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Schistosomiasis, caused by schistosome parasites, is a neglected tropical disease affecting humans and animals. There is no vaccine available yet, and fear of upcoming resistance against the only widely used drug, praziquantel, is omnipresent. Previously, we showed that imatinib (Gleevec), an anticancer drug, affected schistosome physiology and caused the death of adult Schistosoma mansoni in vitro. Here, we present the first known evidence that one effect of imatinib is the induction of autophagy in S. mansoni. Furthermore, worms co-treated with imatinib and bafilomycin A1, a late-phase autophagy inhibitor, reversed imatinib-induced autophagy and its antischistosomal effects as revealed by phenotypic and molecular analyses.

The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.

The parasitic disease schistosomiasis is the cause of more than 200 000 human deaths per year. Although the disease is treatable, there is one major shortcoming: praziquantel has been the only drug used to combat these parasites since 1977. The risk of the emergence of resistant schistosomes is known to be increasing, as a reduced sensitivity of these parasites toward praziquantel has been observed. We developed a new class of substances, which are derived from inhibitors of human aldose reductase, and which showed promising activity against Schistosoma mansoni couples in vitro. Further optimisation of the compounds led to an increase in anti‐schistosomal activity with observed phenotypes such as reduced egg production, vitality, and motility as well as tegumental damage and gut dilatation. Here, we performed structure–activity relationship studies on the carboxylic acid moiety of biarylalkyl carboxylic acids. Out of 82 carboxylic acid amides, we identified 10 compounds that are active against S. mansoni at 25 μm. The best five compounds showed an anti‐schistosomal activity up to 10 μm and induced severe phenotypes. Cytotoxicity tests in human cell lines showed that two derivatives had no cytotoxicity at 50 or 100 μm. These compounds are promising candidates for further optimisation toward the new anti‐schistosomal agents., No flukes with these amides! Schistosomiasis causes more than 200 000 deaths per year and is treated with the single drug praziquantel. We have previously reported biarylalkyl carboxylic acid derivatives as novel chemical entities with in vitro anti‐schistosomal activity. In continuation of these studies, we report here a series of amide derivatives. The best results were obtained with morpholine, thiomorpholine and substituted piperazine derivatives.

Facilitated by the Schistosoma mansoni genome project, multiple transcriptomic studies were performed over the last decade to elucidate gene expression patterns among different developmental stages of the complex schistosome life cycle. While these analyses enable the identification of candidate genes with key functions in schistosome biology, a diverse molecular tool set is needed that allows comprehensive functional characterization at the single gene level. This includes the availability of reliable reference genes to confirm changes in the transcription of genes of interest over different biological samples and experimental conditions. In particular, the investigation of one key aspect of schistosome biology, the pairing-dependent gene expression in females and males, requires knowledge on reference genes that are expressed independently of both pairing and of in vitro culture effects. Therefore, the present study focused on the identification of quantitative reverse transcription (qRT)-PCR reference genes suitable for the investigation of pairing-dependent gene expression in the S. mansoni male. The “pipeline” we present here is based on qRT-PCR analyses of high biological replication combined with three different statistical analysis tools, BestKeeper, geNorm, and NormFinder. Our approach resulted in a statistically robust ranking of 15 selected reference genes with respect to their transcription stability between pairing-unexperienced and -experienced males. We further tested the top seven candidate genes for their transcription stability during in vitro culture of adult S. mansoni. Of these, the two most suitable reference genes were used to investigate the influence of the pairing contact on the transcription of genes of interest, comprising a tyrosine decarboxylase gene Smtdc1, an ebony ortholog Smebony, and the follistatin ortholog Smfst in S. mansoni males. Performing pairing, separation and re-pairing experiments with adult S. mansoni in vitro, our results indicate for the first time that pairing can act as a molecular on/off-switch of specific genes to strictly control their expression in schistosome males.

Schistosomiasis caused by different species of schistosome parasites is one of the most debilitating helminthic diseases of humans worldwide. For decades, chemotherapy is the main method of controlling schistosomiasis. However, the fear of drug resistance has motivated the search for alternatives. It has been demonstrated that the ABL kinase inhibitor imatinib affected the development and survival of Schistosoma mansoni in vitro; however, there is still lack of information on whether imatinib also affects other schistosome species such as Schistosoma japonicum. In the present study, the anti-schistosomal potency of imatinib on adult S. japonicum was investigated in vitro, and the results showed that imatinib had a significant impact on various physiological processes of S. japonicum adult worms. Besides its negative effects on worm motility, pairing stability, and gonad development, imatinib caused pathological changes in the gastrodermis as well as the death of the parasite. Our findings suggest that imatinib is an intriguing candidate for further development as an option to fight S. japonicum.

