Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%). Supported by the Frontier Research Grant of the SCSIO, the Hundred Talents program of Chinese Academy of Sciences, and National Natural Sciences of China projects (Grant No. 40776087)     

Bioassays using Stigeoclonium tenue Kütz. and Scenedesmus quadricauda (Turp.) Bréb. as testorganisms; a comparative study

Three different bioassays, two culture tube test methods with respectively Stigeoclonium tenue or Scenedesmus quadricauda and one bottle test with S. quadricauda, were compared. The yields obtained in the various tests were linearily correlated (r = 0.86, P < 0.001). The same primary limiting nutrient was indicated by the bioassays in most cases. However the algal growth in the tube test using Stigeoclonium was more often P-limited. In the case of S. quadricauda both test methods (tube and bottle) were nearly equally effective. The yields of N-limited samples were significantly correlated with the inorganic-N as well as total-N concentration of the water sample. A significant correlation of the ortho-P as well as total-P concentration with the yield of the P-limited assays was only found for Stigeoclonium tenue. The critical total N/P ratio (by weight) for N or P limitation was approximately 17: 1 for Stigeoclonium tenue and 22: 1 for Scenedesmus quadricauda.     

Parasitoids and other natural enemies of Maruca vitrata feeding on Sesbania cannabina in Taiwan

Natural enemies of Marucavitrata (Geyer) feeding concealed inside leafwhorls of a green manure legume, Sesbaniacannabina, were monitored during the 1996 and1997 summer seasons in Taiwan. A braconid,Apanteles taragamae, accounted for anaverage of 92% of all parasitoid specimensreared from M. vitrata larvae and pupae.Other parasitoid species included two otherbraconids: Bassus asper and Dolichogenidea sp., three ichneumonids: Trichomma sp., Triclistus sp., and Plectochorus sp., and two unidentifiedtachinids. All parasitoid species attacked M. vitrata larvae and emerged as mature larvaefrom host larvae (braconids) or as adults fromhost pupae (ichneumonids and tachinids). Oneunidentified predatory staphylinid and threeentomopathogenic fungi: Fusarium sp.Paecilomyces sp. and Beauveriabassiana were also observed attacking M.vitrata larvae. Apanteles taragamae is a solitary endoparasitoid. Its parasitismreached as high as 63% of M. vitratalarvae found attacking S. cannabina. Theparasitism was higher during June to August andreduced during September to November when otherparasitoid species were more active. Ninespecies of hyperparasitoids, all belonging toHymenoptera, were recovered, mainly from thebraconids.     

Settlement, early growth and survival of Haliotis rubra in response to different algal species

Five diatom species were isolated from settlementplates at Southern Ocean Mariculture, Victoria,Australia (Navicula sp., Naviculajeffreyi, Cylindrotheca closterium, Cocconeis sp., Amphora sp.) and tested insettlement experiments with Haliotis rubralarvae. Settlement was very low on single speciesdiatom films and varied between 1%–6%. Depending onthe species combination larvae preferred to settle onfilms with mixed diatom species than single speciesfilms. The highest settlement was achieved with amixed film of Navicula sp. and Amphora sp.Five and ten-day-old germlings of Sporolithondurum induced settlement of the abalone Haliotisrubra. However, the settlement rate was significantlylower on germlings than on the whole thallus of thealga. Germlings inoculated with the diatom Navicula sp. induced higher settlement than films ofthe diatom species alone. High settlement of up to52% was also achieved with germlings of the greenalga Ulvella lens. Settlement was reduced onU. lens squares inoculated with the diatom Navicula sp. but higher than on films of the diatomalone. The settlement rate was higher if plates withU. lens were previously grazed by juvenileabalone.Post-larval growth rates were higher on monospecificdiatom films than on U. lens or on S.durum. The best growth rate was obtained with Navicula sp. U. lens and S. durum areboth good settlement inducers, but are notsufficient to support rapid growth of young H.rubra post-larvae. Survival was low on U. lensand on the diatom C. closterium. We suggest thatcommercial nursery plates seeded with U. lenswill result in high and consistent settlement, whilean inoculum with Navicula sp. will ensuresufficient food for rapid growth of the post-larvae.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Susceptibility of Delia radicum to steinernematid nematodes

