Testing the predictability of morphological evolution in contrasting thermal environments.

Gaining the ability to predict population responses to climate change is a pressing concern. Using a "natural experiment," we show that testing for divergent evolution in wild populations from contrasting thermal environments provides a powerful approach, and likely an enhanced predictive power for responses to climate change. Specifically, we used a unique study system in Iceland, where freshwater populations of threespine sticklebacks (Gasterosteus aculeatus) are found in waters warmed by geothermal activity, adjacent to populations in ambient-temperature water. We focused on morphological traits across six pairs from warm and cold habitats. We found that fish from warm habitats tended to have a deeper mid-body, a subterminally orientated jaw, steeper craniofacial profile, and deeper caudal region relative to fish from cold habitats. Our common garden experiment showed that most of these differences were heritable. Population age did not appear to influence the magnitude or type of thermal divergence, but similar types of divergence between thermal habitats were more prevalent across allopatric than sympatric population pairs. These findings suggest that morphological divergence in response to thermal habitat, despite being relatively complex and multivariate, are predictable to a degree. Our data also suggest that the potential for migration of individuals between different thermal habitats may enhance nonparallel evolution and reduce our ability to predict responses to climate change.

A warmer environment can reduce sociability in an ectotherm.

The costs and benefits of being social vary with environmental conditions, so individuals must weigh the balance between these trade-offs in response to changes in the environment. Temperature is a salient environmental factor that may play a key role in altering the costs and benefits of sociality through its effects on food availability, predator abundance, and other ecological parameters. In ectotherms, changes in temperature also have direct effects on physiological traits linked to social behaviour, such as metabolic rate and locomotor performance. In light of climate change, it is therefore important to understand the potential effects of temperature on sociality. Here, we took the advantage of a 'natural experiment' of threespine sticklebacks from contrasting thermal environments in Iceland: geothermally warmed water bodies (warm habitats) and adjacent ambient-temperature water bodies (cold habitats) that were either linked (sympatric) or physically distinct (allopatric). We first measured the sociability of wild-caught adult fish from warm and cold habitats after acclimation to a low and a high temperature. At both acclimation temperatures, fish from the allopatric warm habitat were less social than those from the allopatric cold habitat, whereas fish from sympatric warm and cold habitats showed no differences in sociability. To determine whether differences in sociability between thermal habitats in the allopatric population were heritable, we used a common garden breeding design where individuals from the warm and the cold habitat were reared at a low or high temperature for two generations. We found that sociability was indeed heritable but also influenced by rearing temperature, suggesting that thermal conditions during early life can play an important role in influencing social behaviour in adulthood. By providing the first evidence for a causal effect of rearing temperature on social behaviour, our study provides novel insights into how a warming world may influence sociality in animal populations.

Deploying an In Vitro Gut Model to Assay the Impact of the Mannan-Oligosaccharide Prebiotic Bio-Mos on the Atlantic Salmon (Salmo salar) Gut Microbiome.

Alpha mannose-oligosaccharide (MOS) prebiotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and their associated metabolism. Bio-Mos is a commercially available prebiotic currently used in the agri-feed industry, but studies show contrasting results of its effect on fish performance and feed efficiency. Thus, detailed studies are needed to investigate the effect of MOS supplements on the fish microbiome to enhance our understanding of the link between MOS and gut health. To assess Bio-Mos for potential use as a prebiotic growth promoter in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. The microbial communities obtained from ceca compartments from four adult farmed salmon were inoculated in biological triplicate reactors in SalmoSim. Prebiotic treatment was supplemented for 20 days, followed by a 6-day washout period. Inclusion of Bio-Mos in the media resulted in a significant increase in formate (P = 0.001), propionate (P = 0.037) and 3-methyl butanoic acid (P = 0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to Bio-Mos supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to complementin vivo trials of microbiome modulators. IMPORTANCE In this paper we report the results of the impact of a prebiotic (alpha-MOS supplementation) on microbial communities, using an in vitro simulator of the gut microbial environment of the Atlantic salmon. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production by the microbiota from salmon pyloric ceca and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

Genomic analysis of the carboxylesterase family in the salmon louse (Lepeophtheirus salmonis).

