Hasil untuk "Microbial ecology"

Kripal Singh

Junnan Ding

ABSTRACT Biochar soil amendments are attracting attention as one strategy to improve soil microbially ecological environment and regulate the soil nitrogen cycle. This study aimed to evaluate the effects of biochar application on agricultural soil improvement, nitrogen (N) mineralization and nitrification. The experiment was carried out on a typical farmland containing black soil and saline-alkaline soil in Northeast China. Four treatments were undertaken, including the control-treated black soil farmland (CS), the biochar-treated black soil farmland (BCS), the control-treated saline-alkali soil farmland (SAS), and the biochar-treated saline-alkaline soil farmland (BSAS). Basic physical and chemical properties, enzyme activity, and the contents of ammonium-nitrogen (NH4+-N) and nitrate-nitrogen (NO3--N) in the soil were subsequently determined. The co-occurrence networks of bacterial communities of the biochar and control treatment groups were constructed based on high-throughput sequencing data of the 16S rRNA genes. The results showed that the BCS and BSAS treatments significantly increased the contents of soil organic matter, total nitrogen, total phosphorus, and available phosphorus. The application of biochar significantly increased the NH4+-N contents in the black soil and saline-alkaline soil by 81.78 and 80.08 %, respectively, while significantly reducing the soil NH4+-N/NO3--N content, which promoted the transformation of NH4+-N into NO3--N. Subsequently, the released NH4+-N was transformed into NO3--N through nitrification. After the biochar application, the NO3--N contents in the black and saline-alkaline soils could be fixed. The biochar application significantly increased the abundance of gdh, AOA-amoA, AOB-amoA, nirK, nirS, nosZ, and nifH genes, with no significant difference in the abundance of napA genes being found among different treatments. Microbes playing a key role in the co-occurrence network were Proteobateria, Acidobacteria, Bacteroidetes, Actinobacteria, and Chloroflexi. As compared with the CS and SAS treatments, under the BCS+BSAS treatment, the connectors, module hubs, connectedness, and clustering coefficient showed larger parameters, and the networks were more complex. The application of biochar gradually increased the nodes, edges, and average degree of the bacterial co-occurrence network, thus indicating that the interaction between microbial groups in the black and saline-alkaline soils post biochar application may be important in the biogeochemical cycle process in farmland soil.

Carl-Eric Wegner, Raphaela Stahl, Irina Velsko et al.

Abstract Background The terrestrial subsurface is home to a significant proportion of the Earth’s microbial biomass. Our understanding about terrestrial subsurface microbiomes is almost exclusively derived from groundwater and porous sediments mainly by using 16S rRNA gene surveys. To obtain more insights about biomass of consolidated rocks and the metabolic status of endolithic microbiomes, we investigated interbedded limestone and mudstone from the vadose zone, fractured aquifers, and deep aquitards. Results By adapting methods from microbial archaeology and paleogenomics, we could recover sufficient DNA for downstream metagenomic analysis from seven rock specimens independent of porosity, lithology, and depth. Based on the extracted DNA, we estimated between 2.81 and 4.25 × 105 cells × g−1 rock. Analyzing DNA damage patterns revealed paleome signatures (genetic records of past microbial communities) for three rock specimens, all obtained from the vadose zone. DNA obtained from deep aquitards isolated from surface input was not affected by DNA decay indicating that water saturation and not flow is controlling subsurface microbial survival. Decoding the taxonomy and functional potential of paleome communities revealed increased abundances for sequences affiliated with chemolithoautotrophs and taxa such as Cand. Rokubacteria. We also found a broader metabolic potential in terms of aromatic hydrocarbon breakdown, suggesting a preferred utilization of sedimentary organic matter in the past. Conclusions Our study suggests that limestones function as archives for genetic records of past microbial communities including those sensitive to environmental stress at modern times, due to their specific conditions facilitating long-term DNA preservation. Video Abstract

Pranav M. Pande, Hamed Azarbad, Julien Tremblay et al.

