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RAG-1 and IgM genes, markers for early development of the immune system in the gadoid haddock, Melanogrammus aeglefinus, L.

The full sequence of the heavy chain of Immunoglobulin M (IgM) and a partial fragment of the recombination activating gene-1 (RAG-1) gene were sequenced in haddock, Melanogrammus aeglefinus, L. The complete transcript of haddock IgM consisted of 1865 bp and translated into a 572-aa peptide. The RAG-1 fragment was 1776 bp and was identified as the core region of RAG-1. These two immune genes were used in expression studies as markers of early development in haddock larvae. A DIG labelled oligoprobe of the RAG-1 gene was used in whole-mount in situ hybridisation (WISH). A hybridisation signal for RAG-1 was first detected in larvae at 25 days post-hatching (dph) in two bilateral symmetric regions of the head identified as the thymus. Further expression studies were carried out by RT-PCR analysis of RAG-1 and IgM on larval samples obtained during early development, i.e. from fertilisation to weaning. Haddock RAG-1 expression was detected after 21 days post-fertilisation (dpf) whilst IgM transcripts were not detected until 40 dpf, equivalent to day 29 post-hatching. These results suggest that the immune system in haddock starts to develop in larvae of 6-7 mm in length (25-29 dph).

Amino Acid Sequence↗

PCB-containing paint and plaster caused extreme PCB-concentrations in biota from the Sørfjord (Western Norway)--a case study.

The Sørfjord (Western Norway) has a long history of industry and pollution, and has been monitored for several decades. The environmental monitoring has comprised analyses of different contaminants in sea water, mussel, fish, seaweed and sediments. Measurements of polychlorinated biphenyls (PCBs) in blue mussels (Mytilus edulis) some 17 years ago indicated a local source. In 2001 severe concentrations were measured in blue mussels and further investigations disclosed the primary source of PCBs in the Sørfjord. In 2002, extreme PCB-concentrations were found in cod (Gadus morhua) from the same area. However, no induction of EROD activity was detected, indicating the limitations of this assay as marker for PCB contamination. A future scientific challenge will be to understand the specific mechanisms and effects of such PCB accumulation in fish.

Animals↗

Esterase activities and lipid peroxidation levels in offshore commercial species of the NW Mediterranean Sea.

There is a lack of information on monitoring neurotoxicity in offshore commercial species. To help fill this gap, we sampled hake (Merluccius merluccius) and Norway lobster (Nephrops norvegicus) in fishing grounds off the coast of l'Ametlla de Mar (NW Mediterranean) in June 2005 at a depth of 100 m and 400 m. Additionally, at 400 m depth, two other fish species, Micromesistius poutassou and Phycis blennoides were included. Neurotoxicity markers such as Colinesterases (ChEs), namely acethyl- (AChE), butyryl- (BChE), propionyl- (PrChE) and carboxilesterase (CbE) were measured in muscle. Lipid peroxidation (LP), a marker of oxidative damage, was also included. The results are discussed in relation to the animal's sex, size and fishing depth. A comparison of esterases and LP levels between muscle and liver of hake and between muscle and hepatopancreas of Norway Lobster was made. AChE was dominant in muscle and CbE in hepatopancreas. No differences between fish species were seen for AChE. However, N. norvegicus, presented lower levels of ChEs and LP. A size-dependence in ChEs was seen for M. merluccius, with larger animals showing significantly lower activities (p<0.05). Sex-dependence was seen in N. norvegicus for most esterases, except AChE, with males displaying higher activities (p<0.05). A sampling-depth effect was also seen in the crustacea, with animals from 100 m generally presenting lower esterase activities and higher LP levels.

Animals↗

A model for the interaction between gadoid larvae and their nauplii prey.

