Host-gut microbiota interactions shape parasite infections in farmed Atlantic salmon.

Animals and their associated microbiota share long evolutionary histories. However, it is not always clear how host genotype and microbiota interact to affect phenotype. We applied a hologenomic approach to explore how host-microbiota interactions shape lifetime growth and parasite infection in farmed Atlantic salmon (Salmo salar). Multi-omics data sets were generated from the guts of 460 salmon, 82% of which were naturally infected with an intestinal cestode. A single Mycoplasma bacterial strain, MAG01, dominated the gut metagenome of large, non-parasitized fish, consistent with previous studies showing high levels of Mycoplasma in the gut microbiota of healthy salmon. While small and/or parasitized salmon also had high abundance of MAG01, we observed increased alpha diversity in these individuals, driven by increased frequency of low-abundance Vibrionaceae and other Mycoplasma species that carried known virulence genes. Colonization by one of these cestode-associated Mycoplasma strains was associated with host individual genomic variation in long non-coding RNAs. Integrating the multi-omic data sets revealed coordinated changes in the salmon gut mRNA transcriptome and metabolome that correlated with shifts in the microbiota of smaller, parasitized fish. Our results suggest that the gut microbiota of small and/or parasitized fish is in a state of dysbiosis that partly depends on the host genotype, highlighting the value of using a hologenomic approach to incorporate the microbiota into the study of host-parasite dynamics.IMPORTANCEStudying host-microbiota interactions through the perspective of the hologenome is gaining interest across all life sciences. Intestinal parasite infections are a huge burden on human and animal health; however, there are few studies investigating the role of the hologenome during parasite infections. We address this gap in the largest multi-omics fish microbiota study to date using natural cestode infection of farmed Atlantic salmon. We find a clear association between cestode infection, salmon lifetime growth, and perturbation of the salmon gut microbiota. Furthermore, we provide the first evidence that the genetic background of the host may partly determine how the gut microbiota changes during parasite-associated dysbiosis. Our study therefore highlights the value of a hologenomic approach for gaining a more in-depth understanding of parasitism.

Whole genome sequencing in clinical practice.

Whole genome sequencing (WGS) is becoming the preferred method for molecular genetic diagnosis of rare and unknown diseases and for identification of actionable cancer drivers. Compared to other molecular genetic methods, WGS captures most genomic variation and eliminates the need for sequential genetic testing. Whereas, the laboratory requirements are similar to conventional molecular genetics, the amount of data is large and WGS requires a comprehensive computational and storage infrastructure in order to facilitate data processing within a clinically relevant timeframe. The output of a single WGS analyses is roughly 5 MIO variants and data interpretation involves specialized staff collaborating with the clinical specialists in order to provide standard of care reports. Although the field is continuously refining the standards for variant classification, there are still unresolved issues associated with the clinical application. The review provides an overview of WGS in clinical practice - describing the technology and current applications as well as challenges connected with data processing, interpretation and clinical reporting.

Intestinal epigenotype of Atlantic salmon (Salmo salar) associates with tenacibaculosis and gut microbiota composition.

It remains a challenge to obtain the desired phenotypic traits in aquacultural production of Atlantic salmon, and part of the challenge might come from the effect that host-associated microorganisms have on the fish phenotype. To manipulate the microbiota towards the desired host traits, it is critical to understand the factors that shape it. The bacterial gut microbiota composition can vary greatly among fish, even when reared in the same closed system. While such microbiota differences can be linked to diseases, the molecular effect of disease on host-microbiota interactions and the potential involvement of epigenetic factors remain largely unknown. The aim of this study was to investigate the DNA methylation differences associated with a tenacibaculosis outbreak and microbiota displacement in the gut of Atlantic salmon. Using Whole Genome Bisulfite Sequencing (WGBS) of distal gut tissue from 20 salmon, we compared the genome-wide DNA methylation levels between uninfected individuals and sick fish suffering from tenacibaculosis and microbiota displacement. We discovered >19,000 differentially methylated cytosine sites, often located in differentially methylated regions, and aggregated around genes. The 68 genes connected to the most significant regions had functions related to the ulcerous disease such as epor and slc48a1a but also included prkcda and LOC106590732 whose orthologs are linked to microbiota changes in other species. Although the expression level was not analysed, our epigenetic analysis suggests specific genes potentially involved in host-microbiota interactions and more broadly it highlights the value of considering epigenetic factors in efforts to manipulate the microbiota of farmed fish.

