Desmodus rotundus (hematophagous), trapped in two different caves surrounded by primary lowland forest, and Molossus molossus (insectivorous), trapped in forest and urban habitats. These sequences are related to 51 viral families known to infect a wide range of hosts . Most viruses detected reflected the diet of bat species, with a high proportion of plant and insect-related viral families for M. molossus and a high proportion of vertebrate-related viral families for D. rotundus, highlighting its influence in shaping the viral diversity of bats. Lastly, we reconstructed the phylogenetic relationships for five vertebrate-related viral families . The results showed highly supported clustering with other viral sequences of the same viral family hosted by other bat species, highlighting the potential association of viral diversity with the host’s diet. These findings provide significant insight into viral bat diversity in French Guiana belonging to the Amazonian biome and emphasize that habitats and the host’s dietary ecology may drive the viral diversity in the bat communities investigated.Environmental disturbances in the Neotropics  contribute to an increasing risk of cross-species transmission of microorganisms and to disease outbreaks due to changing ecosystems of reservoir hosts. Although Amazonia encompasses the greatest diversity of reservoir species, the outsized viral population diversity (virome) has yet to be investigated. Here, through a metagenomic approach, we identified 10,991 viral sequences in the saliva and feces of two bat species,  Paramyxoviridae, Filoviridae, Lyssavirus, Henipavirus), with viruses detected in bats usually being older than those found in humans or other animals [Bats present a rich viral diversity as compared to other animals and their uniqueness as hosts of pathogenic viruses has animated debates in recent years –3. Their animals . To date animals –17.et al. detected and identified a diversity of paramyxoviruses in several New World bat species [et al. reported novel influenza A viruses in Peruvian flat-faced fruit bats (Artibeus planirostris) [et al. proposed a new genus for two newly characterized polyomaviruses in Tadarida brasiliensis [Amazonia encompasses a great diversity of bat species. Numerous studies, using conventional methods or high-throughput sequencing, have allowed the identification of a number of viruses in this region –21. For  species , Tong etrostris) , and Limiliensis .et al. [et al. [In French Guiana, an Amazonian region, 106 bat species have been listed . During et al.  showed t [et al.  showed tDesmodus rotundus (Phyllostomidae) and Pallas’s mastiff bat, Molossus molossus (Molossidae). Both bat species are sympatric in the forest areas of French Guiana. They both have high plasticity and tolerance to perturbations but present distinct diet and social behavior.The aim of our study was to characterize the virome in the feces and the saliva of two abundant and opportunistic Amazonian bat species: the common vampire bat, D. rotundus bats are hematophagous and live in groups in highly complex social systems, for which Wilkinson et al. showed a strong association between the mating system and genetic structure of populations [D. rotundus also highlighted that their diet associated with social interactions  within colonies contribute to the transmission and maintenance of viruses such as rabies [Desmodus population in Peru identified metapopulation dynamics as a major driver of rabies maintenance [D. rotundus bats trapped in two caves (F and M) located in forest areas, for which no exchange of individuals was observed through a longitudinal mark-recapture study [ulations ,32. Studs rabies ,33. Furtntenance . Turmellntenance . We invere study .M. molossus bats are insectivorous and live in stable social groups that practice social foraging over large spatial scales [M. molossus bats trapped in urban and forest areas.l scales . Studiesl scales . We inveCircoviridae, Herpesviridae, Nairoviridae, Papillomaviridae and Retroviridae (genus Spumavirus)), detected in either high proportion in both species and all habitats or only a distinct bat species and habitat. These findings allowed identifying novel vertebrate-related viral sequences and emphasize the importance of studying the role of habitats and dietary ecology in shaping viral diversity for these two Amazonian bat species.Lastly, focusing on viral families known to infect mammals, we investigated the phylogenetic relationships of viral sequences related to five viral families  located 25 km apart in pristine primary lowland forests, presenting differences in physical patterns, cave-inhabiting bat species as well as population size , and the D. rotundus population ranges from 60 to 100 individuals [i.e., Anoura geoffroyi, Artibeus spp., Carollia perspicillata, Pteronotus rubiginosus and P. sp3, Tonatia saurophila, Trachops cirrhosus and Xophostoma sylvicola), and the D. rotundus population ranges from 120 to 150 individuals [ion size . Cave F ividuals . Cave M ividuals .M. molossus bats were sampled in two sites located in primary lowland rainforests and under the roofs of houses in two sites located in disturbed areas . All animals were trapped with mist nets erected inside roosts or in putative foraging courses. Species were identified on site, using external morphology. Prior to release, 138 saliva swabs were collected from e M only . SamplesM. molossus bats were held in individual sacks secured to a rope line for at least 10 min, allowing enough time for the excretion of 19 fresh feces of about 100 mg each  and three pools for D. rotundus   .et al. [g, 4°C). Eukaryotic and prokaryotic cell-sized particles were removed from supernatants through three successive filtrations , using cellulose acetate membrane filters . A differential centrifugation procedure, described by Prescott et al. [g, 4°C) procedure. Then the pellets were resuspended in nuclease-free water and cleared of persistent high-density particles with a low-speed centrifugation . Lastly, viral particles were pelleted with a 1-h ultracentrifugation step .Samples were processed as previously described by Victoria et al. . Brieflyt et al. , was useg, 4°C). Eukaryotic and prokaryotic cell-sized particles were removed from supernatants through two successive filtrations (0.45 μm and 0.22 μm), using cellulose acetate membrane filters . The filtrates were cleared of persistent high-density particles with low-speed centrifugation , then viral particles were pelleted with a 1-h ultracentrifugation step . All viral pellets were resuspended in 40 μL of nuclease-free water.For saliva samples, swabs were vigorously resuspended and 200 μL of suspension from each collecting tube was used to constitute the pools. Samples were cleared of debris by low-speed centrifugation  [® bio-robot (bioMérieux).Resuspended viral pellets from feces were treated with a mixture of DNases (Turbo DNase from Ambion and Benzonase from Novagen) and RNase One (Promega) to digest nonenveloped nucleic acids (capsids) . Resuspeet al. [For both feces and saliva samples, the RNA virus-only and DNA virus-only libraries were respectively constructed using a whole transcriptome (WTA) or a whole genome (WGA) amplification method previously described by Berthet et al. .™ rRNA depletion kit (Qiagen), following the manufacturer’s recommendations. Then viral RNA amplification was performed as described in the protocol of the QuantiTect® Whole Transcriptome Kit (Qiagen) except for the reverse transcription step. cDNA was synthesized using SuperScript® III Reverse Transcriptase (Invitrogen) and random hexamers (Roche), following the manufacturers’ recommendations.For the RNA virus-only amplification, an aliquot of the extracted viral nucleic acid collected was treated with Turbo DNase to remove viral DNA. Persistent rRNA was depleted with the GeneRead® Whole Genome Kit (Qiagen). To ensure homogeneity and blunt ends at the end of the strands produced, 10 U of Klenow polymerase (Roche) was added to the WTA- and WGA-amplified nucleic acids with 8 μL of random hexamers (Roche), incubated 1 h at 37°C, followed by 10 min at 75°C. Samples were assayed with a Qubit® fluorometric quantitation (Qiagen), using the dsDNA Broad Range and dsDNA High Sensitivity Qubit® assay kits, as recommended by the manufacturer.For DNA virus-only amplification, an aliquot of the extracted viral nucleic acid collected was treated with RNase One to remove viral RNA. Then viral DNA amplification was performed as described in the protocol of the QuantiTectFor both feces and saliva samples, 1 μg of each viral library was pooled together, whenever possible, to construct RNA plus DNA viral libraries. High-throughput sequencing was carried out at the Genomics platform at the Institut Pasteur, Paris. Shotgun libraries were prepared by standard Illumina protocols using 1 μg of total genomic DNA. Each sample  was indexed according to its provenance (species and habitats) using Illumina adaptor-specific primers. Samples were sequenced on a MiSeq sequencer in 300-base paired-end reads.http://hannonlab.cshl.edu/fastx_toolkit/index.html), with a quality threshold of 30 and length criteria of 70%. All cleaned data sets were saved for further analysis. Simultaneously, a three-pass digital normalization was run on each sample file using the Khmer software library [de novo assembled using two assembly algorithms: Velvet [Usearch v.7.0.1090 . A length comparison between the results was performed using a bit score ratio (br = bitscore_blastn_nt/bitscore_blastn_gb_vrl), and only contigs with br > 0.90 were regarded as suspect-viral sequences and conserved for further analysis. Centroids not identified as suspect-viral underwent a BLASTx comparison against a custom-made database, which included all protein sequences of the viral kingdom . To reduce the misidentification bias of false-positive sequences, identified suspect-viral sequences underwent a second BLASTx comparison with the NCBI nr database   with a : 10e-3) . SequencNairoviridae, Circoviridae, Retroviridae, Herpesviridae and Papillomaviridae were used for phylogenetic analysis. Reference genomes or nucleotide sequences of previously identified viruses were downloaded from GenBank. The accession number of viral sequences used to infer the phylogenetic trees are given in the respective analyses. Nucleotide and protein sequence editing were performed with Geneious R9  algorithm was run with four chains with 10 million generations each, with trees sampled every 500 generations and a burn-in of 25%. Validation of the inference was assessed based on the standard deviation of split frequencies, less than the expected threshold value of 0.01 in MrBayes and by inspecting the effective sampling size (ESS > 500) criterion in Tracer version 1.6 [Contigs from five vertebrate-related viral families: ous.com, ). Sequenent tool  includedent tool  and Protent tool , respectsion 1.6 .All virus sequences reported in this study were deposited in the GenBank nucleotide database under accession numbers KX812440 to KX812444, KX812446, KX812447, KX821677 and KX954092. The data from Illumina sequencing were deposited in GenBank Sequence Reads Archive under accession numbers SAMN05725475−SAMN05725481.M. molossus, 33,333,557 raw sequences were generated from both feces and saliva samples and 19,992,037 for D. rotundus. Following the data reduction steps, 10,339,752 non-digitally normalized reads and 4,117,544 digitally normalized reads were used for de novo assembly. SPAdes and Velvet assemblers generated a comparable number of contigs, even between normalized and non-normalized data. Between 50.22% and 67.66% of the contigs were saved after comparison 1  Iridoviridae family and the single-stranded DNA (ssDNA) Parvoviridae family. In contrast, ssDNA viruses (mostly from Parvoviridae) were slightly more numerous for M. molossus trapped in forest habitats compared to RNA viruses