Schistosomiasis is a disease of global importance caused by parasitic flatworms, schistosomes, which cause pathogenicity through eggs laid by the female worm inside the host's blood vessels. Maintenance of cellular homeostasis is crucial for parasites, as for other organisms, and is quite likely important for schistosome reproduction and vitality. We hypothesize a role for autophagy in these processes, an evolutionarily conserved and essential cellular degradation pathway. Here, for the first known time, we shed light on the autophagy machinery and its involvement in pairing-dependent processes, vitality and reproduction of Schistosoma mansoni. We identified autophagy genes by in silico analyses and determined the influence of in vitro culture on the transcriptional expression in male and female worms using quantitative real-time PCR. Among the identified autophagy genes were Beclin, Ambra1, Vps34, DRAM, DAP1, and LC3B, of which some showed a sex-dependent expression. Specifically, the death-associated protein DAP1 was significantly more highly expressed in females compared with males, while for the damage-regulated autophagy modulator DRAM it was the opposite. Furthermore, in-vitro culture significantly changed the transcript expression level of DAP1 in female worms. Next, worms were treated with an autophagy inducer (rapamycin) or inhibitors (bafilomycin A1, wortmannin and spautin-1) to evaluate effects on autophagy protein expression, worm vitality, and reproduction. The conversion of the key autophagy protein LC3B, a marker for autophagic activity, was increased by rapamycin and blocked by bafilomycin. All inhibitors affected worm fitness, egg production, and negatively affected the morphology of gonads and intestine. In summary, autophagy genes in S. mansoni show an interesting sex-dependent expression pattern and manipulation of autophagy in S. mansoni by inhibitors induced detrimental effects, which encourages subsequent studies to identify antischistosomal targets within the autophagy machinery.

Schistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites. Graphical abstract

In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53–68%) than IV-I (93–95%). Unprotected mice developed less sporozoite-specific CD8+ and CD4+ effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8+ dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.

Experimental, chronic Schistosoma infections protect against allergic airway inflammation (AAI), in which splenic, IL-10 producing regulatory B (Breg) cells play a prominent role. Interestingly, we found that pulmonary B cells could also protect against ovalbumin-induced AAI. So far, we excluded a role for IL-10, secreted antibodies or Treg cell-induction by schistosome-induced pulmonary B cells in the observed effect. Phenotypic characterisation revealed a strong up-regulation of the low-affinity IgE receptor CD23, and to a lesser extent of the co-stimulatory molecule CD86, while they produced less cytokines in response to TLR ligands. Co-cultures of naive, OVA-transgenic T cells with OVA-pulsed pulmonary B cells from S. mansoni-infected, allergic mice resulted in a reduced production of the type-2 cytokines IL-4, IL-13 and IL-10 compared to non-infected, allergic controls. This reduction in Th2 cytokine production could not be overcome by a polyclonal stimulation with anti-CD3/28 in co-cultures with naive T cells from wild-type mice. This observation suggests that pulmonary B cells from infected animals are impaired in their capacity to induce Th2 cells regardless of their capacity to present antigens and/or to provide co-stimulation. Until now, blocking of IL-6, IL-10, TGF-β, or CTLA-4 in B cell-T cell co-cultures did not result in a full restoration of Th2 cytokine production. To further study the features of helminth-induced pulmonary B cells, we are currently investigating their gene expression profile by microarray technology. Altogether, these data show that pulmonary B cells protect against AAI and directly modulate T cell-cytokine production.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1., Author summary Infection with helminth parasites is known to be inversely associated with hyper-inflammatory disorders. While Schistosoma (S.) mansoni has been described to exert its down-modulatory effects on inflammation by inducing a network of regulatory immune cells such as regulatory B (Breg), the mechanisms of Breg cell induction remain unclear. Here, we use in vivo and in vitro approaches to show that antigens from S. mansoni eggs, among which the major glycoprotein IPSE/alpha-1, directly interact with splenic marginal zone B cells of mice which triggers them to produce the anti-inflammatory cytokine IL-10 and their capacity to induce regulatory T (Treg) cells. We also found that IPSE/alpha-1 induces IL-10 in human CD1d+ B cells, and that both natural and recombinant IPSE/alpha-1 are equally effective in driving murine and human Breg cells. Our study thus provides insight into the mechanisms of Breg cell induction by schistosomes, and an important step towards the development of helminth-based treatment strategies against hyper-inflammatory diseases.

