Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Rapid groundwater decline and some cases of recovery in aquifers globally.

Groundwater resources are vital to ecosystems and livelihoods. Excessive groundwater withdrawals can cause groundwater levels to decline1-10, resulting in seawater intrusion11, land subsidence12,13, streamflow depletion14-16 and wells running dry17. However, the global pace and prevalence of local groundwater declines are poorly constrained, because in situ groundwater levels have not been synthesized at the global scale. Here we analyse in situ groundwater-level trends for 170,000 monitoring wells and 1,693 aquifer systems in countries that encompass approximately 75% of global groundwater withdrawals18. We show that rapid groundwater-level declines (>0.5 m year-1) are widespread in the twenty-first century, especially in dry regions with extensive croplands. Critically, we also show that groundwater-level declines have accelerated over the past four decades in 30% of the world's regional aquifers. This widespread acceleration in groundwater-level deepening highlights an urgent need for more effective measures to address groundwater depletion. Our analysis also reveals specific cases in which depletion trends have reversed following policy changes, managed aquifer recharge and surface-water diversions, demonstrating the potential for depleted aquifer systems to recover.

Nutritional immunomodulation of Atlantic salmon response to Renibacterium salmoninarum bacterin.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed "switched-diet". Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL-1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the "switched-diet" group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

The Bengal Water Machine: Quantified freshwater capture in Bangladesh.

Global food security depends on the sustainability of irrigated agriculture. Rising groundwater withdrawals from seasonally humid, alluvial plains across tropical Asia have enabled dry-season rice cultivation. This groundwater pumpage increases available subsurface storage that under favorable conditions amplifies groundwater replenishment during the subsequent monsoon. We empirically quantified this nature-based solution to seasonal freshwater storage capture described as the "Bengal Water Machine," revealing its potential and limitations. On the basis of a million piezometric observations from 465 monitoring wells, we show that the collective operation of ~16 million smallholder farmers in the Bengal Basin of Bangladesh from 1988 to 2018 has induced cumulative freshwater capture that volumetrically (75 to 90 cubic kilometers) is equivalent to twice the reservoir capacity of the Three Gorges Dam.

Interacting Effects of Sea Louse (Lepeophtheirus salmonis) Infection and Formalin-Killed Aeromonas salmonicida on Atlantic Salmon Skin Transcriptome.

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis-infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs. PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs. ASAL/no lice); on the contrary, the observed skin transcriptome changes -albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs. PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs. PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs. PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b, bcl6) and synergistic (e.g., hampa, il4r) manner. These results provided insight on the physiological response of the skin of L. salmonis-infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection.

Gill and Liver Transcript Expression Changes Associated With Gill Damage in Atlantic Salmon (Salmo salar).

Gill damage represents a significant challenge in the teleost fish aquaculture industry globally, due to the gill's involvement in several vital functions and direct contact with the surrounding environment. To examine the local and systemic effects accompanying gill damage (which is likely to negatively affect gill function) of Atlantic salmon, we performed a field sampling to collect gill and liver tissue after several environmental insults (e.g., harmful algal blooms). Before sampling, gills were visually inspected and gill damage was scored; gill scores were assigned from pristine [gill score 0 (GS0)] to severely damaged gills (GS3). Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged (i.e., GS2) gill tissue. Rank Products analysis (5% percentage of false-positives) identified 254 and 34 upregulated and downregulated probes, respectively, in GS2 compared with GS0. Differentially expressed probes represented genes associated with functions including gill remodeling, wound healing, and stress and immune responses. We performed gill and liver qPCR for all four gill damage scores using microarray-identified and other damage-associated biomarker genes. Transcripts related to wound healing (e.g., neb and klhl41b) were significantly upregulated in GS2 compared with GS0 in the gills. Also, transcripts associated with immune and stress-relevant pathways were dysregulated (e.g., downregulation of snaclec 1-like and upregulation of igkv3) in GS2 compared with GS0 gills. The livers of salmon with moderate gill damage (i.e., GS2) showed significant upregulation of transcripts related to wound healing (i.e., chtop), apoptosis (e.g., bnip3l), blood coagulation (e.g., f2 and serpind1b), transcription regulation (i.e., pparg), and stress-responses (e.g., cyp3a27) compared with livers of GS0 fish. We performed principal component analysis (PCA) using transcript levels for gill and liver separately. The gill PCA showed that PC1 significantly separated GS2 from all other gill scores. The genes contributing most to this separation were pgam2, des, neb, tnnt2, and myom1. The liver PCA showed that PC1 significantly separated GS2 from GS0; levels of hsp70, cyp3a27, pparg, chtop, and serpind1b were the highest contributors to this separation. Also, hepatic acute phase biomarkers (e.g., serpind1b and f2) were positively correlated to each other and to gill damage. Gill damage-responsive biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon welfare.