Influence of African Swine Fever Virus on Host Gene Transcription within Peripheral Blood Mononuclear Cells from Infected Pigs.

African swine fever virus (ASFV) has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. Peripheral blood mononuclear cells (PBMCs) from pigs infected with ASFV not only express ASFV genes (almost 200 in number) but have altered patterns of host gene expression as well. Both up- and down-regulation of host cell gene expression can be followed using RNAseq on poly(A)+ mRNAs harvested from the PBMCs of pigs collected at different times post-infection. Consistent with the time course of changes in viral gene expression, only few and limited changes in host gene expression were detected at 3 days post-infection (dpi), but by 6 dpi, marked changes in the expression of over 1300 host genes were apparent. This was co-incident with the major increase in viral gene expression. The majority of the changes in host gene expression were up-regulation, but many down-regulated genes were also identified. The patterns of changes in gene expression within the PBMCs detected by RNAseq were similar in each of the four infected pigs. Furthermore, changes in the expression of about twenty selected host genes, known to be important in host defence and inflammatory responses, were confirmed using high-throughput microfluidic qPCR assays.

Extreme freeze-tolerance in cryophilic tardigrades relies on controlled ice formation but does not involve significant change in transcription.

Subzero temperatures are among the most significant factors defining the distribution of organisms, yet, certain taxa have evolved to overcome this barrier. The microscopic tardigrades are among the most freeze-tolerant animals, with selected species reported to survive milli-Kelvin temperatures. Here, we estimate survival of fully hydrated eutardigrades of the species Ramazzottius varieornatus following exposures to -20 °C and  -80 °C as well as -196 °C with or without initial cooling to -80 °C. The tardigrades easily survive these temperatures, yet with a significant decrease in viability following rapid cooling by direct exposure to -196 °C. Hence, post-freeze recovery of R. varieornatus seems to rely on cooling rate and thus controlled ice formation. Cryophilic organisms are renowned for having cold-active enzymes that secure appropriate reaction rates at low temperatures. Hence, extreme freeze-tolerance in R. varieornatus could potentially involve syntheses of cryoprotectants and de novo transcription. We therefore generated a reference transcriptome for this cryophilic R. varieornatus population and explored for differential gene expression patterns following cooling to -80 °C as compared to active 5 °C controls. Specifically, we tested for fast transcription potentially occurring within 25 min of cooling from room temperature to a supercooling point of ca. -20 °C, at which the tardigrades presumably freeze and enter into the ametabolic state of cryobiosis. Our analyses revealed no evidence for differential gene expression. We, therefore, conclude that extreme freeze-tolerance in R. varieornatus relies on controlled extracellular freezing with any freeze-tolerance related genes being constitutively expressed.

Microbiome "Inception": an Intestinal Cestode Shapes a Hierarchy of Microbial Communities Nested within the Host.

The concept of a holobiont, a host organism and its associated microbial communities, encapsulates the vital role the microbiome plays in the normal functioning of its host. Parasitic infections can disrupt this relationship, leading to dysbiosis. However, it is increasingly recognized that multicellular parasites are themselves holobionts. Intestinal parasites share space with the host gut microbiome, creating a system of nested microbiomes within the primary host. However, how the parasite, as a holobiont, interacts with the host holobiont remains unclear, as do the consequences of these interactions for host health. Here, we used 16S amplicon and shotgun metagenomics sequencing to characterize the microbiome of the intestinal cestode Eubothrium and its effect on the gut microbiome of its primary host, Atlantic salmon. Our results indicate that cestode infection is associated with salmon gut dysbiosis by acting as a selective force benefiting putative pathogens and potentially introducing novel bacterial species to the host. Our results suggest that parasitic cestodes may themselves be holobionts nested within the microbial community of their holobiont host, emphasizing the importance of also considering microbes associated with parasites when studying intestinal parasitic infections. IMPORTANCE The importance of the parasite microbiome is gaining recognition. Of particular concern is understanding how these parasite microbiomes influence host-parasite interactions and parasite interactions with the vertebrate host microbiome as part of a system of nested holobionts. However, there are still relatively few studies focusing on the microbiome of parasitic helminths in general and almost none on cestodes in particular, despite the significant burden of disease caused by these parasites globally. Our study provides insights into a system of significance to the aquaculture industry, cestode infections of Atlantic salmon and, more broadly, expands our general understanding of parasite-microbiome-host interactions and introduces a new element, the microbiome of the parasite itself, which may play a critical role in modulating the host microbiome, and, therefore, the host response, to parasite infection.

Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome.

Tardigrades are renowned for their extreme stress tolerance, which includes the ability to endure complete desiccation, high levels of radiation and very low sub-zero temperatures. Nevertheless, tardigrades appear to be vulnerable to high temperatures and thus the potential effects of global warming. Here, we provide the first analysis of transcriptome data obtained from heat stressed specimens of the eutardigrade Ramazzottius varieornatus, with the aim of providing new insights into the molecular processes affected by high temperatures. Specifically, we compare RNA-seq datasets obtained from active, heat-exposed (35 °C) tardigrades to that of active controls kept at 5 °C. Our data reveal a surprising shift in transcription, involving 9634 differentially expressed transcripts, corresponding to >35% of the transcriptome. The latter data are in striking contrast to the hitherto observed constitutive expression underlying tardigrade extreme stress tolerance and entrance into the latent state of life, known as cryptobiosis. Thus, when examining the molecular response, heat-stress appears to be more stressful for R. varieornatus than extreme conditions, such as desiccation or freezing. A gene ontology analysis reveals that the heat stress response involves a change in transcription and presumably translation, including an adjustment of metabolism, and, putatively, preparation for encystment and subsequent diapause. Among the differentially expressed transcripts we find heat-shock proteins as well as the eutardigrade specific proteins (CAHS, SAHS, MAHS, RvLEAM, and Dsup). The latter proteins thus seem to contribute to a general stress response, and may not be directly related to cryptobiosis.

A Comparison of Tools for Copy-Number Variation Detection in Germline Whole Exome and Whole Genome Sequencing Data.

Copy-number variations (CNVs) have important clinical implications for several diseases and cancers. Relevant CNVs are hard to detect because common structural variations define large parts of the human genome. CNV calling from short-read sequencing would allow single protocol full genomic profiling. We reviewed 50 popular CNV calling tools and included 11 tools for benchmarking in a reference cohort encompassing 39 whole genome sequencing (WGS) samples paired current clinical standard-SNP-array based CNV calling. Additionally, for nine samples we also performed whole exome sequencing (WES), to address the effect of sequencing protocol on CNV calling. Furthermore, we included Gold Standard reference sample NA12878, and tested 12 samples with CNVs confirmed by multiplex ligation-dependent probe amplification (MLPA). Tool performance varied greatly in the number of called CNVs and bias for CNV lengths. Some tools had near-perfect recall of CNVs from arrays for some samples, but poor precision. Several tools had better performance for NA12878, which could be a result of overfitting. We suggest combining the best tools also based on different methodologies: GATK gCNV, Lumpy, DELLY, and cn.MOPS. Reducing the total number of called variants could potentially be assisted by the use of background panels for filtering of frequently called variants.

Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

Cyanidin based anthocyanin biosynthesis in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors.

KEY MESSAGE: The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38 mg g-1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.

A comparison of storage methods for gut microbiome studies in teleosts: Insights from rainbow trout (Oncorhynchus mykiss).

Immediate freezing is perhaps the most preferred method used for preserving gut microbial samples, but research on sample preservation has been principally based around samples from mammalian species, and little is known about the advantages or disadvantages relating to different storage methods for fish guts. Fish gut samples may pose additional challenges due to the different chemical and enzymatic profile, as well as the higher water content, which might affect the yield and purity of DNA recovered. To explore this, we took gut content and mucosal scrape samples from 10 rainbow trout (Oncorhynchus mykiss), and tested whether different preservation methods have any effect on the ability to construct high quality genomic libraries for shotgun and 16S rRNA gene sequencing. Four different storage methods were compared for the gut content samples (immediate freezing on dry ice, 96% ethanol, RNAlater and DNA/RNA shield), while two different methods were compared for mucosal scrape samples (96% ethanol and RNAlater). The samples were thereafter stored at -80 °C. Our findings concluded that 96% ethanol outperforms the other storage methods when considering DNA quantity, quality, cost and labor. Ethanol works consistently well for both gut content and mucosal scrape samples, and enables construction of DNA sequencing libraries of sufficient quantity and with a fragment length distribution suitable for shotgun sequencing. Two main conclusions from our study are i) sample storage optimisation is an important part of establishing a microbiome research program in a new species or sample type system, and ii) 96% ethanol is the preferred method for storing rainbow trout gut content and mucosal scrape samples.

