Life cycle complexity, environmental change and the emerging status of salmonid proliferative kidney disease

P>1. Proliferative kidney disease (PKD) is a disease of salmonid fish caused by the endoparasitic myxozoan, Tetracapsuloides bryosalmonae, which uses freshwater bryozoans as primary hosts. Clinical PKD is characterised by a temperature-dependent proliferative and inflammatory response to parasite stages in the kidney.;2. Evidence that PKD is an emerging disease includes outbreaks in new regions, declines in Swiss brown trout populations and the adoption of expensive practices by fish farms to reduce heavy losses. Disease-related mortality in wild fish populations is almost certainly underestimated because of e.g. oversight, scavenging by wild animals, misdiagnosis and fish stocking.;3. PKD prevalences are spatially and temporally variable, range from 0 to 90-100% and are typically highest in juvenile fish.;4. Laboratory and field studies demonstrate that (i) increasing temperatures enhance disease prevalence, severity and distribution and PKD-related mortality; (ii) eutrophication may promote outbreaks. Both bryozoans and T. bryosalmonae stages in bryozoans undergo temperature- and nutrient-driven proliferation.;5. Tetracapsuloides bryosalmonae is likely to achieve persistent infection of highly clonal bryozoan hosts through vertical transmission, low virulence and host condition-dependent cycling between covert and overt infections. Exploitation of fish hosts entails massive proliferation and spore production by stages that escape the immune response. Many aspects of the parasite's life cycle remain obscure. If infectious stages are produced in all hosts then the complex life cycle includes multiple transmission routes.;6. Patterns of disease outbreaks suggest that background, subclinical infections exist under normal environmental conditions. When conditions change, outbreaks may then occur in regions where infection was hitherto unsuspected.;7. Environmental change is likely to cause PKD outbreaks in more northerly regions as warmer temperatures promote disease development, enhance bryozoan biomass and increase spore production, but may also reduce the geographical range of this unique multihost-parasite system. Coevolutionary dynamics resulting from host-parasite interactions that maximise fitness in previous environments may pose problems for sustainability, particularly in view of extensive declines in salmonid populations and degradation of many freshwater habitats.

Non-invasive quantitative estimation of bone density in rats throughout the life cycle and in arthritic osteopenia: preliminary results

A longitudinal bone survey was conducted in 86 female Wistar rats in order to assess mineral density kinetics from young age (5 weeks: 115 g) till late adulthood (64 weeks: 586 g). In vivo quantitative radiographic scanning was performed on the caudal vertebrae, taking trabecular mass as the parameter. Measurements were expressed as Relative Optical Density (ROD) units by means of a high resolution densitometric device. Results showed a progressive increase in mineral density throughout the life cycle, with a tendency to level in the higher weight range, indicating that progressive mineral aposition occurs in rats in dependency of age. This phenomenon, however, should be always considered within the context of continuous skeletal growth and related changes typical of this species. Twelve different animals were also examined following induction of articular inflammation with Freund's adjuvant in six of them. Bone survey conducted 12 to 18 days after inoculation revealed a significant (P less than 0.01) reduction in trabecular bone mass of scanned vertebrae in comparison with the weight-matched untreated controls. It is concluded that the in vivo quantitative assessment of bone density illustrated in this report represents a sensitive and useful tool for the long-term survey of naturally occurring or experimentally induced bone changes. Scanning of the same part of the skeleton can be repeated, thereby avoiding sacrifice of the animal and time-consuming preparation of post-mortem material.

The end of the corporate life cycle

This paper asks how takeover and failure hazards change as listed firms get older. The hypothesis is that they increase because firms gradually run out of growth opportunities. We find the opposite. Both takeover and failure hazard drop significantly with age. The decline in takeover hazard can be explained with Loderer, Stulz, and Waelchli’s (2013) “buggy whip makers” hypothesis: Because old firms are comparatively well-managed and are affected by limited agency problems, on average, they offer little value added potential to acquirers. Failure hazard drops because to learning. The results are robust to various alternative interpretations and cannot be explained by unobserved heterogeneity. While hazards decline with age, they do not go to zero. This explains why, eventually, all listed firms disappear

The end of the corporate life cycle

This paper asks how takeover and failure hazards change as listed firms get older. The hypothesis is that they increase because firms gradually run out of growth opportunities. We find the opposite. Both takeover and failure hazard drop significantly with age. The decline in takeover hazard can be explained with Loderer, Stulz, and Waelchli’s (2013) “buggy whip makers” hypothesis: Because old firms are comparatively well-managed and are affected by limited agency problems, on average, they offer little value added potential to acquirers. Failure hazard drops because to learning. The results are robust to various alternative interpretations and cannot be explained by unobserved heterogeneity. While hazards decline with age, they do not go to zero. This explains why, eventually, all listed firms disappear

The end of the corporate life cycle

This paper asks how takeover and failure hazards change as listed firms get older. The hypothesis is that they increase because firms gradually run out of growth opportunities. We find the opposite. Both takeover and failure hazard drop significantly with age. The decline in takeover hazard can be explained with Loderer, Stulz, and Waelchli’s (2013) “buggy whip makers” hypothesis: Because old firms are comparatively well-managed and are affected by limited agency problems, on average, they offer little value added potential to acquirers. Failure hazard drops because to learning. The results are robust to various alternative interpretations and cannot be explained by unobserved heterogeneity. While hazards decline with age, they do not go to zero. This explains why, eventually, all listed firms disappear

Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle

BACKGROUND: Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. RESULTS: NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. CONCLUSIONS: PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work.

GFP-targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites.

BACKGROUND INFORMATION The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo-erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. RESULTS In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo-erythrocytic schizogony in vitro, leading to impaired parasite maturation. CONCLUSIONS Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red-blood-cell-infective merozoites.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Sublethal neonicotinoid insecticide exposure reduces solitary bee reproductive success

1.Pollinating insects provide crucial and economically important ecosystem services to crops and wild plants, but pollinators, particularly bees, are globally declining as a result of various driving factors, including the prevalent use of pesticides for crop protection. Sublethal pesticide exposure negatively impacts numerous pollinator life-history traits, but its influence on reproductive success remains largely unknown. Such information is pivotal, however, to our understanding of the long-term effects on population dynamics. 2.We investigated the influence of field-realistic trace residues of the routinely used neonicotinoid insecticides thiamethoxam and clothianidin in nectar substitutes on the entire life-time fitness performance of the red mason bee Osmia bicornis. 3.We show that chronic, dietary neonicotinoid exposure has severe detrimental effects on solitary bee reproductive output. Neonicotinoids did not affect adult bee mortality; however, monitoring of fully controlled experimental populations revealed that sublethal exposure resulted in almost 50% reduced total offspring production and a significantly male-biased offspring sex ratio. 4.Our data add to the accumulating evidence indicating that sublethal neonicotinoid effects on non-Apis pollinators are expressed most strongly in a rather complex, fitness-related context. Consequently, to fully mitigate long-term impacts on pollinator population dynamics, present pesticide risk assessments need to be expanded to include whole life-cycle fitness estimates, as demonstrated in the present study using O. bicornis as a model.

Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology

Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

Proteomic analysis of the Theileria annulata schizont

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.