In-situ fluorescence spectroscopy is a more rapid and resilient indicator of faecal contamination risk in drinking water than faecal indicator organisms.

Faecal indicator organisms (FIOs) are limited in their ability to protect public health from the microbial contamination of drinking water because of their transience and time required to deliver a result. We evaluated alternative rapid, and potentially more resilient, approaches against a benchmark FIO of thermotolerant coliforms (TTCs) to characterise faecal contamination over 14 months at 40 groundwater sources in a Ugandan town. Rapid approaches included: in-situ tryptophan-like fluorescence (TLF), humic-like fluorescence (HLF), turbidity; sanitary inspections; and total bacterial cells by flow cytometry. TTCs varied widely in six sampling visits: a third of sources tested both positive and negative, 50% of sources had a range of at least 720 cfu/100 mL, and a two-day heavy rainfall event increased median TTCs five-fold. Using source medians, TLF was the best predictor in logistic regression models of TTCs ≥10 cfu/100 mL (AUC 0.88) and best correlated to TTC enumeration (ρs 0.81), with HLF performing similarly. Relationships between TLF or HLF and TTCs were stronger in the wet season than the dry season, when TLF and HLF were instead more associated with total bacterial cells. Source rank-order between sampling rounds was considerably more consistent, according to cross-correlations, using TLF or HLF (min ρs 0.81) than TTCs (min ρs 0.34). Furthermore, dry season TLF and HLF cross-correlated more strongly (ρs 0.68) than dry season TTCs (ρs 0.50) with wet season TTCs, when TTCs were elevated. In-situ TLF or HLF are more rapid and resilient indicators of faecal contamination risk than TTCs.

Influence of Varying Dietary ω6 to ω3 Fatty Acid Ratios on the Hepatic Transcriptome, and Association with Phenotypic Traits (Growth, Somatic Indices, and Tissue Lipid Composition), in Atlantic Salmon (Salmo salar).

The importance of dietary omega-6 to omega-3 (ω6:ω3) fatty acid (FA) ratios for human health has been extensively examined. However, its impact on fish physiology, and the underlying molecular mechanisms, are less well understood. This study investigated the influence of plant-based diets (12-week exposure) with varying ω6:ω3 (0.4-2.7) on the hepatic transcriptome of Atlantic salmon. Using 44 K microarray analysis, genes involved in immune and inflammatory response (lect2a, itgb5, helz2a, p43), lipid metabolism (helz2a), cell proliferation (htra1b), control of muscle and neuronal development (mef2d) and translation (eif2a, eif4b1, p43) were identified; these were differentially expressed between the two extreme ω6:ω3 dietary treatments (high ω6 vs. high ω3) at week 12. Eight out of 10 microarray-identified transcripts showed an agreement in the direction of expression fold-change between the microarray and qPCR studies. The PPARα activation-related transcript helz2a was confirmed by qPCR to be down-regulated by high ω6 diet compared with high ω3 diet. The transcript expression of two helz2 paralogues was positively correlated with ω3, and negatively with ω6 FA in both liver and muscle, thus indicating their potential as biomarkers of tissue ω6:ω3 variation. Mef2d expression in liver was suppressed in the high ω6 compared to the balanced diet (ω6:ω3 of 2.7 and 0.9, respectively) fed fish, and showed negative correlations with ω6:ω3 in both tissues. The hepatic expression of two lect2 paralogues was negatively correlated with viscerosomatic index, while htra1b correlated negatively with salmon weight gain and condition factor. Finally, p43 and eif2a were positively correlated with liver Σω3, while these transcripts and eif4b2 showed negative correlations with 18:2ω6 in the liver. This suggested that some aspects of protein synthesis were influenced by dietary ω6:ω3. In summary, this nutrigenomic study identified hepatic transcripts responsive to dietary variation in ω6:ω3, and relationships of transcript expression with tissue (liver, muscle) lipid composition and other phenotypic traits.