Isolates of different Steinernema species (S. affine, S. bicornutum, S. feltiae and Steinernema C1) were used in mortality assays with third instar larvae of Delia radicum (L.) (Diptera: Anthomyiidae). The nematode isolates had been obtained by baiting soil regularly grown with cabbage. One isolate (S. feltiae) was the result of a natural infection of a D. radicum puparium. The highest mortality (77%) was obtained with an isolate of S. feltiae (DK1). The isolate DK1 was also used in tests with all larval stages of D. radicum. Mortality around 60% was observed for second and third instar larvae, while first instar larvae showed very low or no susceptibility. Maximum mortality of second and third instar larvae was reached applying only 25 nematodes per larva. Observations of larvae that pupated revealed that some of these puparia contained nematodes. Experiments with hatching puparia showed that a high proportion was infected by nematodes if the flies were prevented from leaving nematode-containing soil. In addition to mortality, the ability of the nematodes to successfully reproduce in the insects was studied. It was found that the species S. feltiae and S. bicornutum reproduced in D. radicum larvae and adults with S. feltiae being the most successful.     

Influence of transgenic cottons with <Emphasis Type="Italic">Bacillus thuringiensis cry1Ac</Emphasis> gene on the natural enemies of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Transgenic cotton has been released for cultivation in several parts of the world to increase crop productivity. However, concerns have been raised regarding the possible undesirable effects of genetically modified crops on non-target organisms in the eco-system. Therefore, we studied the effects of transgenic cottons with cry1Ac gene from Bacillus thuringiensis Berliner (Bt) on the natural enemies of cotton bollworm/legume pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under field and laboratory conditions. There was no apparent effect of transgenic cotton on the relative abundance of predatory spiders (Clubiona sp. and Neoscona sp.), coccinellid (Cheilomenes sexmaculatus Fab.), and the chrysopid (Chrysoperla carnea Stephens). However, the abundance of spiders, coccinellids, and chrysopids was quite low in insecticide protected plots towards end of the cropping season. There was a significant reduction in cocoon formation and adult emergence of the ichneumonid parasitoid, Campoletis chlorideae Uchnida reared on H. armigera larvae fed on the leaves of transgenic cottons before and after parasitization. However, no Bt toxins were detected in H. armigera larvae and the parasitoid cocoons with enzyme linked immunosorbent assay. Reduction in cocoon formation was because of early mortality of the H. armigera larvae due to Bt toxins in the leaves of transgenic cotton. There was a slight reduction in adult weight and fecundity, and prolongation of the larval period when the parasitoid was raised on H. armigera larvae fed on the leaves of transgenic cotton before and after parasitization. Survival and development of C. chlorideae was also poor when H. armigera larvae were fed on the leaves of cotton hybrid Mech 184. The adverse effects of transgenic cotton on survival and development of C. chlorideae were largely due to early mortality, and possibly poor nutritional quality of H. armigera larvae due to toxic effects of the transgene.     

Predation of entomopathogenic nematodes by <Emphasis Type="Italic">Sancassania</Emphasis> sp. (Acari: Acaridae)

Predation of the entomopathogenic nematode, Steinernema feltiae (Rhabditida: Steinernematidae), by Sancassania sp. (Acari: Acaridae) isolated from field-collected scarab larvae was examined under laboratory conditions. Adult female mites consumed more than 80% of the infective juvenile (IJ) stage of S. feltiae within 24 h. When S. feltiae IJs were exposed to the mites for 24 h and then exposed to Galleria mellonella (Lepidoptera: Pyralidae) larvae, the number of nematodes penetrating into the larvae was significantly lower compared to S. feltiae IJs that were not exposed to mites (control). Soil type significantly affected the predation rate of IJs by the mites. Mites preyed more on nematodes in sandy soil than in loamy soil. We also observed that the mites consumed more S. feltiae IJs than Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). No phoretic relationship was observed between mites and nematodes and the nematodes did not infect the mites.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Effect of two microalgae concentrations on body size and egg size of the rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis>

