In vitro spore formation and completion of the asexual life cycle of Neozygites parvispora, an obligate biotrophic pathogen of thrips

Capilliconidia, the asexual secondary spores of Neozygites parvispora (Zygomycetes, Entomophthorales) were produced in vitro either by entrapment of vegetative cells (hyphal bodies) in alginate pellets or after plating them onto water agar. Cultivation of the fungus for 3 days in a medium lacking hemolymph increased spore production 30 to 40-fold, and about 10% of the cells produced capilliconidia. The in vitro produced capilliconidia were infectious to Thrips tabaci and the fungus was reisolated from infected insects, thus completing its asexual life cycle under laboratory conditions. A decrease in capilliconidia production and a modification of the number of nuclei per spore were observed for isolates cultivated in vitro for more than 2 months, but subsequent host passages restored and increased sporulation efficiency without influencing the number of nuclei. Fungal cultures were stored at —80 °C for up to 7 months, and the capability to sporulate and infect T. tabaci was preserved. A bioassay procedure for infecting T. tabaci with N. parvispora is described, the first mycosed insects dying usually after 8 d of incubation.     

Novel aspects in the life cycle and biotrophic interactions in Pezizomycetes (Ascomycota,Fungi)

The ascomycete class Pezizomycetes (single order Pezizales) is known for its cup‐shaped fruit bodies and the evolution of edible truffles and morels, but little is known about the ontogeny and ecology of this large and ecologically diverse fungal group. In this issue of Molecular Ecology, Healy et al. ( 2013 ) make a great leap forward by describing and identifying asexual, anamorphic structures that produce mitotic spores in many ectomycorrhiza‐forming truffle and nontruffle species on soil surfaces worldwide (Fig.  1 ). Although such anamorphic forms have been reported sporadically from certain ectomycorrhizal and saprotrophic Pezizomycetes (e.g. Warcup 1990 ), Healy et al. ( 2013 ) demonstrate that these terricolous asexual forms are both taxonomically and geographically more widespread and, in fact, much more common than previously understood. We anticipate that deeper insight into other substrates, provided by molecular analyses of materials such as dead wood and seeds, is likely to reveal numerous anamorphs of saprotrophic and pathogenic Pezizomycetes as well (see Marek et al. 2009 ).     

Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro

Large numbers (10(4) to greater than 10(5)/ml) of Type I water-resistant Giardia lamblia cysts were produced in vitro under conditions that are characteristic of the human intestinal lumen. We define Type I cyst morphology as oval shaped, smooth, and refractile, with cyst wall, axostyle, and median body visible in relief by Normarski differential interference contrast optics. Human and porcine bile induced higher levels of encystation than bovine bile at the alkaline pH (7.8) which occurs in the human lower small intestine. High-pressure liquid chromatography analysis showed that the porcine bile had a preponderance of hyocholate, rather than cholate, while bovine bile had less chenodeoxycholate and more deoxycholate than human bile. Lactic acid, a major product of bacterial metabolism in the human colon, further stimulated encystation. Growth of the preencystation culture without bile also increased subsequent encystation. More than 90% of Type I cysts produced with porcine bile plus lactic acid were viable as indicated by the uptake and retention of fluorescein diacetate and exclusion of propidium iodide. Biological activity of in vitro-derived water-resistant cysts was demonstrated by the observation that 1 to 9.5% excysted in vitro. The percentage of excystation was greatly decreased following encystation at pH 7.0 or by omission of bile or lactic acid. This is the first quantitative in vitro demonstration of the complete life cycle of G. lamblia from humans.     

Ultrastructural observations on the kinetosomes,and Golgi bodies during the asexual life cycle ofSaprolegnia

Summary At the onset of zoospore cleavage the centrioles ofSaprolegnia ferax reorientate, develop into kinetosomes and become associated with microtubular roots and a striate fibre. After cytoplasmic cleavage a flagellum, with a hitherto undescribed transition zone structure, develops from each kinetosome. Flagellum axonemes occur inside recently encysted primary spores. In vegetative hyphae and germinating cysts most recognizable Golgi bodies are characteristically associated with a cisternum of the endoplasmic reticulum and a mitochondrion but during sporogenesis they all lie adjacent to nuclei where they are apparently active in vesicle production. The structural details of these changes are described and their significance discussed. We wish to acknowledge the numerous helpful discussions with Dr. J. L. Gay. The senior author held a S.R.C. studentship during the course of this work, part of which was submitted in partial fulfillment of the requirements for the degree of Ph. D. at the University of London.     

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its two main hosts, the human and the eel, and how its response to specific environmental parameters determines its survival, its death, or the triggering of an infectious process.     

Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum

Plasmodium parasites are unicellular eukaryotes that undergo a series of remarkable morphological transformations during the course of a multistage life cycle spanning two hosts (mosquito and human). Relatively little is known about the dynamics of cellular organelles throughout the course of these transformations. Here we describe the morphology of three organelles (endoplasmic reticulum, apicoplast and mitochondrion) through the human blood stages of the parasite life cycle using fluorescent reporter proteins fused to organelle targeting sequences. The endoplasmic reticulum begins as a simple crescent-shaped organelle that develops into a perinuclear ring with two small protrusions, followed by transformation into an extensive reticulated network as the parasite enlarges. Similarly, the apicoplast and the mitochondrion grow from single, small, discrete organelles into highly branched structures in later-stage parasites. These branched structures undergo an ordered fission - apicoplast followed by mitochondrion - to create multiple daughter organelles that are apparently linked as pairs for packaging into daughter cells. This is the first in-depth examination of intracellular organelles in live parasites during the asexual life cycle of this important human pathogen.     

