Laboratory assessment of the impacts of transgenic Bt rice on the ecological fitness of the soil non-target arthropod, Folsomia candida (Collembola: Isotomidae)

Transgenic rice expressing Bacillus thuringiensis (Bt) endotoxins (Bt rice) for pest control is considered an important solution to food security in China. However, tests for potential effects on non-target soil organisms are required for environmental risk assessment. The soil collembolan Folsomia candida L. (Collembola: Isotomidae) is a potential non-target arthropod that is often used as a biological indicator in bio-safety assessments of transgenic crops. In the present study, the roots, stems, and leaves of Bt rice were exposed to F. candida under laboratory conditions, with survival, reproduction and growth of the collembolan as ecological fitness parameters. Significant differences in ecological fitness were found among the different treatments, including differences in the plant parts and varieties of non-Bt rice, presumably as the result of three factors: gene modification, plant parts and rice varieties. The fitness of F. candida was less affected by the different diets than by the exposure to the same materials mixed with soil. Our results clearly showed that there was no negative effect of different Bt rice varieties on the fitness of F. candida through either diet or soil exposure.     

The Gut of the Soil Microarthropod Folsomia candida (Collembola) Is a Frequently Changeable but Selective Habitat and a Vector for Microorganisms

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 10² to 2.3 × 10⁶ CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida, gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.     

Chemical Attraction of Conspecifics in <Emphasis Type="Italic">Folsomia candida</Emphasis> (Collembola)

Chemical substances excreted from individuals are regarded as chemo-sensory signals that induce aggregation in Collembola. However, chemical or physiological evidence is required to identify the chemical composition of signals responsible for aggregation behavior. Present study applied solvent extraction and gas chromatograph-mass spectrometer-computer technology to separate and identify the chemical compounds in cuticle extracts from the model Collembola species, Folsomia candida. The occurrence of aggregation behavior was confirmed by conspecific attractiveness using a trap bioassay. Eight chemical compounds were obtained from F. candida’s cuticle solvent. The trap assay detected fatty acid combinations from the body extracts of F. candida that induced attractiveness behavior among conspecific individuals and small molecules with higher concentration induced repellent. However, single sensillum recordings of two thin-wall chemical receptors at the second segment of the distal position of the antenna did not receive any positive responses to fatty acids, either single or in combination. The way F. candida used fatty acid combinations as chemical signal for conspecific aggregating was discussed.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

A new presumably widespread species of Albugo parasitic to Strigosella spp. (Brassicaceae)

White blister rust is one of the most common diseases in Brassicaceae. Recently, molecular approaches revealed that apart from Albugo candida, several other more specialized species of the genus are causing this disease on Brassicaceae and the diversity of this group still remains largely unexplored. All newly described species have so far been sampled only from a limited geographic range, except for Albugo species which followed their invasive host from Europe to other continents. In this study we show that a previously unknown species of Albugo is causing white blister rust disease on two species of Strigosella. This species can be distinguished from other species of the genus Albugo both by its phylogenetic position and its unique oospore ornamentation that might be an adaptation to the harsh environment of the host plants. As a consequence, Albugo arenosa is described and illustrated as a new species, so far known from Strigosella africana in Iran and Spain, and Strigosella brevipes in Iran. Apart from Albugo candida and Albugo lepidii, this is the third hitherto known species of Albugo s. str. with a confirmed native distribution range of several thousand kilometres.     

Scopolamine,anisodamine and hyoscyamine production by Brugmansia candida hairy root cultures in bioreactors

Hyoscyamine, anisodamine (6β-hydroxyhyoscyamine) and scopolamine are tropane alkaloids produced by plants belonging to the Solanaceae family such as Brugmansia candida. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Further therapeutical properties for anisodamine were recently described renewing the interest in these alkaloids. The scaling-up of hairy root cultures is an interesting strategy for the pharmaceutical production of these compounds instead of the isolation from plants. In this work, B. candida hairy roots were cultured in a modified 1.5 L stirred tank in order to analyze an alternative production system of scopolamine and anisodamine. It was found that these cultures produced an increased biomass and alkaloids concentration compared to the processes carried out in Erlenmeyer flasks. Anisodamine was the predominant alkaloid reaching a maximum concentration of 10.05 ± 0.76 mg/g DW in modified bioreactor culture system. The results obtained in this work are potentially applicable for the rational scale-up of the process.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Structure and Cytochemistry of the Stigma and Pollen--Pistil Interaction in Zephyranthes

