photorhabdus luminescens creating the highly pathogenic nematobacterial complex that is able to kill the host within 24 to 48 hours. H. bacteriophora with its bacterial symbionts are able to infect a broad spectrum of insect species. Symbiotic bacteria help to digest host tissues and provide nutrients for themselves and developing nematodes. Drosophila larvae are suitable insect hosts and part of the tripartite model system (Drosophila – EPN - bacteria). In this study we examined transcriptional changes in Drosophila upon nematode infection. We compare gene expression in non- infected and infected fly larvae. We focused on the early time point of nematode infection and therefore infected Drosophila larvae using H. bacteriophora harbouring GFP-labelled P. luminescens bacteria. Infected (GFP positive) larvae were collected 6 hours after infection. We found 642 genes significantly differentially regulated after nematobacterial infection caused by H. bacteriophora and P. luminescens. The majority of them are upregulated upon infection. The most strongly induced fraction comprise antimicrobial humoral response genes and defense response genes. However we observed also a lot of transcripts involved in development, morphogenesis and cell differentiation. Based on Gene Ontology annotation we identified several pathways, which could be involved in sealing and repairing the wound caused by invading nematodes. We compared our results with the available data for other infection types caused by bacteria (both nonpathogenic and pathogenic) and parasitic wasps. A core set of 17 genes was common for all types of infection and approximately 10-25% of genes were overlapping in each pairwise comparison.
Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis are obligate and lethal insect pa... more Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis are obligate and lethal insect parasites. In recent years they have been used increasingly as biological control agents. These EPNs are symbiotically associated with bacteria of the genera Photorhabdus. The bacterial symbionts are essential to kill the host (within 24-48 hours) and digest its tissues to provide nutrients for themselves as well for expanding nematodes. Drosophila larvae are suitable insect hosts and part of the tripartite model system we used before to show the importance of haemolymph clotting and eicosanoids during the infection. We used the well-established tripartite model (Drosophila, nematodes, bacteria), DNA chips and bioinformatic tools to compare gene expression in non-infected and infected fly larvae. We focused on the early time point of nematode infection and therefore infected Drosophila larvae using H. bacteriophora harbouring GFP-labelled P. luminescens bacteria. Infected (GFP positive) larvae were collected 6 hours after infection. We detected approximately 650 genes whose expression was significantly influenced by nematobacterial infection caused by H. bacteriophora and P. luminescens. Most of them are upregulated upon infection including mainly the genes involved in antimicrobial response and development. Based on Gene Ontology annotation we identified several pathways, which could be involved in sealing and repairing the wound caused by invading nematodes. These results we compared with available data for other types infection caused by bacteria and parasitic wasps. Small group of genes were common for all three types of infection and approximately 25 genes were overlapping in each pairwise comparison. We focused on the genes expressed in the hemocytes and fat body, respectively and we subjected selected candidate genes to functional tests. We tested the effect of mutations or knockdown of selected genes for the susceptibility of flies to the nematobacterial infection. The overlap between the protective genes and genes induced by the nematobacterial infection was not complete. Therefore, we assume that only a fraction of the genes involved in the protection of infected larvae from death are induced by the nematobacterial infection. Our research is supported by research grants from the Swedish Research Council (VR-NT 2010-5118), the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) and by grant from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047).
Many lepidopteran larvae produce silk secretions to build feeding tubes and cocoons that play imp... more 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.
Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are parasitic worms, which are ass... more Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are parasitic worms, which are associated with their gut symbiotic bacteria Photorhabdus luminescens. They are generally lethal to insects and used as biocontrol agents. Nematodes can enter to the host via the mouth, anus, spiracles or penetrating through the cuticle. Once they entered into host, they release their gut bacteria and then work in concert with the bacteria to kill the host. Nematode infection in Drosophila is a natural infection, in contrast to many kinds of bacterial infection (through injection). Drosophila lacks an adaptive immune system of the vertebrate style and therefore relies on innate immunity to combat infection against different invaders like bacteria, fungi, viruses and other parasites. Until now, our knowledge on Drosophila immunity mostly comes from studies of bacterial and fungal infections. To obtain a complete picture of Drosophila response to EPN, we performed a genome-wide transcriptional analysis of the Drosophila larvae (approximately 88 hour after egg laying) infected by EPN. We compared the transcriptome profile between infected and non-infected larvae. A total of 642 transcripts were found significantly differentially regulated upon nematode infection. Gene ontology (GO) analysis of the top 100 most significantly upregulated genes identified one-quarter immune-related genes. To test their contribution to the response against nematodes, we used RNAi lines and mutants for candidate genes identified in the arrays and examined the viability of such larvae upon EPN infection. As a criterion for selecting candidate defense genes from the list of induced transcripts we focused on those for which a function in immunity had been established in other infection models, as well as on their paralogs, which had not yet been implicated in immune reactions.
Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired g... more Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G 1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments.
The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived... more The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived from a gene that includes eight exons and spans over 7100 nucleotides (nt). By means of two transcription starts, the gene generates two mRNA isoforms that are present in similar amounts in the larvae, pupae and adults. The A isoform includes exon IA of 115 nt, which is followed by intron 1a extending to position 3457 of the gene. The B mRNA isoform begins with exon IB (100 nt) that occupies positions 3291-3390 within the 1a intron. Exons II to VII are included in both mRNA isoforms. The gene contains 31.6% (36.5% in the translated region) of the GC nucleotides. Two transcription starts and the exon/intron organization distinguish bark beetle G6PD from the homologous genes known in other insects. Two enzyme variants were detected in the protein extracts of individual bark beetles but their relationship to the A and B mRNA isoforms is uncertain.
Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from pred... more Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from predators and parasites for caterpillars and pupae. Yet, the number of lepidopteran species whose silk composition has been studied in detail is very small, because the genes encoding the major structural silk proteins tend to be large and repetitive, making their assembly and sequence analysis difficult. Here we have analyzed the silk of Yponomeuta cagnagella, which represents one of the early diverging lineages of the ditrysian Lepidoptera thus improving the coverage of the order. To obtain a comprehensive list of the Y. cagnagella silk genes, we sequenced and assembled a draft genome using Oxford Nanopore and Illumina technologies. We used a silk-gland transcriptome and a silk proteome to identify major silk components and verified the tissue specificity of expression of individual genes. A detailed annotation of the major genes and their putative products, including their complete sequen...
Seroins are small lepidopteran silk proteins known to possess antimicrobial activities. Several s... more Seroins are small lepidopteran silk proteins known to possess antimicrobial activities. Several seroin paralogs and isoforms were identified in studied lepidopteran species and their classification required detailed phylogenetic analysis based on complete and verified cDNA sequences. We sequenced silk gland-specific cDNA libraries from ten species and identified 52 novel seroin cDNAs. The results of this targeted research, combined with data retrieved from available databases, form a dataset representing the major clades of Lepidoptera. The analysis of deduced seroin proteins distinguished three seroin classes (sn1-sn3), which are composed of modules: A (includes the signal peptide), B (rich in charged amino acids) and C (highly variable linker containing proline). The similarities within and between the classes were 31–50% and 22.5–25%, respectively. All species express one, and in exceptional cases two, genes per class, and alternative splicing further enhances seroin diversity. S...
Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for... more Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers and are produced in the middle part of the silk gland. So far, five genes encoding sericin proteins have been found in Bombyx mori. Sericins 1 and 3 are responsible for silk adhesion in the cocoon, while sericins 2, 4, and 5 are present in non-cocoon spun silk of younger larvae (including the early last instar). We found a new gene, which we named P150/sericin 6, which appears to be an ortholog of the sericin-like protein previously found in Galleria mellonella. The B. mori sequence of the P150/sericin 6 ORF was previously incorrectly predicted and assigned to two smaller, uncharacterized genes. We present a new P150/sericin 6 gene model and show that it encodes a large protein of 467 kDa. It is characterized by repeats with a high proportion of threonine residues and a short conserved region with a cysteine knot motif (CXCXCX) at the C-terminus. Expression analyzes have sho...
Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidop... more Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.
bioRxiv (Cold Spring Harbor Laboratory), Jan 6, 2020
Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mamma... more Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. In the present study, we observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). We altered Ado signaling using genetic tools and found that the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs), were able to minimize mHTT-induced mortality. We also identified the downstream targets of the AdoR pathway, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, we showed that a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. Our study provides important insights into how Ado regulates stress responses in Drosophila.
Developmental profiles of total RNA and four silk protein mRNAs exhibit different correlations wi... more Developmental profiles of total RNA and four silk protein mRNAs exhibit different correlations with ecdysteroid titre changes in course of the 180 hr-long last larval instar. Total RNA and heavy-chain fibroin mRNA increase gradually until 168 hr and 156 hr, respectively, dropping sharply afterwards. The content of sericin1 mRNA rises for 120 hr, remains high until 168 hr and then drops. The amounts of ligh-chain fibroin mRNA and sericin2 mRNA increase sharply at the start of the instar; the former rises until 168 hr, whereas the latter begins to decline already at 120 hr. The pupation-inducing ecdysteroid surge at 144-168 hr causes a drop in all examined RNA classes. Similar drop is induced precociously with exogenous ecdysterone, while a slow decrease of RNA contents to similar bottom levels occurs when the larvae are ligated behind prothorax.
