Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Difference in blood and water diffusion distance in gill lamellae of diploid and triploid tench Tinca tinca (L.)

Image analysis of sagittal sections of gill lamellae of diploid and triploid tench Tinca tinca revealed the blood and water diffusion distance in diploids (2·07 μm) to be significantly higher than that of their triploid siblings (1·46 μm; P < 0·01). Lamellae of diploids compared to triploids were found to be significantly shorter (105·84 v. 132·11 μm) and thicker (18·47 v. 14·21 μm; all at P < 0·05) than those of their triploid siblings but with similar mean sectional areas (1965·44 v. 1910·86 μm²).     

Discrepancies between otoliths of larvae and juveniles of the American eel: is something fishy happening at metamorphosis?

The use of otoliths to interpret early life history in fishes depends upon the assumptions that otoliths record past events accurately and consistently and that records of events in otoliths are continuous. Both the number of growth microincrements ( I ), and the radii ( R ,μm) of otoliths of American eel Anguilla rostrata , leptocephali increased linearly and highly significantly with leptocephalus body length ( L , mm), as expected on the above assumptions ( I , =2·29 L , − 5·75 and R , =1·05 L , + 12·02, r ²,=0·938 and 0·931, n , =20). In contrast, the number of increments and the radii of the leptocephalus growth zones of otoliths of glass-phase American eels were not related to body length, and they were lower than predicted by the relationships developed for leptocephali. Thus, otoliths of American eels apparently violate one or both assumptions. Possibly, the margin of the otolith is resorbed during metamorphosis from leptocephalus to glass eel, perhaps as part of calcium metabolism as skeletal elements are being formed.     

Ultrastructural observations of euspermatozoa and paraspermatozoa in a copulatory cottoid fish Blepsias cirrhosus

Euspermatozoa and paraspermatozoa of a copulatory (internal insemination with external sperm transfer) cottoid fish Blepsias cirrhosus were observed ultrastructurally. Euspermatozoa of B. cirrhosus consisted of an acrosome‐less, thin, disk‐like sperm head (1·6-2·0 μm in length and 1·3-1·6 μm in width), a long middle piece, and a long flagellum ( c . 30 μm). Aberrant spermatids, which were rich in cytoplasm and possessed two nuclei, occurred in testicular lobules. They were also observed in semen and were round (5·0-5·3 μm in diameter) and biflagellate, suggesting that they are released along with euspermatozoa at ejaculation. The nuclei of aberrant spermatids developed into masses of highly electron‐dense globules. Judging from their form, nuclear condition, and connection with normal spermatids by intercellular bridges during spermiogenesis, aberrant spermatids of B. cirrhosus are considered hyperpyrenic paraspermatozoa formed by incomplete cytokinesis at the second meiotic division.     

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).     

A Note on the Diagnosis of Ratoon Stunting Disease of Sugarcane by Negative-Stain Electron Microscopy of the Associated Bacterium

After negatively staining with 1% (w/v) sodium phosphotungstate (pH 6·5) or 1% (w/v) ammonium molybdate (pH 6·5), the cell wall, cytoplasmic membrane and mesosomes of the RSD-associated bacterium obtained from the fibrovascular fluid of infected sugarcane were usually clearly displayed. The cells measured 0·19–0·39 μm (av. 0·27 μm) in width and 0·6–3·4 μm in length. Few mesosomes were visible and the cells were approx. 40% wider (0·27–0·52 μm, av. 0·38 μm) when stained with 1% (w/v) uranyl acetate (pH 3·0–4·2). Freezing and thawing the suspension before negative staining with sodium phosphotungstate did not greatly affect the size of the cells or resolution of the mesosomes. Glycine (0·25 M) as the suspending medium, fixation in 2% (w/w) glutaraldehyde, or placing wet instead of dry specimen grids in the electron microscope resulted in wider cells usually lacking mesosomes.     

Development of bile salt-dependent lipase in larval turbot

Pancreatic bile salt-dependent lipase (BSDL) was present with 0·5 μg BSDL larva⁻¹ from hatching in turbot larvae. The enzyme content increased during the yolk sac phase to 1·1 μg BSDL larva⁻¹. This suggests that larval turbot are able to digest lipids from the start of exogenous feeding. The BSDL synthesis was stimulated first by food about 5 days after the onset of first feeding. The content per larva increased exponentially in fed larvae to 20 μg BSDL larva⁻¹ on day 23 after hatching and decreased in starved larvae. In contrast, the specific content decreased during the first feeding phase, meaning that smaller larvae had a higher content of enzyme related to their biomass than did bigger larvae.     

