Assembly and identification of novel viruses in the Daphnia magna microbiota
Viruses can be important and abundant members of host-associated microbiomes, influencing host fitness directly or indirectly through the microbial communities they cohabit hosts with. Bacteriophages can modulate bacterial species diversity and abundance in the microbiome by introducing virulence factors that may enhance bacterial survival. This can impact host fitness by modifying the host-bacteria interactions present in the microbiome. However, our understanding of bacteriophages in some model systems for microbiome research is limited. By characterizing phages in model systems amenable to experimentation, we can gain a better understanding of multispecies interactions across domains within hosts. Here, we describe and profile potential dsDNA viruses computationally identified in the metagenome of the keystone zooplankton Daphnia magna, an important aquatic model organism. Using a combination of three metagenome-based viral identification tools (VIBRANT, VirSorter, and VirFinder), we find one complete, novel viral genome (a Myoviridae), 24 nearly complete viral genomes (primarily Myoviridae and Siphoviridae), and 508 putative viral contigs in total in a coassembly of 6 Daphnia magna metagenomes. We profiled the novel complete dsDNA virus, finding genes necessary for viral replication and genes obtained from its hosts during lysogeny. Finally, we characterized putative bacterial host-virus interactions by predicting CRISPR-spacer regions across Limnohabitans species, a genus shown to be beneficial to Daphnia fitness. Aligning viral sequences to these CRISPR regions, we found that 1 of 2 Daphnia-associated Limnohabitans genomes had CRISPR-spacer matches to viral contigs, indicating potential tritrophic interactions among bacteriophage, their host bacteria, and the host Daphnia magna. This study highlights the potential importance of viruses in the Daphnia magna metagenome, and can serve as the basis for a complete analysis of the Daphnia magna virome to understand the potential impacts of viruses on microbial abundance and evolution, as well as their impact on the fitness of ecologically important organisms.
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[00:00:02.770]My name is Freddy Gonzalez
[00:00:03.680]and I'll be presenting the assembly and identification
[00:00:06.130]of novel viruses and the Daphnia magna microbiota.
[00:00:10.120]So a bit of the background. Viruses can be an important
[00:00:12.880]and abundant member of host associated microbiomes
[00:00:15.860]and host fitness directly
[00:00:17.810]or indirectly through the microbial communities
[00:00:19.670]that they co-habit hosts with. Specifically,
[00:00:23.410]bacteriophages can modulate bacterial species diversity
[00:00:26.630]and abundance in the microbiome by introducing
[00:00:29.040]virulence factors that may enhance spectral survival.
[00:00:32.510]This can impact host fitness
[00:00:33.660]by modifying the host bacteria interactions present
[00:00:36.620]in the microbiome.
[00:00:39.160]However, our understanding of bacteriophages
[00:00:41.510]and some model systems
[00:00:42.680]for microbiome research is limited by characterizing phages
[00:00:46.240]in model systems that are amiable to experimentation.
[00:00:50.100]We can gain a better understanding
[00:00:51.300]of multi-species interactions across domains within hosts.
[00:00:56.390]For this study, we chose to focus
[00:00:57.610]on the model known as Daphnia magna.
[00:01:01.250]This Daphnia is used in multiple fields,
[00:01:04.370]including equal toxicology studies
[00:01:06.720]and host microbe interactions.
[00:01:10.330]Daphnia bacterial microbiome is well-characterized
[00:01:14.150]but today the biome has not been characterized.
[00:01:16.840]So this is the first study to do so.
[00:01:20.260]So, we had a couple of questions when we were coming
[00:01:21.920]into this project. We were wondering
[00:01:24.520]whether we can describe bacteriophage communities present
[00:01:27.340]in the Daphnia at the family level
[00:01:29.580]using any computational tools that are available to us.
[00:01:34.320]We also wanted to know whether
[00:01:35.470]or not we could profile genes necessary
[00:01:37.290]for viral replication and any genes obtained
[00:01:40.590]from hosts during lysogeny within the phage genomes.
[00:01:45.230]We also wanted to know
[00:01:47.320]if we could characterize putative bacterial host-virus
[00:01:50.050]interactions by predicting CRISPR-spacer regions
[00:01:53.490]across bacterial species.
