Silencing of the DNA methyltransferase 1 associated protein 1 (DMAP1) gene in the invasive ladybird Harmonia axyridis implies a role of the DNA methyltransferase 1-DMAP1 complex in female fecundity.

The invasive harlequin ladybird Harmonia axyridis is a textbook example of polymorphism and polyphenism as the temperature during egg development determines the frequency of melanic morphs and the number and size of black spots in nonmelanic morphs. Recent concepts in evolutionary biology suggest that epigenetic mechanisms can translate environmental stimuli into heritable phenotypic changes. To investigate whether epigenetic mechanisms influence the penetrance and expressivity of colour morphs in H. axyridis, we used RNA interference to silence key enzymes required for DNA methylation and histone modification. We found that neither of these epigenetic mechanisms affected the frequency of different morphs, but there was a significant impact on life-history traits such as longevity and fecundity. Strikingly, we found that silencing the gene encoding for DNA methyltransferase 1 associated protein 1 (DMAP1) severely reduced female fecundity, which correlated with an abundance of degenerated ovaries in DMAP1-knockdown female beetles. Finally, we observed significant differences in DMAP1 expression when we compared native and invasive H. axyridis populations with a biocontrol strain differing in egg-laying capacity, suggesting that the DNA methyltransferase 1-DMAP1 complex may influence the invasive performance of this ladybird.

Knockdown of genes in the Toll pathway reveals new lethal RNA interference targets for insect pest control.

RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well-established model, with a strong and robust RNAi response, which can be used as a high-throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large-scale screening, to encompass others identified using targeted knowledge-based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double-stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal-related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies.

Expression and characterization of a recombinant i-type lysozyme from the harlequin ladybird beetle Harmonia axyridis.

Lysozymes are enzymes that destroy bacterial cell walls by hydrolysing the polysaccharide component of peptidoglycan. In insects, there are two classes of lysozymes, the c-type with muramidase activity and the i-type whose prototypical members from annelids and molluscs possess both muramidase and isopeptidase activities. Many insect genes encoding c-type and i-type lysozymes have been identified during genome and transcriptome analyses, but only c-type lysozymes have been functionally characterized at the protein level. Here we produced one of five i-type lysozymes represented in the immunity-related transcriptome of the invasive harlequin ladybird beetle Harmonia axyridis as recombinant protein. This was the only one containing the serine and histidine residues that are thought to be required for isopeptidase activity. This i-type lysozyme was recombinantly expressed in the yeast Pichia pastoris, but the purified protein was inactive in both muramidase and isopeptidase assays. Transcription and immunofluorescence analysis revealed that this i-type lysozyme is produced in the fat body but is not inducible by immune challenge. These data suggest that i-type lysozymes in insects may have acquired novel and as yet undetermined functions in the course of evolution.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

RNA-sequencing analysis reveals abundant developmental stage-specific and immunity-related genes in the pollen beetle Meligethes aeneus.

The pollen beetle (Meligethes aeneus) is a major pest of oilseed rape (Brassica napus) and other cruciferous crops in Europe. Pesticide-resistant pollen beetle populations are emerging, increasing the economic impact of this species. We isolated total RNA from the larval and adult stages, the latter either naïve or immunized by injection with bacteria and yeast. High-throughput RNA sequencing (RNA-Seq) was carried out to establish a comprehensive transcriptome catalogue and to screen for developmental stage-specific and immunity-related transcripts. We assembled the transcriptome de novo by combining sequence tags from all developmental stages and treatments. Gene expression data based on normalized read counts revealed several functional gene categories that were differentially expressed between larvae and adults, particularly genes associated with digestion and detoxification that were induced in larvae, and genes associated with reproduction and environmental signalling that were induced in adults. We also identified many genes associated with microbe recognition, immunity-related signalling and defence effectors, such as antimicrobial peptides (AMPs) and lysozymes. Digital gene expression analysis revealed significant differences in the profile of AMPs expressed in larvae, naïve adults and immune-challenged adults, providing insight into the steady-state differences between developmental stages and the complex transcriptional remodelling that occurs following the induction of immunity. Our data provide insight into the adaptive mechanisms used by phytophagous insects and could lead to the development of more effective control strategies for insect pests.

More than a colour change: insect melanism, disease resistance and fecundity.

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

Sex pheromone monitoring as a versatile tool for determining presence and abundance of Cydia pomonella (Lep.: Tortricidae) in German apple orchards.

Cydia pomonella (Lep.: Tortricidae), the codling moth, is an apple, pear, quince and walnut pest with considerable impact on horticultural production systems in many parts of the world. In commercial apple production, it is responsible for a yearly damage level of 40 billion dollars. In response to the need of tight codling moth control there are several options for intervention by pest managers in commercially operated orchards. Spray and count methods have been used for decades with success, but at considerable external costs for the integrity of ecological cycles. Also, problems with pesticide residues and with resistant strains are an issue of concern. For environmental reasons, toxicological means are discounted here. Instead, flight curves based on sex pheromone trapping and monitoring are preferred means towards determining the optimal timing of interventions by biotechnical and biological control methods. Finally, ecological reasons are discussed for vastly different population levels of C. pomonella developing in closely neighboring field sections which operated under different environmental management.

Identification of immunity-related genes in the burying beetle Nicrophorus vespilloides by suppression subtractive hybridization.

Burying beetles reproduce on small vertebrate cadavers which they bury in the soil after localization through volatiles emitted from the carcass. They then chemically preserve the carcass and prepare it as a diet for the adults and their offspring. It is predicted that exposure to high loads of soil and/or carrion-associated microbes necessitates an effective immune system. In the present paper, we report experimental screening for immunity-related genes in the burying beetle Nicrophorus vespilloides using the suppression subtractive hybridization approach. A total of 1179 putative gene objects were identified in the Nicrophorus cDNA library, which was enriched for transcripts differentially expressed upon challenge with heat-inactivated bacteria. In addition to genes known to be involved in immunity-related recognition and signalling, we found transcripts encoding for antimicrobial peptides and for an array of enzymes that can be linked to immunity or to stress-induced pathways. We also determined proteins that may contribute to detoxification of toxins produced by microbial competitors. In addition, factors involved in mRNA stability determination and central components of the RNA interference machinery were identified, implying transcriptional reprogramming and potential stress-induced retrotransposon elimination. The identified candidate immune effector and stress-related genes may provide important information about the unusual ecology and evolution of the burying beetles.

Organic electrospun nanofibers as vehicles toward intelligent pheromone dispensers: characterization by laboratory investigations.

Organic nanofibers have a history of technical application in various independent fields, including medical technology, filtration technology, and applications of pharmaceuticals via inhalation into the lungs. Very recently, in a joint effort with polymer chemists, agricultural applications have been added to this list of priorities. The aim is finding novel approaches to insect control. Pheromones, dispensed in a quantifiable way, are being used here in disrupting the mating communication between male and female pest insects, e.g. the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae), where current dispenser technology does not fully meet the high expectations of growers and environmentalists with respect to longevity of constant release, self decomposition, mechanical distribution, renewability as well as sustainability of resources. The methodology of electrospinning is exhaustively covered by Greiner and Wendorff (2007), with technical details reported by Hellmann et al. (2009), Hein et al. (2011), and Hummel et al. (2010). Wind tunnel studies were run within a tunnel with adjustable laminar flow and 0.5 m/sec air velocity. Mass losses of the electrospun fiber bundles were determined with a sensitive analytical balance 2-3 times per week and recorded as time vs. mass change. CLSA experiments were performed with a self developed glass apparatus (Lindner, 2010) based on various suggestions of previous authors. Microgram quantities of volatile pheromone (E,Z)-7,9-Dodecadienylacetate were absorbed on a filter of rigorously purified charcoal and desorbed by repeated micro extraction with a suitable solvent mixture. Aliquots of the solution were subjected to temperature programmed capillary GLC. Retention times were used for identification, whereas the area covered by the pheromone peak originating from a FID detector signal was integrated and compared with a carefully calibrated standard peak. Since these signals were usually in the low nanogram range, several replications were averaged for statistical improvement. - Thermogravimetric analysis between ambient temperature and 500 degrees C provided a series of degradation curves where the diagram contained information on the evaporation of pheromone alone, polymer fiber alone and pheromone included in the fiber.- Microscopic investigations resulted in pictures of nanofibers from which the overall morphology and the fiber dimensions could be quantified. Organic nanofibers loaded with the grapevine moth pheromone have been well characterized by 5 different lab methods, followed by field bioassays reported elsewhere in these communications volumes (HUMMEL et al., 2011). This comprehensive analytical approach to fiber characterization is new and will be further refined. The federal agency JKI Berlin subjected the pheromone loaded organic fibers to various independent toxicological and ecotoxicological tests and found no adverse side effects.

