Cannibalistic behaviour of rock-pool copepods: An experimental approach for space, food and kinship

Studies on cannibalism in harpacticoid copepods are restricted to predation on naupliar larvae in rock-pool harpacticoids of the genus Tigriopus. An earlier experimental study on the Mediterranean copepod Tigriopus fulvus indicated that females recognized their own larvae and preferentially preyed on nauplii other than their own. In a series of laboratory experiments, we tested if there were differences in naupliar predation as a function of crowding, food level and sex in Tigriopus brevicornis and T. fulvus. Results show that cannibalism was restricted to the first larval stages (N1 and N2). Both food availability and adult density significantly affected the predation rate. Contrary to earlier suggestions, adult males also preyed on the nauplii. We found no evidence that adults spare their own offspring, for neither T. fulvus nor T. brevicornis.This is in accordance with what one would expect for species having the life history characteristics of Tigriopus, i.e.: multiple broods and large number of offspring. Earlier results indicating parental care in Tigriopus must be taken with caution.     

The complete mitochondrial genome of the intertidal copepod Tigriopus sp. (Copepoda, Harpactidae) from Korea and phylogenetic considerations

This paper reports on the basic characteristics of the mitochondrial genome of the intertidal copepod Tigriopus sp. from Korea, including its structural organization, base composition of rRNAs and protein-encoding genes, and the secondary structure of tRNAs. We amplified the complete mitochondrial DNA of the intertidal copepod Tigriopus sp. from Korea (sampling site Busan) by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitogenome by primer walking using flanking sequences as sequencing primers. The primer informations were obtained as expressed sequence tags (ESTs) from Tigriopus sp. The resultant Tigriopus sp. mitochondrial DNA sequence was 14,301 bp with a conserved structural organization, compared to that of T. japonicus from Japan with significant differences in several protein-coding regions including rRNAs, although the genomic organization of the mitochondrial genome was identical. In order to investigate biogeographic differences within the genus Tigriopus, we analyzed the CO1 gene by sequencing. This way, we compared several Tigriopus species from Korea, Japan, Hong Kong and Taiwan as well as other related species such as T. californicus, T. brevicornis and T. fulvus. The results further support the notion that the copepods display significantly different genomes within the same genus. These findings provide valuable genomic information for further studies on the population genetics and speciation processes within the genus Tigriopus.     

Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria

The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are “orphan” effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness.     

Male-biased dispersal promotes large scale gene flow in a subterranean army ant, Dorylus (Typhlopone) fulvus

Sex-biased dispersal is a widespread phenomenon in the animal kingdom, which strongly influences gene flow and population structure. Particularly army ants, important key-stone predators in tropical ecosystems, are prone to population fragmentation and isolation due to their extraordinary mating system: queens are permanently wingless, propagate via colony fission, and only the males disperse in mating flights. Here we report on sex-biased dispersal and the genetic population structure of an African subterranean army ant, Dorylus (Typhlopone) fulvus. Using maternally inherited mtDNA markers and bi-parentally inherited nuclear microsatellites we found strong geographical structuring of mtDNA haplotypes, whereas the nuclear genetic population structure was less pronounced. Strong mtDNA (Φ _ST = 0.85), but significantly lower nuclear (F _ST = 0.23) genetic differentiation translated into a more than an order of magnitude larger male migration rate compared to that of queens, reflecting the low motility of queens and strong, promiscuous dispersal by males. Thus, the well flying D. fulvus males appear to be the sex to promote large scale gene flow, and D. fulvus is indeed a species in which sex specific dispersal patterns and the mating system profoundly affect the population structure and phylogeography.     

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

Isolation of Vibrio alginolyticus and Vibrio splendidus from Aquacultured Carpet Shell Clam (Ruditapes decussatus) Larvae Associated with Mass Mortalities

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.     