mostly from the Dicistroviridae and Nodaviridae families. The four viral families  detected for the D. rotundus were likely related to insects flying around feces.Fourteen insect-related viral families and a group of unclassified viruses were identified . PositivLuteoviridae, Bromoviridae and Virgaviridae families, were predominant for M. molossus trapped in urban habitats. In contrast, dsDNA viruses (Phycodnaviridae) and ssDNA viruses (Geminiviridae) were predominant for M. molossus and D. rotundus trapped in forest habitats.Sixteen plant- and protozoan-related viral families and a group of unassigned viruses were identified . ssRNA vAnelloviridae, Circoviridae and Parvoviridae families accounted for the highest proportion of DNA viruses for both samples of D. rotundus, with variation in number according to the sampling sites.Fourteen vertebrate-related viral families were identified . The higCircoviridae and Anelloviridae families were also the most abundant in the sample of M. molossus trapped in urban habitats, with very few viral sequences detected in saliva. In contrast, for M. molossus trapped in forest habitats, viral sequences related to the Herpesviridae, Papillomaviridae and Circoviridae families were the most numerous, with herpesviruses found for the most part in the saliva sample.The Poxviridae in both species, but only the D. rotundus trapped in cave M presented mammal-related poxviruses. These sequences presented high amino acid homology (>90%) with the bovine papular stomatitis virus, suggesting that this virus came directly from cattle and its presence was related to the dietary habit of D. rotundus .We found viral sequences related to Astroviridae, Nairoviridae, Hepeviridae, Picornaviridae and Retroviridae families. Astroviruses were only found in D. rotundus trapped in cave F but in both habitats for M. molossus. In contrast, nairoviruses were only found in D. rotundus trapped in cave F and in M. molossus trapped in forest habitats. Hepeviruses were only found in M. molossus trapped in urban areas. Picornaviruses were found in both habitats for M. molossus, but the only sequence detected in forest habitats was different from those detected in urban habitats.RNA viruses were found in distinct bat species or habitats and belonged to the Circoviridae is known to infect birds, mammals and insects, and comprises two genera, Circovirus and Cyclovirus (ICTV 2016). Their transmission occurs primarily through the fecal–oral route [Circoviridae viruses were detected in both D. rotundus and M. molossus samples, whatever the environment, with a total of 545 contigs  were obtained for M. molossus (n = 1 in urban habitats and n = 3 in forest habitats), and one partial sequence corresponding to the complete replication-associated protein gene for D. rotundus in cave M (n = 1) (Genbank acc. nb.: KX812440−KX812443 and KX954092). The complete genomes presented the archetypal genome organization of CVs, with the two inversely arranged ORFs encoding the Rep and Cap proteins (D. rotundus and forest habitats for M. molossus) formed two distinct monophyletic clades with other bat circoviruses detected in the gastrointestinal tract and feces samples of Chinese bat species [M. molossus was closely related to CVs detected in birds, with a posterior probability of 0.84.The family al route . Here, C contigs . These vproteins . The int species  Fig 3)..CircovirSpumavirus (ICTV 2016) constitutes the only genus of the Spumaretrovirinae subfamily, which belongs to the Retroviridae family. They are highly prevalent in several animal species  and currently six species have been described in the genus. Transmission of spumaviruses between nonhuman primates and cross-species transmission to humans occurs mainly through saliva  [M. molossus in urban habitats, showed 54.88% and 48.48% nucleotide and amino acid identity, respectively, with the pol gene of Rhinolophus affinis foamy virus 1 (Genbank acc. nb.: JQ814855) detected in the feces of Chinese bats [Rhinolophus affinis foamy virus 1, with a high posterior probability value of 0.91  –58. Neve, bites) –61. In tese bats . Phyloge of 0.91 . Bat FVsHerpesviridae family . HVs are organized in three subfamilies–Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. HVs can infect a wide range of hosts, including humans. Most HV-related contigs were found in the saliva sample of M. molossus in forest habitats. We identified a 943-nt-length fragment covering the DNA polymerase gene (Genbank acc. nb.: KX812446). This fragment displayed from 62.46% to 71.31% and from 68.93% to 80.58% nucleotide and amino acid identities, respectively, with other bat HVs. The highest percentage of identity in amino acids was observed with Myotis ricketti herpesvirus 1 (Genbank acc. nb.: JN692429). Phylogenetic analysis showed that the HV identified belonged to the Gammaherpesvirinae subfamily and was related to the Tupaia belangeri gammaherpesvirus 1 and the Myotis ricketti herpesvirus 1, with a posterior probability of 0.68  belong to the  of 0.68 .Nairoviridae family belongs to the order Bunyavirales (ICTV 2016). This family encompasses at least nine serogroups, including the Crimean-Congo hemorrhagic fever serogroup. Nairoviruses (NVs) are primarily tick-borne viruses capable of infecting different vertebrate hosts. Four contigs matching nairoviruses (NVs) were detected in the feces of M. molossus in urban areas (Rhinolophus pearsoni bunyavirus (Genbank acc. nb.: KC154063). Phylogenetic analysis revealed that the NV identified clustered with Rhinolophus pearsoni bunyavirus, with a posterior probability of 0.74  are usually asymptomatic and thought to be rarely transmitted between species. In this study, a total of 235 contigs related to PVs were detected in M. molossus samples, with most of them identified in the feces collected in forest habitats (Miniopterus schreibersii [Eidolon helvum (Germany), Eptesicus serotinus and Rhinolophus ferrumequinum [habitats . The lonhabitats . The phyeibersii , Eidolonmequinum , with a Desmodus rotundus and Molossus molossus bat species, living in sympatry but using distinct microhabitats in French Guiana.A wide spectrum of viruses capable of infecting a wide range of animals, plants, insects and bacteria was detected in bats. Since the 1950s, 28 viral families including more than 50 genera have been described in bats . With thD. rotundus, only 20 were common to both sampling sites (cave F vs. cave M). Likewise, focusing on vertebrate-related viral families, eight of the 11 viral families detected for this species were shared between the populations sampled in the two caves. For the M. molossus species, of the 46 viral families detected, 29 were common to both habitats (forest areas vs. urban areas). Focusing on vertebrate-related viral families, ten of the 12 viral families detected in this species were common to both habitats. Furthermore, we observed differences in the viral genera and species detected, depending on the sampling sites for both species. These results suggest that habitats may play a role in shaping the viral diversity harbored by the bats investigated. This observation was also supported by Hu et al. [e.g., sampling size, type of sample, capture effort, species, feeding strategies) will be necessary to confirm these results. Likewise, population structure analysis at the species level and studies on interactions with other bat species using the same habitat are needed to determine their respective roles in shaping viral diversity.Overall, this study identified 10,991 viral-associated contigs distributed within 51 known viral families of which 14 are known to infect mammals. New mammalian-related viral sequences/variants were discovered, adding knowledge on the bat-borne viral population. The proportion of eukaryote- and prokaryote-related sequences was low . About 0.30% of total contigs were virus-related sequences, with differences in composition depending on the type of sample, the investigated species, habitats, and fine-scale ecological heterogeneity  with avian CVs may result from a cross-species jump from birds to bats, as reported by Hu et al. [et al. [M. molossus showed a high genomic similarity with the FVs detected in pharyngeal and rectal swab samples of Rhinolophus affinis reported by Wu et al. [e.g., equines, felines, bovines, nonhuman primates). Our findings indicate that FVs can indeed infect bats, which could be considered as potential reservoirs and dispersers of this viral genus.We found several novel mammalian-related viral sequences/variants from the  species ,62,65–68 species . For exa species ,69 and cu et al.  and Lima [et al. . LikewisAlpha-, Beta- and Gammaherpesvirinae) in feces, anal swabs, digestive tract and saliva samples of bats [M. molossus from forest habitats were also found in the feces samples, which showed the capacity of these bats to shed HVs in the environment and supported the hypothesis of a potential oral–fecal transmission route. Detected HV-related sequences were novel and showed a high sequence similarity with other bat HVs. Nevertheless, the phylogenetic tree constructed using a Bayesian method revealed that M. molossus herpesvirus 1 (KX812446) did not cluster in a unique group with other known bat gamma-HVs. Rather, it shares a common ancestor with Myotis ricketti and Tupaia belangeri gamma-HVs, stressing the need for further characterization of M. molossus herpesvirus 1 to confirm its taxonomic assignment.Several studies reported the detection of herpesviruses (HVs) of the three subfamilies  naturally infecting bats were reported in a few studies, with some capable of causing intestine and hepatic disorders in other mammals ,78–80. Eet al. , but thePapillomaviridae, indicating that bat species might be associated with a wide diversity of PVs. We described the complete genome of a novel bat PV, MmoPV1 (KX812447), detected in the feces and saliva samples of M. molossus sampled in forest and urban habitats. This is the first detection of PV in this species to date. Comparing MmoPV1 with other PVs showed that it contains the typical PV ORFs coding for four putative early proteins  and two putative late capsid proteins (L2 and L1). The putative early protein E4 –present in other phylogenetically closely-related bat PVs (JQ692938 [Diverse sequences related to papillomaviruses (PVs) were reported in bats ,67,82,83JQ692938 )–was absJQ692938 .M. molossus and D. rotundus, with overlapping distribution but using distinct microhabitats in French Guiana. It provides important insight into the feces and saliva viromes of the two bat species investigated within contrasting habitats.This study is the very first to explore the virome of fecal and saliva samples obtained from the two common Amazonian bat species, D. rotundus and M. molossus, further characterization of their geographic range, foraging-induced interactions, species distribution and gregariousness will be necessary. Indeed, larger geographic ranges encompass greater diversity of ecosystems/habitats, which could lead to a spatial population substructure [However, to fully understand how viral diversity is shaped in tructure . The ovetructure ,35. Liketructure . ConsequS1 Filee novo assembly of processed reads. Contig and sequential BLAST comparisons, with the total number of contigs and viral families identified in D. rotundus and M. molossus samples.Read data reduction steps and d(DOCX)Click here for additional data file.S1 FigThe total number of collected samples per site is given in parentheses. Pie chart indicates the proportion of feces (orange) and saliva (orange dots) samples collected. Detailed characteristics of the different collecting sites are given in (TIF)Click here for additional data file.S2 Figeneious R9.The open-reading frames encoding the putative late (L1 and L2) and early  proteins are shown in green boxes. The genome organization was determined with G(TIF)Click here for additional data file.


