Marine-derived fungi: diversity of enzymes and biotechnological applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Major, trace element and XRF data for sediments and fish tooth samples, from a shallow marine setting with varying redox conditions (Site U1356, Wilkes Land Margin, E.Antarctica)

Fossil fish teeth from pelagic open ocean settings are considered a robust archive for preserving the neodymium (Nd) isotopic composition of ancient seawater. However, using fossil fish teeth as an archive to reconstruct seawater Nd isotopic compositions in different sedimentary redox environments and in terrigenous-dominated, shallow marine settings is less proven. To address these uncertainties, fish tooth and sediment samples from a middle Eocene section deposited proximal to the East Antarctic margin at Integrated Ocean Drilling Program Site U1356 were analyzed for major and trace element geochemistry, and Nd isotopes. Major and trace element analyses of the sediments reveal changing redox conditions throughout deposition in a shallow marine environment. However, variations in the Nd isotopic composition and rare earth element (REE) patterns of the associated fish teeth do not correspond to redox changes in the sediments. REE patterns in fish teeth at Site U1356 carry a typical mid-REE-enriched signature. However, a consistently positive Ce anomaly marks a deviation from a pure authigenic origin of REEs to the fish tooth. Neodymium isotopic compositions of cleaned and uncleaned fish teeth fall between modern seawater and local sediments and hence could be authigenic in nature, but could also be influenced by sedimentary fluxes. We conclude that the fossil fish tooth Nd isotope proxy is not sensitive to moderate changes in pore water oxygenation. However, combined studies on sediments, pore waters, fish teeth and seawater are needed to fully understand processes driving the reconstructed signature from shallow marine sections in proximity to continental sources. This article is protected by copyright. All rights reserved.

Arginine vasotocin regulation of interspecific cooperative behaviour in a cleaner fish

In an interspecific cooperative context, individuals must be prepared to tolerate close interactive proximity to other species but also need to be able to respond to relevant social stimuli in the most appropriate manner. The neuropeptides vasopressin and oxytocin and their non-mammalian homologues have been implicated in the evolution of sociality and in the regulation of social behaviour across vertebrates. However, little is known about the underlying physiological mechanisms of interspecific cooperative interactions. In interspecific cleaning mutualisms, interactions functionally resemble most intraspecific social interactions. Here we provide the first empirical evidence that arginine vasotocin (AVT), a non-mammalian homologue of arginine vasopressin (AVP), plays a critical role as moderator of interspecific behaviour in the best studied and ubiquitous marine cleaning mutualism involving the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus. Exogenous administration of AVT caused a substantial decrease of most interspecific cleaning activities, without similarly affecting the expression of conspecific directed behaviour, which suggests a differential effect of AVT on cleaning behaviour and not a general effect on social behaviour. Furthermore, the AVP-V1a receptor antagonist (manning compound) induced a higher likelihood for cleaners to engage in cleaning interactions and also to increase their levels of dishonesty towards clients. The present findings extend the knowledge of neuropeptide effects on social interactions beyond the study of their influence on conspecific social behaviour. Our evidence demonstrates that AVT pathways might play a pivotal role in the regulation of interspecific cooperative behaviour and conspecific social behaviour among stabilized pairs of cleaner fish. Moreover, our results suggest that the role of AVT as a neurochemical regulator of social behaviour may have been co-opted in the evolution of cooperative behaviour in an interspecific context, a hypothesis that is amenable to further testing on the potential direct central mechanism involved.

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 10(9) to 10(10) cells/mL at the sediment surface to 10(7)-10(9) cells/mL below one meter depth. Based on CARD FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Recruitment patterns and processes of coastal fish species in a temperate rocky reef

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências do Mar, Especialização em Ecologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

The use of fluorescence in situ hybridization in the diagnosis of hidden mosaicism: apropos of three cases of sex chromosome anomalies

FISH has been used as a complement to classical cytogenetics in the detection of mosaicism in sex chromosome anomalies. The aim of this study is to describe three cases in which the final diagnosis could only be achieved by FISH. Case 1 was an 8-year-old 46,XY girl with normal female genitalia referred to our service because of short stature. FISH analysis of lymphocytes with probes for the X and Y centromeres identified a 45,X/46,X,idic(Y) constitution, and established the diagnosis of Turner syndrome. Case 2 was a 21-month-old 46,XY boy with genital ambiguity (penile hypospadias, right testis, and left streak gonad). FISH analysis of lymphocytes and buccal smear identified a 45,X/46,XY karyotype, leading to diagnosis of mixed gonadal dysgenesis. Case 3 was a 47,XYY 19-year-old boy with delayed neuromotor development, learning disabilities, psychological problems, tall stature, small testes, elevated gonadotropins, and azoospermia. FISH analysis of lymphocytes and buccal smear identified a 47,XYY/48,XXYY constitution. Cases 1 and 2 illustrate the phenotypic variability of the 45,X/46,XY mosaicism, and the importance of detection of the 45,X cell line for proper management and follow-up. In case 3, abnormal gonadal function could be explained by the 48,XXYY cell line. The use of FISH in clinical practice is particularly relevant when classical cytogenetic analysis yields normal or uncertain results in patients with features of sex chromosome aneuploidy. Arq Bras Endocrinol Metab. 2012;56(8):545-51

Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae) of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG. The results from the application of the 18S probe confirmed the previous data obtained by silver nitrate staining, identifying a simple nucleolar organizing region system for these species. However, the labeling results from the 5S rDNA probe demonstrated a difference in the number and localization of these sites between the analyzed species. The obtained data allowed inferences on the possible processes involved in the karyotypic evolution of this genus.

