Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10¹⁰ bacterial cells ml⁻¹, a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10⁹ and 10⁸ cells ml⁻¹. Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT₅₀ = 17.1 h) than for Steinernema carpocapsae (RT₅₀ = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect’s food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.     

Effect of temperature on the development of <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> and <Emphasis Type="Italic">Steinernema feltiae</Emphasis> (Nematoda: Rhabditida) in liquid culture

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23°C, 25°C, 27°C, and 29°C for S. carpocapsae and 20°C, 23°C, 25°C, and 27°C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29°C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27°C; whereas, maximum yields were recorded at 25°C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25°C. Optimal culture temperature for both nematode species is 25°C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.     

Pathogenicity,reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest,Dorcadion pseudopreissi (Coleoptera: Cerambycidae)

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have considerable potential as biological control agents of soil-inhabiting insect pests. In the present study, the control potential of the EPNs Steinernema carpocapsae (TUR-S4), S. feltiae (Nemaplus), S. carpocapsae (Nemastar), S. feltiae (TUR-S3) and Heterorhabditis bacteriophora (Nematop) against a new longicorn pest, Dorcadion pseudopreissi Breuning, 1962 (Coleoptera: Cerambycidae), on turf was examined in laboratory studies. Pathogenicity tests were performed at the following doses: 50, 100 and 150 Dauer Juveniles (DJs)/larva at 25°C. Highest mortalities (75–92%) of the larvae were detected at the dose of 150 DJs/larva for all nematodes used. Reproduction capabilities of the used EPNs were examined at doses of 50, 75, 100 and 150 DJs/larva at 25°C. S. carpocapsae (TUR-S4) had the most invasions (32 DJs/larva) and reproduction (28042 DJs/larva) at the dose of 100 DJs, and the highest reproduction (per invaded DJ into a larva) was observed in H. bacteriophora (Nematop) (2402.85 DJs) at a dose of 50 DJs. The foraging behaviour of the nematodes in the presence of D. pseudopreissi and Galleria mellonella L. (Lepidoptera: Galleriidae) larvae was studied using a Petri dish filled with sand at 20°C. All of the used nematodes accumulated near the larvae section of both insect species (32–53% of recovered DJs) with a higher percentage of S. carpocapsae (TUR-S4) (53%) and H. bacteriophora (48%) (Nematop) moving towards larvae of D. pseudopreissi, than the S. feltiae strains.     

Efficacy and persistence of entomopathogenic nematodes for black cutworm control in turfgrass

Field experiments were conducted in turf maintained under golf course fairway conditions in May, June, and August 2009 and in August and September 2010 to evaluate the ability of entomopathogenic nematodes to control larval populations of the black cutworm, Agrotis ipsilon, on golf courses. Commercial products containing the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, S. feltiae, and S. riobrave were applied at 1.0 or 2.5×10⁹ infective juveniles per ha against fourth-instar black cutworms. Larval mortality and turf damage were evaluated at 4 and/or 7 days after treatment (DAT). Steinernema carpocapsae was the best performing species due to a combination of high control rates (average 83%), most consistent results (70–90% range), high speed of kill (average 68% at 4 DAT), and prevention of significant turf damage despite very high larval densities at 0 DAT. Efficacy of S. feltiae and H. bacteriophora was often similar to that of S. carpocapsae but overall less consistent. Short-term persistence of the nematodes was evaluated in four turfgrass sites maintained under golf course putting green, fairway, or rough conditions in June and August 2009 by baiting soil samples at 0, 4, 7, and 14 DAT. Relative to recovery immediately after application, at least 50% of S. feltiae and 25% of S. carpocapsae consistently persisted up to 4 days in one of the greens and up to 7 days in some trials. Our finding suggests that S. carpocapsae and S. feltiae may provide adequate black cutworm control in golf course turf under moderate summer temperatures.     

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed. Received: 27 September 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Cyanobacterial akinete induction and its application as biofertilizer for rice cultivation

Nostoc sp. VICCR1-1 was induced in order to form akinetes on the basis of nutrient modification. Phosphorus and iron were found to be the critical for akinete differentiation, especially when both elements were omitted. The number of akinete cells increased up to 20% when compared with culturing in BG11₀ medium (without N source). In addition, CaCl₂ played a role in heterocyst differentiation, and was able to induce heterocyst ranging between 30% and 46%. In order to prepare akinetes as inoculum, the dried form of akinetes was prepared by mixing it with montmorillonite clay. The inoculum with the amount of 2.8 × 10⁶ cells m⁻² was applied to rice (Oryza sativa) fields. After harvesting, the grain yields from chemical N fertilizer, vegetative cells, and akinete inoculum treatments were not significantly different. To monitor the persistence of Nostoc sp. VICCR1-1 after harvesting, the most probable number-denaturing gradient gel electrophoresis technique using 16S rRNA gene was employed. The results indicated that the remaining population is at 2.5 × 10⁵ and 1.62 × 10⁶ cells m⁻² in treatments supplied with vegetative cells and akinete inocula, respectively. Akinete induction might be one of the appropriate approaches for producing cyanobacterial inoculum.     