In vitro models for analysis of the hepatitis C virus life cycle

Chronic hepatitis C virus (HCV) infection affects approximately 170 million people worldwide. HCV infection is a major global health problem as it can be complicated with liver cirrhosis and hepatocellular carcinoma. So far, there is no vaccine available and the non-specific, interferon (IFN)-based treatments now in use have significant side-effects and are frequently ineffective, as only approximately 50% of treated patients with genotypes 1 and 4 demonstrate HCV clearance. The lack of suitable in vitro and in vivo models for the analysis of HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. The present review focuses on the progress made towards the establishment of such models.     

Biotic and abiotic regulation of resting spore formation in vivo of obligate aphid pathogen Pandora nouryi: Modeling analysis and biological implication

Entomophthoralean fungus Pandora nouryi is an obligate aphid pathogen that enables to produce resting spores (azygospores) for surviving host absence. To explore possible mechanisms involved in the regulation of resting spore formation in vivo, host cohorts consisting of 40-60 nymphs of green peach aphid Myzus persicae produced within 24 h on cabbage leaf discs in petri dishes were exposed to spore showers of P. nouryi at the concentrations (C) from a very few to nearly 2000 conidia/mm² and then reared for 7-11 days at the regimes of 10-25 °C (T) and 8-16 h daylight (H_L) or ambient (17.5 ± 3.1 °C, 13:11 L:D). Aphid mortalities observed from 35-83 cohorts (showered separately) at each regime showed typical sigmoid trend and fit well a general logistic equation (0.79 ? r² ? 0.88), yielding similar LC₅₀ estimates of 1.7-6.1 conidia/mm². The proportions (P) of cadavers forming resting spores in the cohorts also fit the same equation (0.73 ? r² ? 0.85) at all tested regimes except at 10 °C, a low temperature for the host-pathogen interaction. This indicates the dependence of resting spore formation on the spore concentration. The effects of T and H_L on P over C were well elucidated by the fitted modified logistic equations P = 0.578/{1 + exp[1.710 − (0.136 − 0.0053T)C]} and P = 0.534/{1 + exp[1.639 + (0.034 − 0.0053H_L)C]} (both r² = 0.79). Our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight.     

The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum

Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced: resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (<5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1–6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 ± 3.0 μm) had a dormancy period of 1–3 months, whereas temporary asexual cysts (32.5 ± 5.4 μm) germinated in less than 7 days.     

Immunogold localization of THRGP-like epitopes in the haustorial interface of obligate,biotrophic fungi on monocots

Summary Immunoelectron microscopy was used to determine the subcellular distribution of threonine-hydroxyproline-rich glycoprotein (THRGP) epitopes in host-parasite interactions between obligate, biotrophic fungi and cereals. Infection sites of stem rust (Puccinia graminis f. sp.tritici) and leaf rust (Puccinia recondita) on primary leaves of wheat (Triticum aestivum), as well as of powdery mildew (Erysiphe graminis f. sp.hordei) on coleoptiles of barley (Hordeum vulgare), wete probed with a polyclonal antiserum to maize THRGP. A few immunogold particles were found over the cell walls of wheat mesophyll tissue and barley coleoptile epidermis. Unlike previous examples in dicot plants, no enhanced accumulation of THRGP was observed in cereal cell walls adjacent to sites of pathogen ingress. Instead, the most pronounced accumulation of THRGP-like molecules occurred over the extrahaustorial matrix in both incompatible and compatible plant-pathogen interactions. For powdery mildew of barley, immunogold staining was distinctly increased over the center of the penetration sites; however, no labeling was found over papillae that formed during incompatible and compatible interactions. In addition, no cross-reactivity of the anti-THRGP antiserum with intercellularly growing rust pathogens was observed. The highly localized deposition of THRGP-like molecules in the extrahaustorial matrix suggests that the host plant establishes a modified barrier between itself and the pathogen.Abbreviations C chloroplast - EC plant epidermal cell - EM extrahaustorial membrane - EMA extrahaustorial matrix - GO Golgi body - GRP glycine-rich protein - HP high pressure - HRGP hydroxyprolinerich glycoprotein - Hyp hydroxyproline - LT low temperature - PBS phosphate-buffered saline - PBST PBS with Tween-20 - THRGP threonine-hydroxyproline-rich glycoprotein - VA vesicular arbuscular     

Genome size and complexity of the obligate fungal pathogen,Bremia lactucae

The size and organization of the genome of Bremia lactucae, a highly specialized fungal pathogen of lettuce, has been characterized using dot blot genomic reconstructions, reverse genomic blots, and genomic DNA reassociation kinetics. The haploid genome contains 5 × 10⁷ bp of DNA and 65% of the nuclear DNA is repeated. Low copy sequences are interspersed with repeated sequences in a short-period interspersion pattern. This pattern of genome organization is different to that described for other fungi. Although most fungi have been shown to contain some form of repetitive DNA other than the ribosomal repeat, the high percentage of repetitive DNA and the interspersion of low copy and repeated sequences are atypical of fungi characterized previously.