Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes,Cyprinidae) and cross‐species amplification within the family Cyprinidae

Thirteen polymorphic microsatellites were isolated from Leuciscus cephalus, a widespread cyprinid species with great ecological tolerance. Together with the cross‐species amplification of six additional loci originally published for three cyprinid fish species, we optimized a multiplex panel for L. cephalus allowing the genotyping of 19 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 20 fish individuals ranged from two to 16 and from 0.05 to 0.90, respectively. These primers will be useful in determining the population structure of L. cephalus. In addition, successful cross‐amplification was obtained for several species of Cyprinidae.     

Genetic Divergence and Phylogenetic Relationships in Grey Mullets (Teleostei: Mugilidae) Using Allozyme Data

Genetic divergence and phylogenetic relationships among five species of the Mugilidae family (Liza saliens, Liza aurata, Liza ramada, Chelon labrosus, and Mugil cephalus) were investigated, in the present study, on samples taken from Messolongi lagoon in Greece, using allozyme electrophoresis. Ten enzymic systems corresponding to 22 genetic loci were assayed, among which, four were found to be polymorphic in Liza saliens, Liza aurata, and Chelon labrosus, seven in Liza ramada, while only two were polymorphic in Mugil cephalus. Several loci showed different electrophoretic patterns among the species and thus, they can be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. It must be underlined that among them the aGPD-2* locus proved to be species-specific, while the other ones can be also used in various combinations for the same purpose. The observed heterozygosity was found to range from 0.020 to 0.051. Allele frequencies of all loci were used to estimate Nei's (1972) genetic distance, which was found to range between 0.249 and 1.171 among the five species studied. UPGMA and NJ trees, obtained by genetic distance matrix methods, as well as, a tree based on the discrete character parsimony analysis were found to exhibit the same topology. Our result show that the three species of the genus Liza are clustered together, Chelon labrosus being closer to the previous clade, while Mugil cephalus being more distinct.     

Population genetic structure of striped mullet, Mugil cephalus, along the coast of China, inferred by AFLP fingerprinting

The striped mullet, Mugil cephalus, is an economically important species for both aquaculture and commercial fisheries in China. In this study, the amplified fragment length polymorphism (AFLP) technique was employed to analyze population genetic diversity and genetic distance between different populations with the aim of elucidating the population structure and gene flow of M. cephalus along the coast of China. A total of 230 fragments with 150–500 bp were identified from 118 individuals by five AFLP primer combinations. The polymorphic loci within populations varied from 46.52% to 64.78%, with an average of 53.91%, and the average heterozygosity from 0.1829 to 0.2282, with an average of 0.2074. The UPGMA phenograms of 118 individuals were constructed based on Dice similarity coefficients and four clusters were recognized. AMOVA analysis revealed that 60.7% of genetic variations were within populations and 39.3% between populations. The estimated genetic distance (φ_ST) value over all polymorphic loci across the six populations was 0.393 (p < 0.0010), indicating a strong population structuring. The pairwise φ_ST value ranged from 0.1112 to 0.5358, with an average of 0.3693. The population pairwise gene flows (average N_m = 0.73) are low. In addition, the result of the Mantel test showed that there was a significant correlation between geographic and genetic distances (r = 0.5434, p = 0.0050). It was speculated that there exist at least four distinct geographic populational subdivisions of M. cephalus along the Chinese coast. This research has provided new molecular data which will aid our understanding of the genetic structure of this species.     

Phenetic relationships of six species of mullets (<Emphasis Type="Italic">Mugilidae</Emphasis>) from the south Adriatic,as inferred from the study of the visceral and dermal skeleton

This study examines the interspecific variability and phenetic relationships in six southern Adriatic mullet species: Mugil cephalus (Linnaeus, 1758), Chelon labrosus (Risso, 1826), Liza aurata (Risso, 1810), Liza ramada (Risso, 1826), Liza saliens (Risso, 1810), and Oedalechilus labeo (Cuvier, 1829). Twenty-three morphometric characteristics of the visceral skeleton and the dermatocranium of mullets were analyzed. The major distinguishing characters were those of the jaws. M. cephalus was clearly separated from the other species; O. labeo differed to a somewhat smaller extent; and C. labrosus was the least distinguished species. The species of the genus Liza were phenetically most similar, while the other species of mullet differed significantly from them. The greatest distances were between M. cephalus and C. labrosus, as well as between M. cephalus and O. labeo. This text was submitted by the authors in English.     

