Intensive rotifer cultures using chemostats

Continuous production of the rotifer Brachionus plicatilis rotundiformis (S-type) in an intensive chemostat culture system has been investigated. The production dynamics of rotifers in relation to different flow rates and feed regimes show that the growth rate and production depends on the type of algal feed and flow rate utilized in the culture system. It was possible to achieve a mean production of up to 318.84 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 6 1 h^–1 in 100 1 chemostats and up to 261.21 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 40 1 h –1 while using 1 m³ capacity rotifer chemostats as production units. The 3 fatty acid composition of rotifers while using Chlorella and Nannochloropsis in the culture system has been described. The results of this investigation show that the rotifer productivity in the continuous culture system is considerably higher than in any of the conventional culture systems described to date for aquacultural purposes.This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS), Kuwait, under a contract research project code 86-04-02.     

Dependence of temperature on loss rates of rotifers,lipids, and ω3 fatty acids in starved Brachionus plicatilis cultures

Rotifer cultures of Brachionus plicatilis (SINTEF-strain, length 250 m) rich in 3 fatty acids were starved for > 5 days at variable temperature (0–18 °C). The net specific loss rate of rotifer numbers were 0.04 day^–1 (range 0–0.08 day^–1) at 5–18 °C, but reached values up to 0.25 day^–1 at 0–3 °C. The loss rate was independent on culture density (range 40–1000 ind ml^–1), but was to some extent dependent on the initial physiological state of the rotifers (i.e., egg ratio).The loss rate of lipids was 0.02–0.05 day^–1 below 10 °C, where the potential growth rate of the rotifer is low (0–0.09 day^–1). The loss rate of lipids increased rapidly for higher temperatures where the rotifer can maintain positive growth, and reached 0.19 day^–1 at 18 °C. The Q₁₀ for the lipid loss rate versus temperature was higher than the Q₁₀ for respiration found in other strains. This may suggest that other processes than respiration were involved in lipid catabolism. The content of 3 fatty acids became reduced somewhat faster than the lipids (i.e. in particular 22:6 3), but the fatty acid per cent distribution remained remarkably unaffected by the temperature during starvation.The results showed that rotifer cultures could be starved for up to 4 days at 5–8 °C without essential quantitative losses of lipids, 3 fatty acids, and rotifers. The rotifers exhausted their endogenous lipids through reproduction (anabolism) and respiration (including enhanced locomotion) at higher temperatures. At lower temperatures, the mortality rate became very high.     

Use of swimming speed and egg ratio as predictors of the status of rotifer cultures in aquaculture

This study evaluated the use of egg ratio (eggs rotifer^–1) and swimming speed (mm min^–1) as prediction criteria for production and culture quality in mass cultures of the rotifer Brachionus plicatilis. Egg ratio was determined to be a suitable predictor of rotifer growth and production in the cultures. Low egg ratios (i.e., 0–0.17 eggs rotifer^–1) indicate reduced rotifer population over time (i.e., negative net population growth rates). However, at this time egg ratio dynamics are not suitably understood to predict in advance a sudden population collapse.Swimming speed of reproductive, egg-carrying females in the exponential growth phase was 40–45 mm min^–1. During exponential growth swimming speed was independent of the food used. Lower swimming speeds were obtained in late stationary phase (10–25 mm min^–1) when yeast was used as a food source. Both environmental factors (e.g., accumulating metabolites) and changes in nutritional state of the rotifers may have affected the swimming speed, but environmental factors appear to be the most important. We believe that swimming speed has the potential of becoming an accurate predictor of culture quality in mass cultures of rotifers.     

Inoculative releases of Trichogramma ostriniae for suppression of Ostrinia nubilalis (European corn borer) in sweet corn: field biology and population dynamics

The effectiveness of inoculative releases of Trichogramma ostriniae Pang and Chen for suppression of Ostrinia nubilalis (Hübner) in sweet corn was assessed. Early-season, low-density (75,000 females ha⁻¹) releases were made, and establishment, levels of parasitism and sex ratios of emerging T. ostriniae quantified. T. ostriniae established effectively for each season that they were released, but appeared to be unable to overwinter. Parasitism levels tracked egg mass numbers closely, and T. ostriniae persisted in fields even where insecticides were applied. Parasitism by indigenous Trichogramma species was 3%. Field populations of T. ostriniae were distinctly female biased (78%), with males produced in the majority of broods. Numbers of males did not increase linearly with number of O. nubilalis eggs parasitized, but appeared to remain constant above an egg mass size of about 20 eggs. A Type-I functional response to increasing egg and egg mass density was found under field conditions, where the proportion of egg masses parasitized remained constant with increasing egg mass density. A relatively consistent percentage of eggs per egg mass was parasitized, with a linear increase in number of eggs parasitized with increasing number of eggs per egg mass. These results show that T. ostriniae established viable reproductive populations in sweet corn following inoculative release, with the potential to contribute to reduced dependence on insecticides for the control of O. nubilalis in an integrated pest management program.     

