Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers

Pseudomonas aeruginosa K187, a protease- and chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄ and (GlcNAc)₅ were 1.18 mg/mL, 0.76 mg/mL, 1.02 mg/mL, 0.93 mg/mL and 0.90 mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications.     

Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology

Response surface methodology was employed to optimize the composition of medium for the production of avermectin B1a by Streptomyces avermitilis 14-12A in shaker flask cultivation. Corn starch and yeast extract were found to have significant effects on avermectin B1a production by the Plackett–Burman design. The steepest ascent method was used to access the optimal region of the medium composition, followed by an application of response surface. The analysis revealed that the optimum values of the tested variables were 149.57 g/l corn starch and 8.92 g/l yeast extract. A production of 5128 mg/l, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of original level (3528 mg/l), 1.45-fold increase had been obtained.     

Larval hatching in the horseshoe crab, Limulus polyphemus: facilitation by environmental cues

Female horseshoe crabs, Limulus polyphemus (Linnaeus), lay their eggs in nests on sandy beaches near the high water line. Embryos develop within the sand, hatch into trilobite larvae, and enter the water column when the nest is inundated. Given the diversity of tidal and shoreline inundation patterns that populations of L. polyphemus experience throughout their range (semidiurnal and diurnal tides, microtidal, and nontidal), hatching may also be facilitated by environmental triggers that serve to synchronize hatching and larval emergence with periods of high water. The objective of this study was to determine if larval hatching in L. polyphemus is triggered or facilitated by environmental cues. Stage 21 embryos were subjected to one of seven different treatments that simulated conditions experienced during inundation: (1) hydration, (2) agitation, (3) hydration and agitation, (4) hydration and agitation with sand, (5) osmotic shock, (6) terrestrial hypoxia, and (7) aquatic hypoxia. Hatching rates increased significantly under all simulated tidal conditions compared to controls and were highest (96%) for eggs simultaneously exposed to both hydration and agitation with sand. Measurements of the osmolarity of the perivitelline fluid of developing eggs collected from the field indicated that it is hyperosmotic to the ambient seawater and porewater. Thus, when inundated, eggs also experience a hypoosmotic shock, which would likely facilitate hatching by causing the eggs to swell, rupturing the egg membrane and thereby increasing the likelihood that larvae would hatch and enter the water column during periods of high water.     

模拟酸雨对西洋杜鹃生理生态特性的影响

采用盆栽方法,研究了不同pH值条件下的模拟酸雨对西洋杜鹃生长及其叶片主要生理生化特征的影响。结果表明:在酸雨胁迫下,随着酸雨胁迫的增强,西洋杜鹃叶片受害程度逐渐加重;叶片丙二醛含量逐渐升高;可溶性蛋白含量先升后降;过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)活性均呈先升后降的单峰曲线变化,其中pH值为4.3的处理下西洋杜鹃叶片CAT和SOD活性最高,pH值为3.0的处理下其POD活性最高;其叶绿素含量逐渐下降;叶片净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)及水分利用率(WUE)均呈先升后降的趋势,而胞间CO2浓度(Ci)则持续下降,pH值为4.3的处理下其Pn、Tr、Gs和WUE均达到最高。研究表明,大致可以认为pH值≤3.0是酸雨对西洋杜鹃造成隐形伤害的阀值,而酸雨灾害严重地区的降水pH值为2.0—4.0左右,说明西洋杜鹃可以在酸雨灾害较重的地区生长,可作为酸雨灾害严重地区园林绿化及植被构建的物种之一。     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.     

