MALDI-TOF mass spectrometry for rapid differentiation of Tenacibaculum species pathogenic for fish

Tenacibaculosis is a fish disease that limits the culture of a variety of marine fish species of commercial value in the world. The genus Tenacibaculum includes several species, and their discrimination is of clinical interest in order to improve the management of an outbreak of the disease. In this study, a novel proteomic approach based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was evaluated for the identification and differentiation of Tenacibaculum species. The peak mass lists derived from MALDI-TOF-MS analysis were examined for the detection of potential biomarkers, similarity and cluster analysis and principal component analysis (PCA). Culture media used for bacterial growth did not affect the mass fingerprints. Eight genus-specific peaks were found in all the Tenacibaculum species analysed. Moreover, at least one species-specific peak was found in the species Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum dicentrarchi, Tenacibaculum litoreum and Tenacibaculum ovolyticum. These peaks could serve as biomarkers for the rapid identification of these bacterial fish pathogens. The cluster and PCA clearly separated the species T. maritimum, T. soleae, T. dicentrarchi and T. ovolyticum in different clusters. However, species of Tenacibaculum discolor and Tenacibaculum gallaicum were difficult to distinguish based on their protein fingerprints. To our knowledge, this is the first study that deals with the characterization and determination of biomarkers of Tenacibaculum species by MALDI-TOF mass spectrometry. This approach proved to be an effective and reliable technique for the discrimination of the Tenacibaculum species; therefore, it could be integrated as a routine diagnostic tool in microbiological laboratories.

     

Multilocus Sequence Analysis of the Marine Bacterial Genus Tenacibaculum Suggests Parallel Evolution of Fish Pathogenicity and Endemic Colonization of Aquaculture Systems

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.     

Fatty acid analysis as a chemotaxonomic tool for taxonomic and epidemiological characterization of four fish pathogenic Tenacibaculum species

Aims: In this work, fatty acid content and profiles were analysed in order to differentiate the species Tenacibaculum maritimum, Tenacibaculum gallaicum, Tenacibaculum discolor and Tenacibaculum ovolyticum that are pathogenic for cultured marine fish and to assess the potential of fatty acid profiles as a tool for epizootiological typing. Methods and Results: The fatty acid methylesters (FAMEs) were extracted from cells grown on marine agar for 48 h at 25°C and were prepared and analysed according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System. The cellular fatty acid profiles of Tenacibaculum strains tested were characterized by the presence of large amounts of branched (36·1–40·2%) and hydroxylated (29·6–31·7%) fatty acids. The FAME products from the four species significantly (P < 0·05) differed in the content of iso‐C_15:03‐OH, iso‐C_16:03‐OH, iso‐C_15:1G, summed feature 3 (a component that contains C_16:1ω7c and/or iso‐C_15:0 2‐OH), iso‐C_16:0, C_17:1ω6c, C_15:03‐OH, iso‐C_17:03‐OH. Conclusions: Results of present study demonstrated the existence of differences in the fatty acids content between the T. maritimum isolates from different marine fish/geographical origin and between strains of T. maritimum, T. discolor, T. gallaicum and T. ovolyticum. Significance and Impact of the Study: Profiling of fatty acids may be a useful tool to distinguish T. maritimum from other Tenacibaculum species pathogenic for fish as well as for epizootiological differentiation of T. maritimum isolates.     

Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review

Tenacibaculum maritimum is the aetiological agent of an ulcerative disease known as tenacibaculosis, which affects a large number of marine fish species in the world and is of considerable economic significance to aquaculture producers. Problems associated with epizootics include high mortality rates, increased susceptibility to other pathogens, high labour costs of treatment and enormous expenditures on chemotherapy. In the present article we review current knowledge on this bacterial pathogen, focusing on important aspects such as the phenotypic, serologic and genetic characterization of the bacterium, its geographical distribution and the host species affected. The epizootiology of the disease, the routes of transmission and the putative reservoirs of T. maritimum are also discussed. We include a summary of molecular diagnostic procedures, the current status of prevention and control strategies, the main virulence mechanisms of the pathogen, and we attempt to highlight fruitful areas for continued research.     

