The 13th International Workshops on Opportunistic Protists (IWOP13)

The 13th International Workshops on Opportunistic Protists (IWOP‐13) was held November 13–15, 2014 in Seville, Spain. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and; (2) to foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists; e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference which brings together research groups working on these opportunistic pathogens. Progress has been achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune deficient and immune competent hosts and is providing important insights into these emerging and reemerging pathogens. A continuing concern of the participants is the ongoing loss of scientific expertise and diversity in this research community. This decline is due to the small size of these research communities and an ongoing lack of understanding by the broader scientific community of the challenges and limitations faced by researchers working on these organisms, which makes these research communities very sensitive to declines in research funding.     

Highlights and summaries of the 11th International Workshops on Opportunistic Protists

The 11th in the series of International Workshops on Opportunistic Protists (IWOP-11) was held in August 2010 on the Big Island of Hawaii. These meetings are devoted to agents of infections that cause serious problems in AIDS patients and other individuals with defective immune systems. International Workshops on Opportunistic Protists serves as a forum for exchange of current research information on Pneumocystis, Cryptosporidium and the Microsporidia, Toxoplasma, free-living amoebae, kinetoplastid flagellates and other pathogens that are particularly pathogenic in immunodeficient hosts. Studies on interactions between host and pathogen, especially host responses, were highlighted in this year's symposium. The lack of in vitro cultivation methods for luxuriant growth of Pneumocystis, Cryptosporidium and the Enterocytozoon bieneusi remains a major hindrance to understanding the basic biology of these organisms and precludes genetic manipulations. However, slow but steady progress is being achieved by hard work including data mining of some completed or partially completed genome sequencing of several IWOP organisms. Of great concern is evidence for dramatic decline in research funding for these pathogens and the lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms that can provide critical insight into emerging and reemerging pathogens.     

Killing the Dead: Chemotherapeutic Strategies Against Free‐Living Cyst‐Forming Protists (Acanthamoeba sp. and Balamuthia mandrillaris)

The opportunist free‐living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free‐living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst‐forming protists to provide target‐directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases.     

Development and evaluation of an off‐the‐slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

Aims

This study developed and systematically evaluated performance and limit of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts.

Methods and Results

Slide standards containing flow‐sorted (oo)cysts were used to evaluate the off‐the‐slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide.

Conclusions

This study successfully developed and evaluated recovery rates and limits of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide.

Significance and Impact of the Study

This off‐the‐slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.     

High Diversity Revealed in Leaf‐Associated Protists (Rhizaria: Cercozoa) of Brassicaceae

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf‐inhabiting microorganisms, recently termed “phyllosphere microbiome”. Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning‐based approach. The generated sequences revealed a broad diversity of leaf‐associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

Genetic variation in Tertiary relics: The case of eastern‐Mediterranean Abies (Pinaceae)

The eastern‐Mediterranean Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern‐Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the Structure analysis and distance‐based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were distinct but less differentiated. ABC simulations were run for a set of scenarios of phylogeny and past demographic changes. For A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization, the same is true for Abies equi‐trojani: both they represent evolutionary branches of Abies bornmuelleriana.     

Balamuthia mandrillaris: In Vitro Interactions with Selected Protozoa and Algae

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one‐on‐one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.     

Dopaminergic neurotoxicity of S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate,and S‐methyl‐N,N‐diethylthiocarbamate (MeDETC) in Caenorhabditis elegans

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S‐methyl‐N,N‐diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic‐dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.     

Age‐ and temperature‐dependent oviposition model of Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) with Tetranychus urticae as prey

In this study, we developed an oviposition model of Neoseiulus californicus (McGregor) with Tetranychus urticae Koch as prey. To obtain data for the model, we investigated the longevity, fecundity and survivorship of adult female N. californicus at six constant temperatures (16, 20, 24, 28, 32 and 36°C), 60–70% RH and a photoperiod of 16 : 8 (L : D) h. Longevity (average ± SE) decreased as temperature increased and was longest at 16°C (46.7 ± 5.25 days) and shortest at 36°C (12.8 ± 0.75 days). Adult developmental rate (1/average longevity) was described by the Lactin 1 model (r² = 0.95). The oviposition period (average±SE) was also longest at 16°C (29.8 ± 2.93 days) and shortest at 36°C (6.7 ± 0.54 days). Fecundity (average±SE) was greatest at 24°C (43.8 ± 3.23 eggs) and lowest at 36°C (15.9 ± 1.50 eggs). The oviposition model comprised temperature‐dependent fecundity, age‐specific cumulative oviposition rate and age‐specific survival rate functions. The temperature‐dependent fecundity was best described by an exponential equation (r² = 0.81). The age‐specific cumulative oviposition rate was best described by the three‐parameter Weibull function (r² = 0.96). The age‐specific survival rate was best described by a reverse sigmoid function (r² = 0.85).     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.