The rotifer, Brachionus calyciflorus, was grown with two algae species (Chlorella sp. and Scenedesmus obliquus) at different concentrations (0.1, 1 and 10 × 10⁶ cells ml⁻¹). The body size (lorica biovolume) of individual rotifer and their egg size were measured when the populations were roughly in the exponential phase of population growth. The body size of the rotifers differed significantly (P < 0.05) among the two algae species used, however this effect was not observed for egg size. The body size of rotifers fed on higher densities of Chlorella sp. (10 × 10⁶ cells ml⁻¹) was significantly larger than for those fed on lower and medium densities (0.1 and 1 × 10⁶ cells ml⁻¹). Body size and egg size of rotifers fed with different amounts of Scenedesmus did not differ significantly. The egg size was significantly larger at higher food level of Chlorella. A significantly positive correlation was observed between the adult rotifer body size and their egg size.     

Changes in chlorophyll fluorescence quenching and pigment composition in the green alga Chlorococcum sp. grown under nitrogen deficiency and salinity stress

Changes in the in vivo chlorophyll fluorescencequenching, photosynthesis and pigment composition werefollowed in the green alga Chlorococcum sp.during exposure of the culture to nitrogen deficiencyand salinity stress with the aims to study theinterrelations between changes in physiological andphotochemical parameters and xanthophyll-cyclepigments content during adaptation to stress, and toevaluate the capacity of this green alga to producesecondary carotenoids in tubular photobioreactors.Exposure of Chlorococcum to nitrogendeficiency, 0.2 M NaCl and high irradiance outdoorscaused a strong depression of the photosyntheticactivity and of photochemical quantum yield ofPSII (F_v/F_m). These changes wereaccompanied by an increase of the non-photochemicalquenching coefficient (NPQ), of the amount ofxanthophyll-cycle pigments and of thecarotenoid/chlorophyll ratio. As a result of exposureto stress conditions, cell division completelystopped, although an increase in the biomass dryweight could be detected due to an increase in thecell size. These processes were followed, with acertain delay (15–20 h), by massive appearance ofsecondary carotenoids that reached the maximum (about50% total carotenoids) after 2–3 days of cultivation.The results show that despite of the lower carotenoidcontent (2 mg g^-1 dry wt) as compared with Haematococcus, Chlorococcum can be apotentially interesting strain for secondarycarotenoid production because of its higher growthrate.     

Feeding in adult females of Argyrodiaptomus furcatus (Sars,1901), Copepoda-Calanoida,of Lobo Reservoir (Broa), São Carlos,São Paulo,Brazil

The aim of this work was to study the feeding process of Argyrodiaptomus furcatus (Copepoda-Calanoida) in the Lobo Reservoir (São Carlos, SP, Brazil). Non-ovigerous adult females and the ¹⁴C technique were used to measure filtration and assimilation rates. The diet contained the following phytoplankton species: Chlamydomonas sp., Ankistrodesmus gracilis, Melosira italica, Scenedesmus quadricauda and Chlorella zoofingensis.The experiments were carried out using unialgal and mixed cultures during 2-, 4- and 6-h periods. The results of the filtration and assimilation rates were compared.The data obtained by statistical tests showed the highest assimilation rate in Argyrodiaptomus furcatus fed Chlamydomonas sp. in both culture types. However, Chlorella zoofingensis and Scenedesmus quadricauda were the most filtered species in unialgal and mixed cultures, respectively. A higher filtration rate was observed for the 2-h period than for the 4- and 6-h periods.Culture agent was also important. Higher assimilation and filtration rates were obtained during the log phase of Chlamydomonas sp. growth than during the stationary phase.     