Variation in habitat use along the freshwater–marine continuum by grey mullet Mugil cephalus at the southern limits of its distribution

In this study, habitat use by Mugil cephalus was investigated in the waters of the west coast of the North Island of New Zealand by analysing microchemical composition of otoliths (laser‐ablation inductively coupled plasma mass spectrometry) obtained from individuals from commercial fish stocks and research surveys. Results of this study show that M. cephalus at the southern limits of its distribution display highly flexible migratory behaviour with extensive use of freshwater and brackish habitats, potentially enabling them to maximize foraging opportunities. Mugil cephalus can tolerate a wide range of salinities and can therefore utilize higher productivity areas, such as estuaries and eutrophic riverine lakes. Finally, M. cephalus populations across a range of climates and latitudes appear to differ in the extent to which they utilize freshwater and brackish habitats, possibly with increasing penetration of fresh waters with increasing latitude.     

Molecular Phylogeny of Grey Mullets (Teleostei: Mugilidae) in Greece: Evidence from Sequence Analysis of mtDNA Segments

Mitochondrial DNA sequence analysis has been used to explore genetic differentiation and phylogenetic relationships among five species of the Mugilidae family, Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens. DNA was isolated from samples originating from the Messolongi Lagoon in Greece. Three mtDNA segments (12s rRNA, 16s rRNA, and CO I) were PCR amplified and sequenced. Sequencing analysis revealed that the greatest genetic differentiation was observed between M. cephalus and all the other species studied, while C. labrosus and L. aurata were the closest taxa. Dendrograms obtained by the neighbor-joining method and Bayesian inference analysis exhibited the same topology. According to this topology, M. cephalus is the most distinct species and the remaining taxa are clustered together, with C. labrosus and L. aurata forming a single group. The latter result brings into question the monophyletic origin of the genus Liza.     

Saturnius minutus n. sp. and S. dimitrovi n. sp. (Digenea: Hemiuridae) from Mugil cephalus L. (Teleostei: Mugilidae), with a multivariate morphological analysis of the Mediterranean species of Saturnius Manter, 1969

Three species of the bunocotyline genus Saturnius Manter, 1969 are described from the stomach lining of mugilid fishes of the Mediterranean and Black Seas. Two of the species are new: S. minutus n. sp. occurs in Mugil cephalus off the Mediterranean coast of Spain; and S. dimitrovi n. sp., a parasite of M. cephalus off the Bulgarian Black Sea coast and the Spanish Mediterranean coast, was originally described as S. papernai by Dimitrov et al. (1998). In addition, S. papernai Overstreet, 1977 is redescribed from M. cephalus off the Spanish Mediterranean coast and from Liza aurata and L. saliens off the Bulgarian Black Sea coast. The three species are distinguished morphometrically using univariate and multivariate analyses. These results were verified using Linear Discriminant Analysis which correctly allocated all specimens to their species designations based on morphology (i.e. 100% successful classification rate) and assigned almost all specimens to the correct population (locality). The following variables were selected for optimal separation between samples: the length of the forebody, ventral sucker and posterior testis, the length and width of the posteriormost pseudosegment, and the width of the muscular flange at ventral sucker level.     

Morphological and morphometric changes of sagittae otoliths related to fish growth in three Mugilidae species

The aim of this study was to analyze morphologic and morphometric changes of sagittae otoliths throughout the growth of three mullets: Mugil liza, Mugil cephalus and Liza ramada. Fish were collected seasonally between October 2011 and April 2014, and three methods were used: regression analysis of traditional biometric characters; morphological analysis for group identification; and morphometrical analysis of shape indices among groups (circularity, rectangularity, aspect ratio and percentage occupied by the sulcus). In all species, dependence of standard length on otolith length and height were best described as power functions. Two morphological groups were identified for M. liza and M. cephalus, while three for L. ramada. Morphological changes were supported by morphometry differences only for the first two species. Smaller size specimens of M. liza had more rectangular otoliths with more percentage covered by the sulcus while bigger fish tended to have a more circular shape. For M. cephalus smaller size fish had a more elliptical shape. The observed changes may be reflecting life history changes, related to habitat or dietary shifting.     

The relationship of diet to growth and ammonium excretion in salt marsh fish

Synopsis Detritus is an abundant but poor quality food source for consumers in salt marsh ecosystems. Here we present results of feeding experiments to determine the ability of Fundulus heteroclitus, Cyprinodon variegatus, and Mugil cephalus, three major detritivores in Great Sippewissett Marsh, Massachussetts, to assimilate detritus and use it for growth. C. variegatus, the sheepshead minnow, gained weight on a detrital diet, but F. heteroclitus, the marsh killifish, and juvenile M. cephalus, the striped mullet, lost weight and suffered high mortality on detrital diets. C. variegatus is a herbivore with morphological adaptations for ingesting plant material. F. heteroclitus is a carnivore poorly suited to effectively assimilate detritus from the diet. Although adult M. cephalus are adapted for ingesting and assimilating detrital material, the young may lack these adaptations and thus do not assimilate detritus. C. variegatus excretes ammonium at a lower rate than F. heteroclitus when fed animal food, or when starved for short periods of time. This protein sparing effect could be crucial for survival when detritus is the only food available at certain times of the year. While both C. variegatus and M. cephalus are visitors in the marsh and may leave to exploit food sources elsewhere, F. heteroclitus is a year-round resident and is the most abundant species of fish in the marsh. Yet when high quality food sources become scarce in late summer and fall, detritus, although plentiful, is apparently not a suitable alternative.To whom all correspondences should be addressed.     