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK-cell response in mice that required TLR9-positive myeloid DC and IL-12, but no IFN-alpha/beta. Here, we investigated whether IL-15 or IL-18 mediate the activity of IL-12 or function as independent activators of NK cells. In contrast to earlier studies that described IL-15 as crucial for NK-cell priming in response to TLR ligands, the expression of IFN-gamma, FasL, perforin and granzyme B by NK cells in L. infantum-infected mice was completely preserved in the absence of IL-15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN-gamma secretion, cytotoxicity and FasL expression of NK cells from infected IL-18(-/-) mice were significantly reduced compared with controls, but, unlike IL-12, IL-18 was not essential for NK-cell effector functions. Part of the NK-cell-stimulatory effect of IL-12 was dependent on IL-18. We conclude that IL-15 is not functioning as a universal NK-cell priming signal and that IL-18 contributes to the NK-cell response in visceral leishmaniasis. The cytokine requirements for NK-cell activation appear to differ contingent upon the infectious pathogen.

Cercariae of Schistosoma mansoni approach towards their host's skin with a chemokinesis. They respond to human skin extracts by inserting shifts between backward and forward swimming and therewith increase the rate of change of direction (RCD). We identified the host attractants by fractionating human skin surface extracts, exposing the fractions and chemicals to the cercariae and recording cercarial swimming by video tracking. The cercariae responded specifically to three skin surface compounds by increasing the frequency of shifts in swimming direction. (1) Free fatty acids. The effectiveness was limited to chain lengths between 10 and 14 carbon atoms in saturated chains and increased by the number of double bonds. (2) Free L: -arginine at concentrations as low as 100 nM. (3) Small peptides with terminally located L: -arginine at concentrations as low as 50 microM. All chemokinesis stimuli are also involved in subsequent behavioural phases of host invasion: attachment and directed creeping on the skin surface, penetration and navigation within host tissues. The cercariae responded during swimming only to these small molecular water soluble cues and not to cues to which they respond in other behavioural phases of host invasion, but which are unsuitable as chemoattractants due to their chemical properties or distribution within the skin layers. This characterises the chemokinetic responses as adequate adaptation to advance transmission success.

Dermatitis caused by penetrating bird schistosome cercariae is an emerging global public health problem. Infections may be prevented by the use of topical formulations that inhibit cercarial skin penetration. We evaluated nine water resistant formulations by exposing treated arms of volunteers to Trichobilharzia szidati cercariae. Six formulations protected from cercarial invasion. However, after immersion of the treated skin in water (2 x 20 min), only two formulations offered full protection: (1) Safe Sea, a cream protecting against jelly fish, (2) niclosamide in water resistant sun protecting cream formulations at concentrations as low as 0.05%. In an in vitro system Safe Sea and a 0.1% niclosamide formulation caused a high damage rate in T. szidati (92% and 99% after 5 min; only niclosamide with lethal effect) but not in Schistosoma mansoni (1% and 72%; both formulations with lethal effect). However, a 1% niclosamide formulation damaged S. mansoni sufficiently (100% after 5 min) and might offer full penetration protection.

Infection with the helminth Schistosoma mansoni drives the development of IL-10-producing regulatory B cells (Breg cells) which suppress experimental allergic airway inflammation. Here we investigated whether schistosomal egg antigens (SEA) directly interact with B cells and induce development of IL-10-producing Breg cells. Splenic B cell cytokine production and regulatory capacity was analysed after repeated intraperitoneal injection of SEA into C57BL/6 mice. To identify cell types that bind SEA in vivo, mice were intravenously injected with fluorescently labeled SEA and spleens analysed by immunohistochemistry and flow cytometry. For in vitro assays, naive CD19 sorted splenic B cells were cultured with SEA to study binding as well as B cell cytokines and regulatory capacity. SEA treatment upregulated IL-10, CD40 and CD86 expression by splenic marginal zone (MZ) B cells in vivo, and MZ B cells as well as MZ macrophages bound SEA. Importantly, SEA could also induce Breg cell development in vitro which was further increased by CD40 ligation, whereas TLR2- and TLR4-signaling was not involved. SEA-induced Breg cells triggered regulatory T cell development and suppressed T cell IFN-γ production in co-culture experiments. Macrophages were dispensable for Breg induction as shown by macrophage depletion in SEA-treated mice. In conclusion, schistosomal egg antigens can drive Breg cell development by targeting B cells directly. Current work focuses on the identification of relevant molecules in SEA and the role of pattern-recognition receptors to provide new insights in the generation of Breg cells. This may form a basis for novel therapies against allergic disorders by enhancing Breg cell activity.

