Changes in the adenine nucleotide content of cysts of Globodera rostochiensis associated with the hatching of juveniles

The content of AMP, ADP and ATP within single cysts of Globodera rostochiensis (60–80 fig dry weight) was determined as ATP to an accuracy of ± 10^-11 mol by a bioluminescent technique, after microenzymic methods had been used to phosphorylate AMP and ADP to ATP. Results for a total of 120 cysts showed that a change occurs in the adenylate energy charge of their contents after they have been exposed to potato root diffusate. Cysts in water had a mean adenylate energy charge of 0–63 (s.E. ± 0–04), but a randomly selected group of cysts after 24 h treatment with potato root diffusate had a significantly lower mean of 0–49±0–04. In a second, similar experiment, cysts in diffusate for only 8 h had an energy charge of 0*55 ± 0–03, but this value was not significantly less than the corresponding mean of o-6i ±0–03 of cysts that remained in water. The results indicate an effect on the metabolism of the unhatched juveniles that occurs too soon after the addition of diffusate to be directly due to any increase in locomotor activity. Apparently, the primary action of the hatching factor had affected many juveniles within 24 h of the addition of potato root diffusate to the cyst.     

Changes in the second stage juveniles of Globodeva rostochiensis prior to hatching in response to potato root diffusate

cAMP levels in eggs of G. rostochiensis and the diameter of the nucleolus of the nucleus within the dorsal pharyngeal gland cell of the second stage juvenile have been measured as indicators of the response of the nematode to the hatching stimulus in potato root diffusate. The nucleolus increased from 2.72 ± 0.103 μm for unhatched individuals to 3.28 ± 0.14 μm and 3.88 ± 0.15 μm after soaking eggs in potato root diffusate for 3 and 4 days respectively. Juveniles expressed from unstimulated eggs in water to potato root diffusate for 4–5 days showed a similar increase in size of the nucleolus to 3.94 ±0.15 μm but those released into water for this time had smaller nucleoli of 3.20 ± 0.98 μm. The change in diameter of the nucleolus is probably related to the accumulation of secretions in this gland cell before hatching. Preliminary results with dibutyryl analogues of CAMP and cGMP showed some inhibition of hatch in 10% potato root diffusate. Theophylline had a similar effect but NaF was dissimilar in that the effect of this inhibitor was not reversible. A standard radioimmunoassay showed that significant changes in cAMP levels occurred in the unhatched juveniles within cysts after treatment with potato root diffusate for 2.5 or 8 h compared with values for cysts kept in water. This change occurs before other known responses of the juveniles to potato root diffusate and it defines the period of interest for future work on the initial action of hatching factor.     

The uptake of calcium prior to the hatching of the second-stage juvenile of Globodera rostochiensis

Energy dispersive X-ray microanalysis of 83 eggs of G. rostochiensis for calcium content showed that juveniles from eggs exposed to active hatching factor in potato root diffusate for 48 h contained significantly more calcium than those exposed for this time to the same diffusate inactivated by autoclaving, or to water. This occurred despite a slightly greater calcium concentration in the autoclaved than in the untreated diffusate; both contained more calcium than the water. Eggshells removed from stimulated eggs also contained more calcium than those from unstimulated eggs. The calcium content of juveniles and eggshells exposed to inactivated diffusate was similar to their corresponding values for eggs soaked in water. A similar analysis was made of freeze-dried samples of fluid taken from the matrix surrounding the eggs in cysts exposed to water or to active root diffusate. This showed a significantly greater calcium concentration in the stimulated cysts after 48 h exposure. The concentration subsequently decreased over the succeeding 72 h however, suggesting that the rate of calcium uptake by the stimulated eggs exceeded that of diffusion into the cyst. This uptake of calcium by eggs of G. rostochiensis exposed to a hatching stimulus seems pertinent to recent evidence that the active factor in potato root diffusate may initiate hatching through a calcium-mediated process.     

Respiration rates of subitaneous eggs from a marine calanoid copepod: monitored by nanorespirometry

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28°C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean ± SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 ± 0.04 (10°C) to 0.54 ± 0.09 nmol O₂ egg⁻¹ h⁻¹ (28°C). The mean ± SD Q₁₀-value was 2.51 ± 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.     