Hybridisation and selective breeding for improvement of low temperature activity of the entomopathogenic nematode Steinernema feltiae

Steinernema feltiae is used to control overwintering larvae of the codling moth Cydia pomonella L. Application is in autumn when efficacy can be limited by low temperature. The objective of this study was to screen for low temperature activity among wild type populations of S. feltiae, hybridise most active strains and further improve low temperature activity by subjection of a hybrid strain to selective breeding. Significant variation was recorded among 22 S. feltiae strains. The temperature at which 50 % (AT₅₀) and 10 % (AT₁₀) of the dauer juveniles (DJs) were active ranged between 2.9 to 5.8 °C and 0.95 to 3.5 °C, respectively. The mean AT₅₀ of 22 S. feltiae strains was 3.83 °C. The five most active strains were crossed. The hybrid strain HYB01 was more active at low temperature than parental and other hybrid strains with an AT₅₀ of 0.52 °C and an AT₁₀ of 0.09 °C. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella, but was recovered after seven selection cycles with exposure to lowering temperatures. The heritability for the low temperature activity was calculated at h ² = 0.45. Negative trade-off effects on virulence against C. pomonella and reproduction on the same insect were not reported. The most virulent strain was a commercial strain with an LD₅₀ of 30.2 at 8 °C and 37.2 DJs per cocooned instar at 15 °C, followed by the selected hybrid with 48.1 and 47.4 DJs, respectively. Offspring production reached 15.000 DJs per instar at 8 °C and was only half at 15 °C. The results well document the potential of a breeding programme for enhancement of the activity of S. feltiae at lower temperature with the objective to improve the control potential of overwintering codling moth C. pomonella.     

Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR

Using ammonia monooxygenase α-subunit (amoA) gene and 16S rRNA gene, the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in a nitrogen-removing reactor, which was operated for five phases, were characterized and quantified by cloning, terminal restriction fragment length polymorphism (T-RFLP), and quantitative polymerase chain reaction (qPCR). The results suggested that the dominant AOB in the reactor fell to the genus Nitrosomonas, while the dominant AOA belonged to Crenarchaeotal Group I.1a in phylum Crenarchaeota. Real-time PCR results demonstrated that the levels of AOB amoA varied from 2.9 × 10³ to 2.3 × 10⁵ copies per nanogram DNA, greatly (about 60 times) higher than those of AOA, which ranged from 1.7 × 10² to 3.8 × 10³ copies per nanogram DNA. This indicated the possible leading role of AOB in the nitrification process in this study. T-RFLP results showed that the AOB community structure significantly shifted in different phases while AOA only showed one major peak for all the phases. The analyses also suggested that the AOB community was more sensitive than that of AOA to operational conditions, such as ammonia loading and dissolved oxygen.     

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10⁶ to 5.1 × 10⁷ copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10⁴ to 6.2 × 10⁵ copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.     

Effect of temperature on the pathogenicity of entomopathogenic nematodes (Steinernema and Heterorhabditis spp.) to Delia radicum

Susceptibility of last instarlarvae of Delia radicum to Steinernema feltiae, S. carpocapsae,S. arenarium, Heterorhabditismegidis and H. bacteriophora wasevaluated in the laboratory at 10 °C,15 °C and 20 °C. S. feltiaewas the only species that killed the larvae at10 °C; S. carpocapsae, S.arenarium and H. megidis were effectiveat 15–20 °C whereas H.bacteriophora killed the maggot only at20 °C. The temperature significantlyaffected the host searching ability of alltested species. Mobility was reduced at lowtemperatures. Significant effects of the hostpresence on nematode mobility were found forS. feltiae, S. arenarium and H. megidis but not for S. carpocapsaeand H. bacteriophora. The dynamics of theattachment to and penetration into the hostwere monitored for S. feltiae at10 °C, 15 °C or 20 °C and forS. carpocapsae at 20 °C. In theperiod of 6–30 hours after inoculation, S.carpocapsae attached in higher number at20 °C than did S. feltiae at alltemperatures. At 20 °C, S.carpocapsae penetrated the host only after 30hours while S. feltiae penetrated alreadyafter 15, 9, 6 hours at 10 °C,15 °C and 20 °C, respectively.     