Abstract Crops associate with microorganisms that help their resistance to biotic stress. However, it is not clear how the different partners of this association react during exposure to stress. This knowledge is needed to target the right partners when trying to adapt crops to climate change. Here, we grew wheat in the field under rainout shelters that let through 100%, 75%, 50% and 25% of the precipitation. At the peak of the growing season, we sampled plant roots and rhizosphere, and extracted and sequenced their RNA. We compared the 100% and the 25% treatments using differential abundance analysis. In the roots, most of the differentially abundant (DA) transcripts belonged to the fungi, and most were more abundant in the 25% precipitation treatment. About 10% of the DA transcripts belonged to the plant and most were less abundant in the 25% precipitation treatment. In the rhizosphere, most of the DA transcripts belonged to the bacteria and were generally more abundant in the 25% precipitation treatment. Taken together, our results show that the transcriptomic response of the wheat holobiont to decreasing precipitation levels is stronger for the fungal and bacterial partners than for the plant.

Abdulla- Al-Asif, Abu Hena Mustafa Kamal, Mohd Hanafi Idris et al.

The seagrass ecosystems found in the marine and coastal areas, with substantial economic and ecological services and span all over the globe excluding the Antarctic region. The Coral Triangle and Southeast Asia are recognized as a worldwide hotspot of seagrass species and habitats, encompassing 10-21 species of seagrass in every nation, although the study, understanding, and quantity of publications on seagrass ecosystems are rather limited in the region, including Malaysia. Malaysia contains 18 seagrass species from three families, which occupy 16.8 km2 of coastal area, where the study and discovery of seagrass species and meadows began in 1904 with the report of Beccari. All of the published papers reviewed reported on Malaysian seagrass-related research, which was divided into nine topic groups: biology and distribution, carbon sequestration, fauna, remote sensing, impact and pollution genetic study, restoration, microbiological investigation, and others. The extensive study of the seagrass ecosystem began in 1993, and we have identified 183 published papers from Scopus, 141 publications from Web of Science, and 42 from Google Scholar. However, the average trend of the number of publications from 1993 to 1999 was 0.71 ± 0.36, while from 2000 to 2022 was 7.70 ± 1.16 followed by the average trend of the yearly number of publications was 6.78 ± 1.08. The highest number of publications was found on faunal categories (43.17%), followed by biology and distribution (21.85%). The number of articles that were published on Malaysian seagrass meadows each year has been discovered to be rising, which indicates that the trends in seagrass study and publishing were progressively garnering the attention of researchers, academics, and the government. However, to better understand the sustainable ecology and ecosystem services provided by seagrass habitats, an emphasis on certain research niches, such as the genetic study of flora and fauna in seagrass meadows, microbial ecology, and restoration as well as conservation of seagrass species might be helpful.

R. Schmidt, D. Ulanova, L. Wick et al.

Bettina Böttcher, Dominik Driesch, Thomas Krüger et al.

Abstract Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.

Alice Mataigne, Nathan Vannier, Philippe Vandenkoornhuyse et al.