A continuous model for the growth and death of gadoid larvae, including ecological interactions with their nauplii prey, is examined. The present model has a simpler structure than the model due to Cushing and Horwood [D.H. Cushing, J.W. Horwood, The growth and death of fish larvae. J. Plankt. Res. 16 (3) (1994) 291-300] as it does not explicitly incorporate larval metabolism, although indirectly metabolism is included by means of equations for larval growth. Despite this, the model yields related, although not entirely equivalent, results to those obtained by Cushing and Horwood. In the present model, overcompensation (cf. [W.E. Ricker, Stock and Recruitment, J. Fish. Res. Board. Can. 11 (1954) 559-623]) occurs at limited initial food levels, while at infinite food levels, the recruitment curve becomes monotonically increasing towards an upper limit (cf. [R.J.H. Beverton, S.J. Holt, On the dynamics of exploited fish populations. Fish. Invest. Lond. I 19 (1957)]). Moreover, the present study suggests that the duration of the larval stage, the metamorphosis time tau is highly important to the recruitment process, in accordance with Cushing and Horwood. When food is limited the metamorphosis is delayed, causing the larval population to experience (density dependent) mortality for a sufficient long time to make the recruitment curve overcompensatory. It is not necessarily the desire to derive a particular formula for the recruitment curve, as this is probably impossible anyway, except for particular examples. However, reduced versions of the model that in some sense are close to the original model, are examined, and it is argued that many general features of the general model are retained in such examples.

Algorithms↗

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction.

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.

Animals↗

Characterization of parvalbumin, the major allergen in Alaska pollack, and comparison with codfish Allergen M.

Increased fish consumption has led to frequent reporting of fish allergy and adverse reactions. Alaska pollack (Theragra chalcogramma) is a globally important commercial fish species, belonging to the Gadidae family. This family of fish also includes cod whose parvalbumin, Allergen M (Gad c 1), has been thoroughly studied and considered as a reference to sensitization in fish allergy. In the present study, parvalbumin from Alaska pollack, designated The c 1, was purified by use of anion exchange chromatography. To demonstrate the homogeneity of the purified protein, reverse phase high performance liquid chromatography was performed and showed two distinct fractions which had similar IgG and IgE binding capacities. Accordingly, cDNA cloning revealed two isotypic parvalbumin transcripts in pollack muscle. Recombinant parvalbumins of pollack exhibited low IgG and IgE binding capacities, in contrast to the native counterparts, which were almost as potent as cod Gad c 1. The allergenicity of The c 1 was assayed by ELISA inhibition, and compared to cod, the concentration required for obtaining 50% ELISA inhibition (C 50%) was only 18% higher for The c 1.

Allergens↗

Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus).

This paper reports the cloning and sequencing of two pro-inflammatory cytokines, interleukin (IL)-1beta and IL-8, in haddock (Melanogrammus aeglefinus) by homology cloning. The complete transcript of the haddock IL-1beta was sequenced and contained 1043 bp, including a 762 bp open reading frame. The 3' end of the gene includes a polyadenylation signal 13 bp upstream of the poly(A) tail, along with 10 instability motifs. The predicted protein of 253aa revealed the presence of the IL-1 family signature and the absence of an ICE cut site. The cDNA of the chemokine IL-8 was sequenced in haddock and contained 903 bp of which 306 bp are the open reading frame. Interestingly, the predicted protein sequence of 101aa, contains an ELR motif preceding the CXC signature, common in all vertebrate IL-8 molecules but absent in all teleost genes sequenced to date. The expression of both haddock cytokines was studied in four different tissues: head kidney, spleen, liver and gill. Tissues were obtained from both healthy fish and fish stimulated in vivo with four commercial serotypes of LPS, namely Escherichia coli 026:B6, 055:B5, 0111:B4 and 0127:B8 and PMA. Haddock IL-1beta was not constitutively expressed and expression was only observed following stimulation. However, this expression was stimulant dependent and only PMA and LPS 026:B6 induced high levels of expression in the head kidney. The haddock IL-8 gene on the other hand, showed a constitutive expression, that could be up or down-regulated depending on the immunostimulant used, although to a lesser extent than IL-1beta.

Amino Acid Sequence↗

Anisakid parasites of two forkbeards (Phycis blennoides and Phycis phycis) from the Mediterranean coasts of Andalucía (Southern Spain).

