Wim Bert

Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians

Significance Chytridiomycosis has resulted in the serious decline and extinction of >200 species of amphibians worldwide and poses the greatest threat to biodiversity of any known disease. This fungal disease is currently known to be caused by Batrachochytrium dendrobatidis, hitherto the only species within the entire phylum of the Chytridiomycota known to parasitize vertebrate hosts. We describe the discovery of a second highly divergent, chytrid pathogen, Batrachochytrium salamandrivorans sp. nov., that causes lethal skin infections in salamanders, which has resulted in steep declines in salamander populations in northwestern Europe. Our finding provides another explanation for the phenomenon of amphibian biodiversity loss that is emblematic of the current global biodiversity crisis. The current biodiversity crisis encompasses a sixth mass extinction event affecting the entire class of amphibians. The infectious disease chytridiomycosis is considered one of the major drivers of global amphibian population decline and extinction and is thought to be caused by a single species of aquatic fungus, Batrachochytrium dendrobatidis. However, several amphibian population declines remain unexplained, among them a steep decrease in fire salamander populations (Salamandra salamandra) that has brought this species to the edge of local extinction. Here we isolated and characterized a unique chytrid fungus, Batrachochytrium salamandrivorans sp. nov., from this salamander population. This chytrid causes erosive skin disease and rapid mortality in experimentally infected fire salamanders and was present in skin lesions of salamanders found dead during the decline event. Together with the closely related B. dendrobatidis, this taxon forms a well-supported chytridiomycete clade, adapted to vertebrate hosts and highly pathogenic to amphibians. However, the lower thermal growth preference of B. salamandrivorans, compared with B. dendrobatidis, and resistance of midwife toads (Alytes obstetricans) to experimental infection with B. salamandrivorans suggest differential niche occupation of the two chytrid fungi.

Molecular phylogeny of the Tylenchina and evolution of the female gonoduct (Nematoda: Rhabditida)

Tylenchina are a morphologically and functionally diverse group of nematode species that range from free-living bacteriovores, over transitory grazing root-hair feeders to highly specialized plant-parasites with complex host associations. We performed phylogenetic analyses of small subunit rDNA sequences from 97 species including an analysis that account for the RNA secondary structure in the models of evolution. The present study confirms the sister relationship of the bacteriovore Cephalobidae with the predominantly plant-parasitic Tylenchomorpha. All analyses appoint the fungal-feeding Aphelenchidae and Aphelenchoididae as being polyphyletic but the morphology based hypothesis of their monophyly could not be significantly rejected. Within the Tylenchomorpha, the families that exclusively parasitize higher plants are joined in a single clade. However, only the monophyletic position of the (super)families Hoplolaimidae and Criconematoidea were supported; Anguinidae, Tylenchidae, Belonolaimidae and Pratylenchidae appeared to be paraphyletic or polyphyletic. Parsimony and likelihood ancestral state reconstruction revealed that burrowing endoparasitism and sedentary endoparasitism each evolved, respectively, at least six and at least three times independently, mostly from migratory ectoparasitic ancestors. Only root-knot nematodes have evolved from burrowing endoparasitic nematodes. Traditional classifications are partially misled by this convergent evolution of feeding type and associated morphology. Contrastingly, mapping attributes of the gonoduct cellular architecture, including newly obtained data of 18 species belonging to the Aphelenchoidea, Criconematoidea, Anguinidae and Panagrolaimidae, revealed a broad congruence of the gonoduct characters and the molecular phylogenetic hypothesis. Yet, the presence of an offset spermatheca and proliferation of uterus cells has evolved multiple times, the latter associated with derived endoparasitic feeding specialization and resulting reproduction mode. Ancestral state reconstruction further revealed that the gonoduct of the morphologically and ecologically dissimilar tylenchid and cephalobid nematodes evolved from a common ancestor.

Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans

The recent arrival of Batrachochytrium salamandrivorans in Europe was followed by rapid expansion of its geographical distribution and host range, confirming the unprecedented threat that this chytrid fungus poses to western Palaearctic amphibians. Mitigating this hazard requires a thorough understanding of the pathogen’s disease ecology that is driving the extinction process. Here, we monitored infection, disease and host population dynamics in a Belgian fire salamander (Salamandra salamandra) population for two years immediately after the first signs of infection. We show that arrival of this chytrid is associated with rapid population collapse without any sign of recovery, largely due to lack of increased resistance in the surviving salamanders and a demographic shift that prevents compensation for mortality. The pathogen adopts a dual transmission strategy, with environmentally resistant non-motile spores in addition to the motile spores identified in its sister species B. dendrobatidis. The fungus retains its virulence not only in water and soil, but also in anurans and less susceptible urodelan species that function as infection reservoirs. The combined characteristics of the disease ecology suggest that further expansion of this fungus will behave as a ‘perfect storm’ that is able to rapidly extirpate highly susceptible salamander populations across Europe.

Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

Interaction of Aspergillus fumigatus conidia with Acanthamoeba castellanii parallels macrophage-fungus interactions

Aspergillus fumigatus and free-living amoebae are common inhabitants of soil. Mechanisms of A. fumigatus to circumvent the amoebas digestion may facilitate overcoming the vertebrate macrophage defence mechanisms. We performed co-culture experiments using A. fumigatus conidia and the amoeba Acanthamoeba castellanii. Approximately 25% of the amoebae ingested A. fumigatus conidia after 1 h of contact. During intra-amoebal passage, part of the ingested conidia was able to escape the food vacuole and to germinate inside the cytoplasm of A. castellanii. Fungal release into the extra-protozoan environment by exocytosis of conidia or by germination was observed with light and transmission electron microscopy. These processes resulted in structural changes in A. castellanii, leading to amoebal permeabilization without cell lysis. In conclusion, A. castellanii internalizes A. fumigatus conidia, resulting in fungal intracellular germination and subsequent amoebal death. As such, this interaction highly resembles that of A. fumigatus with mammalian and avian macrophages. This suggests that A. fumigatus virulence mechanisms to evade macrophage killing may be acquired by co-evolutionary interactions among A. fumigatus and environmental amoebae.

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

ABSTRACT Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.

A redox signalling globin is essential for reproduction in Caenorhabditis elegans

Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

Phylogeny and Evolution of Nematodes

Many plant-parasitic nematodes including members of the genera Meloidogyne (root-knot nematodes), Heterodera and Globodera (cyst nematodes) and Pratylenchus (lesion nematodes) are studied as they cause major damage to crops such as potato, tomato, soybean and sugar beet. Both for fundamental reasons (Can we reconstruct the evolution of these parasites in the almost complete absence of fossil records?) and for practical reasons (Can we use phylogenetic data to detect specific parasitic species in pools of mainly harmless/beneficial nematodes?) it is important to get an insight into the relationship between these plant-parasitic nematodes and their fungivorous and bacterivorous relatives. In this chapter we will summarize and comment on the rapid increase of molecular (mainly ribosomal DNA) data published over the last decade. The greatest attention will be paid to the Tylenchomorpha (Clade 12), and the relationship within and between root lesion and root-knot nematodes (the genera Pratylenchus and Meloidogyne).

Lack of metabolic ageing in the long-lived flatworm Schmidtea polychroa

Freshwater planarians have a large totipotent stem cell population allowing high rates of cell renewal and morphological plasticity. It is often suggested that they are able to rejuvenate during fission, regeneration and starvation. These features, together with the rapidly expanding molecular toolset, make planarians such as Schmidtea polychroa and S. mediterranea interesting for ageing research. Yet, the basic demographic and physiological data are lacking or still based on fragmentary observations of one century ago. Here, we present the first longitudinal physiological study of the species S. polychroa. Survival, size and metabolic rate, measured by microcalorimetry, of a cohort of 28 individuals were followed over a period of three years. Sexual maturity was reached during the second month after which the worms continued growing up to 5 months. This initial growth phase was followed by alternating periods of synchronised growth and degrowth. Although mass-specific metabolic rates declined during the initial growth phase, no changes were found later in life. The absence of metabolic ageing may be explained by the very high rate of cell renewal during homeostasis and alternating phases of degrowth and growth during which tissues are renewed. Surprisingly, all deaths occurred in pairs of worms that were housed in the same culture recipient, suggesting that worms did not die from ageing. Taking into account the metabolic and demographic data, we suggest that S. polychroa shows negligible ageing. Detailed analyses of size and metabolic rate revealed a remarkable biphasic allometric scaling relation. During the initial growth phase (months 1-5) the allometric scaling exponent b was 0.86 while later in life, it increased to an unusually large value of 1.17, indicating that mass-specific metabolic rate increases with size in adult S. polychroa.

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.