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The helminth endoparasites of many European amphibian species are often known exclusively from morphological descriptions. A molecular library of DNA sequence data linked to morphological identifications is still in its infancy. In this paper, we aim to contribute to such a library on the smooth newt Lissotriton vulgaris, the intermediate and definitive host of 31 helminth parasites, according to evidence published so far. Newts (n = 69) were collected at two study sites in western Germany and examined for the presence of helminths. A total of five helminth species were detected in 56 (81%) of the newts, but only one or two species infected a single host. Four out of five helminth species were identified morphologically and based on DNA sequences as Parastrigea robusta (metacercariae), Oswaldocruzia filiformis, Megalobatrachonema terdentatum (adults and larvae) and Cosmocerca longicauda , and the corresponding sequences were provided subsequently. Oswaldocruzia molgeta was confirmed to be a junior synonym of O. filiformis. Molecular data on a fifth species (a cosmocercid nematode) that could not be identified at species level were added to GenBank. These findings increased the molecular library on morphologically identified smooth newt parasites significantly, from 12 to 15 entries. [ABSTRACT FROM AUTHOR]

Introduction . Fungal infections are relevant health risks for individuals with acquired immunodeficiency in the resource-limited tropics, but available surveillance data are scarce. For Candida auris and Cryptococcus spp., the evolution from environmental reservoirs to human pathogens causing life-threatening diseases is currently discussed as a public health concern in the context of climate change and limited treatment options. Gap statement . Uncovering the gastrointestinal tract as an epidemiological niche of fungi emerging from the environment into individuals for whom fungal infections are not diagnosed. Aim . To contribute to data on the local epidemiology of C. auris and Cryptococcus spp. in Western African Ghana by analysing gastrointestinal samples of Ghanaian individuals. Methodology . Four real-time PCR assays targeting C. auris and five real-time PCR assays targeting Cryptococcus spp. were applied with stool samples of 875 non-age-stratified Ghanaian HIV patients and 30 Ghanaian control individuals without known HIV infection. Also, 664 samples from Ghanaian children under 2 years of age were investigated. The true abundance of the target micro-organism was considered as unlikely in the case of one or fewer positive signals, likely in the case of two to three positive signals and highly likely in the case of four or more positive signals per sample in the real-time PCR assays. Results . The combined application of sensitive, target-specific real-time PCR assays indicates that neither C. auris , Cryptococcus neoformans complex nor Cryptococcus gattii complex were part of the gut microbiota of Ghanaian individuals with or without HIV infection. Conclusion . Despite the significant disease burden from these pathogens in immunosuppressed Ghanaian individuals, detection from gastrointestinal samples was unlikely, which should be taken into account when discussing screening strategies for these fungi of public health concern. In contrast, the detection of these fungi from such samples should not routinely be considered as commensal colonization flora.

Here, we report the simultaneous isolation of Pseudomonas straminea from blood cultures and from a skin ulcer in an elderly woman who suffered from atopic dermatitis and psoriasis and developed acute cellulitis of both arms requiring hospital treatment. To the best of our knowledge, P. straminea has not been previously reported to cause invasive infections in humans. This case highlights how chronic diseases and older age increase the susceptibility to bacterial infections with environmental bacteria of low virulence. Our study describes the microbiological identification of the blood culture isolate, including morpho-molecular characterization and virulence demonstration in a Galleria mellonella model.