Many Drosophila genes exist as members of multigene families and within each family the members c... more Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.
Entomopathogenic nematodes and their associated bacteria comprise together a highly pathogenic co... more Entomopathogenic nematodes and their associated bacteria comprise together a highly pathogenic complex which is able to invade and kill insect host within two days. Both bacteria and nematodes produce a variety of factors interacting with the insect immune system and help to overcome host defences. The tripartite model (Drosophila, nematodes, bacteria) was established and used to show an immune function for candidate genes using different Drosophila mutants or RNAi lines with defects in clotting or other branches of the immune system. Microarray analysis was used to compare gene expression of Drosophila larvae infected by the entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens with non-infected larvae. The role of candidate genes, selected based on genomic comparison, in response towards nematobacterial complex was further evaluated by in vivo infection assays. We demonstrated an immune function during nematode infection for known clotting enzymes and substrates, recognition molecules and eicosanoids. In conclusion, we show that the Heterorhabditis/Photorhabdus infection model is suitable to identify regulators of innate immunity in insects. Our research is supported by research grants from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), by grant from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047) and The Technology Agency of the Czech Republic (TA04020318).
Entomopathogenic nematodes and their associated bacteria comprise together highly pathogenic comp... more Entomopathogenic nematodes and their associated bacteria comprise together highly pathogenic complex able to invade and kill insect host within two days. Both bacteria and nematodes produce variety of factors interacting with insect immune system that help to overcome host defences. The tripartite model (Drosophila, nematodes, bacteria) was established and used to show an immune function for candidate genes using different Drosophila mutants or RNAi lines with defects in clotting or other branches of the immune system. We demonstrated an immune function during nematode infection for known clotting enzymes and substrates, recognition molecules, eicosanoids etc. In conclusion, we show that the Heterorhabditis/Photorhabdus infection model is suitable to identify novel regulators of innate immunity and we bring evidence of coagulation immune function in insects against nematode infection.
Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are obligate and lethal insect par... more Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are obligate and lethal insect parasites. These EPN are symbiotically associated with entomopathogenic bacteria Photorhabdus luminescens creating the highly pathogenic nematobacterial complex that is able to kill the host within 24 to 48 hours. H. bacteriophora with its bacterial symbionts are able to infect a broad spectrum of insect species. Symbiotic bacteria help to digest host tissues and provide nutrients for themselves and developing nematodes. Drosophila larvae are suitable insect hosts and part of the tripartite model system (Drosophila – EPN - bacteria). In this study we examined transcriptional changes in Drosophila upon nematode infection. We compare gene expression in non- infected and infected fly larvae. We focused on the early time point of nematode infection and therefore infected Drosophila larvae using H. bacteriophora harbouring GFP-labelled P. luminescens bacteria. Infected (GFP positive) larvae were collected 6 hours after infection. We found 642 genes significantly differentially regulated after nematobacterial infection caused by H. bacteriophora and P. luminescens. The majority of them are upregulated upon infection. The most strongly induced fraction comprise antimicrobial humoral response genes and defense response genes. However we observed also a lot of transcripts involved in development, morphogenesis and cell differentiation. Based on Gene Ontology annotation we identified several pathways, which could be involved in sealing and repairing the wound caused by invading nematodes. We compared our results with the available data for other infection types caused by bacteria (both nonpathogenic and pathogenic) and parasitic wasps. A core set of 17 genes was common for all types of infection and approximately 10-25% of genes were overlapping in each pairwise comparison.
Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis are obligate and lethal insect pa... more Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis are obligate and lethal insect parasites. In recent years they have been used increasingly as biological control agents. These EPNs are symbiotically associated with bacteria of the genera Photorhabdus. The bacterial symbionts are essential to kill the host (within 24-48 hours) and digest its tissues to provide nutrients for themselves as well for expanding nematodes. Drosophila larvae are suitable insect hosts and part of the tripartite model system we used before to show the importance of haemolymph clotting and eicosanoids during the infection. We used the well-established tripartite model (Drosophila, nematodes, bacteria), DNA chips and bioinformatic tools to compare gene expression in non-infected and infected fly larvae. We focused on the early time point of nematode infection and therefore infected Drosophila larvae using H. bacteriophora harbouring GFP-labelled P. luminescens bacteria. Infected (GFP positive) larvae were collected 6 hours after infection. We detected approximately 650 genes whose expression was significantly influenced by nematobacterial infection caused by H. bacteriophora and P. luminescens. Most of them are upregulated upon infection including mainly the genes involved in antimicrobial response and development. Based on Gene Ontology annotation we identified several pathways, which could be involved in sealing and repairing the wound caused by invading nematodes. These results we compared with available data for other types infection caused by bacteria and parasitic wasps. Small group of genes were common for all three types of infection and approximately 25 genes were overlapping in each pairwise comparison. We focused on the genes expressed in the hemocytes and fat body, respectively and we subjected selected candidate genes to functional tests. We tested the effect of mutations or knockdown of selected genes for the susceptibility of flies to the nematobacterial infection. The overlap between the protective genes and genes induced by the nematobacterial infection was not complete. Therefore, we assume that only a fraction of the genes involved in the protection of infected larvae from death are induced by the nematobacterial infection. Our research is supported by research grants from the Swedish Research Council (VR-NT 2010-5118), the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) and by grant from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047).