Tenacibaculum caenipelagi sp. nov., a member of the family Flavobacteriaceae isolated from tidal flat sediment

A Gram-stain-negative, non-spore-forming, aerobic, non-flagellated, gliding and rod-shaped bacterial strain, designated HJ-26M^T, was isolated from a tidal flat sediment in the Korean peninsula. It grew optimally at 25–30 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that the strain fell within the clade comprising Tenacibaculum species, clustering coherently with the type strains of Tenacibaculum lutimaris and Tenacibaculum aestuarii. Strain HJ-26M^T exhibited the highest 16S rRNA gene sequence similarity values of 98.4 and 98.2 % to T. lutimaris TF-26^T and T. aestuarii SMK-4^T, respectively, and of 94.9–97.4 % to the type strains of the other Tenacibaculum species. Strain HJ-26M^T contained MK-6 as the predominant menaquinone and iso-C_15:0 and iso-C_17:0 3-OH as the major fatty acids. The DNA G+C content of strain HJ-26M^T was 34.5 mol% and its mean DNA–DNA relatedness values with the type strains of T. lutimaris and T. aestuarii were 19 and 23 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain HJ-26M^T is separate from other Tenacibaculum species. On the basis of the data presented, strain HJ-26M^T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum caenipelagi sp. nov. is proposed. The type strain is HJ-26M^T (= KCTC 32323^T = CECT 8283^T).     

Tenacibaculum halocynthiae sp. nov., a member of the family Flavobacteriaceae isolated from sea squirt Halocynthia roretzi

A Gram-negative, non-spore-forming, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated P-R2A1-2^T, was isolated from sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. It grew optimally at 25–28 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that the strain fell within the clade comprising Tenacibaculum species. Strain P-R2A1-2^T exhibited the highest 16S rRNA gene sequence similarity values of 97.6, 97.2 and 97.0 % to Tenacibaculum aestuarii SMK-4^T, T. lutimaris TF-26^T and T. aiptasiae a4^T, respectively, and of 94.5–96.8 % to the type strains of the other Tenacibaculum species. Strain P-R2A1-2^T contained MK-6 as the predominant menaquinone and C_16:1 ω7c and/or iso-C_15:0 2-OH, iso-C_15:0 3-OH and iso-C_15:0 as the major fatty acids. The DNA G + C content of strain P-R2A1-2^T was 30.7 mol % and its DNA–DNA relatedness values with the type strains of T. aestuarii, T. lutimaris and T. aiptasiae were 17 ± 4.2, 21 ± 6.1 and 16 ± 5.2 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that the novel strain is separate from other Tenacibaculum species. On the basis of the data presented, strain P-R2A1-2^T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum halocynthiae sp. nov. is proposed. The type strain is P-R2A1-2^T (=KCTC 32262^T = CCUG 63681^T).     

Toward a preliminary assessment of the diversity and origin of Cyprinid fish genus Carassius in Iran

Introduction of non-native fish species has been criticized as one of the major threats to freshwater ecosystems. During recent years, the cyprinid fish genus Carassius has been the subject of various studies in terms of introduction biology and ecology. The taxonomy and identification of described species in the genus Carassius is still ambiguous. In order to increase the knowledge of introduced species of the genus in Iran, molecular approaches were employed to identify species based on the mitochondrial gene, cytochrome b (Cyt b). In total, 417 specimens across Eurasia and some other parts of the world have been analyzed. Afterwards, investigation of the possible origin and pathways of introduction was done using analysis of haplotype networks based on the Cyt b gene. The results revealed that there are three introduced species i.e., Carassius auratus, Carassius gibelio and Carassius langsdorfii, in inland waters of Iran. Carassius langsdorfii was recorded from Iran and western Asia for the first time. Analysis of haplotype network showed various potential sources of introduction for each species in Iran and regions beyond their native distribution ranges.     