Accumulation of phlorotannins in the abalone Haliotis discus hannai after feeding the brown seaweed Ecklonia cava

Value-added abalone Haliotis discus hannai containing bioactive phlorotannins is produced by simply changing the feed to phlorotannin-rich brown seaweed Ecklonia cava 2 weeks prior to harvesting. We assessed the accumulation of phlorotannins by feeding with the seaweed after 4 days of starvation. Reverse-phase high-performance liquid chromatography afforded isolation of the major phlorotannins, which were identified by mass spectrometry and ¹H-nuclear magnetic resonance to be 7-phloroeckol and eckol. Throughout the E. cava feeding period of 20 days, 7-phloroeckolol accumulated in the flesh (foot muscle tissue), up to 0.85?±?0.21 mg g^?1 dry weight of tissue after 12 days. Eckol reached 0.31?±?0.08 mg g^?1 dry tissue after 14 days. Feeding Laminaria japonica as a control, we detected no phlorotannins in the abalone muscle tissue. Abalone seaweed consumption and growth rate were similar when fed with E. cava or L. japonica for 20 days. Reduction in phlorotannins to half-maximal accumulation took 1.0 and 2.7 days for 7-phloroeckol and eckol, respectively, after replacement of the feed with L. japonica.     

Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate

The potential of several chromatographic methods for isolating hatching factors for potato cyst nematodes from potato root diffusate was investigated using a bioassay based on emergence of juveniles from cysts. Gel filtration provided an overall estimate of molecular weight of 437 Da for the hatching activity and ion exchange chromatography indicated that at least 60% of the recovered activity was anionic in nature. Material less polar than the hatching activity could be removed by passing potato root diffusate through a reversed-phase Sep-Pak C₁₈ cartridge and the elutant showed 83.3 ± 4.4% (mean of 32 cysts) of the initial hatching activity. High performance liquid chromatography (HPLC) with a reversed phase, C₁₈ column and gradient elution (0–80% CH₃OH in water) confirmed that much of the hatching activity was polar and that it was not retained by this method of separation. A weak anion exchange resin achieved slight retention of much of the hatching activity and an ion pairing reagent lowered the polarity sufficiently to allow some retention in subsequent reversed phase HPLC on a C_IS column. Both ion exchange and ion pairing HPLC suggested that hatching activity was not chromatographed as a single compound and indicated that fractions able to influence the nucleolus of the nucleus within the dorsal pharyngeal gland cell did not always show hatching activity.     

Effect of Liquid Nutrient Culture,Vacuum Distillation and Dialysis on Hatching Activity of Sugar Beet Root Diffusate for Heterodera schachtii

Roots of sugar beets grown in liquid culture excrete substances that stimulate egg hatch and emergence of larvae from cysts of Heterodera schachtii. Their hatching effect is comparable to that of sugar beet root diffusate leached from soil-grown sugar beet plants. Consequently, liquid culture provides a way of obtaining H. schachtii hatch-stimulant free of contaminants from soil. Root diffusate, concentrated 50-fold or dried by vacuum distillation, retained hatching activity. The active principle of diffusate is dialyzable with a diffusion rate between those of inorganic salts and compounds with molecular weights greater than 15,000.     

THE EMERGENCE OF LARVAE FROM FREE EGGS OF THE POTATO-ROOT EELWORM HETERODERA ROSTOCHIENSIS (WOLL.)

A technique for conducting hatching experiments on eggs freed from cysts is described. The form of the hatching response was found to be similar to that of eggs contained within cysts, but the response of the free eggs to the hatching stimulus was slightly more rapid. Investigations into factors affecting free egg hatching showed that it was necessary to presoak cysts before extracting the eggs from them for hatching. Eggs taken from dry cysts or from cysts that had been opened or cracked before presoaking did not respond to diffusate. When free eggs and whole cysts were exposed to the same graded series of dilutions of diffusate, the L.A. values (i.e. log concentrations of hatching factor) derived from plotting the hatching curves were in very close agreement.     

The effects of desiccation and root diffusates from potato and mustard on eggs of the white potato cyst nematode Globodera pallida

Following exposures to potato root diftusate of between 6 and 12 h, desiccation killed a proportion of juveniles in eggs of G. pallida and affected the hatching behaviour of survivors. In hatching tests of 9 wk duration, more juveniles hatched in the final wk from cysts, which were soahed and dried alternately for 9 wk than from cysts soaked in tap water for the final 2 wk only. Mustard root diffusate prevented eggs previously stimulated by potato root diffusate from hatching, but it did not alleviate the effects of desiccation.     