Many lepidopteran larvae produce silk secretions to build feeding tubes and cocoons that play imp... more 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.
Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are parasitic worms, which are ass... more Entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora are parasitic worms, which are associated with their gut symbiotic bacteria Photorhabdus luminescens. They are generally lethal to insects and used as biocontrol agents. Nematodes can enter to the host via the mouth, anus, spiracles or penetrating through the cuticle. Once they entered into host, they release their gut bacteria and then work in concert with the bacteria to kill the host. Nematode infection in Drosophila is a natural infection, in contrast to many kinds of bacterial infection (through injection). Drosophila lacks an adaptive immune system of the vertebrate style and therefore relies on innate immunity to combat infection against different invaders like bacteria, fungi, viruses and other parasites. Until now, our knowledge on Drosophila immunity mostly comes from studies of bacterial and fungal infections. To obtain a complete picture of Drosophila response to EPN, we performed a genome-wide transcriptional analysis of the Drosophila larvae (approximately 88 hour after egg laying) infected by EPN. We compared the transcriptome profile between infected and non-infected larvae. A total of 642 transcripts were found significantly differentially regulated upon nematode infection. Gene ontology (GO) analysis of the top 100 most significantly upregulated genes identified one-quarter immune-related genes. To test their contribution to the response against nematodes, we used RNAi lines and mutants for candidate genes identified in the arrays and examined the viability of such larvae upon EPN infection. As a criterion for selecting candidate defense genes from the list of induced transcripts we focused on those for which a function in immunity had been established in other infection models, as well as on their paralogs, which had not yet been implicated in immune reactions.
Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired g... more Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G 1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments.
The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived... more The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived from a gene that includes eight exons and spans over 7100 nucleotides (nt). By means of two transcription starts, the gene generates two mRNA isoforms that are present in similar amounts in the larvae, pupae and adults. The A isoform includes exon IA of 115 nt, which is followed by intron 1a extending to position 3457 of the gene. The B mRNA isoform begins with exon IB (100 nt) that occupies positions 3291-3390 within the 1a intron. Exons II to VII are included in both mRNA isoforms. The gene contains 31.6% (36.5% in the translated region) of the GC nucleotides. Two transcription starts and the exon/intron organization distinguish bark beetle G6PD from the homologous genes known in other insects. Two enzyme variants were detected in the protein extracts of individual bark beetles but their relationship to the A and B mRNA isoforms is uncertain.
Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from pred... more Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from predators and parasites for caterpillars and pupae. Yet, the number of lepidopteran species whose silk composition has been studied in detail is very small, because the genes encoding the major structural silk proteins tend to be large and repetitive, making their assembly and sequence analysis difficult. Here we have analyzed the silk of Yponomeuta cagnagella, which represents one of the early diverging lineages of the ditrysian Lepidoptera thus improving the coverage of the order. To obtain a comprehensive list of the Y. cagnagella silk genes, we sequenced and assembled a draft genome using Oxford Nanopore and Illumina technologies. We used a silk-gland transcriptome and a silk proteome to identify major silk components and verified the tissue specificity of expression of individual genes. A detailed annotation of the major genes and their putative products, including their complete sequen...
Seroins are small lepidopteran silk proteins known to possess antimicrobial activities. Several s... more Seroins are small lepidopteran silk proteins known to possess antimicrobial activities. Several seroin paralogs and isoforms were identified in studied lepidopteran species and their classification required detailed phylogenetic analysis based on complete and verified cDNA sequences. We sequenced silk gland-specific cDNA libraries from ten species and identified 52 novel seroin cDNAs. The results of this targeted research, combined with data retrieved from available databases, form a dataset representing the major clades of Lepidoptera. The analysis of deduced seroin proteins distinguished three seroin classes (sn1-sn3), which are composed of modules: A (includes the signal peptide), B (rich in charged amino acids) and C (highly variable linker containing proline). The similarities within and between the classes were 31–50% and 22.5–25%, respectively. All species express one, and in exceptional cases two, genes per class, and alternative splicing further enhances seroin diversity. S...
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Papers by Michal Zurovec