Effects of Varying Dietary Docosahexaenoic, Eicosapentaenoic, Linoleic, and α-Linolenic Acid Levels on Fatty Acid Composition of Phospholipids and Neutral Lipids in the Liver of Atlantic Salmon, Salmo salar.

Fish oil, the most abundant natural source of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), is a limited resource; however, terrestrial oils are used as an alternative in fish nutrition. The liver of Atlantic salmon is able to synthesize these two long-chain n-3 polyunsaturated fatty acids (n-3LC-PUFAs) from α-linolenic acid (ALA), but the dietary levels of EPA + DHA and the ratios of linoleic acid (LNA) to ALA may affect its abilities. Feeding Atlantic salmon four experimental diets containing EPA + DHA at 0.3 and 1.0% of dietary levels accompanied with high and low LNA/ALA ratios showed that low LNA/ALA ratios increased the proportions of EPA + DHA in phospholipids (PLs) and neutral lipids (NLs). The pattern of PL-to-NL ratios of n-3 LC-PUFA proportions matched the saw tooth pattern of LNA/ALA ratios in diets. Overall, when fish oil is removed from salmon diets, the dietary LNA/ALA ratio must be reduced to stimulate biosynthesis of n-3 LC-PUFAs in the liver.

Transcriptome Profiling of Atlantic Salmon (Salmo salar) Parr With Higher and Lower Pathogen Loads Following Piscirickettsia salmonis Infection.

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon (Salmon salar) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers (campb, hampa, il8a, tlr5a) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. "phagocytosis", "defense response to bacterium", "inflammatory response") and adaptive (e.g. "regulation of T cell activation", "antigen processing and presentation of exogenous antigen") immune processes, while a small number of general physiological processes (e.g. "apoptotic process", development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis-triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen.

Transcriptome Analysis of Atlantic Salmon (Salmo salar) Skin in Response to Sea Lice and Infectious Salmon Anemia Virus Co-Infection Under Different Experimental Functional Diets.

Sea lice (Lepeophtheirus salmonis) are ectoparasitic copepods that cause significant economic loss in marine salmoniculture. In commercial salmon farms, infestation with sea lice can enhance susceptibility to other significant pathogens, such as the highly contagious infectious salmon anemia virus (ISAv). In this study, transcriptomic analysis was used to evaluate the impact of four experimental functional feeds (i.e. 0.3% EPA/DHA+high-ω6, 0.3% EPA/DHA+high-ω6+immunostimulant (IS), 1% EPA/DHA+high-ω6, and 1% EPA/DHA+high-ω3) on Atlantic salmon (Salmo salar) during a single infection with sea lice (L. salmonis) and a co-infection with sea lice and ISAv. The overall objectives were to compare the transcriptomic profiles of skin between lice infection alone with co-infection groups and assess differences in gene expression response among animals with different experimental diets. Atlantic salmon smolts were challenged with L. salmonis following a 28-day feeding trial. Fish were then challenged with ISAv at 18 days post-sea lice infection (dpi), and maintained on individual diets, to establish a co-infection model. Skin tissues sampled at 33 dpi were subjected to RNA-seq analysis. The co-infection's overall survival rates were between 37%-50%, while no mortality was observed in the single infection with lice. With regard to the infection status, 756 and 1303 consensus differentially expressed genes (DEGs) among the four diets were identified in "lice infection vs. pre-infection" and "co-infection vs. pre-infection" groups, respectively, that were shared between the four experimental diets. The co-infection groups (co-infection vs. pre-infection) included up-regulated genes associated with glycolysis, the interferon pathway, complement cascade activity, and heat shock protein family, while the down-regulated genes were related to antigen presentation and processing, T-cell activation, collagen formation, and extracellular matrix. Pathway enrichment analysis conducted between infected groups (lice infection vs. co-infection) resulted in several immune-related significant GO terms and pathways unique to this group, such as "autophagosome", "cytosolic DNA-sensing pathway" and "response to type I interferons". Understanding how experimental functional feeds can impact the host response and the trajectory of co-infections will be an essential step in identifying efficacious intervention strategies that account for the complexities of disease in open cage culture.

