The complete mitochondrial genome of Arctic Calanus hyperboreus (Copepoda,Calanoida) reveals characteristic patterns in calanoid mitochondrial genome

Copepoda is the most diverse and abundant group of crustaceans, but its phylogenetic relationships are ambiguous. Mitochondrial (mt) genomes are useful for studying evolutionary history, but only six complete Copepoda mt genomes have been made available and these have extremely rearranged genome structures. This study determined the mt genome of Calanus hyperboreus, making it the first reported Arctic copepod mt genome and the first complete mt genome of a calanoid copepod. The mt genome of C. hyperboreus is 17,910 bp in length and it contains the entire set of 37 mt genes, including 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. It has a very unusual gene structure, including the longest control region reported for a crustacean, a large tRNA gene cluster, and reversed GC skews in 11 out of 13 protein-coding genes (84.6%). Despite the unusual features, comparing this genome to published copepod genomes revealed retained pan-crustacean features, as well as a conserved calanoid-specific pattern. Our data provide a foundation for exploring the calanoid pattern and the mechanisms of mt gene rearrangement in the evolutionary history of the copepod mt genome.     

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse,Lepeophtheirus salmonis

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Geographic and temporal genetic structure in <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis> from four salmon farms along the northwest and west coasts of Ireland: results from a microsatellite analysis

The marine ectoparasitic copepod of salmonids, Lepeophtheirus salmonis (Krøyer), is a major pest of farmed Atlantic salmon (Salmo salar L.) causing great economic impact. The spatial scales over which L. salmonis populations in different salmon farms are typically connected, and the temporal scales over which L. salmonis from the same farm typically undergo genetic change are largely unknown. These questions were posed in a small-scale geographic study of population structure in L. salmonis from four salmon farms, along the northwest and west coasts of Ireland, using two outgroups from Norway and Canada. The temporal stability of genetic composition was also studied in samples collected quarterly during one year from one salmon farm in Ireland. Genetic composition in L. salmonis was characterised using four nuclear microsatellites. Significant but low genetic differentiation was observed between all sites (F _ST = 0.08), with no evidence that differentiation was correlated with geographic distance. Temporal genetic differentiation was also evident (F _ST = 0.07). An analysis of all L. salmonis samples except the ones from Norway detected two separate clusters. Each cluster contained both geographical and temporal samples. These results are consistent with a population model in which L. salmonis in salmon farms along the northwest and west coasts of Ireland are not isolated, but are potentially subject to (i) localised ecological factors at the particular farm sites or (ii) selection post-settlement or a combination thereof.     

Infestations of wild adult Atlantic salmon (Salmo salar L.) by the ectoparasitic copepod sea louse Lepeophtheirus salmonis Krøyer: prevalence,intensity and the spatial distribution of males and [2pt] females on the host fish

The copepod Lepeophtheirus salmonis Krøyer is a specific ectoparasite of North Atlantic and Pacific salmonids in their marine phases. We compared infestations of L. salmonis on wild Atlantic salmon (Salmo salarL.) captured in estuarine (Firth of Tay, east Scotland; 1995, 1996) and marine coastal waters (Strathy Point, north Scotland; 1998, 1999). Host fish from the Tay were caught by sweep netting, whilst those from Strathy Point were trapped in anchored bagnets. Fish capture method and exposure of the parasites to brackish conditions may both have detrimental effects on the retention of L. salmonis by the host, and hence possibly lead to their being under-estimated on returning adult fish. At Strathy Point, we recorded (i) an infestation prevalence of 100%, (ii) mean log abundances of pre-adult + adult L. salmonis at 19 (1998) and 24 (1999) per fish, (iii) 85/93% of all L. salmonis as being adults and (iv) overall 68/69% females. Fish caught in the upper Firth of Tay showed significantly lower prevalences, intensities and abundances of L. salmonis and probably had lost part or all of their lice burdens prior to capture, whereas those sampled from Strathy Point were apparently minimally affected by capture method or brackish water influences. The loss of parasites for the Tay fish was not markedly biased to males or females, or to pre-adult versus adult developmental stages. There were significantly greater abundances of L. salmonis on two sea-winter fish (30 lice per fish) than on one sea-winter fish (17 lice per fish) sampled at Strathy Point in 1998. There are several possible explanations for such age-related patterns of abundance, but the indications are that (i) initial infestation of smolts occurs in coastal waters, (ii) infestation of hosts in the open ocean is a persistent event, and (iii) oceanic reinfestation outweighs mortality losses of L. salmonis. This parasite typically occupies rather few zones on the host fish covering only a small percentage of the total available body surface area. Female predominance appears to be characteristic of L. salmonisinfestations of wild Atlantic salmon; this is in marked contrast to reports of extreme male dominance on farmed stocks. Adult females predominated on the epidermis adjacent to, and posterior of, the insertion of the anal fin and along the posterior dorsal midline between the dorsal and caudal fins. Males predominated on the sides of the head and along the dorsal midline between the head and the dorsal fin. Mate guarding/precopulatory pairs are formed between pre-adult II females and adult males. The significant correlation between the distribution of pre-adult females and adult males may be indicative of pre-adult females actively seeking out adult males, but more likely is due to the (large) adult females competitively ousting all smaller life stages (female and male) from those preferred zones. Given the relatively low fecundity of adult females, and observations of 100% prevalence of L. salmonis, the infective planktonic copepodid stage evidently is extremely efficient at locating and establishing upon its host fish in the pelagic environment.     

Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains

The salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) is an ectoparasitic copepod which represents a major pathogen of wild and farmed salmonid fishes in the marine environment. In order to facilitate research on this ecologically and economically important parasite, a hatchery and culturing system permitting the closure of the life-cycle of L. salmonis in the laboratory was developed. Here, the hatchery system, breeding practices, and selected louse strains that have been maintained in culture in the period 2002–2009 are presented. The hatchery and culture protocol gave rise to predictable hatching of larvae and infections of host fish, permitting the cultivation of specific strains of L. salmonis for 22 generations. Both in- and out-bred lice and mutant colour strains have been established, and some of these strains were characterised by microsatellite DNA markers confirming their pedigree. No evidence of inbreeding depression, fitness or morphological changes was observed in any of the strains cultured. It is suggested that the culturing system, and the strains produced represent a significant resource for future research on this parasite.     

Systematic identification and characterization of stress-inducible heat shock proteins (HSPs) in the salmon louse (<Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>)

Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis.     

Genomic Resources for Sea Lice: Analysis of ESTs and Mitochondrial Genomes

Sea lice are common parasites of both farmed and wild salmon. Salmon farming constitutes an important economic market in North America, South America, and Northern Europe. Infections with sea lice can result in significant production losses. A compilation of genomic information on different genera of sea lice is an important resource for understanding their biology as well as for the study of population genetics and control strategies. We report on over 150,000 expressed sequence tags (ESTs) from five different species (Pacific Lepeophtheirus salmonis (49,672 new ESTs in addition to 14,994 previously reported ESTs), Atlantic L. salmonis (57,349 ESTs), Caligus clemensi (14,821 ESTs), Caligus rogercresseyi (32,135 ESTs), and Lernaeocera branchialis (16,441 ESTs)). For each species, ESTs were assembled into complete or partial genes and annotated by comparisons to known proteins in public databases. In addition, whole mitochondrial (mt) genome sequences of C. clemensi (13,440 bp) and C. rogercresseyi (13,468 bp) were determined and compared to L. salmonis. Both nuclear and mtDNA genes show very high levels of sequence divergence between these ectoparastic copepods suggesting that the different species of sea lice have been in existence for 37–113 million years and that parasitic association with salmonids is also quite ancient. Our ESTs and mtDNA data provide a novel resource for the study of sea louse biology, population genetics, and control strategies. This genomic information provides the material basis for the development of a 38K sea louse microarray that can be used in conjunction with our existing 44K salmon microarray to study host–parasite interactions at the molecular level. This report represents the largest genomic resource for any copepod species to date.     

In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

Akirins in sea lice: First steps towards a deeper understanding

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.     

Phylogeny,Evidence for a Cryptic Plastid,and Distribution of Chytriodinium Parasites (Dinophyceae) Infecting Copepods

Spores of the dinoflagellate Chytriodinium are known to infest copepod eggs causing their lethality. Despite the potential to control the population of such an ecologically important host, knowledge about Chytriodinium parasites is limited: we know little about phylogeny, parasitism, abundance, or geographical distribution. We carried out genome sequence surveys on four manually isolated sporocytes from the same sporangium, which seemed to be attached to a copepod nauplius, to analyze the phylogenetic position of Chytriodinium based on SSU and concatenated SSU/LSU rRNA gene sequences, and also characterize two genes related to the plastidial heme pathway, hemL and hemY. The results suggest the presence of a cryptic plastid in Chytriodinium and a photosynthetic ancestral state of the parasitic Chytriodinium/Dissodinium clade. Finally, by mapping Tara Oceans V9 SSU amplicon data to the recovered SSU rRNA gene sequences from the sporocytes, we show that globally, Chytriodinium parasites are most abundant within the pico/nano‐ and mesoplankton of the surface ocean and almost absent within microplankton, a distribution indicating that they generally exist either as free‐living spores or host‐associated sporangia.     

Comparative Analysis of the Complete Genome Sequence of the California MSW Strain of Myxoma Virus Reveals Potential Host Adaptations

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Annual changes in the population size of the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae) on high-seas Pacific Salmon (Oncorhynchus spp.), and relationship to host abundance

The population size of the salmon louse, Lepeophtheirus salmonis, was monitored annually in the summers of 1991–1997 by examining six species of Pacific salmon (Oncorhynchus spp.) caught by surface long-lines in oceanic offshore waters of the North Pacific Ocean and Bering Sea. The annual copepod population size on all salmonids caught was estimated by combining the calculated number of copepods carrying on each salmonid species. The copepod population fluctuated markedly from year to year, which resulted largely from marked annual changes in abundance of pink salmon (O. gorbuscha). Since pink salmon were most frequently and heavily infected and since their abundance changed every year, the copepod population was high in the years when this salmonid species was abundant, but low when it was rare. On the contrary, chum salmon (O. keta) did not show high prevalence and intensity of infection, but the annual abundance of this host species was consistently high, i.e. chum salmon carried many copepods every year. Copepods on other salmonid species (sockeye salmon O. nerka, coho salmon O. kisutch, chinook salmon O. tshawytscha, and steelhead trout O. mykiss) constantly formed a small percentage of the total copepod population. Both chum and pink salmon are the most important hosts in terms of their substantial contribution to support the copepod population, but the importance as hosts of each species is definitely different between the species. Chum salmon is a stable important host supporting the copepod population at a relatively high level every year, while the number of copepods on pink salmon annually exhibits marked fluctuations, and this salmonid species is regarded as an unstable important host.     

Staphylococcus lugdunensis IsdG Liberates Iron from Host Heme

Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization.