Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

The effect of artificial photoperiod on growth and reproduction in the land snail Cepaea nemoralis

Summary

Specimens of Cepaea nemoralis were raised from egg to adult in the laboratory for 15.5 months in two artificial photoperiods: short-day (LD 8:16) and long-day (LD 16:8). Over 20% of the snails in each photoperiod were functional adults by the end of the experiment and had fully developed male and female reproductive systems as determined both by dissection and by oviposition. A total of 925 eggs were laid by animals reared in a long-day photoperiod, whereas animals in a short-day photoperiod laid 677 eggs. Animals reared in a long-day photoperiod grew slightly faster (2.04 mg/day compared to 1.73 mg/day), began laying eggs about 18 days sooner, and suffered higher mortality (19% compared to 11%) than animals reared in a short-day photoperiod. These results clearly establish for C. nemoralis that gametogenesis and the development of a functional hermaphroditic reproductive system are neither prevented nor significantly retarded by exposing the animals to a short-day photoperiod for their entire pre-reproductive life.     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

Relationship between saprotrophic growth in soil of different biotypes of Pochonia chlamydosporia and the infection of nematode eggs

The ecology of Pochonia chlamydosporia in soil and its interaction with both plant and nematode hosts are important for the successful exploitation of the fungus as a biological control agent. Differences in saprotrophism and parasitism were assessed for biotypes of P. chlamydosporia, which had originated from the eggs of cyst or root‐knot nematodes. Colonisation in soils of different textures (compost, sandy loam and loamy sand) measured by the numbers of colony‐forming units, differed greatly. Most biotypes were more abundant in sterilised soil of the different textures compared with non‐sterilised soils. The proportion of nematode eggs parasitised in a baiting technique demonstrated that biotypes had host preferences. Those biotypes that originated from root‐knot nematodes (RKN‐biotypes) infected significantly more Meloidogyne hapla eggs than Globodera pallida eggs, whereas biotypes from cyst nematodes (CN‐biotypes) parasitised more G. pallida eggs than M. hapla eggs. Differences in virulence between biotypes in an in vitro assay in which the fungi were placed directly onto the egg masses of M. hapla and those differences observed in the baiting technique showed similar trends. There was a negative linear correlation between the growth of the eight biotypes in soil and the proportion of eggs they infected in compatible interactions (i.e. fungal biotype originated from the same nematode genus as the target eggs). Those biotypes that infected most nematode eggs colonised soil the least extensively, suggesting that virulence may have a fitness cost. However, the relationship between saprotrophic growth and virulence is complex. The relative abundance of the different biotypes in soil in Petri dish assays was similar to that under glasshouse conditions using potato but not tomato as the plant host. Chlamydospores of some biotypes applied to soil significantly reduced (>50%) the population densities of M. hapla on tomato and of G. pallida on potato plants. Some biotypes that were both effective and virulent are good candidates for biological control of specific nematode pests. Data presented here and elsewhere indicate that RKN‐biotypes have different host preferences to CN‐biotypes; the specific primers based on the vcp1 gene from P. chlamydosporia rapidly confirmed the host origin of seven of the eight biotypes.     

Chemical Composition,Anthelmintic Activity,and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC₅₀=0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.     

Mode of action studies of the acaricidal agent,epingaione

Summary

Activity directed isolation studies of the ethanol extract of Bontia daphnoides L (Myoporaceae) leaves and stems led to the isolation of epingaione, a sesquiterpene furan. This compound was previously described by Chinnock et al. (1987) from the same plant. The compound showed growth regulatory activities on gravid adult female Boophilus microplus Canestrini (the southern cattle tick). The dose required for inhibiting the hatching of B. microplus eggs by 50% (Fid₅₀)was 0.4±0.06 mg/g of the tick body weight. The Fid₅₀ dose inhibited the sequestration of protein into eggs oviposited by 80%. These eggs were non-agglutinated and wrinkled. Histological examination of ovarian sections from the treated ticks revealed significant degeneration of funicle cells and reduction in yolk content.     