Phylogeny of leafcutter ants in the genus Atta Fabricius (Formicidae: Attini) based on mitochondrial and nuclear DNA sequences

Leafcutting ants of the genus Atta are the most conspicuous members of the tribe Attini, the fungus-growing ants. Atta species have long attracted the attention of naturalists, and have since become a common model system for the study of complex insect societies as well as for the study of coevolutionary dynamics due to their numerous interactions with fungi and other microbes. Nevertheless, systematics and taxonomy of the 15 species in the genus Atta have proven challenging, due in part to the extreme levels of worker polymorphism these species display, leading to disagreements about the validity of as many as five different subgenera and calling into question the monophyly of the genus. Here, we use DNA sequence information from fragments of three mitochondrial genes (COI, tRNA leucine and COII) and one nuclear gene (EF1-αF1), totaling 1070 base pairs, to reconstruct the phylogenetic relationships of Atta species using maximum parsimony, maximum likelihood and Bayesian inference techniques. Our results provide support for monophyly of the genus Atta, and suggest that the genus is divided into four monophyletic groups, which correspond to four of the five previously erected Atta subgenera: Atta sensu stricto and Archeatta, each with species composition identical to earlier proposals; Neoatta and Epiatta, with major differences in species composition from earlier proposals. The current geographic ranges of these species suggest that the historical separation of South America from Central and North America has played a role in speciation within this genus.     

Length–weight relationships of five fish species from the Nujiang River (Salween basin), southwest China

This study provides the total length–weight relationships (LWRs) for five fish species collected from the Nujiang River, southwest China. Samples were obtained in May 2015 and May 2016 using drift net and electroshock fishing techniques. A total of 502 specimens belonging to five fish species were analyzed; their LWRs were calculated. The five fish were Schizothorax nukiangensis, Akrokolioplax bicornis, Creteuchiloglanis kamengensis, Channa gachua and Glyptothorax trilineatus. Prior to this study, the length‐weight relationship for S. nukiangensis is unknown to FishBase and new maximum lengths are recorded for A. bicornis and C. kamengensis.These results are useful for fishery research, conservation and management in the Nujiang River.     

The Lepocreadiidae (Digenea) of fishes from the north-east Atlantic: Profundivermis intercalarius n. g., n. sp. from the marine fish Coryphaenoides (Nematonurus) armatus (Hector) (Macrouridae) from the Porcupine Abyssal Plain

A new genus and species, Profundivermis intercalarius, is described from the deep-sea fish Coryphaenoides (Nematonurus) armatus from 4,850 m deep on the Porcupine Abyssal Plain, NE Atlantic. The genus is characterized by an inter-testicular ovary and is the only member of the subfamily Lepidapedinae to show this feature. The relationships of the members of the subfamily are analysed phylogenetically. Members of the subfamily are often found in deep-sea gadiforms.     

A multilocus phylogeny of the fish genus Poeciliopsis: Solving taxonomic uncertainties and preliminary evidence of reticulation

The fish genus Poeciliopsis constitutes a valuable research system for evolutionary ecology, whose phylogenetic relationships have not been fully elucidated. We conducted a multilocus phylogenetic study of the genus based on seven nuclear and two mitochondrial loci with a thorough set of analytical approaches, that is, concatenated (also known as super‐matrix), species trees, and phylogenetic networks. Although several relationships remain unresolved, the overall results uncovered phylogenetic affinities among several members of this genus. A population previously considered of undetermined taxonomic status could be unequivocally assigned to P. scarlli; revealing a relatively recent dispersal event across the Trans‐Mexican Volcanic Belt (TMVB) or Pacific Ocean, which constitute a strong barrier to north–south dispersal of many terrestrial and freshwater taxa. The closest relatives of P. balsas, a species distributed south of the TMVB, are distributed in the north; representing an additional north–south split in the genus. An undescribed species of Poeciliopsis, with a highly restricted distribution (i.e., a short stretch of the Rio Concepcion; just south of the US‐Mexico border), falls within the Leptorhaphis species complex. Our results are inconsistent with the hypothesis that this species originated by “breakdown” of an asexual hybrid lineage. On the other hand, network analyses suggest one or more possible cases of reticulation within the genus that require further evaluation with genome‐wide marker representation and additional analytical tools. The most strongly supported case of reticulation occurred within the subgenus Aulophallus (restricted to Central America), and implies a hybrid origin for P. retropinna (i.e., between P. paucimaculata and P. elongata). We consider that P. balsas and P. new species are of conservation concern.     

