The effect of exposure to farmed salmon on piscine orthoreovirus infection and fitness in wild Pacific salmon in British Columbia, Canada

Alexandra Morton , Richard Routledge , Stacey Hrushowy , Molly Kibenge , Frederick Kibenge Published: December 13, 2017https://doi.org/10.1371/journal.pone.0188793 Figures Abstract The disease Heart and Skeletal Muscle Inflammation (HSMI) is causing substantial economic losses to the Norwegian salmon farming industry where the causative agent, piscine orthoreovirus (PRV), is reportedly spreading from farmed to wild Atlantic salmon (Salmo salar) with as yet undetermined impacts. To assess if PRV infection is epidemiologically linked between wild and farmed salmon in the eastern Pacific, wild Pacific salmon (Oncorhynchus sp.) from regions designated as high or low exposure to salmon farms and farmed Atlantic salmon reared in British Columbia (BC) were tested for PRV. The proportion of PRV infection in wild fish was related to exposure to salmon farms (p = 0.0097). PRV was detected in: 95% of farmed Atlantic salmon, 37–45% of wild salmon from regions highly exposed to salmon farms and 5% of wild salmon from the regions furthest from salmon farms. The proportion of PRV infection was also significantly lower (p = 0.0008) where wild salmon had been challenged by an arduous return migration into high-elevation spawning habitat. Inter-annual PRV infection declined in both wild and farmed salmon from 2012–2013 (p ≤ 0.002). These results suggest that PRV transfer is occurring from farmed Atlantic salmon to wild Pacific salmon, that infection in farmed salmon may be influencing infection rates in wild salmon, and that this may pose a risk of reduced fitness in wild salmon impacting their survival and reproduction. Figures Fig 5Table 3Fig 1Table 1Fig 2Fig 3Fig 4Table 2Fig 5Table 3Fig 1Table 1Fig 2 Citation: Morton A, Routledge R, Hrushowy S, Kibenge M, Kibenge F (2017) The effect of exposure to farmed salmon on piscine orthoreovirus infection and fitness in wild Pacific salmon in British Columbia, Canada. PLoS ONE 12(12): e0188793. https://doi.org/10.1371/journal.pone.0188793 Editor: Uwe Fischer, Friedrich-Loeffler-Institut Bundesforschungsinstitut fur Tiergesundheit, GERMANY Received: July 18, 2017; Accepted: November 13, 2017; Published: December 13, 2017 Copyright: © 2017 Morton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are included within the paper and its supporting information files. Funding: This work was supported by NSERC Discovery Grant, T and K Allard, E Borak, D and V Bradshaw, Y Chouinard, J Corbett, Eden Conservation Trust, S Haney, The Jaymac Foundation, Patagonia, R North, B. Wheeler, The Tides Foundation, The Vancouver Foundation, Vancity Credit Union and many smaller donors. Competing interests: I have read the journal's policy, and the authors of this manuscript have the following competing interests: Alexandra Morton is on the board of the Pacific Coast Wild Salmon Society, Drs. Molly and Frederick Kibenge are married. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction Infectious viruses are imposing a significant impact on the global salmon farming industry [1], where high host density can elevate both pathogen production and virulence above levels generally found in wild salmon [2]. Reduction in wild salmon productivity has been related to the scale [3] and presence [4] of salmon farms, and pathogen surveillance provides useful insight into declining wild salmon populations [5]. Nonetheless, few studies have examined the relationship between exposure to salmon farms and the proportion of wild salmon infected with specific viruses [6]. The piscine orthoreovirus (PRV), discovered in 2010 [7], belongs to the family Reoviridae, subfamily Spinareovirinae [8], and is now considered ubiquitous in marine farmed Atlantic salmon (Salmo salar) in Norway and British Columbia (BC), Canada [9, 10]. PRV is the causative agent of the disease heart and skeletal muscle inflammation (HSMI) [11], which causes specific lesions in the heart and skeletal muscle and can result in anorexia and abnormal swimming behavior in affected fish [9, 10, 11]. An HSMI outbreak can cause 100% morbidity in a salmon farm [11, 12] with associated mortality between 0 and 20% [12]. Stressors, such as sea lice treatment, bacterial infection, and algae blooms, appear to trigger the development of HSMI in PRV-infected fish [13, 14]. PRV infection is also associated with melanized foci in white muscle in Atlantic salmon in Norway [15]. A PRV variant (genotype II) is associated with HSMI-like disease in farmed coho salmon (Onchorhynchus kisutch) in Chile [16] and rainbow trout (O. mykiss) in Norway [17, 18]. Recently, another related orthoreovirus (PRV-2) was demonstrated as the etiologic agent of erythrocytic inclusion body syndrome (EIBS), a condition associated with mass mortality in farmed juvenile coho salmon in Japan [19]. PRV sequences have also been detected in rainbow trout in Chile that were affected by idiopathic syndrome of rainbow trout (ISRT) [20], and another potential member of the PRV group was associated with epidemic mortality in wild largemouth bass (Micropterus salmoides) in the USA [21]. PRV and related orthoreoviruses of fish are therefore not only of major economic concern to the salmon aquaculture industry worldwide, but also with significant consequences for conservation and fisheries of wild salmon. Recent virus challenge studies with Atlantic salmon show that initially PRV causes a transient acute infection of the erythrocytes (red blood cells), which are nucleated in fish, where it replicates rapidly infecting up to 50% of the red blood cell population [9, 22, 23, 24]. PRV becomes detectable in other organs subsequent to this initial blood-borne infection [11, 22, 23]. HSMI is not detectable within the first 8–10 weeks post challenge [25]. The science on the infection dynamics of PRV in wild fish populations is still emerging. Garseth et al. [5] provide molecular-based evidence that salmon farms play a significant role in the long-distance transport and transmission of PRV in Norway, speculating that pathogen exchange solely between wild salmon during the at-sea migration phase likely plays a minor role in PRV dispersal. While PRV infection in Norwegian sea trout (Salmo trutta) is low (1.9–3.0%), the species’ persistence in the nearshore environment elevates exposure to salmon aquaculture. This heightens the possibility that sea trout could serve as an intermediary host for aquaculture-source PRV through habitat overlap with salmon during the freshwater spawning and juvenile rearing phases [26]. While no evidence of HSMI was detected in Norwegian wild salmonids [26], the researchers postulated that the impact of severe heart and skeletal muscle damage on a salmon’s cardiovascular capacity could decrease the likelihood of an infected fish entering the riverine habitat where sampling was conducted. It is widely observed that diseased wild fish are typically difficult to sample because they are preferentially removed from the population by predators [27]. Most BC marine salmon farms, which are distributed in clusters along the southern half of the BC coast (Fig 1), raise Atlantic salmon, while steelhead (O. mykiss) are farmed in BC lakes. Although the Atlantic salmon eggs that entered BC may not have come directly from Norway [28], the dominant strain of Atlantic salmon farmed in BC is the Norwegian ‘Mowi’ strain [29]. thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Map of BC with salmon farms and regions sampled. This map shows the following: (1) locations of salmon farms (red dots), (2) the 9 regions where wild salmon were sampled, (3) three lakes discussed in the text, (a) Oweekeno (elevation 15 m) (b) Chilko (elevation 1172 m) and (c) Cultus (elevation 47 m), and three river systems also discussed, the Fraser, Skeena, and Nass. Region color corresponds to the cluster analysis in Fig 4. The blue arrow represents the major Fraser River salmon migration route [38]. https://doi.org/10.1371/journal.pone.0188793.g001 The BC Ministry of Agriculture has reported that approximately 80% of BC farmed Atlantic salmon are infected with PRV [30]). Previous research on PRV infection in wild salmon in BC includes failure to detect the virus in 200 wild salmon collected in 2008 [31], but also a later report that the virus has been common in BC farmed and wild salmon at statistically similar rates of infection since 1987 [32]. Siah et al. [33] also reported a well-established PRV presence in wild and farmed salmon from Alaska through BC to Washington State. Experimental challenge studies show that PRV will transmit readily from Atlantic salmon to conspecifics through cohabitation [9, 22, 23, 34], as well as sockeye (O. nerka) and chinook salmon (O. tshawytscha). These infections result in high viral loads in the erythrocytes and kidney [9, 22, 23, 24, 34, 35]. However, evidence of immune activation in response to PRV infection is mixed. Dahle et al. [34] and Haatveit et al. [9] find that PRV infection strongly induces a wide number of interferon-regulated antiviral and MHC class I genes in Atlantic salmon red blood cells. Garver et al. [23] report only a modest antiviral immune response in Atlantic salmon red blood cells, and they fail to find this response in head kidney tissues. Similarly, Polinski et al. [24], find no upregulated innate immune gene expression in sockeye salmon head kidney tissues. The cause for this discrepancy is unclear. However, Dahle et al. [34] and Haatveit et al. [9] challenged with PRV-infected tissues from a field outbreak of HSMI in Norway, while Garver et al. [23] and Polinski et al. [24] performed their studies with a strain of PRV from BC. The samples used in the present study have yielded 14 PRV isolates [16, 36]. The discrepancies between published findings as they relate to the induction of immune responses in host salmon have not been resolved. While Garver et al. [23] reported that western North American PRV fails to cause HSMI, Di Cicco et al. [13] reported on two HSMI outbreaks in a salmon farm in BC. Hence, while earlier work reported that HSMI does not occur in BC [23, 32, 33], it is now understood that HSMI does occur in BC. However, HSMI has not been reported in wild or captive Pacific salmon. Here, we report the results of PRV screening of a broad collection of wild salmonids sampled throughout much of BC in 2012 and 2013, and samples of farmed Atlantic salmon and steelhead reared in BC net pen facilities from the same time period. We assess these data for evidence of (i) a potential epidemiological link between farmed and wild salmon and (ii) potential impact of PRV infection on wild fish. In addition, we also present data, sampled from Oweekeno Lake between 2014 and 2016, on PRV infection status of wild salmonids including an endangered sockeye salmon population (S1 and S2 Tables). Materials and methods Sampling As per restricted direct access to farm-specific Atlantic salmon, samples were obtained from markets selling fresh farmed salmon reared in BC marine net pen facilities. In 2012–2013, gill and head kidney samples were collected from 262 fresh farmed BC Atlantic salmon and 35 farmed Steelhead reared in freshwater net pens purchased from 10 BC market chains located in southwestern BC on 93 different dates. The fish suppliers confirmed that these farm salmon had been reared in the pens sited on the BC coast. There was no information as to the specific farm each sample was from. The “Best Before” date was used to select for the freshest samples. In 2012–2013, gill, heart, head kidney, and spleen tissues were extracted from 601 wild Pacific salmonids (Oncorhynchus spp.) (Table 1) collected from marine and freshwater throughout southern British Columbia from the numbered Regions in Fig 1. Another 402 salmonids were sampled 2014–2016 from Oweekeno Lake, Region 2a (Fig 1, S1 Table). Because these were sampled during different years, they were analyzed separately. thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. Numbers of wild salmon and trout collected in 2012 and 2013 by species and life stage. Numbers inside brackets are for the subset “exposed” to salmon farms, i.e. from Regions 5, 6, 7, 8 and 9. https://doi.org/10.1371/journal.pone.0188793.t001 We note that the sampling did not constitute an extensive, structured surveillance of wild salmonids in BC. Hence, we have not attempted to construct precise estimates of PRV prevalences in wild salmon with tight confidence limits. Our study, aimed at exploring potential geographic patterns and generating epidemiological evidence providing provisional support for key hypotheses, was more akin to those reported in [5] and [37]. The 192 juvenile salmon sampled in 2012–2013 were obtained from weekly beach seines conducted to monitor the spring outmigration through the near shore marine environment in Regions 5 and 6. These were collected under Fisheries and Oceans Canada scientific collection permits. The juvenile salmon collected from Oweekeno Lake in 2014–2016 were obtained via fixed trap nets, purse seining, and surface trawling. Adult salmon collected in 2012–2013 were opportunistically collected from marine sport and commercial fisheries in Regions 3, 5 and 6, and were obtained as freshly dead specimens from rivers in Regions 1, 2, 3, 4, 5, 6, 8, and 9. The Kokanee (O. nerka) sampled 2012–2013 from Region 7 and the trout sampled in the same years from Regions 2a, 7 and 8c were obtained from sport fisheries. One to ten fish were taken from each sampling event. The adult fish sampled in Oweekeno in 2014–2016 were collected via angling and gillnetting under BC Provincial licenses and from aboriginal food fisheries. Percussion to the head was used to euthanize live fish. Tissues were extracted within hours after specimens were obtained, whether live-caught, from fisheries or purchased from markets, using aseptic technique, including fresh, disinfected tools (a separate set for external vs. internal sample removal), and disposable work surfaces for each fish. Tissue samples were preserved in RNAlater® and shipped on ice to the Atlantic Veterinary College laboratory. No accompanying information on specific site identification or exposure classification was provided to laboratory analysts in order to minimize any bias. Cross contamination