The effect of intraspecific competition on the development and quality of Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae)

The effect of intraspecific competition on the development and reproduction of the slug parasitic nematode Phasmarhabditis hermaphrodita was studied in a series of laboratory experiments. Different inocula concentrations (1, 10, 25, 50, 100, 250, 500 and 1000) of P. hermaphrodita dauer juveniles (DJ) were applied to 0.02 g of homogenised slug Deroceras reticulatum on agar in multi-well plates and the development time, yield, lipid content, and body length of females and DJs were recorded. There was an inverse relationship between dose and recorded female length or lipid content. In newly emerged DJs the body length and lipid content were relatively invariable up to a dose of 250 DJs (ranging between 850 and 925 µm, 6.8 and 7.4, respectively), and decreased at higher doses (500 and 1000 DJs). The yield increased with increasing dosage to its optimum at a dose of 100 (200,000 DJs per 1 g of substrate) DJs and thereafter decreased. In the treatment with the highest dose (1000 DJs), only a minor portion of the inoculum developed to adult stage and completed their reproduction cycle while the rest remained at the larval stage. It might, therefore, be concluded that P. hermaphrodita tolerates intraspecific competition to a certain level at which crowding becomes too severe and a part of the inoculum do not develop and probably leave the site to avoid the fatal overcrowding. In conclusion, we show that intraspecific competition negatively affects the yield and quality of DJs of P. hermaphrodita, but it seems that this nematode can partly prevent overcrowding by avoiding occupied sites.     

Efficacy of <Emphasis Type="Italic">Steinernema feltiae</Emphasis> against the leek moth <Emphasis Type="Italic">Acrolepiopsis assectella</Emphasis> in laboratory and field conditions

The susceptibility of larvae of the leek moth, Acrolepiopsis assectella Zeller (Lepidoptera: Acrolepiidae) to different concentrations of an autochthonous strain of Steinernema feltiae (Rhabditida: Steinernematidae) was examined in laboratory experiments using Petri dishes. The efficacy of this strain in pots and field experiments was also evaluated. High mortality (80%–100%) of leek moth larvae was observed when these larvae were exposed to low concentrations (3 × 10³ to 1 × 10⁴ IJs/m²) of S. feltiae under laboratory conditions. Foliar application of 30,000 IJs/leek in pot experiments caused a 98% reduction in leek moth larvae. Field experiments showed a 87.7% reduction of leek moth larvae with the nematode treatment, significantly higher than the 22% reduction with the Bacillus thuringiensis treatment. The efficacy of the treatments with S. feltiae in relation to the microhabitat of the leek moth larvae between the interfolded leaves of the leek is discussed.     

Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes

Mortality of larval, pupal, and adult western cherry fruit fly, Rhagoletis indifferens (Tephritidae) exposed to the steinernematid nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema intermedium, was determined in the laboratory and field. Larvae were the most susceptible stage, with mortality in the three nematode treatments ranging from 62 to 100%. S. carpocapsae and S. feltiae were equally effective against larvae at both 50 and 100 infective juveniles (IJs)/cm². S. intermedium was slightly less effective against larvae than the other two species. Mortalities of R. indifferens larvae at 0, 2, 4, and 6 days following their introduction into soil previously treated with S. carpocapsae and S. feltiae at 50 IJs/cm² were 78.6, 92.5, 95.0, and 77.5% and 87.5, 52.5, 92.5, and 70.0%, respectively, and at 100 IJs/cm² were 90.0, 92.0, 100.0, and 84.0% and 90.0, 50.0, 42.0, and 40.0%, respectively. There was no decline in mortality caused by S. carpocapsae as time progressed, whereas there was in one test with S. feltiae. Larval mortalities caused by the two species were the same in a 1:1:1 vermiculite:peat moss:sand soil mix and a more compact silt loam soil. In the field, S. carpocapsae and S. feltiae were equally effective against larvae. Pupae were not infected, but adult flies were infected by all three nematode species in the laboratory. S. carpocapsae was the most effective species at a concentration of 100 IJs/cm² and infected 11–53% of adults that emerged. The high pathogenicity of S. carpocapsae and S. feltiae against R. indifferens larvae and their persistence in soil as well as efficacy in different soil types indicate both nematodes hold promise as effective biological control agents of flies in isolated and abandoned lots or in yards of homeowners.     

