Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

Synaptonemal Complex Analysis in Oocytes and Spermatocytes of Threespine Stickleback Gasterosteus Aculeatus (Teleostei, Gasterosteidae)

A surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of stickleback, Gasterosteus aculeatus, in order to visualize the process of chromosome synapsis. The mean SC length was 150±18m in three males and 143±12m in one female analyzed. A representative SC karyotype with 21bivalents was also presented. Each SC had lateral elements of equal length. No bivalent displaying the atypical synaptic behaviour which is often associated with heteromorphic sex chromosomes was observed neither in males nor in the female analyzed.     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

Seed dispersal curve of a Mediterranean myrmecochore: Influence of ant size and the distance to nests

Euphorbia characias is a Mediterranean spurge with a diplochorous dispersal system: after a ballistic dispersal that scatters the seeds, some ant species find and retrieve the seeds to their nest (myrmecochorous dispersal). The seed dispersal curve generated by ants in an abandoned field was described and partitioned according to ant size and to the distance to nest entrance from where seeds fell after ballistic dispersal. Both variables (ant size, distance to nest) affected dispersal distance. The seed dispersal curve showed a peak at short distance (median=1m) and a tail extending to 9.4m. The peak and the tail are explained differently. Short distances are usually generated by small ants (Pheidole pallidula and Tapinoma nigerrimum; 0.56±0.41m [n=48]) both from the nearest or farther nest entrances. The tail of the curve is generated disproportionately by big ants (Aphaenogaster senilis and Messor barbarus; 2.09±1.71m [n=61]) from farther nests. Seeds have a much greater probability (P=0.734) of being transported to nests which are not the nearest. This effect is largely due to transportation by big ants.     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Biology and Egg Morphology of The Dalmatian Barbelgudgeon Aulopyge Huegeli, An Endangered Endemic Species in Croatia

This paper describes patterns of annual and diurnal activity in the endangered endemic cyprinid Aulopyge huegeli from Croatia. The species is largely nocturnal. Spawning in the laboratory begins at temperatures of 20°C. Aulopyge huegeli is a rock and gravel brood hider. The female deposits eggs in various fissures with a special ovipositor. The eggs are whitish and have a diameter between 1.5 and 2mm. The zona radiata is relatively thin (3.2–5.5m) and provides the eggs with the necessary plasticity. The surface is covered with an attaching substance and with short attachment extension around the vegetal pole. This correlates with features of the spawning microhabitats. Aulopyge huegeli has a very small micropyle which consists of a pit with a diameter of 7.5m and a canal with a diameter of 2.5m (micropyle type I).     

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of <Emphasis Type="Italic">npt</Emphasis>II-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20M paromomycin was used, although up to 40M streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7M kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.     

Inoculation of sugar mill by-products compost with N2-fixing bacteria

Inoculation of sugar mill by-products compost with N₂-fixing bacteria may improve its quality by increasing total N and available P. Compost was inoculated with Azotobacter vinelandii(ATCC 478), Beijerinckia derxii (ATCC 49361), and Azospirillumsp. TS8, each alone and all three together. Numbers of all N₂-fixing bacteria in compost declined from an initial population of 5×10⁵cellsg^–1 during incubation. The population of Azotobacter declined to approximately 2×10²cellsg^–1 and the population of Beijerinckia and Azospirillum declined to approximately 9×10³ and 3.5×10⁴cellsg^–1 respectively, at day 50. Inoculation with N₂-fixing bacteria increased acetylene reduction, total N by 6–16 and available P by 25–30% in comparison to the uninoculated control. Increasing the N content and P availability of compost increases its value and there may be additional benefit from providing N₂ fixing bacteria.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Relations between hydrological variables and year-class strength of sportfish in eight Florida waterbodies

Hydrological variables have influenced fish recruitment in lakes, reservoirs and rivers. We evaluated how annual and seasonal hydrological variables were related to year-class strength (i.e., residuals from catch curves) of sportfish across eight Florida waterbodies (four rivers and four lakes). Multiple regression equations computed for black bass Micropterus spp. were combined across rivers and year-class strength was negatively related to spring median flow rates and in some cases positively related to winter median flow rates (all p0.10). Conversely, Lepomis spp. residuals combined from the rivers indicated that year-class strength was positively related to median flow rates in the fall prior to spawning and negatively related to post-spawn fall median flow rates (all p0.10). Fish recruitment combined across lakes were not related to water levels in this study, although within lake relationships did occur in some instances. Ecological implications of this work include regulations such as minimum flows and levels (MFLs) regarding sportfish species. Impacts of hydrology on year-class strength of sportfish were stronger in rivers than in lakes for these Florida systems. High flows at least once every 3years in the fall may allow inundation of floodplain habitat, providing favorable environmental conditions for Lepomis spp. reproduction. Setting MFLs during periods of drought (i.e., 3years or more) should consider impacts to short-lived species such as Lepomis spp.     

