Encystment <Emphasis Type="Italic">in vitro</Emphasis> of the Cercariae <Emphasis Type="Italic">Himasthla elongata</Emphasis> (Trematoda: Echinostomatidae)

Trigger and toxic effects of Mytilus edulis (Bivalvia) hemolymph on encystment of cercariae Himasthla elongata obtained from infestated Littorina littorea (Prosobranchia) was evaluated as a result of 24-h experiments in vitro. The contact of H. elongata larvae with the whole hemolymph or mussel acellular plasma led to an intensive transformation of cercariae into metacercariae. In both tested media, the cercariae had to complete the encystment phase as fast as for 2 h, otherwise the risk of the larvae injury by humoral and cellular components of the mussel hemolymph would increase dramatically. The cercaria mortality after 24 h in the whole hemolymph was twice higher than in plasma (40% and 20%, respectively) and much higher than in the control medium (sea water). Both toxic and trigger effects of plasma was revealed to depend on its concentration, with the maximal larva mortality in the undiluted medium and with the highest number of successful transformations in the medium diluted more than 4 times. There is shown both the strong individual variability of toxicity of the individual mussel hemolymph for cercariae and the variability of the resistance to the toxic factors of the cercariae obtained from various L. littorea individuals. These experiments not only offer a method of the massive encystment of H. elongata cercariae, but also propose a perspective model for the study of the systemic defensive response of Bivalvia to invasion of multicellular parasite.     

Study of protein metabolism of herring gulls (<Emphasis Type="Italic">Larus argentatus</Emphasis> Pontop.) infected by trematode <Emphasis Type="Italic">Himasthla larina</Emphasis> (Trematoda: Echinostomatidae)

The values and dynamics of some indices of protein metabolism were studied in herring gulls Larus argentatus infected with trematode Himasthla larina in natural populations and in experiment. These indices were compared in infected and uninfected birds. Trematode infection considerably affected host protein metabolism irrespective of the age; however, the changes were more pronounced in nestlings. Increased concentration of γ-globulins, modified albumin, and circulating immune complexes was observed in plasma of infected herring gulls. The experiments demonstrated the most significant changes in protein metabolism of herring gulls 8–11 days after infection with trematode H. larina.     

Surface display of lipolytic enzyme,Lipase A and Lipase B of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spore

For the enhancement of lipase stability in organic solvent containing reaction, live immobilization method, using Bacillus subtilis spore as a display vehicle was attempted. Bacillus subtilis coat protein cotE was used as an anchoring motif for the display of lipA and lipB of Bacillus subtilis. Using this motif, lipolytic enzyme Lipase A and Lipase B were functionally displayed on the surface of Bacillus subtilis spore. Purified spore displaying CotE-LipB fusion protein showed higher lipolytic activity compared to that of CotE-LipA fusion protein. The surface localization of Lipase B was verified with flow cytometry and protease accessibility experiment. Spore displayed lipase retained its activity against acetone and benzene which completely deactivated free soluble lipase in the same reaction condition.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Adaptive evolution of osmoregulatory-related genes provides insight into salinity adaptation in Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

Osmoregulation is an important mechanism by which euryhaline crustaceans regulate osmotic and ionic concentrations. The Chinese mitten crab (Eriocheir sinensis) is a strong osmoregulating animal model among crustacean species, as it can maintain its hemolymph composition and survives well in either seawater or freshwater. Osmoregulation by E. sinensis during physiological adaptation has been studied extensively. However, the genetic basis of osmoregulation in E. sinensis for acclimating to changing salinities remains unclear. The current study investigated five genes involved in E. sinensis osmoregulation and compared them with a representative marine crab Portunus trituberculatus to test whether adaptive evolution has occurred changing salinity conditions. The results showed that carbonic anhydrase (CA), cytochrome P450 4C (CYP4C), glutamate dehydrogenase (GDH), and the Na⁺/H⁺ exchanger (NHE) have undergone positive selection (i.e., directional selection) in E. sinensis. Thus, the positive selection in CA and NHE suggests that E. sinensis has enhanced capacity for maintaining systemic acid-base balance and ion regulation. GDH and CYP4C also demonstrated positive selection in E. sinensis, suggesting that E. sinensis might have acquired an enhanced capacity to metabolize glutamate and synthesize ecdysteroids in response to a change in osmotic concentration. The present study provides new insight into the molecular genetic basis of salinity adaption in E. sinensis.     