Larval host plant affects fitness consequences of egg size variation in the seed beetle Stator limbatus

Egg size variation often has large effects on the fitness of progeny in insects. However, many studies have been unable to detect an advantage of developing from large eggs, suggesting that egg size variation has implications for offspring performance only under adverse conditions, such as during larval competition, periods of starvation, desiccation, or when larvae feed on low-quality resources. We test this hypothesis by examining the consequences of egg size variation for survivorship and development of a seed-feeding insect, Stator limbatus, on both a low-quality (Cercidium floridum) and a high-quality (Acacia greggii) host plant. Our results are consistent with the hypothesis. S. limbatus larval performance was affected by egg size only when developing on the poor-quality host (C. floridum); larvae from large eggs survived better on C. floridum than those from small eggs, while there was no evidence of an effect of egg size on progeny development time, body weight, or survivorship when larvae developed on A. greggii. These results indicate intense selection for large eggs within C. floridum-associated populations, but not in A. greggii-associated populations, so that egg size is predicted to vary among populations associated with different hosts. Our results also support this hypothesis; females from a C. floridum-associated population (Scottsdale) laid larger eggs than females from an A. greggii-associated population (Black Canyon City).     

A proteinase inhibitor from Nicotiana alata inhibits the normal development of light-brown apple moth, Epiphyas postvittana in transgenic apple plants

Insecticidal proteins are a potential resource to enhance resistance to insect pests in transgenic plants. Here, we describe the generation and analysis of the apple cultivar ‘Royal Gala’ transgenic for Nicotiana alata (N. alata) proteinase inhibitor (PI) and the impact of this PI on the growth and development of the Epiphyas postvittiana (light-brown apple moth). A cDNA clone encoding a proteinase inhibitor precursor from N. alata (Na-PI) under the control of either a double 35S promoter or a promoter from a ribulose-1,5-bisphosphate carboxylase small sub-unit gene (rbcS-E9 promoter) was stably incorporated into ‘Royal Gala’ apple using Agrobacterium-mediated transformation. A 40.3 kDa Na-PI precursor protein was expressed and correctly processed into 6-kDa proteinase inhibitors in the leaves of transgenic apple lines. The 6-kDa polypeptides accumulated to levels of 0.05 and 0.1% of the total soluble protein under the control of the rbc-E9 promoter and the double 35S promoter, respectively. Light-brown apple moth larvae fed with apple leaves expressing Na-PI had significantly reduced body weight after 7 days of feeding and female pupae were 19–28% smaller than controls. In addition, morphological changes such as pupal cases attached to the wing, deformed wings, deformed body shape, and pupal cases and curled wings attached to a deformed body were observed in adults that developed from larvae fed with apple leaves expressing Na-PI, when compared to larvae fed with the non-transformed apple leaves.     

Influence of thrips prey species on the life-history and behaviour of Orius albidipennis

The effects of two diets, i.e., cowpea leaves plus Megalurothrips sjostedti larvae versus cowpea leaves alone, on nymphal development and mortality of Orius albidipennis were studied under controlled laboratory conditions in Benin, West Africa. Nymphal development was longer when O. albidipennis was fed with cowpea leaves only, compared to the diet complemented with M. sjostedti larvae (17.6 days versus 14.8, respectively). Nymphal mortality was very high (78.6%) if fed with cowpea leaves only, and still relatively high if the diet was complemented with thrips larvae (44.4%).In a separate study on the longevity and fecundity of O. albidipennis adult females feeding on M. sjostedtilarvae, an average of 6.8 eggs per day (maximum 16 eggs) and a mean total fecundity of 61.1 eggs were observed. The females lived on average 13.5 days.Finally, observations on the predation by O. albidipennis adults on three different thrips species revealed that M. sjostedti larvae were killed at a lower rate than larvae of Ceratothripoides cameroni and Frankliniella schultzei. Higher predation rates were measured using unmated females and males than in mated females. However, the rate of first attack, measured as first larva attacked in dual-choice assays, was higher for M. sjostedti when a F. schultzei larva was offered simultaneously, and not different when a C. cameroni or Sericothrips adolfifridericilarva was offered.The results of this study are discussed with regard to the lack of efficacy of O. albidipennis as biological control agent for M. sjostedti.     