The stigma in Zephyranthes candida and Z. citrina is of thedry type with a continuous cuticle—pellicle. In some papillae,however, the terminal portion of the cuticle—pellicleis lifted upwards and occasionally even disrupted by the accumulationof a secretion product below it. Both non-specific esterasesand acid phosphatases are present on the stigma surface. Thestyle is solid with a central core of transmitting tissue whichhas conspicuous intercellular spaces containing a matrix thatincludes proteins, polysaccharides and pectic substances. Zephyranthes citrina is self-compatible while Z. candida isself-incompatible. Followng incompatible pollination in Z. candida,pollen germination is normal but pollen tube growth is inhibitedat the junction of the stigma and style. Self-incompatibilitycan be overcome by bud pollination. Protein synthesis is necessaryfor pollen germination in both species. Concanavalin A bindsto the stigma surface of both species, but does not affect pollentube penetration in Z. candida. In crosses between the two speciestypical unilateral incompatibility is observed when Z. candidais used as the pistillate parent. Zephyranthes, stigma-surface enzymes, dry stigma, pollen-pistil interaction, self-incompatibility, unilateral-incompatibility     

Distribution of subfossil ostracod assemblages in lacustrine profundal sediments of north-eastern Poland

The distribution of ostracod valves was studied in seven short (< 50 cm) sediment cores collected from the sublittoral-profundal zones (depth > 12 m) of four lowland, dimictic lakes (Kamedu?, Szelment Ma?y, Szelment Wielki and Szurpi?y) situated in the Eastern Suwa?ki Lakeland of NE Poland. These postglacial channel lakes are still moderate in trophy level, but to some extent, they differ from each other in the increase of anthropogenic alterations, especially in their hypolimnion. The studied sediment sequences, corresponding roughly to the period of the last ca. 300 years, yielded ca. 5500 valves of 20 ostracod species, out of which only seven are here considered as the autochthonous profundal component of the fossil assemblages: Candona candida, C. neglecta, Fabaeformiscandona protzi, F. tricicatricosa, Bentocypria curvifurcata, Cytherissa lacustris and Limnocytherina sanctipatricii. Valves of the remaining species were most probably subjected to postmortem transport from shallow water deposits or from lakeside and/or subaqueous springs, and thus were not deposited in situ at the deep bottom. By clustering classification and multi-dimensional scaling ordination, three major assemblage types were recognised in the moderately eutrophicated parts of the studied lakes: (a) assemblage dominated by C. candida, (b) assemblage dominated by C. lacustris and (c) the most diverse assemblage, in which four species (C. lacustris, C. candida, F. protzi and C. neglecta) occur in more or less equal percentages. On the other hand, sediments of the most eutrophic basins of the lakes (especially in Kamedu? and Szelment Ma?y) are characterised by the decline of total valve abundances and complete extinction of C. lacustris, a species indicative of low trophy, so that, in extreme cases, a few valves (if any) of more eurytopic or only allochthonous species could be found in some of the uppermost layers. Successional transitions of the recognised assemblage types are discussed according to the generalised model of the deep lacustrine ostracod succession previously established for the Polish lakes on the country scale. The recorded assemblages correspond well with the trophic diversity of the studied lakes, confirming inferences based on previously published data on other trophy indices from this area. Finally, additional data on modern profundal ostracod fauna from dredge samples of the studied lakes are also provided.     

Appendage patterning in the primitively wingless hexapods Thermobia domestica (Zygentoma: Lepismatidae) and Folsomia candida (Collembola: Isotomidae)

Arthropod appendages are among the most diverse animal organs and have been adapted to a variety of functions. Due to this diversity, it can be difficult to recognize homologous parts in different appendage types and different species. Gene expression patterns of appendage development genes have been used to overcome this problem and to identify homologous limb portions across different species and their appendages. However, regarding the largest arthropod group, the hexapods, most of these studies focused on members of the winged insects (Pterygota), but primitively wingless groups like the springtails (Collembola) or silverfish and allies (Zygentoma) are underrepresented. We have studied the expression of a set of appendage patterning genes in the firebrat Thermobia domestica and the white springtail Folsomia candida. The expressions of Distal-less (Dll) and dachshund (dac) are generally similar to the patterns reported for pterygote insects. Modifications of gene regulation, for example, the lack of Dll expression in the palp of F. candida mouthparts, however, point to changes in gene function that can make the use of single genes and specific expression domains problematic for homology inference. Such hypotheses should therefore not rely on a small number of genes and should ideally also include information about gene function. The expression patterns of homothorax (hth) and extradenticle (exd) in both species are similar to the patterns of crustaceans and pterygote insects, but differ from those in chelicerates and myriapods. The proximal specificity of hth thus appears to trace from a common hexapod ancestor and also provides a link to the regulation of this gene in crustaceans.     

Gallic acid as a natural inhibitor of flowering in Kalanchoe blossfeldiana

Gallic acid (I) has been isolated as a specific flowering-inhibitory substance from leaves of vegetative Kalanchoe blossfeldiana. It is also present in leaves of flowering Kalanchoe, apparently in an inactive, non-dialysable form. Gallic acid acts as a flowering inhibitor when applied to Kalanchoe, a short-day plant. Its detection and isolation was facilitated by use of a bioassay based on tissue culture of partially induced apices of the long-day plant Viscaria candida.     

Molecular mapping reveals two independent loci conferring resistance to Albugo candida in the east European germplasm of oilseed mustard Brassica juncea

White rust caused by Albugo candida (Pers.) Kuntze is a major disease of the oilseed mustard Brassica juncea. Almost all the released varieties of B. juncea in India are highly susceptible to the disease. This causes major yield losses. Hence, there is an urgent need to identify genes for resistance to white rust and transfer these to the existing commercial varieties through marker-assisted breeding. While the germplasm belonging to the Indian gene pool is highly susceptible to the disease, the east European germplasm of B. juncea is highly resistant. In the present study, we have tagged two independent loci governing resistance to A. candida race 2V in two east European lines, Heera and Donskaja-IV. Two doubled haploid populations were used; the first population was derived from a cross between Varuna (susceptible Indian type) and Heera (partially resistant east European line) and the second from a cross between TM-4 (susceptible Indian type) and Donskaja-IV (fully resistant east European line). In both the resistant lines, a single major locus was identified to confer resistance to white rust. In Heera, the resistance locus AcB1-A4.1 was mapped to linkage group A4, while in Donskaja-IV, the resistant locus AcB1-A5.1 was mapped to linkage group A5. In both the cases, closely linked flanking markers were developed based on synteny between Arabidopsis and B. juncea. These flanking markers will assist introgression of resistance-conferring loci in the susceptible varieties.     

Morphological and molecular confirmation of Albugo resedae (Albuginales; Oomycota) as a distinct species from A. candida

The genus Albugo s.str. causes white blister rust on four families of the Brassicales, Brassicaceae, Capparaceae, Cleomaceae, and Resedaceae. Recent phylogenetic studies have revealed that several host specific lineages are present within Albugo on Brassicales, while it was also confirmed that Albugo candida has an exceptionally wide host range which extends from Brassicaceae to Cleomaceae and Capparaceae. The Albugo species infecting the Resedaceae was attributed in monographic studies as well as local floras to either A. resedae or, applying a broader species concept, to A. candida. In the present study, A. resedae specimens were morphologically and molecularly compared to the five Albugo species so far confirmed from Brassicales, A. candida, A. koreana, A. laibachii, A. lepidii, and A. voglmayrii. Both morphological differences of oospore ornamentation and phylogenetic analysis of cox2 mtDNA sequences provided evidence that A. resedae is distinct from A. candida and from the additional four species so far described from Brassicaceae. It thus seems possible that so far unknown factors restrict Albugo candida to Brassicaceae and its sister families, Cleomaceae and Capparaceae.     

6α-Acetoxy-16β,22-dihydroxyhopan-24-oic acid,a triterpene from the fern Notholaena candida var. copelandii

Farinose exudates on fronds of gymnogrammoid ferns generally consist of flavonoid aglycones. In Notholaena candida var. copelandii a new triterpene was found as a major component of the farina besides galangin 3-methylether and kaempferol 3-methylether. Extensive mass spectral and NMR studies revealed this triterpene to be a new natural product, 6α-acetoxy-16β,22-dihydroxyhopan-24-oic acid.     

Wolbachia endosymbiont is essential for egg hatching in a parthenogenetic arthropod

Wolbachia pipientis can induce a range of sex ratio distortions including parthenogenesis. Recently Wolbachia has been detected in the diploid, parthenogenetic, collembolan species Folsomia candida. In this paper we address the effect of Wolbachia on reproduction in F. candida. Wolbachia infection was removed by antibiotic and heat treatment, and quantitative PCR techniques confirmed the success of the treatments. Complete loss of Wolbachia-infection led to the production of normal clutch sizes, but was associated with full egg hatching failure. Our results demonstrate that F. candida is strictly dependent on Wolbachia to produce viable offspring. This is one of the few cases of obligatory Wolbachia infection in arthropods. Our data suggest a unique mechanism underlying Wolbachia-dependence of egg development. One of our more salient results is that hatching success increased in consecutive egg clutches of antibiotic-treated individuals, probably due to restoration of bacterial densities over time. These observations suggest that reproduction in F. candida is a threshold effect requiring a critical Wolbachia density as is hypothesized by the bacterial dosage model. Quantitative PCR analysis showed that heat or antibiotic treated individuals with egg hatching failure had low average bacterial densities, but bacterial densities were not significantly different from those of treated individuals with successfully eclosing eggs. Additional experiments with partially cured F. candida are needed to prove the dosage model.     

Morphological variability of the water lily (<Emphasis Type="Italic">Nymphaea</Emphasis>) in the Polesie Zachodnie region,Eastern Poland

Morphological analysis of the water lily (Nymphaea) was carried out to verify distribution of two closely related species Nymphaea alba and N. candida in the large swampy area in Eastern Poland, Polesie Zachodnie. A total of 25 lakes were investigated across the region. Data were collected from 20 lakes of different origin. Based on morphological characters of flowers and leaves the specimens varied continuously from “pure” N. candida to N. alba with high number of plants with mixed morphology, commonly known as N. × borealis. The intermediate morphotypes were common in all over the study area, whereas plants with exact morphology of N. candida were found mostly in humic lakes, in the eastern part of the region. The results provide evidences that N. candida occurs beyond formerly recognized southern border of its range in the country.