Characterization of the Cassava Mycobiome in Symptomatic Leaf Tissues Displaying Cassava Superelongation Disease.

Superelongation disease (SED) is a fungal disease that affects cassava in the Caribbean. The symptoms include the appearance of dry necrotic spots and lesions on the leaves, which may severely affect the plant yield. However, the primary causal pathogen is difficult to culture and isolate in the lab because of its slow growth and potential contamination from faster-growing organisms. In addition, the leaf symptoms can be confused with those caused by other pathogens that produce similar necrotic spots and scab-like lesions. There is also little or no information on the contribution of endophytes, if any, to disease symptoms in cassava, a plant where the disease is prevalent. Therefore, this study aimed to characterize the fungal communities in cassava associated with SED symptoms by analyzing gross fungal morphology and performing metagenomics profiling. First, several individual pathogenic fungi were isolated and cultured from diseased cassava leaf tissues from seven locations in Barbados (BB). Both culture isolation and molecular community analyses showed the presence of several other fungi in the disease microenvironment of symptomatic cassava leaves. These included Fusarium, Colletotrichum, and Alternaria species and the suspected species Elsinoë brasiliensis synonym Sphaceloma manihoticola. Additionally, a community analysis using ITS2 amplicon sequencing of 21 symptomatic leaf tissues from BB, St. Vincent and the Grenadines (SVG), Trinidad and Tobago (TT), and Jamaica (JA) revealed that the disease symptoms of superelongation may also result from the interactions of fungal communities in the mycobiome, including Elsinoë species and other fungi such as Colletotrichum, Cercospora, Alternaria, and Fusarium. Therefore, we suggest that examining the pathobiome concept in SED in the future is necessary.

Incense Burning is Associated with Human Oral Microbiota Composition.

Incense burning is common worldwide and produces environmental toxicants that may influence health; however, biologic effects have been little studied. In 303 Emirati adults, we tested the hypothesis that incense use is linked to compositional changes in the oral microbiota that can be potentially significant for health. The oral microbiota was assessed by amplification of the bacterial 16S rRNA gene from mouthwash samples. Frequency of incense use was ascertained through a questionnaire and examined in relation to overall oral microbiota composition (PERMANOVA analysis), and to specific taxon abundances, by negative binomial generalized linear models. We found that exposure to incense burning was associated with higher microbial diversity (p < 0.013) and overall microbial compositional changes (PERMANOVA, p = 0.003). Our study also revealed that incense use was associated with significant changes in bacterial abundances (i.e. depletion of the dominant taxon Streptococcus), even in occasional users (once/week or less) implying that incense use impacts the oral microbiota even at low exposure levels. In summary, this first study suggests that incense burning alters the oral microbiota, potentially serving as an early biomarker of incense-related toxicities and related health consequences. Although a common indoor air pollutant, guidelines for control of incense use have yet to be developed.

Adaptation of the Human Gut Microbiota Metabolic Network During the First Year After Birth.

Predicting the metabolic behavior of the human gut microbiota in different contexts is one of the most promising areas of constraint-based modeling. Recently, we presented a supra-organismal approach to build context-specific metabolic networks of bacterial communities using functional and taxonomic assignments of meta-omics data. In this work, this algorithm is applied to elucidate the metabolic changes induced over the first year after birth in the gut microbiota of a cohort of Spanish infants. We used metagenomics data of fecal samples and nutritional data of 13 infants at five time points. The resulting networks for each time point were analyzed, finding significant alterations once solid food is introduced in the diet. Our work shows that solid food leads to a different pattern of output metabolites that can be potentially released from the gut microbiota to the host. Experimental validation is presented for ferulate, a neuroprotective metabolite involved in the gut-brain axis.

Metabolic adaptation in the human gut microbiota during pregnancy and the first year of life.