Development of eggs,larvae and juveniles of the puffer,Takifugu chrysops,reared in the laboratory

The artificial fertilization of the puffer,Takifugu chrysops (Hilgendorf), was carried out at Sajima in Yokosuka City on May 22, 1984. Hatched larvae were reared for a period of about 150 days. The spawning period seems to extend from mid to late May in the eastern part of Sagami Bay. The eggs were spherical, pale milky white and semitransparent, demersal and adhesive in nature, measuring 1.32±0.04 mm in diamter, and with a cluster of small oil-globules. The incubation period was about 162 hours at a water temperature of 17.4 to 21.8°C. During embryonic development, the only pigment cells that appeared on the embryo were the black chromatophores. The newly hatched larvae measured from 2.72 to 3.06 mm TL, averaging 2.87±0.1 mm TL, and 22–23 (9 + 13?14) myomeres. At yolk absorption, 4 days after hatching, the larvae attained 3.64–3.79 mm TL. On the 11th day, postlarvae averaged 4.69±0.24 mm TL. Larval finfolds disappeared and rudimental dorsal, anal and caudal fins were formed. There were two large clusters of melanophores, one on the back, exteding from the mid-base of the dorsal fin to the caudal peduncle region, the other along the anal fin base. The color of the body began to turn pale green to brownish-orange and spinelike scales appeared on the belly. Eighteen days after hatching (7.02±0.27 mm TL), the caudal notochord began to turn up and a “constriction” appeared on the posterior margin of the caudal fin membrane. This notch moved upwards as the notochord upturning advances. The larvae attained full fin ray counts and reached the juvenile stage at 9.1-9.5 mm TL, 24 days after hatching. Characteristic black blotches on the back and specific brownish orange body color appeared at the stage of 20 mm TL, 24 days after hatching. The growth during the larval stage and early juvenile stage (24 to 51 days after hatching) were expressed by the following equations, wherey is total length (mm) andx is days after hatching.y ₁=2.8424× 1.0509⁹ (0≦x≦24)y ₂ = 3.7872×1.0372^x (24≦x≦51)     

The response of eggs of the white potato cyst nematode Globodera pallida to diffusate from potato and mustard roots

Brief exposure of eggs of Globodera pallida to potato root diffusate not only initiated hatching but also caused the majority of unhatched juveniles to respond more rapidly to subsequent treatment with diffusate. Eggs previously exposed to diffusate had a peak hatch after 1 or 2 days compared with 4 days for untreated eggs. Mustard root diffusate prevented hatch, and further stimulation with potato root diffusate was necessary to re-initiate it. Eggs previously treated with potato root diffusate for 24 h were much more sensitive to drought than untreated eggs. These results are discussed in relation to the theory that potato root diffusate alters the permeability of the eggshell as an initial step in the hatching process.     

Evidence for the involvement of calcium in the hatching of Globodera rostochiensis

Low concentrations of ruthenium red and lanthanum chloride inhibited hatching of juveniles from eggs in cysts of Globodera rostochiensis in potato root diffusate. Pro-bit analysis for 31 cysts at seven concentrations of ruthenium red showed that 50% inhibition with 95% fiducial limits occured at 47 ± 23 μm; a similar value of 59 ± 14 μm was obtained using eggs removed from cysts. Results for 10 to 20 cysts at six concentrations of lanthanum chloride suggested a somewhat higher value for 50% inhibition of 110 ± 83 μM. In contrast hatching of eggs in cysts of Heterodera schachtii in water was unaffected by 5 ITIM lanthanum chloride and 625 μM ruthenium red, concentrations which cause over 90% inhibition of hatch in G. rostochiensis.
Two calcium ionophores synergised hatching of a 1971 population of G. rostochiensis in dilute diffusate. Optimal concentrations of 2 μM for A23187 and 10 μM for BrX537A increased the hatch from 17 ± 3–6 juveniles/cyst to 114 ± 44 juveniles/cyst and 138 ± 40 juveniles/cyst respectively. Ionophores in the absence of diffusate hatched very few eggs of this population but caused a greater hatch in a second (1975) population which gave a high hatch in water of 43 ± 10 juveniles/cyst. This was increased by A23187 to 181 ± 41 juveniles/cyst. The results with both the inhibitors and the ionophores suggest that hatching in G. rostochiensis might be a calcium-mediated process.     

Temporal changes in body mass, body composition and metabolism of gibel carp Carassius auratus gibelio during food deprivation

