Observations on egg production in the monogenean Entobdella soleae

Egg production in the monogenean Entobdella soleae increases as adult parasites grow. Medium-sized adults (anterior hamulus length 550–600 μm; total body length approx. 5 mm) produced about 30 eggs per day at 12°C. Parasites with anterior hamuli exceeding 650 μm in length (total body length about 6 mm) may produce more than 60 eggs per day. In smaller adults, eggs tend to spend longer in the ootype after assembly of the shell and the ootype remains empty for longer periods of time. The time taken to assemble a single egg is relatively constant (4–6 min) in adults of all sizes and there is evidence of a small but significant increase in egg size as parasites increase in size. Some eggs were retained in the uterus for as long as 168 min but others spent less than 5 s in the uterus, indicating that the uterus is no more than a passageway for these eggs to the outside world. The egg cell (or zygote) precedes the vitelline cells as they progress along the ovo-vitelline duct to the ootype.     

Infection of adult migratory river shrimps,Macrobrachium ohione,by the branchial bopyrid isopod Probopyrus pandalicola

Abstract. Macrobrachium ohione is a migratory (amphidromous) river shrimp (Decapoda, Caridea) that may be parasitized by the branchial parasite Probopyrus pandalicola (Isopoda, Bopyridae). The parasite disrupts gonadal maturation and spawning in female shrimps, resulting in the total loss of reproduction. Shrimps are usually infected by bopyrid parasites during the late zoeal or early postlarval stages; in this study, we investigated the apparent parasite infection of adult shrimps. We analyzed the relationships between parasite body size (total length) and host shrimp body size (carapace length) to test the hypothesis that parasite infection of adult shrimps occurs during the shrimps' reproductive migrations. The results presented here indicate that infection of adult shrimps is common in M. ohione in the Atchafalaya and Mississippi Rivers, Louisiana, USA. In the two upriver sites sampled, Butte La Rose (BLR) and River Bend (RB), parasite size was not associated with host body size. In these locations, many parasitized adult M. ohione were infected with immature P. pandalicola (40.3% in BLR and 51.2% in RB), indicating that the shrimps were adults at the time of infection. A possible explanation is that when female shrimps enter the estuary to hatch larvae, they molt and spawn another brood. The smaller male shrimps that accompany the females downstream are also assumed to molt and continue growth. The intermediate host of the parasite is an estuarine copepod, and thus the parasite cryptoniscus larva that infects the host shrimp is primarily estuarine as well. Newly molted shrimps have soft cuticles, which may facilitate their infection by parasite cryptonisci. Our conclusion is that most infections of adult shrimps occur during their migration into estuarine waters, the primary habitat of infective parasite larvae, and that host vulnerability is probably increased following host ecdysis.     

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp^? parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines.     

Infection of Sphyrion laevigatum (Copepoda: Sphyriidae) on Genypterus blacodes (Pisces: Ophidiidae) from the Falkland Islands,South Atlantic

The degree of parasitism by the parasitic copepod Sphyrion laevigatum on kingclip Genypterus blacodes in the Falkland Islands was investigated. In a sample of 719 kingclip, ranging in size from 35 to 145 cm total length, the prevalence of infection was 49%. The number of parasites per fish ranged from 0 to 24, with only 2% of the sample having more than 5 parasites. There was a significant increase in mean abundance of parasites with increasing fish age and length (P < 0.01 and P < 0.05, respectively). In addition, there was a significant difference in the prevalence in 1 of 3 geographical areas for the largest size class.     

Experimental Infection of Dogs with Leishmania and Saliva as a Model to Study Canine Visceral Leishmaniasis

Background

Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches.

Methodology/Principal Findings

In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1×10⁷ parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35×10⁵ parasites in their lymph nodes. An increase of IFN-γ and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection.

Conclusion

The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.     

Scale‐dependent variation in visual estimates of grassland plant cover

Abstract. Plant cover was visually estimated by five observers, independent of each other, in a species‐rich grassland in the Bílé Karpaty Mts., southeastern Czech Republic, in seven plots ranging from 0.001 to 4 m². Variation of total plant cover among the observers was high at small scales: 0.001–0.016 m²; coefficient of variation, CV = 35 to 45%, but much lower at larger scales: 0.06–4 m²; CV = 7 to 15%. Differences between visual estimates of plant cover of individual species made by different observers were affected by plot size, total cover and morphology of particular plants. CV of the cover of individual species ranged from 0 to 225% and decreased with increasing plot size. For abundant plants the CV attained ca. 50%, independent of plot size. In spite of a very high number of sterile plants with similar leaf morphology and colour, the observed variation in cover estimates in the studied grassland was comparable with results reported from other vegetation types. Differences between estimates by individual observers were often larger than usual year to year changes in undisturbed grasslands. Therefore, I suggest that to avoid difficulties in the interpretation of results based on plant cover data obtained from visual estimates, several observers should always work together, adjusting their extreme estimates.     

