Life Cycle of Tetranychus macfarlanei Baker and Pritchard (Acari: Tetranychidae): A New Pest of Medicinal Plants in West Bengal

The life cycle of Tetranychus macfarlanei Baker and Pritchard was studied on two different medicinal plants, Clitoria ternatea L. and Justicia adhatoda L. Nees, in BOD at 32.5 °C and 75 % RH during April 2007 to May 2007. Observations towards duration of different stages like egg, larva, protonymph, deutonymph, adult, total life cycle, preoviposition, oviposition, postoviposition periods, longevity of female and male, and fecundity, sex ratio were recorded. Total developmental period of T. macfarlanei from egg to adult was 6.4 ± 0.37 (Mean ± SE) and 10.6 ± 0.56 days on C. ternatea and J. adhatoda, respectively. On C. ternatea, the fecundity in case of fertilized and unfertilized female were 91.6 ± 11.61 and 80 ± 21.64 eggs, respectively and longevity of fertilized and unfertilized female was 16.4 ± 1.44 and 8.6 ± 2.32 days, respectively. The corresponding figure on J. adhatoda for fecundity in case of fertilized and unfertilized female were 39 ± 2.85 and 19.8 ± 3.90 eggs, respectively and for longevity was 16 ± 0.37 and 11 ± 086 days, respectively. Among the two hosts, C. ternatea appears to be more preferred to J. adhatoda because life cycle was completed in shorter time and fecundity and female longevity were for longer duration.     

An elastase-like enzyme in the oocytes of Arbacia Punctulata.

An elastase-like enzyme was demonstrated in the unfertilized eggs of Arbacia punctulata. The enzyme was discovered in the fertilization product of A. punctulata, but was also found to be present in the seawater surrounding unfertilized eggs. Isolation of the elastase-like enzyme was accomplished by preparation of a 100,000g supernatant from a homogenization of unfertilized eggs. Its presence and specificity were determined by assay using a synthetic peptide substrate. The elastase-like activity was nondialyzable, heat labile, and unstable at pH 4. The enzyme was inhibited by antipain, elastatinal, and DFP, but not by leupeptin or soybean trypsin inhibitor. Eggs were fertilized and developed normally in the presence of 1.0 mM elastatinal. Trypsin-like and chymotrypsin-like enzymes were also found in the seawater surrounding fertilized eggs. The trypsin-like enzyme was isolated from this source and characterized by inhibitor profile.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Reproductive mode of the geographic parthenogenetic mayfly Ephoron shigae, with findings from some new localities (Insecta: Ephemeroptera, Polymitarcyidae)

The burrowing polymitarcyid mayfly Ephoron shigae is distributed widely in Japan. Some populations are bisexual, others are unisexual, and the distributions of the two types overlap broadly. Experimental evidence of parthenogenetic reproduction, long suspected in unisexual populations, is presented here, based on a comparative analysis of the developmental rate of fertilized and unfertilized eggs. The developmental rate of fertilized eggs from 20 mated females in a bisexual population was 98.4% ± 0.73% (mean ± SD), and no unfertilized eggs from 20 virgin females in that population developed. The developmental rates of unfertilized eggs in two unisexual populations were 89.0% ± 4.59% and 84.2% ± 1.96%, respectively. This article presents experimental evidence of geographic parthenogenesis in E. shigae and provides support for the previous interpretation. In addition, we discuss the relationship between the sex ratio of each population and the developmental rate of fertilized versus unfertilized eggs from the females in those populations.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

延伸因子-1α-A和β-肌动蛋白启动子在青鳉转基因胚胎中的表达（简报）

转基因技术是二十世纪八十年代初发展起来的一项生物领域高新技术。近年来,外源基因经显微注射导入哺乳类、两栖类、昆虫类以及鱼类的受精卵或胚胎,从而使人们对在整个动物的系统发育期间外源基因表达的研究更加深入。与哺乳类、两栖类以及昆虫类相比,鱼作为在脊椎动物进化的低级阶段,更适合在受精卵或胚胎期的显微操作。转基因鱼模型的研究为鱼类基因工程育种奠定了理论基础,基因导入方法的成熟、胚胎干细胞技术的发展以及基因组学理论的应用则为鱼类基因工程育种提供     