薇甘菊萎蔫病毒感染对薇甘菊光合特性和4种酶活性的影响

为明确薇甘菊萎蔫病毒(Mikania micrantha wilt virus,MMWV)对薇甘菊(Mikania micrantha)叶片生理生化的影响,探讨了MMWV侵染对薇甘菊叶片超氧化物岐化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、多酚氧化酶(polyphenol oxidase,PPO)和苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)活性及叶绿素总含量和光合特性的影响.结果表明:MMWV感染薇甘菊16d内叶片的SOD活性均比对照组高,其中第16天达最大值,但接种后第24和32天SOD活性分别比对照低了13.28％和25.37％;POD活性在接种后16-32d均显著高于对照.PPO和PAL变化趋势相似,在接种后第8天这两个酶的活性分别比对照高了77.75％和23.58％,而在第32天分别比对照减少了14.27％和20.53％.随着侵染时间的增加,感病薇甘菊叶片中叶绿素含量减少,叶绿素a/b值逐步降低;同时最大净光合速率(Pmax)和光饱和点(LSP)分别比健康对照减少了32.34％和12.52％,而对暗呼吸速率(Rd),光补偿点(LCP)和表观量子效率(AQY)无显著影响.表明MMWV侵染可减低薇甘菊叶片光合作用的效率.     

Impact of the flower stalk-boring moth Neoschoenobia testacealis (Lepidoptera: Crambidae) and water-level fluctuations on the flower and fruit production of the yellow water lily Nuphar subintegerrima (Nymphaeaceae) in irrigation ponds of western Japan

The flower and fruit production of the yellow water lily Nuphar subintegerrima and the seasonal occurrence of the flower stalk-boring moth Neoschoenobia testacealis were surveyed in two irrigation ponds in western Japan. Water-level fluctuations at each pond were measured. Flowering was observed from late May to September, and fruiting from late May to October. The yellow water lily produced cumulative numbers of 189 and 181 flowers and 121 and 129 fruits in the two ponds’ quadrats. The fruit setting rates at the two ponds were 0.64 and 0.71, and decreased in the summer. The percentage of bored flower stalks was 16 and 5%. Mature larvae bored flower stalks severely during the summer. Whenever the boring larvae attacked the flower stalks, the flower buds and flowers were aborted. There was a significant negative correlation between the fruit setting rate and the boring rate per month (number of bored flower stalks/number of all flower stalks). Whenever abruptly rising water levels (about 10 cm) submerged the flowers, the flowers were aborted too and could not set fruits. The percentages of non-setting fruit due to the rising water levels at the two ponds were 6 and 8%. Overall, our results indicated that flower stalk boring was a more important factor than water-level fluctuation in the sexual reproduction of N. subintegerrima and that N. subintegerrima was well suited to small irrigation ponds.     

Different responses to selection in two stocks of the bay scallop, Argopecten irradians irradians Lamarck (1819)

Two different stocks (A and B) of the bay scallop Argopecten irradians irradians (Lamarck, 1819) were used to test mass selection on growth. Stock A was a descending stock from the initial introduction from U.S.A. in 1982, which had been cultured in China for about 20 years. Stock B was the third generation from a recent introduction from U.S.A. in 1999. Truncation selection was conducted by selecting the largest 11% scallops in shell length from Stock A and the largest 12.7% scallops from Stock B as parents for the respective selected groups. Before the removal of parents for truncation selection, equal numbers of scallops were randomly chosen from Stock A and B to serve as parents for the control groups. Offspring from the four groups were reared under the same hatchery, nursery, and grow-out conditions. Values of response to selection and realized heritability at larvae, spat and grow-out stages for Stock B were all significantly (P<0.001) higher than its counterpart for Stock A. For Stock A, no significant response to selection was observed (P>0.05) at any stage, and the realized heritability for shell length was 0.015±0.024 for larvae, 0.040±0.027 for spat, and 0.080±0.009 for grow-out, respectively. For Stock B, however, significant (P<0.05) response to selection was observed, and the realized heritability for shell length was 0.511±0.010 for larvae, 0.341±0.022 for spat, and 0.338±0.015 for grow-out. On average, responses to selection at the three stages for Stock B was 30×, 7.1×, and 3× higher than its counterpart for Stock A, respectively. Accordingly, realized heritability at above stages for Stock B was 33×, 7.5×, and 3.2× higher than its counterpart for Stock A, respectively. Differences in response to selection and realized heritability between the two stocks are presumably due to differences in genetic variability. As the 20th generation from the initial introduction consisted of only 26 scallops, Stock A is known to be highly inbred, while inbreeding in Stock B is negligible.     

