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Melanization is regulated by the prophenoloxidase cascade and functions as a response to intruding microorganisms in invertebrates. When injecting dsRNA of the lepidopteran immune protein hemolin in pupae of Hyalophora cecropia (Lepidoptera: Saturniidae), we observed a significant reduction in phenoloxidase activity after 24 h, but not after 72 h. The link between hemolin and the prophenoloxidase system suggests that hemolin is a pattern recognition protein important for the triggering of the prophenoloxidase cascade in the defence against bacterial infections.

Background The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes. Results Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2 % of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82 % of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes. Conclusions Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0475-8) contains supplementary material, which is available to authorized users.

In this study, sugar-feeding was investigated as a possible means of re-introducing bacteria into mosquito midguts with the aim of identifying bacteria that are suitable for creating paratransgenic mosquitoes. In a paratransgenic approach, bacteria are utilised to deliver effector molecules capable of inhibiting pathogen development in the midgut of the vector. To determine if mosquitoes discriminate between sterile sugar solutions and sugar solutions with bacteria, a method for screening mosquito feeding preferences was developed. This method was tested for Aedes aegypti, Anopheles arabiensis and An. gambiae s.s. mosquitoes and is based on a dual-choice test of solutions labelled with food dyes. Three different tests (dye/colour detection, sugar detection and sugar-concentration detection) were performed to evaluate the method, after which bacteria previously isolated from mosquitoes were used in the experiments. It was shown that mosquitoes do not discriminate between sugar solutions with or without these bacteria indicating that sugar-feeding is a possible means to introduce bacteria into mosquitoes. Furthermore, two different setups of the method were used, enabling us to differentiate between tactile/taste and olfactory responses. The method described in this paper is easy to use, cost-effective and allows broad screening of mosquito sugar-feeding preferences.

The pine weevil Hylobius abietis is a severe pest of conifer seedlings in reforestation areas. Weevils lay eggs in the root bark or in the soil near roots of recently dead trees and cover the eggs with frass (feces combined with chewed bark), possibly to avoid conspecific egg predation. The aim of the present investigation focused on isolation, identification, and volatile production of fungi from pine-weevil feces and frass. Fungi were isolated from weevil frass and feces separately, followed by identification based on ITS sequencing. Fifty-nine isolates belonging to the genera Penicillium, Ophiostoma, Mucor, Leptographium, Eucasphaeria, Rhizosphaera, Debaryomyces, and Candida were identified. Volatile organic compounds (VOCs) produced by the fungal community and fungal isolates cultured on weevil-frass broth were identified by SPME-GCMS. Major VOCs emitted from the fungal community and pure isolates were species- and strain specific and included isopentylalcohol, styrene, 3-octanone, 6-protoilludene, methyl salicylate, 3-methylanisole, 2-methoxyphenol, and phenol. Some of these are known to influence the orientation of pine weevils when tested among highly attractive newly planted conifer seedlings.

Two Gram-negative, rod-shaped strains, T2.1T and W5.1.1T, isolated from larvae of the mosquito Anopheles arabiensis, were investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strains T2.1T and W5.1.1T were shown to belong to the genus Thorsellia , both showing 97.8 % similarity to the type strain of Thorsellia anophelis , with 98.1 % similarity to each other. Chemotaxonomic data supported the allocation of the strains to the genus Thorsellia : their major fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0 and they harboured a ubiquinone Q-8 quinone system and a polyamine pattern with the major compound 1,3-diaminopropane. Qualitative and quantitative differences in their polar lipid profiles distinguished strains T2.1T and W5.1.1T from each other and from T. anophelis . Average nucleotide identity (ANI), DNA–DNA hybridization, multilocus sequence analysis (MLSA) as well as physiological and biochemical tests allowed T2.1T and W5.1.1T to be distinguished both genotypically and phenotypically from each other and from the type strain of T. anophelis . Thus, we propose that these isolates represent two novel species of the genus Thorsellia , named Thorsellia kenyensis sp. nov. (type strain T2.1T = CCM 8545T = LMG 28483T = CIP 110829T) and Thorsellia kandunguensis sp. nov. (type strain W5.1.1T = LMG 28213T = CIP 110794T). Furthermore, phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the genus Thorsellia forms a separate branch, distinct from the families Enterobacteriaceae , Pasteurellaceae and Orbaceae . As a consequence, a new family Thorselliaceae fam. nov. is proposed. An emended description of Thorsellia anophelis is also provided.

