Microscopio and PCR-based detection of microsporidia spores in human stool samples

Abstract Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Resumen Los microsporidios son hongos intracelulares obligados con una notable capacidad para infectar una amplia gama de hospedadores invertebrados y vertebrados. Enterocytozoon bieneusi es el microsporidio más frecuentemente reportado en todo el mundo, principalmente tricrómicaasociado con diarrea crónica y síndrome debilitante en pacientes con sida. Las técnicas dedetección basadas en microscopía y PCR son útiles para el diagnóstico y la identificación deespecies y genotipos, pero estos métodos deben estar estandarizados en cada laboratorio.En este estudio evaluamos técnicas de microscopía y PCR anidada, con secuenciación de losproductos, para detectar E. bieneusi en muestras de heces humanas. Estas técnicas, usadas con-juntamente, podrían ser útiles para su aplicación en el diagnóstico de microsporidiosis intestinaly para realizar estudios epidemiológicos de esta afección en Argentina.

Diversity structure of the microbial communities in the guts of four neotropical termite species.

The termite gut microbiome is dominated by lignocellulose degrading microorganisms. This study describes the intestinal microbiota of four Argentinian higher termite species with different feeding habits: Microcerotermes strunckii (hardwood), Nasutitermes corniger (softwood), Termes riograndensis (soil organic matter/grass) and Cornitermes cumulans (grass) by deep sequencing of amplified 16S rRNA and ITS genes. In addition, we have performed a taxonomic and gut community structure comparison incorporating into the analysis the previously reported microbiomes of additional termite species with varied diets. The bacterial phylum Spirochaetes was dominant in the guts of M. strunckii, N. corniger and C. cumulans, whereas Firmicutes predominated in the T. riograndensis gut microbiome. A single bacterial genus, Treponema (Spirochaetes), was dominant in all termite species, except for T. riograndensis. Both in our own sequenced samples and in the broader comparison, prokaryotic α-diversity was higher in the soil/grass feeders than in the wood feeders. Meanwhile, the ß-diversity of prokaryotes and fungi was highly dissimilar among strict wood-feeders, whereas that of soil- and grass-feeders grouped more closely. Ascomycota and Basidiomycota were the only fungal phyla that could be identified in all gut samples, because of the lack of reference sequences in public databases. In summary, higher microbial diversity was recorded in termites with more versatile feeding sources, providing further evidence that diet, along with other factors (e.g., host taxonomy), influences the microbial community assembly in the termite gut.

Comparative Pathogenesis of Generalist AcMNPV and Specific RanuNPV in Larvae of Rachiplusia nu (Lepidoptera: Noctuidae) Following Single and Mixed Inoculations.

The South American soybean pest, Rachiplusia nu (Guenée), is naturally infected by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Rachiplusia nu nucleopolyhedrovirus (RanuNPV). We compared their pathogenicity to fourth-instar R. nu larvae, by evaluating time to death and virus spread throughout the tissues in single and mixed infections. Bioassays showed that generalist AcMNPV had a faster speed of kill than specific RanuNPV, while the mixed-virus treatment did not statistically differ from AcMNPV alone. Histopathology evidenced similar tissue tropism for both viruses, but co-inoculation resulted in mostly AcMNPV-infected cells. In sequential inoculations, however, the first virus administered predominated over the second one. Implications on baculovirus interactions and biocontrol potential are discussed.

Microscopic and PCR-based detection of microsporidia spores in human stool samples.

Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrhea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Neotropical termite microbiomes as sources of novel plant cell wall degrading enzymes.

In this study, we used shotgun metagenomic sequencing to characterise the microbial metabolic potential for lignocellulose transformation in the gut of two colonies of Argentine higher termite species with different feeding habits, Cortaritermes fulviceps and Nasutitermes aquilinus. Our goal was to assess the microbial community compositions and metabolic capacity, and to identify genes involved in lignocellulose degradation. Individuals from both termite species contained the same five dominant bacterial phyla (Spirochaetes, Firmicutes, Proteobacteria, Fibrobacteres and Bacteroidetes) although with different relative abundances. However, detected functional capacity varied, with C. fulviceps (a grass-wood-feeder) gut microbiome samples containing more genes related to amino acid metabolism, whereas N. aquilinus (a wood-feeder) gut microbiome samples were enriched in genes involved in carbohydrate metabolism and cellulose degradation. The C. fulviceps gut microbiome was enriched specifically in genes coding for debranching- and oligosaccharide-degrading enzymes. These findings suggest an association between the primary food source and the predicted categories of the enzymes present in the gut microbiomes of each species. To further investigate the termite microbiomes as sources of biotechnologically relevant glycosyl hydrolases, a putative GH10 endo-ß-1,4-xylanase, Xyl10E, was cloned and expressed in Escherichia coli. Functional analysis of the recombinant metagenome-derived enzyme showed high specificity towards beechwood xylan (288.1 IU/mg), with the optimum activity at 50 °C and a pH-activity range from 5 to 10. These characteristics suggest that Xy110E may be a promising candidate for further development in lignocellulose deconstruction applications.