Biomarkers have been used extensively to provide the connection between external levels of contaminant exposure, internal levels of tissue contamination, and early adverse effects in organisms.To present a three-step protocol for identifying suitable biomarkers to assess fish health in coastal and marine ecosystems, using Gladstone Harbour  as a case study.Prior to applying our protocol, clear working definitions for biomarkers were developed to ensure consistency with the global literature on fish health assessment. First, contaminants of concern were identified based on the presence of point and diffuse sources of pollution and available monitoring data for the ecosystem of interest. Second, suitable fish species were identified using fisheries dependent and independent data, and prioritised based on potential pathways of exposure to the contaminants of concern. Finally, a systematic and critical literature review was conducted on the use of biomarkers to assess the health of fish exposed to the contaminants of concern.We present clear working definitions for bioaccumulation markers, biomarkers of exposure, biomarkers of effect and biomarkers of susceptibility. Based on emission and concentration information, seven metals were identified as contaminants of concern for Gladstone Harbour. Twenty out of 232 fish species were abundant enough to be potentially suitable for biomarker studies; five of these were prioritised based on potential pathways of exposure and susceptibility to metals. The literature search on biomarkers yielded 5,035 articles, of which 151met the inclusion criteria. Based on our review, the most suitable biomarkers include bioaccumulation markers, biomarkers of exposure , and biomarkers of effect .Our protocol outlines a clear pathway to identify suitable biomarkers to assess fish health in coastal and marine ecosystems, which can be applied to biomarker studies in aquatic ecosystems around the world. Globally, the coastal environment is the ultimate sink for many contaminants and their associated breakdown products . Contamiearly warning’ signals that have the potential to detect an effect in target biota prior to one being observed at the population, community or ecosystem level . Lo. Lo31]. Finally, organochlorine and organophosphorus pesticides, herbicides, carbamate pesticides and insecticides have not been detected in the sediments of Gladstone Harbour  27, 31], 3127, 3Sillago ciliata), as earlier surveys report this as the most prevalent fish caught by recreational fishers between 1990 and 2004  –48 46–48http://www.fishbase.org/; http://australianmuseum.net.au/). Maximum sizes for individual species ranged from 20 cm Standard Length (SL) (Ambassis marianus) to 200 cm Total length (TL) . Many of the 14 species for which weight and/or age information is available have maximum ages of 10 years or more. Labile patterns of sexual development such as protandry and protogony occur in at least five of the 20 species. This information, combined with our systematic review on fish biomarkers (see next section), was used to identify fish species suitable for biomarker studies that could be used to assess fish health in Gladstone Harbour.Information on life history characteristics was not as readily available for these 20 fish species  46–48] –48 46–48Our systematic review of the literature identified a total of 5,862 publications, including 832 duplicate records ; S1 TextA. australis, A. berda, E. coioides, L. equulus, M. cephalus, P. fuscus and P. indicus pyrene. Both EROD and AHH are sensitive biomarkers when measured in the livers of fish, especially when used in conjunction with other biomarkers such as CYP1A and CYP1A mRNA. The other sub units of the cytochrome 450 system are measured using the following assays pentoxyresorufin-O-depentylase (PROD) measures the catalysis of cytochrome P450 CYP2B, 7-benzyloxyresorufin-O-depentylase (BROD) measures the catalytic activity of the cytochrome P450 subfamily CYP3A and 7-methoxyresorufin-O-methoxyresorufin (MROD) cytochrome P450 CYP1A2.Solea senegalensis) exposed to sediment contaminated with metals and PAHs showed a significant decrease, correlated to heavy metals in preference to PAHs , 9686, 9Metallothioneins (MT). Metallothioneins (MTs) are a family of proteins essential for the regulation, sequestration and detoxification of metals including Cu, Zn, Cd, Hg and have been shown to be induced by Co, Ni, Bi and Ag. MTs act by intercepting and binding metal ions as they are being taken up by the cell and also by the removal of metals by non-thionein ligands. MTs can be measured by quantitative assays of the protein via IEC-AAS, metal substitution assays and polarographic and immunochemical techniques. In fish, MT activity is greatest in tissues that are most closely involved in uptake, storage and excretion .Aphanius fasciatus, Atherinops affnis, Gobius niger, Sparus aurata and T. jarbua with increasing Cd concentrations [T. jarbua and A. latus but not in their gills [MTs are one of the most studied biomarkers for assessing the effect of metal contamination in fish, with a total of 32 studies reviewed . Thirty-trations , 136–139trations , 136–139trations , 140. Coir gills . CombineS. aurata did not change following aqueous exposure to Cu, and the response varied with tissue following dietary uptake of Cu [The copper transporter gene (Ctr1) mediates the transport of copper into eukaryotes for metabolic processes such as a cofactor for enzymes. The single study identified in our review showed that this biomarker in ke of Cu . Hence, Multixenobiotic resistance (MXR). Multixenobiotic resistance (MXR) prevents the accumulation of both xenobiotic and endogenous compounds inside the cell, by removing them via an energy-dependent transport protein, Transmembrane P-glycoprotein (PGP). This biomarker is not further considered as none of the studies reviewed used MXR.Haematological parameters. Haematological parameters offer the ability to conduct non-lethal or non-destructive tests. Haematological parameters may be less chemical specific than some other biomarkers, but provide other insights into the general health and physiology of the organism. A number of haematological parameters have been used to monitor pollution induced stress in fish, including plasma enzyme activity, erythrocyte counts, haemoglobin content and haematocrit (matocrit .Serum transaminases. Measurement of enzymes in the serum (extracellular fluid or plasma) is considered a sensitive indicator of cellular damage. Alanine transferases (ALT or GPT) and aspartate transaminase (AST or GOT) are a group of enzymes responsible for the conversion of amino acids and α-ketoacids. One of the two studies identified in our review  gene encodes for the δ-aminolevulinic acid dehydratase enzyme also known as porphobilinogen synthase [synthase . The ALASodium (Na) and chloride (Cl) are the main ions in the extracellular fluid. Maintaining these ion concentrations is necessary so that membrane potential is maintained across the cell. Membrane potential is critical for generating energy, transmission of nerve impulses and muscle movement. Only one study measured Na and Cl in fish after metal exposure and found no significant change  S18 Tab. Hence tHaematocrit is the percentage by volume of red blood cells in the blood, whereas erythrocyte counts refer to the absolute number of red blood cells in given volume of blood. Haemoglobin is an iron based oxygen transport molecule that makes up a large proportion of the red blood cell, it can be easily measured and is expressed as g/dL of blood. All of these parameters have been shown to change in the presence of certain contaminants and can be used as indicators of stress or toxicity . HaematoAlkaline phosphatase (ALP) is an enzyme that catalyses the dephosphorylation (hydrolysis of phosphate group) of a range of molecules such as nucleotides and proteins. Two studies used ALP activity in fish , with a G. morhua or P. flesus when exposed to water containing metals. Due to the limited data on these biomarkers, combined with apparent species-specific responses, they are not recommended for assessing fish health in Gladstone Harbour.Intermediary metabolism parameters in the serum, such as glucose, albumin and total protein, are frequently analysed to examine the nutritional status of fish. Two field studies reported different responses for all three biomarkers to metal exposure , 120 S1. Omar etImmunological parameters. Immunological parameters have not been used often as biomarkers in fish exposed to metal contamination, with the most common ones being proliferating cell nuclear antigen (PCNA) [Coris julis [D. labrax [P. hasta [D. labrax did not respond to Cu exposure (in antifouling nets) [n (PCNA) , 146, can (PCNA) , 129  . Other bng nets)  suggestiA. anguilla, an increase in P. flesus, and a mixed response in S. maximus [G. morhua and S. senegalensis following exposure to sediment contaminated with metals, including when exposed to the same sediment under different conditions [Additional immunological parameters that have been used include % leukocytes and % thrombocytes , 127, st maximus , 148. Sinditions , 127. Innditions , 147, annditions , 148. Onnditions . Due to Neurotoxic parameters. Cholinesterase (ChE) are enzymes relevant to neural functions; those with a high affinity for acetylcholin (AChE), and those with affinity for butyrylcholin (BChE) [L. calcarifer muscle differed across rivers with different levels of contamination, but was not clearly linked with aqueous or sediment metal concentrations [S. senegalensis in gill and muscle tissue, but no response in brain tissue [n (BChE) . Fish mun (BChE) , 147, win (BChE) , 147, whn (BChE) , 106. Actrations . The resn tissue . The resn tissue , 146. ThD. labrax exposed to sediment contaminated with metals and hydrocarbons [C. julis exposed to contaminated sediment [C. julis but no response in D. labrax [The neurotransmitters choline acetyltransferase (ChAT) and serotonin, as well as the enzymes tyrosine hydroxylase (TH), involved dopamine synthesis and the serotonin receptor (5-HT3) have been used as fish biomarkers , 146 S1. Levels sediment . The res. labrax , 146. GiReproductive and endocrine parameters. Pollution stress has well-known effects on reproductive and endocrine parameters in fish [vtg transcripts assessed on real-time PCR were higher in male A. fasciatus collected from a site contaminated with heavy metals compared to a more pristine site [P. flesus varied considerable between sites including reference sites [vtg has been isolated for one of the priority species identified for Gladstone Harbour, namely L. calcarifer [ in fish , however in fish . VTG lev in fish . Only twine site . In contce sites . The spelcarifer –154. HenThe endocrine receptor, thyroid receptor alpha (Trα) has also been used as a biomarker to assess fish health but the levels varied with exposure time . Given tGenotoxic parameters. Genotoxic parameters refer to any changes to the genetic material of an organism caused by exposure to a contaminant. A number of genotoxic parameters have been used to monitor pollution induced stress in fish, including DNA adducts, secondary DNA modifications, and irreversible genotoxic events . DNA strand breaks can be assessed using the comet assay and has been examined in 14 studies [E. coioides [M. cephalus [S. senegalensis following Cd exposure in toxicity testing conditions [ studies , 158–162 studies , 159–162cephalus . DNA damcephalus . Seasonacephalus . No channditions . Given tnditions  will neeP. flesus [L. calcarifer exposed to contaminated sediment [DNA unwinding was assessed in two field studies only, showing no effect of metal contamination in . flesus , but higsediment   is an enzyme in the plasma membrane involved in ion transport and osmoregulation, and has been used in five studies to assess the response to metal exposure [A. anguilla [D. labrax [Fundulus heteroclitus exposed to Zn [Squalus acanthias exposed to Pb [exposure , 163(S21anguilla  but not . labrax . Activit. labrax . In laboed to Pb . Given tThe enzyme lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate in anaerobic glycolysis, and has been used in five studies , 128 S2. Four of2+-ATPase, trimethylamine