Chronic low-grade inflammation associated with obesity contributes to insulin resistance and type 2 diabetes. Helminth parasites are the strongest natural inducers of type 2 immune responses, and short-lived infection with rodent nematodes was reported to improve glucose tolerance in obese mice. Here, we investigated the effects of chronic infection (12 weeks) with Schistosoma mansoni, a helminth that infects millions of humans worldwide, on whole-body metabolic homeostasis and white adipose tissue (WAT) immune cell composition in high-fat diet-induced obese C57BL/6 male mice. Our data indicate that chronic helminth infection reduced body weight gain (-62%), fat mass gain (-89%), and adipocyte size; lowered whole-body insulin resistance (-23%) and glucose intolerance (-16%); and improved peripheral glucose uptake (+25%) and WAT insulin sensitivity. Analysis of immune cell composition by flow cytometry and quantitative PCR (qPCR) revealed that S. mansoni promoted strong increases in WAT eosinophils and alternatively activated (M2) macrophages. Importantly, injections with S. mansoni-soluble egg antigens (SEA) recapitulated the beneficial effect of parasite infection on whole-body metabolic homeostasis and induced type 2 immune responses in WAT and liver. Taken together, we provide novel data suggesting that chronic helminth infection and helminth-derived molecules protect against metabolic disorders by promoting a T helper 2 (Th2) response, eosinophilia, and WAT M2 polarization.

Toll-like receptors (TLRs) are key components for the recognition of microorganisms, for the initiation of innate immunity, and for promoting adaptive immune responses. TLR signaling in B cells, in addition to B cell receptor or CD40 ligation, plays an important role in B cell differentiation and activation. In contrast, various infectious agents and/or TLR ligands can also prime B cells to induce tolerance and downregulate inflammatory reactions; those B cells are called regulatory B (Breg) cells and are characterized by a dominant IL-10 production. Several studies have suggested that Breg cells are impaired in patients with autoimmune diseases and allergic asthma. However, the role for TLR ligands in the induction of Breg cells as a potential therapy for some of these inflammatory diseases has not yet been investigated. Here, we provide detailed instructions on how to analyze and validate cytokine production in human and mouse B cells in response to various TLR ligands. Furthermore, we describe an assay to investigate the suppressive properties of TLR-induced B cells to confirm their regulatory B cell status.

Natural killer (NK) cells are important components of a protective immune response against intracellular pathogens such as Leishmania parasites, which reside within myeloid cells. Previous in vivo studies in murine cutaneous or visceral leishmaniasis showed that NK cells are activated by conventional dendritic cells in a Toll-like receptor 9-, interleukin-12 (IL-12)-, and IL-18-dependent manner during the early phase of infection and help to restrict the tissue parasite burden by unknown mechanisms. Here, we tested whether NK cells contribute to the control of Leishmania infections by lysing or by activating infected host cells. Coculture experiments revealed that activated NK cells from poly(I:C)-treated mice readily killed tumor target cells, whereas Leishmania infantum - or L. major -infected macrophages or dendritic cells remained viable. Infection with Leishmania did not significantly alter the expression of NK cell-activating molecules (retinoic acid early transcript alpha [Rae-1α], mouse UL16-binding protein-like transcript 1 [MULT-1], CD48) or inhibitory molecules (major histocompatibility complex [MHC] class I, nonclassical MHC class 1b molecule Qa-1) on the surface of myeloid cells, which offers an explanation for their protection from NK cell cytotoxicity. Consistent with these in vitro data, in vivo cytotoxicity assays revealed poor cytolytic activity of NK cells against adoptively transferred infected wild-type macrophages, whereas MHC class I-deficient macrophages were efficiently eliminated. NK cells activated by IL-12 and IL-18 stimulated macrophages to kill intracellular Leishmania in a cell contact-independent but gamma interferon-, tumor necrosis factor-, and inducible nitric oxide synthase-dependent manner. We conclude that Leishmania parasites, unlike viruses, do not render infected myeloid cells susceptible to the cytotoxicity of NK cells. Instead, soluble products of NK cells trigger the leishmanicidal activity of macrophages.

Metabolic disorders, such as obesity and insulin resistance, are caused by progressive impairment in whole-body energy homeostasis, leading to increased risk for type 2 diabetes. Obesity is often associated with chronic low-grade inflammation and alterations of immune cells composition in metabolic organs, especially in adipose tissues (AT).