Variability in the impact of an introduced predator (Asterias amurensis: Asteroidea) on soft-sediment assemblages

The introduction and establishment of the predatory asteroid Asterias amurensis in coastal waters of southern Australia is considered a major threat to benthic marine assemblages and commercial bivalve species. We compare the impact of the seastar in experiments on three soft-sediment assemblages. The experiments were carried out immediately beyond the current range of the seastar in southeast Tasmania. This allows us to assess the repeatability, and hence predictability, of the type and magnitude of the impact of A. amurensis on soft-sediment assemblages. Responses to manipulations at the species level were dissimilar across the three experiments, reflecting marked initial differences in the three assemblages at both the species and functional group levels. However, at the functional group level, there were notable similarities in the impact of the seastar on the different assemblages. When potential prey taxa were separated into functional groups reflecting ecological availability (e.g. surface dwelling vs. deep burrowing bivalves), seastar effects were largely restricted to the surface bivalves. While the effect of seastar predation on surface bivalves was common in all three assemblages, the magnitude of the impact varied both among and within assemblages. Variability in the impact of A. amurensis at both levels appeared to be largely due to differences in relative availability of prey species. Overall, the results of these experiments indicate that while the impact of the seastar is broadly predictable at the functional group level, the exact nature of seastar effects is likely to be site- and time-specific given the inherent natural variability in soft-sediment assemblages and the seastar's responses to them.     

石榴园西花蓟马种群动态及其与气象因素的关系

2007-2008年,对云南省建水石榴园西花蓟马种群动态进行了系统调查,并采用回归分析(逐步回归分析、通径分析)、主成分分析及灰色系统分析就气象因子对该虫种群动态的影响进行了系统分析。结果表明,西花蓟马在建水石榴园常年发生,冬季较低,夏季最高,成虫全年种群消长呈单峰型,高峰期为5月份。相关性分析结果表明,西花蓟马种群数量与月相对湿度间呈极显著正相关性(P<0.01),与月均气温和月最低气温间呈显著正相关性(P<0.05),与月最高气温、月均降雨量和月均蒸发量间无相关性(P>0.05)。回归分析结果表明,石榴园西花蓟马种群动态的决定因子中影响最大的气象因素是月最低气温,而月均气温和月相对湿度是影响种群数量变动的主要因素。主成分分析表明,月最低气温是主要成分,其累积方差贡献率达73.03%。灰色系统分析结果表明,影响石榴园6种蓟马种群动态最关键的因子是月相对湿度;年度间影响最大的是年总降雨量;石榴花期各蓟马的种群数量与气象因素间关联度最大的是月最低温;果期各种蓟马的种群数量与气象因素间关联度最大的是月均降雨量。     

Identification of Pseudomonas aeruginosa in water-bottling plants on the basis of procedures included in ISO 16266:2006

ISO 16266:2006 provides a standardized procedure for the isolation and identification of Pseudomonas aeruginosa in waters. In some cases the method described in this ISO is not conclusive enough to confirm or discard the presence of this opportunistic human pathogen. In this study the capacity of the procedure described in ISO 16266:2006 to identify presumptive P. aeruginosa isolates was evaluated.Forty-one presumptive P. aeruginosa strains, previously isolated from water-bottling plants following ISO 16266:2006, were submitted to all the tests recommended by ISO 16266:2006 (Cetrimide agar with nalidixic acid, King B agar, Acetamide broth and Oxidase test). Additional tests that have been widely used for the identification of P. aeruginosa were also performed (Asparagine broth and King A agar). Furthermore, we also conducted the non-compulsory ISO 16266:2006 assay to study the capacity of the strains to grow at 4 °C and 42 °C. Finally, all the strains were biochemically phenotyped with PhP-48 plates (Bactus AB, Sweden) and API 20NE galleries (Biomérieux, France), and their 16 rRNA gene was sequenced.ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained.Growth in Asparagine broth should be discarded as a confirmative test as it showed false negatives and false positives. In contrast, API 20NE galleries correctly identified all the confirmed isolates.King A medium and growth tests at 4 °C and 42 °C correctly discriminated all the studied strains, even the two that were not identified with the basic ISO 16266:2006 tests.Given that King A medium and growth tests at 4 °C and 42 °C are straightforward, rapid, and inexpensive, it is strongly recommended that they be used for routine confirmation of P. aeruginosa when applying ISO 16266:2006.     

