Relationships between larval nutritional experience, larval growth rates, juvenile growth rates, and juvenile feeding rates in the prosobranch gastropod Crepidula fornicata

Planktonic larvae experiencing short periods of starvation or reduced food supply often grow and develop more slowly, have poor survival, fail to metamorphose, metamorphose at smaller sizes, or grow slowly as juveniles. In this study, we examined the impact of short periods of food limitation at various stages of larval development on larval and juvenile growth in Crepidula fornicata. In addition, we considered whether juveniles that were stressed as larvae grew poorly because of reduced rates of food collection due to impaired gill function. For 5 experiments, larvae were either starved for several days beginning within 12 h of hatching or were starved for the same number of days following 1 or more days of feeding at full ration (cells of the naked flagellate Isochrysis galbana, clone T-ISO, at 18×10⁴ cells ml⁻¹). In one experiment, larvae were transferred for 2 or 4 days to seawater with extremely low phytoplankton concentration (1×10⁴ cells ml⁻¹). In all experiments, larvae were returned to full ration following treatment. Control larvae were fed full ration from hatching to metamorphosis. When larvae reached shell lengths of about 900 μm they were induced to metamorphose and then reared individually at full ration in glass bowls, with phytoplankton suspension replenished daily. Larval and juvenile growth rates were determined by measuring changes in shell length (longest dimension) over time. Juvenile feeding rates were determined by monitoring changes in phytoplankton concentration over 2–3 h at the end of the growth rate determinations. In general, larval growth rates for the first 2 days after the resumption of feeding were inversely proportional to the length of time that larvae were starved. However, larval growth rates ultimately recovered to control levels in most treatments. Starving the larvae caused a significant reduction in initial juvenile growth rates (first 3–4 days post-metamorphosis) in most experiments even when larval growth rates had recovered to control levels prior to metamorphosis. Juvenile growth rates were not significantly reduced when larvae were subjected to reduced food availability (1×10⁴ cells ml⁻¹), even for treatments in which larval growth rates were compromised. Mean weight-specific filtration rates for juveniles were significantly reduced (p<0.05) following larval feeding experience in only one or possibly 2 of the 4 experiments conducted. Our data suggest that although larvae of C. fornicata may fully recover from early nutritional stress, the resulting juveniles may exhibit poor initial growth due to impaired gill function, reduced digestive capability, or reduced assimilation efficiency.     

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Growth hormone (GH, 0.0025 and 0.025 I.U. ml⁻¹) and γ-aminobutyric acid (GABA, 50 μg ml⁻¹) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10⁶ Nannochloropsis oculata cells ml⁻¹ or at low free ammonia level of 2.4 μg ml⁻¹, the F₁ offspring of rotifers treated with GH at 0.0025 I.U. ml⁻¹ had significantly higher population growth rate (r) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10⁵ cells ml⁻¹ or at high free ammonia level of 3.1 μg ml⁻¹, rotifers treated with GABA at 50 μg ml⁻¹ had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F¹ and F² generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.     

Feeding on dinoflagellates by intermediate and late stage crab zoeae raised in the laboratory and collected from the field

Hatching stage crab larvae will ingest algae, including non-toxic and toxic dinoflagellates. We determined that later zoeal stages, obtained from both laboratory-raised larvae and natural assemblages, also ingest dinoflagellates and we measured the effects of prey density, prior feeding history and time of exposure to prey on incidence of ingestion. Both stage 1 and later stage larvae exposed to algal prey were examined using epifluorescence for the presence of chl a. Both stage 1 and stage 3 laboratory-raised Cancer oregonensis (Dana) and Hemigrapsus nudus (Dana) ingested both the non-toxic dinoflagellate Prorocentrum micans Ehrenberg and the toxic Alexandrium andersoni Balech, with no difference between the stages. Both species showed higher ingestion of P. micans than A. andersoni. Ingestion of both prey types occurred at prey densities as low as 200 cell ml^− 1 in C. oregonensis and 50 cells ml^− 1 in H. nudus. Samples collected in summer, 2004, provided both stage 1 and late stage Lophopanopeus bellus (Stimpson); stage 1, intermediate, and late stage Fabia subquadrata Dana; and an unidentified porcellanid. Stage 1 L. bellus ingested both prey, while late stage zoeae did not, although the latter apparently were not actively feeding. F. subquadrata fed on both prey, with no difference between early and late larvae. Both stages ingested P. micans more readily than A. andersoni. First evidence of ingestion of P. micans at 600 cells ml^− 1 occurred after only 0.5 h, while it took 2 h for ingestion at 50 cells ml^− 1. The model of larval feeding involving both omnivory and prey discrimination described previously for the hatching stage is sustained throughout zoeal development and is, perhaps, an adaptation to an uncertain prey environment, one that trades opportunism for inefficiency.     

Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae

Since the collapse of populations of northern quahogs (hard clam), Mercenaria mercenaria, in Long Island bays, brown tide blooms have been proposed to pose a barrier to recovery. We tested whether the brown tide alga, Aureococcus anophagefferens, affects survivorship, development or growth in the larvae of M. mercenaria. There was no effect of A. anophagefferens (clone CCMP1708) on survivorship of hard clam larvae, even at bloom concentrations. Under most experimental conditions, larvae fed a mixed diet of Isochrysis galbana (T-Iso) and A. anophagefferens or a single species diet of A. anophagefferens, developed faster than those fed a single species diet of Isochrysis. A mixed diet of I. galbana and A. anophagefferens either had no effect on larval growth, or produced enhanced growth at moderate cell densities (8 × 10⁴ cells ml⁻¹ of A. anophagefferens). Similarly, moderate cell densities of a single food diet of A. anophagefferens (1.6 × 10⁵ cells ml⁻¹) generally had no effect on the growth of larvae. When fed bloom concentrations (10⁶ cells ml⁻¹) of A. anophagefferens, larvae developed faster, but growth was reduced, compared to those fed an equal biovolume of Isochrysis. Larvae fed slow growing or near stationary phase cultures of A. anophagefferens experienced reduced growth and slowed development. These data suggest a qualitative difference between slow or stationary phase and fast growing cultures of the brown tide alga. They also suggest that impacts of A. anophagefferens, when present, are likely to be due to the nutritional quality of this alga as a food source for hard clam larvae, which could have a lasting legacy through ontogeny. Additional studies are needed to test whether our findings apply to more recently isolated strains of A. anophagefferens.     

Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas

The shore crab, Carcinus maenas L. (Portunidae), is a coastal and estuarine species, which can live and reproduce under brackish water conditions; freshly hatched larvae have been observed in the field at salinities below 15‰. In the present laboratory study, the tolerance of hypo-osmotic stress was experimentally investigated in early larvae of a marine (North Sea) population of C. maenas reared at four different salinities (15, 20, 25, 32‰). Two and 4 days after hatching, the Zoea I larvae were moult-staged microscopically, and their rates of respiration and growth (changes in dry weight, W, carbon, C, nitrogen, N, and hydrogen, H) were measured. Survival and development were monitored until the megalopa was reached: 15‰ did not allow for development beyond the first zoeal stage, while metamorphosis to the megalopa was reached at salinities ≥20‰. At 20‰, development was significantly delayed and mortality enhanced as compared with 25 and 32‰. Rates of growth and respiration decreased during exposure to reduced salinities ≤25‰. Hence, the suppression of growth could not be explained as a consequence of enhanced metabolic losses per larva. Instead, a partial C budget indicates that the Zoea I larvae suffered from decreased capabilities of assimilating ingested and subsequently converting assimilated matter to tissue growth. Net growth efficiency (K₂, C-based) was at 25 and 32‰ initially high (>60% during the postmoult and intermoult stages of the Zoea I moult cycle), but decreased during the later stages (down to ≤30% in premoult). An inverse pattern of C partitioning was observed at ≤20‰, with initially low K₂ values (≤21% during the first 2 days of the moult cycle), and a later increase (up to ≥46% in premoult). Thus, larval growth was initially suppressed under conditions of reduced salinity, but this was later (during premoult) partially compensated for by an increase in C assimilation and K₂. Our observations indicate that Zoea I shore crab larvae react during the late stages of their moulting cycle less sensitively against reduced salinities than during postmoult and intermoult. This suggests that the transition between moult cycle stages C and D₀ may be a critical point for effects of hypo-osmotic stress, similarly as already known in relation to effects of nutritional stress. Negative effects were found also when freshly hatched Zoea I shore crab larvae were exposed only transitorily (for 24–72 h) to 20‰, with significantly lower rates of survival, development, growth, respiration, and K₂. These effects increased with increasing duration of initial exposure to reduced salinity.     