Parasitoids acquired byColeophora parthenica [Lepidoptera: Coleophoridae] ten years after its introduction into southern California for the biological control of Russian thistle

Parasitism of the stem and branch-boring mothColeophora parthenica Meyrick [Lepidoptera: Coleophoridae], introduced into California for the biological control of Russian thistle,Salsola australis R. Brown [Chenopodiaceae] was studied in the Coachella Valley of southern California during 1985–1986. Eight parasitoid species were reared from overwintering larvae ofC. parthenica, but none from the F₁ larvae, and just 2 individuals of one species from the F₂ summer generation. The level of parasitism of overwintering larvae was positively correlated with branch diameter, and ranged from 2% in the primary (main) branches to 45% in the tertiary branches in the spring 1985 sample, and from 2% to 19% in the spring 1986 sample, respectively. Rates of parasitism>20% were only found at sites with higher plant cover and chenopod diversity, but no other plant source or alternate hosts of the parasitoids ofC. parthenica were found. The 2 dominant species, the solitary, hymenopterous ectoparasitoids,Norbanus perplexus (Ashmead) [Pteromalidae] andEurytoma strigosa Bugbee [Eurytomidae], are both congeners of native parasitoids ofC. parthenica in Pakistan. The 2 other species of parasitoids ofC. parthenica in southern California for which other hosts are known are polyphagous and external on the larvae. No specialized endoparasitoid Braconidae, like those which dominate the native parasitoid complex in Pakistan and the U.S.S.R., have transferred toC. parthenica during its first 10 years in southern California.      

Does body size difference in the leeches Glossiphonia complanata (L.) and Helobdella stagnalis (L.) contribute to co-existence?

The effect of predator and prey body size on the feeding success of the British lake-dwelling leeches Glossiphonia complanata and Helobdella stagnalis was examined in the laboratory, and any involvement of size difference between the leeches in allowing coexistence in the field assessed. G. complanata breeds in advance of H. stagnalis and maintains a body size advantage throughout their annual life-cycle. In experiments, conducted at 14 °C and a photoperiod of 16 hrs L: 8 hrs D, three size classes of leeches of each species were each exposed to each of three size classes of each of five prey species, viz. Tubifex sp., Chironomus sp., Asellus aquaticus, Lymnaea peregra and Potamopyrgus jenkinsi. For each prey species, three different types of experiments were performed: one leech exposed to four prey individuals; four leeches of the same species with sixteen prey; and two leeches of each species with sixteen prey. In the first experiment, all sizes of G. complanata were capable of feeding on all sizes of the prey types offered; the same was true for H. stagnalis with exceptions of feeding on large A. aquaticus and large L. peregra. For both species, but especially for G. complanata, there was a trend within each size class of leech for decreasing proportions of fed leeches with increasing prey size, and within each size class of prey for an increasing proportion of fed leeches with increasing leech size; however there were several exceptions to these trends. Both leeches fed extensively on Tubifex sp. but there were significant differences in the proportions feeding on other prey types; G. complanata fed more on A. aquaticus and the two snail species, and less on Chironomus, than H. stagnalis. The effect of increasing the number of leech individuals from one to four individuals, of the same or mixed species, had little effect on the proportion of leeches which had fed. It is concluded that large G. complanata will have access to large individuals of certain prey taxa denied H. stagnalis, which may lessen the intensity of interspecific competition.     

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.