Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin.

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

Chemical Profiling and Biological Activity of Extracts from Nine Norwegian Medicinal and Aromatic Plants.

There is an increased interest in identifying beneficial compounds of plant origin that can be added to animal diets to improve animal performance and have a health-promoting effect. In the present study, nine herb species of the Norwegian wild flora or which can be cultivated in Norway were selected for phytogenic evaluation (hops, maral root, mint, oregano, purslane, rosemary, roseroot, sweet wormwood, yarrow). Dried herbs were sequentially extracted with dichloromethane (DCM), ethanol (EtOH) and finally water (H2O) by ultrasound-assisted extraction (UAE). The UAE protocol was found to be more rational than conventional Soxhlet with respect to DCM extraction. Total extraction yield was found to be highest for oregano (Origanum vulgare) with 34.4 g 100-1 g dry matter (DM). H2O-extracts gave the highest yields of the three solvents, with up to 25 g 100-1 g DM for purslane (Portulaca oleracea ssp. sativa) and mint (Mentha piperita). EtOH- and H2O-extracts were the most efficient extracts with respect to free radical scavenging capacity (ABTS (=2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and oregano, mint, hops (Humulus lupulus) and maral root-leaves (Leuzea carthamoides) were found to be the most efficient antioxidant sources. Hops (EtOH-extract) contained α- and ß-acids, xanthohumols, chlorogenic acid and the hitherto unreported 3-O-glucosides of kaempferol and quercetin. Maral root-leaves contained among other compounds hexosides of the 6-hydroxy- and 6-methoxy-kaempferol and -quercetin, whereas roseroot (Rosea rhodiola) revealed contents of rosavin, rhodiosin and rhodionin. Sweet wormwood (Artemisia annua) contained chlorogenic acid and several derivatives thereof, scopoletin and poly-methylated flavones (eupatin, casticin, chrysoplenetin). Antimicrobial potential of different plant extracts was demonstrated against Gram-positive and Gram-negative bacteria using the indicator organisms Staphylococcus aureus, and Escherichia coli, and the Atlantic salmon bacterial pathogens Moritella viscosa, Tenacibaculum finnmarkense and Aliivibrio wodanis. DCM extracts possessed the highest activities. Data demonstrate the potential ability of herb extracts as natural antimicrobials. However, future safety studies should be performed to elucidate any compromising effect on fish health.

Feed microbiome: confounding factor affecting fish gut microbiome studies.

There is an increasing interest in the impact of feed on the fish gut microbiome. Most of the studies are based on sequencing the bacterial housekeeping gene 16S rRNA from extracted total DNA, including resident and non-resident live bacteria as well as dead bacteria. It has not been a common practice to include the feed as control, although it contains various nutritious ingredients that microorganisms can use before or after feed preparation. Thus, study designs using digesta as a proxy for the intestinal microbiome raise the concern that composition of the gut microbiome might be biased by carry-over of microbial DNA from the feed itself. Here we report analysis of 15 feeds and representative intestinal digesta of Atlantic salmon (Salmo salar) from five independent case studies. This allowed us to identify "feed microbiomes" that were microbially diverse and shared taxa with digesta microbiomes. Digesta-specific microbiomes were identified, though they were mainly enriched by a few taxa, such as Mycoplasma and Ruminococcaceae. Overall, findings are consistent with a model wherein gut microbial profiles are to a different degree influenced by bacterial DNA present in the feed itself through a "feed microbiome" carry-over effect.

Transcriptome Responses of Atlantic Salmon (Salmo salar L.) to Viral and Bacterial Pathogens, Inflammation, and Stress.

Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima's Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes - VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections - wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA (SAV, IPNV, PRV, and ISAV), and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families (gig and several trim families) and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress.

Phylogenetic Revision of the Genus Aliivibrio: Intra- and Inter-Species Variance Among Clusters Suggest a Wider Diversity of Species.

Genus Aliivibrio is known to harbor species exhibiting bioluminescence as well as pathogenic behavior affecting the fish farming industry. Current phylogenetic understanding of Aliivibrio has largely remained dormant after reclassification disentangled it from the Vibrio genus in 2007. There is growing evidence of wider diversity, but until now the lack of genomes and selective use of type strains have limited the ability to compare and classify strains firmly. In this study, a total of 143 bacterial strains, including 51 novel sequenced strains, were used to strengthen phylogenetic relationships in Aliivibrio by exploring intra-species and inter-species relations. Multilocus sequence analysis (MLSA), applying the six housekeeping genes 16S ribosomal RNA (rRNA), gapA, gyrB, pyrH, recA, and rpoA, inferred 12 clades and a singular branch in Aliivibrio. Along with four new phylogenetic clades, the MLSA resolved prior inconsistencies circumscribing Aliivibrio wodanis and formed a unique clade we propose as the novel species Aliivibrio sp. "friggae." Furthermore, phylogenetic assessment of individual marker genes showed gyrB, pyrH, and recA superior to the 16S rRNA gene, resolving accurately for most species clades in Aliivibrio. In this study, we provide a robust phylogenetic groundwork for Aliivibrio as a reference point to classification of species.

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks.

Atlantic salmon skin barrier functions gradually enhance after seawater transfer.

Atlantic salmon farming operates with high production intensities where skin integrity is recognized as a central factor and indicator for animal health and welfare. In the described trial, the skin development and its immune status in healthy Atlantic salmon reared in two different systems, a traditional open net-pen system and a semi-closed containment system, were investigated. Freshwater smolts were compared to post-smolts after 1 and 4 months in seawater. Growth performance, when adjusted for temperature, was equal between the systems. Skin analyses, including epidermis and dermis, showed that thickness and mucus cell numbers increased in pace with the growth and time post seawater transfer (PST). Gene expression changes suggested similar processes with development of connective tissue, formation of extracellular matrix and augmented cutaneous secretion, changes in mucus protein composition and overall increased immune activity related to gradually enforced protection against pathogens. Results suggest a gradual morphological development in skin with a delayed recovery of immune functions PST. It is possible that Atlantic salmon could experience increased susceptibility to infectious agents and risk of diseases during the first post-smolt period.

Pan genome and CRISPR analyses of the bacterial fish pathogen Moritella viscosa.

BACKGROUND: Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M. viscosa strains. Matches between M. viscosa clustered, regularly interspaced, short palindromic, repeats and associated cas genes (CRISPR-Cas) were analysed to correlate CRISPR-Cas with adaptive immunity against MGEs. RESULTS: The comparative genomic analysis of M. viscosa isolates from across the North Atlantic region and from different fish species support delineation of M. viscosa into four phylogenetic lineages. The results showed that M. viscosa carries two distinct variants of the CRISPR-Cas subtype I-F systems and that CRISPR features follow the phylogenetic lineages. A subset of the spacer content match prophage and plasmid genes dispersed among the M. viscosa strains. Further analysis revealed that prophage and plasmid-like element distribution were reflected in the content of the CRISPR-spacer profiles. CONCLUSIONS: Our data suggests that CRISPR-Cas mediated interactions with MGEs impact genome properties among M. viscosa, and that patterns in spacer and MGE distributions are linked to strain relationships.

Complete genome sequence of Halomonas sp. R5-57.

The marine Arctic isolate Halomonas sp. R5-57 was sequenced as part of a bioprospecting project which aims to discover novel enzymes and organisms from low-temperature environments, with potential uses in biotechnological applications. Phenotypically, Halomonas sp. R5-57 exhibits high salt tolerance over a wide range of temperatures and has extra-cellular hydrolytic activities with several substrates, indicating it secretes enzymes which may function in high salinity conditions. Genome sequencing identified the genes involved in the biosynthesis of the osmoprotectant ectoine, which has applications in food processing and pharmacy, as well as those involved in production of polyhydroxyalkanoates, which can serve as precursors to bioplastics. The percentage identity of these biosynthetic genes from Halomonas sp. R5-57 and current production strains varies between 99 % for some to 69 % for others, thus it is plausible that R5-57 may have a different production capacity to currently used strains, or that in the case of PHAs, the properties of the final product may vary. Here we present the finished genome sequence (LN813019) of Halomonas sp. R5-57 which will facilitate exploitation of this bacterium; either as a whole-cell production host, or by recombinant expression of its individual enzymes.

Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis.

BACKGROUND: Aliivibrio wodanis and Moritella viscosa have often been isolated concurrently from fish with winter-ulcer disease. Little is known about the interaction between the two bacterial species and how the presence of one bacterial species affects the behaviour of the other. RESULTS: The impact on bacterial growth in co-culture was investigated in vitro, and the presence of A. wodanis has an inhibitorial effect on M. viscosa. Further, we have sequenced the complete genomes of these two marine Gram-negative species, and have performed transcriptome analysis of the bacterial gene expression levels from in vivo samples. Using bacterial implants in the fish abdomen, we demonstrate that the presence of A. wodanis is altering the gene expression levels of M. viscosa compared to when the bacteria are implanted separately. CONCLUSIONS: From expression profiling of the transcriptomes, it is evident that the presence of A. wodanis is altering the global gene expression of M. viscosa. Co-cultivation studies showed that A. wodanis is impeding the growth of M. viscosa, and that the inhibitorial effect is not contact-dependent.