BACKGROUND: The relationship between the gut microbiome and the human host is dynamic and we may expect adjustments in microbiome function if host physiology changes. Metatranscriptomic approaches should be key in unraveling how such adjustments occur. METHODS: We employ metatranscriptomic sequencing analyses to study gene expression in the gut microbiota of infants through their first year of life, and of their mothers days before delivery and one year afterwards. FINDINGS: In infants, hallmarks of aerobic metabolism disappear from the microbial metatranscriptome as development proceeds, while the expression of functions related to carbohydrate transport and metabolism increases and diversifies, approaching that observed in non-pregnant women. Butyrate synthesis enzymes are overexpressed at three months of age, even though most butyrate-producing organisms are still rare. In late pregnancy, the microbiota readjusts the expression of carbohydrate-related functions in a manner consistent with a high availability of glucose. INTERPRETATION: Our findings suggest that butyrate production may be ensured in the gut of young infants before the typical butyrate synthesizers of the adult gut become abundant. The late pregnancy gut microbiota may be able to access the high levels of blood glucose characteristic of this period. Moreover, late pregnancy gut bacteria may reach stationary phase, which may affect their likelihood of translocating across the intestinal epithelium. FUNDS: This work was supported by grants CSD2009-00006 (CONSOLIDER Program) and SAF2009-13032-C02-02 from MICINN (Ministry of Science and Innovation, Spain), and by grant SAF2012-31187 from MINECO (Ministry of Economics and Competitiveness, Spain).

Air Pollution, Early Life Microbiome, and Development.

PURPOSE OF REVIEW: We review how an altered microbiome in early life impacts on immune, metabolic, and neurological development, focusing on some of the most widespread diseases related to each of these processes, namely atopic disease, obesity, and autism. RECENT FINDINGS: The early development of the microbial communities that inhabit the human body is currently challenged by factors that range from reduced exposure to microbes, antibiotic use, and poor dietary choices to widespread environmental pollution. Recent work has highlighted some of the long-term consequences that early alterations in the establishment of these microbiotas can have for different aspects of human development and health. The long-term consequences of early microbiome alterations for human development and health are only beginning to be understood and will require in-depth investigation in the years to come. A solid understanding of how present day environmental conditions alter microbiome development, and of how an altered microbiome in early life impacts on life-long health, should inform both public health policies and the development of dietary and medical strategies to counteract early microbiota imbalances.

Types of tobacco consumption and the oral microbiome in the United Arab Emirates Healthy Future (UAEHFS) Pilot Study.

Cigarette smoking alters the oral microbiome; however, the effect of alternative tobacco products remains unclear. Middle Eastern tobacco products like dokha and shisha, are becoming globally widespread. We tested for the first time in a Middle Eastern population the hypothesis that different tobacco products impact the oral microbiome. The oral microbiome of 330 subjects from the United Arab Emirates Healthy Future Study was assessed by amplifying the bacterial 16S rRNA gene from mouthwash samples. Tobacco consumption was assessed using a structured questionnaire and further validated by urine cotinine levels. Oral microbiome overall structure and specific taxon abundances were compared, using PERMANOVA and DESeq analyses respectively. Our results show that overall microbial composition differs between smokers and nonsmokers (p = 0.0001). Use of cigarettes (p = 0.001) and dokha (p = 0.042) were associated with overall microbiome structure, while shisha use was not (p = 0.62). The abundance of multiple genera were significantly altered (enriched/depleted) in cigarette smokers; however, only Actinobacillus, Porphyromonas, Lautropia and Bifidobacterium abundances were significantly changed in dokha users whereas no genera were significantly altered in shisha smokers. For the first time, we show that smoking dokha is associated to oral microbiome dysbiosis, suggesting that it could have similar effects as smoking cigarettes on oral health.

Microbial succession in the gut: directional trends of taxonomic and functional change in a birth cohort of Spanish infants.