The effects of <Emphasis Type="Italic">β</Emphasis>-amino-butyric acid on resistance of cucumber against root-knot nematode, <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

In this study, the effects of β-amino-butyric acid (BABA) on root-knot nematode(Meloidogyne javanica) infection of cucumber and accumulation of total phenolic compounds, hydrogen peroxide and activity of some enzymes related to plant defense mechanisms, i.e., guaiacol peroxidase (GPOX), polyphenol oxidase (PPO), catalase (CAT) in cucumber roots infected with nematode were investigated. Results of this study show that treating the cucumber seedlings with the above elicitor significantly reduces the nematode infection level (the nematode galls, number of egg masses per plant and number of eggs per individual egg mass) compared to control. Additionally, treatment of cucumber roots by BABA and BABA + nematode, significantly increased peroxidase, polyphenol oxidase and catalase activities in root tissues, 1 day after nematode inoculation in comparison to nematode inoculated plants as control and sterile water-treated plants. Enzyme activities reached to a maximum level at 4, 4 and 3 days after nematode inoculation, respectively. Additionally, the amount of H₂O₂, a product of oxidative stress, was significantly increased in the BABA and BABA + nematode treatments in comparison to control. Such increases have occurred in two phases and maximum levels of it were observed at 5 days after inoculation. Inoculation of cucumber plants by BABA also significantly increased accumulation of total phenol in comparison to control and maximum level of it was observed at 7 days after nematode inoculation. The results suggest that the inhibitory effect of BABA on the root-knot nematode (M. javanica) may be related to its ability to enhance defense responses in the cucumber roots.     

Assessment of three strategies for the management of Meloidogyne arenaria on carrot in Mexico using Pochonia chlamydosporia var. mexicana under greenhouse conditions

Three control strategies for management of Meloidogyne arenaria were evaluated on carrot under greenhouse conditions. The control strategies tested were: i) incorporation of fresh broccoli; ii) carbofuran (Furadan®); and iii) Pochonia chlamydosporia var. mexicana, isolate Pcp21. Each strategy was evaluated separately and in combination (16 treatment combinations), each with three replicates. The experiment was done between February and April 2014 and repeated over the same time period in 2015. Each replicate experimental unit consisted of a 1?kg pot filled with tyndallised soil, to which broccoli, fungus and carbofuran were added according to treatment. Carrot seeds were sown into each pot and inoculated with M. arenaria eggs according to treatment. Pots were then maintained in the glasshouse for 12 weeks at 25?±?5°C. After this time the fresh root weight, root length, percent reduction in root galling, P. chlamydosporia var. mexicana colony forming units (CFU)/g of soil, and CFU/g root were all measured. Results showed that isolate Pcp21 of P. chlamydosporia var. mexicana significantly reduced root galling percentage by 50 and 78% in 2014 and 2015, respectively. Application of the fungus in combination with broccoli or carbofuran also reduced root galling. The number of CFU/ g of soil or root remained the same over the 12 weeks of the experiment. This isolate of P. chlamydosporia significantly reduced damage caused by M. arenaria on carrot and is a promising agent for management of this nematode.     

Biological control of root-knot nematode (Meloidogyne javanica) disease by Pseudomonas fluorescens (Chao)

Plant growth-promoting Rhizobacteria is currently developed as an biocontrol agent against many plant pathogens. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Pseudomonas fluorescens was investigated in greenhouse and laboratory experiments. Results showed that 10⁹?(CFU/ml) of P. fluorescens decreased nematode infection and other parameters significantly, compared to the control. P. fluorescens was able to cause destruction of nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX) and phenylalanine ammonia lyase (PAL), increased significantly in P. fluorescens-inoculated plants. Maximum activities of POX and PAL were observed at the 5?days after inoculation, respectively. Results suggested that the destruction of eggs and plant defence mechanisms leading to systemic resistance are two main suppression mechanisms used by P. fluorescens against nematode.     

Isolation of Mn-SOD and Low Active Fe-SOD from Methylomonas J; Consisting of Identical Proteins

Cultures of Methylomonas J. an aerobic methylotrophic bacterium, were grown both in Mn-rich and Fe-rich media. Crude extracts of the cultures from the Mn-rich and Fe-rich medium showed a specific activity of 12.2 and 0.6 units/mg by a cytochrome c-xanthine oxidase method and 19.4 and 1.3 units/mg by an ESR method, respectively. We isolated Mn-SOD and Fe-SOD from the bacteria grown in the Mn-rich and Fe-rich mediums, respectively. Specific activity and metal contents of the Mn-enzyme were 2,250 units/mg/g-atom Mn and Mn = 0.98 and Fe = 0.12 (g-atoms/mol dirner), while those of the Fe-enzyme were 61 units/mg/g-atom Fe and Mn = 0.02 and Fe = 1.08. No difference of physicochemical properties of the Fe-and Mn-enzymes were detected. Furthermore, enzyme activity was restored by dialysis of an apoprotein obtained from the Fe-enzyme with either manganese sulfate or ferrous ammonium sulfate.     