An epidemiological survey of anisakids was carried out on 209 specimens of greater forkbeard (Phycis blennoides) and 58 of forkbeard (Phycis phycis) captured off the Mediterranean coasts of Andalucía (southern Spain). Four species of nematodes were identified: Anisakis simplex s.l., Anisakis physeteris, Hysterothylacium aduncum and Hysterothylacium fabri. The total prevalence was 62.06% in the forkbeard and 58.85% in the greater forkbeard. The highest values of prevalence (56.90%), mean intensity (5.21) and mean abundance (2.96) were all obtained for H. fabri in the forkbeard. The most frequent parasite in the greater forkbeard was H. aduncum with 51.20% prevalence and values of 3.00 and 1.53 for mean intensity and mean abundance, respectively. The infestation parameters were also analyzed according to the host length, observing a maximum of parasitized fish (91.67%) in samples of P. blennoides longer than 40 cm, while in P. phycis, the highest prevalence (82.35%), conditioned by H. fabri parasitization, was found in fish with lengths between 30 and 35 cm.

Animals↗

Temporal trends of organochlorine contaminants in burbot and lake trout from three selected Yukon lakes.

Historical studies have demonstrated that organochlorine (OC) concentrations in top predators can vary considerably from lake to lake within a small geographic region but temporal trends of these contaminants have rarely been monitored in a sub-Arctic area for a long period of time. This study examined OC concentrations, including chlordane (CHL), DDT, hexachlorocyclohexane (HCH), toxaphene (CHB), PCB and chlorinated benzenes (CBz) in lake trout and burbot, from three Yukon lakes (Laberge, Kusawa, Quiet), over a span of 11 years (1992-2003). Temporal and spatial differences continue to exist in the OC concentrations of burbot and lake trout between these lakes. There is strong evidence that these contaminants are declining at various rates in lake trout (Salveninus namaycush) in Laberge, Kusawa and Quiet Lakes. For example, SigmaDDT concentrations have decreased 39%, 85% and 84% in Kusawa, Quiet and Laberge Lakes, respectively. Conversely, no consistent trends were observed in OC concentrations for burbot (Lota lota). For example, there is no evidence of a decline in toxaphene concentrations of Kusawa burbot yet a 58% decrease was observed in Laberge samples. Increases were also observed in the SigmaHCH levels of Kusawa Lake burbot, as well as increases in all OC groups (except SigmaHCH) for the Quiet Lake burbot samples. Decreases in burbot were evident in SigmaHCH and SigmaCHB for Lake Laberge fish and in SigmaCHL for Kusawa Lake samples. Spatial variations in OC levels are quite evident as Lake Laberge trout and burbot continued to maintain the highest levels over the eleven-year period from 1992 to 2003 followed by Kusawa Lake and then Quiet Lake. These differences were related to a variety of factors especially the species morphological characteristics such as log age, log weights and fish lipid content. A decreasing trend in Quiet and Laberge Lake trout lipid content, coupled with fluctuating condition factors and increases in body masses, suggest biotic changes may be occurring within the food webs due to fish population variations related to the cessation of commercial fishing or potentially an increase in lake plankton productivity related to annual climate variation. It is suspected that biotic factors rather than atmospheric inputs are the primary factors affecting the contaminant concentrations in lake trout and burbot in the study lakes.

Animals↗

Phylogeny of the Gadidae (sensu Svetovidov, 1948) based on their morphology and two mitochondrial genes.