Calcium signalling in malaria parasites

Ca²⁺ is a ubiquitous intracellular messenger in malaria parasites with important functions in asexual blood stages responsible for malaria symptoms, the preceding liver‐stage infection and transmission through the mosquito. Intracellular messengers amplify signals by binding to effector molecules that trigger physiological changes. The characterisation of some Ca²⁺ effector proteins has begun to provide insights into the vast range of biological processes controlled by Ca²⁺ signalling in malaria parasites, including host cell egress and invasion, protein secretion, motility and cell cycle regulation. Despite the importance of Ca²⁺ signalling during the life cycle of malaria parasites, little is known about Ca²⁺ homeostasis. Recent findings highlighted that upstream of stage‐specific Ca²⁺ effectors is a conserved interplay between second messengers to control critical intracellular Ca²⁺ signals throughout the life cycle. The identification of the molecular mechanisms integrating stage‐transcending mechanisms of Ca²⁺ homeostasis in a network of stage‐specific regulator and effector pathways now represents a major challenge for a meaningful understanding of Ca²⁺ signalling in malaria parasites.     

Purinergic signalling is involved in the malaria parasite <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca²⁺ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca²⁺ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca²⁺ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca²⁺ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca²⁺]_c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca²⁺]_c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Diel vertical movements,and effects of infection by the cestode <Emphasis Type="Italic">Schistocephalus solidus</Emphasis> on daytime proximity of three-spined sticklebacks <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis> to the surface of a large Alaskan lake

We conducted a field study in Iliamna Lake, Alaska, to test the hypothesis that proximity of three-spined sticklebacks Gasterosteus aculeatus to the lake’s surface during the daytime varies with macroparasitic cestode parasite Schistocephalus solidus infection in a manner consistent with enhanced vulnerability to avian predators. Extensive sampling in the lake and likelihood-based modeling revealed that sticklebacks displayed a diel vertical migration, being closer to the surface at night than during the evening and early morning. Additional sampling, also coupled with a likelihood-based modeling approach, showed that fish caught at the surface of the lake during the day were more often parasitized (76 vs. 65%), more heavily parasitized (26.8 vs. 22.7% of their body mass), and had larger individual parasites (0.24 vs. 0.20 g) than those caught at night. Parasite infection was related, non-linearly, to fish size, which also differed between day and night sampling at the surface. We performed statistical competitions among nested hierarchies of models that accounted for this effect of length. The most likely models indicated that fish captured during the day had greater parasite prevalence, higher parasite burdens, and larger parasites than did fish captured at night. Proximity to the surface during the day in this very clear lake would likely increase the vulnerability of sticklebacks to predation from birds, enabling completion of the parasite’s lifecycle.     

Size at first sexual maturity,fecundity, length–weight and length–length relationships of Puntius sophore (Cyprinidae) in Bangladeshi waters

The present study describes the size at first sexual maturity, fecundity, length–weight (LWRs) and length–length relationships (LLRs) of the pool barb, Puntius sophore, using data obtained from different geographical locations in Bangladesh. A total of 905 specimens were caught by traditional fishing gear from March 2010 to February 2011. Additionally, a total of 121 females were collected from a commercial catch of the Padma River during June–July 2011 to estimate size at first maturity and to determine fecundity. Total length (TL), fork length (FL) and standard length (SL) were measured with digital slide calipers. Individual body weights (BW) were determined for all specimens, and gonad weights (GW) from 121 females were weighed to an accuracy of 0.001 g. The female gonadosomatic index (GSI) was calculated as [GSI (%) = (GW/BW) × 100]. Female size at first maturity was estimated using GSI and TL as indicators, and estimated as 5.00 cm TL in the Padma River. Specimens larger than 5.00 cm TL were used to determine fecundity. Mean total fecundity was 5300 ± 2700, ranging from 1580 to 16590. A positive exponential correlation was recorded between total fecundity and total length (r² = 0.421). Relative fecundity ranged from 466 to 4036 (mean 1100 ± 580) in the Padma River. The LWR of pooled data for sexes combined was estimated as BW = 0.0155 TL^2.98 as ancova revealed no significant differences in LWRs between rivers (P > 0.05). All LLRs were highly correlated (r² > 0.983; P < 0.001), and ancova analyses further indicated that LLRs did not differ between rivers (P > 0.05). These results will help in further studies on the population assessment of the species.     