A direct effect of some rifamycin derivatives on the morphology of mammalian mitochondria

Protein synthesis has been investigated in cell-free preparations from mature ovarian oocytes, unfertilized and fertilized eggs, and early embryos of Drosophila melanogaster. Preparations from unfertilized eggs have a specific activity that is 5- to 6-fold higher than the activity of fractions from ovarian oocytes. There is an additional small increase in activity of preparations from fertilized eggs. The specific activity that is rapidly attained in the fertilized egg remains essentially constant for 2 to 2.5 h after fertilization, decreases sharply during blastoderm formation, and again increases during gastrulation. The activities of unfertilized eggs decline slightly during the first 2 h after oviposition, and then decrease more sharply. About 35 % of the ribosomes in preparations from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients, whereas no polyribosomes could be detected in preparations from ovarian oocytes. In both ovarian oocytes and fertilized eggs, less than 1 % of the ribosome populations were present as subunits. Additional ribonucleoprotein material of buoyant densities different from those of ribosomal subunits or ribosomes was found throughout the sucrose gradients. About 3.5 % of the ribosomes were found to be membrane-bound in preparations from both unfertilized and fertilized eggs.     

Culture-independent analysis of Pseudomonas community structures in fertilized and unfertilized agricultural soils

Pseudomonas community structures were investigated by analyzing 16S rRNA clone libraries derived from fertilized and unfertilized soil plots under corn–alfalfa rotation in a long-term experiment. Amplified 16S rRNA fragments derived by polymerase chain reaction (PCR) were cloned and sequenced. A total of 729 clone sequences were analyzed, of which 51 were possible chimeras and discarded. The remaining clone sequences (678) belonged to γ-proteobacteria with 61.8 % (419) classified to the genus Pseudomonas. Unclassified Gammaproteobacteria accounted for 23.4 % of total clones sequences. Rarefaction analyses showed a more diverse community structure of both Gammaproteobacteria and Pseudomonas in unfertilized than fertilized field soils irrespective of plant types under cultivation. Bacterial or Pseudomonas community structures differed significantly between fertilized and unfertilized soil plots. Clone sequences that are affiliated to Pseudomonas putida and P. oryzihabitans were more prominent in libraries from fertilized plots, while those that clustered with Pseudomonas frederiksbergensis were more often retrieved from unfertilized soil plots. A strong influence of fertilizer applications on community structure was supported by principal component analysis. We conclude that long-term use of mineral fertilizers could influence Pseudomonas community structure.     

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the ?148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the ?148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

Adaptation of cultured human lymphoblasts to growth in citrulline

DNA synthesis is initiated in unfertilized sea urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus) by exposing them to NH₄OH-sea water (ordinary sea water titrated to pH 9–9.1 with NH₄OH). The eggs are considered to be unfertilized eggs by visual and electro-biological criteria and because they can later be fertilized and then do give visible and electrobiological fertilization reactions. The incorporation of ³H-thymidine proceeds in rounds, the magnitude increasing in successive rounds. It is also reported that the treatment with NH₄OH activates the uptake of thymidine by the eggs, although the internal thymidine builds up more slowly in unfertilized eggs treated with NH₄OH than it does in fertilized eggs. The magnitude of the incorporation of exogenously supplied labelled thymidine into DNA is lower in the NH₄OH-treated unfertilized eggs than in normal fertilized eggs. This difference is not attributed to differences in the amount of DNA synthesized and the explanation is sought in thymidine uptake and nucleotide pathways.     