Effect of Water Extracts of Carob Pods, Tannic Acid, and Their Derivatives on the Morphology and Growth of Microorganisms

The effect of aqueous extracts of carob (Ceratonia siliqua) pods, gallotannic acid, gallic acid, and catechol on several microorganisms was studied. Carob pod extract and tannic acid showed a strong antimicrobial activity toward some cellulolytic bacteria. On the basis of tannin content, to which antimicrobial effect was related, carob pod extracts inhibited Cellvibrio fulvus and Clostridium cellulosolvens at 15 μg/ml, Sporocytophaga myxococcoides at 45 μg/ml, and Bacillus subtilis at 75 μg/ml. The inhibiting concentrations for tannic acid were found to be 12, 10, 45, and 30 μg/ml, respectively. Gallic acid and catechol were much less effective. Tannic acid and the tannin fraction of carob extract exerted both bacteriostatic and bactericidal effects on C. fulvus. Respiration of C. fulvus in the presence of bactericidal concentrations of tannic acid or tannin fraction of carob extract was inhibited less than 30%. A partial formation of “protoplasts” by C. fulvus was obtained after 2 hr of incubation in a growth medium to which 20% sucrose, 0.15% MgSO₄·7H₂O, and 10 to 50 μg/ml of tannic acid or 500μg/ml of penicillin, or both, had been added. Tannic acid and the tannin fraction of carob extract protected C. fulvus from metabolic lysis in sucrose solution. Although the growth of other microorganisms tested was only slightly affected, the morphology of some of them was drastically changed in the presence of subinhibitory concentrations of carob pod extracts of tannic acid. It is suggested that the site of action of tannins on sensitive microorganisms is primarily the cell envelope.     

A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels (Spermophilus: Rodentia, sciuridae)

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Changes in apparent water permeability during the moulting cycle in the western rock lobster

The kinetics of water exchange, as measured by ³H₂O fluxes, were examined in the western rock lobster, Panulirus longipes (Milne Edwards) during stages C₄, D₃, D₄, D₄-E and B₁ of the moulting cycle. A series of samples of blood and external water was taken during each experiment and the inward and outward rate constants found using the SAAM 25 computer program, assuming a simple 2-compartment model with reversible exchange. Both inward and outward turnover rates remained constant up to the swelling prior to ecdysis when there was an increase of 203% inwards compared with 133% outwards. The latter increased in stage B₁, so that both rates were comparably high, probably due to the increased permeability of the soft integument.Further analysis showed that the rock lobster does not behave as a single compartment with respect to tritiated water. Two compartments were resolved by graphical analysis, one with fast and the other with slow exchange. It is suggested that blood and tissue ‘free’ water comprise one compartment and chemically ‘bound’ water the other, with fast exchange between the free and bound water and relatively slow exchange between free body water and the external medium.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Quantitative study, identification and antibiotics sensitivity of someVibrionaceae associated to a marine fish hatchery

This study characterises the bacteria associated with a marine hatchery in Tunisian coastal marine waters. Presumptive vibrios (TCBS agar) and heterotrophic aerobic microflora (CFU) were studied at different stages within the hatchery: seawater, batches of algal cultures, rotifers andArtemia culture tanks. The bacterial strains were isolated on TCBS Agar plates and described using different bacteriological tests (standardised micromethods “API 20 E Strips”, exoenzymes production, growth at different temperatures, pH and salinity, vibriostatic agent O/129 and antibiotics susceptibility). Two dominant genera of bacteria were found (Vibrio andAeromonas) associated with some strains of thePseudomonadaceae family.Vibrio alginolyticus was the dominant bacteria (75% of total isolates) found in rotifers (Brachionus plicatilis) andArtemia cultures (Artemia salina). In larvae rearing tanks, an increase ofVibrionaceae was noted after larvae were fed withArtemia. Most of the studied bacteria used the skin mucus ofSparus aurata larvae as their sole source of carbon. All theV. alginolyticus strains were β-haemolytic, hydrolyse the DNA and were susceptible to several tested antibiotics.     

A new parasitic copepod species of Tautochondria (Siphonostomatoida: Hyponeoidae), found in the narrowmouthed catshark Schroederichthys bivius from the Strait of Magellan

Hyponeoidae is a copepod family that is rare around the world, with only three species described until now. Recently, a hyponeoid copepod was found in the narrowmouthed catshark, Schroederichthys bivius, from Chile, which has not been formally described. The objective of this study is to describe morphologically a new species of Hyponeoidae, from samples of the Strait of Magellan, Southern Chile. Also, a genetic analysis, based on the COI gene, was used to determine the relatedness of the new hyponeoid species to other copepods from Siphonostomatoida, which were available in the GenBank platform. The new species belongs to the genus Tautochondria and is here described as T. magellanica n. sp. This species differs from T. dolichoura mainly in the presence of long process at each side of the buccal cone, the absence of lobes on the head and short processes on the genital complex. According to the genetic sequences, T. magellanica n. sp. was not closely related to any other species. Therefore, this result confirms that Hyponeoidae is a separate family. However, the relatedness to other genera in Siphonostomatoida is still unknown.     