Effects of copulation temperature on female reproductive output and longevity in the wolf spider Pardosa astrigera (Araneae: Lycosidae)

The copulation duration of male wolf spider Pardosa astrigera, was significantly influenced by environmental temperature, as had been found in some insect species. Therefore, temperature during male courtship and copulation may influence the amount of sperm and seminal fluids transferred during copulation, which in turn could influence female fitness. In order to test this hypothesis, we subjected pairs of male and female P. astrigera to five temperature groups from 16 to 32 °C at an interval of 4 °C, and investigated whether and to what extent the various temperatures during male courtship and copulation influenced female reproductive output and female adult longevity under controlled laboratory conditions. With the increase of copulation temperature, females were more likely to lay egg sacs. The total egg sacs and lifetime fecundity of female were positively influenced by copulation temperature, whereas female lifetime spiderlings and adult longevity were independent of copulation temperature.     

Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: Evaluation by two molecular markers

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.     

Fractionation of stable isotopes in the Arctic marine copepod Calanus glacialis: Effects on the isotopic composition of marine particulate organic matter

Fractionation of δ¹³C and δ¹⁵N between food, consumer, and faecal pellets was studied in the Arctic marine copepod Calanus glacialis Jaschnov, fed with isotopically distinct algal monocultures. Temporal variations in δ¹³C and δ¹⁵N of copepods that were fed ice algae and phytoplankton followed those of a control group consisting of starved animals. There were no significant trends in the δ¹³C and δ¹⁵N values of copepods that were starved for 42 days, suggesting that the isotopic composition of non-lipid body tissues is unaffected by the metabolic processes during prolonged periods of starvation. The stable isotopic composition of starved copepods therefore seems to reflect food consumed during the previous period of feeding and growth. Faecal pellets produced by feeding copepods were depleted in ¹³C and ¹⁵N by 6.3-11.2‰ and 0.7-9.1‰, respectively, relative to the food ingested. These results indicate that faecal pellet production is an important pathway for the trophic fractionation of δ¹³C, whereas other fractionation pathways, such as excretion of ammonia, may be relatively more important for δ¹⁵N. The strong depletion of ¹³C in faecal pellets compared to the food suggests that grazing by herbivorous copepods on primary production adds to the variability of δ¹³C in marine particulate organic matter.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q_A) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield F_M, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH₂ by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH₂ starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of F_M was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl₂ that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F_M. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F₀ allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F₀ level (Q₀) and to compare it with the fractional quenching at the F_M level (Q_M). The experimentally determined Q₀/Q_M ratios were found to be equal to the corresponding F₀/F_M ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Fasciola hepatica: Screening of chemical compounds on immature stages in the mouse: I. Pathology and method of investigation

Examinations in albino mice under rigorously standardized conditions demonstrated that the most marked reactions resulting from fascioliasis are: increase of the weight of the liver and spleen, lowering of the hemoglobin content and of the hematocrit. The effect of the extent of infection on these parameters was investigated. In addition, the effect of the intensity of infection with regard to the average number and the length of the parasites was examined, as well as the mortality in the host and the occurrence of blood in the abdominal cavity.     

The long and the short of gene flow and reproductive isolation: Inter-Simple Sequence Repeat (ISSR) markers support the recognition of two floral forms in Pelargonium reniforme (Geraniaceae)

The current taxonomy of Pelargonium reniforme recognises two subspecies on the basis of habit and vegetative characters, but excludes floral characters. However, populations of P. reniforme in the wild tend to belong to easily discernable floral groups based on floral colour and hypanthium length. The aim of this study was to determine the genetic diversity within and between populations of both subspecies using the Inter-Simple Sequence Repeat (ISSR) method of DNA fingerprinting. Ninety individuals from eight populations were sampled. Both phenetic and Neighbor-Net analyses reveal that populations are genetically discernable, but that there is no genetic evidence to support the recognition of the two currently defined subspecies of P. reniforme. Instead, the analyses resolved all individuals that possess long hypanthia into a single group, and the role of different pollinators in driving reproductive isolation of this long-tubed form is suggested.