between samples from fisheries is expected and was minimized by sampling between different boats. In the case of sport-caught fish only 1–2 fish were sampled per boat. Regions While the regional source of the farmed salmon could only be identified as the southern half of BC where salmon farms are established, the wild salmonids were collected from nine distinct geographic regions across BC (Fig 1). These regions, shown in Fig 1, are grouped into two categories which differ with respect to exposure to Atlantic salmon farms. Regions 1 and 2 are distant from salmon farms, while Regions 3 and 4, though closer to salmon farms, are directly flushed by open-ocean water. Collectively, Regions 1–4 were classified as experiencing low exposure to Atlantic salmon farms (369 fish). Regions 5 and 6 are inshore archipelago environments with high fish farm density and retentive marine circulation [39]. Region 7 is a lake that is inaccessible to anadromous fish, where a steelhead farm is sited. Regions 8 and 9, divide the lower and upper Fraser River at the strong rapids in the Fraser Canyon. A large percentage of sockeye, the second most numerically abundant salmon species in the Fraser River system [40], migrate through Region 6 as they approach the river to spawn [41]. Salmon from Regions 5–9 were therefore classified as having a high exposure to farmed Atlantic salmon (233 fish). Migration challenge Fish sampled from the upper reaches of substantial watersheds (the Fraser, Skeena, and Nass, Fig 1) were deemed to have overcome significant migration challenges. The two largest of these watersheds are the Fraser and Skeena. For the Fraser, the most significant restriction is at Hells Gate in the Fraser Canyon (elevation about 100 m, but with the majority of the samples above this restriction taken from elevations of over 300 m). The primary salmon rivers in the Skeena watershed are the Babine, with major restriction in the vicinity of the 1951 Babine Slide (elevation around 400 m) [42], and the Bulkley, with major restriction at Moricetown Canyon (elevation around 380 m). Fish sampled from above these restrictions, and from above 300 m in another tributary, were placed in the high-challenge category. All fish sampled from the Nass were obtained from the Meziadin Lake watershed above the rapids in the Nass River, and were therefore also placed in this category. Viral screening The laboratory was provided with a unique identification code for each sample which did not include information on the site or exposure classification. When the laboratory returned the results for the statistical analysis, the identification codes were used to link viral status to sampling location, species, and life stage. RNA was manually extracted from fish tissues and quality was based on the OD A260/A280 ratio and quantitative reverse transcription polymerase chain reaction (RT-qPCR) amplification of either Atlantic salmon ELF-1α (GenBank accession number BT072490) or chinook salmon ELF-1α (GenBank accession number FJ890356) as an internal control. RNA was considered suitable for viral testing if amplification of ELF-1α yielded cycle threshold (Ct) values <30. Primers, probes, and RT-qPCR thermal cycling parameters were as described in Kibenge et al. [36]. All samples were screened for PRV targeting the L1 gene segment as described in Kibenge et al. [36]. In brief, Ct values ≤ 40 were considered positive. Statistical analyses The data files used in the following analyses are available in S3 and S4 Tables. The relationship between the viral screening results and exposure to salmon farms was first examined using a cluster analysis on the proportions of PRV-positive test results within farmed fish (Atlantic salmon and steelhead) and the nine wild fish regions (all species combined). Additionally, logistic regression analyses were used to: (a) probe for potential underlying causes for the geographic patterns in these proportions, (b) generate leads for further investigation, and (c) check for the potential that any apparent patterns could be attributable to other causes. The focus in the logistic regression analysis was on levels of exposure to salmon farms, return migration challenge, and host species. Lastly, the proportions of Atlantic salmon testing positive for PRV were assessed for inter-annual variation using likelihood-based inference. Because so little is known about the potential epidemiological interactions between farmed and wild salmon in the North Pacific, an exploratory approach to our analyses was used. Thus, in keeping with the spirit of exploratory data analysis [43], we adopted a flexible approach to the selection of statistical methods and models, and put forward our conclusions as hypotheses worthy of further attention. Cluster analysis. To perform the cluster analysis on the regional proportions of PRV results we applied the agglomerative, hierarchical clustering method based on cluster centroids as implemented in the SAS® CLUSTER procedure, SAS software, Version 9.4. In keeping with commentary in SAS 2013, the centroid method was selected to avoid giving too much influence to the much larger proportion of PRV positive fish in the farmed Atlantic salmon category. Logistic regression analysis. The logistic regression analysis was conducted solely on the wild fish. The factors of primary interest were: salmon farm exposure, migration challenge, and host species. The number of categories was restricted to avoid the potential for over-parameterization. Farm exposure and migration challenge were categorized as low or high as described above. Host species were reduced to four taxonomic units among the wild fish by combining lineages that had not diverged prior to approximately 7.5 million years ago [44, 45]–chinook-coho salmon with 168 samples, chum-pink salmon with 175 samples, sockeye salmon with 220 samples, and rainbow-cutthroat trout, with 38 samples. Two other factors, life stage and year, were included in the logistic regression analysis to probe for potential confounding effects. The wild salmon life stages were divided into 2 categories: juveniles (192 fish) and adults (409 fish). Observations used in the logistic regression analysis were limited to 2012 and 2013, the years for which farmed and wild salmon were concurrently sampled in sufficient numbers. There was insufficient data to extend formal inferences to other years. There were too few degrees of freedom, and the standard assumption of independence between years that underlies the usual models for random effects would have been compromised if, for example fish returning at ages 4 and 5 from the same cohort were both exposed to the same PRV source at an earlier life stage. Furthermore, Taksdal [46] highlights the potential both for differences in virulence between virus subtypes, and for relatively abrupt changes in viral-subtype presences that could produce sudden jumps in the proportions of positive tests. Both of these events would reduce the comparability of years in which only farmed Atlantic or wild salmon were collected. Such complex behavior calls for more elaborate modelling. Hence, inferences have been limited to 2012–2013, year effects were treated as fixed, and Oweekeno Lake data was not included in the analysis. Furthermore, there were sufficient numbers of observations to assess the main effects of each of the factors, but not necessarily for interactions between them (see S1 File for further explanation). Finally, a random effect associated with the within-cluster correlation of fish obtained from the same location and year was included to account for potential dependency in PRV presence among fish sampled from the same effective host population. This term additionally compensates for cross-contamination within a sampling event, as this would have had a comparable impact to the contagious spread of virus within a school of fish before they were caught. A more formal description of the statistical model is provided in S1 Text. Model selection. We used a stepwise approach to our logistic regression (starting with a full model) to screen for potentially influential factors. To reduce the likelihood of deleting potentially important variables in this exploratory analysis, we planned to remove, at each deletion step, the variable with the highest p-value from the model only if its p-value exceeded 0.10. Competing methods based on AIC and other similar measures of goodness of fit were complicated by occasional cases of missing information on some variables. Hence, the stepwise approach was more appropriate, and generated a preferred model after only two deletion steps. All mixed-effects logistic regression inferences were performed using the SAS GLIMMIX procedure as implemented in SAS software, Version 9.4. Comparative analysis of test results on farmed Atlantic salmon. We also formally compared the proportions of PRV-positive tests for the farmed Atlantic salmon between 2012 and 2013. Because multiple fish were purchased from the same outlet on the same day, we needed to account for potential dependence within such clusters of sampled fish generated by factors such as a common farm of origin and cross-contamination in processing and handling during harvest. We did so by incorporating a random effect term similar to that used in the logistic regression model. Details are provided in S2 Text. Results The farmed fish generated slightly higher ELF-1α Ct values, in keeping with the unavoidable delay in tissue preservation of market-sourced fish; however, tissue quality was suitable for RT-qPCR testing across all samples (Fig 2). thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Internal control ELF-1α Ct values indicating sample quality. Ct values <30 are considered of sufficient quality for RTqPCR viral screening. https://doi.org/10.1371/journal.pone.0188793.g002 Raw proportions of PRV-positive tests are shown in Fig 3. PRV infection was highest among the farmed salmon categories; Atlantic salmon (95%) and steelhead (69%). The highest proportions of PRV-infected wild salmonids were from the high exposure regions, i.e., Regions 5–8, including the lake with a steelhead farm and the highly exposed inshore archipelago environments (37–50%). The proportion of PRV infection declined between the highly exposed lower (41%) and upper (22%) Fraser River. The lowest proportions were in Regions 1 and 2, furthest from salmon farms (5%). In addition, Cultus Lake trout were highly infected with PRV (76%) (Lake c, Fig 1), while only 3% of the salmonids in Oweekeno Lake were infected with PRV (Lake a, Fig 1, S1 Table). thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Proportions of PRV RT-qPCR-positive results. Results are arranged in decreasing order. The “Wild” designations reflect the Region numbers in Fig 1; i.e., Wild 1 is from Region 1. Numbers of fish sampled are provided in parentheses on the horizontal axis labels. Relevant estimates and confidence limits for key differences in this figure were generated by the logistic regression modelling where the effects of potential confounding variables could be filtered out (Fig 5). https://doi.org/10.1371/journal.pone.0188793.g003 A complementary perspective emerged from the cluster analysis on these proportions (Fig 4). The two farmed fish species each formed distinct, single-element clusters. All high exposure regions, except Region 9 (post high migration challenge) appear in the yellow cluster. The green, less homogeneous cluster includes the high migration challenge Region 9 with all the low exposure regions. thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. Hierarchical cluster analysis of test results by region and farmed categories. https://doi.org/10.1371/journal.pone.0188793.g004 Details of the stepwise logistic regression procedure are summarized in S5 Table Summary of stepwise regression process. Two factors were dropped in the stepwise regression: “species group” and “life stage”, and then the algorithm terminated. All of the fixed factors in the preferred model (year, farm exposure, and migration challenge) were significant (p < 0.01, Table 2). thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Table 2. Summary of results for the preferred model. The SAS-generated table shows results of tests generated by dropping each factor from the model containing all three factors, with each factor replaced in the model before the next deletion. Degrees of freedom were calculated by SAS with a Satterthwaite correction. Estimates of the odds ratios were obtained by exponentiating the estimated coefficients for the log-odds ratios. https://doi.org/10.1371/journal.pone.0188793.t002 The proportion of PRV-infection in both wild and farmed salmon declined substantially between 2012 and 2013. For highly exposed wild salmon that had not faced a major migration challenge, the least-squares mean estimate of this proportion declined from 0.564 in 2012 to 0.129 in 2013 (Fig 5). The corresponding decline for farmed Atlantic salmon, from 0.974 to 0.790, was also strongly significant (p = 0.002 from the modelling procedure outlined in S2 Text). thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. Least-squares mean proportions for RT-qPCR positive test results. The black squares provide a reference estimate for fish at high farm exposure level, and low migration challenge level in 2012 (the ‘common reference’). The blue circles are least-squares mean proportions with each of these factors switched in turn to the opposite level, with the other factors left at the common reference level. The vertical bars cover approximately 2 standard errors. Where a vertical bar does not span the gap between the two estimates, the difference is significant at approximately the 5% level in a test against a two-sided alternative. https://doi.org/10.1371/journal.pone.0188793.g005 In addition to year, the effects of the other two factors, exposure and migration challenge, were also estimated to be large, though with substantial standard errors (Fig 5). The estimated effect of the most significant of all three factors, migration challenge, was also the largest. Fig 5 shows that, for high-exposure wild salmon in 2012, there was over a six-fold decline in the estimated proportion of PRV-positive test results from (a) fish in the low-challenge category to (b) those in the high-challenge category. This