Septic injury-inducible genes in medicinal maggots of the green blow fly Lucilia sericata.

Lucilia sericata maggots are used world-wide in biosurgery for the medical treatment of nonhealing wounds because they ingest necrotic tissues and significantly promote healing. To gain further insight into interdependencies between ecological adaptation and molecular evolution of innate immunity in Diptera, we used the suppression subtractive hybridization method to screen for genes that are differentially expressed in response to septic wounding of sterile second instar larvae of L. sericata. This approach resulted in the identification of 65 novel Lucilia genes including potential signalling proteins (e.g. inhibitor of apoptosis 2 protein) and a number of digestive enzymes including lipases and proteinases. Additionally, we found numerous putative antimicrobial peptides (AMPs), such as a potential Lucilia defensin, diptericin and three novel proline-rich AMPs. The identified genes may facilitate access to both peptides and proteins within the beneficial excretions, secretions and haemolymph of medicinal maggots and provide novel insights into the evolution of innate immunity in Diptera.

Wounding-mediated gene expression and accelerated viviparous reproduction of the pea aphid Acyrthosiphon pisum.

Most insects mount a potent antimicrobial defence upon contact with microbes or microbe-associated pattern molecules. Using a combined set of methods for analysis of insect innate immunity, we report here that piercing of the pea aphid Acyrthosiphon pisum with a bacteria-contaminated needle elicits lysozyme-like activity in the haemolymph but no detectable activities against live bacteria. Confirming these results, we found no homologues of known antimicrobial peptides in our cDNA library generated by using the suppression subtractive hybridization method or in over 90,000 public expressed sequence tag (EST) sequences, but lysozyme genes have recently been described in A. pisum. Interestingly, we discovered that production of viviparous offspring was significantly accelerated upon wounding. Therefore, we postulate that aphids may increase terminal reproductive investment and limit antibacterial defence in response to a threat to their survival.

A single amphioxus and sea urchin runt-gene suggests that runt-gene duplications occurred in early chordate evolution.

Runt-homologous molecules are characterized by their DNA binding runt-domain which is highly conserved within bilaterians. The three mammalian runt-genes are master regulators in cartilage/bone formation and hematopoiesis. Historically these features evolved in Craniota and might have been promoted by runt-gene duplication events. The purpose of this study was therefore to investigate how many runt-genes exist in the stem species of chordates, by analyzing the number of runt-genes in what is likely to be the closest living relative of Craniota-amphioxus. To acquire further insight into the possible role of runt-genes in early chordate evolution we have determined the number of runt-genes in sea urchins and have analyzed the runt-expression pattern in this species. Our findings demonstrate the presence of a single runt-gene in amphioxus and sea urchin, which makes it highly likely that the stem species of chordates harbored only a single runt-gene. This suggests that runt-gene duplications occurred later in chordate phylogeny, and are possibly also associated with the evolution of features such as hematopoiesis, cartilage and bone development. In sea urchin embryos runt-expression involves cells of endodermal, mesodermal and ectodermal origin. This complex pattern of expression might reflect the multiple roles played by runt-genes in mammals. A strong runt-signal in the gastrointestinal tract of the sea urchin is in line with runt-expression in the intestine of nematodes and in the murine gastrointestinal tract, and seems to be one of the phylogenetically ancient runt-expression domains.

Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach.

In this study we have analyzed bacterial lipopolysaccharide (LPS) induced genes in hemocytes of the Lepidopteran species Galleria mellonella using subtractive hybridization, followed by suppressive PCR. We have found genes that show homologies to molecules, such as gloverin, peptidoglycan recognition proteins and transferrin known to be involved in immunomodulation after bacterial infection in other species. In addition, a few molecules previously not described in the innate immune reactions were detected, such as a RNA binding molecule and tyrosine hydroxylase. Furthermore, the full-length cDNA of a LPS-induced molecule with six toxin-2-like domains is described to be a promising candidate to further elucidate the relationship between toxin- and defensin-like domains in arthropod host defense.

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses.

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.

Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.

Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella.

The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 micrograms per larvae caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.

Antimicrobial activity of exocrine glandular secretions, hemolymph, and larval regurgitate of the mustard leaf beetle phaedon cochleariae

Larvae and adults of the mustard leaf beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) possess exocrine glands with secretions which are used in defense against predators. This study addressed the question whether these defensive secretions also display antimicrobial activity. Additionally, the effects of larval and adult hemolymph and larval regurgitate toward microorganisms were examined. The larval glandular secretion showed growth-inhibitory activity against the gram-negative bacterium Escherichia coli, but no lytic effect against cell walls of the gram-positive bacterium Micrococcus luteus. Growth of the yeast Saccharomyces cerevisiae and of the entomopathogenic fungus Beauveria bassiana was also inhibited by the larval secretion. The antibacterial and antifungal activity of the larval secretion was found to be due to its main component, the iridoid monoterpene, (epi)chrysomelidial. The mechanism of its antifungal activity was examined by different bioassays and compared to the commercially available fungicide nystatin. The antifungal activity of the larval secretion is not due to a loss of intracellular potassium in treated fungal cells, while high potassium efflux from treated cells is the mode of action of common fungicides. The larval secretion exhibited direct cytotoxicity against both fungal cells and plasmatocytes isolated from the greater wax moth Galleria mellonella. In contrast to the larval secretion, the adult glandular secretion of P. cochleariae showed lytic activity against the cell walls of the gram-positive bacterium M. luteus, but no activity against the gram-negative bacterium E. coli and eukaryotic cells. Hemolymph and larval regurgitate of P. cochleariae displayed the same activity as the tested glandular secretion of adults. Copyright 1998 Academic Press.

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella.

In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor.

Detection of a P-glycoprotein related pump in Chironomus larvae and its inhibition by verapamil and cyclosporin A.

A membrane associated ATP-dependent efflux pump, similar in function to mammalian P-glycoprotein, was detected in anal papillae of Chironomus riparius larvae. Immunohistochemical analysis of larval tissues, using monoclonal antibodies against P-glycoprotein, was supplemented by functional in vivo and in vitro assays which confirmed the existence of a mechanism for transporting xenobiotic substances. The in vitro ATPase activity of homogenate fractions increased in the presence of typical P-glycoprotein substrates (vinblastine, actinomycin D or ivermectin). This increase was unaffected by inhibitors of other membrane ATPases (sodium azide, EGTA, ouabain), but sensitive to vanadate, cyclosporin A and verapamil which inhibit mammalian P-glycoprotein mediated ATP-consumption. Sublethal concentrations of specific P-glycoprotein-inhibitors such as verapamil or cyclosporin A synergistically enhanced the mortality of C. riparius towards ivermectin. Although cyclosporin A originates from entomopathogenic fungi, its mode of action in insects and its function during infection are not understood. Our results lend some credit to the hypothesis that this compound is possibly released to promote poisoning of the infected host by xenobiotics which are normally removed by a P-glycoprotein related pump. The putative role of insect P-glycoprotein homologues in the context of multiple resistance towards insecticides in discussed.