Antagonistic Potential of Bacillus pumilus L1 Against Root‐Knot Nematode,Meloidogyne arenaria

This study was conducted to estimate the potential of Bacillus pumilus L1 against root‐knot nematode, Meloidogyne arenaria, in both in vitro and in vivo conditions. B. pumilus L1 was found to produce both protease and chitinase. When various concentrations (1–10%) of the bacterial culture (BC) or 0.02–0.11 mg/ml of the crude enzymes produced by B. pumilus L1 were used to treat M. arenaria eggs and second‐stage juveniles (J2), inhibition of hatching and J2 mortality were significantly increased under in vitro conditions. In addition, the hatching inhibition and J2 mortality rate were improved with increasing concentrations of BC and the crude enzymes. Similarly, these effects also increased over time after treatment with BC. Moreover, the crude enzymes caused partial degradation of the eggshell and juvenile body when treated at 0.11 mg/ml. The pot experiment also demonstrated that the application of BC to potted soil caused significant reduction in the number of galls and egg masses in the plant roots and of the J2 population as compared to the untreated control 6 weeks after M. arenaria infestation. In addition, the simultaneous application of BC upon nematode inoculation proved more effective than application 2 days postinoculation with nematode. B. pumilus L1 inoculation (BC, BCs and BC2) also promoted tomato plant growth as compared to the controls (TW, Ne, GM and NeT). Thus, our results demonstrated the ability of B. pumilus L1 as a potential biocontrol agent against root‐knot nematode, with additional activity as a plant growth promoter for tomato.     

Nematicidal effect of an Amaranthus hypochondriacus L. cystatin (AhCPI) on the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood

The root-knot nematode Meloidogyne incognita is one of the most damaging plant parasitic nematodes in the world. In this study, the effect of cystatin from Amaranthus hypochondriacus (AhCPI) as a potential control agent for M. incognita was explored. In vitro bioassays demonstrated that AhCPI affects the growth and development of eggs and the infectivity of juveniles (J2) of M. incognita, such as mortality and slower development, showing characteristic tissue damage. Mortality levels were quantified by Probit analysis, estimating LC₅₀s of 1.4 mg/mL for eggs and 0.028 mg/mL for J2. In planta bioassays showed that infected tomato seedlings treated with 0.056 mg/mL of AhCPI showed a 60% reduction in the number of galls, as compared with untreated J2-inoculated seedlings. Under greenhouse conditions, three applications of 10 mL of AhCPI (1.4 mg/mL) in the soil around the stem of M. incognita-infected tomato plants, reduced the number of galls by 93 ± 8%, as compared to the control M. incognita-infected plants. The application of AhCPI to the infected plants increased the yield (10.7%) of harvested tomato fruits, as compared to infected plants. These results show the potential of AhCPI for the control of M. incognita in tomato plants.     

The pathogenicity and increase of Heterodera rostochiensis on tomato cultivars, self-rooted or grafted on to rootstocks.

Self-rooted tomatoes ‘Ailsa Craig’ and ‘Kingley Cross’, and scions of ‘Ailsa Craig’ grafted on to different rootstocks, were grown in soil infested with different numbers of Heterodera rostochiensis (Mainly pathotype A). Three rootstocks (RS₄, RSKN, ISKVF) tolerated damage from nematode populations of up to 125 eggs/g soil, but nematodes increased up to x45 on them. Two nematode-resistant F₁ hybrid rootstocks, B66₃₃ (ex Lycopersicon pimpinellifolium B6₁₇₃) and B6₀₁₅ (ex L. hirsutum var. glabratum B6₀₁₃), tolerated up to 125 eggs/g soil; nematode populations declined as much as 80% under B66₃₃, and increased little under B6₀₁₅. On the self-rooted ‘Ailsa Craig’ and ‘Kingley Cross' the numbers of nematodes increased markedly (maximum increase x40), and greatly affected growth and yield so that initial populations greater than 62 eggs/g soil caused crop failure.     

