Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.

A comparative study of the intercellular connections between parasite/host in two Gelidiocolax/Gelidium algal parasitic systems

Comparative ultrastructural study of the intercellular connections between parasite and host cells in two algal parasitic systems, Gelidiocolax christianae Feldmann and Feldmann/Ge/iV/ium spathulatum (Kutz.) Bornet and Gelidiocolax deformans Seoane Camba/Gelidium sesquipedale (Clem.) Thur, shows quantitative and structural differences. The number of free conjunctor cells (before fusión with the adjacent host cells) differs between the two parasitic systems and is inversely related to the number of complex pit connections. The fibrillar cell wall structure of the conjunctor cells and the lamellar structure of the complex pit plugs in the two systems are also different A hypothesis concerning the different activity of the conjuntor cell wall in the two parasitic systems, related with the different structural appearance, is proposed.

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.

Signs of a vector's adaptive choice: on the evasion of infectious hosts and parasite-induced mortality

Laboratory and field experiments have demonstrated in many cases that malaria vectors do not feed randomly, but show important preferences either for infected or non-infected hosts. These preferences are likely in part shaped by the costs imposed by the parasites on both their vertebrate and dipteran hosts. However, the effect of changes in vector behaviour on actual parasite transmission remains a debated issue. We used the natural associations between a malaria-like parasite Polychromophilus murinus, the bat fly Nycteribia kolenatii and a vertebrate host the Daubenton's bat Myotis daubentonii to test the vector's feeding preference based on the host's infection status using two different approaches: 1) controlled behavioural assays in the laboratory where bat flies could choose between a pair of hosts; 2) natural bat fly abundance data from wild-caught bats, serving as an approximation of realised feeding preference of the bat flies. Hosts with the fewest infectious stages of the parasite were most attractive to the bat flies that did switch in the behavioural assay. In line with the hypothesis of costs imposed by parasites on their vectors, bat flies carrying parasites had higher mortality. However, in wild populations, bat flies were found feeding more based on the bat's body condition, rather than its infection level. Though the absolute frequency of host switches performed by the bat flies during the assays was low, in the context of potential parasite transmission they were extremely high. The decreased survival of infected bat flies suggests that the preference for less infected hosts is an adaptive trait. Nonetheless, other ecological processes ultimately determine the vector's biting rate and thus transmission. Inherent vector preferences therefore play only a marginal role in parasite transmission in the field. The ecological processes rather than preferences per se need to be identified for successful epidemiological predictions.

Trophic structure in a seabird host-parasite food web: insights from stable isotope analyses

Ecological studies on food webs rarely include parasites, partly due to the complexity and dimensionality of host-parasite interaction networks. Multiple co-occurring parasites can show different feeding strategies and thus lead to complex and cryptic trophic relationships, which are often difficult to disentangle by traditional methods. We analyzed stable isotope ratios of C (13C/12C, δ13C) and N (15N/14N, δ15N) of host and ectoparasite tissues to investigate trophic structure in 4 co-occurring ectoparasites: three lice and one flea species, on two closely related and spatially segregated seabird hosts (Calonectris shearwaters). δ13C isotopic signatures confirmed feathers as the main food resource for the three lice species and blood for the flea species. All ectoparasite species showed a significant enrichment in δ15N relatively to the host tissue consumed (discrimination factors ranged from 2 to 5 depending on the species). Isotopic differences were consistent across multiple host-ectoparasite locations, despite of some geographic variability in baseline isotopic levels. Our findings illustrate the influence of both ectoparasite and host trophic ecology in the isotopic structuring of the Calonectris ectoparasite community. This study highlights the potential of stable isotope analyses in disentangling the nature and complexity of trophic relationships in symbiotic systems.

Spermiogenesis and spermatozoon ultrastructure of the dilepidid cestode Molluscotaenia crassiscolex (von Linstow, 1890), an intestinal parasite of the common shrew Sorex araneus

Spermiogenesis in Molluscotaenia crassiscolex begins with the formation of a differentiation zone containing two centrioles. One of the centrioles develops a flagellum directly into the cytoplasmic extension. The nucleus elongates and later migrates along the spermatid body. During advanced stages of spermiogenesis, a periaxonemal sheath appears in the spermatid. Spermiogenesis finishes with the appearance of a single helicoidal crested body at the base of the spermatid and, finally, the narrowing of the ring of arched membranes causes the detachment of the fully formed spermatozoon. The mature spermatozoon of M. crassiscolex exhibits a partially detached crested body in the anterior region of the spermatozoon, one axoneme, twisted cortical microtubules, a periaxonemal sheath, and a spiralled nucleus. The anterior spermatozoon extremity is characterized by the presence of an electron-dense apical cone and a single spiralled crested body, which is attached to the sperm cell in the anterior and posterior areas of region I, whereas in the middle area it is partially detached from the cell. This crested body is described for the first time in cestodes. The posterior extremity of the male gamete exhibits only the disorganizing axoneme. Results are discussed and compared particularly with the available ultrastructural data on dilepidids sensu lato.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