In spite of its major impact on life-long health, the process of microbial succession in the gut of infants remains poorly understood. Here, we analyze the patterns of taxonomic and functional change in the gut microbiota during the first year of life for a birth cohort of 13 infants. We detect that individual instances of gut colonization vary in the temporal dynamics of microbiota richness, diversity, and composition at both functional and taxonomic levels. Nevertheless, trends discernible in a majority of infants indicate that gut colonization occurs in two distinct phases of succession, separated by the introduction of solid foods to the diet. This change in resource availability causes a sharp decrease in the taxonomic richness of the microbiota due to the loss of rare taxa (p = 2.06e-9), although the number of core genera shared by all infants increases substantially. Moreover, although the gut microbial succession is not strictly deterministic, we detect an overarching directionality of change through time towards the taxonomic and functional composition of the maternal microbiota. Succession is however not complete by the one year mark, as significant differences remain between one-year-olds and their mothers in terms of taxonomic (p = 0.009) and functional (p = 0.004) microbiota composition, and in taxonomic richness (p = 2.76e-37) and diversity (p = 0.016). Our results also indicate that the taxonomic composition of the microbiota shapes its functional capacities. Therefore, the observed inter-individual variability in taxonomic composition during succession is not fully compensated by functional equivalence among bacterial genera and may have important physiological consequences. Finally, network analyses suggest that positive interactions among core genera during community assembly contribute to ensure their permanence within the gut, and highlight an expansion of complexity in the interactions network as the core of taxa shared by all infants grows following the introduction of solid foods.

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Additional duplicated Hox genes in the earthworm: Perionyx excavatus Hox genes consist of eleven paralog groups.

Annelida is a lophotrochozoan phylum whose members have a high degree of diversity in body plan morphology, reproductive strategies and ecological niches among others. Of the two traditional classes pertaining to the phylum Annelida (Polychaete and Clitellata), the structure and function of the Hox genes has not been clearly defined within the Oligochaeta class. Using a PCR-based survey, we were able to identify five new Hox genes from the earthworm Perionyx excavatus: a Hox3 gene (Pex-Hox3b), two Dfd genes (Pex-Lox6 and Pex-Lox18), and two posterior genes (Pex-post1 and -post2a). Our result suggests that the eleven earthworm Hox genes contain at least four paralog groups (PG) that have duplicated. We found the clitellates-diagnostic signature residues and annelid signature motif. Also, we show by semi-quantitative RT-PCR that duplicated Hox gene orthologs are differentially expressed in six different anterior-posterior body regions. These results provide essential data for comparative evolution of the Hox cluster within the Annelida.

The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant.

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tc(r)) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tc(r) genes. Fecal fosmid libraries were functionally screened for Tc(r), and further PCR-screened for specific Tc(r) genes. Tc(r) fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tc(r), although encoded by different genes and organisms. Tc(r) organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tc(r) gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tc(r) was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tc(r) bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual.

Crawling through time: Transition of snails to slugs dating back to the Paleozoic, based on mitochondrial phylogenomics.

Sea slugs (Gastropoda: Opisthobranchia) are characterized by extensive morphological homoplasy. In particular, reduced or absent shells are predominant throughout the group. This trend towards shell loss has resulted in a poor fossil record. DNA-based phylogenies have been helpful in improving our understanding of the evolution of this group and major clades are emerging. We report 13 new complete opisthobranch mitochondrial genomes that provide robust support for some of these emerging nodes. We name three new clades within the Opisthobranchia, the Actopleura (Acteonoidea plus Nudipleura), Placoesophaga (Cephalaspidea plus Anaspidea), and Siphoglossa (Sacoglossa plus the Siphonaria). Finally we use molecular clock dating that suggests an earlier opisthobranch divergence than previously reported. The implications of this evolutionary scenario are discussed.

Evolutionary dynamics of the wnt gene family: a lophotrochozoan perspective.

The wnt gene family encodes a set of secreted glycoproteins involved in key developmental processes, including cell fate specification and regulation of posterior growth (Cadigan KM, Nusse R. 1997. Wnt signaling: a common theme in animal development. Genes Dev. 11:3286-3305.; Martin BL, Kimelman D. 2009. Wnt signaling and the evolution of embryonic posterior development. Curr Biol. 19:R215-R219.). As for many other gene families, evidence for expansion and/or contraction of the wnt family is available from deuterostomes (e.g., echinoderms and vertebrates [Nusse R, Varmus HE. 1992. Wnt genes. Cell. 69:1073-1087.; Schubert M, Holland LZ, Holland ND, Jacobs DK. 2000. A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate amphioxus. Mol Biol Evol. 17:1896-1903.; Croce JC, Wu SY, Byrum C, Xu R, Duloquin L, Wikramanayake AH, Gache C, McClay DR. 2006. A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus. Dev Biol. 300:121-131.]) and ecdysozoans (e.g., arthropods and nematodes [Eisenmann DM. 2005. Wnt signaling. WormBook. 1-17.; Bolognesi R, Farzana L, Fischer TD, Brown SJ. 2008. Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum. Curr Biol. 18:1624-1629.]), but little is known from the third major bilaterian group, the lophotrochozoans (e.g., mollusks and annelids [Prud'homme B, Lartillot N, Balavoine G, Adoutte A, Vervoort M. 2002. Phylogenetic analysis of the Wnt gene family. Insights from lophotrochozoan members. Curr Biol. 12:1395.]). To obtain a more comprehensive scenario of the evolutionary dynamics of this gene family, we exhaustively mined wnt gene sequences from the whole genome assemblies of a mollusk (Lottia gigantea) and two annelids (Capitella teleta and Helobdella robusta) and examined them by phylogenetic, genetic linkage, intron-exon structure, and embryonic expression analyses. The 36 wnt genes obtained represent 11, 12, and 9 distinct wnt subfamilies in Lottia, Capitella, and Helobdella, respectively. Thus, two of the three analyzed lophotrochozoan genomes retained an almost complete ancestral complement of wnt genes emphasizing the importance and complexity of this gene family across metazoans. The genome of the leech Helobdella reflects significantly more dynamism than those of Lottia and Capitella, as judged by gene duplications and losses, branch length, and changes in genetic linkage. Finally, we performed a detailed expression analysis for all the Helobdella wnt genes during embryonic development. We find that, although the patterns show substantial overlap during early cleavage stages, each wnt gene has a unique expression pattern in the germinal plate and during tissue morphogenesis. Comparisons of the embryonic expression patterns of the duplicated wnt genes in Helobdella with their orthologs in Capitella reveal extensive regulatory diversification of the duplicated leech wnt genes.

Group II introns break new boundaries: presence in a bilaterian's genome.

Group II introns are ribozymes, removing themselves from their primary transcripts, as well as mobile genetic elements, transposing via an RNA intermediate, and are thought to be the ancestors of spliceosomal introns. Although common in bacteria and most eukaryotic organelles, they have never been reported in any bilaterian animal genome, organellar or nuclear. Here we report the first group II intron found in the mitochondrial genome of a bilaterian worm. This location is especially surprising, since animal mitochondrial genomes are generally distinct from those of plants, fungi, and protists by being small and compact, and so are viewed as being highly streamlined, perhaps as a result of strong selective pressures for fast replication while establishing germ plasm during early development. This intron is found in the mtDNA of an annelid worm, (an undescribed species of Nephtys), where the complete sequence revealed a 1819 bp group II intron inside the cox1 gene. We infer that this intron is the result of a recent horizontal gene transfer event from a viral or bacterial vector into the mitochondrial genome of Nephtys sp. Our findings hold implications for understanding mechanisms, constraints, and selective pressures that account for patterns of animal mitochondrial genome evolution.

Lophotrochozoan mitochondrial genomes.

Progress in both molecular techniques and phylogenetic methods has challenged many of the interpretations of traditional taxonomy. One example is in the recognition of the animal superphylum Lophotrochozoa (annelids, mollusks, echiurans, platyhelminthes, brachiopods, and other phyla), although the relationships within this group and the inclusion of some phyla remain uncertain. While much of this progress in phylogenetic reconstruction has been based on comparing single gene sequences, there are also higher order features of genomes, such as the relative order of genes, that have contributed, and this seems likely to be even more fruitful in the future. Even though tremendous progress is being made on the sequence determination of whole nuclear genomes, the dataset of choice for genome-level characters for many animals across a broad taxonomic range remains mitochondrial genomes. We review here what is known about mitochondrial genomes of the lophotrochozoans and how comparisons of some of these features may be useful in discerning the phylogeny of this group.