Abstract Background From a theoretical ecology point of view, microbiomes are far more complex than expected. Besides competition and competitive exclusion, cooperative microbe-microbe interactions have to be carefully considered. Metabolic dependencies among microbes likely explain co-existence in microbiota. Methodology In this in silico study, we explored genome-scale metabolic models (GEMs) of 193 bacteria isolated from Arabidopsis thaliana roots. We analyzed their predicted producible metabolites under simulated nutritional constraints including “root exudate-mimicking growth media” and assessed the potential of putative metabolic exchanges of by- and end-products to avoid those constraints. Results We found that the genome-encoded metabolic potential is quantitatively and qualitatively clustered by phylogeny, highlighting metabolic differentiation between taxonomic groups. Random, synthetic combinations of increasing numbers of strains (SynComs) indicated that the number of producible compounds by GEMs increased with average phylogenetic distance, but that most SynComs were centered around an optimal phylogenetic distance. Moreover, relatively small SynComs could reflect the capacity of the whole community due to metabolic redundancy. Inspection of 30 specific end-product metabolites (i.e., target metabolites: amino acids, vitamins, phytohormones) indicated that the majority of the strains had the genetic potential to produce almost all the targeted compounds. Their production was predicted (1) to depend on external nutritional constraints and (2) to be facilitated by nutritional constraints mimicking root exudates, suggesting nutrient availability and root exudates play a key role in determining the number of producible metabolites. An answer set programming solver enabled the identification of numerous combinations of strains predicted to depend on each other to produce these targeted compounds under severe nutritional constraints thus indicating a putative sub-community level of functional redundancy. Conclusions This study predicts metabolic restrictions caused by available nutrients in the environment. By extension, it highlights the importance of the environment for niche potential, realization, partitioning, and overlap. Our results also suggest that metabolic dependencies and cooperation among root microbiota members compensate for environmental constraints and help maintain co-existence in complex microbial communities. Video Abstract

Richard Mugani, Fatima El Khalloufi, El Mahdi Redouane et al.

Research on Plant Growth-Promoting Bacteria (PGPB) has focused much more on rhizospheric bacteria. However, PGPB associated with toxic cyanobacterial bloom (TCB) could enter the rhizosphere through irrigation water, helping plants such as <i>Pisum sativum</i> L. (pea) overcome oxidative stress induced by microcystin (MC) and improve plant growth and nutritional value. This study aimed to isolate bacteria associated with toxic cyanobacteria, test PGPB properties, and inoculate them as a consortium to pea seedlings irrigated with MC to investigate their role in plant protection as well as in improving growth and nutritional value. Two bacterioplankton isolates and one rhizosphere isolate were isolated and purified on a mineral salt medium supplemented with 1000 μg/L MC and identified via their 16S rRNA gene. The mixed strains were inoculated to pea seedlings in pots irrigated with 0, 50, and 100 μg/L MC. We measured the morphological and physiological parameters of pea plants at maturity and evaluated the efficiency of the plant’s enzymatic and non-enzymatic antioxidant responses to assess the role and contribution of PGPB. Both bacterioplankton isolates were identified as <i>Starkeya</i> sp., and the rhizobacterium was identified as <i>Brevundimonas aurantiaca</i>. MC addition significantly (<i>p</i> < 0.05) reduced all the growth parameters of the pea, i.e., total chlorophyll content, leaf quantum yield, stomatal conductance, carotenoids, and polyphenol contents, in an MC concentration-dependent manner, while bacterial presence positively affected all the measured parameters. In the MC treatment, the levels of the pea’s antioxidant traits, including SOD, CAT, POD, PPO, GST, and ascorbic acid, were increased in the sterile pots. In contrast, these levels were reduced with double and triple PGPB addition. Additionally, nutritional values such as sugars, proteins, and minerals (Ca and K) in pea fruits were reduced under MC exposure but increased with PGPB addition. Overall, in the presence of MC, PGPB seem to positively interact with pea plants and thus may constitute a natural alternative for soil fertilization when irrigated with cyanotoxin-contaminated water, increasing the yield and nutritional value of crops.

Ahmed Mohammed, Jiaying Hu, Raj Murugesan et al.