Analysis of marine bivalve shellfish from the fish market in Santos city, Sao Paulo state, Brazil, for Toxoplasma gondii

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, Sao Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of Sao Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Glycogen-accumulating organisms in laboratory-scale and full scale wastewater treatment processes

Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OPU (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92 % of the Q sludge bacteria and 28 % of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two fullscale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'.

Experimental evidence that partner choice is a driving force in the payoff distribution among cooperators or mutualists: the cleaner fish case

Supply and demand largely determine the price of goods on human markets. It has been proposed that in animals, similar forces influence the payoff distribution between trading partners in Sexual selection, intraspecific cooperation and interspecific mutualism. Here we present the first experimental evidence supporting biological market theory in it study on cleaner fish, Labroides dimidiatus. Cleaners interact with two classes of clients: choosy client species with access to several cleaners usually do not queue for service and do not return if ignored, while resident client species with access to only one cleaning station do queue or return. We used plexiglas plates with equal amounts of food to stimulate these behaviours of the two client classes. Cleaners soon inspected 'choosy' plates before 'resident' plates. This supports previous field observations that suggest that client species with access to several cleaners exert choice to receive better(immediate) service.

Geographic variation in the behaviour of the cleaner fish Labroides dimidiatus (Labridae)

Geographical variation in the outcome of interspecific interactions has a range of proximate ecological causes. For instance, cleaning interactions between coral reef fishes can result in benefits for both the cleaner and its clients. However, because both parties can cheat and because the rewards of cheating may depend on the local abundance of ectoparasites on clients, the interaction might range from exploitative to mutualistic. In a comparative analysis of behavioural measures of the association between the cleaner fish Labroides dimidiatus and all its client species, we compared cleaning interactions between two sites on the Great Barrier Reef that differ with respect to mean ectoparasite abundance. At Heron Island, where client fish consistently harbour fewer ectoparasites, client species that tended to pose for cleaners were more likely to receive feeding bites by cleaners than client species that did not pose for cleaners. This was not the case at Lizard Island, where ectoparasites are significantly more abundant. Client fish generally spent more time posing for cleaners at Lizard Island than their conspecifics at Heron Island. However, fish at Heron Island were inspected longer on average by cleaners than conspecifics at Lizard Island, and they incurred more bites and swipes at their sides per unit time from cleaners. These and other differences between the two sites suggest that the local availability of ectoparasites as a food source for cleaners may determine whether clients will seek cleaning, and whether cleaners will feed on parasites or attempt to feed on client mucus. The results suggest that cleaning symbiosis is a mosaic of different outcomes driven by geographical differences in the benefits for both participants.

Cleaner fish drives local fish diversity on coral reefs

Coral reefs are one of the most diverse habitats in the world [1], yet our understanding of the processes affecting their biodiversity is limited [1-3]. At the local scale, cleaner fish are thought to have a disproportionate effect, in relation to their abundance and size, on the activity of many other fish species, but confirmation of this species' effect on local fish diversity has proved elusive. The cleaner fish Labroides dimidiatus has major effects on fish activity patterns [4] and may indirectly affect fish demography through the removal of large numbers of parasites [5, 6]. Here we show that small reefs where L. dimidiatus had been experimentally excluded for 18 months had half the species diversity of fish and one-fourth the abundance of individuals. Only fish that move among reefs, however, were affected. These fish include large species that themselves can affect other reef organisms [2, 7]. In contrast, the distribution of resident fish was not affected by cleaner fish. Thus, many fish appear to choose reefs based on the presence of cleaner fish. Our findings indicate that a single small [8] and not very abundant [9] fish has a strong influence on the movement patterns, habitat choice, activity, and local diversity and abundance of a wide variety of reef fish species.

Allopodocotyle skoliorchis n. sp. (Opecoelidae: Plagiorporinae) from Parequula melbournensis (Castelnau) (Gerreidae), a temperate marine fish in Australian waters

A new species of Allopodocotyle Pritchard, 1966 is described from the intestine and pyloric caeca of Parequula melbournensis (Gerreidae) caught from the waters off South and Western Australia. The new species is distinguished from other species by its larger eggs, broader form, pre-bifurcal genital pore and a number of other measurable features that are discussed. Of the species that share morphological similarities with Allopodocotyle skoliorchis n. sp., it is the only species known from a gerreid; all the other species are from serranids.

A new species of Podocotyloides Yamaguti, 1934 (Digenea: Opecoelidae) from a Western Australian temperate marine fish

A new species of Podocotyloides is described from Sillago bassensis caught off the coast of Western Australia. This is the second report of a species of this genus from Australian waters but the first of a new species. P. victori n. sp. is one of four species whose vitelline follicles extend into the forebody. It is distinguished from the other three species with vitelline follicles in the forebody by its relatively shorter forebody, smaller eggs and bipartite seminal vesicle. Pedunculotrema Fischthal & Thomas, 1970 is reduced to synonymy with Podocotyloides Yamaguti, 1934.

Mapping multi-species habitat use for marine conservation planning

Tese de Doutoramento, Ciências do Mar (Biologia Marinha)