estimated decline is commensurate with the observed declines (i) between Regions 8 and 9 (the lower and upper Fraser River areas) and (ii) between the lower and higher elevations in Region 1 in northern BC (Table 3). Fig 5 also shows that, for low-challenge wild salmon in 2012, there was over a two-fold decline in the estimated proportion of PRV-positive test results from (a) fish in the high-exposure category to (b) those in the low-exposure category. thumbnail Download: PPT PowerPoint slide PNG larger image TIFF original image Table 3. Observed proportions of positive PRV tests by migration challenge level for regions with substantial numbers of migration-challenged fish (low exposure region 1 and high exposure regions 8 & 9). Numbers in brackets reflect numbers of positive tests per fish sampled. https://doi.org/10.1371/journal.pone.0188793.t003 Discussion The results of this work suggest that exposure to salmon farms has a strong association with increased risk of PRV infection in wild salmonids, and that the proportion of PRV-infected wild vs. farmed salmon can vary synchronously between years. In addition, the decline in PRV infection between the low and high migration challenge groups suggests that PRV infection may reduce a host’s capacity to complete a challenging upriver migration, thereby reducing reproductive fitness. We stress the correlational nature of the present findings, but believe, in keeping with the Precautionary Principle, that they warrant further research attention due to the high ecological, economic, and cultural value of wild Pacific salmon. The hierarchical cluster analysis of PRV-infection proportions showed a clear separation between the more highly infected farmed Atlantic salmon and all categories of wild Pacific salmon (Fig 4). This demonstrates the significantly greater potential infection pressure imposed by farmed salmon in comparison to wild salmon. Further, the logistic regression analysis has demonstrated that higher exposure to farmed salmon is associated with a significant increase in the proportion of PRV infected wild salmon. This is a plausible result as 79–95% of farmed salmon tested positive for PRV and wild fish in the regions where salmon farms operate among retentive currents would likely experience a higher contact rate with infectious PRV particles than wild salmon elsewhere. This result is in keeping with other research findings [6,47]. Deterministic modeling of water-borne infectious particles demonstrates that a high number of shedding hosts elevates the localized concentration of infectious particles thereby increasing the rate of infection in susceptible hosts [47]. This model is well supported by the empirical evidence that farmed salmon epizootics tend to cluster in both space and time (as reviewed in [6]). PRV has also been shown to be highly infectious both among and between species with transmission occurring from Atlantic salmon to both Atlantic and Pacific salmon through experimental cohabitation challenges [9, 22, 23, 24, 34]. While the exact mechanism of PRV transmission remains unknown, Hauge et al. [48] show that faecal virus shedding may release a significant amount of infectious particles into the water. While the heightened proportions of PRV-infection in wild salmon from high exposure regions provides some epidemiological evidence of PRV transmission between farmed and wild fish, the presence of PRV in low exposure populations suggests that transmission may also occur between individuals of wild populations in the open Pacific, though perhaps at a lower frequency. Together, these findings raise the concern that point-source pathogen release from aquaculture facilities may affect both populations directly exposed, and those that are not directly exposed to salmon farms. Siah et al. [33] also suggested that wild-to-wild transmission best explains the homogenous distribution of PRV S1 sequence types in the eastern Pacific. By contrast in Norway, Garseth et al. [5] proposed that PRV transmission between low density wild Atlantic salmon during their at sea phase likely plays a minor role in infection rates. However, it is possible that the more abundant wild salmon populations in the northeastern Pacific may provide better opportunities for PRV transmission. Our data provides some evidence for PRV transmission between wild fish, as low exposure populations also carry PRV. However, the higher PRV infection rates among those wild salmon in closer contact with Atlantic salmon also provides provisional evidence of PRV transmission in at least one direction between wild and farmed salmon. Additionally, the significant effect of year on the PRV infected proportion, which acts in the same direction for both wild and farmed salmon, also appears to corroborate the hypothesis that PRV prevalence in wild salmon is epidemiologically linked to prevalence in farmed Atlantic salmon. Garver et al.’s [23] findings that PRV can be transmitted from Atlantic salmon to Pacific salmon but not vice versa provides support for a dominant farmed-to-wild transmission route. Additionally, this study demonstrates strong evidence generated collectively from two regions in BC of a negative association between increased migratory challenge and PRV-positive proportions in return-migrating wild adult salmon. Fewer infected adults of any species were detected at higher vs. lower elevations in the Fraser River, as well as tributaries of the Skeena and Nass rivers in northern BC. This association points to a cost of infection from PRV to the fitness of wild Pacific salmon. While the pathogenicity of PRV in wild Pacific salmon has been questioned (e.g., [26, 32]), PRV-associated disease states (i.e., HSMI [13] and Jaundice Syndrome [16, 17]) are characterized by lethargy and erratic swimming behaviour [12], which would have more serious consequences for wild Pacific salmon than for farmed Atlantic salmon in net pens. The statistical modelling performed accounted for potential confounding effects from year, exposure level, salmonid host species, and life stage, and still found strong evidence of a decline in the infected proportion of salmon at higher elevations. However, it is possible that some other factor not included in the model could account for this change in proportions. Further investigations employing the tracking of the in-river fates of individual salmon by biotelemetry, as has been demonstrated by Jeffries et al. [49], and Miller et al. [37, 50], can better resolve confounding variation possibly associated with the migration timing of specific stocks and the timing of sampling events. However, with the geographic scale and numbers of fish used in the present study, it was infeasible to employ such technologies. Nonetheless, the evidence of lower PRV presence in salmon at higher elevations has important potential implications regarding fitness costs and population impacts of PRV on wild salmon. Similar findings were also reported by Miller et al. [37], who found PRV infection to be significantly associated with en-route migration losses for Chilko Lake sockeye salmon, which are challenged by an arduous 1,172m elevation gain in their return migration (Region 9, Lake b, Fig 1). In contrast, these authors reported that PRV infection was not significantly associated with migration losses into the lower elevation Shuswap Lake watershed (elevation 350m). The PRV infections detected in salmonids in low-lying lakes, Cultus (elevation 47 m) and Oweekeno (elevation 15 m), and in particular the higher proportion of positives in Cultus Lake trout where anadromous salmon entering the lake have been highly exposed to farmed salmon potentially on both seaward and return migrations, provide a contrast to the observed reduction in PRV in fish sampled at higher elevations. This contrast suggests the following hypotheses for future research: (i) PRV-infected wild fish are less able to meet the challenge of migrating into higher elevations above sea level, (ii) easily accessed, low-lying lakes lack the infection filtering effects of return migrations with greater challenges and may be more vulnerable to the introduction of aquaculture-source viruses via infected anadromous salmonids than high elevation habitat, and (iii) resident trout or other fish species in these lakes may act as viral reservoirs increasing the complexity of PRV transmission dynamics and potentially exposing successive generations of salmonids to infection. PRV has previously been shown to have a broad host range among salmonids in the Pacific and Atlantic [this study, 5, 23, 26, 32, 33, 36], including a first report in this study of a positive test for Dolly Varden char (from Oweekeno Lake, Fig 1, Lake a, S1 Table). Positive PRV results have been reported for some non-salmonid marine fish in coastal Norway as well [51]. The consequences of these potentially complex host-pathogen dynamics for sustaining infections in wild salmon populations are unknown, but their prospect raises important questions regarding the vulnerability of low-elevation salmonid populations to viral disease. Future work should attempt to identify competent host species, and to characterize viral reservoirs in addition to Atlantic salmon farms, particularly in light of the collapse of both the low-lying Cultus and Oweekeno Lake sockeye salmon populations to less than 1% of their historic spawner returns with no clear cause, despite significant restoration efforts [52, 53]. Recent research on PRV points to mechanisms through which the virus might impact the capacity of a salmon to complete a challenging migration to reach its spawning grounds [13, 22]. PRV has been found to proliferate in the erythrocytes, with possible implications for oxygen transport and swimming performance [22]. Research on PRV infection in Atlantic salmon hosts has also shown that PRV has a transient acute infection stage during which innate antiviral pathways are strongly upregulated [9]. Activation of these immune system pathways has been shown to have both direct and indirect energetic costs to a host [54]. While a similar level of immune activation in response to PRV infection has not been shown for Pacific salmon species [24], this could have other explanations beyond a total lack of pathogenicity, specifically: differences in pathogenicity among described and uncharacterized PRV strains, host species/virus strain interactions, and inferential complications arising from the current inability to culture PRV in fish cell lines. Histopathological examination of samples has value in confirming disease state and reinforcing the association between a condition and any impact to fitness; however, this approach was not employed in the present study as it is considered unlikely that wild salmon will progress to clinical disease before being targeted by predation [55]. A potentially more profitable approach employed by Miller et al. [55] uses modern molecular methods to predict the pathogenic outcomes of infection for salmon at early stages of infection. These authors have found that gene expression biomarkers for active virus infection can differentiate between both Atlantic salmon with HSMI and Pacific salmon species with Jaundice Syndrome (also strongly associated with PRV [16, 17]) from virus-negative fish and from fish with clinical diseases caused by other pathogen types [55]. It is hoped that greater numbers of future studies will take this approach in order to strengthen or refute the associations found herein, and more fully understand the consequences of viral pathogens like PRV for the fate of infected wild salmonids. Conclusions This study provides the first evidence that (i) exposure to farmed Atlantic salmon is associated with infection of wild Pacific salmon with PRV, a virus of significant concern to both the aquaculture industry and wild fisheries management, and (ii) that PRV infection may impair the capacity of wild salmon to complete a challenging spawning migration, with the potential for population-level impacts. The evidence, based solely on molecular screening tests from this observational study, and constrained by limited access to farmed Atlantic salmon samples of known provenance, cannot be definitive. Nonetheless, we view it as providing an early warning sign of a potentially serious problem that warrants immediate and ongoing research. Research into the fitness impacts to wild Pacific salmonids of farmed salmon pathogens is needed in wild fish populations in addition to controlled laboratory environments, and could provide valuable insights useful for the management of critically declining wild salmon populations. Supporting information S1 Table.docx S1 Table.Oweekeno Lake salmonid samples.Juvenile Sockeye salmon from Oweekeno include both freshwater fry and saltwater smolt stages. SpeciesNumberscreenedNumberpositivePercent (%) PRVpositiveSockeye*AdultsJuvenilesTotal742703442682.7%2.2%2.3%Chinook4112.4%Cutthroat trout19210.5%Dolly Varden (Salvelinus malma)10110.0%*All salmonids other than sockeye salmon were > 1 year old.1234 figshare 1 / 8 download Oweekeno Lake salmonid samples. (DOCX) S1 Table. Oweekeno Lake salmonid samples. https://doi.org/10.1371/journal.pone.0188793.s001 (DOCX) S2 Table. Oweekeno Lake data file. https://doi.org/10.1371/journal.pone.0188793.s002 (XLSX) S3 Table. Wild salmonid data file. https://doi.org/10.1371/journal.pone.0188793.s003 (XLSX) S4 Table. Farmed fish data file. https://doi.org/10.1371/journal.pone.0188793.s004 (XLSX) S5 Table. Summary of stepwise regression process. https://doi.org/10.1371/journal.pone.0188793.s005 (DOCX) S1 File. Numbers of 2012–3 sampled salmonids by species group and migration challenge categories. https://doi.org/10.1371/journal.pone.0188793.s006 (DOCX) S1 Text. Logistic regression model summary. https://doi.org/10.1371/journal.pone.0188793.s007 (DOCX) S2 Text. Technical details of comparative analysis on farmed Atlantic salmon. https://doi.org/10.1371/journal.pone.0188793.s008 (DOCX) Acknowledgments We thank T and K Allard, D and V Bradshaw, E Borak, Y Chouinard, J Corbett, Eden Conservation Trust, S Haney, The Jaymac Foundation, Patagonia, R North, B Wheeler, The Tides Foundation, The Vancouver Foundation, Vancity Credit Union, S Bodrug, F Campbell, T and R Campbell, B Crowther, R Dunlop, R Erikson, J Eriksson, N Gerbrandt, D and B Mackay, N Mackay, A Reed, D Rolston, M Woods, T and D Walkus, S Woodworth for help sampling and the Boston Bar, Dzawada’enuxw, Heiltsuk, Kwikwasut’inuxw Haxwa’mis, Lake Cowichan, Mowachaht/Muchalaht, Namigis, Nicola, N'Quatqua, Seton Lake, Shuswap, Snuneymuxw, Splatsin, St’at’imc, Stellaquo, Stolo, Takla Lake, Xeni Gwet'in, and the Wuikinuxv Nations for sampling assistance and access to wild salmon in their territories. References 1. Taranger GL, Karlsen Ø, Bannister RJ, Glover KA, Husa V, Karlsbakk E, et al. Risk assessment of the environmental impact of Norwegian Atlantic salmon farming. ICES J Mar Sci J. 2015; 72: 997–1021. View Article PubMed/NCBI Google Scholar 2. Mennerat A, Nilsen F, Ebert D, Skorping A. Intensive farming: Evolutionary Implications for Parasites and Pathogens. Evol Biol. 2010; 37: 59–67. pmid:21151485 View Article PubMed/NCBI Google Scholar 3. Hansen LP, Windsor ML. Interactions between Aquaculture and Wild Stocks of Atlantic Salmon and other Diadromous Fish Species: Science and Management, Challenges and Solutions: An introduction by the Conveners. ICES J Mar Sci. 2006; 63: 1159–1161. https://doi.org/10.1016/j.icesjms.2006.05.003 View Article PubMed/NCBI Google Scholar 4. Ford JS, Myers RA. A global assessment of salmon aquaculture impacts on wild salmonids, PLoS Biol. 2008; 6: 411–417. View Article PubMed/NCBI Google Scholar 5. Garseth ÅH, Ekrem T, Biering E. Phylogenetic evidence of long distance dispersal and transmission of piscine reovirus (PRV) between farmed and wild Atlantic salmon. 