Comparative homology modeling-inspired protein engineering for improvement of catalytic activity of Mugil cephalus epoxide hydrolase

The epoxide hydrolase (EH) of a marine fish, Mugil cephalus, was engineered to improve the catalytic activity based on comparative homology modeling. The 3-D crystal structure of the EH from Aspergillus niger was used as a template. A triple point mutant, F193Y for spatial orientation of the nucleophile (D199), W200L for removing electron density overlap between W200 and Y348, and E378D for good charge relay in the active site, was developed. The initial hydrolysis rate, the reaction time to reach 98 %ee, and yield were enhanced up to 35-fold, 26-fold and 32%, respectively, by homology modeling-inspired site-directed mutagenesis of M. cephalus EH.     

Genetic divergence and phylogenetic relationships in grey mullets (Teleostei: Mugilidae) based on PCR-RFLP analysis of mtDNA segments

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, the first one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.     

Phylogenetic relationships of nine mullet species (Mugilidae) in the Mediterranean Sea

Phylogenetic relationships among four genera and nine species (Mugil cephalus, Mugil soiuy, Liza ramada, Liza aurata, Liza abu, Liza saliens, Liza carinata, Chelon labrosus, Oedalechilus labeo) of the Mediterranean mullets (Mugilidae) were investigated by means of allozyme electrophoresis using a seven-enzyme system (CK*, GAPDH*, G3PDH*, IDHP*, ME*, MDH*, PGM*) comprising eleven putative loci. The highest genetic divergence was 1.299, detected between M. cephalus and L. aurata and the lowest (0.280) was found between L. carinata and L. saliens. The amount of genetic divergence between the genera Chelon and Oedalechilus did not appear to be high (0.285). In a UPGMA tree, all nine species were grouped in two main branchings. In the first branch, C. labrosus and O. labeo clustered as closest taxa and were sister group to L. ramada. The other four Liza species produced two sub-branching in this group; L. carinata branched together with L. saliens, and L. aurata branched together with L. abu. In the second branch the two species of the genus Mugil, M. cephalus and M. soiuy, clustered together and were clearly isolated from the other three genera.     

THE DISTRIBUTION AND ECOLOGY OF TWO MULLET SPECIES IN SOME FRESH WATER RIVERS IN THE EASTERN CAPE,SOUTH AFRICA

SUMMARY

Data on the relative abundance, penetration and breeding biology of the freshwater mullet Mugil cephalus and the flathead mullet Mugil cephalus in the freshwater reaches of some Eastern Cape coastal rivers are described. The differences found between the two species indicate that Myxus capensis is more specialized for a catadromous life history in an unstable riverine environment. Evidence showing the importance of the freshwater phase for the latter species is given and the disastrous effects of the erection of barriers to fish movement are stressed.     

Identification of the sex‐determining region in flathead grey mullet (Mugil cephalus)

Elucidation of the sex‐determination mechanism in flathead grey mullet (Mugil cephalus) is required to exploit its economic potential by production of genetically determined monosex populations and application of hormonal treatment to parents rather than to the marketed progeny. Our objective was to construct a first‐generation linkage map of the M. cephalus in order to identify the sex‐determining region and sex‐determination system. Deep‐sequencing data of a single male was assembled and aligned to the genome of Nile tilapia (Oreochromis niloticus). A total 245 M. cephalus microsatellite markers were designed, spanning the syntenic tilapia genome assembly at intervals of 10 Mb. In the mapping family of full‐sib progeny, 156 segregating markers were used to construct a first‐generation linkage map of 24 linkage groups (LGs), corresponding to the number of chromosomes. The linkage map spanned approximately 1200 cM with an average inter‐marker distance of 10.6 cM. Markers segregating on LG9 in two independent mapping families showed nearly complete concordance with gender (R² = 0.95). The sex determining locus was fine mapped within an interval of 8.6 cM on LG9. The sex of offspring was determined only by the alleles transmitted from the father, thus indicating an XY sex‐determination system.     

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.     