Saliva of hematophagous insects contains many different compounds, mainly acting as anticoagulants. Investigating the bacteriolytic compounds of the saliva of the bloodsucking Triatoma infestans photometrically between pH 3 and pH 10 using unfed fifth instars and nymphs up to 15 days after feeding, we found bacteriolytic activity against lyophilized Micrococcus luteus was stronger at pH 4 and pH 6. After feeding, the activity level at pH 4 was unchanged, but at pH 6 more than doubled between 3 and 7 days after feeding. In zymographs of the saliva and after incubation at pH 4, bacteriolytic activity against Micrococcus luteus was present at eight lysis zones between 14.1 and 38.5 kDa, showing the strongest activity at 24.5 kDa. After incubation at pH 6, lysis zones only appeared at 15.3, 17, and 31.4 kDa. Comparing zymographs of the saliva of unfed and fed nymphs, bacteriolytic activity at 17 kDa increased after feeding. In total nine lysis bands appeared, also at >30 kDa, so far unreported in the saliva of triatomines. Reverse transcription polymerase chain reaction using oligonucleotides based on the previously described lysozyme gene of T. infestans, TiLys1, verified expression of genes encoding TiLys1 and TiLys2 in the salivary glands, but also of an undescribed third lysozyme, TiLys3, of which the cloned cDNA shares characteristics with other c-type lysozymes of insects. While TiLys1 was expressed in the tissue of all three salivary glands, transcripts of TiLys2 and of TiLys3 seem to be present only in the gland G1 and G3, respectively., (© 2023 The Authors. Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC.)

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant Escherichia coli isolated between 2007 and 2016 from German soldiers after deployments, with a particular focus on the African Sahel region. A total of 51 third-generation cephalosporine-resistant E. coli isolated from 51 military returnees from deployment collected during the assessment period between 2007 and 2016 were subjected to short-read next-generation sequencing analysis. Returnees from the Sahel region (Djibouti, Mali, South Sudan, Sudan, Sudan, and Uganda) comprised a proportion of 52.9% (27/51). Repeatedly isolated sequence types according to the Warwick University scheme from returnees from the Sahel region were ST38, ST131, and ST648, confirming previous epidemiological assessments from various sub-Saharan African regions. Locally prevalent resistance genes in isolates from returnees from the Sahel region associated with third-generation resistance were bla CTX-M-15 , bla CTX-M-27 , bla CTX-M-1 , bla TEM-169 , bla CTX-M-14 , bla CTX-M-99 -like, bla CTX-M-125 , bla SHV-12 , and bla DHA-1 , while virulence genes were east1 , sat , and tsh in declining order of frequency of occurrence each. In line with phenotypically observed high resistance rates for aminoglycosides and trimethoprim/sulfamethoxazole, multiple associated resistance genes were observed. A similar, slightly more diverse situation was recorded for the other deployment sites. In summary, this assessment provides first next-generation sequencing-based epidemiological data on third-generation cephalosporine-resistant E. coli imported by deployed German soldiers with a particular focus on deployments to the Sahel region, thus serving as a small sentinel. The detected sequence types are well in line with the results from previous epidemiological assessments in sub-Saharan Africa.

The study was performed to provide an overview of the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in Afghanistan isolated by the German military medical service during the Afghanistan conflict. A total of 18 isolates were collected between 2012 and 2018 at the microbiological laboratory of the field hospital in Camp Marmal near Mazar-e Sharif, Afghanistan, from Afghan patients. The isolates were subjected to phenotypic and genotypic differentiation and antimicrobial susceptibility testing as well as to a core genome multi-locus sequence typing (cgMLST) approach based on whole-genome next-generation sequence (wgNGS) data. Next to several sporadic isolates, four transmission clusters comprising strains from the international clonal lineages IC1, IC2, and IC9 were identified. Acquired carbapenem resistance was due to bla OXA-23 in 17/18 isolates, while genes mediating resistance against sulfonamides, macrolides, tetracyclines, and aminoglycosides were frequently identified as well. In conclusion, the assessment confirmed both the frequent occurrence of A. baumannii associated with outbreak events and a variety of different clones in Afghanistan. The fact that acquired carbapenem resistance was almost exclusively associated with bla OXA-23 may facilitate molecular resistance screening based on rapid molecular assays targeting this resistance determinant.

Species of MegalobatrachonemaYamaguti, 1941 (Ascaridida: Cosmocercoidea) are important nematode parasites in amphibians and reptiles. However, the phylogenetic relationship of its included two subgenera Megalobatrachonema and Chabaudgolvania remains unclear. In the present study, a new species of Megalobatrachonema, M. (Chabaudgolvania) wangi sp. nov., was described based on the specimens collected from the lesser spiny frog Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2)] and mitochondrial [12S small subunit ribosomal DNA and cytochrome c oxidase subunit 1 (cox1)] target regions of the new species and M. (Chabaudgolvania) terdentatum, together with the 12S region of M. (Megalobatrachonema) hainanensis, were amplified and sequenced for molecular identification and phylogeny. Moreover, in order to clarify the systematic position of the new species and the phylogenetic relationship of the two subgenera Megalobatrachonema and Chabaudgolvania, phylogenetic analyses based on 28S + ITS1-5.8S-ITS2 + 12S sequence data were performed using maximum likelihood (ML) inference and Bayesian inference (BI). The molecular phylogenetic results conflicted with the current classification and challenged the validity of the subgenus Chabaudgolvania, that should be a synonym of the subgenus Megalobatrachonema. The presence or absence of valves in the oesophageal bulb as a key criterion for delimitation of the two subgenera Megalobatrachonema and Chabaudgolvania seems to be unreliable., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses.

Ixodes ricinus is a well-known vector of different human pathogens including Rickettsia helvetica. The role of wild mammals in the distribution and probable maintenance of Rickettsia in nature is still to be determined. We therefore investigated various parasites from different wild mammals as well as companion animals for the presence of Rickettsia. A total of 606 I. ricinus, 38 Cephenemyia stimulator (botfly larvae), one Dermacentor reticulatus, 24 Haematopinus suis (hog lice) and 30 Lipoptena cervi (deer flies) were collected from free-ranging animals during seasonal hunting, and from companion animals. Sample sites included hunting leases at three main sampling areas and five additional areas in West and Central Germany. All collected parasites were screened for Rickettsia spp. and I. ricinus were investigated for tick-borne encephalitis virus (TBEV) in addition. While no TBEV was detected, the minimum infection rate (MIR) of I. ricinus with Rickettsia was 4.1% referring to all sampling sites and up to 6.9% at the main sampling site in Koblenz area. Sequencing of a fragment of the ompB gene identified R. helvetica. Approximately one third (29.5%) of the animals carried Rickettsia-positive ticks and the MIR in ticks infesting wild mammals ranged from 4.1% (roe deer) to 9.5%. These data affirm the widespread distribution of R. helvetica in Germany. One botfly larva from roe deer also harboured R. helvetica. Botfly larvae are obligate parasites of the nasal cavity, pharynx and throat of cervids and feed on cell fragments and blood. Based on this one might hypothesise that R. helvetica likely induces rickettsemia in cervids thus possibly contributing to maintenance and distribution of this rickettsia in the field., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Acanthamoeba spp. are frequently the etiological agents of a severe form of sight-threatening keratitis, called Acanthamoeba keratitis. The contact lens storage solution of a patient with keratitis of unknown genesis was screened using our diagnostic tools to detect potentially pathogenic free-living amoebae (FLA). Culture methods and a triplex quantitative real-time polymerase chain reaction (qPCR) targeting Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris were used in context of this routine screening. While no amoebae were detected by culture, qPCR specifically detected DNA of B. mandrillaris. This FLA is known as the etiological agent of a fatal form of encephalitis in humans and other mammals, Balamuthia amoebic encephalitis (BAE). A fragment of the 18S rDNA gene was amplified from the sample and showed 99 % sequence identity to B. mandrillaris sequences from GenBank. To the best of our knowledge, this is the first report of B. mandrillaris found in association with contact lenses. Although no viable amoeba was obtained by culturing efforts, the verification of B. mandrillaris DNA in the contact lens storage solution demonstrates how easily this pathogen might come into close contact with humans.