To investigate the response to starvation, gibel carp Carassius auratus gibelio [12.5 ± 0.03 g (mean ± SE, n = 24)] were deprived of food at 25.8 ± 0.2°C (mean ± SE, n = 56) for 56 days. Body mass, proximate composition in whole body and muscle, and respiration were measured at 7‐day intervals. Body mass decreased with prolongation of deprivation, with a significant decline recorded after 7 days deprivation. Fish lost 22% of their fresh mass and 34% of dry mass after 56 days. Fish lost 38% of the body lipid over the first 7 days, and lost body lipid at a rate of 0–11% per week over the remaining 49 days. Body protein was lost at 1–5% per week throughout deprivation. Compared with the initial composition, body lipid concentration was lower and ash concentration higher on day 7. Water as a percentage of body mass was higher after 28 days, and protein concentration lower after 42 days, than at the start of deprivation. Muscle lipid and protein concentration was lower, and % water higher, after 7 days than at the start of deprivation, whereas muscle ash concentration was relatively constant during deprivation. After 56 days, fish lost body water by 18%, body lipid by 84%, body protein by 30%, and body energy by 45%. Oxygen consumption rate dropped from day 1 to day 3, increased from day 4 to day 14, gradually decreased from day 15 to day 35, and maintained a relatively constant level from day 36 to day 56. Results of the present experiment reveal that gibel carp utilize body lipid as a major energy source in the first 7 days of food deprivation, then turn to body protein as an energy fuel when lipid reserves are heavily depleted. Oxygen consumption is maintained at a relatively low and constant level when most lipid reserves are exhausted.     

Oxygen consumption of human blood platelets. I. Effect of thrombin

The effect of thrombin on the oxygen consumption of washed human platelets was measured polarographically with the Clark oxygen electrode. The average basal respiratory rate was 18±1.6 (mean ±S.E.) natoms oxygen per min per 10⁹ platelets. Thrombin (1.9 units/ml) caused a 4–13-fold increase in the rate of oxygen consumption (138±14 (mean ±S.E.) natoms oxygen per min per 10⁹ platelets). The thrombin-stimulated increase of oxygen consumption was transient, lasting from 1 to 1.5 min before returning to the respiratory rate observed before the thrombin addition. Release of platelet constituents appeared to precede the stimulation of oxygen consumption. These results may provide a basis for explaining the discrepancy in the literature concerning the effects of thrombin on platelet respiration.     

Oxygen consumption by eggs and larvae of striped mullet, Mugil cephalus, in relation to development, salinity and temperature

Oxygen consumption rates during embryonic and the first 38 days of larval development of the striped mullet were measured at 24° C by differential respirometry. Measurements were obtained at the blastula, gastrula and four embryonic stages, and at the yolk-sac, preflexion, flexion and post-flexion larval stages.
Oxygen uptake rates of eggs increased linearly from 0.024 μl O₂ per egg h^-1 (0·323 μl O₂ mg^-1 dry wt h^-1) by blastulae to 0·177 μlO₂ per egg h^-1 (2·516 μlO₂mg ¹dry wth^-1) by embryos prior to hatching. Respiration rates did not vary significantly among four salinities (20,25, 30, 35%₀).
Larval oxygen consumption increased in a curvilinear manner from 0·243 μl O₂ per larva h^-1 shortly after hatching to 18·880 μl O₂ per larva h^-1 on day 38. Oxygen consumption varied in direct proportion to dry weight. Mass-specific oxygen consumption rates of preflexion, flexion, and postflexion larvae did not change with age (10·838 μl O₂ mg ¹dry wt h^-1).
Larval oxygen consumption rates did not vary significantly among salinities 10–35%. Acute temperature increases elicited significant increases in oxygen consumption, these being relatively greater in yolk-sac larvae ( Q₁₀ = 2·75) than in postflexion larvae ( Q₁₀ = 1·40).     

Primary infection with mouse-derived cysticercoids of Hymenolepis nana prepared from baby or adult mice and secondary infections with eggs or cysticercoids

Oral infection with mouse-derived cysticercoids (cysts) of Hymenolepis nana on day 0 did not make the 5 ± 1 week old mouse host immune to egg challenge by day 7 of the prepatent period, although the number of cyst-derived tapeworms was 1000 times greater than that of egg-derived tapeworms sufficient to make the host immune by day 7. Neither cysts recovered from immunologically competent 5 ± 1 week old donor mice, which should have become immune within 2 days of egg inoculation, nor those from incompetent 5–7 day-old baby mice given eggs when 0–2 days old made the host immune. The time course of differentiation of cysts in baby mice was not different from that in 5 ± 1 week-old mice. Mice infected twice with cysts on days 0 and 4 did not become immune either. Rapid protective immunity against egg challenge was acquired by inoculation exclusively with eggs but not with cysts. Apparently cysts differ from oncospheres in their immunogenicity. The importance of cysts for analysing the mouse—H. nana system from the immunological point of view is discussed.     