Influence of Dietary Long-Chain Polyunsaturated Fatty Acids and ω6 to ω3 Ratios on Head Kidney Lipid Composition and Expression of Fatty Acid and Eicosanoid Metabolism Genes in Atlantic Salmon (Salmo salar).

The interaction of dietary eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) levels with omega-6 to omega-3 ratios (ω6:ω3), and their impact on head kidney lipid metabolism in farmed fish, are not fully elucidated. We investigated the influence of five plant-based diets (12-week exposure) with varying EPA+DHA levels (0.3, 1.0, or 1.4%) and ω6:ω3 (high ω6, high ω3, or balanced) on tissue lipid composition, and transcript expression of genes involved in fatty acid and eicosanoid metabolism in Atlantic salmon head kidney. Tissue fatty acid composition was reflective of the diet with respect to C18 PUFA and MUFA levels (% of total FA), and ω6:ω3 (0.5-1.5). Fish fed 0.3% EPA+DHA with high ω6 (0.3% EPA+DHA↑ω6) had the highest increase in proportions (1.7-2.3-fold) and in concentrations (1.4-1.8-fold) of arachidonic acid (ARA). EPA showed the greatest decrease in proportion and in concentration (by ~½) in the 0.3% EPA+DHA↑ω6 fed fish compared to the other treatments. However, no differences were observed in EPA proportions among salmon fed the high ω3 (0.3 and 1.0% EPA+DHA) and balanced (1.4% EPA+DHA) diets, and DHA proportions were similar among all treatments. Further, the transcript expression of elovl5a was lowest in the 0.3% EPA+DHA↑ω6 fed fish, and correlated positively with 20:3ω3, 20:4ω3 and EPA:ARA in the head kidney. This indicates that high dietary 18:3ω3 promoted the synthesis of ω3 LC-PUFA. Dietary EPA+DHA levels had a positive impact on elovl5a, fadsd5 and srebp1 expression, and these transcripts positively correlated with tissue ΣMUFA. This supported the hypothesis that LC-PUFA synthesis is positively influenced by tissue MUFA levels in Atlantic salmon. The expression of pparaa was higher in the 0.3% EPA+DHA↑ω6 compared to the 0.3% EPA+DHA↑ω3 fed fish. Finally, significant correlations between head kidney fatty acid composition and the expression of eicosanoid synthesis-related transcripts (i.e., 5loxa, 5loxb, cox1, cox2, ptges2, ptges3, and pgds) illustrated the constitutive relationships among fatty acids and eicosanoid metabolism in salmon.

Minimizing marine ingredients in diets of farmed Atlantic salmon (Salmo salar): effects on liver and head kidney lipid class and fatty acid composition.