We studied the skin invasion of Schistosoma mansoni cercariae by placing gamma-irradiated and nonirradiated cercariae onto the living human skin and timing the behavior of 53 individuals. The skin invasion of S. mansoni was less efficient compared to the bird schistosome Trichobilharzia szidati. S. mansoni cercariae crept longer on the skin after attachment until they started penetration movements (median of 43 s [range of 15 s-6.58 min]; T. szidati, median of 8 s [range of 0-80 s]). Subsequent to this longer exploratory phase, 74% penetrated into wrinkles (T. szidati 84%), 22% into the smooth skin surface (T. szidati 0%), and 4% into hair follicles (T. szidati 16%). The S. mansoni cercariae needed, on average, 6.58 min (range of 1.57-13.13 min) for full entry, while T. szidati needed 4.0 min (range of 1.38-13.34 min); the fastest S. mansoni cercaria entered the skin within 94 s, while T. szidati entered within 83 s. Sixty percent of the S. mansoni cercariae had the tails still attached when the bodies disappeared in the skin whereas all T. szidati cercariae shed their tails within 0-105 s after the onset of penetration movements. The faster invasion of T. szidati may result from the more sophisticated host-finding mechanisms of this species. Regarding S. mansoni, cercarial dermatitis, as immediate skin response, developed after a sensitization period of 19 days.

Schistosoma mansoni cercariae recognize the human host with a sequence of behavioral responses particularly to chemical host cues. After attaching to the skin surface, cercariae are stimulated by so far unknown skin components to hold enduring contact with the skin and to start creeping towards entry sites. We studied the chemical stimulus of human skin for the cercarial enduring contact response by fractionation of human and pig skin surface extracts and offering the fractions to the cercariae via membrane filters. Enduring contact was stimulated exclusively by ceramides, specific lipids of the uppermost skin layers. This chemical cue differs from the 6 chemical host signals used by S. mansoni cercariae in other behavioral steps of host invasion, and thus underlines the specialization of S. mansoni cercariae particularly in chemical host signals. All together, the enduring contact response of the cercariae is, like the other phases of host invasion, well adapted to the chemical properties of human skin.

SUMMARYCercariae of Diplostomum spathaceum penetrate the skin of fish, and then migrate along blood vessels and tissues towards the head and the eye-lens. We studied their orientation behaviour in tail fins of guppies and in chemical concentration gradients within agar-filled choice chambers. In fins, they entered veins and orientated cranially, independent of the blood flow and living cells. In choice chambers, they were attracted by a small molecular fraction of fish serum, D-glucose (at 1, 10, and 1000 μm), D-mannose, D-maltotriose and Cl-ions, whereas D-glucosamine repelled them (even at 1·0 nm). Amino acids were not attractive, but arginine in tetrapeptides attracted at concentrations as low as 1 μm and melatonin at 0·4–4·3 pm. We suggest a preliminary model for the behaviour of diplostomula in fish fins and attracting (+) or repelling (−) host cues: (1) migration towards deeper skin layers and avoidance of skin surface, cues: Cl-ions (+ and −), glucose (+), glucosamine (−), light radiation (−); (2) orientation in cranial direction, cue: Cl-ions (+); (3) localization of blood vessels, cues: glucose (+), arginine-residues (+); (4) localization of the retina, cue: melatonin (+). A comparison with the navigation mechanisms of tissue-migrating schistosomules and hookworm larvae reveals an enormous diversity of strategies.

Leishmaniasis is caused by protozoa of the genus Leishmania and transmitted by sand flies from mammalian reservoirs to humans. In recent years, a northward spread of L. infantum from highly endemic Mediterranean countries into previously non-endemic Central European areas has been suspected based on presumed sporadic cases of autochthonous leishmaniasis. Here, we investigated whether sand flies are prevalent in Bavaria in Southern Germany, a federal state in which autochthonous cases have previously been reported. Considering the present and future climatic conditions, we determined whether Bavaria is suitable for five sand fly species with assumed spreading tendencies towards Central Europe: Phlebotomus ariasi, P. neglectus, P. perfiliewi and P. perniciosus that are known vectors for Leishmania in Europe, and P. mascittii, a suspected but not proven vector. Within Bavaria we defined sampling regions based on their climatic suitability and their spatial distance to the sites of the autochthonous cases and/or to areas of reported sand fly detection in states adjacent to Bavaria. At 155 locations in 7 sampling regions, CDC light traps were placed during 38 nights in the summers of 2009 and 2010, resulting in 202 trap-nights. All traps were negative for sand flies. The results suggest that Bavaria is not yet endemic for sand flies, but do not exclude the possibility of sporadic cases of autochthonous human or zoonotic Leishmania infections. This study, which combined methodological approaches from different disciplines, serves as reference for future surveys and risk analyses of sand flies and leishmaniasis in so far non-endemic areas of Europe.