2013a; PLoS One. 8: https://doi.org/10.1371/journal.pone.0082202 View Article PubMed/NCBI Google Scholar 6. Johansen LH, Jensen I, Mikkelsen H, Bjørn P-A, Jansen PA, Bergh Ø. Disease interaction and pathogens exchange between wild and farmed fish populations with special reference to Norway. Aquaculture. 2011; 315: 167–186. View Article PubMed/NCBI Google Scholar 7. Palacios G, Lovoll M, Tengs T, Hornig M, Hutchison S, Hui J, et al. Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus. PLoS ONE. 2010; 5: e11487. https://doi.org/10.1371/journal.pone.0011487 pmid:20634888 View Article PubMed/NCBI Google Scholar 8. Attoui H, Mertens PPC, Becnel J, Belaganahalli S, Bergoin M, Brussaard CP, et al: Family Reoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Academic Press; 2011. pp. 541–637. 9. Haatveit HM, Wessel Ø, Markussen T, Lund M, Thiede B, Nyman IB, et al. Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells. Viruses. 2017; 9: 49. pmid:28335455 View Article PubMed/NCBI Google Scholar 10. Løvoll M, Alarcón M, Bang Jensen B, Taksdal T, Kristoffersen AB, Tengs T. Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production. Dis Aquat Organ. 2012; 99; 7–12. pmid:22585298 View Article PubMed/NCBI Google Scholar 11. Wessel Ø, Braaen S, Alarcon M, Haatveit H, Roos N, Markussen T, et al. Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon. PLoS ONE. 2017; 12(8): e0183781. https://doi.org/10.1371/journal.pone.0183781 pmid:28841684 View Article PubMed/NCBI Google Scholar 12. Kongtorp RT, Taksdal T, Lyngøy A. Pathology of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar. Dis Aquat Org. 2004; 59: 217–224. pmid:15264718 View Article PubMed/NCBI Google Scholar 13. Di Cicco E, Ferguson HW, Schulze AD, Kaukinen KH, Li S, Vanderstichel R, et al. Heart and skeletal muscle inflammation (HSMI) disease diagnosed on a British Columbia salmon farm through a longitudinal farm study. PLoS One. 2017; 12(2): e0171471. https://doi.org/10.1371/journal.pone.0171471 pmid:28225783 View Article PubMed/NCBI Google Scholar 14. Biering E, Garseth, ÅH. Heart and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) and the associated Piscine reovirus (PRV). ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish. 2012; 58: 6pp. http://www.ices.dk/sites/pub/Publication%20Reports/Disease%20Leaflets/Sheet%20no.%2058.pdf. 15. Bjørgen H, Wessel Ø, Fjelldal PG, Hansen T, Sveier H, Saebø HR, et al. Piscine orthoreovirus (PRV) in red and melanised foci in white muscle of Atlantic salmon (Salmo salar). BMC Vet Res. 2015; 46: 89. pmid:26346256 View Article PubMed/NCBI Google Scholar 16. Godoy MG, Kibenge MJT, Wang Y, Suarez R, Leiva C, Vallejos F, Kibenge FSB. First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV. Virol J. 2016; 13: 98. pmid:27296722 View Article PubMed/NCBI Google Scholar 17. Olsen AB, Hjortaas M, Tengs T, Hellberg H, Johansen R. First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV). PLoS ONE. 2015; 10: e0131638. https://doi.org/10.1371/journal.pone.0131638 pmid:26176955 View Article PubMed/NCBI Google Scholar 18. Hauge H, Vendramin N, Taksdal T, Olsen AB, Wessel Ø, Mikkelsen SS, et al. Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids. PLoS ONE. 2017; 12: e0180293. https://doi.org/10.1371/journal.pone.0180293 pmid:28678799 View Article PubMed/NCBI Google Scholar 19. Takano T, Nawata A, Sakai T, Matsuyama T, Ito T, Kurita J, et al. Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus. PLoS ONE. 2016; 11: e0165424. https://doi.org/10.1371/journal.pone.0165424 pmid:27788206 View Article PubMed/NCBI Google Scholar 20. Cartagena J, Tambley C, Sandino AM, Spencer E, Tello M. Detection of a virus related to Piscine orthoreovirus (PRV) in O. mykiss affected by idiopathic syndrome of rainbow trout (ISRT). 2016; Database: GenBank [internet]. https://www.ncbi.nlm.nih.gov/nuccore/KX844958.1; Accession Nos. KX844958-KX844965 21. Sibley SD, Finley MA, Baker BB, Puzach C, Armien AG, Giehtbrock D, et al. Novel reovirus associated with epidemic mortality in wild Largemouth Bass (Micropterus salmoides). J Gen Virol. 2016; 97: 2482–2487. pmid:27488948 View Article PubMed/NCBI Google Scholar 22. Finstad ØW, Dahle MK, Lindholm TH, Nyman IB, Løvoll M, Wallace C, Olsen CM, Storset AK, Rimstad E. Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes. Vet Res. 2014; Apr 3: 45:35. pmid:24694042 View Article PubMed/NCBI Google Scholar 23. Garver KA, Johnson SC, Polinski MP, Bradshaw JC, Marty GD, Snyman HN, et al. Piscine Orthoreovirus from Western North America Is Transmissible to Atlantic Salmon and Sockeye Salmon but Fails to Cause Heart and Skeletal Muscle Inflammation. PLoS ONE. 2016; 11: e0146229. pmid:26730591 View Article PubMed/NCBI Google Scholar 24. Polinski MP, Bradshaw JC, Inkpen SM, Richard JR, Fritsvold C, Poppe TT, Rise ML, Garver KA, Johnson SC. De novo assembly of Sockeye salmon kidney transcriptomes reveal a limited early response to piscine reovirus with or without infectious hematopoietic necrosis virus superinfection. BMC Genomics. 2016; 17:848. pmid:27806699 View Article PubMed/NCBI Google Scholar 25. Johansen LH, Dahle MK, Wessel Ø, Timmerhaus G, Løvoll M, Røsæg M, Jørgensen SM, Rimstad E, Krasnov A. Differences in gene expression in Atlantic Salmon parr and smolt after challenge with Piscine orthoreovirus (PRV). Mol Immunol. 2016; 73:138–150. pmid:27101566 View Article PubMed/NCBI Google Scholar 26. Garseth ÅH, Fritsvold C, Opheim M, Skjerve E, Biering E. Piscine reovirus (PRV) in wild Atlantic salmon, Salmo salar L., and seatrout, Salmo trutta L., in Norway. J Fish Dis. 2013b; 36: 483–493. View Article PubMed/NCBI Google Scholar 27. Bergh Ø. The dual myths of the healthy wild fish and the unhealthy farmed fish, Dis Aquat Organ. 2007; 75: 159–164. pmid:17578255 View Article PubMed/NCBI Google Scholar 28. Fisheries and Oceans Canada (DFO). Public Reporting on Aquaculture—Salmon Egg Imports. 2012. https://web.archive.org/web/20170304051933/http://www.pac.dfo-mpo.gc.ca/aquaculture/reporting-rapports/egg-oeuf-eng.html. 29. Withler RE, Supernault KJ, Miller KM. Genetic variation within and among domesticated Atlantic salmon broodstocks in British Columbia, Canada. Anim Genet. 2005; 36: 43–50. pmid:15670130 View Article PubMed/NCBI Google Scholar 30. Marty G. Piscine reovirus information sheet. 2013; pp. 1–3. http://marineharvest.ca/globalassets/canada/pdf/other-pdfs/piscine-reovirus-prv-information-sheet_gary-marty_2013.pdf 31. Saksida SM, Marty GD, Jones SRM, Manchester HA, Diamond CL, Bidulka J. St-Hilaire, S Parasites and hepatic lesions among pink salmon, Oncorhynchus gorbuscha (Walbaum), during early seawater residence. J Fish Dis. 2014; 35: 137–151. View Article PubMed/NCBI Google Scholar 32. Marty GD, Morrison DB, Bidulka J, Joseph T, Siah A. Piscine reovirus in wild and farmed salmonids in British Columbia, Canada: 1974–2013. J Fish Dis. 2015; 38: 713–728. pmid:25048977 View Article PubMed/NCBI Google Scholar 33. Siah A, Morrison DB, Fringuelli E, Savage P, Richmond Z, Johns R, et al. Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast. PLoS ONE. 2015; 10: e0141475. https://doi.org/10.1371/journal.pone.0141475 pmid:26536673 View Article PubMed/NCBI Google Scholar 34. Dahle MK, Wessel O, Timmerhaus G, Nyman IB, Jørgensen SM, Rimstad E, Krasnov A. Transcriptome analysis of Atlantic Salmon (Salar salmo L.) erythrocytes infected with piscine orthoreovirus (PRV), Fish Shellfish Immunol. 2015; 45:780–790. pmid:26057463 View Article PubMed/NCBI Google Scholar 35. Garver KA, Marty GD, Cockburn SN, Richard J, Hawley LM, Müller A, et al. Piscine reovirus, but not Jaundice Syndrome, was transmissible to Chinook Salmon, Oncorhynchus tshawytscha (Walbaum), Sockeye Salmon, Oncorhynchus nerka (Walbaum), and Atlantic Salmon, Salmo salar L. J Fish Dis. 2015; 39: 117–128. pmid:25630226 View Article PubMed/NCBI Google Scholar 36. Kibenge MJT, Iwamoto T, Wang Y, Morton A, Godoy MG, Kibenge FSB. Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes. Virol J. 2013; 10: 230–250. pmid:23844948 View Article PubMed/NCBI Google Scholar 37. Miller KM, Teffer A, Tucker S, Li S, Schulze AD, Trudel M, et al. Infectious disease, shifting climates, and opportunistic predators: cumulative factors potentially impacting wild salmon declines. Evol Appl. 2014; 7: 812–855. pmid:25469162 View Article PubMed/NCBI Google Scholar 38. Welch DW, Melnychuk MC, Rechisky ER, Porter AD, Jacobs MC, Ladouceur A, et al. Freshwater and marine migration and survival of endangered Cultus Lake sockeye salmon (Oncorhynchus nerka) smolts using POST, a large-scale acoustic telemetry array. Can J Fish Aquat Sci. 2009; 66: 736–750. https://doi.org/10.1139/F09-032 View Article PubMed/NCBI Google Scholar 39. Foreman MGG, Stucchi DJ, Garver KA, Tuele D, Isaac J, Grime T, Guo M, Morrison J. A Circulation Model for the Discovery Islands, British Columbia. Atmos-Ocean. 2012; 50: 301–316. View Article PubMed/NCBI Google Scholar 40. LaPointe, M, Cooke, SJ, Hinch, SG, Farrell, AP, Jones, S, MacDonald, S, et al., Late-run Sockeye Salmon in the Fraser River, British Columbia, are Experiencing Early Upstream Migration and Unusually High Rates of Mortality—What is Going On? In T.W. Droscher and D.A. Fraser (eds). Proceedings of the 2003 Georgia Basin/Puget Sound Research Conference. Mar 31-Apr 3; Vancouver, BC; 2003. http://www.psat.wa.gov/03_proceedings/start.htm. 41. Groot C, Quinn TP. Homing migration of sockeye salmon, Oncorhynchus nerka, to the Fraser River. Fish Bull. 1987; 85: 455–469. View Article PubMed/NCBI Google Scholar 42. Godfrey H, Hourston WR, Stokes JW, and Withler FC. Effects of a rock slide on Babine River salmon. J Fish Res Board Can. Bulletin no. 101, Ricker, WE, Editor. Ottawa (ON), Canada; 1954. View Article PubMed/NCBI Google Scholar 43. Tukey JW. We need both exploratory and confirmatory. Am Stat. 1980; 34: 23–5. View Article PubMed/NCBI Google Scholar 44. McKay SJ, Devlin RH, Smith MJ. Phylogeny of Pacific salmon and trout based on growth hormone type-2 and mitochondrial NADH dehydrogenase subunit 3 DNA sequences. Can J Fish Aquat Sci. 1996; 53: 1165–1176. https://doi.org/10.1139/f96-042. View Article PubMed/NCBI Google Scholar 45. Kitano T, Matsuoka N, Saitou N. Phylogenetic relationship of the genus Oncorhynchus species inferred from nuclear and mitochondrial markers. Genes Genet Syst. 1997; 72: 25–34. pmid:9248045 View Article PubMed/NCBI Google Scholar 46. Taksdal T, Olsen AB, Bjerkaas I, Hjortaas MJ, Dannevig BH, Graham DA, Mcloughlin MF. Pancreas disease in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway. J Fish Dis. 2007; 30: 545–558. pmid:17718709 View Article PubMed/NCBI Google Scholar 47. Murray AG. Using simple models to review the application and implications of different approaches used to simulate transmission of pathogens among aquatic animals. Prev Vet Med. 2009; 88: 167–177. pmid:18930326 View Article PubMed/NCBI Google Scholar 48. Hauge H, Dahle M, Moldal T, Thoen E, Gjevre AG, Weli S, et al. Piscine orthoreovirus can infect and shed through the intestine in experimentally challenged Atlantic salmon (Salmo salar L.). Vet Res. 2016; 47. pmid:27216404 View Article PubMed/NCBI Google Scholar 49. Jeffries KM, Hinch SG, Gale MK, Clark TD, Lotto AG, Casselman MT, et al. Immune response genes and pathogen presence predict migration survival in wild salmon smolts. Mol Ecol. 2014; 23: 5803–5815. pmid:25354752 View Article PubMed/NCBI Google Scholar 50. Miller KM, Li S, Kaukinen KH, Ginther N, Hammill E, Curtis JMR, et al. Genomic signatures predict migration and spawning failure in wild Canadian salmon. Science. 2011; 331:214–217. pmid:21233388 View Article PubMed/NCBI Google Scholar 51. Wiik-Nielsen CR, Løvoll M, Sandlund N, Faller R, Wiik-Nielsen J, Bang Jensen B. First detection of piscine reovirus (PRV) in marine fish species. Dis Aquat Organ. 2012; 97: 255–258. pmid:22422096 View Article PubMed/NCBI Google Scholar 52. Bradford MJ, Hume JMB, Withler RE, Lofthouse D, Barnetson S, Grant S, et al. Status of Cultus Lake sockeye salmon. DFO Can Sci Advis Sec Res Doc. 2011; 123: 44. Available from: http://www.dfo-mpo.gc.ca/Library/342579.pdf. View Article PubMed/NCBI Google Scholar 53. Fisheries and Oceans Canada. Escapement of Owikeno Lake sockeye salmon 1950–2014; 1999 [cited 2017 Jul 16]. Database: NuSEDS-New Salmon Escapement Database System [internet]. http://open.canada.ca/data/en/dataset/c48669a3-045b-400d-b730-48aafe8c5ee6. 54. Lochmiller RL, Deerenberg C. Trade-offs in evolutionary immunity: just what is the cost of immunity? Oikos. 2000; 88: 87–98. View Article PubMed/NCBI Google Scholar 55. Miller KM, Günther OP, Li S, Kaukinen KH, Ming TJ. Molecular indices of viral disease development in wild migrating salmon. Cons Physiol. 2017; 5(1). View Article PubMed/NCBI Google Scholar

Geographical distribution of the red howler monkey (Alouatta seniculus) and yellow fever in Colombia.