The aim of this study was to examine if synbiotics present similar efficiency to a common antibiotic used in poultry production under heat stress (HS) conditions. Two hundred and forty-one-day-old male Ross 708 broiler chicks were distributed among 3 treatments with 8 pens per treatment of 80 birds each for a 42-day trial. From day 15, birds were heat stressed (32°C for 9 h daily, HS) and fed the basal diet (CONT), the basal diet mixed with an antibiotic (Bactiracin Methylene Disalicylate) (0.05 g/kg of feed, BMD) or a synbiotic (0.5 g/kg of feed, SYN). The treatment effects on bird behavior, production performance, jejunal histomorphology, and cecal microbial ecology were examined. Behavioral observation was recorded by using instantaneous scan sampling technique. Production parameters were measured on day 14, 28, and 42. Cecal microbial populations of Escherichia coli and Lactobacilli and jejunal histomorphological parameters were measured at day 42. The results showed that, SYN birds exhibited more feeding and preening but less drinking and panting behaviors compared with both BMD and CONT birds (P < 0.05). The SYN birds also had higher body weight (BW) at both day 28 and 42 compared to CONT birds (P < 0.05). At the end of the experiment, the counts of Escherichia coli of SYN birds were at the similar levels of BMD but were lower than that of CONT birds (P < 0.05); while there were no treatment effects on the populations of Lactobacilli (P > 0.05). In addition, SYN birds had greater villus height compared with both CONT and BMD birds (P < 0.05). These findings suggest that the dietary synbiotic supplement has significant performance and welfare benefits, with the potential to be used as an alternative to antibiotics for poultry meat production, especially during hot seasons.

D. Karl

Antonio Reverter, Maria Ballester, Pamela A. Alexandre et al.

Abstract Background Analyses of gut microbiome composition in livestock species have shown its potential to contribute to the regulation of complex phenotypes. However, little is known about the host genetic control over the gut microbial communities. In pigs, previous studies are based on classical “single-gene-single-trait” approaches and have evaluated the role of host genome controlling gut prokaryote and eukaryote communities separately. Results In order to determine the ability of the host genome to control the diversity and composition of microbial communities in healthy pigs, we undertook genome-wide association studies (GWAS) for 39 microbial phenotypes that included 2 diversity indexes, and the relative abundance of 31 bacterial and six commensal protist genera in 390 pigs genotyped for 70 K SNPs. The GWAS results were processed through a 3-step analytical pipeline comprised of (1) association weight matrix; (2) regulatory impact factor; and (3) partial correlation and information theory. The inferred gene regulatory network comprised 3561 genes (within a 5 kb distance from a relevant SNP–P < 0.05) and 738,913 connections (SNP-to-SNP co-associations). Our findings highlight the complexity and polygenic nature of the pig gut microbial ecosystem. Prominent within the network were 5 regulators, PRDM15, STAT1, ssc-mir-371, SOX9 and RUNX2 which gathered 942, 607, 588, 284 and 273 connections, respectively. PRDM15 modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency and regulates the production of Th1- and Th2-type immune response. The signal transducer STAT1 has long been associated with immune processes and was recently identified as a potential regulator of vaccine response to porcine reproductive and respiratory syndrome. The list of regulators was enriched for immune-related pathways, and the list of predicted targets includes candidate genes previously reported as associated with microbiota profile in pigs, mice and human, such as SLIT3, SLC39A8, NOS1, IL1R2, DAB1, TOX3, SPP1, THSD7B, ELF2, PIANP, A2ML1, and IFNAR1. Moreover, we show the existence of host-genetic variants jointly associated with the relative abundance of butyrate producer bacteria and host performance. Conclusions Taken together, our results identified regulators, candidate genes, and mechanisms linked with microbiome modulation by the host. They further highlight the value of the proposed analytical pipeline to exploit pleiotropy and the crosstalk between bacteria and protists as significant contributors to host-microbiome interactions and identify genetic markers and candidate genes that can be incorporated in breeding program to improve host-performance and microbial traits. Video Abstract

Manon Boxberger, Valérie Cenizo, Nadim Cassir et al.

Abstract The skin is the exterior interface of the human body with the environment. Despite its harsh physical landscape, the skin is colonized by diverse commensal microbes. In this review, we discuss recent insights into skin microbial populations, including their composition and role in health and disease and their modulation by intrinsic and extrinsic factors, with a focus on the pathobiological basis of skin aging. We also describe the most recent tools for investigating the skin microbiota composition and microbe-skin relationships and perspectives regarding the challenges of skin microbiome manipulation. Video abstract

Shin‐ichiro Oka, Hideyuki Doi, Kei Miyamoto et al.

Abstract An environmental DNA (eDNA) metabarcoding approach has been widely used for biodiversity monitoring of fishes, although it has rarely been applied to tropical and subtropical aquatic ecosystems, where species diversity is remarkably high. This study examined the extent to which species richness can be estimated in a small coral reef lagoon (1,500 × 900 m) near Okinawa Island, southern Japan, where the surrounding waters are likely to harbor more than 1,500 species of fish. During 2015–2017, a total of 16 capture‐based surveys were conducted to create a faunal list of fish species, followed by eDNA metabarcoding based on seawater samples taken from 11 sites in the lagoon on a day in May 2019. We also tested whether eDNA metabarcoding could detect differences between adjacent fish communities inhabiting the offshore reef edge and shore‐side seagrass beds within the lagoon. A total of 217 fish species were confirmed by the capture‐based samplings, while 291 fish species were detected by eDNA metabarcoding, identifying a total of 410 species distributed across 119 families and 193 genera. Of these 410 species, only 96 (24% of the total) were commonly identified by both methods, indicating that capture‐based surveys failed to collect a number of species detected by eDNA metabarcoding. Interestingly, two different approaches to estimate species richness based on eDNA data yielded values close to the 410 species, including one that suggested an additional three or more eDNA surveys from 11 sites (36 samples) would detect 90% of the 410 species. In addition, nonmetric multidimensional scaling for fish assemblages clearly distinguished between the fish communities of the offshore reef edge and those of the shore‐side seagrass beds. This study demonstrates that an eDNA metabarcoding approach is useful for estimating species richness and detection of habitat segregation even in ecosystems with remarkably high species diversity.

Tanvi Taparia, Tanvi Taparia, Marjon Krijger et al.

Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: “Pseudomonas gingeri,” Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (&gt;0.95), amplification efficiency (&gt;90%) and magnitude of amplification signal (&gt;2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.

Aman S. Gill, Kai Purnell, Matthew I. Palmer et al.

Functional and biogeographical properties of soil microbial communities in urban ecosystems are poorly understood despite their role in metabolic processes underlying valuable ecosystem services. The worldwide emergence of engineered habitats in urban landscapes—green roofs, bioswales, and other types of soil-based green infrastructure—highlights the importance of understanding how environmental changes affect the community assembly processes that shape urban microbial diversity and function. In this study we investigated (1) whether engineered green roofs and bioswales in New York City had distinct microbial community composition and trait-associated diversity compared to non-engineered soils in parks and tree pits, and (2) if these patterns were consistent with divergent community assembly processes associated with engineered specifications of green infrastructure habitats not present in conventional, non-engineered green infrastructure; specifically, tree pit and park lawn soils. We found that green roofs and bioswales each had distinct bacterial and fungal communities, but that community composition and diversity were not significantly associated with geographic distance, suggesting that the processes structuring these differences are related to aspects of the habitats themselves. Bioswales, and to a lesser extent green roofs, also contained increased functional potential compared to conventional GI soils, based on the diversity and abundance of taxa associated with nitrogen cycling, biodegradation, decomposition, and traits positively associated with plant growth. We discuss these results in the context of community assembly theory, concluding that urban soil microbial community composition and diversity in engineered habitats are driven largely by environmental filtering, whereas stochastic processes are more important among non-engineered soils.

Himanshu Kumar, Maria Carmen Collado, Harm Wopereis et al.