Although Codfishes are probably one of the most studied groups of all teleost fishes worldwide owing to their great importance to fisheries, their phylogeny and classification are still far from being firmly established. In this study, we present phylogenetic relationships of 19 out of 22 genera traditionally included in the Gadidae based on the analysis of entire cytochrome b and partial cytochrome oxidase I genes (1530 bp). Maximum Parsimony, Maximum Likelihood, and Bayesian analyses all recovered five main clades that correspond to traditionally recognized groupings within Gadoids. The same clades were recovered with MP analysis based on 30 morphological characters (collected from the literature). Given these findings, we propose a revised provisional classification of Gadoids: one suborder Gadoidei containing two families, the Merlucciidae (1 genus) and the Gadidae (21 genera) distributed into four subfamilies: the Gadinae (12 genera), the Lotinae (3 genera), the Gaidropsarinae (3 genera), and the Phycinae (3 genera). Lastly, nuclear inserts of mitochondrial DNA (Numts) were identified in two species, i.e., Gadiculus argenteus and Melanogrammus aeglefinus.

Animals↗

Mitochondrial DNA analyses of the Cape hakes reveal an expanding, panmictic population for Merluccius capensis and population structuring for mature fish in Merluccius paradoxus.

The Cape hake species, Merluccius capensis and Merluccius paradoxus are the most important resource of the South African and Namibian demersal fishery, but it is unclear whether there is a single population of each shared by both countries. We analysed the population structure and evolutionary history of these two species using the variable 5' region of the mtDNA control region for 311 specimens of M. capensis and 333 specimens of M. paradoxus sampled between Lüderitz (southern Namibia) to south of Cape Point (South Africa). 107 haplotypes for M. capensis and eight haplotypes for M. paradoxus were recovered. AMOVA and pairwise Phi(st) analyses revealed no structure in M. capensis, however significant genetic differentiation between Namibian and South African 'populations' was detected for M. paradoxus. This was only restricted to mature fish older than 3 and 4 years and not for juvenile fish younger than 3 years. Analyses reveal that M. capensis has undergone population expansion (Fu's Fs=-26.65, P<0.001), possibly within the last 4500-23,000 years, whereas M. paradoxus has not. Our study highlights the utility of genetic markers to unravel the evolutionary history of sympatric species, as well as addressing management issues within regions where commercially valuable fish stocks are shared between nations.

Animals↗

Food-protein-induced enterocolitis syndrome caused by fish.

INTRODUCTION: Food protein-induced enterocolitis syndrome (FPIES) occurs in infants. Typical symptoms include profuse vomiting and/or diarrhea several hours after ingestion of a given food. The disorder is a non-IgE mediated food hypersensitivity. The most frequently involved foods are milk and soy, but some cases of FPIES induced by solid foods have been described. We report 14 patients with FPIES due to fish protein. MATERIAL AND METHODS: History and physical examination, skin prick test (SPT) with fish allergens and Anisakis simplex, prick-by-prick test with implicated fish and determination of specific IgE antibodies against fish were performed. In eight children atopy patch test (APT) were also performed. In nine patients an open oral food challenge with the implicated fish was carried out. RESULTS: There were six boys and eight girls, aged from 9 to 12 months at diagnosis, with between two and six reactions to the offending fish proteins before the diagnosis was established. Four patients had a previous history of atopy. Presenting symptoms included diarrhea in two patients, profuse vomiting in six patients, and recurrent vomiting and subsequent diarrhea in three patients. In addition to these symptoms, associated septic appearance, apathy and lethargy were present in the remaining three patients. Onset of symptoms occurred a few minutes after fish ingestion in two patients and from 60 minutes to 6 hours in the 12 remaining patients. SPT to fish were negative in all patients. Serum food-specific IgE antibodies were negative in all patients except one. APT was positive in three patients. Open oral challenge (OC) was performed in nine infants and was positive in all. The patients were followed-up for between 1 and 7 years after diagnosis, and follow-up OC tests were performed after fish had been eliminated from the patients' diet for 3-4 years. Four patients became clinically tolerant to the causal food. Three patients currently tolerate only one type of fish (swordfish). CONCLUSIONS: We report 14 patients with FPIES caused by fish protein. The symptoms suggest a form of cell-mediated, non-IgE mediated food hypersensitivity. The gold standard for diagnosis is OC, although caution should be exercised in infants with several reactions or a recent diagnosis. After a period of elimination of the causal food from the diet, tolerance can develop.

Animals↗