Effect of irradiance, temperature and salinity on growth and toxin production by Nodularia spumigena

The object of this work was to determine, using a full-factorial experiment, the influence of temperature, irradiance and salinity on growth and hepatotoxin production by Nodularia spumigena, isolated from Lake Alexandrina in the south-east of South Australia. Higher levels of biomass (determined as particulate organic carbon, POC), toxin production and intracellular toxin concentration per mg POC were produced under light limited conditions (30 mol m^–2 s^–1) and at salinities equal to or greater than those experienced in Lake Alexandrina. Both highest biomass and total toxin production rates were recorded at temperatures equal to or greater than those of the lake (20 and 30°C). The temperature at which maximum biomass and toxin production was recorded decreased from 30°C for cultures grown at 30 mol m^–2 s^–1 to 20°C when grown at 80 mol m^–2 s^–1. In contrast, intracellular toxin per mg POC was highest at the lowest growth temperature, 10°C, at both 30 and 80 mol m^–2 s^–1. It appears that the optimum temperature for biosynthetic pathways used in the production of toxin is lower than the optimum temperature for those pathways associated with growth. Intracellular toxin levels were higher in cells cultured at 10°C/30 mol m^–2 s^–1 whereas the majority of the toxin was extracellular in cells grown at 30°C/30 mol m^–2 s^–1. This implies that the highest concentration of toxin in lake water would occur under high temperature and high irradiance conditions. Individual environmental parameters of salinity, irradiance and temperature were all shown to influence growth and toxin production. Notwithstanding, the overall influence of these three parameters on toxin production was mediated through their effect upon growth rate.     

Light utilization and competition between Echinacea purpurea, Panicum virgatum and Ratibida pinnata under greenhouse and field conditions

Competitive interactions were compared under field and greenhouse conditions for three representative tallgrass prairie species, Echinacea purpurea, Panicum virgatum and Ratibida pinnata. These were planted in monoculture and in mixtures of two species using a replacement series design with groups of four or eight plants. Competition was determined from shoot dry weight data collected during 120days in the greenhouse and after 415days in the field. Yields declined with increased density of a single species in the greenhouse from 40days onward and in the field. Relative yields were up to 100% higher in mixtures than in monocultures for all species early on in the greenhouse experiment. Later in the experiment and in the field relative yields of E.purpurea decreased in the presence of P.virgatum and R.pinnata, whereas relative yields of these two species increased in the presence of E.purpurea. There were correlations in relative yield between the field and the greenhouse experiment at 80 and 120days. In the greenhouse P.virgatum maintained higher net assimilation rates than the other species. Relative growth rates of all species were higher in monoculture and in mixtures up to 40days, after which they declined, especially for E.purpurea in mixtures. In the field, higher light intensities occurred in pure stands of E.purpurea than in mixed stands with other species. The order of competitive ability that was apparent from these field and greenhouse studies, P.virgatum=R.pinnata>E.purpurea, could be partially explained by photosynthetic rates in relation to canopy light interception.     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

Life Cycle of the Tick Haemaphysalis Leporis-palustris (Acari: Ixodidae) Under Laboratory Conditions

The life cycles of two separate populations (colonies A and B) of the rabbit tick, Haemaphysalis leporis-palustris, were studied under laboratory conditions. Domestic New Zealand rabbits, Oryctolagus cuniculus, and wild rabbits, Sylvilagus brasiliensis, were used as hosts for ticks from colony B and only O. cuniculus rabbits were used as hosts for ticks from colony A. Developmental periods were observed in an incubator at 27±1°C and RH 90±5%. Larvae from colonies A and B fed for 8.0±3.7 days and 8.5±1.3 days, respectively, on O. cuniculus. On S. brasiliensis larvae from colony B fed for 7.2±1.3 days. Nymphs from colony A fed for 8.1±1.4 days on O. cuniculus and nymphs from colony B fed for 8.1±1.0 days on S. brasiliensis. Only one engorged nymph from colony B was recovered from O. cuniculus. Females from colony A fed for 20.9±5.9 days on O. cuniculus and females from colony B fed for 18.6±2.4 days on O. cuniculus and 18.7±3.7 days on S. brasiliensis. Engorged larvae from colony A required 13.7±3.7 days to molt while engorged larvae from colony B required 11.8±3.0 and 11.5±1.8 days to molt, after having fed on O. cuniculus and S. brasiliensis, respectively. Engorged nymphs from colonies A and B required 16.3±1.9 days and 14.7±1.4 days to molt, respectively. Engorged females from colonies A and B required 4–7 and 3–5 days, respectively, to start oviposition. Mean egg incubation periods lasted for 33–34 days. For ticks from colony B, host species accounted for significant differences (p<0.05) in larval and nymphal feeding periods, oviposition weights and CEIs. Significant differences (p<0.05) between the two colonies when ticks fed on O. cuniculus were observed for larval and nymphal feeding and premolt periods, engorged female and oviposition weights and conversion efficiency indexes (CEI). S. brasiliensis were always a more suitable host for H. leporis-palustris than O. cuniculus. Significantly more larvae and nymphs engorged and molted when fed on S. brasiliensis (p<0.001). Females fed S. brasiliensis were more successful to lay fertile eggs and showed the highest engorged and egg mass weights, and the highest CEIs. Data of H. leporis-palustris fed on wild rabbits (one of its natural host species) are reported for the first time.