The pyrethroid deltamethrin and the macrocyclic lactone emamectin benzoate (EMB) are used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis. While the efficacy of both compounds against Atlantic populations of the parasite has decreased as a result of the evolution of resistance, the molecular mechanisms of drug resistance in L. salmonis are currently not fully understood. The functionally diverse carboxylesterases (CaE) family includes members involved in pesticide resistance phenotypes of terrestrial arthropods. The present study had the objective to characterize the CaE family in L. salmonis and assess its role in drug resistance. L. salmonis CaE homologues were identified by homology searches in the parasite's transcriptome and genome. The transcript expression of CaEs predicted to be catalytically competent was studied using quantitative reverse-transcription PCR in drug susceptible and multi-resistant L. salmonis. The above strategy led to the identification of 21 CaEs genes/pseudogenes. Phylogenetic analyses assigned 13 CaEs to clades involved in neurodevelopmental signaling and cell adhesion, while three sequences were predicted to encode secreted enzymes. Ten CaEs were identified as being potentially catalytically competent. Transcript expression of acetylcholinesterase (ace1b) was significantly increased in multi-resistant lice compared to drug-susceptible L. salmonis, with transcript abundance further increased in preadult-II females following EMB exposure. In summary, results from the present study demonstrate that L. salmonis possesses fewer CaE gene family members than most arthropods characterized so far. Drug resistance in L. salmonis was associated with overexpression of ace1b.

BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression.

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

Genome-wide survey of cytochrome P450 genes in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837).

BACKGROUND: The salmon louse (Lepeophtheirus salmonis) infests farmed and wild salmonid fishes, causing considerable economic damage to the salmon farming industry. Infestations of farmed salmon are controlled using a combination of non-medicinal approaches and veterinary drug treatments. While L. salmonis has developed resistance to most available salmon delousing agents, relatively little is known about the molecular mechanisms involved. Members of the cytochrome P450 (CYP) superfamily are typically monooxygenases, some of which are involved in the biosynthesis and metabolism of endogenous compounds, while others have central roles in the detoxification of xenobiotics. In terrestrial arthropods, insecticide resistance can be based on the enhanced expression of CYPs. The reported research aimed to characterise the CYP superfamily in L. salmonis and assess its potential roles in drug resistance. METHODS: Lepeophtheirus salmonis CYPs were identified by homology searches of the genome and transcriptome of the parasite. CYP transcript abundance in drug susceptible and multi-resistant L. salmonis was assessed by quantitative reverse transcription PCR, taking into account both constitutive expression and expression in parasites exposed to sublethal levels of salmon delousing agents, ecdysteroids and environmental chemicals. RESULTS: The above strategy led to the identification of 25 CYP genes/pseudogenes in L. salmonis, making its CYP superfamily the most compact characterised for any arthropod to date. Lepeophtheirus salmonis possesses homologues of a number of arthropod CYP genes with roles in ecdysteroid metabolism, such as the fruit fly genes disembodied, shadow, shade, spook and Cyp18a1. CYP transcript expression did not differ between one drug susceptible and one multi-resistant strain of L. salmonis. Exposure of L. salmonis to emamectin benzoate or deltamethrin caused the transcriptional upregulation of certain CYPs. In contrast, neither ecdysteroid nor benzo[a]pyrene exposure affected CYP transcription significantly. CONCLUSIONS: The parasite L. salmonis is demonstrated to possess the most compact CYP superfamily characterised for any arthropod to date. The complement of CYP genes in L. salmonis includes conserved CYP genes involved in ecdysteroid biosynthesis and metabolism, as well as drug-inducible CYP genes. The present study does not provide evidence for a role of CYP genes in the decreased susceptibility of the multiresistant parasite strain studied.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis).

BACKGROUND: Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS: Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS: The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis. © 2018 Society of Chemical Industry.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma).

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.

Maternal inheritance of deltamethrin resistance in the salmon louse Lepeophtheirus salmonis (Krøyer) is associated with unique mtDNA haplotypes.