An in vitro Cr release assay for Ascaris suum infective larvae

This report describes the use of ⁵¹Cr release as a measure of the viability of Ascaris suum inefective larvae after treatment with sodium hypochlorite or kalafungin. Inefective larvae of A. suum were treated with varying dilutions of these compounds and percent ⁵¹Cr release and percent visual larval damage were recorded. An 8% solution of sodium hypochlorite released >90% of the ⁵¹Cr and produced >90% visual larval damage. Kalafungin released >32% ⁵¹Cr and produced >60% visual larval damage at a concentration of 1 μg·ml⁻¹. The results demonstrate that both assays are equal indicators of the viability of A. suum infective larvae when the cytotoxic agent is sodium hypochlorite. Kalafungin, an inhibitor of mitochondrial function, is detected more efficiently using percent visual larval damage when compared to ⁵¹Cr release.     

Zooplankton feeding ecology: bacterivory by metazoan microzooplankton

Bacterivory by the rotifer Brachionus plicatilis Müller, nauplii and copepodites of the copepods Centropages Krøyer sp. and Acartia tonsa Dana, and the tintinnid Favella panamensis Kofoid & Campbell was examined using fluorescently labelled bacteria (FLB) and epifluorescence microscopy. FLB were < 1 μm in diameter, and were offered at environmental concentrations (1.47−9.08 × 10⁶ cells·ml⁻¹). FLB were visible within rotifers, nauplii, copepodites, and tintinnids, confirming ingestion. Rotifer clearance rates (32–418 μl·animal⁻¹·h⁻¹) exhibited no relation with FLB concentration. In some cases rates of clearance of FLB by rotifers were different with alternative phytoplankton food (Nanochloris Naumann sp.) than in replicates with FLB alone, whereas in other cases presence of alternative food exhibited no clear effects on rates of ingestion of FLB. Clearance rates for all six naupliar stages of A. tonsa nauplii (0–320 μl·animal⁻¹·h⁻¹) were stage-related, with higher rates by NIII-VI nauplii than NI-II nauplii. Nauplii had higher rates of clearance of FLB in the absence of alternative phytoplankton food (Isochrysis Parke sp.). Clearance rates of FLB by a single stage of Centropages sp. nauplii, A. tonsa CI copepodites and F. panamensis (each obtained at only a single food concentration of either 1.5 or 5.0 × 10⁶ cells·ml⁻¹) were within the range of 85–142 μl·animal⁻¹·h⁻¹. These ranges were similar to those of rotifers and A. tonsa nauplii. This is the first report of FLB ingestion by metazoan marine microzooplankton. Although rotifers and ciliates might be expected to ingest small particles such as FLB using ciliary induced feeding currents, the means by which nauplii and copepodites eat FLB is less clear. We propose that they may “eat” bacteria as they “drink” to osmoregulate.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation

Experiments were carried out to evaluate the influence of rearing temperature and food concentration (20 and 30 °C, 1×10⁵ and 2×10⁵ cells ml⁻¹) on the starvation threshold and nucleic acid content of the larvae of Balanus amphitrite. The larvae were also field-reared using micro-enclosures. Laboratory-reared larvae were larger in size than the field-reared larvae. An increase in size, DNA content and instar index of the starved II instar larvae was observed indicating that the absence of food may not be fatal to this early instar. The temperature at which larvae were raised and the food concentration had variable influence on the capacity to withstand starvation. Exposure to increased temperatures during starvation eliminated the effect of doubling food concentration during their feeding period prior to starvation. The larvae reared at 20 °C had comparatively lower nucleic acid content. The laboratory-reared larvae had ca. 1.7 times greater RNA:DNA ratio than larvae raised at comparable temperature in the field.     

Biostoning of denims by Penicillium occitanis (Pol6) cellulases

The Pol6 mutant of Penicillium occitanis, secreting a large quantity of cellulases, was cultivated in fermentor using a local paper pulp as an inducer substrate. A high titer of extracellular cellulase activity was reached after a fed batch process: 23 IU ml⁻¹ filter paper activity, 21 IU ml⁻¹ CMCases activity (endoglucanase units) and 25 mg ml⁻¹ of proteins. Various tests were done to compare the action of the P. occitanis cellulases with those commercially available and with the traditional stonewashing process. This cellulase preparation was successfully applied in a biostoning process at an industrial scale. The abrasive effect of the P. occitanis cellulases was very uniform and with an efficiency comparable to that obtained by the commercial ones.     