In this article, the results of a long effort to derive valuable phylogenetic data about an extraordinary spore-like infectious particle (endocytobiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis are presented. The development of these endocytobionts has already been demonstrated with electron microscopic photo sequences, leading to a relevant model of its development presented here. The molecular biological investigation following the discovery of two other Pandoravirus species within aquatic sediments in 2013 led to the taxonomic affiliation of our endocytobiont with the genus Pandoravirus. A range of endocytobionts (intracellular biofilms) have been found in recent years, among which are several viruses which obligatorily proliferate within free-living amoebae. In human medicine, foreign objects which are placed in or on humans cause problems with microorganisms in biofilms. Contact lenses are especially important, because they are known as a source of a rapid formation of biofilm. These were the first Pandoraviruses described, and because this is additionally the first documented association with humans, we have clearly demonstrated how easily such (viral) endocytobionts can be transferred to humans. This case counts as an example of parasites acting as vectors of phylogenetically different microorganisms especially when living sympatric within their biocoenosis of biofilms. As the third part of the "Pandoravirus trilogy", it finally reveals the phylogenetic nature of these "extraordinary endocytobionts" within Acanthamoebae.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

During a small-scale surveillance project to identify possible routes of entry for invasive mosquitoes into Germany, 14 adult Aedes (Stegomyia) albopictus (Skuse) were discovered between July and October 2012. They were trapped at three different service stations in Bavaria and Baden-Wuerttemberg located along two motorways that connect Germany with southern Europe. This indicates regular introduction of A. albopictus into Germany and highlights the need for a continuous surveillance and control programme.

Background: During the last decades, population densities of Ixodes ricinus and prevalences of Borrelia burgdorferi s.l. have increased in different regions in Europe. In the present study, we determined tick abundance and the prevalence of different Borrelia genospecies in ticks from three sites in the Siebengebirge, Germany, which were already examined in the years 1987, 1989, 2001 and 2003. Data from all investigations were compared., Methods: In 2007 and 2008, host-seeking I. ricinus were collected by monthly blanket dragging at three distinct vegetation sites in the Siebengebirge, a nature reserve and a well visited local recreation area near Bonn, Germany. In both years, 702 ticks were tested for B. burgdorferi s.l. DNA by nested PCR, and 249 tick samples positive for Borrelia were further genotyped by reverse line blotting., Results: A total of 1046 and 1591 I. ricinus were collected in 2007 and 2008, respectively. In comparison to previous studies at these sites, the densities at all sites increased from 1987/89 and/or from 2003 until 2008. Tick densities and Borrelia prevalences in 2007 and 2008, respectively, were not correlated for all sites and both years. Overall, Borrelia prevalence of all ticks decreased significantly from 2007 (19.5%) to 2008 (16.5%), thus reaching the same level as in 2001 two times higher than in 1987/89 (7.6%). Since 2001, single infections with a Borrelia genospecies predominated in all collections, but the number of multiple infections increased, and in 2007, for the first time, triple Borrelia infections occurred. Prevalences of Borrelia genospecies differed considerably between the three sites, but B. garinii or B. afzelii were always the most dominant genospecies. B. lusitaniae was detected for the first time in the Siebengebirge, also in co-infections with B. garinii or B. valaisiana., Conclusions: Over the last two centuries tick densities have changed in the Siebengebirge at sites that remained unchanged by human activity since they belong to a nature reserve. Abiotic and biotic conditions most likely favored the host-seeking activity of I. ricinus and the increase of multiple Borrelia infections in ticks. These changes have led to a potential higher risk of humans and animals to be infected with Lyme borreliosis.

Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Triatomines inhibit the clotting of ingested blood in the stomach (anterior midgut). After verifying this phenomenon in Panstrongylus megistus using coagulation assays, a full-length cDNA encoding a Kazal-like inhibitor was amplified by PCR. The open reading frame encodes a putative precursor protein of 412 amino acid residues, which was named PmStKaz and contains seven Kazal-like domains forming four Kazal-type inhibitors. A single domain inhibitor and three double-domain inhibitors possess sequence identities of up to 91% to the respective domains of Kazal-type inhibitors from other triatomines. The gene is expressed in the stomach (anterior midgut) but not in the small intestine (posterior midgut), salivary glands or haemocytes. After hydrophobic interaction chromatography of the stomach contents, four fractions (numbers 1-4) inhibited the activity of trypsin, fraction 2 that of subtilisin A, fractions 1, 3 and 4 that of plasmin, and fractions 3 and 4 that of thrombin. After ion exchange chromatography, MALDI-TOF-MS analysis of the intact proteins in fractions 3 and 4 showed diverse masses correlating to PmStKaz IV-V and PmStKaz II-III, respectively. Both proteins seem to be present in several isoforms with variant amino- and carboxy-terminal ends. In reverse zymography of the proteins of the stomach contents after separation by isoelectric focusing and non-reducing SDS-PAGE, much higher concentrations of isoforms of PmStKaz II-III and IV-V were evident than of PmStKaz I and VI-VII., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The outbreak of bluetongue disease in Central Europe necessitates new approaches in the identification of vectors to follow-up changes of populations of species and not of complexes. Since females of species of the complex of Culicoides obsoletus are difficult to be identified according to morphological criteria, we applied a polymerase chain reaction (PCR)-based strategy targeting the mitochondrial cytochrome oxidase subunit I to differentiate between the species Culicoides obsoletus s.s. and Culicoides scoticus. Catches of culicoids obtained from May to November 2007 in an ultraviolet lamp trap at a cattle farm in Rhineland-Palatinate, Southern Germany were surveyed for changes of the abundance of both species. Only in May 2007, the samples contained similar proportions of both species. Afterwards, C. scoticus dominated with up to 88%. Calculating the number of specimens of both species within the total catches of culicoids, the numbers of C. obsoletus s.s. slightly decreased from May to July and increased to a little maximum in August. C. scoticus seemed to have three maxima in this period of time, the strongest one in August, presumably due to different generations and not to climatic conditions. These results indicate that the applied PCR strategy can be used for a detailed analysis of culicoids as basis for the estimation of the transmission risk of the bluetongue virus by different species of the Obsoletus complex.

Within the entomological monitoring program of the German federal ministry of food, agriculture, and user protection (BMELV), at 12 cattle farms in Rhineland-Palatinate and two in Saarland, ultraviolet lamp traps were used to monitor the distribution and seasonal appearance of potential vectors of the bluetongue virus, with special consideration of species of Culicoides. Using the traps during the first seven nights of each month from April 2007 to May 2008, 5,000-120,000 ceratopogonids were caught at different locations, in total about 500,000 and mainly females. Ninety-four percent belonged to the genus Culicoides, and of these, 90% were Culicoides obsoletus s.l., 6% were Culicoides pulicaris s.l., and 4% were other species of this genus. In all traps, the first ceratopogonids were caught in April 2007, the total number peaking in August 2007. After a reduction in September, a lower peak occurred in October. During the whole winter, some ceratopogonids were active. At nearly all locations, the total numbers of C. obsoletus s.l., C. pulicaris s.l., and of other ceratopogonids were significantly correlated with the temperatures, and higher population densities of C. obsoletus s.l. seemed to occur at altitudes of about 300 m above sea level.

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.

In photoautotrophic organisms, the expression of nuclear genes encoding plastid proteins is known to be regulated at various levels. In this study, we present the analysis of two non-photosynthetic mutants (CC1051 and TR72) from the unicellular green alga Chlamydomonas reinhardtii. Both mutant strains show a defect in the processing of chloroplast psaA mRNA, and therefore they are assumed to be defective in photosystem I (PSI) assembly. We have performed macroarray experiments with trans-splicing mutants CC1051 and TR72 in order to analyse putative pleiotropic effects of nuclear-located mutations leading to a non-functional PSI. To the best of our knowledge, this is the first example of Chlamydomonas cDNA macroarray analysis comparing the transcriptional regulation of nuclear genes in wild-type and photosystem I mutants. The macroarray results demonstrated a transcriptional downregulation of members of the Lhcb gene family more than 2-fold in both mutant strains. In addition, real-time RT-PCR experiments found a 4- to 16-fold reduction in transcript levels of several Lhca genes in TR72; whereas in CC1051, no significant change in transcript levels was observed. Taken together, our data suggest that a signal is transmitted from the chloroplast to the nucleus that serves to regulate the level of light harvesting polypeptides in the organelle.

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.