Local diversity reduces infection risk across multiple freshwater host‐parasite associations

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Theileria parva: Kinetics of infection in the lymphoid system of cattle

The kinetics of infection with Theileria parva in cattle were studied by examining the total cellularity and numbers of parasites in a range of lymphoid organs from animals killed at intervals during the course of the infection. With the dose of T. parva stabilate used, macroschizonts were initially detected in the drainage lymph node about 7 days after inoculation and death of the host resulted on Day 18–19. Associated with the initial detection of parasites, there was a marked increase in cellularity of the drainage lymph node and a more gradual and less pronounced increase in cellularity of the other lymphoid organs. From about Day 12 onward, there was a gradual decrease in the cellularity in all of the lymphoid organs, so that in animals examined in the terminal stages of the infection there was often cellular depletion. The pattern of these cellular changes was similar in groups of Boran and Friesian cattle, although both the increase in cellularity and the terminal depletion were more marked in the Friesians. Blood leukocyte counts in infected Boran started to drop as early as Day 7 of infection and by Day 14 had reached values less than 25% of normal. Quantitation of parasitic schizonts indicated that the numbers of parasites in the lymphoid organs do not increase in a simple exponential manner. Rather, there appears to be an early rapid increase in parasite numbers followed by a phase of less rapid multiplication. Because of the marked changes which occured in total cellularity of the lymphoid organs during the course of the infection, a significant discrepancy was found between the replication rate of the parasite as calculated using total numbers of parasites and that obtained using schizont index (SI). These results indicated that the use of SI, as described in previous studies, is not a reliable method of determining the replication rate of the parasite.     

The effect of Schistocephalus solidus infection on meal size of three‐spined stickleback

The effect of infection with the pseudophyllidean cestode Schistocephalus solidus on the meal size of individually housed three‐spined stickleback Gasterosteus aculeatus was quantified. Infected fish harboured plerocercoid loads that contributed from 1·1 to 33·9% of their total mass. Across this range of infection levels, the presence of S. solidus infection had no significant directional effect on standard length ( L _S) corrected meal size of host three‐spined sticklebacks. Amongst multiply infected fish there was a significant negative relationship between L _S‐corrected meal size and the proportion of host mass contributed by S. solidus parasites. This relationship, however, did not hold for singly‐infected fish. Furthermore, the data suggest that multiply‐infected fish that harbour a combined mass of parasites contributing < c . 15% to host body mass might exhibit meal sizes that exceed those of length‐matched uninfected fish. The results suggest that although heavy infections can significantly reduce the meal size of heavily infected three‐spined sticklebacks, in the early stages of multiple S. solidus infections host food intake may increase. The probable causes of these differential effects on meal size and their consequences for the host‐parasite system are discussed.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Ecological implications of parasites in natural <Emphasis Type="Italic">Daphnia</Emphasis> populations

In natural host populations, parasitism is considered to be omnipresent and to play an important role in shaping host life history and population dynamics. Here, we study parasitism in natural populations of the zooplankton host Daphnia magna investigating their individual and population level effects during a 2-year field study. Our results revealed a rich and highly prevalent community of parasites, with eight endoparasite species (four microsporidia, one amoeba, two bacteria and one nematode) and six epibionts (belonging to five different taxa: Chlorophyta, Bacillariophyceae, Ciliata, Fungi and Rotifera). Several of the endoparasites were associated with a severe overall fecundity reduction of the hosts, while such effects were not seen for epibionts. In particular, infections by Pasteuria ramosa, White Fat Cell Disease and Flabelliforma magnivora were strongly associated with a reduction in overall D. magna fecundity. Across the sampling period, average population fecundity of D. magna was negatively associated with overall infection intensity and total endoparasite richness. Population density of D. magna was negatively correlated to overall endoparasite prevalence and positively correlated with epibiont richness. Finally, the reduction in host fecundity caused by different parasite species was negatively correlated to both parasite prevalence and the length of the time period during which the parasite persisted in the host population. Consistent with epidemiological models, these results indicate that parasite mediated host damages influence the population dynamics of both hosts and parasites.     

Male rescue maintains low frequency parthenogenesis-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> infection in <Emphasis Type="Italic">Trichogramma</Emphasis> populations

Parthenogenesis-inducing (PI) Wolbachia bacteria are reproductive parasites that cause infected (W ⁺) female haplodiploid parasitoids to produce daughters without fertilization by males. Theoretically, PI Wolbachia infection should spread to fixation within Trichogramma populations as males are no longer required to produce female offspring. Infections in some naturally occurring Trichogramma populations are, however, maintained at frequencies ranging from 4 to 26%. Here we describe discrete equation models to examine if the PI Wolbachia infection in Trichogramma populations can be maintained at relatively low frequencies by mating regularity. Model outcomes suggest the probability of W ⁺ females mating could stabilize Wolbachia infection frequency at low levels in Trichogramma populations. The primary mechanism maintaining low-level PI Wolbachia infection in Trichogramma populations is reducing the survivorship from egg to adult in infected relative to uninfected females. The model successfully demonstrates that the relatively low PI Wolbachia infection frequency in host populations can be maintained by fertilization, or male rescue, of infected eggs, which avoids potentially hazardous gamete duplication that occurs during Wolbachia-induced parthenogenesis.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Imaging liver‐stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High‐speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver‐stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.