Numerical analysis of phenotypic properties,genomic fingerprinting,and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

Purpose

The aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae).

Methods

The genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB, and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria.

Results

Both BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium. Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB, and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum.

Conclusion

L. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil.

     

New monogeneans from the bathydemersal southern African endemic catshark,Holohalaelurus regani (Gilchrist, 1922)

The catshark genus Holohalaelurus Fowler is currently represented by five species distributed off the southern and eastern parts of the African coast. Very few parasitological records exist for any of these five species, representing a significant knowledge gap. We report the first monogenean species from the bathydemersal species Holohalaelurus regani (Gilchrist): a new species of Microbothriidae Price, 1936, Leptomicrobothrium holohalaelure n. sp. from the dorsal skin surface, and a new species of Hexabothriidae Price, 1942 representing a new genus, Scyliorhinocotyle narvaezae n. gen., n. sp., from the gill lamellae. Both monogenean records represent the first for any member of the catshark genus Holohalaelurus. Previously we focused on the identification of monogenean taxa of emerging veterinary importance for public aquaria. We now begin a focused effort to document a generally unexplored monogenean biodiversity from diverse marine habitats off South Africa.

     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

A carboniferous chondrichthyan assemblage from residues within a Triassic karst system at Cromhall quarry,Gloucestershire, England

Abstract. Sixteen different Lower Carboniferous (Tournaisian Courceyan to Chadian age, Mississippian) chondrichthyan teeth types have been extracted from Triassic erosional/aeolian fills in shallow karst systems found in the limestone quarry at Cromhall, Gloucestershire, England. These Carboniferous teeth have been found within a much larger assemblage of disarticulated bones and teeth belonging to small late Triassic terrestrial reptiles, for which Cromhall quarry is famous. The Carboniferous teeth are derived fossils, released during subaerial dissolution of the surrounding limestones. Owing to low specimen numbers and uncertainty of intraspecific character, 10 types of teeth are left in open nomenclature, although it is likely they represent new taxa. They belong to Jalodus, Ctenacanthiformes, Protacrodus, Orodus, Chomatodus, Petalodontidae, Euchondrocephali?, Helodus and Psephodus. A new genus and species, Cromhallia parvunda, of indeterminate phylogenetic relationships, is also erected. The assemblage includes also an identified suite comprising Thrinacodus ferox, Bransonella cf. nebraskensis and Stethacanthus cf. altonensis. The identification of all fish teeth found in the Cromhall assemblages as derived fossils from the Carboniferous removes any ambiguity regarding the fully terrestrial nature of the late Triassic fauna preserved in the residue‐bearing karst systems.     

Filling the gap: Length–weight and length–length relationships of intertidal endemic fishes of the Brazilian Province Oceanic Islands

Length‐weight and length‐length relationships were determined for nine fish species of Rocas Atoll, Fernando de Noronha and Trindade Island. Samples were conducted in 2014 (Rocas) and 2015 (Noronha and Trindade) in tidepools using anaesthetic clove oil and hand nets to collect fish fauna. Four species (Stegastes rocasensis, Scartella itajobi, Starksia multilepis and Bathygobius brasiliensis) are endemic from the Noronha‐Rocas ridge, two are endemic from the Trindade‐Martin Vaz insular complex (Scartella poiti and Malacoctenus brunoi), and three species have a widespread distribution. All relationships are novel for science.     

Description of <Emphasis Type="Italic">Gosztonyia antarctica</Emphasis>, a new genus and species of Zoarcidae (Teleostei: Perciformes) from the Antarctic Ocean

A new genus and species of zoarcid fish, Gosztonyia antarctica, is described on the basis of four specimens collected from the Bellingshausen Sea, Antarctic Ocean, at a depth of 615 m. Gosztonyia is placed in the subfamily Lycodinae and can be distinguished from all other zoarcid genera by the following combination of characters: seven branchiostegal rays, interdigitating ceratohyal–epihyal articulation, palatal arch reduced, posterior hyomandibular ramus longer than anterior, cranium narrowed, supratemporal commisure and occipital pores absent. A new species, Gosztonyia antarctica, is described and the relationships of the new genus are discussed.     