Diurnal changes in leaf gas exchange and chlorophyll fluorescence in tropical tree species with contrasting light requirements

Interspecific ecophysiological differences in response to different light environments are important to consider in regeneration behavior and forest dynamics. The diurnal changes in leaf gas exchange and chlorophyll fluorescence of two dipterocarps, Shorea leprosula (a high light-requiring) and Neobalanocarpus heimii (a low light-requiring), and a pioneer tree species (Macaranga gigantea) growing in open and gap sites were examined. In the open site, the maximum net photosynthetic rate (P_n), photosystem II (PSII) quantum yield (; F/Fm), and relative electron transport rate (r-ETR) through PSII at a given photosynthetic photon flux density (PPFD) was higher in S. leprosula and M. gigantea than in N. heimii, while non-photochemical quenching (NPQ) at a given PPFD was higher in N. heimii. The maximum values of net photosynthetic rate (P_n) in M. gigantea and S. leprosula was higher in the open site (8–11 mol m^–2 s^–1) than in the gap site (5 mol m^–2 s^–1), whereas that in N. heimii was lower in the open site (2 mol m^–2 s^–1) than in the gap site (4 mol m^–2 s^–1), indicating that N. heimii was less favorable to the open site. These data provide evidence to support the hypothesis that ecophysiological characteristics link with plants regeneration behavior and successional status. Although P_n and stomatal conductance decreased at midday in M. gigantea and S. leprosula in the open site, both r-ETR and leaf temperature remained unchanged. This indicates that stomatal closure rather than reduced photochemical capacity limited P_n in the daytime. Conversely, there was reduced r-ETR under high PPFD conditions in N. heimii in the open site, indicating reduced photochemical capacity. In the gap site, P_n increased in all leaves in the morning before exposure to direct sunlight, suggesting a relatively high use of diffuse light in the morning.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

Development and survival of Telenomus lobatus, a parasitoid of chrysopid eggs: effect of host species

Telenomus lobatus Johnson & Bin (Hymenoptera: Scelionidae), previously known only from the eggs of a berothid species, emerged from field-collected eggs of the chrysopid Mallada macleodi. In the laboratory, host species differentially influenced development, survival, and size of the parasitoid. Females parasitized between 55 and 65% of the available eggs of Chrysoperla (C. carnea, C. harrisii, and C. rufilabris), and these yielded 98 to 100% adult parasitoids within an average of 13 days. In contrast, eggs of Chrysopa were parasitized less frequently, (C. chi: 50%; C. oculata: 16%; C. quadripunctata: 1.3%) and relatively few (C. chi: 5.6%; C. oculata: 21%) or no (C. quadripunctata) parasitoids survived to adult emergence. Preimaginal development of T. lobatus in eggs of C. chi and C. oculata was approximately two days longer than in eggs of Chrysoperla spp. Although eggs of Meleoma dolicharthra were parasitized infrequently (approx. 16%), they yielded 100% adult parasitoids. Anomalochrysa maclachlani eggs were parasitized at low rates (1.8%) and parasitoid survival was intermediate (40.7%). Among all species, size of the host egg was positively related to the size of the emerging parasitoid adult.

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Résumé T. lobatus Johnson & Bin (Hym. Scelionidae) connu jusqu'ici uniquement comme parasitoïde des ufs de Berothidae, a étè obtenu d'ufs du chrysope, Mallada macleodi récoltés dans la nature. Au laboratoire, l'espèce de l'hôte influe sur le développement, la survie et la taille du parasitoïde. Les femellles ont parasité 55 à 65% des ufs disponibles de Chrysoperla (C. carnea, C. harrisii, C. rufilabris) et ceux-ci ont donné 98 à 100% de parasitoïdes adultes en 13 jours en moyenne. Par contre, les ufs de Chrysopa étaient moins souvent parasités,-C. chi: 50%; C. oculata: 16%; C. quadripunctata: 1,3%-, et relativement peu de parasitoïdes avaient survecu jusqu'à l'émergence,-C. chi: 5,6%; C. oculata: 21%; C. quadripunctata: 0. Le développment préimaginal est approximativement 2 jours plus long dans les ufs de C. chi et C. oculata que dans ceux de Chrysoperla spp. Bien que les ufs de Meleoma dolicharthra aient été peu parasités,-environ 16%-, 100% ont donné des parasitoïdes adultes. Les ufs de Anomalochrysa maclachlani ont été peu parasités,-1,8%-, et le taux de survie a été moyen: 40,7%. Pour toutes les espèces, la taille de l'uf de l'hôte était liée positivement à la taille du parasitoïde obtenu.