Extraction and characterisation of alginate from brown seaweeds (Fucales,Phaeophyceae) collected from Port Dickson,Peninsular Malaysia

Four species of brown seaweeds, namely Sargassum baccularia, Sargassum binderi, Sargassum siliquosum and Turbinaria conoides, harvested from Port Dickson, Negeri Sembilan, Malaysia were analysed for ash content, alginate yield and alginate properties. Seaweeds calcined at 450°C were found to have low amount of non-combustible residue as these were not contaminated by calcareous animals. Alginate was extracted from these seaweeds by two methods: hot and cold. In the hot method, the storing time was 3 h and the processing temperature was 50°C, whilst in the cold method, the sample was stored overnight at room temperature. Higher yield of alginate was obtained by the hot method compared to the cold method, but alginate extracted by the cold method gave higher molecular weight. In the hot method, 49.9% of alginate was extracted from S. siliquosum, followed by T. conoides (41.4%), S. binderi (38.9%) and S. baccularia (26.7%). Alginate extracted from T. conoides has an average molecular weight, M _w, of 8.06 × 10⁵ g mol⁻¹, whereas alginate from S. siliquosum was the lowest in M _w (4.81 × 10⁵ g mol⁻¹) when the extraction was done at room temperature. Alginate extracted from S. baccularia was found to be very heat-sensitive. Its M _w has dropped more than 83%, from 7.52 × 10⁵ to 1.23 × 10⁵ g mol⁻¹, when the extraction temperature was raised. The effect of heat on the extent of depolymerisation of the alginate molecule of the other three brown seaweed species was less significant, with decrease in molecular weight ranging between 13% and 16%.     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

Isolation of algicidal compounds from the red alga <Emphasis Type="Italic">Corallina pilulifera</Emphasis> against red tide microalgae

We determined the structure of two compounds, namely, 5,8,11,14,17-eicosapentaenoic acid (EPA) and di-n-octylphthalate (DnOP), which have algicidal activity against the toxic dinoflagellate, Cochlodinium polykrikoides. The polyunsaturated fatty acid EPA and the anthropogenic DnOP were isolated from the MeOH extract of the red alga Corallina pilulifera. We also found that a commercial EPA has algicidal activity identical to that of the EPA purified from C. pilulifera. At low inoculum (5.0 × 10² cells mL⁻¹), the highest algicidal activity of a commercial EPA exhibited approximately 92.6% algicidal activity after 1 h and 96.8% after 6 h treatment at 6 μg mL⁻¹, respectively. At high inoculum (1.0 × 10⁴ cells mL⁻¹), the strongest algicidal activity of EPA showed 69.5% after 1 h and 75.5% algicidal activity after 6 h treatment at 6 μg mL⁻¹, respectively. However, EPA did not show algicidal activity against several microalgae used in aquaculture such as Pavlova lutheri, Tetraselmis suecica, Isochrysis galbana, and Nannochloris oculata for 6 h treatment at 6 μg mL⁻¹. The algicidal activity of the five red tide strains to EPA (3 μg mL⁻¹) showed about 86.6%, 86.6%, and 67.3% algicidal activity against Skeletonema costatum, Chaetoceros curvisetus, and C. polykrikoides after 1 h treatment at low inoculum (5.0 × 10² cells mL⁻¹), respectively, but not against Prorocentrum minimum and Scrippsiella trochoidea. We concluded that EPA might be useful as a controlling agent of harmful algal blooms.     

Penguins, fur seals, and fishing: prey requirements and potential competition in the South Shetland Islands, Antarctica

Antarctic and sub-Antarctic seabirds, marine mammals, and human fisheries concentrate their foraging efforts on a single species, Antarctic krill (Euphausiasuperba). Because these predators may have a significant effect on krill abundance, we estimated the energy and prey requirements of Adelie (Pygoscelisadeliae), chinstrap (Pygoscelisantarctica), and gentoo (Pygoscelispapua) penguins and female Antarctic fur seals (Arctocephalusgazella) breeding on the South Shetland Islands, Antarctica and compared these estimates with catch statistics from the Antarctic krill fishery. Published data on field metabolic rate, population size, diet, prey energy content, and metabolic efficiency were used to estimate prey requirements of these breeding, adult, land-based predators and their dependent offspring. Due to their large population size, chinstrap penguins were the most significant krill predators during the period examined, consuming an estimated 7.8 × 10⁸ kg krill, followed by Adelie penguins (3.1 × 10⁷ kg), gentoo penguins (1.2 × 10⁷ kg), and Antarctic fur seals (3.6 × 10⁶ kg). Total consumption of all land-based predators on the South Shetland Islands was estimated at 8.3 × 10⁸ kg krill. The commercial krill fishery harvest in the South Shetland Island region (1.0 × 10⁸ kg) was approximately 12% of this. Commercial harvest coincides seasonally and spatially with peak penguin and fur seal prey demands, and may affect prey availability to penguins and fur seals. This differs from the conclusions of Ichii et al. who asserted that the potential for competition between South Shetland predators and the commercial krill fishery is low. Received: 26 August 1997 / Accepted: 16 December 1997     

Transfer of megaplasmid pKB1 from the rubber-degrading bacterium <Emphasis Type="Italic">Gordonia westfalica</Emphasis> strain Kb1 to related bacteria and its modification

Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10⁻⁸ events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 10³, 1.9 × 10³, and 8.3 × 10² transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.