We have with great interest read the paper by Tuuminen et al. ([1][1]) published in the July 2014 issue of this journal where the pine weevil (Hylobius abietis) is incriminated as a vector of the potential human pathogen Capnocytophaga canimorsus. The authors base their conclusion on the fact that

Hemolin is one of the haemolymph proteins most strongly induced upon bacterial infection in Lepidoptera. When we applied RNA interference (RNAi) to suppress Hemolin expression in the Chinese oak silk moth Antheraea pernyi, we discovered that Hemolin is induced by double-stranded RNA (dsRNA) per se. As dsRNA is recognized as a virus pattern molecule, we then investigated the effect of a baculovirus (ApNPV) infection. We found that Hemolin is induced and expressed with similar kinetics as upon dsRNA injection. Notably, no Attacin gene expression or antibacterial activity was recorded. When baculovirus and high amounts of dsRNA were coinjected, the viral symptoms appeared earlier with Hemolin dsRNA than with GFP dsRNA. This indicates that silencing of hemolin affected the progress of the viral infection.

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite-specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence. Part of urn:nbn:se:su:diva-224

There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso-ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria-inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi-females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality.

Ticks were collected from pastures and domestic and wild vertebrates during the rainy seasons of 1994 and 1996 in Guinea-Bissau. We collected the following species: from pastures Rhipicephalus lunulatus Neumann, R. muhsamae Morel & Vassiliades, R. senegalensis Koch, and R. sulcatus Neumann; from reptiles Amblyomma nuttalli Donitz and Aponomma flavomaculatum (Lucas); and from mammals Amblyomma paulopunctatum Neumann, Amblyomma splendidum Giebel, Amblyomma variegatum (F.), Hyalomma truncatum Koch, R. muhsamae, R. sanguineus (Latreille), R. senegalensis, and R. sulcatus. New host records are as follows: Amblyomma nuttalli on Senegal flap shelled turtle (Cyclanorbis senegalensis Dumeril & Bibron), Aponomma flavomaculatum on West African dwarf crocodile (Osteolaemus tetraspis Cope), and R. sulcatus on bushbuck (Tragelaphus scriptus Pallas). We reviewed the literature for earlier records of ticks from Guinea-Bissau. The regional distributions of the 21 tick species hitherto recorded in Guinea-Bissau are listed.

The increasing use of pesticides demands a world-wide control of residue levels in food and the environment. However, pesticide residue analysis is challenging and needs both experience and instrumentation. Although much can be done with fairly simple means, many countries with limited resources can only perform certain analyses. Also in technologically more advanced countries, different laboratories may specialize in certain kinds of analyses and there is also a need to confirm results with an exchange of samples between laboratories. There are usually several technical difficulties connected with the transport of pesticide samples, in addition to the risk of transmission of pests and diseases and related customs control; and in many areas of the world transport of pesticide samples is not feasible at all. A way to circumvent these difficulties is presented here. Even if a laboratory lacks the basic requirements to purchase, run and maintain instrumentation to analyze pesticide residues, there are often possibilities to extract the pesticide samples. The extracts are taken to dryness in the presence of a ‘keeper’ substance, and the residues can then be sent in apparently empty glass vials. We have investigated the use of polyethylene glycol as a keeper at harsh conditions, for up to three weeks at 44°C. This was done to find out whether the residues remain in the vials at simulated transport conditions. In all, 64 pesticides were tested and of these 56 substances give recoveries of at least 70% after storage at 44°C for 3 weeks. Thus, according to the results it would be possible to send pesticide samples as evaporated extracts even in a hot climate. In conclusion, the results presented here clearly show that a much simplified method of transfer of samples between laboratories is adequate, which enables monitoring of pesticide residues in any part of the world.

Pattern recognition receptors play an important role in insect immune defense. We cloned the β-1,3-glucan recognition protein, lectin-5 and C-type lectin 1 genes of Antheraea pernyi and examined the expression profiles of immune-stimulated pupae. After infection with Bacillus subtilis, Escherichia coli, Antheraea pernyi nuclear polyhedrosis virus (ApNPV) and Saccharomyces cerevisiae, respectively, the pupae showed different gene expression levels in the different tissues examined (midgut, fatbody, epidermis, testis, and hemocytes). ApβGRP and Aplectin-5 was induced by all the microorganisms, and mainly in epidermis and hemocytes, but not in testis; Aplectin-5 was also expressed in fatbody. Ap C-type lectin 1 was, on the contrary, highly expressed in testis and also in fatbody, but not in hemocytes. Unlike ApβGRP and Aplectin-5, Ap C-type lectin 1 was not induced by Gram-positive bacteria. The results suggest that the cloned lectins may have different functions in different tissues of A. pernyi.