Overexpression of calmodulin gene fragment from Antarctic notothenioid fish improves chilling tolerance in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Calmodulin (CaM) is a highly conserved calcium sensor protein associated with chilling tolerance in living organisms. It has four EF-hand domains for binding of four Ca²⁺, two of them located in the N-terminus, and the other two in the C-terminus. A notothenioid CaM gene fragment (CaMm), which only codes for N-terminus of CaM (with two EF-hand domains), was introduced into Nicotiana benthamiana. Effects of its overexpression on chilling tolerance in plants were explored. During 4?C or 0?C chilling treatment, both CaMm and CaM transgenic plants showed higher PSII maximum quantum yield, actual quantum yield, and soluble protein content, lower electrolyte leakage and malondialdehyde content than that of the control. The changes in these physiological indices were comparable between the CaMm and CaM transgenic plants during the treatments. These results indicate that the N-terminus of calmodulin is likely the key functional domain involved in the adaptive response to cold stress.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

The bitter crab syndrome in commercial crabs in the Western part of the Bering and Chukchi Seas

We studied the distribution of the “bitter-crab” syndrome, a disease caused by the parasitic dinoflagellate Hematodinium sp., in eight commercial species of crabs in the west of the Bering and Chukchi seas. The crabs (25 388 individuals) were sampled during bottom trawl surveys of July?September 2010 and October?November 2012. The disease was first identified visually by a color change of the exoskeleton and the hemolymph of the animals and then using microscope analysis of hemolymph samples. Infestation was detected in crabs of three species, Chionoecetes opilio, C. bairdi, and Paralithodes platypus. The prevalence of the disease (the percent of infected individuals relative to all of those examined) in C. bairdi and P. platypus was very low, 0.1 and 0.3%, respectively. Infestation was widespread among C. opilio, its peak in the Bering Sea was in the fall. The average prevalence of the crab disease in different areas of the Bering Sea ranged from 0.8 to 10.8%. A high rate of crab infection was recorded in the Korfa Bay. In the Chukchi Sea, the average prevalence was 2%. Infestation by Hematodinium sp. was not revealed in the deep-sea snow crabs Chionoecetes tanneri and C. angulatus, and in three species of lithodid crabs, Paralithodes camtschaticus, P. brevipes, and Lithodes couesi. This can be explained by the small sample volume and/or ecology of these species, since the disease was registered in other areas in four of them.     

Toxicity and kinetics of spinosad in different developmental stages of the endoparasitoid <Emphasis Type="Italic">Hyposoter didymator</Emphasis> (Hymenoptera: Ichneumonidae) and its host <Emphasis Type="Italic">Spodoptera littoralis</Emphasis> larvae (Lepidoptera: Noctuidae)

The toxicity of spinosad was evaluated using the RaPID Assay^® Spinosad immunosorbent assay in different developmental stages of the parasitoid, Hyposoter didymator, and in its host, fourth-instar larvae of the cotton leafworm Spodoptera littoralis. Spinosad was applied directly to pupae and adults of H. didymator (ingestion or topical application) or to the immature stages of the parasitoid via the host larvae. Low amounts of spinosad were recovered from S. littoralis host larvae after topical treatment, and the compound was mainly retained in the hemolymph. Amounts of spinosad detected in third-instar larvae of H. didymator, pulled out from the hemolymph of parasitized S. littoralis larvae, were 85 pg (3.57 ng a.i./g body weight) in dead larvae, and 82 pg (3.42 ng a.i./g body weight) in alive individuals. After topical treatment of H. didymator cocoons, most of the compound was retained in the silken cocoon, preventing contamination of the pupa. Also in the parasitoid adults, relatively low amounts of spinosad were accumulated in the body overall, but half of all the insecticide recovered was found in the ovaries. The kinetic results obtained help to better understand the toxicity of spinosad in the complex S. littoralis–H. didymator, and to ascertain the compatibility between spinosad and the parasitoid for optimizing the control of lepidopteran pests.     