Desiccation and egg viability of the ragwort flea beetle,Longitarsus flavicornis (Coleoptera: Chrysomelidae)

A study was conducted to investigate the effect of desiccation on the survival of eggs of Longitarsus flavicornis. Eclosion of L. flavicornis eggs in laboratory trials decreased with increasing desiccation time between 0 days (93% hatching) and 42 days (no egg hatching) at 50±2% relative humidity and 23±2°C. Probit analysis indicated that 25, 50 and 99% mortality of L. flavicornis eggs occurred after 5.7, 9.3 and 50.4 days desiccation, respectively. Egg development varied between a minimum of 8 days at 7 days desiccation to a maximum of 15 days at 28 days desiccation. Hatching span did not differ between treatments with all eggs hatching within 12 days of each other. A relative humidity of 88–100% was measured under ragwort rosettes in non-drought field conditions. This would be expected to facilitate successful egg eclosion. However, the occurrence of summer drought could be detrimental to egg survival.     

Assessment of egg quality and realised fecundity of whiting Merlangius merlangus L. in captivity

Potential predictors of egg quality were assessed in whiting Merlangius merlangus L. permitted to spawn in a tank from which eggs were collected. These included fertilisation rate, the proportion of viable buoyant eggs, egg diameter, and egg wet and dry weights; all were influenced by temporal effects and were negatively correlated with days from start of spawning. The spawning period was protracted, from February to June. Mean daily egg production per female was 2.74 ± 2.43 g and 2338 ± 2075 eggs, equivalent to 14.6 ± 13.1 g kg^?1 day^?1 female^?1. Egg diameter was 1.21 ± 0.04 mm, egg wet weight 1.20 ± 0.21 mg, dry weight 0.10 ± 0.02 mg, and mean fertilisation rate and hatching rates were 76.8 and 73.3%, respectively. The incubation period ranged from 72 to 80 degree days and was dependent on temperature (x) and described by the equation y = 25.92 e^?0.1219x. Realised fecundity was also assessed to determine if this gave a more accurate measure of reproductive potential, and this was compared with potential fecundity estimated from predictive regressions on fish length from fisheries data. Realised egg production of 20 females of 185 g mean weight and 256 mm fork length was 4 444 360 (95% CL 4 093 961–4 743 018), similar to predicted seasonal egg production based on gravimetric fecundity measurements of wild caught fish.     

Oxygen consumption and ammonia excretion rates in Octopus maya,loligo forbesi and Lolliguncula brevis (Mollusca: Cephalopoda)

Oxygen consumption and ammonia excretion rates were investigated in young Octopus maya (hatching to 139 days old; 0.11–81.23 g wet body weight, BW; 22.5–23.9°C), young squids of Loligo forbesi (hatching to 45 days old; 9.4–115.3 mg BW; 12.3–13.1°C) and young squids of Lolliguncula brevis (2.00–39.98 g BW; 23.8–24.7°C). Except at hatching, oxygen consumption and ammonia excretion rates on an individual basis (M) of these three cephalopods increased linearly with increasing body weight (BW) expressed as M = aBW^b . Values of b for oxygen consumption were 0.900, 0.910 and 0.848 and for ammonia excretion were 0.744, 0.809 and 0.751 for O. maya, L. forbesi and L. brevis, respectively. Among the three species the value a varied widely, while b was similar for both oxygen consumption and ammonia excretion rates. Based upon these data, metabolism for hatchlings of O. maya and L. forbesi was estimated to be relatively lower than that of older juveniles. The O/N ratios for hatchlings of O. maya and L. forbesi were relatively high and indicate an apparent dependence upon lipids in the immediate post‐hatching period, followed by standard protein energy utilization thereafter.     

The alien false limpet (Siphonaria pectinata) in the Bizerte channel (northern Tunisia): spawning,development and growth under laboratory conditions

Spawning, development and growth of Siphonaria pectinata in the laboratory were studied and described in detail during a one-year study period. Egg ribbons were observed in February, March, April, June, July, August and September, with a peak in number of ribbons per individual in July. On average, individuals laid 9.0 ± 5.1 egg ribbons at a spawning frequency of one egg ribbon day^?1. The number of eggs per ribbon ranged from 752 to 50,400 depending on ribbon length. Embryonic development studied in February (13–15 °C), April (15–17 °C) and July (25–27 °C), reached hatching within 8–16 days with average larval lengths of 76.7 ± 5.9, 83.0 ± 11.3 and 78.3 ± 9.0 μm, respectively. Massive mortality was registered a few days after hatching, with larval longevity depending on the study period. Larval settlement occurred within 36–38 days after hatching, but only in the spawn deposited in February. Larval growth was slow during the first three weeks (18–26 μm week^?1) and then accelerated until the sixth week (40–67 μm week^?1). The present study contributes knowledge on the spawning, development and growth of S. pectinata, an alien species recently spreading throughout the Tunisian coast.