     

Continuous culture of the rotifer Brachionus plicatilis fed recycled algal diets

A culture system for the rotifer Brachionus plicatilis was designed to maintain higher food conversion rates and stable population densities. Two 2001 plastic tanks were employed in the culture experiments, tank A for feedback culture and tank B for a control culture. The experiments were carried out for 70 days at 24 °C, light intensity, 1500 lux, and a photoperiod of L:D 15:9. B. plicatilis were fed once a day on baker's yeast and Chlorella.Food conversion rates in tanks A and B were 24.7% and 10.1%, respectively. Population density of B. plicatilis in tank A was consistently stable at 100–150 ind. ml^–1 throughout the culture period. Density in tank B, however, showed large fluctuations after 40 or 50 days and by the end of the experiment, declined to zero.     

Ingestion of the cyanobacterium Trichodesmium spp. by pelagic harpacticoid copepods Macrosetella,Miracia and Oculosetella

Trichodesmium is a filamentous, colonial nitrogen fixing cyanobacteria, ubiquitous in tropical and subtropical regions of the world's oceans. Trichodesmium fixes atmospheric nitrogen and can comprise a significant fraction of total primary production in oceanic surface waters. Therefore, the consumption and fate of Trichodesmium has important consequences for understanding carbon and nitrogen cycling in the open ocean. The pelagic harpacticoid copepod Macrosetella gracilis uses Trichodesmium not only as a physical substrate for juvenile development, but also as a food source. Several different types of pelagic copepods (including several species of calanoids, harpacticoids and a poecilostomatoid species) were tested for ingestion of Trichodesmium by labelling the cyanobacteria with ¹⁴C. Only the pelagic harpacticoids ingested Trichodesmium. Here we report the first grazing rates based on ¹⁴C-uptake measurements for Macrosetella gracilis (0.173 µg C copepod^–1 h^–1), and the first quantitative measurements of both Miracia efferata (0.402 µg C copepod^–1 h^–1) and Oculosetella gracilis (0.126 µg C copepod^–1 h^–1) ingesting this cyanobacteria. Ingestion rates of M. gracilis and M. efferata on the two different species of Trichodesmium, T. thiebautii and T. erythraeum, as well as the two different colonial morphologies of T. thiebautii, spherical-shaped (puffs) and fusiform (tufts), were also compared. Both Miracia and Macrosetella had higher ingestion rates on the puff colonies than the tuft colonies of T. thiebautii.. Both also had higher ingestion rates of T. erythraeum than T. thiebautii. Trichodesmium thiebautii contains a previously reported neurotoxin which may be an important factor in determining trophodynamic interactions. Our results suggest that pelagic harpacticoid copepods can be quantitatively important in determining the fate of Trichodesmium carbon and nitrogen.     

Interspecific variation in temperature dependence of egg development of five congeneric stonefly species (Protonemura Kempny, 1898, Nemouridae,Plecoptera)

Embryonal development of the five congeners Protonemura auberti Illies, 1954, P. hrabei Rauser, 1956, P. meyeri (Pictet, 1841), P. nitida (Stephens, 1835), and P. praecox (Morton, 1894) was studied under various laboratory temperatures and different photoperiods.Mean number of eggs in field collected batches was between 470 (P. praecox) and 1211 (P. auberti). Spring species had smaller egg batches than autumn species (Table 1). Mean hatching success in the laboratory was 50–100% at 2–18 °C. In most species hatching success decreased slightly with increasing temperature (Figs. 1a-e). None of the eggs incubated at 24 °C developed. Hatching pattern followed an asymmetric frequency distribution. In general, the hatching periods were the shorter the higher the incubation temperature.Embryonic development of all five species was inversely temperature dependent (Figs. 2a-e), and well described by a power law relationship (Figs. 3a-e). Interspecific differences in incubation periods were notable at nearly all temperatures (Fig. 4). There was a distinct interspecific sequence in length of incubation period (with steps of about 4 days), which was the same as can be seen in the flight periods: The later the species flies the longer the incubation period. Temperature fluctuations and variations in photoperiod had no influence on incubation and hatching periods or hatching success.The thermal demand of the egg stage neither explains the recent geographical distribution of the Protonemura species, nor does it directly correspond to the field temperatures common during their egg development. However, it is optimal in respect to resource partitioning between the five species, with the consequence of temporal displacement of life cycles.Derived from Brittain's (in press) proposal to compare the two constants a and b of the regressions describing the temperature dependence of embryonal development, a new index (Integral Development Time, IDT) indicating the thermal demand was created for easier comparison of numerous species (Table 2). Evaluation of the IDT for various species of Plecoptera (Fig. 5) suggests that species belonging to the family group Systellognatha generally have higher thermal requirements in the egg stage than species belonging to the Euholognatha.     