International audience; Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments. (C) 2010 Elsevier Ltd. All rights reserved.

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.

Immunity in insects has largely focused on responses towards bacteria and fungi, but recently the study of immune responses against viral infections has also received attention. In Lepidoptera, phagocytosis and encapsulation mediated by hemocytes, and apoptosis are part of the response against virus infection; however, many studies also suggest the presence of unknown factors involved in the anti-viral defense. An up-regulation of the lepidopteran-specific pattern recognition protein Hemolin after baculovirus infection in the Chinese oak silkmoth and discovery of putative virus responsive elements in the up-stream regions of Hemolin in the Cecropia moth and the Tobacco horn worm could suggest that Hemolin is involved in virus defense. In this paper, a number of studies investigating baculovirus pathogenesis, and others analyzing Hemolin expression have been revisited leading to the speculation that Hemolin could be engaged in several anti-viral processes.

We investigated whether exposure of rat brain to microwaves (MWs) of global system for mobile communication (GSM) induces DNA breaks, changes in chromatin conformation and in gene expression. An exposure installation was used based on a test mobile phone employing a GSM signal at 915 MHz, all standard modulations included, output power level in pulses 2 W, specific absorption rate (SAR) 0.4 mW/g. Rats were exposed or sham exposed to MWs during 2 h. After exposure, cell suspensions were prepared from brain samples, as well as from spleen and thymus. For analysis of gene expression patterns, total RNA was extracted from cerebellum. Changes in chromatin conformation, which are indicative of stress response and genotoxic effects, were measured by the method of anomalous viscosity time dependencies (AVTD). DNA double strand breaks (DSBs) were analyzed by pulsed-field gel electrophoresis (PFGE). Effects of MW exposure were observed on neither conformation of chromatin nor DNA DSBs. Gene expression profiles were obtained by Affymetrix U34 GeneChips representing 8800 rat genes and analyzed with the Affymetrix Microarray Suite (MAS) 5.0 software. In cerebellum from all exposed animals, 11 genes were upregulated in a range of 1.34-2.74 fold and one gene was downregulated 0.48-fold (P < .0025). The induced genes encode proteins with diverse functions including neurotransmitter regulation, blood-brain barrier (BBB), and melatonin production. The data shows that GSM MWs at 915 MHz did not induce PFGE-detectable DNA double stranded breaks or changes in chromatin conformation, but affected expression of genes in rat brain cells.

A Gram-positive, aerobic, non-motile strain, H2.16BT, isolated from the midgut of the mosquito Anopheles arabiensis was investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strain H2.16BT was shown to belong to the genus Janibacter, being most closely related to Janibacter melonis (98·3 %), Janibacter terrae (98·5 %) and Janibacter limosus (98·5 %). Chemotaxonomic data (meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall and major fatty acids of iso-C16 : 0, C17 : 1 ω8c and C17 : 0) supported the allocation of the strain to the genus Janibacter. The results of DNA–DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain H2.16BT from closely related species. Thus, H2.16BT represents a novel species of the genus Janibacter, for which the name Janibacter anophelis sp. nov. is proposed. The type strain is H2.16BT (=CCUG 49715T=CIP 108728T).

A Gram-negative, rod-shaped organism (CCUG 49520T) was isolated from the midgut of the mosquito Anopheles arabiensis. 16S rRNA gene sequence analysis demonstrated that this isolate is unique, showing Enterobacteriaceae and Vibrionaceae. The quinone system consisted exclusively of ubiquinone Q-8; the polar lipid profile consisted of the major compounds phosphatidylethanolamine and phosphatidylglycerol, a moderate to minor amount of two unknown aminophospholipids, an unknown phospholipid and two unknown polar lipids; the polyamine pattern was characterized by the predominant compound 1,3-diaminopropane and showed some significant differences when compared with members of the Enterobacteriaceae and Vibrionaceae. On the basis of 16S rRNA gene sequence analysis in combination with chemotaxonomic data, strain CCUG 49520T is considered to represent a new genus and species, for which the name Thorsellia anophelis gen. nov., sp. nov. is proposed. The type strain is CCUG 49520T (=CIP 108754T).

Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus , were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides , is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito- Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

The Chinese oak silk moth Antheraea pernyi is an important silk producer. To understand microbial resistance of this moth, we cloned Hemolin, encoding a multifunctional immune protein belonging to the immunoglobulin superfamily, and examined the expression in gonads and fat body. The ApHemolin amino acid sequence was compared to other Hemolin sequences in order to predict functional sites. Several sites were conserved; among them a phosphate binding site, which according to 3D structure modelling does not appear in neuroglian, the phylogenetically closest related protein. In addition, two conserved KDG sequences in the C-C' loop of immunoglobulin domains 1 and 3, give rise to gamma-turns, which is a common motif in the C'-C'' loop of the hypervariable region L2 in vertebrate immunoglobulins. The comparisons also show variable regions of specific interest for future studies of hemolin and its interaction with microbial entities.

The successful detection of early signs of dementia in people with Down syndrome could form a basis for useful early support and for drug treatment. This report describes the development and preliminary application of an interview and test instrument for the assessment of dementia among people with intellectual disability, as well as a framework for diagnosis that combines the findings of an interview and a test with the diagnostic criteria of ICD-10, DSM-IV and NINCDS-ADRDA. From among the number of tests and interview questions developed, those showing the most significant differences between participants in three groups of differing levels of intellectual disability and estimated dementia were kept. Reported are the assumptions for the items used, descriptions of the process and items used, and the associations of test items with predicting the presence of dementia. The authors conclude that a protocol combining testing and interview has promise and potential for detecting early signs of dementia in this population and could prove feasible for use in practice.

SummaryMalaria, which is caused by Plasmodium parasites, is transmitted by anopheline mosquitoes. When gametocytes, the precursor cells of Plasmodium gametes, are transferred to a mosquito, they fertilize and proliferate, which render the mosquito infectious to the next vertebrate host [1]. Although the fertilization of malaria parasites has been considered as a rational target for transmission-blocking vaccines [2], the underlying mechanism is poorly understood. Here, we show that the rodent malaria parasite gene Plasmodium berghei GENERATIVE CELL SPECIFIC 1 (PbGCS1) plays a central role in its gametic interaction. PbGCS1 knockout parasites show male sterility, resulting in unsuccessful fertilization. Because such a male-specific function of GCS1 has been observed in angiosperms [3, 4], this indicates, for the first time, that parasite sexual reproduction is controlled by a machinery common to flowering plants. Our present findings provide a new viewpoint for understanding the parasitic fertilization system and important clues for novel strategies to attack life-threatening parasites.

Genetic strategies for reducing populations of vector mosquitoes or replacing them with those that are not able to transmit pathogens benefit greatly from molecular tools that allow gene manipulation and transgenesis. Mosquito genome sequences and associated EST (expressed sequence tags) databases enable large-scale investigations to provide new insights into evolutionary, biochemical, genetic, metabolic, and physiological pathways. Additionally, comparative genomics reveals the bases for evolutionary mechanisms with particular focus on specific interactions between vectors and pathogens. We discuss how this information may be exploited for the optimization of transgenes that interfere with the propagation and development of pathogens in their mosquito hosts.

The pine weevil, Hylobius abietis, is a severe forest pest insect as it feeds on newly planted conifer seedlings. To identify and develop an antifeedant could be one step towards the protection of seedlings from feeding damage by the pine weevil. With the aim to trace the origin of the antifeedants previously found in feces of the pine weevil, we investigated the culturable bacteria associated with the gut and identified the volatiles they produced. Bacterial isolates were identified by 16S ribosomal RNA gene analysis. The volatile emissions of selected bacteria, cultivated on NB media or on the grated phloem of Scots pine twigs dispersed in water, were collected and analyzed by solid-phase microextraction gas chromatography-mass spectrometry. The bacterial isolates released a variety of compounds, among others 2-methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, dimethyl disulfide, and dimethyl trisulfide. A strong antifeedant effect was observed by 2-phenylethanol, which could thus be a good candidate for use to protect planted conifer seedlings against feeding damage caused by H. abietis.