Influence of egg vitamin A status and egg incubation temperature on subsequent development of the early vertebral column in Atlantic salmon fry

The effect of egg vitamin A (VA) status and egg incubation temperature on the development of spinal disorders was investigated in Atlantic salmon Salmo salar fry. Atlantic salmon eggs were sorted into two groups with high VA (3·3 ± 0·1 μg retinol g⁻¹ dry mass) and low VA (2·2 ± 0·3 μg retinol g⁻¹ dry mass) status before fertilization and incubated at high (14° C) or low (8° C) temperature from 133 day degrees until the onset of feeding. High egg incubation temperatures increased the concentration of retinol in the eggs: the high VA and high temperature group displayed a significantly higher retinol concentration than the high VA and low temperature group ( P  = 0·001). After hatching, all experimental groups increased their retinol concentration. The source of the increased retinol levels was probably retinal, although astaxanthin may also be a VA precursor after hatching. Atlantic salmon fry incubated at high temperatures had increased amounts of notochord tissue. When measuring morphogenic activity in the notochord using the expression of sonic hedgehog ( shh , mRNA), however, no significant difference was found between the experimental groups. No clear effect of VA status or incubation temperature could be found on the formation of the early vertebral column although Atlantic salmon fry incubated at low temperatures had less regular constrictions of the prospective vertebral column than fry incubated at high temperatures.     

Morphology and fine structure of the heart of the burbot, a cold stenothermal fish

The ventricle of the burbot Lota lota heart comprised 0·148 ± 0·006% of the body mass which is nearly two-fold heavier than the relative ventricular mass ( M _V) of other similarly sized teleosts. The shape of the ventricle is pyramidal and the wall is exclusively composed of spongious muscle without a distinct compact layer. The atrium forms 0·017 ± 0·002% of the body mass. Length, width, sarcolemmal surface area and volume of enzymatically isolated myocytes from burbot ventricle were 147·2 ± 10·2 μm, 6·3 ± 0·4 μm, 2440·8 · 251·5 μm² and 2356·8 ± 316·6 μm³, respectively. The myofibrils were peripherally located and their volume density was remarkably high: 65 ± 2 and 68 ± 3% in ventricle and atrium, respectively ( P >0·05). Although not particularly conspicuous, some nonjunctional and junctional sarcoplasmic reticulum (SR) was present in both atrial and ventricular myocytes. The SR formed peripheral couplings with the sarcolemma and the junctional clefts were frequently occupied by foot processes. These findings suggest that cold-adaptation is achieved by cardiac enlargement, high volume density of myofibrils and well-developed peripheral couplings in the SR in the heart of stenothermal burbot.     

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient‐enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post‐hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± s.d. total length (L_T) = 5·13 ± 0·37 cm, range 5–6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± s.d. L_T = 14·92 ± 0·55 cm, range 13–16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l^?1 per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l^?1 per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± s.d. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.     

Erythrocyte nuclear measurements of diploid and triploid channel catfish, Ictalurus punctatus (Rafinesque)

Minor axis, major axis, and volume measurements of erythrocyte nuclei were compared by discriminant analysis to determine which variable best predicted ploidy levels. Predicted correct classification percentages for minor axis, volume, major axis, and all variables combined were 81·33, 86·21, 92·36 and 92·65%, respectively. Minor axis and volume measurements were not as accurate in predicting ploidy levels because nuclear shape changed as the ploidy level changed from the diploid to triploid state. Mean major axis measurements alone classified 92·36% of the fish correctly with a discriminant level of 4·726 μm.     

Myxobolus dahomeyensis infection in ovaries of Tilapia species from Benin (West Africa)

The prevalence of Myxobolus dahomeyensis in ovaries of Tilapia zillii was 31·6% and 18·3% in Sarotherodon melanotheron melanotheron . The parasite induced destruction of the oocytes and host castration. Its ovoid spore was 6·5–12·0 × 6·0–8·0 μm and the polar capsules averaged 3·6 × 2·2 μm ( n = 30).     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Leidyana canadensis N. Sp. (Apicomplexa: Eugregarinida) from Larval Eastern Hemlock Looper, Lambdina fiscellaria fiscellaria (Lepidoptera: Geometridae)