[00:01:58.660]The methods for this study.
[00:02:01.480]So to identify any putative viruses
[00:02:04.976]in the Daphnia microbiota,
[00:02:06.640]we used six previously collected samples.
[00:02:10.770]DNA was extracted from these Daphnia samples
[00:02:14.020]and shotgun sequencing was subsequently done using Illumina.
[00:02:19.030]The metagenome was then assembled.
[00:02:21.360]We trimmed low quality DNA fragments
[00:02:25.890]and adapter sequences
[00:02:27.210]from the pooled rates of six Daphnia magna samples
[00:02:30.570]using a program called "Trimmomatic."
[00:02:33.740]MetaSPAdes, another program, was used to generate
[00:02:40.840]So viral content identification. To identify the contigs
[00:02:44.800]and the metagenome assembly,
[00:02:46.760]we profiled putative viral contigs
[00:02:48.880]from metagenomes using three programs called VIBRANT,
[00:02:52.330]VirSorter, and VirFinder.
[00:02:54.460]And we highlighted contigs that were identified
[00:02:56.810]by two or more tools.
[00:02:59.250]So we use a consensus based method
[00:03:01.830]from these identification tools.
[00:03:06.050]So if a contig was identified by all three tools
[00:03:10.180]we gave it a high, the highest ranking of 3.
[00:03:15.260]If it was only identified in one tool
[00:03:16.960]it received the lowest ranking
[00:03:18.270]which was a ranking of 1.
[00:03:23.863]We ended a taxonomic identification.
[00:03:26.650]We chose to focus
[00:03:27.483]on one single contig for the most part, NODE 49.
[00:03:31.860]The reason we chose to focus on this contig
[00:03:34.800]for the majority of the study was because it was identified
[00:03:37.400]as a complete phage by all three tools.
[00:03:43.356]So as far as mapping out virus-host interactions,
[00:03:47.760]Limnohabitans is a genus
[00:03:49.400]of bacteria within the Daphnia microbiome.
[00:03:52.990]It's the most abundant as it's the most abundantly
[00:03:57.350]characterized bacterial species in this microbiome.
[00:04:02.500]And it is also very beneficial to the host
[00:04:05.350]as it has direct impacts on fecundity, lifespan and so on.
[00:04:10.860]So, because the species is abundant
[00:04:13.290]they end up on a constituent
[00:04:15.636]in the Daphnia magna microbiota.
[00:04:17.000]We aim to assess which identified viruses may interact
[00:04:20.430]with the Limnohabitans species.
[00:04:24.040]So, we first downloaded all available
[00:04:26.660]Limnohabitans assemblies available on jenmink
[00:04:29.460]and combined them with two Daphnia associated
[00:04:32.110]metagenome assembled genomes that were generated
[00:04:34.370]in a previous study within the lab.
[00:04:37.880]And we also included
[00:04:43.350]other free-living species
[00:04:44.480]which were identified in a different study as an out group.
[00:04:52.680]The results for this study were interesting.
[00:04:57.270]So, double-stranded DNA viruses were identified
[00:05:00.270]in the Daphnia magna metagenome,
[00:05:03.790]specifically Myoviridae and Siphoviridae
[00:05:06.143]were the most represented viral families.
[00:05:09.050]We found 511 viral contigs with the total length
[00:05:12.320]of over a million base pairs from the filtered metagenome.
[00:05:17.540]And these were identified by at least one
[00:05:19.910]of the three tools.
[00:05:22.610]And 24 were identified by at least two tools,
[00:05:26.260]giving them the certainty of 2,
[00:05:28.693]and 2 were identified by all three tools,
[00:05:30.870]which was NODE 49 and NODE 652.
[00:05:39.466]So then we decided to predict the coding regions
[00:05:41.430]of phage NODE 49.
[00:05:44.450]The majority of genes were identified as Myoviridae,
[00:05:48.630]but several were annotated with
[00:05:49.930]hypothetical bacterial origin from Limnohabitans
[00:05:52.610]and Actinobacteria, indicating that it had picked up
[00:05:55.990]some sort of genes, indicating that it had picked up genes
[00:06:00.330]from its bacterial hosts during lysogeny.