Doridicola indistinctus n. sp. (Copepoda: Poecilostomatoida: Rhynchomolgidae) associated with the soft coral Gersemia fruticosa Sars (Octocorallia: Alcyonacea: Nephtheidae) from the White Sea

A new species of poecilostomatoid copepod, Doridicola indistinctus n. sp. (Rhynchomolgidae), is described from specimens found in association with the soft coral Gersemia fruticosa Sars (Alcyonacea: Nephtheidae), collected from the White Sea. The new species is distinguished from its congeners by the combination of the following features in the female: (i) antenna tipped, with two subequal large claws which are about as long as the segment bearing them; (ii) two naked, extremely unequal setae on the middle segment of the maxilliped, the short, medial seta less than half-length of the outer seta; and (iii) free segment of leg 5 bears the basal swelling and is ornamented with spinules on the outer surface. This is the first report of a copepod occurring in symbiosis with nephtheid corals from the Arctic Zone. It also constitutes the northernmost record for a species of Doridicola Leydig, 1853, which is the largest genus of the Rhynchomolgidae Burmeister, 1835 comprising 52 species, including the present new species.     

Inhibition of carotenoid synthesis in Myxococcus fulvus (Myxobacterales)

1. The inhibitory effects of CPTA, nicotine, DPA, and San 6706 on carotenogenesis in Myxococcus fulvus were investigated.
2. The effects of CPTA, D-nicotine, and L-nicotine were very similar. The action of the drugs wasadditive. The cyclization was inhibited at low doses, the introduction of the hydroxyl group at C-1′ at higher doses. Lycopene accumulated at high drug concentration. The mode of action of the inhibitors is discussed.
3. In a carotenoid mutant of M. fulvus a stimulation of the “7,8-dehydrogenase” by CPTA was observed.
4. The specific carotenoid content of bacteria was increased by DPA due to an enhanced formation of phytoene. At low doses of DPA small amounts of an intermediate carotenoid glucoside ester, a 7,8-dihydro derivative, were detected.
5. DPA was taken up by the plasma membrane. Quantitative removal of DPA by washing was not possible.
6. San 6706 specifically and reversibly blocked the desaturation of phytoene.

     

Distribution of some virulence related-properties of Vibrioalginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibriocholerae virulence genes

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibrio cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.     

Pathogenic analysis of Vibrio alginolyticus infection in a mouse model

Vibrio alginolyticus is a Gram-negative halophilic bacterium and has been recognized as an opportunistic pathogen to both humans and marine animals. So far, most studies have been focused on marine animals and few reports have been aimed at mammals, including human. In this study, we first established a mouse model to understand the pathogenic mechanism of V. alginolyticus infection. After infection via intraperitoneal injection, hematological and liver function indicators were evaluated and serum interleukin (IL)-1β and IL-6 expression were detected by ELISA. Furthermore, we compared the virulence of two V. alginolyticus strains, ATCC17749T and E0666. The results demonstrated that V. alginolyticus infection causes robust lung and liver damage and induces changes in IL-1β, IL-6, hematological, and liver indicators. In addition, the ATCC17749T strain appeared to be more virulent than the E0666 strain. Better understanding of the pathogenic mechanism of V. alginolyticus infection should guide effective prevention and therapy for V. alginolyticus infection.     

Flexibacter elegans and Myxococcus fulvus: Aerobic gram-negative bacteria containing menaquinones as the only isoprenoid quinones

The aerobic Gram-negative gliding bacteria, Flexibacter elegans (Cytophagales) and Myxococcus fulvus (Myxobacterales) contain exclusively menaquinones as isoprenoid quinones. Each strain produces 3 different menaquinones. The menaqinone content of Flexibacter elegans was 1.5 μmol/g dry cells, with MK-7 as the main compound (>99%). MK-7 was identified by mass and n.m.r. spectroscopy. Myxococcus fulvus contained 2.1 μmol menaquinones per g dry mass, with more than 90% being MK-8.     

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.