Three new genera of Leptophlebiidae (Ephemeroptera) from New Zealand

Abstract

Three new genera—Austroclima, Mauiulus, and Cryophlebia—are established for species of Leptophlebiidae from New Zealand. The following new combinations are included: Austroclima sepia (Phillips) and Cryophlebia aucklandensis (Peters). Atalophlebioides sepia is redescribed as Austroclima sepia and a neotype is designated. Two new species, Austroclima jollyae and Mauiulus luma, are described. All life stages are described, and the relationships of each genus are discussed. Keys are given to male and female imagos, subimagos, and nymphs of each species, and to all New Zealand genera with species previously placed in Atalophlebioides.     

Marine-derived Phoma—the gold mine of bioactive compounds

The genus Phoma contains several species ubiquitously present in soil, water, and environment. There are two major groups of Phoma, viz., terrestrial and marine. After 1981 researchers all over the world have focused on marine-derived Phoma for their bioactive compounds. The marine Phoma are very rich sources for novel bioactive secondary metabolites, which could potentially be used as drugs. Recently, a large number of structurally unique metabolites with potential biological and pharmacological activities have been isolated from the marine Phoma species particularly Phoma herbarum, P. sorghina, and P. tropica. These metabolites mainly include diterpenes, enolides, lactones, quinine, phthalate, and anthraquinone. Most of these compounds possess antimicrobial, anticancer, radical scavenging, and cytotoxic properties. The present review has been focused on the general background of Phoma, current approaches used for its identification and their limitations, difference between terrestrial and marine Phoma species. In addition, this review summarizes the novel bioactive compounds derived from marine Phoma and their biological activities.

     

Phylogenetic relationships among species of the genus Oligoryzomys (Rodentia,Cricetidae) from Central and South America

González‐Ittig, R. E., Salazar‐Bravo, J., Barquez, R. M. & Gardenal, C. N. (2010). Phylogenetic relationships among species of the genus Oligoryzomys (Rodentia, Cricetidae) from Central and South America. —Zoologica Scripta, 39, 511–526. The genus Oligoryzomys includes several species very similar in external morphology, which has resulted in a confusing specific taxonomy. Accurate species identification is particularly important because several species of Oligoryzomys act as natural hosts of hantaviruses affecting humans. Here, we assign specific status to individuals from a wide geographical area of Argentina and Chile using sequences of the mtDNA control region. We also compare cytochrome b sequences of 14 species recognized from Central and South America to infer the phylogenetic relationships within the genus. In addition, the results were analysed using available data on chromosome numbers, and the host–parasite relationships reported for the genus Hantavirus. We confirm the geographical distribution of Oligoryzomys longicaudatus (Argentina, Chile), Oligoryzomys nigripes (Argentina, Paraguay, Brazil), Oligoryzomys chacoensis (Argentina, Bolivia, Paraguay), Oligoryzomys fornesi (Argentina, Paraguay), Oligoryzomys destructor (Argentina, Bolivia) and Oligoryzomys microtis (Bolivia, Brazil). Oligoryzomys longicaudatus is strongly related to the Oligoryzomys flavescens complex, which comprises four clades; O. nigripes is closely related to Oligoryzomys stramineus, and Oligoryzomys vegetus, to Oligoryzomys fulvescens from Central America. Oligoryzomys chacoensis, O. destructor, O. fornesi, O. longicaudatus, O. microtis, O. nigripes, O. stramineus, Oligoryzomys moojeni, Oligoryzomys rupestris, O. fulvescens and O. vegetus are confirmed as valid species, whereas O. flavescens, Oligoryzomys magellanicus, Oligoryzomys griseolus, Oligoryzomys victus, Oligoryzomys andinus and Oligoryzomys arenalis need exhaustive revision. The sister species to all the remaining entities of the genus was O. microtis, suggesting an Amazonian origin for the genus.