Utility of fish scales from stock assessment surveys in stable isotope analysis for initial assessments of trophic relationships in riverine fish communities

The utility of using fish scales collected during stock assessment exercises to assess the trophic relationships of riverine fishes using their stable isotopes of d¹³C and d¹⁵N was tested using three riverine fish communities in England (Rivers Great Ouse, Ivel and Goyt). In each river, European barbel Barbus barbus was an important species, with other cyprinid species, including chub Squalius cephalus, present. Stable isotope analyses was completed using relatively small sample sizes per species (<11) from fish samples collected in 2001, 2005 and 2006 when up to 5 scales were collected from each fish. The calculation of standard ellipse areas (as a measure of trophic niche size) revealed that relative to other fishes, B. barbus occupied high trophic positions with minimal overlap in their trophic niche with other species, especially S. cephalus. As the analysed fish samples comprised species of various length ranges and as length has strong ontogenetic consequences for fish diet composition, generalized linear models were developed in which length was the covariate; model outputs included length‐adjusted mean δ¹³C and δ¹⁵N for each species. In each fish community, significant differences in δ¹³C and δ¹⁵N were apparent between B. barbus and S. cephalus, but were less apparent between B. barbus and other fishes. Thus, whilst the utility of using fish scales from stock assessments in stable isotope analyses are limited due to the differing length ranges of the sampled fishes, they can be useful in identifying trophic differences between species when methods such as stomach content analyses are unavailable.     

Motile aeromonas infection of striped (grey) mullet Mugil cephalus

Infection of striped mullet (Mugil cephalus) with Aeromonas hydrophila results in an acute septicemic disease. The disease can be experimentally induced by intramuscular injection, skin or gill scarification or by the oral route using pellets purposely seeded with bacteria. The organism was isolated from the blood 1–2 days after infection and from all organs 24 hr or longer after infection. The disease is characterized by early inflammatory and proliferative changes and later necrotic changes. Enteritis and hepatic necrosis are constant findings in aeromonad disease of M. cephalus but surface lesions are not pathognomic for these infections in mullet. Death of infected fish may be attributed to bacterial toxins which cause necrosis of parenchymal organs and soft tissue structure.     

Cloning,expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, <Emphasis Type="Italic">Mugil cephalus</Emphasis>

The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of α/β-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.     

Critical thermal maxima of three species of Hawaiian estuarine fish: a comparative study*

Crticial thermal maxima (CTM) of three species of Hawaiian estuarine fishes were determined in relation to acclimation temperature and time of day. The species studied were Mugil cephalus L., Chelon engeli (Bleeker) and Kuhlia sandvicensis (Steindachner). Critical thermal maxima of all three species were significantly affected by acclimation temperature and time. In relation to acclimation temperature, Mugil cephalus and Chelon engeli have similar critical thermal maxima with Kuhlia sandvicensis having a critical thermal maxima about 1° C below that of the other two species studied. Mugil cephalus and Chelon engeli showed an increasing critical thermal maxima toward midday which decreased therafter. Critical thermal maximum of Kuhlia sandvicensis increased throughout the day.     

Multiple Pleistocene refugia and post‐glacial colonization in the European chub (Squalius cephalus) revealed by combined use of nuclear and mitochondrial markers

Aim To analyse patterns of nuclear and mitochondrial genetic variation in the European chub, Squalius cephalus (Linnaeus, 1758), in order to understand the evolutionary history of this species and to test biogeographical hypotheses for the existence of co‐distributed European freshwater fish species. Location Rivers in Europe (Finland, Poland, Czech Republic, France, Bulgaria, Spain, Italy). Methods We genotyped 12 polymorphic microsatellite markers derived from 310 individuals collected from across the distribution of S. cephalus in Europe (including a total of 15 populations) and sequenced mitochondrial DNA (mtDNA) from a subset of 75 individuals. Sequences of mtDNA cytochrome b were analysed using both phylogenetic (median‐joining networks) and population genetic methods (tests for demographic history, mismatch distributions, Bayesian coalescent analysis). Geographical structure in microsatellite loci was examined using a distance method (F_ST), factorial correspondence analysis (FCA) and a Bayesian clustering method (structure ). Results The mtDNA network showed a clear split into four different haplogroup lineages: Western (separated into Atlantic and Danubian sublineages), Eastern, Aegean (occurring in two distinct sublineages in the Balkans and in Spain) and Adriatic. Our results indicate recent population expansion in the Eastern and Western Atlantic lineages and the admixture of two previously separate sublineages (Atlantic and Danubian) in the Western lineage. Bayesian structure analysis as well as FCA results roughly corresponded to the mtDNA‐based structure, separating the sampled individuals into almost non‐overlapping groups. Main conclusions Our results support hypotheses suggesting origins of extant lineages of freshwater fishes in multiple refugia and the subsequent post‐glacial colonization of Europe via different routes. We confirmed the previously proposed two‐step expansion scenario from the Danube refuge, the existence of a secondary (Atlantic) refuge during the last glaciation (probably in the Rhone River) and population expansion of this lineage. Conspicuous divergences among Mediterranean populations reflect their different origin, as well as their low contribution to the recent genetic pool of chub in central Europe.