Host specificity and clade dependent distribution of putative virulence genes in Moritella viscosa.

Moritella viscosa is the aetiological agent of winter-ulcer disease in farmed salmonids in the North Atlantic. Previously, two major (typical and variant) genetic clades have been demonstrated within this bacterial species, one of which is almost solely related to disease in Atlantic salmon (Salmo salar). In the present study infection trials demonstrated that 'typical' M. viscosa isolated from Norwegian Atlantic salmon was highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. 'Variant' M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species and a number of other Moritella spp. were investigated for the presence/absence of putative virulence related homologs. All isolates were positive for DNA sequences coding for; the Type VI secretion ATPase (clpV), hemolysin co-regulated protein (hcp), bacterioferritins (bfrA and bfrB), lectin (hemG), phospholipase D (pld), multifunctional autoprocessing repeats-in-toxin (martxA), aerolysin (aer), invasin (inv), and cytotoxic necrotizing factor (cnf), with the exception of one isolate in which cnf could not be confirmed. The product of an ABC transporter metal-binding lipoprotein (mat) was consistently detected although 11 isolates, all phylogenetically related, appear to produce a truncated version. A putative insecticidal toxin complex (mitABC) was detected almost exclusively in 'typical' Atlantic salmon isolates, and our data indicate that this complex of genes is expressed and co-transcribed. Transmission electron microscopy investigation revealed pili and flagella surface structures on nine M. viscosa representing both typical and variant isolates. Our results provide strong support for the existence of host specificity/high virulence in 'typical' M. viscosa related to Atlantic salmon. The gene distribution also provides further support for the genetic division within M. viscosa, and constitutes a basis for further study of the importance of the mitABC complex in winter-ulcer pathogenesis.

Co-infection of Atlantic salmon (Salmo salar), by Moritella viscosa and Aliivibrio wodanis, development of disease and host colonization.

Two species of bacteria are repeatedly isolated from farmed fish with winter-ulcer disease. Moritella viscosa is the aetiological agent of the disease; the significance of Aliivibrio wodanis is uncertain but has not been related to the primary pathogenesis. A cell culture infection model showed that A. wodanis adhered to, but did not invade the fish cells. Exposure to culture supernatant of A. wodanis caused the fish cells to vacoulate, retract, round up and detach from the surface, and rearrange the actin filaments of the cytoskeleton. These observations suggest that the bacterium secretes toxins into the extracellular environment. Any pathologic effect of A. wodanis and the effect of co-culturing with M. viscosa was studied in Atlantic salmon (Salmo salar) bath challenged with; only M. viscosa or only A. wodanis or both bacteria together. Both M. viscosa and A. wodanis were re-isolated from external surfaces and internal organs from live and deceased co-infected fish. It is further hypothesized that A. wodanis colonization might influence the progression of a M. viscosa infection. This is to our knowledge the first study that reproduces field observations where both bacteria infect Atlantic salmon.

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection.

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome.

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.

Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase.

This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process. The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His(6)-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 degrees C and not at 28 degrees C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE's own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart. In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.

Motility and flagellin gene expression in the fish pathogen Vibrio salmonicida: effects of salinity and temperature.

The success of several Vibrio species, including Vibrio cholerae, Vibrio anguillarum and Vibrio fischeri in colonizing their symbiont, or causing infection is linked to flagella-based motility. It is during early colonization or the initial phase of infection that motility appears to be critical. In this study we used Vibrio salmonicida, a psychrophilic and moderate halophilic bacterium that causes cold-water vibriosis in seawater-farmed Atlantic salmon (Salmo salar), to study motility and expression of flagellins under salt conditions mimicking the initial and later phases of an infection. Our results, which are based on motility in semi-solid agar, membrane protein proteomics, quantitation of flagellin gene expression, challenge infection of fish, and microscopy, show that V. salmonicida is highly motile, expresses elevated levels of flagellins, and typically contains several polar flagella under salt conditions that are seawater-like. In contrast, V. salmonicida cells are non-motile and express significantly lower levels of flagellins under physiological-like salt conditions.

Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.

Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.