Parasitic infections by the salmon louse, Lepeophtheirus salmonis (Krøyer), cause huge economic damage in salmon farming in the northern hemisphere, with combined treatment costs and production losses in 2014 having been estimated at US$ 350 million for Norway (annual production 1.25 million tonnes). The control of L. salmonis relies significantly on medicinal treatments, supplemented by non-pharmacological approaches. However, efficacy losses have been reported for several delousing agents, including the pyrethroid deltamethrin. The aim of the present study was to analyse the genetic basis of deltamethrin resistance in L. salmonis. Deltamethrin median effective concentrations (EC50) were 0.28 µg L-1 in the drug susceptible L. salmonis strain IoA-00 and 40.1 µg L-1 in the pyrethroid resistant strain IoA-02. IoA-00 and IoA-02 were crossed to produce families spanning one parental and three filial generations (P0, F1-F3). In three families derived from P0 crosses between an IoA-00 sire and an IoA-02 dam, 98.8% of F2 parasites (n = 173) were resistant, i.e. remained unaffected after exposure to 2.0 µg L-1 deltamethrin. F3 parasites from these crosses showed a deltamethrin EC50 of 9.66 µg L-1. In two families of the inverse orientation at P0 (IoA-02 sire x IoA-00 dam), 16.7% of F2 parasites were resistant (n = 84), while the deltamethrin EC50 in F3 animals was 0.26 µg L-1. The results revealed a predominantly maternal inheritance of deltamethrin resistance. The 15,947-nt mitochondrial genome was sequenced and compared among six unrelated L. salmonis strains and parasites sampled from wild salmon in 2010. IoA-02 and three further deltamethrin resistant strains, established from isolates originating from different regions of Scotland, showed almost identical mitochondrial haplotypes. In contrast, the mitochondrial genome was variable among susceptible strains and L. salmonis from wild hosts. Deltamethrin caused toxicity and depletion of whole body ATP levels in IoA-00 but not IoA-02 parasites. The maternal inheritance of deltamethrin resistance and its association with mitochondrial haplotypes suggests that pyrethroid toxicity in L. salmonis may involve molecular targets encoded by mitochondrial genes.

Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice.

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere. While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains. STATEMENT OF RELEVANCE: Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance.

Effects of 17α-ethinyl estradiol exposure on estrogen receptors α and ß and vitellogenins A, B and C mRNA expression in the liver of sand goby (Pomatoschistus minutus).

This study aims to characterize the estrogen receptor (er) in sand goby (Pomatoschistus minutus) and determine the temporal effects of 17α-ethinyl estradiol (EE2) on erα and vitellogenin (vtg) gene expression in males. Two partial cDNA sequences (erα and erß1) are presented showing conserved structural features with ers of other species. Transcript levels for both ers were low in control fish but EE2 exposure (11 ng/L, for 29 days) increased both to a pattern similar to vitellogenic females. The relative expression of three vtg genes (vtga, vtgb and vtgc) along with erα was determined in control and male fish exposed to EE2 (11 ng/L) at multiple time-points over 29 days. All four transcripts were significantly induced due to exposure and expression rose during the time course with distinct temporal patterns and vtga reached a substantially higher level at the end of the time course coinciding with rapid elevation in erα expression.

Characterisation of genes transcriptionally upregulated in the liver of sand goby (Pomatoschistus minutus) by 17α-ethinyloestradiol: identification of distinct vitellogenin and zona radiata protein transcripts.

The sand goby (Pomatoschistus minutus), is a marine and estuarine teleost that is used in environmental, reproductive and behavioural studies of oestrogenic endocrine disruption. The xeno-oestrogen, 17α-ethinyloestradiol (EE2), induces expression of egg proteins vitellogenin (VTG) and zona radiata protein (ZRP) in male fish and impairs reproduction. Multiple forms of VTG and ZRP genes are found in other teleost species, yet the characteristics of VTG and ZRP in the sand goby are unknown. In this investigation, Suppressive Subtractive Hybridization was used to isolate cDNA fragments from liver, identified as belonging to 11 distinct sand goby genes, suggesting that these genes are transcriptionally upregulated by EE2. Assembly of these fragments revealed three VTG genes which shared homology with VTG classes A, B and C in other fish and two ZRP genes sharing homology with ZRP classes Ba and Bb. RTqPCR of RNA from the sand goby liver was used to show that these VTGs and ZRPs were present in low levels in control males and high levels in mature females. Exposure of males to a concentration of 11 ngL(-1) EE2 caused a significant increase in all VTG and ZRP transcript levels. The identification of these egg protein transcripts and the development of validated assays for their quantification will facilitate future work with this useful model species.