Reproduction of the calanoid copepod Calanus propinquus in the southern Weddell Sea,Antarctica: observations in laboratory

Reproduction of the dominant Antarctic copepod Calanus propinquus was studied in February–April, 1989 aboard the R.V Dmitry Mendeleev during cruise N° 43 to the Weddell Sea. Single females were kept at 0 °C in the laboratory for 56 days with abundant food concentration (above 300 µg C l^–1 of Platymonas viridis). Females released clutches at night at 2–3 day intervals. Most clutches contained from 10 to 40 eggs, mean 37.3 eggs female^–1. Average carbon content of an egg was 0.37 ± 0.05 µg C. The maximum daily egg production rate of 30–50 eggs female^–1 d^–1 was observed for the first 3 days of the laboratory incubation, corresponding to 3.7–6.2% body C. The state of gonadal development of females showed the decline of the reproductive season in late February. The data suggest that egg laying in the region under study starts in December and lasts until March. The state of ovarian maturation, changes in vertical distribution and biochemical body composition of females suggest the possibility of two-year life cycle in C. propinquus in the southern Weddell Sea.     

Diurnal vertical distribution of rotifers (Rotifera) in the Chara zone of Budzyńskie Lake,Poland

Diurnal vertical distribution of rotifers was investigated in the Chara bed and the water immediately above it in the shallow region (ca. 1 m depth) of Budzyskie Lake (Wielkopolski National Park, Poland) in early September 1998. Eighty one rotifer species were identified – 71 among Chara and 59 in the open water. Significant differences in rotifer densities were observed in the Chara, with highest numbers during the day (2316 ind. l^–1) and lowest numbers early morning (521 ind. l^–1) and at dusk (610 ind. l^–1). Above the Chara, the numbers of rotifers did not change significantly (615–956 ind. l^–1). Littoral- or limnetic-forms differed in their diel vertical distribution between both zones. One group of littoral species was characterized by increased densities in the Chara in the daytime, while a second group increased in density during the night. The densities of limnetic species, which were much higher in open water, decreased in the morning or daytime in this zone. These differences in the diel behaviour of particular groups of rotifers may be dependent on microhabitat and may also be related to different kinds of predation, the exploitative competition for shared food resources between rotifers and crustaceans, as well as typical adaptation to littoral or limnetic life.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

The water velocity preferences of Austrosimulium furiosum and Simulium ornatipes (Diptera: Simuliidae) larvae,and the implications for micro-habitat partitioning

The aquatic larvae of two simuliid species, Austrosimulium furiosum (Skuse) and Simulium ornatipes Skuse, which often occur together in Victorian streams, were shown in laboratory experiments to have preferences for different water velocities: larvae of A. furiosum preferred water velocities of 0.2–0.3 m s^–1, and S. ornatipes preferred water velocities of 0.9–1.3 m s ^–1 . Final instar larvae of both species selected slow water speeds of less than 0.25 m s ^–1 prior to pupation. Flow patterns around a cylinder in a laboratory stream were mapped, and the distribution of A. furiosum larvae within the wake, paired vortices and horseshoe vortex was recorded. Larvae selected areas with suitable water velocities and aligned with the flow, providing flow visualization of micro-current speed and direction. The potential for micro-habitat partitioning is discussed in relation to benthic sampling strategies.     

Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris

A 1.2 kb DNA fragment coding for the pro-peptide and mature keratinase from Bacillus licheniformis PWD-1 (kerA) was cloned into vectors pPICZA and pGAPZA for extracellular expression in the methylotrophic yeast, Pichia pastoris. Recombinant keratinase was secreted by the pPICZA-kerA transformants 24 h after methanol induction of shake-flask cultures, and reached a final yield of 124 mg l^–1 (285 U ml^–1) 144 h after the induction. The recombinant keratinase was glycosylated ( 39 kDa), and was optimal between pH 8.5–9.5 and between 55°C –60°C using azokeratin as substrate. The enzyme degraded bovine serum albumin, collagen, and soy protein concentrate. In conclusion, P. pastoris can be used as an efficient host to express keratinase for nutritional and environmental applications.     

Functional analysis of linker insertions and point mutations in the α-Amy2/54 GA-regulated promoter

Functional analysis of a gibberellin-regulated wheat -amylase promoter, -Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease Bsm I we have shown that nucleotides –119 and –109 within the GARE ^–121GTAACAGAGTCTGG^–108 and nucleotide –152 within the proposed element ^–156GATTGACTTGACC^–144 are essential for high level expression from this promoter.     

Egg size and egg mass of Daphnia magna: response to food availability

The influence of different food availability on egg size and egg mass in Daphnia magna Straus was studied in long-term experiments using a flow-through system. Daphnia were either kept a constant high or low food levels or subjected to alternating periods of high food and starvation. Some animals were starved continuously after they had deposited their first clutch of eggs. Eggs were measured and weighed and their density (dry mass per volume) was determined. The results support the model of Glazier (1992), which defines a region of reproductive constraint at very low food concentrations and a region of adaptive response as food concentration increase. Egg sizes were largest under continuously low food concentrations (0.1 mg Cl^–1), which indicates that the maximum of Glazier's non-linear response curve is at very low food levels. Eggs produced during starvation were small, probably as a result of reproductive constraints. Egg density was about 0.37 mg dry weight mm^–1 and did not differ between treatments.     

Growth of Albugo candida (race unidentified) on Brassica juncea callus cultures

An obligate fungus Albugo candida (Pers. ex Lév.) Ktze. (race unidentified) was successfully grown on host callus tissues of Brassica juncea cv. Varuna. Of the various type of diseased explants used, young (green) hypertrophied inflorescence axis bearing non-erumpent zoosporangial blisters allowed the fungus to multiply asexually over the host calli on modified MS-medium (Murashige and Skoog, 1962). The dual cultures were maintained up to 6–8 subcultures without loss of viability of zoosporangia on MS-medium supplemented with 10.0 mg L^–1 IBA, 0.05 mg L^–1 kinetin, 25.0 mg L^–1 AA, 1.0 mg L^–1 biotin, 1.0 mg L^–1 thiamine-HCl and 1.0 g L^–1 casein hydrolysate. The fungus grew only on the callus cells and not axenically on the medium. Pathogenicity test and histopathology of cultures proved the existence of the viable fungus in vitro.Abbreviations AA ascorbic acid - BAP 6-benzyl aminopurine - CH casein hydrolysate acid hydrolysed - 2,4-D-2,4 dichlorophenoxy acetic acid - FAA formaldehyde acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - HgCl₂ mercuric chloride - Kinetin 6-furfuryl aminopurine - MS Murashige and Skoog (1962) - NAA alpha naphthalene acetic acid - rh relative humidity - sdw sterile distilled water - wt. weights     

Effect of unionized ammonia,viscosity and protozoan contamination on reproduction and enzyme activity of the rotifer Brachionus rotundiformis

We determined the effect of environmental stressors on the physiological condition of Brachionus rotundiformis. For two morphologically distinct B. rotundiformis strains: Hawaii (average lorica length = 222 m) and Langkawi strains (average lorica length 180 m), neonates hatched from resting eggs were exposed to different levels of unionized ammonia (0.7–9.8 mg l^–1), viscosity (relative viscosity against natural seawater = 1–1.17) and Euplotes sp. (protozoan) contamination (1–40 cells ml^–1). Increasing stress decreased fecundity and lifespan of both rotifer strains. Glucosidase and phospholipase activities were correlated with reproductive responses of both the strains exposed to unionized ammonia. When culture water viscosity was changed, the activity of esterase and phospholipase was correlated with reproductive responses of the Hawaiian strain, and glucosidase activity was correlated with those of Langkawi strain.With the protozoan contamination, esterase and glucosidase activities were correlated only with reproductive responses of the Hawaiian strain, while activity of all three enzymes was correlated to those of the Langkawi strain. Glucosidase activity proved to be a reliable indicator of stress for cultured B. rotundiformis.