oxide (TMAO), urea, PaO2, PaCO2, arterial pH, K, Ca, NH4, Na and Cl [Single studies were identified using other osmoregulatory and respiratory markers, such as aquaporins (AQPs), Caa and Cl , 163 (S2Carbohydrate and aerobic metabolism. Pyruvate kinase (PK) is involved in the generation of energy for the cell due to its role in transferring a phosphate to ADP in the final step of glycolysis. Two laboratory toxicity studies examined the effects of Cd exposure , malic dehydrogenase (MD), hepatic lipase (HL), lipoprotein lipase (LPL), isocitrate dehydrogenase (IDH), hexokinase (HK), citrate synthase (CS), cytochrome c oxidase (COX), 9-cis retinic acid receptor (RXRα) and nuclear receptor binds xenochemical compounds termed estrogen like molecules (Erα) , 123 S2. These wHistopathology. Histopathology of a range of fish tissues following metal exposure has been assessed in 19 studies [M. cephalus, L. calcarifer and E. coioides [ studies , 164–170 studies , 168–170coioides . While hcoioides , 170. ThGross indices. Gross indices reflect fish condition as determined by morphology, appearance and other gross characteristics [eristics . These bLiver somatic index (LSI). The hepatic somatic index (HIS) or liver somatic index (LSI) is used to identify possible liver diseases and is estimated using the equation;L. calcarifer [Eleven studies examined changes in HSI/LSI in fish exposed to metals , 171, wilcarifer . Seasonalcarifer , 171. ThGonadosomatic index (GSI). The gonadosomatic index (GSI) is used as an indicator of reproductive condition and is estimated using the equation;M. cephalus [Six of the studies reviewed used GSI to assess fish health following metal exposure , 161. Thcephalus , 161, tocephalus , 98, 156Spleen somatic index (SSI). The spleen somatic index (SSI) is an indicator of overall spleen health and is calculated using the equation;Symphodus melops and no response for G. morhua [Two studies used SSI to assess fish health following metal exposure, with a decline reported for . morhua , 108. ThVisceral somatic index VSI. The visceral somatic index is an indicator of the overall health of a fish’s viscera. VSI is calculated using the equation;One toxicity test used VSI and reported no change in fish exposed to Cd . Hence, Condition factor (CF). The condition factor (CF) is used to assess the general condition of fish, and is estimated using the following equation;Eleven out of the fifteen studies that used CF to assess fish health following metal exposure reported no change or a mixed response , 167–169Fulton’s condition index (K index). The Fulton’s condition index (K index) is a measure of fish health based on standard weight, and is estimated using the equation;L. calcarifer, showed no change in their K index [Nine studies used the K index to assess fish health following exposure to sediment contaminated with metals , 172–175 K index , 124. On K index . Six fis K index . The varGrowth index. Specific growth rates (in % day) in length and weight have been used to estimate growth indices for both length and weight, using the following equations;D. labrax, but not in S. maximus, caged in a polluted harbour [S. maximus [Only one field study used the growth index showing a significant decrease in juvenile  harbour . A contr maximus . These iRNA: DNA ratio. The RNA:DNA ratio is used as an indicator of nutritional condition, but its response to metal exposure is highly variable [L. calcarifer [variable , 175. Sovariable , 172. Twvariable  and in Llcarifer . In addilcarifer , 172. GiOther biomarkers of effect. A range of other biomarkers of effect have been used to assess the response in fish following metal exposure, including metabolism and nutrition related biomarkers, parasites, neuromasts in lateral line tissue, prey capture and food intake, phototactic responses and epidermal diseases  to the quantity of sterols  (TAG:ST) [S. maximus under controlled laboratory conditions [D. labrax and S. maximus caged in a polluted harbour [P. flesus in field conditions [S. hasta following Cd exposure [E. coioides following Cu toxicity [E. coioides following Cu exposure [A. fasciatus deformed following Cd exposure, compared to normal fish under field conditions, and corresponded with increased collagen fibres [(TAG:ST) , 175. Fonditions , in juvenditions . The totnditions , 173, winditions  to a decexposure , but dectoxicity . Total ptoxicity , 144, whexposure . In the exposure . Collagen fibres . In contn fibres . OverallParasites. The abundance of parasites has been assessed as a fish biomarker of sediment metal concentrations in the North Sea [P. flesus was influenced by sediment metal concentrations, with the abundance of several parasites negatively correlated with the concentration of individual metals [orth Sea , 167. Thl metals . Parasitl metals . No chanl metals , and theLateral line tissue. The lateral line of fish is composed of neuromasts and is involved in their behaviour such as detecting predators and avoiding obstacles [D. labrax, resulting in a 10% decrease in fish escape rate [bstacles . The oneape rate . Damage Food intake and prey capture. Food intake was examined in one laboratory toxicity study, showing a decrease with increasing Cd concentrations in A. affinis [Fundulus heteroclitus captured in contaminated sites showed a lower prey capture ability in laboratory experiments than fish from reference sites [ affinis . Prey ca affinis . Funduluce sites , 178.Thece sites , 178. GiPhototactic response. The phototactic response of fish larvae can be used as indicator of their physical condition [ondition . This biondition . Given tEpidermal diseases. The incidence of epidermal diseases in fish exposed to contaminants in mesocosm experimental systems increased in both contaminated and clean mesocosms tanks [ms tanks . Howeverms tanks . Given tTraditionally, a suite of individual biomarkers has been used to assess biological impacts of contaminants on fish. New techniques such as microarray, proteomics and RNA sequencing (RNASeq) take fish health assessments beyond individual biomarkers to a global analysis of how all genes or proteins within an individual fish respond to contaminants , 156. ApMicroarray and proteomics. Our systematic review identified four papers which assessed fish health using microarray or proteomics [P. flesus exposed to reference and contaminated sediments showed significant changes in the hepatic abundance of 241 transcripts and 18 metabolites [S. aurata exposed to contaminated sediment [S. aurata exposed to Cu under laboratory conditions, 10 proteins were differentially expressed between controls and Cu exposed fish [oteomics , 156. Miabolites , and difsediment . Based osed fish . The tensed fish . Hence msed fish , potentiRNASeq. RNASeq is based on measuring the abundance of transcripts, but unlike microarray, is not limited to commercially available array chips or customising expensive individual chips, and does not require pre-existing knowledge of the fish genome [L. calcarifer [L calcarifer transcriptome [h genome . This meh genome , such aslcarifer . Our revcriptome . HoweverBased on the metals being identified as contaminant of concern , our revL. calcarifer [L. carponotatus [A. berda [L. calcarifer [L. carponotatus [M. cephalus [M. cephalus [L. calcarifer [L. calcarifer [L. calcarifer [M. cephalus [Acanthopagrus australis (Yellowfin Bream), Lates calcarifer (Barramundi), and Mugil cephalus (Sea Mullet), and the pelagic Eleutheronema tetradactylum (Blue Threadfin), and Scomberomorus queenslandicus (School Mackerel).Our review highlighted that the pathway of metal exposure is critical in causing an effect, particularly whether metals are taken up through the water or the sediment. Hence, fish species recommended for Gladstone Harbour need to include both pelagic and demersal species. In addition, several biomarkers have already been developed for certain species identified as potentially suitable , albeit lcarifer , 181 andonotatus , 183;  VTlcarifer –154; (v)lcarifer , and  as a case study . To ensuhttp://www.fishbase.org/; http://australianmuseum.net.au/) , Lates calcarifer (Barramundi), Mugil cephalus (Sea Mullet), Eleutheronema tetradactylum (Blue Threadfin), and Scomberomorus queenslandicus (School Mackerel). For two of these species , many of the potentially suitable biomarkers have already been developed but will need to be verified for Gladstone Harbour conditions. For the other fish species, the potentially suitable biomarkers will need to be developed and subsequently verified for Gladstone Harbour.Our systematic review has identified various biomarkers that could be suitable for assessing fish health in Gladstone Harbour . In addinet.au/) , and Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.S14 Table(DOCX)Click here for additional data file.S15 Table(DOCX)Click here for additional data file.S16 Table(DOCX)Click here for additional data file.S17 Table(DOCX)Click here for additional data file.S18 Table(DOCX)Click here for additional data file.S19 Table(DOCX)Click here for additional data file.S20 Table(DOCX)Click here for additional data file.S21 Table(DOCX)Click here for additional data file.S22 Table(DOCX)Click here for additional data file.S23 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Giardia duodenalis is a common human and animal pathogen. It has been increasingly reported in wild and captive non-human primates (NHPs) in recent years. However, multilocus genotyping information for G. duodenalis infecting NHPs in southwestern China is limited. In the present study, the prevalence and multilocus genotypes (MLGs) of G. duodenalis in captive NHPs in southwestern China were determined. We examined 207 fecal samples from NHPs in Sichuan and Guizhou provinces, and 16 specimens were positive for G. duodenalis. The overall infection rate was 7.7%, and only assemblage B was identified. G. duodenalis was detect positive in northern white-cheeked gibbon , crab-eating macaque  and rhesus macaques . Multilocus sequence typing based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) revealed nine different assemblage B MLGs (five known genotypes and four novel genotypes). Based on a phylogenetic analysis, one potentially zoonotic genotype of MLG SW7 was identified in a northern white-cheeked gibbon. A high degree of genetic diversity within assemblage B was observed in captive northern white-cheeked gibbons in Southwestern China, including a potentially zoonotic genotype, MLG SW7. To the best of our knowledge, this is the first report using a MLGs approach to identify G. duodenalis in captive NHPs in Southwestern China. Giardia duodenalis is the etiological agent of giardiasis, a gastrointestinal infection that is typically asymptomatic, but may also be severe in some individuals [G. duodenalis (A-H), assemblages A and B frequently infect humans and animals, assemblages C and D have been described in domestic and wild canines, assemblage E have been widely reported in ruminants but sporadically detected in NHPs and humans, assemblage F in cats, assemblage G in rodents and assemblage H in seals and gulls [ividuals –3. At prnd gulls . Assemblnd gulls –6.G. duodenalis have a monoxenous life cycle and can spread rapidly in captive NHPs [G. duodenalis has been widely investigated in NHPs. Assemblages A, B and E are found in NHPs and assemblage B is dominant [NHPs are valuable wildlife resources. Owing to their high genetic homology to humans, NHPs are important experimental models for clinical research and public health research. ive NHPs . Geneticdominant , 6. Moledominant , 5.G. duodenalis infecting NHPs based on multi-locus genotyping. Molecular analyses to date have typically focused on a single genetic locus [G. duodenalis, multi-locus genotyping (MLG) employing beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) has been used for genotyping and subtyping G. duodenalis in humans and animals [G. duodenalis in captive NHPs in Southwestern China. These findings improve our understanding of the genetic diversity and the transmission routes of G. duodenalis in NHPs.However, little is known about genetic variation in ic locus , 8, 9. Iic locus , 10. To  animals –13. The This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-S20156703. Prior to the collection of fecal specimens from NHPs, permission was obtained from owners.From March to May 2016 and September to November 2016, 207 fecal specimens from NHPs were collected from Sichuan and Guizhou provinces. Fresh fecal specimens were collected immediately after defecation on the ground and separately stored in 50-mL centrifuge tubes. The specimens were kept cool during transport and arrival at the Sichuan Agricultural University. Specifically, 101 samples were obtained from rhesus macaques from the National Experimental Macaque Reproduce Laboratory in Southwest China (n = 31), Chengdu Gaoxin rhesus macaque farm (n = 30), Chengdu zoo (n = 20) and Bifengxia zoo (n = 20). Thirty-six samples were from northern white-cheeked gibbons from zoos in Guiyang (n = 30), Chengdu (n = 2) and Bifengxia (n = 4). Nine samples were from Golden snub-nosed monkeys in the Chengdu zoo. Sixty samples were from crab-eating macaques in the National Experimental Macaque Reproduce Laboratory in Southwest China and the Chengdu Gaoxin rhesus macaque farm . Samples® DNA Isolation Kit  following the manufacturer`s instructions. DNA samples were stored in 100 μL of the kit's Solution Buffer at 20°C until use.Before extracting DNA, the fecal samples were washed with distilled water until potassium dichromate was removed. Genomic DNA was extracted using the PowerSoilG. duodenalis by nested PCR amplification of the beta-giardin (bg) gene [bg-positive specimens were further characterized by PCR amplification of the tpi and gdh genes [Each specimen was examined for bg) gene , The bg-dh genes . SecondaG. duodenalis assemblage, the sequences were aligned with sequences downloaded from the GenBank database based on a BLAST analysis (http://blast.ncbi.nlm.nih.gov) using ClustalX. For the phylogenetic analysis, sequences obtained in this study were used to construct a neighboring-joining tree using Mega 5 (http://www.megasoftware.net/). A total of 1000 replicates were used for the bootstrap analysis.The amplified products of the expected size were sequenced by Invitrogen . To determine the Differences in infection rates among NHPs and among animals in different areas were assessed using the chi-square test implemented in SPSS version 17.0 . P < 0.05 was considered significant.bg-based PCR analysis of 207 specimens from 4 NHP species, 16 (7.7%) samples from 3 species were positive for G. duodenalis. All the positive specimens were successfully amplified and sequenced for the bg, tpi and gdh genes. Sequences were deposited in the GenBank database under the accession numbers KY696790-KY696821.In the G. duodenalis. Northern white-cheeked gibbons showed the highest infection rate . All golden snub-nosed monkeys (n = 9) were negative for G. duodenalis. The difference in infection rates among 4 species was significant (P<0.05). In China, six studies have examined G. duodenalis infection in NHPs in parks, zoos, farms and laboratories to date, and the overall infection rate was between 1.3% and 18.6% in these studies [G. duodenalis infection is common in wild and captive NHPs and has a wide geographic distribution in China.The infection rates ranged from 0% to 38.9% in the 4 species . Specifi studies , 15–19.  studies , and was studies  and two  studies , 18. OurG. duodenalis infection in NHPs showed a similar trend to that observed in China. The overall infection rate of G. duodenalis in NHPs is between 2.2% and 47.0% [In other countries, nd 47.0% , indicatnd 47.0%  and Thaind 47.0% , and it nd 47.0%  and Croand 47.0% . This rend 47.0% , 8, 20.G. duodenalis, which was also higher than the infection rate in a previous study  [G. duodenalis than wild animals. This might be explained by the single-host life cycle and the resilient infectious cysts of G. duodenalis [In this study, the infection rate for captive NHPs in Sichuan province was 0.6% (1/106), which is almost identical to that in a comprehensive parasite study performed in 2009–2015 in Sichuan  . The infodenalis . Captiveodenalis , 15.G. duodenalis may explain the low infection diversity of assemblages [To date, assemblages A, B and E have been detected in NHP species in China. Assemblage A and B have both been found in captive and free-range NHPs, but assemblage E has only been found in captive NHPs . In thisemblages , and sugemblages , assemblgdh sequences. All of the 16 sequences were 100% similar to sequences of human isolates from Brazil  [bg and tpi loci showed high levels of sequence polymorphism. 5 and 6 subtypes were identified in the 16 strains, including two new subtypes. At the bg locus, four known subtypes Bb-4 (KJ888977) [tpi locus, five known subtypes B7 (JQ863259) [No genetic variation was observed among the 16 F507682) . The bg J888977) , BIII-1 J888977) , and B1 J888977)  were fouQ863259) , WB8 (KFQ863259) , BIV (ABQ863259)  and WB6 A total of 16 NHPs specimens  were classified as assemblage B and nine MLGs were identified among the 16 positive specimens. The subtype identities and geographical and host distributions of the nine MLGs are listed in G. duodenalis between animals [G. duodenalis from this species [Phylogenetic analyses showed that MLG SW7 belonged to a zoonotic group. Given the zoonotic potential of this subtype, epidemiological and source tracking investigations as well as strict surveillance in captive NHPs in southwestern China are needed. MLG SW9 was closely related to the sequences obtained from NHPs in other studies . MLG SW1 animals . Other M species .G. duodenalis in captive NHPs in Southwestern China. One genotype of the potentially zoonotic assemblage B of MLG SW7 strain was identified in a northern white-cheeked gibbon. This suggests that the zoonotic transmission of Giardia might occur between the northern white-cheeked gibbon and humans. Additionally, high degree genetic diversity of assemblage B MLGs (7 MLGs) was detected in captive northern white-cheeked gibbons in Southwestern China. Additional MLGs studies of captive NHPs are needed, to better characterize genetic diversity and the routes of transmission of G. duodenalis between NHPs and humans or other animals.The results of the present study confirm previous findings that assemblage B is dominant in northern white-cheeked gibbons. We first used a MLGs approach to identify


Chryseobacterium bovis DSM 19482T  is a Gram-negative, rod shaped, non-motile, facultative anaerobe, chemoorganotroph bacterium. C. bovis is a member of the Flavobacteriaceae, a family within the phylum Bacteroidetes. It was isolated when psychrotolerant bacterial communities in raw milk and their proteolytic and lipolytic traits were studied. Here we describe the features of this organism, together with the draft genome sequence and annotation. The DNA G + C content is 38.19%. The chromosome length is 3,346,045 bp. It encodes 3236 proteins and 105 RNA genes. The C. bovis genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes study. Chryseobacterium bovisDSM 19482T , was isolated by Hantsis-Zacharov and Halpern [Chryseobacterium. Chryseobacterium bovis proliferates at low temperatures and produce heat-stable proteolytic and lipolytic enzymes which remain active after the milk pasteurization process. This may be a limiting factor in maintaining the flavor quality of fluid milk and its products [C. bovis H9TDSM 19482T was isolated in April 2004 from a modern farm equipped with automated milking facilities in northern Israel [Chryseobacterium species were isolated and identified from raw milk in the same study [C. bovis, C. haifense and C. oranimense [Chryseobacterium [Flavobacteriaceae and currently consists of about 100 species with Chryseobacterium gleum as the type species. Species belonging to this genus exist in diverse environments such as milk, water, sludge, soil, animals, insects, plants and human samples [acterium  is a mem samples .C. bovis, together with the permanent draft genome sequence description and annotation of the type strain (DSM 19482T).Here we describe a summary classification and a set of the features of the species C. bovis strain DSM 19482T shares typical characteristics of Chryseobacterium such as Gram-negative staining, occurrence as chemoheterotrophic rods and positive catalase and oxidase reactions. The strain contains flexirubin-type pigments, which are also typical for Chryseobacterium [DSM 19482T belongs to Chryseobacterium genus , esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, ß-galactosidase and cystine arylamidase activities are present [Growth is observed under anaerobic conditions on SPC agar containing 0.1% (w/v) potassium nitrate but not on SPC agar with the addition of 0.5% glucose (indicating that glucose is not fermented) . The str15:0; antesio-C15:0 and iso-C17:0 3OH. Some strains in this species also possess iso-C17:0 ω9c as a major fatty acid [The major fatty acids of the type strains are: iso-Ctty acid .Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project [http://jgi.doe.gov/) using state of the art sequencing technology [This organism was selected for sequencing based on its phylogenetic position  and is p project . The goa project . The pro project  and the chnology . A summaDSM 19482T was grown aerobically in DSMZ medium 381 [A culture of dium 381  at 28 °Cdium 381 .http://www.jgi.doe.gov). All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts [https://github.com/lh3/wgsim), (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r46652) [The draft genome was generated at the DOE Joint genome Institute (JGI) using the Illumina technology . An Illurtifacts . Followirtifacts , (2) 1–3 r46652) . ParametWalnut Creek, CA, USA.Genes were identified using Prodigal , as partThe assembly of the draft genome sequence consists of 96 scaffolds amounting to 3,346,045 bp, and the G + C content is 38.19% , phytoene desaturase (neurosporene-forming), phytoene desaturase (zeta-carotene-forming), all-trans-zeta-carotene desaturase and beta-carotene 3-hydroxylase.C. bovisDSM 19482T was able to grow under anaerobic conditions when nitrate was provided. This ability is supported by the presence of the following genes: MFS transporter, NNP family, nitrate/nitrite transporter (two genes) and assimilatory nitrate reductase catalytic subunit.Bacteroidetes phylum such as gliding motility-associated lipoprotein GldK and gliding motility-associated lipoprotein GldH. Another gene that supports the motility feature is the chemotaxis protein MotB gene.Gliding motility properties are reflected by the presence of the genes that are exclusive to the C. bovisDSM 19482T genome are genes for resistance to different components. For example a gene for multidrug resistance protein, MATE family. Members of the Multi-Antimicrobial Extrusion (MATE) family function as drug/sodium antiporters. These proteins mediate resistance to a wide range of cationic dyes, fluroquinolones, aminoglycosides and other structurally diverse antibodies and drugs. These proteins are predicted to have twelve alpha-helical transmembrane regions. The Strain DSM 19482T genome, also possesses a gene for cobalt-zinc-cadmium resistance protein CzcA. CzcA has a low cation-transport activity for cobalt and is essential for the expression of cobalt, zinc and cadmium resistance. Another gene found in the genome is a tellurite resistance protein TerC. TerC has been implicated in resistance to tellurium, and may be involved in efflux of tellurium ions. The quaternary ammonium compound-resistance protein SugE gene that is found in C. bovisDSM 19482T genome encodes an efflux pump which confers resistance to cetylpyridinium, cetyldimethylethyl ammonium and cetrimide cations.Among the genes found in Resistance to antibiotics is revealed by the following genes: glycopeptide antibiotics resistance protein (plays a role in resistance to glycopeptide antibiotics such as vancomycin); MFS transporter, DHA1 family; tetracycline resistance protein gene; and Fusaric acid resistance protein-like gene, which is involved in the resistance (detoxification) of the fungal toxin Fusaric acid.C. bovisDSM 19482T to produce a capsule, capsular exopolysaccharide family protein and polysaccharide export outer membrane protein.A gene for putative auto-transporter adhesin head GIN domain demonstrates the function of cell adhesion. Two genes indicate the possibility of C. bovis strain DSM 19482T that was isolated from raw cow milk [C. bovis is a psychrotolerant bacterium which can grow at 7 °C, although its optimal growth temperature is higher (30–32 °C). After milk collection, the milk is kept in cold storage, and psychrotolerants dominate the bacterial flora. These bacteria possess extracellular enzymes, mainly proteases and lipases which contribute to the spoilage of dairy products, as their enzymes can resist pasteurization [C. bovisDSM 19482T genome demonstrates that indeed, this genome encodes proteases and lipases which may play a role in milk products spoilage.In the current study we characterized the genome of C. bovis strain DSM 19482T produces a carotenoid pigment, a feature that was also observed for C. haifense [haifense , but notC. bovisDSM 19482T genome demonstrated the strains' potential to produce a multidrug-resistance protein, resistance to cobalt, zinc, cadmium, tellurite, cetylpyridinium, cetyldimethylethyl ammonium and cetrimide cations as well as resistance to glycopeptide antibiotics, tetracycline and resistance to the fungal toxin fusaric acid. The whole-genome sequence of C. oranimense G311, a strain that was isolated from a cystic fibrosis patient, also demonstrated multi-drug resistance [C. bovisDSM 19482T and C. oranimense G311. Sharma et al. [C. oranimense G311 to colistin maybe due to the production of capsular polysaccharides.sistance . Indicata et al.  suggeste


Mus musculus domesticus origin. These two inbred strains were selected based on their geographic origin, karyotype, and use in ongoing research. We generated 14× and 18× coverage sequence, respectively, and discovered over 1.1 million novel variants, most of which are private to one of these strains. This report expands the number of wild-derived inbred genomes in the Mus genus from six to eight. The sequence variation can be accessed via an online query tool; variant calls (VCF format) and alignments (BAM format) are available for download from a dedicated ftp site. Finally, the sequencing data have also been stored in a lossless, compressed, and indexed format using the multi-string Burrows-Wheeler transform. All data can be used without restriction.Wild-derived mouse inbred strains are becoming increasingly popular for complex traits analysis, evolutionary studies, and systems genetics. Here, we report the whole-genome sequencing of two wild-derived mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of  Mus musculus (the house mouse) is among the most commonly used scientific model organisms  translocations involving many different autosomes . Most taxa, including the M. m. domesticus wild-derived inbred strains: LEWES/EiJ, which is derived from mice trapped in Lewes, DE, with the standard M. musculus diploid chromosome number of 40; and ZALENDE/EiJ, which is derived from mice trapped in the Poschiavinus Valley , and has a 26-chromosome karyotype due to fixation of seven Rb translocations.Here, we report the whole-genome sequencing of two M. m. domesticus strains sequenced, and thus provides a clearer picture of intrasubspecific variation. LEWES/EiJ and ZALENDE/EiJ were among the top 10 inbred strains recommended for resequencing based on their potential to increase the catalog of known mouse variants  is associated with losses or gains on the centromeric ends of the chromosomes involved.In addition, Rb translocations have been implicated in the evolution of the mammalian karyotype, and are subject to meiotic drive during female meiosis in both mouse and humans . Our labmesticus . We wereM. m. domesticus × M. m. musculus hybrids  in multiple independent mouse populations  R2d2 allele, while LEWES/EiJ—derived from mice trapped only ∼100 km away from Centreville—has the low-copy  R2d2 allele. We hypothesized that comparison of the WSB/EiJ genome, which was sequenced previously  to Eva M. Eicher at The Jackson Laboratory in 1996.Derived from mice trapped in the Poschiavinus Valley  by Richard D. Sage. Mice from Sage’s colony were transferred to Michael Potter in 1981. A single pair of mice was sent from Michael Potter to Eva M. Eicher at The Jackson Laboratory in 1982. ZALENDE/EiJ is homozygous for seven Rb translocations .High molecular weight DNA from one ZALENDE/EiJ male was obtained from The Jackson Laboratory. High molecular weight DNA from a LEWES/EiJ female was prepared from tissues samples from a colony maintained in the Pardo-Manuel de Villena laboratory in the past , pooled, and sequenced across multiple lanes and multiple flowcells on an Illumina HiSeq 2000 instrument. After a small pilot run with single-end 50-bp reads, and a mixture of single- and paired-end 100-bp reads were generated for each sample. Base calling and demultiplexing were performed using the Casava 1.8 pipeline. We obtained 498,668,400 reads for LEWES/EiJ and 573,861,165 reads for ZALENDE/EiJ.http://broadinstitute.github.org/picard).Integrity of raw sequencing reads was confirmed using FastQC . Reads wSNV and short indel variants were called using the Sanger Mouse Genomes Project pipeline, the current version of which is described in detail elsewhere (https://github.com/holtjma/msbwt/wiki/Converting-to-msbwt’s-RLE-format.The multi-string Burrows-Wheeler transform (BWT) for each whole genome dataset was individually constructed in-memory using the ropebwt2 program . After chttp://www.sanger.ac.uk/science/data/mouse-genomes-project). Aligned reads (in BAM format): ftp://ftp-mouse.sanger.ac.uk/current_bams; web interface for querying variants: http://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1505; bulk download of variants (VCF format): ftp://ftp-mouse.sanger.ac.uk/current_snps). BWTs are available for download at http://csbio.unc.edu/WildDerived.Raw reads have been deposited in the European Nucleotide Archive (accession #PRJEB15190). All processed data are available from the Sanger Mouse Genomes Project  where unambiguous alignment of short reads is not possible.We sequenced one female LEWES/EiJ individual to median 14× coverage , and one male ZALENDE/EiJ individual to median 18× coverage . Genetic sex was confirmed by comparing relative read depth on the X chromosome to the autosomes. Coverage profiles across the nuclear genome are shown in M. m. domesticus origin identifies at least one order of magnitude more variants that sequencing classical laboratory strains (Sequence variants (SNVs and short indels < 10 bp in size) were ascertained using the Sanger Mouse Genomes Project pipeline , with a  strains  , 39 (private to LEWES/EiJ), and 67 (private to ZALENDE/EiJ) are predicted to disrupt gene function. We observe a similar picture for small indels . As expeD2hgdh and Gal3st2, are also present in other sequenced strains , PWK/PhJ (M. m. musculus) and CAST/EiJ (M. m. castaneus)—as representatives for their subspecies, and SPRET/EiJ (Mus spretus) as an outgroup. We classified alleles as ancestral or derived based on the pattern of sharing with the outgroup. After masking the 19% of the genome with known intersubspecific introgression or contamination in these strains (M. m. domesticus) over 92 and 94% of the genome, respectively . We found no evidence for private CNVs in any of these centromere-proximal regions . Based oData Availability in Materials and Methods). In particular, we provide access to the BWT of the sequencing reads generated from each of these strains. A BWT is a compact and lossless data structure that allows rapid interrogation of the reads in the absence of alignment.Novel alleles presented here have already been used to increase the utility of SNP arrays for genotyping of wild mice (M. musculus genetic variation will provide a valuable resource to researchers using mouse models to study a wide variety of biological processes, including karyotype evolution (Integration of these two novel genome sequences with the growing catalog of known volution , protein


Rickettsia felis rickettsiosis include pathogenesis and transmission. Detection of R. felis DNA in a number of arthropod species has been reported, with characterized isolates, R. felis strain LSU and strain LSU-Lb, generated from the cat flea, Ctenocephalides felis, and the non-hematophagous booklouse, Liposcelis bostrychophila, respectively. While it is realized that strain influence on host biology varies, the rickettsial response to these distinct host environments remained undefined. To identify a panel of potential rickettsial transmission determinants in the cat flea, the transcriptional profile for these two strains of R. felis were compared in their arthropod hosts using RNAseq. Rickettsial genes with increased transcription in the flea as compared to the booklouse were identified. Genes previously associated with bacterial virulence including LPS biosynthesis, Type IV secretion system, ABC transporters, and a toxin-antitoxin system were selected for further study. Transcription of putative virulence-associated genes was determined in a flea infection bioassay for both strains of R. felis. A host-dependent transcriptional profile during bloodfeeding, specifically, an increased expression of selected transcripts in newly infected cat fleas and flea feces was detected when compared to arthropod cell culture and incubation in vertebrate blood. Together, these studies have identified novel, host-dependent rickettsial factors that likely contribute to successful horizontal transmission by bloodfeeding arthropods.Crucial factors influencing the epidemiology of  Rickettsia have emerged as human pathogens, including Rickettsia felis, a Gram-negative bacterium predominately described in the cat flea, Ctenocephalides felis. Biological characteristics and genomic analysis have led to the classification of R. felis as a transitional group Rickettsia, containing attributes of both insect-borne typhus and tick-borne spotted fever group Rickettsia [Rickettsia and R. felis DNA in patients’ sera coincides with clinical features including fever, marked fatigue, headache, generalized maculopapular rash [R. felis has been recognized as a cosmopolitan pathogen [R. felis was detected in a number of flea species [R. felis rickettsiosis is associated primarily with suburban foci corresponding with the detection of rickettsial DNA in a variety of peri-domestic vertebrate hosts and their fleas [In the last three decades, several species of ckettsia . Human ilar rash . Since ipathogen –7. Highl species –10 and r species , 11, 12.ir fleas –16.Rickettsia prowazekii, the agent of epidemic typhus, to vertebrate hosts by bloodfeeding arthropods occurs through inoculation of infectious insect feces into the vertebrate host [Rickettsia typhi, the agent of endemic typhus, involves pathogen transmission to vertebrate hosts via infected flea feces, with subsequent oral transmission during feeding made possible by a sustained extrinsic incubation period in the flea host [R. felis with cat fleas has facilitated a number of studies examining the transmission mechanisms employed. Indeed, the presence of R. felis in cat flea salivary glands [R. felis-infected flea feces in vertebrate or inter-arthropod transmission is undefined, detection of transcriptionally active rickettsiae by qPCR in cat flea feces post-exposure to an infectious bloodmeal suggests that fecal transmission is also employed by this flea-borne pathogen [Transmission of insect-borne rickettsial pathogens such as louse-borne ate host . Similarlea host . The assy glands  supportsy glands , 21 and y glands , 23. Altpathogen . DespiteR. felis have been identified in a number of other arthropod species [Liposcelis bostrychophila, also called psocids or common booklice, the presence of vertically maintained R. felis infection is associated with reproductive manipulation (parthenogenesis) and a positive fitness effect for the arthropod host [R. felis is highly variable; a fitness effect has not been described [R. felis str. LSU)- and booklouse (R. felis str. LSU-Lb)-derived isolates revealed that the strains are closely related. While host-specific differences were evident, no distinct gene or set of genes that could be attributed to pathogenicity or horizontal transmission were identified [R. felis transcription profile. Using next generation sequencing, specifically RNAseq, differentially transcribed genes were identified and host-specific transcription profiles by R. felis in hematophagous (cat flea) and non-hematophagous (booklice) arthropod hosts described. The expression of a subset of identified genes was further examined in both R. felis strains in a flea infection bioassay . A distinct transcription profile was associated with both R. felis strains in the flea host, but not in culture or blood. The host-dependent transcription profile of R. felis provides novel insight into the molecular basis for rickettsial virulence and horizontal transmission.In addition to its emergence as a flea-borne rickettsial pathogen, several genotypes of  species . In Lipopod host , 27. Conescribed . Thus, ientified . In the L. bostrychophila) and cat flea (C. felis colonies) were maintained at LSU-SVM as previously described [R. felis str. LSU and R. felis str. LSU-Lb, which were originally isolated from the Louisiana State University cat flea colony [Ixodes scapularis-derived cell line (ISE6) [R. felis was cultured in ISE6 cells in a humidified incubator with 5% CO2 at 32°C. A modified L15B medium containing 10% tryptose phosphate broth (Sigma) and 10% heat-inactivated fetal bovine serum (HyClone) was used for cell maintenance. Viability of R. felis was determined using the BacLight viability stain kit (Life Technologies) prior to cat flea infection. For infection of cat fleas with R. felis, partially purified R. felis from ISE6 cells were resuspended in bovine bloodmeal (1 ml) at the concentration of 1 x 1010 rickettsiae per ml and were used for feeding cat fleas as previously described [R. felis, infected bloodmeal, feces and cat fleas were collected. Cat fleas were cleaned by sequential surface washing with 10% bleach for 5 min, 70% ethanol for 5 min repeated 3 times, followed by sterile distilled water. Flea salivary glands and guts were dissected out in sterile PBS and the tissues were stored in TRIzol (500 ml) at -80°C until RNA isolation.Both booklice  . As prevescribed , 22. BriR. felis mRNA were validated by qPCR using specific primers for 18s rRNA [R. felis 17 kDa surface antigen [Total RNA from flea salivary glands and guts or booklice was isolated with TRIzol (Invitrogen) according to manufacturer’s protocol followed by phenol/chloroform purification. The RNA extracted from cat flea tissues and booklice were quantified by spectrophotometry. Eukaryotic and bacterial rRNA were depleted from the total RNA by using the Ribo-Zero Magnetic Gold Kit and Ribo-Zero Magnetic Kit-Gram Negative Bacteria (Epicentre). The remaining RNA was further treated with a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) to deplete eukaryotic mRNA. The resulting RNA was ethanol precipitated and resuspended in 14 μl of RNase-free water. Removal of rRNA and presence of 18s rRNA  and sali antigen ). An estR. felis str. LSU infecting cat fleas are archived under accessions SRR2049306, SRR2051064, and SRR2051066. Sequencing files from R. felis str. LSU-Lb infecting booklice are archived under accessions SRR2051067, SRR2051068, and SRR2051069.Three independent infection assays were carried out in fleas and compared to three independent preparations of constitutively infected psocids by RNAseq. All data sets have been deposited into the NCBI Sequence Read Archive under bioproject accession PRJNA285354. Sequencing files from R. felis URRWXCal2 was performed with TMAP using default settings. Transcript assembly was performed using Cufflinks v2.0.2 (76) and differential transcript analyses comparing rickettsial transcription in cat fleas versus booklice were performed using CuffDiff v2.0.2 [http://usegalaxy.org) [Analysis of the resulting reads was facilitated by the Ion Torrent pipeline v2.2.1. After determining quality metrics and filtering low quality reads, alignment to the reference genome for f v2.0.2 . Tuxedo axy.org) –35. CuffTable 1. Each 35 μL qPCR reaction was composed of 5 μL of cDNA template, 17.5 μL of LightCycler 480 SYBR Green I Master (Roche), 1 μL of each 10 mM primer and DNase/RNase-free, distilled water. 10 μL of PCR reaction mix was assessed in triplicate on a 384-well plate and amplified using a Light Cycler 480 Real-Time PCR system (Roche). The cycling parameters were 95°C for 10 min, then 45 cycles at 95°C for 10 sec, 55°C for 30 sec, and 72°C for 1 sec, followed by cooling down to 40°C for 30 sec. No reverse transcriptase controls (water was added instead of reverse transcriptase) were performed to confirm the absence of genomic gDNA. Serial dilutions of the pCR4-Topo plasmid containing a portion of individual target genes were assessed in parallel, serving as a standard for concentration analysis. To determine the quantity of rickettsiae in the samples, DNA was extracted from the same samples after RNA isolation and the number of rickettsial genomic equivalents  was quantified by qPCR [Rf17kDa gene, providing the cDNA copy number per individual rickettsia [Selected significantly expressed rickettsial transcripts identified by RNAseq with previous virulence association in other bacteria were confirmed quantitatively by qPCR. Infection experiments for qPCR validation were performed separately from the RNAseq infection experiments. Briefly, total RNA was extracted using TRIzol (Invitrogen) according to manufacturer’s instructions, treated with TurboDNase (Ambion), cleaned using the Zymo Clean and Concentrator-5 kit (Zymo). Total purified RNA (50 ng) was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad). cDNA was amplified using specific primers for individual target genes. Sequences of primers (Integrated DNA Technologies) for genes of interest (GOI) are listed in  by qPCR . The relckettsia . Data arR. felis transcription profiles in hematophagous and non-hematophagous arthropod hosts, three independent infection assays were carried out in fleas and compared to three independent preparations of constitutively infected psocids. The sequencing libraries resulted in 79.6-145bp average read lengths. For each analysis, an average of 30,145 reads  were acquired for R. felis str. LSU in fleas compared to an average of 13,265 reads  acquired for R. felis str. LSU-Lb in psocids. Combined, 115 differentially expressed transcripts in fleas were identified with an average run coverage between 11–27% of the total genome and with an average depth between 0.2–1.8x. Among the 115 differentially expressed genes, 63 genes had higher transcriptional levels in R. felis within fleas in comparison to the booklice. These differentially expressed genes in fleas were blasted against the R. felis genome URRWXCal2 and putative assignments were given to 80.9% (51/63) of the genes with the remainder consisting of hypothetical proteins (S1 Table). Putatively assigned and hypothetical proteins were further characterized by cluster of orthologous group classification .To investigate R. felis str. LSU infecting flea-associated transcripts to R. felis str. LSU-Lb infecting psocid-associated transcripts. Of the commonly identified upregulated transcripts in fleas, seven genes  were chosen for both validation by qPCR and for further analysis in the flea infection bioassay based on previous virulence association in other bacteria . The qPCR transcript analysis confirms that these target R. felis transcripts are upregulated in hematophagous fleas, compared to non-hematophagous psocids, validating the RNAseq differential transcription analysis.Further analysis identified common transcripts significantly upregulated among three RNAseq replicates, comparing Table 2) is specific to the infection of the hematophagous arthropod, transcript levels of R. felis in adult fleas and also viable R. felis excreted by infected fleas in feces were compared to R. felis transcription when rickettsiae were maintained in arthropod cell culture or incubated with blood alone. RNA was isolated from rickettsiae at each stage of the flea infection bioassay . Transcription was then quantified by qPCR, and expression at each stage was compared to transcription in ISE6 culture. As shown in Fig 3, transcription of all the genes remain unchanged in the blood as compared to culture. In cat fleas exposed to an infectious bloodmeal of R. felis str. LSU for 36 hours, lpxB, RF0211, RF0580, virB4_2, trmE, uvrA and relB2 transcription was significantly increased when compared to culture. Likewise, transcription of the seven selected genes was significantly increased in feces from R. felis str. LSU exposed fleas compared rickettsiae maintained in cell culture.To determine if transcription of differentially regulated genes . Considered with the results for R. felis str. LSU in the flea infection bioassay, the increased transcript expression by R. felis LSU-Lb in fleas suggest that R. felis transcription of selected genes occurs in a host-dependent manner and is independent of the origin of the R. felis strain.The two strains of ct hosts , 31. TheR. felis rickettsiosis [R. felis has been detected within multiple arthropod hosts including other species of fleas, and of particular interest, the non-hematophagous booklouse L. bostrychophila [R. felis is maintained 100% vertically and acts as a reproductive manipulator inducing parthenogenesis, and removal of the bacteria from the host decreases booklouse fitness [R. felis, specifically the variable vertical transmission of R. felis in cat fleas, suggests the necessity of bacterial reintroduction into cat flea populations [R. felis in multiple arthropods may be facilitated by feeding on a rickettsemic host or indirectly cofeeding with infected arthropods on vertebrate hosts [R. felis-like organisms (RFLOs) in cofeeding arthropods confound the epidemiology of R. felis [R. felis and RFLOs in arthropod populations. Most notably missing from rickettsial biology is the identification of transmission determinants differentially encoded in the genomes of Rickettsia or expressed differentially in specific hosts to facilitate transmission. Recent genome sequencing and comparison of R. felis str. LSU and R. felis str. LSU-Lb assessed the genomic diversity of two R. felis strains isolated from arthropods with different transmission modes [e.g. plasmids) were identified which would explain known transmission and pathogenic differences. As an extension of the genomic sequencing, we utilized next-generation sequencing methodologies to compare the transcription of R. felis str. LSU and R. felis str. LSU-Lb in cat fleas and booklice, respectively, seeking to identify a panel of putative transmission determinants.The combination of pathogenicity of bacterial isolates and transmission modalities in the maintenance of pathogens in arthropod vectors are crucial factors which influence the epidemiology of the emerging ttsiosis –10. Varittsiosis , 22. Addchophila , 29. Boo fitness , 27. Hosulations , 24. White hosts , the isoR. felis . There ion modes . While gAmblyomma aureolautum, the yellow dog tick, infected with Rickettsia rickettsii [R. rickettsii [Anaplasma phagocytophilum in the salivary glands of the tick species I. scapularis after 48 hours of feeding [A. phagocytophilum revealed a pattern of increased transcription and translation machinery coupled with reduced replication and energetics machinery during a time when horizontal transmission most likely occurs. Overall, transcription and translation of proteins targeted to the cell membrane of Anaplasma were increased. Together, these two transcriptomic studies demonstrate the complex relationship between Rickettsiales and their bloodfeeding arthropod hosts. The current study builds on these previous studies in order to move towards identifying potential horizontal transmission determinants by comparing the transcription of two similar, but divergent strains of R. felis in their respective arthropod hosts.The interaction between rickettsial pathogens and their arthropod hosts has been examined previously with the use of microarrays and next-generation sequencing. Microarrays were first used to determine transcriptional differences associated with temperature change and bloodfeeding of ckettsii . Brieflyckettsii . Also, n feeding . Using sR. felis str. LSU and str. R. felis LSU-Lb infected fleas and booklice, respectively, the transcriptional profiles of strains of bacteria infecting their arthropod hosts were compared. The sequencing runs yielded a coverage and depth which is comparable to the Illumina sequencing of A. phagocytophilum [R. felis strain RNAseq differential analysis yielded a total of 115 significantly differentially expressed transcripts. For the purpose of identifying potential R. felis virulence and transmission determinant associated with the hematophagous arthropod, our subsequent analyses focused on transcripts significantly upregulated in the flea. Specifically there were 63 transcripts significantly upregulated by R. felis str. LSU in fleas as compared to booklice constitutively infected with R. felis str. LSU-Lb. These genes were analyzed into COGs with the most represented groups relating to i) cell wall/membrane/envelope biogenesis, ii) energy production and conversion, iii) translation, ribosomal structure and biogenesis, iv) replication, recombination and repair, and those with v) general predicted function or hypothetical proteins. From this panel, selected genes of interest include R. felis genes differentially upregulated in fleas as compared to booklice that have a previous virulence association in other bacteria. These factors expressed by R. felis in the cat flea are potentially required for virulence and horizontal transmission. Putative virulence associated genes which were upregulated included lipopolysaccharide (LSP) biosynthesis genes, a Type IV secretion system component and putative effector, ABC transporter systems, and toxin-antitoxin system components. The expression of hypothetical proteins is a common factor in all expression experiments to date. Collectively, 27% of the genes identified in this study have either a generalized function or no function  assigned in the reference URRWXCal2 genome (accessions: CP000053.1 and CP000054.1). This high percentage of differentially transcribed, but still poorly annotated genes, is a hindrance to the determination of stage-specific and arthropod-specific virulence determinants of Rickettsia. In this study, all of the hypothetical genes expressed by R. felis in fleas had no other features which might point to a function such as the presence of conserved domains or Blast homology. These genes are best described as genes of unknown function, and experimental evidence is needed for a better understanding of their function in virulence and transmission.Using an RNAseq analysis of tophilum . The R. Yersinia pestis under conditions most similar to the flea vector [Francisella in mice [mltE [Neisseria meningitidis, Erwinia amylovaora and Pseudomonas syringae, aiding in pathogenesis [Rickettsia [lpxB lipid-A disaccharide synthase, a putative polysaccharide polymerase, and mltE, by R. felis infecting cat fleas suggest a role for LPS synthesis and rearrangement in horizontal transmission by the arthropod host.Virulence associated with the expression of LPS biosynthesis machinery has been widely studied in the field of vector-borne diseases. While LPS is recognized by the host immune system, the expression of LPS machinery by bacteria can still confer benefits. Known protective functions of LPS expression include protection from host immune response components such as complement, and the evasion of TLR4 immune recognition through the expression of modified forms of LPS (reviewed in ). Modifia vector , 42. Add in mice . Proteince [mltE , have beogenesis . Additiockettsia . The uprRickettsia, and function in the transport DNA, conjugation, and the secretion of effector proteins [R. felis genome encodes 22 ankyrin repeat-containing ORFs [R. felis str. LSU and R. felis str. LSU-Lb during infection of fleas. While the function of RF0580 and other ankyrin repeat-containing proteins in R. felis has not been characterized experimentally, the secretion of ankyrin repeat-containing proteins has been characterized in other Rickettsiales. Secretion of ankyrin repeat-containing protein AnkA through a Type IV secretion system which facilitates intracellular infection in conjunction with tyrosine phosphorylation was identified for A. phagocytophilum [Orientia tsutsugamushi was determined to be expressed within mammalian host cells and secreted in a heterologous Escherichia coli secretion system. Ectopically expressed Anks showed localization to host cell secretion machinery including the endoplasmic reticulum [R. felis strains in the flea may aid in increased pathogenesis of the flea host facilitating transmission and virulence; thus requiring further study.Type IV secretion systems and secreted effector proteins have been characterized in intracellular bacteria and aid in growth, infection, and virulence. These secretion systems are present in a diverse set of bacteria, including proteins , 48. Theing ORFs , includitophilum . More reeticulum . TogetheR. felis str. LSU within flea host. While the function of the four upregulated transporter genes  of R. felis in fleas is not well characterized, ABC transporters have been associated with virulence in flea-borne Y. pestis [Salmonella, by facilitating the movement of effector proteins across the phagolysosome membrane aiding in pathogenesis [R. felis in fleas. Similar patterns of expression have been previously observed in Rickettsia conorii [Ehrlichia chaffeensis infecting arthropod-derived AAE2 and ISE6 cells [R. felis strains in cat fleas is still poorly understood; future studies would be necessary to fully elucidate their involvement in maintenance and transmission of this pathogen.ABC transporters and other membrane synthesis associated proteins are involved in the biogenesis of membrane components and the movement of phospholipids, LPS, and other proteins to the outer membrane of gram-negative bacteria . ATPase . pestis . Additioogenesis . In the  conorii  and alsoE6 cells . The funRickettsia spp. and their activity, presenting molecular evidence of apoptotic activity corresponding to bacterial pathogenesis when TAS were overexpressed in E. coli [relB2 and relE identified in our RNAseq analyses have been associated with human eschars and vertical transmission of Rickettsia in vectors [Mycobacterium infections, where RelB functions as an antitoxin that neutralizes the toxin RelE. Expression of RelBE has been characterized in Mycobacterium tuberculosis, when stress, such as nutrient deprivation, and expression of the RelBE complex together trigger a reduced translational phenotype leading to cell cycle arrest and increased survival of the bacterium [Another further mechanism of persistence, stress response, and virulence of bacteria is through the expression of toxin-antitoxin systems (TAS). Recent studies have explored the presence of TAS in the genomes of  E. coli . Additio vectors . The Relacterium . The reguvrA and trmE were expressed in a flea specific manner by both flea- and booklouse-derived R. felis strains. These genes are involved in the coordination of recombination and DNA repair. UvrA is an exinuclease which aids in repair of DNA. For organisms such as Mycobacterium, UvrA, in concert with other repair proteins such as UvrD, recognize and repair DNA damaged by nitric oxide and other host cell response which is a function required for bacteria survival and growth in vertebrate hosts [trmE is a GTPase that mainly functions to modify tRNA molecules. A targeted mutagenesis screen identified TrmE of Francisella tularensis as a regulator of the expression of genes present on the Francisella pathogenicity island involved in sensing changes in nutritional and oxidative stress in eukaryotic cells and regulating genes which induce expression of virulence factors [Rickettsia to environmental stresses experienced during bloodfeeding and transmission may indirectly affect the expression of virulence determinants and aid in successful transmission through known or alternative protein functions.While not previously associated with virulence in other vector-borne systems, genes such as te hosts . The gen factors . Genes wR. felis during bloodfeeding, fleas were exposed to strains of R. felis and transcription was assessed and compared to transcription of R. felis in ISE6 cell culture. Compared to standard in vitro culture, expression of selected R. felis str. LSU genes did not change when incubated in the bovine bloodmeal, however, expression increased in the flea and in the flea feces. These results suggest transcript regulation in R. felis str. LSU is dependent on interaction with the hematophagous host, specifically the adult flea and fecal stages. The role of fecal transmission has not been well characterized in R. felis infection, but has been in other flea borne-bacteria [R. typhi fecal transmission by the Oriental rat flea is the primary route of transmission to vertebrates [R. felis is known to be excreted in the feces of cat fleas, suggesting that fecal transmission could act as an alternative route of transmission to vertebrates and arthropod populations [R. felis sustained vertical transmission in cat fleas and co-feeding horizontal transmission, potential non-transovarial, vertical transmission via cat flea larva ingestion of infected adult flea feces may aid in the maintenance of R. felis in cat flea populations [Rickettsia, such as R. typhi and R. prowazekii. While functional characterization is required, heightened expression of putative virulence associated genes during flea shedding may point to a greater role of fecal transmission in the overall ecology of R. felis rickettsiosis.After qPCR validation of a selected subset of differentially expressed genes in the flea host, seven genes were selected for further transcriptional analysis in the flea infection bioassay. In order to characterize host-dependent transcription of bacteria , 63. Spetebrates . Viable ulations . When coR. felis str. LSU-Lb expressed lpxB, RF0211, RF0580, virB4_2, trmE, uvrA, and relB2 in a manner similar to the R. felis str. LSU during the flea infection cycle, increasing transcription in an infection-dependent manner in the hematophagous flea. Expression of the seven target genes suggests a role in transmission in the flea host, as increased transcription is dependent on flea bloodfeeding and is significantly increased as compared to expression in cell culture or in blood alone. The flea imparts pressures far different than those during infection in cell culture or infection in the vertebrate host. Thus, future transcriptional studies must consider vertebrate and arthropod stages, as well as arthropod biology, for a more complete understanding of rickettsial transcription.To support the potential for host-associated transcriptional requirements, the booklouse associated Rickettsia in cell culture without corresponding assessment of transcription in competent vectors may lead to an underestimation of the range of rickettsial activity. To better characterize the dynamic response of R. felis strains in the hematophagous cat flea and non-hematophagous booklouse, we used RNAseq methodologies to determine and compare the transcription profiles in arthropod hosts. Increased transcription of putative virulence factors including Type IV secretion system factors, LPS synthesis machinery, toxin/antitoxin modules, and proteins in the competent flea vector increases the likelihood for their role in transmission events, providing candidates for targeted experimentation in the cat flea vector. Additional studies are required to determine the complex relationship between Rickettsia and arthropod hosts to understand the arthropod-dependent cues responsible for virulence and horizontal transmission.Rickettsial strain dynamics may be more complex than is currently understood. Previous assessment of the responses of S1 TableRickettsia open reading frame (ORF), gene name, gene annotation and Cluster of Orthologous Groups (COG) annotations were retrieved from the URRWXCal2 R. felis genome . Log2(Fold-change) is reported as the greatest change determined by RNAseq with significant digits determined by the number of reads quantified by Cufflinks. *Expression of relB2 in booklice was below the limit of detection, but the positive fold change was determined statistically significant by CuffDiff.(DOCX)Click here for additional data file.