Limited fish meal and fish oil supplies have necessitated research on alternatives for aquafeeds. Seven dietary treatments with different protein and lipid sources were formulated for farmed Atlantic salmon, and their effects on liver and head kidney lipid class, fatty acid, and elemental composition were studied. Fish meal, fish oil, and EPA + DHA content ranged from 5-35%, 0-12%, and 0.1-3%, respectively. Elemental analysis showed that the C to N ratio was higher in the head kidney than in the liver, which is consistent with higher content of total lipids in the head kidney compared with the liver. There was a greater susceptibility to dietary lipid alterations in the liver compared with the head kidney despite liver having a greater proportion of phospholipid and a much lower proportion of triacylglycerol. So long as fish oil levels were 5% or more of the diet, arachidonic acid (ARA) and docosahexaenoic acid (DHA) proportions were the same for each tissue as with feeding the marine diet with 12% fish oil; however, livers and head kidneys from fish fed the lowest amount of fish meal and fish oil had the lowest levels of eicosapentaenoic (EPA) and DHA and the highest ARA levels. Removal of fish oil and reduction of fish meal to 5% in diets of farmed Atlantic salmon affected elemental and lipid compositions of the liver and head kidney tissues potentially increasing susceptibility to inflammation. However, with 10% of the diet comprising fish meal and fish oil, lipid contents were comparable with fish fed marine-based diets.

Tryptophan-like and humic-like fluorophores are extracellular in groundwater: implications as real-time faecal indicators.

Fluorescent natural organic matter at tryptophan-like (TLF) and humic-like fluorescence (HLF) peaks is associated with the presence and enumeration of faecal indicator bacteria in groundwater. We hypothesise, however, that it is predominantly extracellular material that fluoresces at these wavelengths, not bacterial cells. We quantified total (unfiltered) and extracellular (filtered at < 0.22 µm) TLF and HLF in 140 groundwater sources across a range of urban population densities in Kenya, Malawi, Senegal, and Uganda. Where changes in fluorescence occurred following filtration they were correlated with potential controlling variables. A significant reduction in TLF following filtration (ΔTLF) was observed across the entire dataset, although the majority of the signal remained and thus considered extracellular (median 96.9%). ΔTLF was only significant in more urbanised study areas where TLF was greatest. Beneath Dakar, Senegal, ΔTLF was significantly correlated to total bacterial cells (ρs 0.51). No significant change in HLF following filtration across all data indicates these fluorophores are extracellular. Our results suggest that TLF and HLF are more mobile than faecal indicator bacteria and larger pathogens in groundwater, as the predominantly extracellular fluorophores are less prone to straining. Consequently, TLF/HLF are more precautionary indicators of microbial risks than faecal indicator bacteria in groundwater-derived drinking water.

Interaction between ω6 and ω3 fatty acids of different chain lengths regulates Atlantic salmon hepatic gene expression and muscle fatty acid profiles.

Atlantic salmon smolts (approx. 20-months old) were fed experimental diets with different combinations of omega-6:omega-3 fatty acids (FAs) (high-ω6, high-ω3, or balanced) and eicosapentaenoic acid plus docosahexaenoic acid (EPA + DHA) levels (0.3, 1.0 or 1.4%) for 12 weeks. Muscle FA (% total FA) reflected dietary C18-polyunsaturated FA; however, muscle EPA per cent and content (mg g-1) were not different in salmon fed high-ω3 or balanced diets. Muscle DHA per cent was similar among treatments, while DHA content increased in fish fed 1.4% EPA + DHA, compared with those fed 0.3-1.0% EPA + DHA combined with high-ω6 FA. Muscle 20:3ω6 (DGLA) content was highest in those fed high-ω6 with 0.3% EPA + DHA. Quantitative polymerase chain reaction analyses on liver RNA showed that the monounsaturated FA synthesis-related gene, scdb, was upregulated in fish fed 1.0% EPA + DHA with high-ω6 compared to those fed 0.3% EPA + DHA. In high-ω3-fed salmon, liver elovl2 transcript levels were higher with 0.3% EPA + DHA than with 1.0% EPA + DHA. In high-ω6-fed fish, elovl2 did not vary with EPA + DHA levels, but it was positively correlated with muscle ARA, 22:4ω3 and DGLA. These results suggest dietary 18:3ω3 elongation contributed to maintaining muscle EPA + DHA levels despite a two- to threefold change in dietary proportions, while 18:2ω6 with 0.3% EPA + DHA increased muscle DGLA more than arachidonic acid (ARA). Positive correlations between hepatic elovl2 and fabp10a with muscle ω6:ω3 and EPA + DHA + ARA, respectively, were confirmed by reanalysing data from a previous salmon trial with lower variations in dietary EPA + DHA and ω6:ω3 ratios. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

In-situ fluorescence spectroscopy indicates total bacterial abundance and dissolved organic carbon.