Molecular Evolution of Metazoan Hypoxia-inducing Factors

Most metazoans rely on aerobic energy production, which is dependent on adequate oxygen supply. In the case of reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors named hypoxia-inducible factors (HIF alpha). These proteins are post-translationally regulated by prolyl-4-hydroxylase (PHD) enzymes that are direct “sensors” of cellular oxygen levels. This thesis examines the molecular evolution of metazoan HIF systems. In early metazoans the HIF system emerged from pre-existing PHD oxygen sensors and early bHLH-PAS transcription factors. In invertebrates our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics. An early branching vertebrate, the epaulette shark (Hemiscyllium ocellatum) was chosen for sequencing and hypoxia preconditioning studies of HIF alpha and PHD genes. As no quantitative PCR reference genes were available, this thesis includes the first study of reference genes in cartilaginous fish species. Applying multiple statistical analysis we also discoveredthat commonly used reference gene software may perform poorly with some data sets. Novel reference genes allowed accurate measurements of the mRNAlevels of the studied target genes. Cartilaginous fishes have three genomic duplicates of both HIF alpha and PHD genes like mammals and teleost fishes. Combining functional divergence and selection analyses it was possible to describe how sequence changes in both HIF alpha and PHD duplicates may have contributed to the differential oxygen sensitivityof HIF alphas. Additionally, novel teleost HIF-1 alpha sequences were produced and used to reveal the molecular evolution of HIF-1 alpha in this lineage rich with hypoxia tolerant species.

Host Parasite Interactions in Damselflies. From Individuals to Populations

The main goal of this thesis is to increase understanding on evolutionary and ecological factors that have contributed to differences in parasite numbers in insects. Furthermore, the thesis addresses the effects of parasites on their hosts. The most important findings were: The Northern damselfly’s (Coenagrion hastulatum) immune response to artificial pathogen increased with increasing parasite numbers (Article I). Marginal, more isolated C. hastulatum populations on the edge of distribution have fewer parasites when compared to distribution’s core populations (Article II). The Banded damselfly Calopteryx splendens individuals with higher homozygosity have more parasites, however, the rate of homozygosity did not differ between populations (Article III). Parasite prevalence was affected by whether the host species occurred in allopatric or sympatric population: sympatric C. splendens populations with sister species the Beautiful damselfly Calopteryx virgo harbored more parasites (Article IV). Parasites were associated with the wing spot size, an ornament under sexual selection, and thus may play an important role in character displacement, i.e. the size of the wing spot (Article V). To conclude with, this thesis brings about new information on the parasite infection patterns in insects, proposing several factors to contribute to these patters, as well as it addresses the effects of parasites on their hosts, from individual to population level.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

Apoptosis in parasites and parasite-induced apoptosis in the host immune system: a new approach to parasitic diseases

Apoptosis, a form of programmed cell death (PCD), has been described as essential for normal organogenesis and tissue development, as well as for the proper function of cell-renewal systems in adult organisms. Apoptosis is also pivotal in the pathogenesis of several different diseases. In this paper we discuss, from two different points of view, the role of apoptosis in parasitic diseases. The description of apoptotic death in three different species of heteroxenic trypanosomatids is reviewed, and considerations on the phylogenesis of apoptosis and on the eventual role of PCD on their mechanism of pathogenesis are made. From a different perspective, an increasing body of evidence is making clear that regulation of host cell apoptosis is an important factor on the definition of a host-pathogen interaction. As an example, the molecular mechanisms by which Trypanosoma cruzi is able to induce apoptosis in immunocompetent cells, in a murine model of Chagas' disease, and the consequences of this phenomenon on the outcome of the experimental disease are discussed.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

Interruption of the blood-stage cycle of the malaria parasite, Plasmodium chabaudi, by protein tyrosine kinase inhibitors

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37ºC with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 µM - 75%), staurosporine (1 µM - 58%), R03 (1 µM - 75%), and tyrphostins B44 (100 µM - 66%) and B46 (100 µM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.