Nano-silver modifies the vase life of cut herbaceous peony (<Emphasis Type="Italic">Paeonia lactiflora</Emphasis> Pall.) flowers

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-grade cut flower because of higher ornamental value. However, its short flowering time severely restricts the production and application of cut P. lactiflora flowers. In this study, nano-silver (NS) was applied to prolong the vase life of cut P. lactiflora flowers. Under the NS treatment, related physiological indices including relative electrical conductivity (REC), malondialdehyde (MDA), superoxide anion free radical (O₂^·?), hydrogen peroxide (H₂O₂) and free proline contents, and protective enzyme activities including superoxide dismutase (SOD), peroxidase (POD) and ascorbic acid peroxidase (APX) all increased in cut P. lactiflora flowers except soluble protein. Meanwhile, NS treatment increased relative water uptake (RWU) and Ag⁺ distribution. Moreover, the observation of microstructures indicated that the stem-ends without NS treatment were blocked by microbes which were identified as Alternaria sp. and Phoma sp., and NS effectively inhibited their growth by antibacterial efficacy observation. Additionally, three aquaporin genes (AQPs) including two plasma membrane intrinsic protein genes (PlPIP1;2, PlPIP2;1) and one NOD26-like intrinsic protein gene (PlNIP) were isolated, PlPIP1;2, and PlPIP2;1 that were induced by NS treatment took common effects on maintaining the water balance of cut P. lactiflora flowers. Consequently, the vase life of cut P. lactiflora flowers was prolonged and flower fresh weight together with flower diameter was well kept because of these above factors. These results would provide a theoretical basis for prolonging the vase life and improving the ornamental quality of cut P. lactiflora flowers with NS application.     

Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1?, yro2?, and mrh1?yro2?) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.     

Fish Heat Shock Cognate 70 Derived AMPs <Emphasis Type="Italic">Cs</Emphasis>HSC70 A1 and <Emphasis Type="Italic">Cs</Emphasis>HSC70 A2

Heat shock cognate 70 (HSC70) is an important evolutionary conserved protein that plays a major role in maintaining the homeostasis and immunity of many organisms. In this study, a HSC70 from Channa striatus was identified from its cDNA library and characterized using bioinformatics and molecular biology tools. CsHSC70 cDNA was 1953 base pair (bp) in length along with an open reading frame which encoded a polypeptide of 650 amino acid residues. Tissue distribution results showed that CsHSC70 was considerably expressed in gill, to a lesser extent in head kidney, blood, spleen and liver and at low level in other tissues. Using C. striatus gill as cell model, effects of fungal, bacterial and poly I:C stimulant on the mRNA levels of CsHSC70 was examined. We also described the antimicrobial features of two peptides namely CsHSC70 A1and CsHSC70 A2 derived from the N-terminal of CsHSC70 protein. CsHSC70 A1 peptide (40 µg/ml) exhibited potent bactericidal activity against Micrococcus luteus cells. Flow cytometric analysis revealed that the M. luteus cells stained with propidium iodide, upon treated with CsHSC70 A1 at the concentration of 40 µM/ml showed 38% survival compared to its control (99.6%). It seems that CsHSC70 A1 peptide shows antimicrobial activity against M. luteus through membrane disruption. Additionally, scanning electron microscope (SEM) observation confirmed that CsHSC70 A1 peptide treatment completely damaged and destructed the M. luteus cells. Taken together, these findings suggest that CsHSC70 A1 peptide could be a safe and potential therapeutic molecule substitute to antibiotics in various clinical fields.     

Seed protein fraction electrophoresis in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) accessions and wild species

Total seed storage proteins were studied in 50 accessions of A. hypogaea (11 A. hypogaea ssp. hypogaea var hypogaea, 13 A. hypogaea ssp. hypogaea var hirsuta, 11 A. hypogaea ssp. fastigiata var fastigiata and 15 A. hypogaea ssp. fastigiata var. vulgaris accessions) in SDS PAGE. These accessions were also analysed for albumin and globulin seed protein fractions. Among the six seed protein markers presently used, it was found that globulin fraction showed maximum diversity (77.2%) in A. hypogaea accessions followed by albumin (52.3%), denatured total soluble protein fraction in embryo (33.3%) and cotyledon (28.5%). The cluster analysis based on combined data of cotyledons, embryos, albumins and globulins seed protein fractions demarcated the accessions of two subspecies hypogaea and fastigiata into two separate clusters supported by 51% bootstrap value, with few exceptions, suggesting the genotypes to be moderately diverse. Native and denatured total soluble seed storage proteins were also electrophoretically analysed in 27 wild Arachis species belonging to six sections of the genus. Cluster analysis using different methods were performed for different seed proteins data alone and also in combination. Section Caulorrhizae (C genome) and Triseminatae (T genome) formed one, distantly related group to A. hypogaea and other section Arachis species in the dendrogram based on denatured seed storage proteins data. The present analysis has maintained that the section Arachis species belong to primary and secondary genepools and, sections Procumbenetes and Erectoides belong to tertiary gene pools.     

VeA of <Emphasis Type="Italic">Aspergillus niger</Emphasis> increases spore dispersing capacity by impacting conidiophore architecture

Aspergillus species are highly abundant fungi worldwide. Their conidia are among the most dominant fungal spores in the air. Conidia are formed in chains on the vesicle of the asexual reproductive structure called the conidiophore. Here, it is shown that the velvet protein VeA of Aspergillus niger maximizes the diameter of the vesicle and the spore chain length. The length and width of the conidiophore stalk and vesicle were reduced nearly twofold in a ΔveA strain. The latter implies a fourfold reduced surface area to develop chains of spores. Over and above this, the conidial chain length was approximately fivefold reduced. The calculated 20-fold reduction in formation of conidia by ΔveA fits the 8- to 17-fold decrease in counted spore numbers. Notably, morphology of the ΔveA conidiophores of A. niger was very similar to that of wild-type Aspergillus sydowii. This suggests that VeA is key in conidiophore architecture diversity in the fungal kingdom. The finding that biomass formation of the A. niger ΔveA strain was reduced twofold shows that VeA not only impacts dispersion capacity but also colonization capacity of A. niger.     

A RAPD fingerprinting of sibling species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

A comparative analysis of the sibling species of Drosophila virilis was performed by RAPD-PCR technique using a set of random primers. The degree of relatedness was studied by cluster analysis (UPGMA) and multidimensional scaling. The resulting pattern of species relationships contradicts the classical taxonomy. The main result of the cluster analysis is that D. virilis does not cluster with the remaining three species of its phylad, while according to multidimensional scaling, D. virilis is equidistant from all the species of its group, from both the species of its phylad and the species of the montana phylad. The montana phylad is extremely heterogeneous; moreover, the species D. littoralis, D. ezoana, and D. kanekoi appear to be closer to the virilis phylad than to the other species of the montana phylad, wherein these species are traditionally included. The phylogenetic relationships between the studied species discovered using RAPD fingerprinting comply with the results obtained using protein markers and quantitative traits.     

Comparative analysis of otolith morphology in three species of <Emphasis Type="Italic">Scomber</Emphasis>

Geometric morphometrics is a quick and reliable approach to differentiate fish stocks based on the variation of otolith shapes. In this study, morphometric analysis of otolith shapes was used to differentiate three species of Scomber. The sagittae morphology of S. scombrus otolith is totally different from that of S. japonicus and S. australasicus. Multivariate analysis consistently showed that S. japonicus was morphologically similar to S. australasicus, whereas a significant difference in otolith shapes was detected between S. scombrus and other two species of Scomber. The rostrum, antirostrum, excisural notch and dorsal-posterior margin of the otolith reflect the main variations between the three species of Scomber. Shape indices and Fourier coefficients were used to discriminate fish species using analysis of variance and Fisher discriminant analysis. The shape indices successfully differentiated 100%, 95.7% and 96.4% of otoliths in S. japonicus, S. australasicus and S. scombrus, respectively, while the Fourier coefficients only discriminated 70.0%, 61.9% and 91.3% of the sagittae in S. japonicus, S. australasicus and S. scombrus. This study indicates that the shape analysis on the sagittae morphometrics of otoliths is a better method to differentiate species of Scomber.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

A Novel Little Membrane Protein Confers Salt Tolerance in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.     

Delivery of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HpaA to gastrointestinal mucosal immune sites using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its immune efficacy in mice

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.