ABSTRACT. Leidyana canadensis n. sp. (Apicomplexa: Eugregarinida) is described from larvae of the eastern hemlock looper, Lambdina fiscellaria (Lepidoptera: Geometridae) collected near St. Stephen, Charlotte County, New Brunswick, Canada. Gamonts solitary, located between host ventricular peritrophic membrane and ventricular epithelium. Protomerite very broadly ellipsoid with transverse posterior margin; length 43.7 μm, width 42.7 μm; protomerite-deutomerite septum strongly constricted. Deutomerite narrowly obovoid with anterior transverse margin; length 186.6 μm, width 58.7 μm; total length 227.1 μm. Nucleus spherical to broadly ellipsoid; length 26.3 μm, width 20.2 μm; placement abaxial in the posterior 2/3 of the deutomerite. Nucleus often obscured in late gamonts. Association late, caudofrontal, ephemeral and leading directly to syzygy. Gametocysts roughly spherical; diameter 216.7 μm; hyaline coat increasing diameter to 359.1 μm. Gametocysts mature and dehisce through six short spore tubes within 48 hours. Oocysts axially symmetric, dolioform in dorsal aspect, compressed in the plane perpendicular to the major axis, very uniform in size and shape; length along major axis 5.2 μm, terminal width 1.8 μm, medial width 3.8 μm; extruded in chains.     

Validation of daily increment formation in otoliths of juvenile and adult European anchovy

The otoliths of juveniles and adults of European anchovy Engraulis encrasicolus held in aquaria were marked by immersion in oxytetracycline hydrochloride (OTC) at concentrations between 350 and 410 mg l⁻¹ for 12 h. Counts of microincrements between fluorescent bands validated the daily otolith increment formation. The otolith increments were easily readable at ×400 with average increment widths of c . 1·1 µm. Validation was successfully demonstrated in juveniles and adults maintained for short periods in the aquaria in the summer. For European anchovy captured as juvenile and reared to adults, however, increment formation appeared less than daily. The daily periodicity of the otoliths in juvenile European anchovy implies that counting of microincrements can be used to study their birth dates. The application of this technique to adults, however, may lead to the underestimation of actual age and further research needs to be done to clarify the reasons for the apparent loss of the daily rhythm over long periods.     

Sexual differences in the increase of white muscle fibres in Argentine hake, Merluccius hubbsi, from the San Matias Gulf (Argentina)

Growth dynamics of white fibres from axial muscle has been investigated in Argentine hake, Merluccius hubbsi , discriminating between sexes for the first time. The frequency distributions of fibre diameters are remarkably different in both sexes at sizes between 42 and 63·9 cm total length (T.l.). Males have a much lower proportion of newly recruited fibres (0–10 μm) than females; at 52–53·9 cm T.l., females have 4% of fibres in that category and males 0·5%. It appears that, from this size interval onwards, the increase in muscle mass is due only to the increase in diameter of individual fibres, which may exceed 300 μm. The lower recruitment rate of new fibres in males, and its relationship to lower growth rates and smaller final sizes, are discussed, and possible effects of reproductive activity are considered.     

Viable ultramicrocells in drinking water

Aims:  To examine the diversity of cultivable 0·2 micron filtrate biofilm forming bacteria from drinking water systems.
Methods and Results:  Potable chlorinated drinking water hosts phylogenetically diverse ultramicrocells (UMC) (0·2 and 0·1  μ m filterable). UMC (starved or dwarf bacteria) were isolated by cultivation on minimal medium from a flow system wall model with polyvinyl chloride (PVC) pipes. All cultivated cells (25 different isolates) did not maintain their ultra-size after passages on rich media. Cultured UMC were identified by their 16S ribosomal DNA sequences. The results showed that they were closely related to uncultured and cultured members of the Proteobacteria, Actinobacteria and Firmicutes. The isolates of phylum Actinobacteria included representatives of a diverse set of Actinobacterial families: Micrococcaceae, Microbacteriaceae, Dermabacteraceae, Nocardiaceae and Nocardioidaceae.
Conclusions:  This study is the first to show an abundance of cultivable UMC of various phyla in drinking water system, including a high frequency of bacteria known to be involved in opportunistic infections, such as Stenotrophomonas maltophilia, Microbacterium sp., Pandoraea sp. and Afipia strains.
Significance and Impact of the Study:  Chlorinated tap water filtrate (0·2 and 0·1  μ m) still harbours opportunistic micro-organisms that can pose some health threat.     

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0·0%; T2 = 1·1%; T3 = 1·7%; T4 = 4·0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH‐TR reaction and strong acid‐stable mATPase activity, and a deep white one, most of the muscle mass, formed by fibres with weak NADH‐TR reaction and strong alkali‐stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid‐stable or acid‐labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1–T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters ≤8 μm was higher in the lysine treatments, and with diameters between 16 and 24 μm, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 μm and in T1–T4 were between 8 and 16 μm. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy.