Assessment of effect of partial sterility on mating performance in sweetpotato weevil (Coleoptera: Curculionidae)

The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations. Although the effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females, the use of gamma radiation to induce sterility negatively impacts reproductive cells as well as somatic cells. Consequently, sterilization by irradiation drastically diminishes mating performance over time. In the current study, we evaluated the effect of irradiation dose intensity on fertility, mating propensity, and mating competitiveness in sweetpotato weevil, Cylas formicarius elegantulus (Summers) (Coleoptera: Curculionidae), for 16 d after irradiation. Although the mating propensity of males irradiated with 200 Gy, the dose currently used to induce complete sterility of C. f. elegantulus in the SIT program in Okinawa Prefecture, was equal to that of nonirradiated weevils for the first 6 d, the mating propensity of males irradiated with doses between of 75 and 150 Gy was maintained for the first 12 d. The potential fertilization ability of weevils was highly depressed compared with the control weevils, even in those treated with 75 Gy. Mating performance was severely compromised in weevils that were irradiated with a dose of 100 Gy or more. These results demonstrate that partial sterilization can be highly advantageous in eradication programs for the sweetpotato weevil. We discuss the advantages of the application of partial irradiation in insect eradication programs.     

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.     

The neogregarine protozoan Farinocystis sp. reduces longevity and fecundity in the West Indian sweet potato weevil, Euscepes postfasciatus (Fairmaire)

The number of West Indian sweet potato weevils, Euscepes postfasciatus, being mass-reared in a facility for use in sterile insect technique (SIT) eradication programs has undergone a drastic reduction. A neogregarine protozoan pathogen Farinocystis sp. (an undescribed species) was detected in vivo in the mass-reared E. postfasciatus. We investigated the effects of this disease on the longevity and fecundity of host weevils and the incubation time of the disease in the host body under mass-rearing conditions. Our results demonstrated that infection by this Farinocystis sp. decreased both longevity and fecundity in E. postfasciatus. In particular, the pathogen severely limited the production of progeny by infected females compared to healthy females. Therefore, we consider this protozoan infection to be the major cause of the decreased E. postfasciatus production in the mass-rearing facility.     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.     

Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide

Calreticulin (CRT), a Ca²⁺-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H₂O₂, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca²⁺ ([Ca²⁺]_i) were significantly increased by H₂O₂, whereas in controls, only a slight increase was observed. The H₂O₂-induced apoptosis was enhanced by the increase in [Ca²⁺]_i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca²⁺ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca²⁺]_i in the sensitivity to H₂O₂-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca²⁺]_i in the CRT-overexpressing cells treated with H₂O₂ compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H₂O₂ through a change in Ca²⁺ homeostasis and the regulation of the Ca²⁺-calpain-caspase-12 pathway in myocardiac cells. apoptosis; calcium; endoplasmic reticulum     

DNA binding affinity and sequence permutation preference of the telomere protein from Euplotes crassus

Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA x EcTEBP and DNA x EcTEBP-N complexes were comparable: K(D-DNA) = 38 +/- 2 nM for the full-length protein and K(D-DNA) = 60 +/- 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 +/- 4 microM(-1) s(-1), kd = 0.10 +/- 0.02 s(-1)) relative to that of the N-terminal domain (ka = 18 +/- 1 microM(-1) s(-1), kd = 0.15 +/- 0.01 s(-1)). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55-1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5'-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.     

Suppressing effect of gamma-irradiated weevils on progeny production in the West Indian sweetpotato weevil Euscepes postfasciatus (Coleoptera: Curculionidae)

The West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) is a major pest of sweet potato Ipomoea batatas (L.) in the tropical and subtropical regions. The sterile insect technique (SIT) could be used as one of the most effective methods for suppression or eradication of the weevil. The effectiveness of SIT depends on the ability of the released sterile males to mate with and inseminate wild females. However, the effect of sterile weevils on the fitness of E. postfasciatus has not been evaluated on natural density. Here, we investigated the effect of gamma-irradiated weevil density on the number of weevil progeny. When irradiated weevils were released in numbers equal to those of non-irradiated weevils, the number of progeny was reduced by half of that in the control treatment, and it remained at this state for 2?weeks. Our results show that irradiated weevils ensure adequate and efficient suppression of wild weevils. We conclude that the SIT programs will be employed as effective eradication method for E. postfasciatus.     

Rigidity matching between cells and the extracellular matrix leads to the stabilization of cardiac conduction

Biomechanical dynamic interactions between cells and the extracellular environment dynamically regulate physiological tissue behavior in living organisms, such as that seen in tissue maintenance and remodeling. In this study, the substrate-induced modulation of synchronized beating in cultured cardiomyocyte tissue was systematically characterized on elasticity-tunable substrates to elucidate the effect of biomechanical coupling. We found that myocardial conduction is significantly promoted when the rigidity of the cell culture environment matches that of the cardiac cells (4 kiloPascals). The stability of spontaneous target wave activity and calcium transient alternans in high frequency-paced tissue were both enhanced when the cell substrate and cell tissue showed the same rigidity. By adapting a simple theoretical model, we reproduced the experimental trend on the rigidity matching for the synchronized excitation. We conclude that rigidity matching in cell-to-substrate interactions critically improves cardiomyocyte-tissue synchronization, suggesting that mechanical coupling plays an essential role in the dynamic activity of the beating heart.