[00:06:05.376]The blast in search of the entire genome
[00:06:06.550]found several low similarity matches
[00:06:08.670]to other uncultured phage genome assemblies,
[00:06:11.170]with the most similar match only covering about 14%
[00:06:14.130]of the query, giving us an 81% sequence identity
[00:06:19.530]to another phage on a blast.
[00:06:23.640]Prediction of the gene coding regions
[00:06:25.960]and NODE 49's genome indicated
[00:06:29.060]that there were 86 potential genes.
[00:06:32.930]However, after annotating the genome,
[00:06:35.720]the majority of these genes were
[00:06:36.984]unannotated for, or hypothetical in function.
[00:06:40.560]And only 14 of these gene coding regions were annotated
[00:06:43.330]with known functions, functions that included host benefit,
[00:06:49.290]host-virus benefit, or virus replication and structure.
[00:06:56.150]So some of these genes that were identified were again
[00:06:59.130]mostly of unknown function, but known viral genes
[00:07:02.270]included the head completion protein,
[00:07:07.200]putative, phage-specific DNA primase, repressor proteins,
[00:07:11.830]which were expected.
[00:07:16.084]And they tell you right resistance protein,
[00:07:18.760]which was most likely picked up
[00:07:20.820]from the host during lysogeny at some point.
[00:07:25.870]So, to map out the Limnohabitans virus associations,
[00:07:31.689]we found variation in the number
[00:07:32.750]of regions across assemblies. For example,
[00:07:37.190]Liminohabitans species 2KL-3,
[00:07:40.910]which is right here, had 62 predicted CRISPR spacers
[00:07:44.900]but 29 assemblies had no predictive CRISPR spacers at all.
[00:07:50.320]Only one of the Daphnia associated Limnohabitans
[00:07:52.543]metagenome assembled genomes had identified CRISPR regions.
[00:07:56.760]This was Daphnia-associated species 1,
[00:07:59.700]which is contained within our lab.
[00:08:03.150]This one contained about 18 hits for this NODE 49,
[00:08:07.060]indicating that it had been infected
[00:08:08.690]at some point by this phage,
[00:08:10.250]and that it had developed
[00:08:12.700]some sort of an immunity toward it.
[00:08:16.400]So in this study we used the code assembly
[00:08:18.010]of six Daphnia magna metagenomes to identify dsDNA
[00:08:22.340]bacteriophages in the Daphnia magna microbiome.
[00:08:25.300]We found out then its potential roles
[00:08:26.680]of viruses in the system as well.
[00:08:29.770]We assembled a complete novel dsDNA viral genome,
[00:08:32.450]which is known as NODE 49.
[00:08:34.360]We annotated its genome to find genes necessary
[00:08:36.610]for replication and host infection.
[00:08:40.100]We also identified putative host-virus relationships
[00:08:42.870]by examining CRISPR spacer regions across genomes
[00:08:46.242]of Limnohabitans species, a bacterial genus
[00:08:49.140]which is present in high abundances in Daphnia magna
[00:08:51.970]and is also beneficial to Daphnia magna fitness.
[00:08:57.820]Matching Limnohabitans CRISPR spacers to viral sequences,
[00:09:01.490]we found one Daphnia-associated Limnohabitans
[00:09:05.090]species that contained CRISPR sequences specific
[00:09:08.700]to NODE 49.
[00:09:11.230]Several other putative viruses matched
[00:09:13.310]to CRISPR spacers and other free living
[00:09:15.690]Limnohabitans species, suggesting some sort
[00:09:18.030]of longterm evolutionary relationship
[00:09:20.290]between these viruses and their Limnohabitans host.
[00:09:24.773]This study indicates that tri-tierific interactions
[00:09:26.780]among Daphnia magna, its associated bacterial microbiome
[00:09:29.850]and viruses that infect those bacteria are present.
[00:09:33.730]This study again is the first to look
[00:09:36.962]at viruses within Daphnia.
[00:09:38.300]I thank you care for letting me conduct this project
[00:09:42.235]and for the Cressler lab as well.
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