Extensive microbial colonization of the infant gastrointestinal tract starts after parturition. There are several parallel mechanisms by which early life microbiome acquisition may proceed, including early exposure to maternal vaginal and fecal microbiota, transmission of skin associated microbes, and ingestion of microorganisms present in breast milk. The crucial role of vertical transmission from the maternal microbial reservoir during vaginal delivery is supported by the shared microbial strains observed among mothers and their babies and the distinctly different gut microbiome composition of caesarean-section born infants. The healthy infant colon is often dominated by members of the keystone genus <i>Bifidobacterium</i> that have evolved complex genetic pathways to metabolize different glycans present in human milk. In exchange for these host-derived nutrients, bifidobacteria’s saccharolytic activity results in an anaerobic and acidic gut environment that is protective against enteropathogenic infection. Interference with early-life microbiota acquisition and development could result in adverse health outcomes. Compromised microbiota development, often characterized by decreased abundance of <i>Bifidobacterium</i> species has been reported in infants delivered prematurely, delivered by caesarean section, early life antibiotic exposure and in the case of early life allergies. Various microbiome modulation strategies such as probiotic, prebiotics, synbiotics and postbiotics have been developed that are able to generate a bifidogenic shift and help to restore the microbiota development. This review explores the evolutionary ecology of early-life type <i>Bifidobacterium</i> strains and their symbiotic relationship with humans and discusses examples of compromised microbiota development in which stimulating the abundance and activity of <i>Bifidobacterium</i> has demonstrated beneficial associations with health.

E. González-Toril, Enrique Llobet-Brossa, E. Casamayor et al.

Weizhi Song, Bernd Wemheuer, Shan Zhang et al.

Abstract Background Metagenomic datasets provide an opportunity to study horizontal gene transfer (HGT) on the level of a microbial community. However, current HGT detection methods cannot be applied to community-level datasets or require reference genomes. Here, we present MetaCHIP, a pipeline for reference-independent HGT identification at the community level. Results Assessment of MetaCHIP’s performance on simulated datasets revealed that it can predict HGTs with various degrees of genetic divergence from metagenomic datasets. The results also indicated that the detection of very recent gene transfers (i.e. those with low levels of genetic divergence) from metagenomics datasets is largely affected by the read assembly step. Comparison of MetaCHIP with a previous analysis on soil bacteria showed a high level of consistency for the prediction of recent HGTs and revealed a large number of additional non-recent gene transfers, which can provide new biological and ecological insight. Assessment of MetaCHIP’s performance on real metagenomic datasets confirmed the role of HGT in the spread of genes related to antibiotic resistance in the human gut microbiome. Further testing also showed that functions related to energy production and conversion as well as carbohydrate transport and metabolism are frequently transferred among free-living microorganisms. Conclusion MetaCHIP provides an opportunity to study HGTs among members of a microbial community and therefore has several applications in the field of microbial ecology and evolution. MetaCHIP is implemented in Python and freely available at https://github.com/songweizhi/MetaCHIP.

David Paez-Espino, Jinglie Zhou, Simon Roux et al.

Abstract Background Virophages are small viruses with double-stranded DNA genomes that replicate along with giant viruses and co-infect eukaryotic cells. Due to the paucity of virophage reference genomes, a collective understanding of the global virophage diversity, distribution, and evolution is lacking. Results Here we screened a public collection of over 14,000 metagenomes using the virophage-specific major capsid protein (MCP) as “bait.” We identified 44,221 assembled virophage sequences, of which 328 represent high-quality (complete or near-complete) genomes from diverse habitats including the human gut, plant rhizosphere, and terrestrial subsurface. Comparative genomic analysis confirmed the presence of four core genes in a conserved block. We used these genes to establish a revised virophage classification including 27 clades with consistent genome length, gene content, and habitat distribution. Moreover, for eight high-quality virophage genomes, we computationally predicted putative eukaryotic virus hosts. Conclusion Overall, our approach has increased the number of known virophage genomes by 10-fold and revealed patterns of genome evolution and global virophage distribution. We anticipate that the expanded diversity presented here will provide the backbone for further virophage studies.