Influence of a growth hormone transgene on the genetic architecture of growth-related traits: A comparative analysis between transgenic and wild-type coho salmon.

Genetic engineering has been increasingly applied to many commercially important plant and animal species, generating phenotypic changes that are not observed in natural populations and creating genetic interactions that have not experienced natural selection. The degree to and way in which such human-induced genetic variation interacts with the rest of the genome is currently largely unknown. Integrating such information into ecological and risk assessment frameworks is crucial to understand the potential effects of genetically modified organisms in natural environments. Here, we performed QTL mapping to investigate the genetic architecture of growth-related traits in nontransgenic (NT) and growth hormone transgenic (T) coho salmon with large changes in growth and related physiology, with the aim of identifying how an inserted transgene might influence the opportunity for selection. These fish shared the same parental genetic background, thus allowing us to determine whether the same or different loci influence these traits within the two groups. The use of over 1,700 loci, derived from restriction site-associated DNA sequencing, revealed that different genomic regions were linked with growth over time between the two groups. Additionally, the effect sizes of detected QTL appear to have been influenced by the transgene. Direct comparison of QTL between the T and NT fish during two size-matched periods identified little overlap in their location. Taken together, the results showed that the transgene altered the genetic basis of growth-related traits in this species. The study has important implications for effective conservation and management of wild populations experiencing introduction of transgenes. Evolutionary changes and their ecological consequences may occur at different rates and in different directions in NT versus T individuals in response to selection. Thus, assessments of phenotypic change, and hence ecological risk, should be determined periodically to evaluate whether initial estimates made with founder strains remain valid.

Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq.

BACKGROUND: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.) can accumulate large quantities of anthocyanin in their storage roots, the regulatory genes responsible for their biosynthesis are not well characterized. The current study aimed to analyze global transcription profiles based on RNA sequencing (RNA-Seq), and mine MYB, bHLH and WD40 genes that may function as positive or negative regulators in the carrot anthocyanin biosynthesis pathways. RESULTS: RNA was isolated from differently colored calli, as well as tissue samples from taproots of various black carrot cultivars across the course of development, and gene expression levels of colored and non-colored tissue and callus samples were compared. The expression of 32 MYB, bHLH and WD40 genes were significantly correlated with anthocyanin content in black carrot taproot. Of those, 11 genes were consistently up- or downregulated in a purple color-specific manner across various calli and cultivar comparisons. The expression of 10 out of these 11 genes was validated using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CONCLUSIONS: The results of this study provide insights into regulatory genes that may be responsible for carrot anthocyanin biosynthesis, and suggest that future focus on them may help improve our overall understanding of the anthocyanin synthesis pathway.

Applied Hologenomics: Feasibility and Potential in Aquaculture.

Aquaculture will play an essential role in feeding a growing human population, but several biological challenges impede sustainable growth of production. Emerging evidence across all areas of life has revealed the importance of the intimate biological interactions between animals and their associated gut microbiota. Based on challenges in aquaculture, we leverage current knowledge in molecular biology and host microbiota interactions to propose an applied holo-omic framework that integrates molecular data including genomes, transcriptomes, epigenomes, proteomes, and metabolomes for analyzing fish and their gut microbiota as interconnected and coregulated systems. With an eye towards aquaculture, we discuss the feasibility and potential of our holo-omic framework to improve growth, health, and sustainability in any area of food production, including livestock and agriculture.

Mapping of quantitative trait loci for temporal growth and age at maturity in coho salmon: Evidence for genotype-by-sex interactions.