Differential susceptibility of marine invertebrate larvae: Laboratory predation of sand dollar, Dendrasterexcentricus (Eschscholtz), embryos and larvae by zoeae of the red crab, Cancer productus Randall

Predation on eggs, embryos, and larvae of the sand dollar, Dendraster excentricus (Eschscholtz) was investigated in a series of laboratory feeding experiments. Dendraster susceptibility to predation by zoea larvae of the red crab, Cancer productus Randall was strongly dependent on developmental stage and ontogenetic differences in motility. Clearance rates by C. productus were highest for eggs and averaged 0.551·zoea⁻¹·day⁻¹. Embryos and prism larvae of Dendraster were consumed at an intermediate rate, while pluteus larvae were captured at a relatively low rate. Clearance rates decreased from 0.18 to 0.031·zoea⁻¹·day⁻¹ during the transition of prism larvae into echinoplutei. Differences in Dendraster susceptibility to predation cannot be attributed to increasing prey body size because dwarf plutei were captured at the same rate as normal plutei. Reduced capture rates by Cancer productus zoeae are dependent on the development of Dendraster swimming behavior. Periodic reversals in the direction of ciliary beating and backwards swimming effectively remove Dendraster plutei from the immediate capture sphere of Cancer productus. Reversed swimming appears to function as a post-contact encounter response that reduces the mortality rates of Dendraster plutei.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Early development of milkfish: effects of salinity on embryonic and larval metabolism, yolk absorption and growth

During embryogenesis of Chanos chanos , more than half of the yolk was consumed and the majority of it was converted into larval tissue. Salinity affected both yolk absorption and embryonic and larval growth. Larvae hatched in 20% had larger yolk reserves but were smaller and grew more slowly than larvae in 35 and 50%. Larvae hatched in 35 and 50% had equal amounts of yolk but those from 35% were larger. Oxygen consumption rates increased during development (from 0.06 ± 0.01 μl O₂ egg^–1 h^–1 by blastulae to 0.37 ± 0-01 μl O₂ egg^–1 h^–1 by prehatch embryos and 0–43 ± 0–03 μl O₂ larva ^–1 h ^–1 by newly-hatched larvae) and were significantly affected by salinity. Eggs and yolk-sac larvae incubated in 35% consumed more oxygen than those in the low and high salinities. Salinity affected both the rate and pattern of yolk utilization but salinity-related differences in metabolism, yolk absorption, and growth were not related directly to the osmotic gradient. Low salinity retarded yolk absorption while high salinity reduced yolk utilization efficiencies. Differences in oxygen consumption rates were probably related to variations in the relative amounts of metabolically active embryonic and larval tissue and/or higher activity levels rather than differential osmoregulatory costs. 35% is probably the most suitable salinity for incubation and larval rearing of milkfish.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Effects of larvae ontogeny, turbidity, and turbulence on prey attack rate and swimming activity of Atlantic herring larvae

The effect of turbulence, light level, and ontogeny on herring larva's attack rate and swimming activity was tested in a previous study. However, during larval seasons (spring and autumn), water clarity is frequently impaired by alga blooms, which also most probably will affect larva feeding rate. Therefore, this study was to investigate the effects of turbidity, turbulence, and ontogeny on the attack rate and swimming activity of herring larvae. By adding diatomaceous earth (DE) to the water, three turbidity levels were established: 0, 35, and 80 Jackson Turbidity Unit [JTU; which coincide with a beam attenuation (c) of 0.1, 2.5, and 4.8 m⁻¹, respectively]. An unfavourable (8×10⁻⁶ W/kg) and a favourable turbulence level (1×10⁻⁶ W/kg) were chosen based on results from the earlier study. The results show that intermediate turbidity (35 JTU) has a positive effect on the attack rate of smaller larvae (20 mm), while high turbidity (80 JTU) has a negative effect on attack rate of all tested larvae size groups. In general, attack rate was lower at the highest turbulence compared to the low level, independent of turbidity level. However, there was one exception, when turbidity was at the highest, the largest larvae (29 mm) seemed to gain from feeding in the highest turbulence level. The overall activity level was higher in the presented study than in the earlier study without turbidity. The favourable turbidity level (35 JTU) coincides with turbidity levels normally found at the equivalent depth during spring and autumn blooms in the area of where the experimental larvae originate. In addition, turbidity's effect on light absorbtion and how it influences the maximum feeding depth of the larva are discussed.     

