Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.     

东海原甲藻(Prorocentrum donghaiensis)对原生动物群落结构影响的现场船基实验

2005年在长江口赤潮频发海域调查期间,分别于4月27日、5月4日和5月8日,在zzf1、zc18a和ra5站位利用现场船基培养的方法,研究了添加到赤潮密度10^6cells L^-1的东海原甲藻（Prorocentrum donghaiense）对原生动物群落结构的影响。结果发现,赤潮密度的东海原甲藻抑制了小型无壳纤毛虫的种群数量,而没有抑制中大型砂壳纤毛虫和夜光虫（Noctiluca scintillans）的种群数量,从而使得原生动物群落向中大型种类演替,这种影响的程度与原生动物本身的群落组成和浮游植物的组成密切相关。添加东海原甲藻72h后,在以小型无壳纤毛虫管游虫（Cyrtostrombidium sp．）为优势种的zzf1站位,演替为以大型原生动物夜光虫为优势种的群落;在以中大型原生动物百乐拟铃虫（Tintinnospsis beroidea）和夜光虫为优势种的zc18a站位,仍然是以此为优势种,且大型原生动物夜光虫在群落中的比例上升;在以小型无壳纤毛虫急游虫2（Strombidium sp．2）为优势种的ra5站位,演替为以中大型原生动物百乐拟铃虫和亚速岛网纹虫（Favella azorica）为优势种的群落。zzf1和ra5站位实验组中原生动物的总丰度都呈下降趋势,而zc18a站位变化不明显,这是由于前两个站位的最优势种管游虫（zzf1站位）和急游虫2（ra5站位）的丰度迅速下降,而zc18a站位的优势种百乐拟铃虫和夜光虫的丰度比较稳定造成的。在zzf1和zc18a站位,对照组和实验组中原生动物的总生物量在实验前后都没有大的变化,而在ra5站位却均呈下降的趋势。这可能与浮游植物的组成有关,zzf1和zc18a站位是以无毒的锥状斯氏藻（Scrippsiella trochoidea）和螺旋环沟藻（Gyrodinium spirale）为主,而ra5站位是以有毒的亚历山大藻（Alexandriumsp．）为主。综上可见,可预测当东海大规模赤潮爆发时,会使原生动物群落向中大型种类演替,进而可能影响海洋生态系统的结构和功能。     

Investigating how clathrin adaptor complex AP-2 participates in Giardia lamblia encystation

The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease.     

TNF but not Fas ligand provides protective anti-L. major immunity in C57BL/6 mice

The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNF-deficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF(-/-), B6.gld and C57BL/6.gld x TNF(-/-) (B6.gld.TNF(-/-)) double-negative mice. Visceral, fatal disease was only observed in B6.TNF(-/-) mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF(-/-) mice were similar to those of B6.TNF(-/-) mice. B6.gld.TNF(-/-) mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF(-/-) mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen.     

Leishsmania (Leishmania) amazonensis infection: Muscular involvement in BALB/c and C3H.HeN mice

Recent studies have provided some insights into Leishsmania (Leishmania) amazonensis muscular infection in dogs, although, muscular disease due to leishmaniasis has been poorly documented. The aim of our study was to evaluate involvement of Leishmania in muscular infection of two distinct mouse strains (BALB/c and C3H.He), with different genetic backgrounds. BALB/c mice, susceptible to Leishmania infection, showed, at the beginning of infection, a great number of infected macrophages among muscle fibers; however, in C3H.He resistant mice, muscle fibers were less damaged than in BALB/c mice, but some parasitized macrophages could be seen among them. A follow up of the infection showed an intense inflammatory infiltrate mainly composed of infected macrophages in BALB/c muscles and the presence of amastigotes within muscle fibers; while C3H.He mice exhibited a moderate inflammatory infiltrate among skeletal muscle fibers and an absence of amastigotes. Total destruction of muscles was observed in BALB/c mice in the late phase of infection (day 90) while C3H.He mice showed a process of muscle repair. We concluded that: (1) the muscles of BALB/c mice were more affected by leishmaniasis than those of C3/H.He mice; (2) Leishmania amastigotes are capable of infecting muscular fibers, as observed in BALB/c mice; (3) as inflammatory infiltrate is less intense in C3H.He mice these animals are capable of restoring muscular fibers.     

Role of Plasmodium berghei cGMP-dependent Protein Kinase in Late Liver Stage Development

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.     

Epibiont protozoan communities on Caridina lanceolata (Crustacea, Decapoda) from the Malili lakes of Sulawesi (Indonesia)

The epibiont protozoan communities living on the freshwater shrimp Caridina lanceolata Woltereck, 1937a from the three major lakes (Towuti, Matano and Mahalona) of the Malili lake system (Sulawesi, Indonesia) were studied. The number of epibionts varied between 2 and 971 per shrimp. Seven protozoan ciliate species were found: Acineta sulawesiensis n. sp., Cothurnia sp., Zoothamnium sp. (in all three lakes), Vorticella sp. (Lake Mahalona and Lake Matano), Opercularia sp. (Lake Mahalona), Epistylis sp. (Lake Mahalona and Lake Matano), and Podophrya sp. (Lake Mahalona). Although these ciliates had been found previously on other crustaceans, they have not been observed as epibionts on Caridina H. Milne Edwards, 1837. The distribution of the different epibiont species on the anatomical units of the shrimp was analyzed in each lake. There is a statistical significant difference between the three lakes in respect to the number of epibionts on each anatomical unit of all analyzed shrimps. The total and mean densities of each epibiont species on the different analyzed shrimps showed a significant difference between the three lakes; i.e., the presence of each epibiont species on the population of C. lanceolata varied from one lake to another. In Lake Towuti the highest density of epibionts was found on the anterior part of the shrimp body (rostrum, antennae, antennulae and eyes) (32.41%), while in the other two lakes, the highest colonization corresponded to the maxillipeds (31.56% Lake Matano, 40.89% Lake Mahalona). In Lake Towuti the rest of epibionts colonized mainly maxillipeds and pleopods (both 45.76% of epibionts). In Lake Matano, other epibionts were distributed principally on the anterior part of the body and pleopods (in total 57.18% of epibionts). In Lake Mahalona, other epibionts were divided among the anterior part of the body, pereiopods and pleopods (in total 57.39% of the epibionts). Uropods and telson were the units less colonized in Lake Matano (3.64%) and Lake Mahalona (1.72%), while in Lake Towuti, they presented a moderate density (13.18% of the epibionts). Taking into account the distribution of epibionts along the antero-posterior axis of the shrimp, considering the different anatomical units, there was a significant correlation between the three lakes. This fact indicates that, in the three lakes, the colonization on C. lanceolata followed a similar distribution pattern, independently of the epibiont species present. The comparison between the distributions of the same epibiont species along the longitudinal axis of the shrimp on the diverse lakes showed that they correlated respect to their density values on the anatomical units of the shrimp. Diverse aspects of the colonization patterns are discussed. Morphological features, taxonomic identification, and particular distribution of the epibiont species in each lake are also included.