椰子木蛾的产卵节律及其对寄主植物的产卵选择性

【背景】椰子木蛾是近年来新入侵我国棕榈科植物的害虫,研究其产卵习性可为监测和防治该虫提供参考。【方法】在室内条件下,观察、记录了椰子木蛾雌成虫的产卵节律及其对不同寄主植物的产卵选择性。【结果】椰子木蛾最高日产卵量可达34.4粒·头-1,且主要集中在羽化后4 d内产卵,占总产卵量的54.1%;产卵活动主要发生在夜间23:00到次日8:00;在椰子、蒲葵、大王棕、槟榔和散尾葵等寄主植物上的产卵量无显著差异,为89.3~147.7粒·头-1,但产卵位置存在差别。【结论与意义】椰子木蛾雌成虫具有较强的繁殖能力和产卵节律性,且在不同寄主植物上的产卵量一致。     

Laboratory rearing of potato cyst nematode; a method suitable for pathotyping and biological studies

A method is described for the laboratory rearing of potato cyst nematode, Globodera rostochiensis (Wollenweber, 1923) Mulvey & Stone, 1976 and G. pallida (Stone, 1973) Mulvey & Stone, 1976. Potato root systems were grown in 500 ml of sand in closed, clear plastic canisters. The enclosed system inhibited foliage growth and stabilised moisture balance. Root systems were sustained by the seed tubers, but supplementary nutrient was added. Potato cyst nematode eggs were inoculated into the sand after root initiation. At optimum rearing temperatures of 15–20°c the life cycle was completed within 8–12 weeks. The cyst‐to‐cyst multiplication factor was 10–20‐fold. The method has the advantage of not requiring light, and rearing canisters can be stacked in standard incubators. Its high reliability renders the method particularly suited to pathotype analysis.     

Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination

For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 μmol/g fresh mass to 5-8 μmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 μmol/g fresh mass to 5.57 μmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination.     

An artificial diet for larvae of the potato tuber moth

Potato tuber moth, Phthorimaea ( = Gnorimoschema) operculella (Zeller) (Lepidoptera: Gelechiidae), was reared from egg to adult for 3 generations on an artificial diet. At 30±2°c with a 16‐h photoperiod the total life cycle was completed in 23 days, compared with 18.5 days on potato tubers. Average life cycle data from the 3 generations reared on diet (with corresponding data from tubers in parentheses) are: larval period, 13.0(11.8) days; pupal period, 5–6(4–5) days; survival to adult, 76.1(100)%; fecundity, 102(169) eggs/?; pupal wt‐ ? 8.3(11.4) mg, ? 7.7(9.1) mg.     

Host status of some plants for Xiphinema diversicaudatum (Micol.) and their susceptibility to viruses transmitted by this species

Forty plant species were grown in pots containing viruliferous Xiphinema diversicaudatum (Micol.) for 15 wk to assess the host range of the nematode in relation to infection with arabis mosaic (AMV) and strawberry latent ring-spot (SLRV) viruses. Host status for the nematode was determined mainly from changes in total populations, but the presence of eggs in the uteri of females and changes in the numbers of adults provided additional criteria. The nematode multiplied on relatively more woody perennials than on herbaceous crop plants or weeds. Chrysanthemum coronarium was the only plant on which numbers declined significantly below those on the controls. Most plant species became infected with either AMV or SLRV. Neither virus was detected in eight out of thirteen species of trees and shrubs although four were good hosts for the nematode. Galling or distortion of the terminal region of fine feeder roots, associated with X. diversicaudatum feeding, was seen on many of the experimental plants.     

Comparison of temporal changes in established cardiovascular biomarkers after acute coronary syndrome between Caucasian and Chinese patients with diabetes mellitus

Abstract

Background: Population means of conventional cardiovascular biomarkers are known to differ between ethnic groups. In this study we performed detailed comparisons in the temporal pattern of these biomarkers between Caucasian and Chinese diabetic patients with acute coronary syndrome (ACS).

Methods: We studied differences in temporal changes of established cardiovascular biomarkers, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol, cardiac Troponin T (TnT), NT-proBNP and C-reactive protein (CRP), in 48 Chinese and 48 clinically matched Caucasian patients with type 2 diabetes mellitus who were admitted for ACS. Blood samples were collected at regular time intervals during 30?days to 1?year after the index ACS.

Results: In the >30?day post ACS period, mean serum levels of LDL (2.16 vs. 1.47?mmol/L; p-value <0.001), total cholesterol (4.08 vs. 3.11?mmol/L; p-value <0.001), TnT (11.0 vs. 7.76?ng/L; p-value 0.010) and CRP (2.0 vs. 0.78?mg/L; p-value <0.001) were systematically higher in Caucasian than in Chinese patients. HDL and NT-proBNP levels were similar.

Conclusions: Our study showed clinically relevant differences in levels of established cardiovascular biomarkers between Caucasian and Chinese post ACS patients. Further cross-ethnic studies are warranted to determine secondary prevention treatment biomarker targets in specific populations.