Fecundity and fertility of ladybird beetle Harmonia axyridis after prolonged cold storage

Some species of ladybird beetles (Coleoptera: Coccinellidae) mate both before and after overwintering. The purpose of the pre-diapause mating was studied in the alien invasive ladybird Harmonia axyridis (Pallas 1773). Our study demonstrates the persistence of high fecundity (daily oviposition rate of 21 eggs per fertilized female during the first month of reproduction) and fertility (85 % of eggs hatching) of females of H. axyridis after long storage (up to eight months) at low temperature (6 °C). The females were not mated after activation in spring and had to rely on the sperm supply maintained from the pre-winter period (58 % of females were fertilized). Unfertilized females also laid eggs but in low numbers (an average of 345 eggs by virgin females during an individual’s lifetime, 1,174 eggs by females fertilized before winter) and after a longer pre-oviposition period (2–5 weeks in comparison to 7–8 days for fertilized females). We show that the unfertilised eggs were not trophic eggs. The high sperm survival ability observed questions the need for the high levels of sexual activity generally observed in Coccinellidae. Fertilized females of H. axyridis may found large colonies after dispersal to new areas even without males, which contributes to the striking invasive ability of this species.     

Fertilization-induced changes in concanavalin A binding to mouse eggs

The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml^?1) the fertilized egg shows a higher affinity for [³H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 10⁸ ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.     

Field Note: Phytoremediation of Petroleum Sludge Contaminated Field Using Sedge Species,Cyperus Rotundus (Linn.) and Cyperus Brevifolius (Rottb.) Hassk

The aim of this study was to degrade total petroleum hydrocarbon (TPH) in a petroleum sludge contaminated site (initial TPH concentration of 65,000–75,000 mg.kg^–1) with two native sedge species namely Cyperus rotundus (Linn.) and Cyperus brevifolius (Rottb.) Hassk. Fertilized and unfertilized treatments were maintained separately to record the influence of fertilizer in TPH degradation. The average biomass production (twenty plants from each treatment) of C. rotundus was 345.5 g and that of C. brevifolius was 250.6 g in fertilized soil during 360 days. Decrease in soil TPH concentration was higher in fertilized soil (75% for C. rotundus and 64% for C. brevifolius) than in unfertilized soil (36% for C. rotundus and 32% for C. brevifolius). In unvegetated treatments, decrease in soil TPH concentration in fertilized (12%) and unfertilized soil (8%) can be attributed to natural attenuation and microbial degradation. TPH accumulation in roots and shoots was significantly higher in fertilized soil in comparison to unfertilized soils (p < 0.05). Most probable number (MPN) in planted treatments was significantly higher than in unplanted treatments (p < 0.05).     

On protein fractions and inorganic ions in sea urchin eggs,unfertilized and fertilized

Frozen and dried unfertilized or fertilized eggs were subjected to extraction by 1-N KCl solution according to the method inaugurated by Mirsky.In confirmation of Mirsky's results a certain protein fraction seemed to become insoluble in normal fertilization of the egg.The differences proved almost to vanish if the eggs prior to fertilization were subjected to trypsin treatment which removes the vitelline membrane and thus inhibits the formation of a continuous fertilization membrane.An obvious correlation was found between the percentage of extracted proteins and the salt content of the egg samples (fig. 1). This is particularly high in frozen and dried fertilized eggs with the fertilization membrane normally formed. This probably accounts entirely for the previously reported differences between unfertilized and fertilized eggs.Extraction of the frozen and thawed eggs with distilled water gave the same general picture as extraction with N KCl. Here also the solubility of the proteins is correlated with the salt content of the samples.The dialyzed water extracts gave a strong precipitation upon addition of Ca (optimum at 0.0125-0.025 M). Freezing and thawing of the extracts likewise cause a precipitation of a certain fraction.The fraction which becomes insoluble in the presence of high salt content of the sample and that precipitated from extracts by additions of Ca or by freezing and thawing seem to be identical. It contains the soluble nucleoprotein of the eggs.No changes in Ca or Mg content was found to occur upon fertilization of the egg.