Biomedica. 2016 Feb 11;36(0):116-24. doi: 10.7705/biomedica.v36i0.2929. Piedrahita-Cortés J1, Soler-Tovar D. Author information 1 Grupo de Epidemiología y Salud Pública, Universidad de La Salle, Bogotá, D.C., Colombia. jmpcvet@gmail.com. Abstract INTRODUCTION: Colombia is a country with an important diversity of non-human primates, of which the red howler monkey (Alouatta seniculus) stands out because of its distribution and the role it plays in the occurrence of yellow fever. OBJECTIVE: To describe the geographic co-occurrence of Alouatta seniculus and the reported presence of yellow fever. MATERIALS AND METHODS: We conducted a descriptive study. The reported presence of yellow fever in Colombia was obtained from the reports and bulletins issued by the Instituto Nacional de Salud, and the study by Segura, et al. (2013). The occurrence of A. seniculus was determined based on the data from the Global Biodiversity Information Facility and the Colombian Biodiversity Information System. A map of the occurrence was developed using the DIVA-GIS program, and the ecological niche model under current conditions was created with the Maxent program. RESULTS: The departments with the highest occurrence of A. seniculus were Antioquia, Meta and Casanare; 69.5% of the departments with reported history of yellow fever had co-occurrence with A. seniculus. The ecological niche model showed that Antioquia, Bolívar, La Guajira, Magdalena, Meta, Santander, Norte de Santander and Vichada had geographical portions with a probability rate nearing to 0.9 (90%). CONCLUSIONS: In 69.5% of the departments with a history of yellow fever there was co-occurrence with A. seniculus, which is relevant because non-human primates play a well-known role as natural reservoirs of the virus, and they might contribute to the occurrence of the yellow fever, which makes them very useful as sentinels. KEYWORDS: Alouatta; Flavivirus; Latin America; epidemiology; primate diseases; tropical medicine; yellow fever; zoonoses

what my Trini relatives saw on the cocoa estate on 25/12/17

apparently it more than farted (pooped) in their general direction. Not going to sharpen the photo since it ain't mine.

Grandmother Misao and her beloved cat Fukumaru, by photographer and granddaughter Miyoko Ihara

#womensart #MerryChristmasEveryone pic.twitter.com/4eZDoNyy2I

Phytochemicals as inhibitors of NF-κB for treatment of Alzheimer's disease

Pharmacol Res. 2017 Nov 24. pii: S1043-6618(17)31134-9. doi: 10.1016/j.phrs.2017.11.030. [Epub ahead of print] . Seo EJ1, Fischer N1, Efferth T2. Author information 1 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany. 2 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany. Electronic address: efferth@uni.mainz.de. Abstract Alzheimer's disease (AD) is the most prevalent form of dementia. The exact pathophysiology of this disease remains incompletely understood and safe and effective therapies are required. AD is highly correlated with neuroinflammation and oxidative stress in brain causing neuronal loss. Nuclear factor of activated B-cells (NF-κB) is involved in physiological inflammatory processes and thus representing a promising target for inflammation-based AD therapy. Phytochemicals are able to interfere with the NF-κB pathway. They inhibit the phosphorylation or the ubiquitination of signaling molecules, and thus, inhibit the degradation of IκB. The translocation of NF-κB to the nucleus and subsequent transcription of pro-inflammatory cytokines are inhibited by the actions of phytochemicals. Additionally, natural compounds preventing the interaction of NF-κB can block NF-κB's transcriptional activity by inhibiting its binding to target DNA. Many polyphenols including curcumin, resveratrol, pterostilbene, punicalagin, macranthoin G, salidroside, 4-O-methylhonokiol, lycopene, genistein, obovatol and gallic acid were reported as potent NF-κB inhibitors for AD treatment. Several alkaloids such as galantamine, glaucocalyxin B, tetrandrine, berberine, oridonin, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in vivo. Besides, vitamins, tanshinone IIA, artemisinin, dihydroasparagusic acid, geniposide, xanthoceraside, l-theranine, 1,8-cineole and paeoniflorin were described as promising NF-κB inhibitors. In conclusion, natural products from plants represent interesting candidates for AD treatment. They may qualify as promising compounds for the development of derivatives providing enhanced pharmacological features. KEYWORDS: Botanicals; Cognitive impairment; Medicinal plants; Natural products; Neurodegenerative diseases; Small molecule inhibitor PMID: 29179999 DOI: 10.1016/j.phrs.2017.11.030

Determination of sugars and cyclitols isolated from various morphological parts of Medicago sativa L

J Sep Sci. 2017 Dec 18. doi: 10.1002/jssc.201701147. [Epub ahead of print] . Al-Suod H1,2, Ratiu IA2,3, Ligor M1, Ligor T1,2, Buszewski B1,2. Author information 1 Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland. 2 Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University, Toruń, Poland. 3 Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering, Cluj-Napoca, Romania. Abstract Plant research interest has increased all over the world, and a large body of evidence has been collected to show the huge potential of medicinal plants in various disease treatments. Medicago sativa L., known as alfalfa, is a rich source of biologically active components and secondary metabolites and was frequently used from the ancient times both as fodder crop and as a traditional medicine in the treatment of various diseases. Cyclitols, naturally occurring in this plant, have a particular interest for us due to their significant anti-diabetic, antioxidant, anti-inflammatory and anti-cancer properties. In the present study we revealed the isolation, the identification and the quantification of some cyclitols and sugars extracted from different morphological parts of alfalfa plant. Soxhlet extraction and solid phase extraction were used as extraction and purification methods, while for the analyses derivatization followed by gas chromatography with mass spectrometry was involved. The obtained results showed significant differences in the quantities of cyclitols and sugars found in the investigated morphological parts, ranging between 0.02 and 13.86 mg/g of plant in case of cyclitols, and in the range of 0.09 and 40.09 mg/g of plant for sugars. However, roots have the richest part of cyclitols and sugars in contrast to the leaves. This article is protected by copyright. All rights reserved. KEYWORDS: Alfalfa; Cyclitols; Gas chromatography-mass spectrometry; Solid-phase extraction; Sugars PMID: 29250921 DOI: 10.1002/jssc.201701147

Anti-aging activities of extracts from Tunisian medicinal halophytes and their aromatic constituents

EXCLI J. 2017; 16: 755–769. Published online 2017 May 22. doi: 10.17179/excli2017-244 PMCID: PMC5547381 A. Jdey,1,2,3 H. Falleh,2 S. Ben Jannet,2 K. Mkadmini Hammi,2 X. Dauvergne,3 C. Magné,*,3 and R. Ksouri2 Author information ► Article notes ► Copyright and License information ► Go to: Abstract Six medicinal halophytes widely represented in North Africa and commonly used in traditional medicine were screened for pharmacological properties to set out new promising sources of natural ingredients for cosmetic or nutraceutical applications. Thus, Citrullus colocynthis, Cleome arabica, Daemia cordata, Haloxylon articulatum, Pituranthos scoparius and Scorzonera undulata were examined for their in vitro antioxidant (DPPH scavenging and superoxide anion-scavenging, β-carotene bleaching inhibition and iron-reducing tests), antibacterial (microdilution method, against four human pathogenic bacteria) and anti-tyrosinase activities. Besides, their aromatic composition was determined by RP-HPLC. H. articulatum shoot extracts exhibited the strongest antioxidant activity and inhibited efficiently the growth of Salmonella enterica and Escherichia coli. P. scoparius and C. arabica inhibited slightly monophenolase, whereas H. articulatum was the most efficient inhibitor of diphenolase activity. Furthermore, H. articulatum exhibited the highest aromatic content (3.4 % DW), with dopamine as the major compound. These observations suggest that shoot extract of H. articulatum, and to a lesser extent of C. arabica, could be used as antioxidant, antibiotic as well as new natural skin lightening agents. Also, possible implication of aromatic compounds in anti-tyrosinase activity is discussed. Keywords: anti-aging, aromatic composition, biological activities, ethnobotany, medicinal halophytes Go to: Introduction Consumers are currently demanding less use of chemicals or minimally processed plant-derived food, so more attention had been paid to search for naturally occurring substances to treat illnesses or for health care. This is particularly true for plant materials that act as alternative antimicrobial or antioxidant sources. Therefore, the antioxidant phytochemicals from vegetables, fruits and medicinal plants have received increasing concern in the last decades for their potential role in preventing human diseases. Besides, more attention is being paid to the use of natural plant extracts in the cosmetic industry (Rangkadilok et al., 2007[42]; Shen at al., 2010[47]). Here, the use of natural products as antioxidant, antimicrobial and anti-aging agents provides a better alternative to the wide and injudicious use of synthetic agents. Halophytes are salt-tolerant plants living in hostile natural environment characterized with a number of stressful conditions (e.g. drought, salinity, UV radiations, and extreme temperatures). In those conditions, they experience a strong and permanent oxidative stress and produce harmful ROS. As a consequence, they are constitutively equipped with a powerful antioxidant system that includes enzymatic and non-enzymatic components (Ksouri et al., 2012[27]; Falleh et al., 2013[17]). Among the latter, aromatic compounds are secondary metabolites that may participate in cell protection against the harmful action of xenobiotics or UV radiations through their capacity to quench reactive oxygen species (ROS) and to regulate oxidative pathways including melanogenesis (Rice-Evans et al., 1997[43]; Panich et al., 2010[38]). These properties make halophyte aromatic compounds of great potential for cosmetic and pharmaceutical uses. Accordingly, investigations have been addressed to identify novel and natural bioactive sources among medicinal halophytes, which could be used for skin-protective applications. With the aim at setting out new natural sources of cosmetic interest, six medicinal halophytes were, for the first time, tested for their ability to inhibit tyrosinase, as well as for their antioxidant and antimicrobial capacities. Moreover, aromatic compounds were analyzed in these species, as possible bioactive compounds responsible for those activities. Go to: Materials and Methods 1. Plant material Since traditional ethnomedicinal use of plant is recognized as an important potential source for compounds used in mainstream medicine, six halophytic species widely used in African folk medicine have been selected for scientific investigations: Citrullus colocynthis (L.) Schrad., Cleome arabica (L.), Daemia cordata (Forssk.) R. Br. ex Schult., Haloxylon articulatum (Cav.) Bunge, Pituranthos scoparius (Coss. & Durieu) Benth. and Scorzonera undulata Vahl. Main characteristics of the selected plants are resumed in Table 1(Tab. 1). Table 1 Table 1 Characteristics of the six studied species Fruit of Citrullus colocynthis have long been used for the treatment of diabetes, cancers, microbial infection, ulcer, asthma, inflammation, jaundice, and urinary disease in Asian and African countries (Ziyyat et al., 1997[57]; Qureshi et al., 2010[40]; Rajamanickam et al., 2010[41]). Moreover, its dried fruit is a strong laxative and an abortifacient (Alkofahi et al., 1996[2]; Madari and Jabobs, 2004[30]), and it may be used against arthritis, parasitic worms and skin diseases. Is has been shown to contain colocynthin and vegetative organs colocynthetin, two bioactive glycosides. Roots contain elaterin, a poisonous cathartic substance used as a fat burner in Asian herbal, and the cytotoxic diterpenoid hentriacontane. Cleome arabica has long been used as folk medicine in North Africa to treat scabies and inflammation (Ahmad et al., 1990[1]; Tsichritzis et al., 1993[52]) as well as rheumatic pains (Bouriche and Arnhold, 2010[7]). It also possesses antimicrobial (Takhi et al., 2011[50]), antioxidant (Alkofahi et al., 1996[2]; Selloum et al., 1997[45]), acricidal (Kemassi et al., 2012[24]), and cytotoxic activities (Nagaya et al., 1997[35]). Daemia cordata (= Pergularia tomentosa, Telosma tomentosa) leaves and roots are prepared as an infusion, decoction, or powder, and taken orally or applied externally on the skin. It is reputed for diverse folk uses as an antirheumatic, laxative, abortive or hepatoprotective agent. It is also efficient in treatment of some skin diseases or against asthma and bronchitis (Gohar et al., 2000[18]; Suresh Kumar and Mishra, 2008[49]). Haloxylon articulatum (= Caroxylon articulatum, Salsola articulata, Hammada articulata) grows in sandy habitats in subtropical regions from North Africa to China. Although it has no interest as forage, it is important for soil conservation. Eight alkaloids (mainly isoquinolins and beta-carboline derivatives) have been reported from H. articulatum aerial parts (Benkrief et al., 1990[4]; El-Shazly and Wink, 2003[16]), and it has some traditional uses in Saharian folkloric medicine against scorpion stings, cooling, indigestion, and hypertension (Hammiche and Maiza, 2006[19]). In traditional medicine, stems and leaves of Pituranthos scoparius have been used in the treatment of measles, fever, rheumatism, asthma, jaundice, digestive difficulties, urinary infections, diabetes, hepatitis and postpartum spasms or pains (Boukef, 1986[6]; Shaker et al., 1999[46]). They are also applied locally as a poultice against snake and scorpion bites (Boukef, 1986[6]; Hammiche and Maiza, 2006[19]). Scorzonera species are used against pulmonary diseases, colds, for the treatment of wounds and gastro-intestinal disorders, as well as for their stomachic, diuretic, galactogogue, antipyretic and appetizing effects in European traditional medicine (Zidorn et al., 2003[56]; Tsevegsuren et al., 2007[51]). 