Potential of predatory Neotropical ladybirds and minute pirate bug on strawberry aphid.

Laboratory trials were performed to determine the impact of three Neotropical predatory coccinellids (Cycloneda sanguinea, Eriopis connexa and Coleomegilla quadrifasciata) and a minute pirate bug (Orius insidiosus) on Chaetosiphon fragaefolii, an important strawberry aphid pest. The predation on C. fragaefolii nymphs and adults, as well as the time to the first attack of all predators were compared with predation on Aphis gossypii. Predator preferences for prey and aphid defensive behavior were also evaluated. Moreover, the effect of coccinellids on C. fragaefolii population growth was assessed in experimental greenhouse conditions. The predation rate varied among predators, being significantly lower for O. insidiosus than for the coccinellids. Consumption was higher on A. gossypii than on C. fragaefolii, regardless of the aphids developmental stage. The time to the first attack of all predators was longer in the presence of C. fragaefolii. Walking away and cornicle secretion were the most common antipredator behaviors of aphid against coccinellids and O. insidiosus, respectively. Coccinellids preferred A. gossypii over C. fragaefolii, while O. insidiosus showed indifference. Cycloneda sanguinea and E. connexa exhibited the highest suppression effect on the growth rate of C. fragaefolii. Thus, the four predators evaluated could contribute to reduce strawberry aphid populations, especially C. sanguinea and E. connexa.

Biological and molecular characterization of Rachiplusia nu single nucleopolyhedrovirus, a promising biocontrol agent against the South American soybean pest Rachiplusia nu.

Rachiplusia nu (Lepidoptera: Noctuidae) is a key soybean pest in Argentina. Current management of this moth relies mainly on the use of synthetic insecticides and transgenic plants. In search of biological control-based alternatives, a baculovirus from R. nu (hereafter RanuNPV) was characterized and its insecticidal properties tested under laboratory conditions. RanuNPV occlusion bodies (OBs) were nearly tetrahedral, averaging 1.0 ±â€¯0.2 µm in their longest edge and containing singly enveloped nucleocapsids. Histopathology of infected late-instar larvae revealed broad tissue tropism, where fat bodies and epidermis were the most affected organs. Phylogenetic analysis of concatenated polh, lef-8 and lef-9 partial sequences classified RanuNPV as a new species that clusters with other group II alphabaculoviruses infecting larvae of Plusiinae. Bioassays performed with R. nu neonates determined the median lethal dosage to be approximately 2.5 OBs/larva; most insects died within 4-5 days post inoculation showing typical baculovirus-induced liquefaction. No effects were observed in other lepidopteran species assayed, including Spodoptera frugiperda, Cydia pomonella and Diatraea saccharalis. High pathogenicity and host specificity make RanuNPV a good candidate for controlling R. nu.

Evidencia molecular de la ocurrencia de bacterias celulolíticas del género Cohnella en especies neotropicales de Nasutitermitinae colectadas en el NEA / Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.

Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina.

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14 µM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30 nm and 100-120 nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

Further Description of Helicoverpa zea (Lepidoptera: Noctuidae) Male Genitalia and New Genetic Evidence of Synonymy With Respect to the Anomalous Form, "Heliothis stombleri".

The Helicoverpa/Heliothis complex can cause serious damage to agricultural crops. Phenotypic similarity makes it difficult to discriminate between closely related Helicoverpa species. Currently, morphology of the male genitalia complemented with molecular techniques constitutes the best approach for species identification. In this work, a broad microscopic examination of adult Helicoverpa zea (Boddie) males (n = 200) captured in central Argentina was carried out in order to provide a detailed description of the valvae and the phallus. A considerable degree of variability was recorded. Both rounded and sharp valve apices were observed and valvae were not always parallel-sided. Most evident differences were detected concerning the number of cornuti on the phallus. A range of 15-21 cornuti per phallus was recorded, the mode being 18. A significant minority of the samples (3.5%) displayed an abnormal genital condition showing a constricted phallus lacking cornuti, and pointed valvae. This form was initially attributed to a distinct species, Heliothis stombleri, and later proposed as a synonym of H. zea based on additional morphological observations and molecular studies. Here, a phylogenetic analysis combining mitochondrial (cytochrome oxidase subunit I) and nuclear (elongation factor -1 alpha) genes was performed on these and other Helicoverpa specimens collected in the same geographical region, in order to further verify the taxonomic status of H. stombleri. The tree topology clearly grouped H. stombleri with H. zea, supporting the assumption that the former represents, in fact, an anomalous form of the latter. Further experiments are needed to clarify the etiology of this anomaly and its persistence over time.