Ciliates and their picophytoplankton-feeding activity in a high-altitude warm-monomictic saline lake

The impact of feeding on autotrophic picoplankton (APP) on the ciliate composition of the assemblage was surveyed monthly along a depth gradient in the maar crater, athalassohaline, warm monomictic Lake Alchichica (Puebla, Mexico) from June 2003 to December 2005. Numbers of APP were evaluated from their autofluorescence. DAPI staining and the Fluorescently Labeled Bacteria technique were employed to count ciliates and estimate their feeding rates. A total of 38 taxa of ciliates have been identified using Quantitative Protargol Staining. Peritrichs followed by minute spirotrichs (particularly Halteria grandinella) often numerically dominated the ciliate assemblage and emerged as the most efficient APP feeders. A maximum of 54 ciliate cells ml⁻¹ was observed in the surface layer at the end of the mixing period, during the development of diatoms (Cyclotella alchichicana), the cyanobacterial bloom (Nodularia sp.) and its decay. Vorticellids (Pelagovorticella natans, Vorticella sp.) had the highest APP uptake (median 130 APP cil⁻¹ h⁻¹). Mixotrophic Euplotes cf. daidaleos were important APP grazers near the oxycline. Scuticociliates (Cyclidium glaucoma, Uronema nigricans and an anaerobic cf. Isocyclidium globossum), were numerically dominant within the hypolimnetic assemblages and did not ingest APP. Generally, APP were not an important food source for the majority of the ciliate assemblage, being positively selected by a few species during the APP decay in aerobic and microaerobic conditions.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.     

Identification of gonadotropin-releasing hormone and associated binding substances in the blood serum of a holocephalan (Hydrolagus colliei)

The identity of the gonadotropin-releasing hormone (GnRH) form and the presence of GnRH-binding substances in the blood serum of the holocephalan, spotted ratfish (Hydrolagus colliei), were investigated. The GnRH-like peptides in the serum were identified on the basis of relative hydrophobicity using reverse-phase HPLC. [His⁵,Trp⁷,Tyr⁸]GnRH (chicken GnRH-II) was the only GnRH form detected in the serum. It has been previously shown to be the only GnRH form in the brain of this species. The presence of GnRH-binding substances was inferred by anomalous HPLC elution of GnRH, ultrafiltration behavior, and by the direct binding of iodinated GnRH analogues by blood serum components. The mean GnRH concentration in the extracted blood serum was 125 ± 11 pg ml⁻¹ (n = 5) in males and 64 ± 48 pg ml⁻¹ (n = 4) and 155 ± 26 (n = 4) in two separate groups of females. Measurement of GnRH in the blood serum is complicated by the presence of GnRH-binding substances, which may cause the coprecipitation of GnRH during extraction with organic solvents. The high concentration of GnRH and the presence of GnRH-binding substances suggest that systemic blood is the route by which GnRH reaches the gonadotropes and/or that GnRH may have a hormonal role in H. colliei.     

Sporulation and sterilization method for axenic culture of Gelidium canariensis

A sporulation and sterilization procedure was used to establish axenic cultures of sporelings of Gelidium canariensis. Sporangial branchlets excised from the thallus were rinsed in distilled water twice and in 1% sodium hypochlorite (2 min). The branchlets were cultivated overnight in multiwell plates with 0.3 ml of autoclaved seawater to promote spore liberation in 90% of the cultivated branchlets. The branchlets were transferred to an antibiotic solution made of ampicillin, penicillin, rifampicin, nystatin (0.2 mg ml⁻¹ each) and 0.1 g ml⁻¹ of GeO₂ in liquid PES for 45 days, during which clusters of spores (85–100 spores) were observed on the surface of the branchlet. After 55 days, they became axenic sporelings with the prostrate and erect system characteristic of Gelidium canariensis.