2. Sampling and extraction Aerial parts of the six xerohalophytic species were harvested in summer 2014 from Sidi-Bouzid. The site is located at the middle-east of Tunisia (latitude 35°2'24''N; longitude 9°30'0''E), in a region characterized with arid bioclimatic stage. All samples were rinsed with distilled water, freeze-dried and ground in a Mettler AE 200 blender. Extraction was performed by magnetic stirring of 2.5 g dry powder in 25 ml aqueous ethanol (50 %, v/v) for 16 h at 4 °C. Then, the mixture was filtered through a Whatman No 4 filter paper and evaporated under vacuum to dryness. The dry residue was resuspended in 50 % ethanol at a concentration of 100 mg/ml and stored at -27 °C until analyses. 3. Determination of antioxidant activities 3.1. DPPH scavenging activity The hydrogen atom or electron donation ability of the extracts was measured from the bleaching of purple colored methanol solution of 1,1-diphenyl-2picrylhydrazyl (DPPH) according to the method described by Sokmen et al. (2004[48]). One milliliter of various concentrations of the ethanol extracts was added to 250 µl of 0.2 mM DPPH radical solution in methanol. The mixtures were shaken vigorously and allowed to stand for 30 min in the dark. The absorbance of the resulting solutions was measured at 517 nm and butylated hydroxytoluene (BHT) was used as a positive control. Inhibition of DPPH radical was calculated as follows: DPPH• scavenging effect (%) = [100 * (A0 - A1) / A0] (1) where A0 and A1 are the absorbance values of the control and of the sample at 30 min, respectively. The antiradical activity was expressed as IC50 (µg.ml−1), a low IC50 value corresponding to a high antioxidant activity. All samples were analyzed in triplicate. 3.2. β-carotene bleaching test A slightly modified method of that described by Koleva et al. (2002[26]) was employed to estimate shoot extract capacity to inhibit the β-carotene bleaching. Two milligrams of β-carotene were dissolved in 20 ml of chloroform, and to 4 ml of this solution linoleic acid (40 mg) and Tween 40 (400 mg) were added. Chloroform was evaporated under vacuum at 40 °C and 100 ml of oxygenated water were added, then the fresh emulsion was vigorously shaken. An aliquot (150 µl) of the β-carotene/linoleic acid emulsion was distributed in 96-well microtiter plates (NUNC microplate, Fisher Bioblock) and methanol solutions of the test samples or authentic standards (10 µl) were added. Three replicates were prepared for each concentration. The absorbance of all wells was measured at 470 nm using a microtiter reader (Multiskan EAR 400, Labsystems), both immediately (t = 0 min) and after 120 min of incubation at 50 °C. The antioxidant activity of the BHT standard and of plant extracts was calculated in percentage of β-carotene bleaching inhibition as follows: % inhibition = (S − C120 / C0 − C120) * 100 (2) where C0 and C120 are the absorbances of the control at 0 and 120 min, respectively, and S is the sample absorbance at 120 min. Results were expressed as IC50 values (µg.ml−1). 3.3. Superoxide anion radical-scavenging activity Superoxide scavenging capacity was assessed according to Duh et al. (1999[14]). The reaction mixture contained 0.2 ml of shoot extracts at different concentrations, 0.2 ml of 60 mM PMS, 0.2 ml of 677 mM NADH and 0.2 ml of 144 mM NBT, all in phosphate buffer (0.1 M, pH 7.4). After 5 min of incubation at room temperature, the absorbance was read at 560 nm against blank. The inhibition percentage of superoxide anion generation was calculated using the previous formula (2). As for the antiradical activity, the antioxidant activity in shoot extracts was expressed as IC50 in µg.ml-1. 3.4. Iron reducing power The iron reducing power was determined based on the transformation of Fe3+ to Fe2+ induced by the plant extracts according to the method of Oyaizu (1986[37]). Sample solutions at different concentrations were prepared in 1 ml of 0.2 M phosphate buffer (pH 6.6) and mixed with 1 ml of potassium ferricyanide (1 %, w/v). Tube was incubated at 50 °C for 20 min. Afterwards, 1 ml of TCA (10 %) were added and the mixture was centrifuged for 10 min at 1000 g. Supernatant (1 ml) was mixed with distilled water (1 ml) and 0.1 ml of ferric chloride (0.1 %, w/v), and the absorbance was read at 700 nm. Higher absorbance values of the reaction mixture indicate a greater reducing power. EC50 value (µg.ml−1) is the effective concentration of the extract at which the absorbance was reduced by 50 % and it was obtained from linear regression analysis. All samples were analyzed in triplicate. 4. Evaluation of the antibacterial activity 4.1. Microorganisms tested Antibacterial activity was screened against four human pathogenic bacteria including the Gram positive Micrococcus luteus (ATCC 10240) and Staphylococcus aureus subsp. aureus (ATCC 33862), and the Gram negative Escherichia coli (ATCC 4157) and Salmonella enterica subsp. arizonae (ATCC 13314). 4.2. Antibacterial bioassay Strains were grown in liquid nutrient broth (Difco Surrey, England) at 37 °C for 24 h before being used. A microplate-bioassay (microdilution) was used to study the antimicrobial activities of plant extract. An aliquot of each extract, corresponding to 100 µg plant dry matter, was dropped in sterile 96-well plates. After complete evaporation of the solvent, 100 µl of microorganism suspensions (102 cells.ml−1) obtained by dilution from the culture tube (108 cells.ml−1) were added to each well. Microbial suspension was used alone as positive control or in the presence of antibiotic mixture (5 mg.ml-1 streptomycin and 10 mg.ml−1 penicillin G) as negative control. Then, the microplate was aseptically sealed, agitated and incubated at 30 °C for 24 h. Finally, microorganism growth was estimated by reading the absorbance in each well at 405 nm with a microplate spectrophotometer (Multiskan MCC/340, Titertek). Antibacterial activity was expressed both in percentage of growth inhibition and in microbial susceptibility index (MSI, proportion of extracts inhibiting one microorganism). The absorbance data allowed us to calculate the percentage of growth inhibition using the following formula: Growth inhibition (%) = 100 − [100 * (Asample-ASC) / (AGC-ASC)] (3) where ASC and AGC are the absorbances of the sterility control (negative control) and of the growth control (positive control), respectively. Then, the microbial susceptibility index (MSI) was calculated using the following formula: MSI = 100 * (No. of extracts inhibiting one strain / Total No. of extracts) (4) Thus, MSI values for a particular strain range from '0' (resistant) to '100' (susceptible). 5. Evaluation of tyrosinase inhibition properties Tyrosinase inhibition activity was determined as described by Momtaz et al. (2008[34]), with L-3,4-dihydroxyphenylalanine (L-DOPA) or L-tyrosine as substrates. Samples were dissolved in dimethyl sulfoxide (DMSO), and further diluted in potassium phosphate buffer (50 mM, pH 6.5). Assays were carried out in a 96-well microtiter plate and absorbances were red on a Multiskan FC microplate reader (Thermo scientific technologies, China). Each prepared sample (70 μl) was mixed with 30 μl of tyrosinase (333 Units.ml-1 in phosphate buffer, pH 6.5). After 5 min of incubation at room temperature, 110 μl of substrate (2 mM L-tyrosine or 12 mM L-DOPA) were added and the reaction mixture was incubated further for 30 min. Kojic acid was used as a positive control, as well as a blank containing all the components except L-tyrosine or L-DOPA. Absorbance was measured at 492 nm, and the percentage of tyrosinase inhibition was calculated as follows: % inhibition = [(Acontrol - Asample) / Acontrol] * 100 (5) where Acontrol and Asample are the absorbances of the blank and of the test reaction mixture (containing extract or kojic acid), respectively. The IC50 values of extracts and kojic acid were calculated. 6. Aromatic compound determination The separation of aromatic compounds from plant extracts was done using HPLC Agilent 1260 system (Agilent technologies, Germany) equipped with a reversed phase C18 analytical column of 4.6 x 100 mm, and 3.5 μm particle size (Zorbax Eclipse XDB C18). Diode array detector was set to a scanning range of 200-400 nm and column temperature was maintained at 25 °C. The injected volumes were 5 μl and the flow-rate of mobile phase was 0.4 ml.min-1. Mobile phase was constituted of a mixture of two solvents, methanol (A) and 0.1 % formic acid (B), with the following gradient program: 10 % A 90 % B (0-5 min), 20 % A 80 % B (5-10 min), 30 % A 70 % B (10-15 min), 50 % A 50 % B (15-20 min), 70 % A 30 % B (20-25 min), 90 % A 10 % B (25-30 min), 50 % A 50 % B (30-35 min) and 10 % A 90 % B (35-36 min). Chromatograms were monitored at 254 nm and peak identification was obtained by comparing the retention time and the UV spectra of each peak with those of pure standards (purity > 99%) purchased from Sigma (Table 2(Tab. 2)). Moreover, individual aromatic contents were obtained from calibration curve with standards. Table 2 Table 2 Retention times (RT) of aromatic standards determined by RP-HPLC (see 'Materials and Methods' section for details) 7. Statistical analyses All extractions and assays were conducted in triplicate. The means were compared by using one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests performed by the ''Statistica v 5.1'' software (Statsoft, 2008). The differences between individual means were deemed to be significant at P < 0.05. Moreover, a correlation study was performed between all measured parameters in order to show possible relationships between activities and aromatic contents. Go to: Results 1. Antioxidant activities of halophyte shoot extracts The six studied halophytes differed significantly in their capacity to stabilize DPPH radical (Figure 1A(Fig. 1)). Of the six species, H. articulatum exhibited the highest antiradical activity, with the lowest IC50 value (71.5 µg.ml-1), followed by D. cordata. Conversely, P. scoparius and S. undulata showed a limited activity, as compared to the BHT standard. Figure 1 Figure 1 DPPH radical-scavenging (A), β-carotene bleaching inhibition (B) and superoxide anion radical-scavenging (C) activities of halophyte shoot extracts (50 % ethanol). All activities were expressed as IC50 (µg.ml-1). Means of three replicates ... The efficiency of the six halophytic extracts to inhibit the auto-oxidation of polyunsaturated fatty acids was evaluated using the β-carotene bleaching test. As shown in Figure 1B(Fig. 1), H. articulatum extract exhibited the highest inhibition (IC50 = 62.5 µg.ml−1), followed by C. arabica. The other four species showed only a moderate antioxidant activity using this bioassay, as compared to the standard BHT. Results from the superoxide-scavenging test showed that the six species quench superoxide anion to significantly different extents (Figure 1C(Fig. 1)). Here again, H. articulatum extract exhibited the highest antiradical potential (IC50 = 109 µg.ml−1), representing a 2.5 fold higher activity than D. cordata and S. undulata. The other three species were almost inactive compared to BHT. The six halophytes studied here differed significantly in their iron-reducing power (Figure 2(Fig. 2)). H. articulatum shoots exhibited the highest reducing capacity, with the lowest EC50 value (380 µg.ml-1). P. scoparius and D. cordata exhibited moderate activity, whereas the other three halophytes were rather ineffective compared to the BHT standard (130 µg.ml-1). Figure 2 Figure 2 Iron reducing power, expressed as EC50 (µg.ml-1), of halophyte shoot extracts (50 % ethanol). Means of three replicates followed by different letters are significantly different at P < 0.05. 2. Antimicrobial activity of halophyte shoot extracts As shown above for their antioxidant capacity, the antimicrobial activity of the studied halophytes was much contrasted. Haloxylon articulatum shoot extract was found to possess the most powerful antibacterial activity, as it inhibited efficiently the growth of all the tested strains (Table 3(Tab. 3)). Noteworthy, the growth of Salmonella enterica and Escherichia coli was completely stopped and that of Micrococcus luteus was inhibited by 80 %. Moreover, S. undulata, C. arabica, P. scoparius and C. colocynthis exhibited a strong antibacterial activity, as they inhibited efficiently (by more than 50 %) the growth of three strains, particularly S. aureus (for S. undulata and C. arabica), S. enterica (for P. scoparius) and E. coli (for C. colocynthis). Finally, D. cordata extract was the less active halophyte, with only light effects on E. coli, S. aureus and S. enterica growth. Overall, bacterial strains may be classified on the basis of their sensitivity to halophyte extracts: E. coli, S. aureus and S. enterica were the most susceptible bacteria while M. luteus exhibited the strongest resistance toward halophytic extracts. Table 3 Table 3 In vitro evaluation of antibacterial activity of halophyte extract against four pathogenic bacteria. Data are expressed as percent of growth inhibition induced by plant extract at the concentration of 1 mg.mL-1. Microbial Susceptibility Index (MSI) of ... 3. Tyrosinase inhibiting activities of halophyte shoot extracts Inhibition of the two activities of tyrosinase, i.e. monophenolase (=cresolase) and diphenolase (=catechol oxidase), by halophyte shoot extracts was assessed through dopachrome formation. The obtained results showed significant (P < 0.05) differences in the inhibitory concentrations among the tested extracts (Table 4(Tab. 4)). The most active halophytes against monophenolase were Cleome arabica and Pituranthos scoparius (IC50 = 125 µg.ml-1), whereas the other four species were rather ineffective (IC50 > 1000 µg.ml-1). Conversely, every plant extract inhibited diphenolase activity, with H. articulatum exhibiting the strongest effect. Table 4 Table 4 Anti-tyrosinase activities of halophyte shoot extracts (50 % ethanol). Activity against monophenolase and diphenolase were expressed as IC50 (µg.mL-1). In the same column, means ± SD of three replicates followed by different letters differ ... 4. Aromatic composition of halophyte shoot extracts Individual aromatic compounds were determined and assayed in ethanolic shoot extracts of the six halophytes. Nineteen compounds from different aromatic classes (amines, phenolic acids, flavonoids and diphenols) were identified and appeared to be diversely distributed in the studied plants (Table 5(Tab. 5)). Our results highlighted the richness of Haloxylon articulatum, and to a lesser extent C. arabica, in total aromatics. Thus, H. articulatum aromatic level (3.4 % DW) was 2.5 to 190 fold higher than that of the other species, D. cordata and S. undulata being particularly poor in aromatics. Table 5 Table 5 Aromatic compound distribution (mg.g-1DW) in shoot extracts of the studied halophytes, as analyzed by high-performance liquid chromatography (HPLC-DAD) Taking into consideration aromatic compound distribution, C. colocynthis and S. undulata displayed the highest diversity, with 6 molecules identified, while only four compounds were detected in H. articulatum shoots. Isorhamnetin-3-o-glucoside was found to be the major compound (1.6 mg.g-1 DW) in P. scoparius, luteolin7-o-glucoside (7.7 mg.g-1 DW) in C. arabica, and dopamine in H. articulatum and C. colocynthis (31.3 and 5 mg.g-1 DW, respectively). On a qualitative basis, whereas phenolic acids and flavonoids were detected in every species, H. articulatum and C. colocynthis shoot extract were characterized by the presence of the aromatic amine dopamine. Besides, the former and S. undulata could be distinguished by the presence of the diphenol catechol and poor levels of flavonoids. Go to: Discussion Since traditional ethnomedicinal use of plant is recognized as an important potential source for compounds used in mainstream medicine, six halophytic species widely used in African folk medicine have been selected for scientific investigations. Thus, antioxidant, antibacterial and anti-tyrosinase activities, as well as aromatic compound levels, were investigated in the selected species. Antioxidant activities of the studied species differed greatly. Whatever the bioassay used (DPPH, β-carotene, superoxide, FRAP), Haloxylon articulatum showed the best antioxidant capacity as compared to the other halophytes. These results are in agreement with a recent report showing that H. articulatum possesses a strong ability to neutralize free radicals, as compared to other species including Solenostemma oleifolium (Asclepiadaceae) and Echium pycnanthum (Boraginaceae) (Chaouche et al., 2014[9]). Moreover, the antioxidant potential displayed by H. articulatum is the highest reported hitherto among Chenopodiaceae species (Hupel et al., 2011[22]; Magné, unpublished results). It is well known that phenolic compounds are powerful antioxidants in plant tissues. Accordingly, of the six halophytes studied, H. articulatum could be distinguished by its high level in gallic acid and catechol, two well-known strong antioxidants (Miura et al., 1998[33]; Kim et al., 2007[25]), and the adrenergic catechol derivative dopamine. The latter was found for the first time as the major compound in that species, thus could contribute, at least in part, to the strong antioxidant capacity of the halophyte (Yen and Hsieh, 1997[55]; Miura et al., 1998[33]). Besides, it is interesting to note that antioxidant capacity of each species depended on the test used. Daemia cordata showed a strong capacity to neutralize DPPH radical, as compared to the others species. These results are consistent with a recent report on D. cordata plants from other regions (Yakubu et al., 2015[54]). On this point, the halophyte D. cordata showed comparable antioxidant activity to other Asclepiadaceae species, for example Solenostemma oleifolium (Chaouche et al., 2014[9]). Besides, Cleome Arabica showed the best capacity to inhibit linoleic acid oxidation, as compared to the other species. As far as we know, this is the first study reporting such activity in C. arabica. It confirms that this species possesses strong antioxidant activities (Djeridane et al., 2010[13]). Finally, Scorzonera undulata was the most powerful quencher of superoxide anion. Literature on antioxidant properties of this species is scarce. Nevertheless, earlier study showed that it has an interesting antiradical activity with DPPH test (Harkati et al., 2013[20]). Pituranthos scoparius, although exhibiting a lower antioxidant activity than that of H. articulatum, showed higher antioxidant levels as compared to other Apiaceae species (Namjooyan et al., 2010[36]). Similar observations could be made with the Capparaceae C. arabica and the Asclepiadaceae D. cordata. The antibacterial activities of shoot extracts were tested against four human pathogenic bacteria. The microplate bioassay results showed a significant variability in microbial growth inhibition depending on the plant species. As for the antioxidant activity, H. articulatum showed the best efficiency since it was the only halophyte inhibiting all tested bacterial strains. Earlier studies have supported that H. articulatum has an antibacterial activity against Staphylococcus aureus (Lamchouri et al., 2012[28]). With that respect, El-Shazly and Wink (2003[16]) showed that H. articulatum plants from Algeria are rich in antimicrobial molecules, including isoquinoline derivatives and β-carboline alkaloids. In our study, it could be that the strong antimicrobial activity of that species is the result of the abundance of phenolic compounds (Cueva et al., 2010[12]). In particular, such action might be due to dopamine since the aromatic amine and its derivatives have been reported to inhibit microbial growth (Pattan et al., 2009[39]; Maji et al., 2010[31]). Besides, the other halophyte extracts inhibited strongly one bacterial strain, and more weakly two others. Citrullus colocynthis showed a significant inhibition of E. coli, in good agreement with a previous study using agar diffusion method on several Gram-bacteria (Bnyan et al., 2013[5]). Shoot extract of Pituranthos scoparius exhibited a strong antibacterial activity against Salmonella enteritidis (Gram-), confirming a recent report on P. scoparius plants from Algeria, toward Salmonella typhimurium growth (Houria et al., 2014[21]). Scorzonera undulata showed a strong inhibitory effect on Staphylococcus aureus, confirming a recent study using agar diffusion method on several Staphylococcus and Pseudomomonas strains (Kargol et al., 2013[23]). Cleome arabica caused a significant inhibition of Gram+ bacteria, especially S. aureus. This result, obtained for the first time with the accurate microdilution method, does not confirm earlier works which reported no antimicrobial activity of C. arabica plants from Algeria (Takhi et al., 2011[50]). At last, shoot extract of D. cordata, studied here for the first time, showed only a weak inhibitory effect against the tested strains (and no effect at all against Micrococcus luteus). The antibacterial activity is commonly related to extract composition. Interestingly, the most active halophyte against the studied bacteria accumulated the highest level of aromatic compounds. Accordingly, phenolic composition of the halophytes studied here (in particular phenolic acids, flavanones, flavanols and flavonols) could account for their antibacterial activities (Cowan, 1999[11]; Barber et al., 2000[3]). It is well known that tyrosinase plays a critical role in catalyzing the melanogenic pathway since it promotes the production of reactive metabolites in the process of melanin formation (Sanchez-Ferrer et al., 1995[44]). The anti-tyrosinase effects were determined here for the first time in halophyte extracts, by assessing the hydroxylation of L-tyrosine to L-DOPA (monophenolase) and the oxidation of L-DOPA to DOPAquinone (diphenolase) according to the in vitro mushroom tyrosinase assay. Thus, Cleome arabica and Pituranthos scoparius extracts appeared as the sole active halophytes against monophenolase. Interestingly, the monophenolase inhibition capacity of these two species (IC50=125 µg.ml-1) was higher than that of many plants used for cosmetic purpose, particularly in skin whitening. These plants include Erigeron annuus, Albizzia julibrissin, Cornus macrophylla and Maackia floribunda (Kim et al., 2007[25]). Besides, H. articulatum exhibited a strong inhibition of diphenolase activity. That capacity is close to those previously reported in several Aloe species, which are commonly used in skin-lightening preparations (Mapunya et al., 2012[32]). Overall, the differing inhibitory capacity of monophenolase and diphenolase activities observed within the same plant is likely due to the different mechanisms of tyrosinase inhibition. For example, as a catalyzer of oxidative reactions, tyrosinase may be inhibited by reducing agents such as phenolic compounds, which show a good affinity for the enzyme, thus reducing its catalytic capacity (Chang, 2009[8]). Alternatively, specific tyrosinase inactivators, such as mechanism-based inhibitors, form covalent bond with the enzyme, thus irreversibly inactivating the enzyme during catalytic reaction. They inhibit tyrosinase activity by inducing “suicide reaction”. Interestingly, Choi et al. (2008[10]) and Lee et al. (2009[29]) reported a positive correlation between antioxidant activity and tyrosinase inhibition. In our study, although no significant relationship could be found between anti-monophenolase and antioxidant activities, a positive correlation appeared between diphenolase inhibition and some antioxidant activities tested (e.g. FRAP or β-carotene bleaching inhibition) (Table 6(Tab. 6)). Table 6 Table 6 Correlation coefficients between total aromatic (TA) level, antioxidant and anti-tyrosinase (monophenolase and diphenolase inhibition) activities Such correlation may be explained by the fact that tyrosinase increases the oxidative burst in different physiological systems and may, therefore, be counteracted by antioxidant molecules (Sanchez-Ferrer et al., 1995[44]; Wang et al., 2011[53]). In order to explain the