We explore in-situ fluorescence spectroscopy as an instantaneous indicator of total bacterial abundance and faecal contamination in drinking water. Eighty-four samples were collected outside of the recharge season from groundwater-derived water sources in Dakar, Senegal. Samples were analysed for tryptophan-like (TLF) and humic-like (HLF) fluorescence in-situ, total bacterial cells by flow cytometry, and potential indicators of faecal contamination such as thermotolerant coliforms (TTCs), nitrate, and in a subset of 22 samples, dissolved organic carbon (DOC). Significant single-predictor linear regression models demonstrated that total bacterial cells were the most effective predictor of TLF, followed by on-site sanitation density; TTCs were not a significant predictor. An optimum multiple-predictor model of TLF incorporated total bacterial cells, nitrate, nitrite, on-site sanitation density, and sulphate (r2 0.68). HLF was similarly related to the same parameters as TLF, with total bacterial cells being the best correlated (ρs 0.64). In the subset of 22 sources, DOC clustered with TLF, HLF, and total bacterial cells, and a linear regression model demonstrated HLF was the best predictor of DOC (r2 0.84). The intergranular nature of the aquifer, timing of the study, and/or non-uniqueness of the signal to TTCs can explain the significant associations between TLF/HLF and indicators of faecal contamination such as on-site sanitation density and nutrients but not TTCs. The bacterial population that relates to TLF/HLF is likely to be a subsurface community that develops in-situ based on the availability of organic matter originating from faecal sources. In-situ fluorescence spectroscopy instantly indicates a drinking water source is impacted by faecal contamination but it remains unclear how that relates specifically to microbial risk in this setting.

Diet-Induced Physiological Responses in the Liver of Atlantic Salmon (Salmo salar) Inferred Using Multiplex PCR Platforms.

The simultaneous quantification of several transcripts via multiplex PCR can accelerate research in fish physiological responses to diet and enable the development of superior aquafeeds for farmed fish. We designed two multiplex PCR panels that included assays for 40 biomarker genes representing key aspects of fish physiology (growth, metabolism, oxidative stress, and inflammation) and 3 normalizer genes. We used both panels to assess the physiological effects of replacing fish meal and fish oil by terrestrial alternatives on Atlantic salmon smolts. In a 14-week trial, we tested three diets based on marine ingredients (MAR), animal by-products and vegetable oil (ABP), and plant protein and vegetable oil (VEG). Dietary treatments affected the expression of genes involved in hepatic glucose and lipid metabolism (e.g., srebp1, elovl2), cell redox status (e.g., txna, prdx1b), and inflammation (e.g., pgds, 5loxa). At the multivariate level, gene expression profiles were more divergent between fish fed the marine and terrestrial diets (MAR vs. ABP/VEG) than between the two terrestrial diets (ABP vs. VEG). Liver ARA was inversely related to glucose metabolism (gck)- and growth (igfbp-5b1, htra1b)-related biomarkers and hepatosomatic index. Liver DHA and EPA levels correlated negatively with elovl2, whereas ARA levels correlated positively with fadsd5. Lower hepatic EPA/ARA in ABP-fed fish correlated with the increased expression of biomarkers related to mitochondrial function (fabp3a), oxidative stress (txna, prdx1b), and inflammation (pgds, 5loxa). The analysis of hepatic biomarker gene expression via multiplex PCR revealed potential physiological impacts and nutrient-gene interactions in Atlantic salmon fed lower levels of marine-sourced nutrients.