Late Oligocene to Recent benthic foraminifers from the northeastern North Atlantic

Deep-sea benthic foraminiferal faunas were studied from Sites 608 (depth 3534 m, 42°50'N, 23°05'W) and 610 (depth 2427 m, 53°13'N, 18°53'W). The sampling interval corresponded to 0.1 to 0.5 m.y. at Site 608 and in the sections of Site 610 from which core recovery was continuous. First and last appearances of benthic foraminiferal taxa are generally not coeval at the two sites, although the faunal patterns are similar and many species occur at both sites. Major periods of changes in the benthic faunas, as indicated by the numbers of first and last appearances and changes in relative abundances, occurred in the early Miocene (19.2-17 Ma), the middle Miocene (15.5-13.5 Ma), the late Miocene (7-5.5 Ma), and the Pliocene-Pleistocene (3.5-0.7 Ma). A period of minor changes in the middle to late Miocene (10-9 Ma) was recognized at Site 608 only. These periods of faunal changes can be correlated with periods of paleoceanographic changes: there was a period of sluggish circulation in the northeastern North Atlantic from 19.2 to 17 Ma, and the deep waters of the oceans probably cooled between 15.5 and 13.5 Ma, as indicated by an increase in delta18O values in benthic foraminiferal tests. The period between 10 and 9 Ma was probably characterized by relatively vigorous bottom-water circulation in the northeastern Atlantic, as indicated by the presence of a widespread reflector. The faunal change at 7 to 5.5 Ma corresponds in time with a worldwide change in delta13C values, and with the Messinian closing of the Mediterranean. The last and largest faunal changes correspond in time with the onset and intensification of Northern Hemisphere glaciation.

Benthic foraminifera off West Africa (1°N to 32°S)

Recent benthic foraminifera (> 125 µm) were investigated from multicorer samples on a latitudinal transect of 20 stations between 1°N and 32°S along the upper slope off West Africa. Samples were selected from a narrow water depth interval, between 1200 and 1500 m, so that changes in water masses are minimized, but changes in surface productivity are important and the only significant environmental variable. Live (Rose Bengal stained) benthic foraminifera were counted from the surface sediment down to a maximum of 12 cm. Dead foraminifera were investigated in the top 5 cm of the sediment only. Five live and five dead benthic foraminiferal assemblages were identified using Q-mode principal component analysis, matching distinct primary productivity provinces, characterized by different systems of seasonal and permanent upwelling. Differences in seasonality, quantity, and quality of food supply are the main controlling parameters on species composition and distribution of the benthic foraminiferal faunas. To test the sensitivity of foraminiferal studies based on the uppermost centimeter of sediment only, a comparative Q-mode principal component analysis was conducted on live and dead foraminiferal data from the top 1 cm of sediment. It has been demonstrated that, on the upper slope off West Africa, most of the environmental signals as recorded by species composition and distribution of the 'total' live and dead assemblages, i.e., including live and dead foraminifera from the surface sediment down to 12 cm and 5 cm, respectively, can be extracted from the assemblages in the top centimeter of sediment only. On the contrary, subsurface abundance maxima of live foraminifera and dissolution of empty tests strongly bias quantitative approaches based on the calculation of standing stocks and foraminiferal numbers in the topmost centimeter.

Sustained swimming performance and muscle structure are altered by thermal acclimation in male mosquitofish

Males of the eastern mosquito fish (Gambusia holbrooki) possess one of the widest reproductively active temperature ranges for any ectotherm, ranging across seasons from at least 18degreesC to 34degreesC. In this study, we tested the ability of male G. holbrooki to acclimate their sustained swimming performance following long-term exposure to 18degreesC or 30degreesC. We also investigated some of the possible physiological mechanisms associated with thermal acclimation responses in swimming performance, including changes in slow muscle fibre size and abundance and the expression of myosin heavy chains (MyHC). We found that U-crit, of 18degreesC-acclimated G. holbrooki was 20% greater at 18degreesC than 30degreesC-acclimated fish, and the Ucrit of the 30degreesC-acclimated group was more than 15% greater at 30degreesC. Slow, fast and intermediate muscle fibres were identified on the basis of their myosin ATPase staining reaction. Although the number of slow and intermediate muscle fibres was similar between groups, the total cross-sectional area of aerobic fibre types was 40% greater in 18degrees-than 30degreesC-acclimated fish, reflecting an increase in the average fibre diameter. An S58 antibody raised against chicken slow skeletal muscle myosin stained a sub-set of the slow fibres identified by myosin ATPase staining. The number of S58-positive muscle fibres was 50% greater in 30degreesC-than 18degreesC-acclimated fish, implying that different MyHCs are being expressed in cold and warm acclimated individuals. Given the sexually coercive mating system of this species, increases in the sustained swimming performance via thermal acclimation may benefit the ability of males to maintain a high rate of sneaky copulations. (C) 2004 Elsevier Ltd. All rights reserved.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaccae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins

The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved alpha-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.

Variations in cyclotide expression in Viola species

Cyclotides, a family of approximately 50 mini-proteins isolated from various Violaceae and Rubiaceae plants, are characterized by their circular peptide backbone and six conserved cysteine residues arranged in a cystine knot motif. Cyclotides show a wide range of biological activities, making them interesting targets for both pharmaceutical and agrochemical research, but little is known about their natural function and the events that trigger their expression. An investigation of the geographical and seasonal variations of cyclotide profiles has been performed, using the native Australian violet, Viola hederacea, and the Swedish sweet violet, Viola odorata, as model plants. The results showed that in the Australian violet the relative peptide levels of some cyclotides remained almost constant throughout the year, while other cyclotides were present only at certain times of the year. Therefore, it appears that V. hederacea expresses a basic armory of cyclotides as well as special add-ons whose levels are influenced by external factors. In the Swedish violet, cyclotide levels were increased up to 14 times during the warmest period of the year. The larger variation in expression levels of the Swedish plants may be a reflection of a greater climatic variation.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Two new hemiurine species (Digenea : Hemiuridae) from Spratelloides robustus Ogilby (Clupeiformes : Clupeidae) off south-western Australia and records of Parahemiurus merus (Linton, 1910) from Australian and New Caledonian waters

Two new species of hemiurine hemiurid are described from Spratelloides robustus off Woodman Point in southern Western Australia. Hemiurus lignator n. sp. differs from its congeners by a combination of similar-sized suckers, long sinus-sac, tandem testes, relatively elongate shape and unthickened seminal vesicle wall. Parahemiurus xylokopos n. sp. differs from its congeners in a combination of its squat form, its distinctly lobed vitellarium and the proximity of the gonads to the ventral sucker. P. merus (Linton, 1910) is reported from Acanthopagrus australis, Pomatomus saltatrix and Trachinotus coppingeri off northern New South Wales, Caranx sexfasciatus, Scorpis lineolata, Siganus nebulosus, Thunnus tonggol and T. coppingeri off southern Queensland, Cephalopholis boenak and Euthynnus affinis off Heron Island, southern Great Barrier Reef, P. saltatrix off southern Western Australia and Priacanthus hamrur off New Caledonia.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Temperature influences the coercive mating and swimming performance of male eastern mosquitofish

Although the functional consequences of temperature variation have been examined for a wide range of whole-animal performance traits, the implications of thermal variation for reproductive behaviour or performance are poorly known. I examined the acute effects of temperature on the mating behaviour and swimming performance of male eastern mosquitofish, Gambusia holbrooki, which rely on a coercive strategy to obtain matings and are routinely exposed to wide daily temperature fluctuations. Males showed reproductive behaviours across the entire test temperature range of 14-38 degrees C, representing one of the widest reproductively active temperature ranges for any ectotherm. Both the time spent in pursuit of females and the total number of mating attempts increased with temperature to a plateau that started at approximately 22-26 degrees C. However, males maintained a constant level of copulations at 18-34 degrees C, the temperature range they routinely experience in southeast Queensland. In contrast, maximum swimming performance and approach speeds during copulations were highly thermally dependent across this temperature range. Thus, acute temperature variation has important fitness implications for male G. holbrooki, but mating performance was significantly limited only at extreme temperatures. (c) 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.