anti-tyrosinase activities of the studied halophytes, we investigated aromatic composition of each halophyte, and found a high level of aromatics in H. articulatum and, to a lesser extent, in Cleome arabica. Thus, the most aromatic-rich halophytes are also the most powerful diphenolase inhibitory species, which was confirmed by a strong positive correlation between total aromatic (TA) level and diphenolase inhibition (Table 6(Tab. 6)). To our knowledge, such relationship has never been reported hitherto. Furthermore, H. articulatum and C. colocynthis appeared to accumulate the aromatic amine dopamine. Since these two halophytes inhibit strongly diphenolase activity, but not monophenolase one, it could be that dopamine, being the result of DOPA decarboxylation, retro-inhibits dopachrome formation. Further experiments are under progress to ascertain that hypothesis, by investigating the in vitro effect of pure dopamine on both tyrosinase activities. Apart from this possible skin lightening action, a literature survey indicates that dopamine could be used as a valuable adrenergic, antimyocontractant, cardiotonic, diuretic, or hypertensive molecule, as well as to treat neurodegenerative troubles such as Alzheimer or Parkinson diseases (Duke, 1992[15]). Also, dopamine has been shown to possess antioxidant properties (Yen and Hsieh, 1997[55]). Therefore, Haloxylon articulatum could prove to be a valuable source of new drug against oxidative stress-associated syndromes. Go to: Conclusion This study is the first to report simultaneously the antioxidant, antibacterial and anti-tyrosinase activities of six halophytes from Tunisian inland: Citrullus colocynthis, Cleome arabica, Daemia cordata, Haloxylon articulatum, Pituranthos scoparius and Scorzonera undulata. These species differ markedly in shoot antioxidant, antimicrobial, anti-tyrosinase activity, and their aromatic fingerprint was elucidated. Overall, our result highlights the strong potential of H. articulatum as a source of antioxidants, antimicrobials, as well as of whitening agents. With that respect, bioassay-guided fractionation of the ethanol extract of H. articulatum is under progress and will allow us to isolate the active compounds, making the extract or purified fractions promising sources of natural ingredients for cosmetic or food applications. Besides, the contribution of its principle compound dopamine to the biological activities described here will be elucidated. Go to: Acknowledgements This work was supported by Campus France in the framework of the PHC CMCU program (n° 15G0812). Go to: Conflict of interest The authors declare that they have no conflict of interest. Go to: References 1. Ahmad I, Malik MI, Iqbal K, Ahmed K, Naz S. Efficacy of formalinized liver-organ-vaccine against Angara disease in broilers. Veterinarski Arhiv. 1990;60:131–8. 2. Alkofahi A, Batshoun R, Owais W, Najib N. Biological activity of some Jordanian medicinal plant extracts. Fitoterapia. 1996;67:435–42. 3. Barber MS, McConnell VS, DeCaux BS. Antimicrobial intermediates of the general henylpropanoid and lignin specific pathways. Phytochemistry. 2000;54:53–6. [PubMed] 4. Benkrief R, Brum-Bousquet M, Tillequin F, Koch M. Alkaloids and flavonoid from aerial parts of Hammada articulata ssp. scoparia. Ann Pharmaceut Franç 1990;48:219–24. [PubMed] 5. Bnyan I, Hasan H, Ewadh M. Antibacterial activity of Citrullus colocynthis against different types of bacteria. Adv Life Sci Technol. 2013;7:48–51. 6. Boukef MK. Les plantes dans la médecine traditionnelle tunisienne. Paris: Agence de coopération culturelle et technique; 1986. 7. Bouriche H, Arnhold J. Effect of Cleome arabica leaf extract treated by naringinase on neutrophil degranulation. Acta Hortic. 2010;854:15–22. [PubMed] 8. Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci. 2009;10:2440–75. [PMC free article] [PubMed] 9. Chaouche TM, Haddouchi F, Ksouri R, Atik-Bekkara F. Evaluation of antioxidant activity of hydromethanolic extracts of some medicinal species from South Algeria. J Chin Med Assoc. 2014;77:302–7. [PubMed] 10. Choi HK, Lim YS, Kim YS, Park SY, Lee CH, Hwang KW, et al. Free-radical-scavenging and tyrosinase-inhibition activities of Cheonggukjang samples fermented for various times. Food Chem. 2008;106:564–8. 11. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12:564–82. [PMC free article] [PubMed] 12. Cueva C, Moreno-Arribas MV, Martín-Álvarez PJ, Bills G, Vicente MF, Basilio A, et al. Antimicrobial activity of phenolic acids against commensal, probiotic and pathogenic bacteria. Res Microbiol. 2010;161:372–382. [PubMed] 13. Djeridane A, Yousfi M, Brunel JM, Stocker P. Isolation and characterization of a new steroid derivative as a powerful antioxidant from Cleome arabica in screening the in vitro antioxidant capacity of 18 Algerian medicinal plants. Food Chem Toxicol. 2010;48:2599–606. [PubMed] 14. Duh PD, Tu TT, Yen GC. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat) LWT-Food Sci Technol. 1999;32:269–277. 15. Duke JA. Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL: CRC Press; 1992. 16. El-Shazly A, Wink M. Tetrahydroisoquinoline and β-carboline alkaloids from Haloxylon articulatum (Cav.) Bunge (Chenopodiaceae) Z Naturforsch. 2003;58c:477–480. [PubMed] 17. Falleh H, Msilini N, Oueslati S, Ksouri R, Magné C, Lachaâl M, et al. Diplotaxis harra and Diplotaxis simplex organs: Assessment of phenolics and biological activities before and after fractionation. Industr Crops Prod. 2013;45:141–7. 18. Gohar AA, El-Olemy MM, Abdel-Sattar E, El-Said M, Niwa M. Cardenolides and β-sitosterol glucoside from Pergularia tomentosa L. Nat Prod Sci. 2000;6:142–146. 19. Hammiche V, Maiza K. Traditional medicine in Central Sahara: pharmacopoeia of Tassili N’ajjer. J Ethnopharmacol. 2006;105:358–67. [PubMed] 20. Harkati B, Akkal S, Bayet C, Laouer H, Dijoux-Franca MG. Evaluation of antioxidant activity, free radical scavenging and CUPRAC of two compounds isolated from Scorzonera undulata ssp deliciosa. Adv Environ Biol. 2013;7:591–4. 21. Houria A, Esma F, Rabah D, Dounia M. In vitro antibacterial activity of Pituranthos scoparius from Algeria. Int J Biol Chem Sci. 2014;8:2095–2108. 22. Hupel M, Poupart N, Ar Gall E. Development of a new in vitro method to evaluate the photoprotective sunscreen activity of plant extracts against high UV-B radiation. Talanta. 2011;86:362–71. [PubMed] 23. Kargol HS, Elgadi HM, Gadamsi MT, Shubar HM, Geroushi AM. Pharmacognostical, antimicrobial and laxative study of Scorzonera undulata in Libya. Int Res J Pharm. 2013;4:96–99. 24. Kemassi A, Boual Z, Lebbouz I, Daddi Bouhoun M, Saker ML, Ould El Hadj-Khelil A, et al. Étude de l’activité biologique des extraits foliaires de Cleome arabica L. (Capparidaceae) Lebanese Sci J. 2012;13:81–97. 25. Kim SS, Hyun CG, Lee J, Lim J, Kim JY, Park D. In vitro screening of Jeju medicinal plants for cosmeceutical materials. J Appl Biol Chem. 2007;50:215–220. 26. Koleva II, van Beek TA, Linssen JP, de Groot A, Evstatieva LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem Anal. 2002;13:8–17. [PubMed] 27. Ksouri R, Megdiche W, Jallali I, Debez H, Magné C, Isoda H, et al. Medicinal halophytes: potent source of health promoting biomolecules with medical, nutraceutical and food applications. Crit Rev Biotechnol. 2002;32:289–326. [PubMed] 28. Lamchouri F, Benali T, Bennani B, Toufik H, Hassani LIM, Bouachrine B, et al. Preliminary phytochemical and antimicrobial investigations of extracts of Haloxylon scoparium. J Mater Environ Sc. 2012;3:754–9. 29. Lee KH, Abdel Aziz FH, Syahida A, Abas F, Shaari K, Israf DA, et al. Synthesis and biological evaluation of curcumin-like diarylpentanoid analogues for anti-inflammatory, antioxidant and anti-tyrosinase activities. Eur J Med Chem. 2009;44:3195–200. [PubMed] 30. Madari H, Jacobs RS. An analysis of cytotoxic botanical formulations used in the traditional medicine of ancient Persia as abortifacients. J Nat Prod. 2004;67:1204–10. [PubMed] 31. Maji S, Maji HS, Chakraborty P, Dastidar SG. Potential of dopamine hydrochloride as a novel antimicrobial agent. Int J Biomed Pharm Sci. 2010;4:70–75. 32. Mapunya MB, Nikolova RV, Lall N. Melanogenesis and antityrosinase activity of selected South African plants. Evid-Based Compl Alt Med. 2012:e374017. [PMC free article] [PubMed] 33. Miura T, Muraoka S, Ogiso T. Antioxidant activity of adrenergic agents derived from catechol. Biochem Pharmacol. 1998;55:2001–6. [PubMed] 34. Momtaz S, Mapunya BM, Houghton PJ, Edgerly C, Hussein A, Naidoo S, et al. Tyrosinase inhibition by extracts and constituents of Sideroxylon inerme L. stem bark, used in South Africa for skin lightening. J Ethnopharmacol. 2008;119:507–12. [PubMed] 35. Nagaya H, Tobita Y, Nagae T, Itokawa H, Takeya K, Halim AF, et al. Cytotoxic triterpenes from Cleome africana. Phytochemistry. 1997;44:1115–9. [PubMed] 36. Namjooyan F, Azemi ME, Rahmanian VR. Investigation of antioxidant activity and total phenolic content of various fractions of aerial parts of Pimpinella barbata (DC.) Boiss. Jundishapur J Nat Pharm Prod. 2010;5:1–5. 37. Oyaizu M. Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn J Nutr Diabetics. 1986;44:307–315. 38. Panich U, Kongtaphan K, Onkoksoong T, Jaemsak K, Phadungrakwittaya R, Thaworn A, et al. Modulation of antioxidant defense by Alpinia galanga and Curcuma aromatica extracts correlates with their inhibition of UVA-induced melanogenesis. Cell Biol Toxicol. 2010;26:103–16. [PubMed] 39. Pattan SR, Kekare P, Dighe NS, Nirmal SA, Musmade DS, Parjane SK, et al. Synthesis and biological evaluation of some 1,3,4-thiadiazoles. J Chem Pharm Res. 2009;1:191–198. 40. Qureshi R, Bhatti GR, Memon RA. Ethnomedicinal uses of herbs from northern part of Nara desert, Pakistan. Pak J Bot. 2010;42:839–51. 41. Rajamanickam E, Gurudeeban S, Ramanathan T, Satyavani K. Evaluation of anti-inflammatory activity of Citrullus colocynthis. Int J Curr Res. 2010;2:67–69. 42. Rangkadilok N, Sitthimonchai S, Worasuttayangkurn L, Mahidol C, Ruchirawat M, Satayavivad J. Evaluation of free radical scavenging and antityrosinase activities of standardized longan fruit extract. Food Chem Toxicol. 2007;45:328–36. [PubMed] 43. Rice-Evans C, Miller N, Paganga G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997;2:152–9. 44. Sanchez-Ferrer A, Rodriguez-Lopez JN, García-Cánovas F, García-Carmona F. Tyrosinase: A comprehensive review of its mechanism. Biochim Biophys Acta. 1995;1247:1–11. [PubMed] 45. Selloum L, Sebihi L, Mekhalfia A, Mahdadi R, Senator A. Antioxidant activity of Cleome arabica leaves extract. Biochem Soc Trans. 1997;25:S608. [PubMed] 46. Shaker AN, Laouer H, Dahia M. Usages de plantes médicinales dans la région de Bou-Saada (Algérie) Rev Méd Pharmacol Afr. 1999;13:81–89. 47. Shen JL, Man KM, Huang PH, Chen WC, Chen DC, Cheng YW, et al. Honokiol and magnolol as multifunctional antioxidative molecules for dermatologic disorders. Molecules. 2010;15:6452–65. [PubMed] 48. Sokmen A, Gulluce M, Askin Akpulat H, Daferera D, Tepe B, Polissiou M, et al. The in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic Thymus spathulifolius. Food Control. 2004;15:627–34. [PubMed] 49. Suresh Kumar SV, Mishra SH. In vitro evaluation of hepatoprotective activity of Pergularia daemia. Pharmacogn Mag. 2008;3:187–191. 50. Takhi D, Ouinten M, Yousfi M. Study of antimicrobial activity of secondary metabolites extracted from spontaneous plants from the area of Laghouat, Algeria. Adv Environ Biol. 2011;5:469–76. 51. Tsevegsuren N, Edrada R, Lin W, Ebel R, Torre C, Ortlepp S, et al. Biologically active natural products from Mongolian medicinal plants Scorzonera divaricata and Scorzonera pseudodivaricata. J Nat Prod. 2007;70:962–7. [PubMed] 52. Tsichritzis F, Abdel-Mogib M, Jakupovic J. Dammarane triterpenes from Cleome africana. Phytochemistry. 1993;33:423–5. 53. Wang BS, Chang LW, Wu HC, Huang SL, Chu HL, Huang MH. Antioxidant and antityrosinase activity of aqueous extracts of green asparagus. Food Chem. 2011;127:141–6. 54. Yakubu R, Jibril FM, Lukman A, Sheikh F. Trends for antioxidant power of phytochemicals from Pergularia tomentosa L. (Asclepiadaceae) whole plant. Sch Acad J Pharm. 2015;4:74–80. 55. Yen GC, Hsieh CL. Antioxidant effects of dopamine and related compounds. Biosci Biotechnol Biochem. 1997;61:1646–9. [PubMed] 56. Zidorn C, Ellmerer EP, Sturm S, Stuppner H. Tyrolobibenzyls E and F from Scorzonera humilis and distribution of caffeic acid derivatives, lignans and tyrolobibenzyls in European taxa of the subtribe Scorzonerinae (Lactuceae, Asteraceae) Phytochemistry. 2003;63:61–7. [PubMed] 57. Ziyyat A, Legssyer A, Mekhfi H, Dassouli A, Serhrouchni M, Benjelloun W. Phytotherapy of hypertension and diabetes in oriental Morocco. J Ethnopharmacol. 1997;58:45–54. [PubMed] Articles from EXCLI Journal are provided here courtesy of Leibniz Research Centre for Working Environment and Human Factors