Description of 'Candidatus Phytoplasma meliae', a phytoplasma associated with Chinaberry (Melia azedarach L.) yellowing in South America.

China tree yellows (ChTY) phytoplasma is associated with the yellowing disease of the China tree (Melia azedarach) in Argentina. According to partial 16S rRNA gene analysis, ChTY phytoplasma belongs to the 16Sr XIII group, subgroup G. Strains of species of ChTY have 98-99 % 16S rDNA gene sequence similarity with 16SrXIII-group phytoplasmas, and less than 97.5 % when compared to all 'CandidatusPhytoplasma' described so far, except for the novel 'CandidatusPhytoplasma hispanicum'. However, strains of species of ChTY are differentiated from the latter due to having additional molecular and biological attributes. The presence of unique features in the 16S rDNA sequence distinguishes ChTY from all species of 'CandidatusPhytoplasma' currently described. The in silico RFLP profile of 16S rDNA (1.2 kb) and rpLV-rpsC (1.3 kb) genes distinguished ChTY, as in the 16SrXIII-G subgroup within the 16SrXIIII group. The phylogenetic analyses, based on 16S rDNA, rpLV-rpsC and secA gene sequences, in addition to the restricted host range, characteristic symptoms and geographical distribution, confirm that the collective strains of the species ChTY represent a distinct lineage within the phytoplasma clade and support the description of a novel species of 'CandidatusPhytoplasma meliae' with the reference strain being ChTY-Mo3 (Montecarlo, Argentina).

Molecular Identification of Helicoverpa armigera (Lepidoptera: Noctuidae: Heliothinae) in Argentina and Development of a Novel PCR-RFLP Method for its Rapid Differentiation From H. zea and H. gelotopoeon.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae: Heliothinae) is among the most voracious global pests of agriculture. Adults of this species were identified recently in northern Argentina by dissection of male genitalia. In this work, a rapid and simple molecular tool was designed to distinguish H. armigera from the morphologically similar indigenous bollworms Helicoverpa zea (Boddie) and Helicoverpa gelotopoeon (Dyar), regardless of the life stage. Amplification of partial COI gene with a new primer pair, and subsequent digestion with endonuclease HinfI, yielded different RFLP profiles for the three main Helicoverpa pests currently present in South America. The method was validated in Helicoverpa specimens collected across Argentina, whose identity was further corroborated by COI sequencing and phylogenetic analysis. The data reported here constitute the first molecular confirmation of this pest in the country. The survey revealed the occurrence of H. armigera in northern and central Argentina, including the main soybean- and maize-producing area.

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites.

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.

Molecular identification of the secondary endosymbiont Hamiltonella defensa in the rose-grain aphid Metopolophium dirhodum.

This is the first report of the association between the rose-grain aphid Metopolophium dirhodum, a potentially important cereal pest and the facultative symbiont Hamiltonella defensa. The infection with this gamma-proteobacterium was determined by PCR in laboratory-reared and field-collected specimens of an Argentinian population of the aphid. Partial bacterial 16S, IGS and 23S rRNA genes were sequenced and compared to other available Hamiltonella sequences by phylogenetic analysis. the present study provides new information on previously unknown M. dirhodum microbiota.

Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells.

Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.

Transcriptomic analysis of intestinal genes following acquisition of pea enation mosaic virus by the pea aphid Acyrthosiphon pisum.

Viruses in the family Luteoviridae are strictly transmitted by aphids in a non-propagative, circulative and persistent mode. Virions ingested by aphids successively cross the gut and the accessory salivary gland epithelia before being released, together with saliva, into the plant vasculature. Virion transport through aphid cells occurs by a transcytosis mechanism. This study conducted a transcriptomic analysis of intestinal genes of the pea aphid Acyrthosiphon pisum following uptake of pea enation mosaic virus. Among the 7166 transcripts analysed, 128 were significantly regulated (105 genes downregulated and 23 upregulated). Of these genes, 5 % were involved in intracellular trafficking, endocytosis and signal transduction, three important steps in the internalization and transport of virions. The limited levels of downregulation (maximum of 3.45-fold) and upregulation (maximum of 1.37-fold) suggest that the virus hijacks a constitutive endocytosis-exocytosis mechanism without heavily perturbing cell metabolism. Although limited to about 20 % of the pea aphid genes, this work represents the first large-scale analysis of aphid gene regulation following virus acquisition. A better knowledge of this virus-vector interaction will be possible only when tools representing the complete genomic capacity of the aphid become available.