Phenotypic differences between males and females are ubiquitous throughout the animal kingdom. Therefore, investigating the extent to which sex-dependent genetic effects contribute to phenotypic variation is important in understanding the evolutionary response of each sex to natural and artificial selection. Sex-specific differences in growth patterns and age at sexual maturity have been observed in a number of anadromous salmonid fishes. In these species, faster growing individuals in a given cohort often mature earlier than conspecifics, and earlier maturing individuals are often males. The aim of this study was to determine whether sex-dependent genetic effects contribute to phenotypic variation in age at sexual maturity and growth in coho salmon reared through juvenile stages to first maturity. To achieve this aim, quantitative trait loci (QTL) underlying growth-related traits and age at first maturity were mapped across four families, and interactions between offspring sex and trait were examined by investigating the significance of genotype-by-sex (QTL×sex) interactions. Several temporally expressed growth-related QTL mapped to the same position, suggesting that these regions affected growth across many months. QTL×sex interactions were widespread, indicating that the effect of QTL on age at sexual maturity and growth over the course of development in coho salmon may be under sex-specific genetic control. We also found evidence for epistatic interactions between some growth traits. Our results provide insights into the genetic architecture underlying growth-related traits in coho salmon, and have implications for understanding the genetic and evolutionary basis of important fitness-related traits.

Comparative mapping between Coho Salmon (Oncorhynchus kisutch) and three other salmonids suggests a role for chromosomal rearrangements in the retention of duplicated regions following a whole genome duplication event.

Whole genome duplication has been implicated in evolutionary innovation and rapid diversification. In salmonid fishes, however, whole genome duplication significantly pre-dates major transitions across the family, and re-diploidization has been a gradual process between genomes that have remained essentially collinear. Nevertheless, pairs of duplicated chromosome arms have diverged at different rates from each other, suggesting that the retention of duplicated regions through occasional pairing between homeologous chromosomes may have played an evolutionary role across species pairs. Extensive chromosomal arm rearrangements have been a key mechanism involved in re-dipliodization of the salmonid genome; therefore, we investigated their influence on degree of differentiation between homeologs across salmon species. We derived a linkage map for coho salmon and performed comparative mapping across syntenic arms within the genus Oncorhynchus, and with the genus Salmo, to determine the phylogenetic relationship between chromosome arrangements and the retention of undifferentiated duplicated regions. A 6596.7 cM female coho salmon map, comprising 30 linkage groups with 7415 and 1266 nonduplicated and duplicated loci, respectively, revealed uneven distribution of duplicated loci along and between chromosome arms. These duplicated regions were conserved across syntenic arms across Oncorhynchus species and were identified in metacentric chromosomes likely formed ancestrally to the divergence of Oncorhynchus from Salmo. These findings support previous studies in which observed pairings involved at least one metacentric chromosome. Re-diploidization in salmon may have been prevented or retarded by the formation of metacentric chromosomes after the whole genome duplication event and may explain lineage-specific innovations in salmon species if functional genes are found in these regions.

Temporal variation in selection on body length and date of return in a wild population of coho salmon, Oncorhynchus kisutch.

BACKGROUND: A number of studies have measured selection in nature to understand how populations adapt to their environment; however, the temporal dynamics of selection are rarely investigated. The aim of this study was to assess the temporal variation in selection by comparing the mode, direction and strength of selection on fitness-related traits between two cohorts of coho salmon (Oncorhynchus kisutch). Specifically, we quantified individual reproductive success and examined selection on date of return and body length in a wild population at Big Beef Creek, Washington (USA). RESULTS: Reproductive success and the mode, direction and strength of selection on date of return and body length differed between two cohorts sampled in 2006 and 2007. Adults of the first brood year had greater success over those of the second. In 2006, disruptive selection favored early and late returning individuals in 2-year-old males, and earlier returning 3-year-old males had higher fitness. No evidence of selection on date of return was detected in females. In 2007, selection on date of return was not observed in males of either age class, but stabilizing selection on date of return was observed in females. No selection on body length was detected in males of both age classes in 2006, and large size was associated with higher fitness in females. In 2007, selection favored larger size in 3-year-old males and intermediate size in females. Correlational selection between date of return and body length was observed only in 2-year-old males in 2006. CONCLUSIONS: We found evidence of selection on body length and date of return to the spawning ground, both of which are important fitness-related traits in salmonid species, but this selection varied over time. Fluctuation in the mode, direction and strength of selection between two cohorts was likely to be due to factors such as changes in precipitation, occurrence of catastrophic events (flooding), the proportion of younger- versus older-maturing males, sex ratio and densities of spawners.