Ultrastructure of the marine predatory flagellate <Emphasis Type="Italic">Metromonas simplex</Emphasis> Larsen et Patterson, 1990 (Cercozoa)

The ultrastructure of the marine predatory flagellate Metromonas simplex Larsen et Patterson was studied. The cell is surrounded by a low-contrast fibrous layer composed of thin hairs covered by a thin bilayer membrane and an outer layer of thin short fibers. The plasmalemma lies under these layers. The predator captures whole cells of the prey, usually bodonids or chrysomonads. The cytostome as a cell pocket is undetectable. The long flagellum bears very thin mastigonemes (hairs) with lengths of 0.8–1.0 μm; the short flagellum is naked and reduced in length. The transitional zone lacks spirals or other additional elements. The transversal plate is elevated on the cell surface. The flagellar root system is very simple and has one microtubular band which originates near the kinetosomes. The latter are parallel to each other and interconnected by fibrous bridges. The vesicular nucleus, Golgi apparatus, and endoplasmic reticulum are of typical structures. The oval mitochondria of 0.6–2.5 μm contain lamellar cristae. The cylindrical extrusomes (trichocysts) found in the cytoplasm have lengths of 1.0–1.4 μm and diameters of 0.12–0.08 μm. The trichocysts have a wheel-shaped structure with 13 spokes visible in cross-sections. The contractile vacuole is absent. The similarity that M. simplex shares with Metopion fluens Larsen et Patterson, cryothecomonads, and other predatory flagellates is discussed.     

Development of ovarioles and nurse-cell cytoskeleton in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg (Diptera: Calliphoridae)

We have examined ovariole morphology and their development in Calliphora erythrocephala Mg. (Diptera: Calliphoridae). It has been shown that several regions can be distinguished in C. erythrocephala germarium. In these regions, cyst morphogenesis subsequently continues until the separation of the formed egg chamber. The germarium has variously branching fusomes. Two egg chambers develop in the C. erythrocephala vitellarium. The distribution of actin filaments and the formation of ring canals was studied in C. erythrocephala nurse cells. The diameter of the ring canal increases in the germarium zone from 1.5 μm to 14.7 μm during the third and fourth studies of oogenesis. A scheme is presented that illustrates the irregular stretching of actin rollers, which are formed during middle and late oogenesis around ring canals, which joins the proximal and neighboring parts and dramatically increases the size of nurse cells.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.     

Light and electron microscopic study of <Emphasis Type="Italic">Pelomyxa stagnalis</Emphasis> sp. n. (Archamoebae,pelobiontida)

The structure of a new pelomyxa species was investigated on the level fo light and electron microscopy. The length of locomotive forms of Pelomyxa stagnalis reaches 800 μm. The thin layer of amorphous glycocalyx is located on the cell surface. Numerous nonfunctioning flagellae are revealed predominantly in the uroidal zone. The axoneme has a nonstable set of microtubules. No additional structures are present in the transition zone. The length of P. stagnalis flagella kinetosomes does not exceed 150 nm. Fifteen to twenty microtubules extend from the side surface of each kinetosome at a small angle to the cell surface. One of main components of the P. stagnalis cytoplasm are structural vacuoles. Glycogen bodies in cells are surrounded by flattened ER cisterns, which are often filled with electron-dense material. Cells of P. stagnalis were found to contain two species of prokaryote endobionts that differ in the peculiarities of their fine structure. The number of nuclei in cells of the P. stagnalis adult individuals can reach 50 or more. The nuclei are surrounded by a bilayer envelope formed by the multilaminar layer and by the outer layer composed of vesicles often filled with an electron-dense material. The nucleolus is usually single and is located in the center of the nucleus. In nuclei, predominantly in connection with nucleoli, bodies are formed that are formed by interlacing electron-dense strands.     

Identification,distribution and occurrence of the ascomycete <Emphasis Type="Italic">Metschnikowia typographi</Emphasis> in the great spruce bark beetle, <Emphasis Type="Italic">Dendroctonus micans</Emphasis>

This is the first report on the ascomycete Metschnikowia typographi from the adults and larvae of the great spruce bark beetle Dendroctonus micans in Turkey. In total, 910 of 1928 adults and 44 of 149 larvae investigated during the two years were infected by the pathogen. In a fresh smear the asci of the pathogen measure 18.5 ± 2.05 μm (14.7–22.3) in length and 2.1 ± 0.4 μm in width (n = 35). The ascospores are about 2 μm shorter than asci, having an average length of 16.4 ± 1.5 μm (14.2–18.0). The total infection rate of D. micans was 47.2 %. The prevalence of M. typographi infections differed between localities and years. Different infection rates of male and female beetles of D. micans were not recognized.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> roxb

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microtuttings on a peat moss, soil, and compost mixture (1∶1∶1). About 52% of plantlets were successfully acclimatized and established in pots.     

Plant regeneration,origin, and development of shoot buds from root segments of <Emphasis Type="Italic">Melia azedarach</Emphasis> L. (<Emphasis Type="Italic">Meliaceae</Emphasis>) seedlings

Summary In vitro regeneration of plants from root culture of Melia azedarach seedlings was obtained. The origin and mode of development of the regenerated shoot buds were studied by means of histological analysis and scanning electron microscopy (SEM). Maximum shoot bud regeneration was achieved when root segments were cultured on Murashige and Skoog (MS) medium at quarter strength with 3% sucrose and 0.44 μM benzyladenine (BA) and kept under light (116 μmol m⁻² s⁻¹). Shoot bud elongation was achieved on MS with 0.44 μM BA, 0.46 μM kinetin (KIN), and 3.26 μM adenine sulphate (AD). Regenerated shoots were rooted on MS with 12.26 μM indole-3-butyric acid (IBA) for 4 d and subsequently in MS lacking plant growth regulators for 26 d. Plants were established in a potting substrate. Histological analysis of roots from intact seedlings (without treatment) demonstrated that during the early life of the roots, M. azedarach lacks preformed buds. In contrast, when the roots were excised and cultured in vitro, the histology and SEM observations revealed that buds originated from meristematic groups of cells, which had been formed from the pericycle and several layers beneath. These meristematic groups of cells grew towards the periphery of the cortex by crushing the outer layer of cortical cells. Further develoment led to the differentiation of leaf primordia and a shoot apical meristem.     

In vivo light-microscopic documentation for primary calcification processes in the hermatypic coral Stylophora pistillata

Skeletogenesis in the hermatypic coral Stylophora pistillata was studied by using the lateral skeleton preparative (LSP) assay, viz., a coral nubbin attached to a glass coverslip glued to the bottom of a Petri dish. Observations on tissue and skeletal growth were made by polarized microscopy and by using vital staining. The horizontal distal tissue edges developed thin transparent extensions of ectodermal and calicoblastic layers only. Four stages (I-IV) of skeletogenesis were observed at these edges, underneath the newly developed tissue. In stage I, a thin clear layer of coral tissue advanced 3–40 μm beyond the existing LSP peripheral zone, revealing no sign of spiculae deposition. At stage II, primary fusiform crystals (1 μm each) were deposited, forming a primary discontinuous skeletal front 5–30 μm away from the previously deposited skeleton. During stage III, needle-like crystals appeared, covering the primary fusiform crystals. Stage IV involved further lengthening of the needle-like crystals, a process that resulted in occlusion of the spaces between adjacent crystals. Calcification stages I-III developed within hours, whereas stage IV was completed in several days to weeks. Two basic skeletal structures, “scattered” and “laminar” skeletons, were formed, integrating the growth patterns of the needle-like crystals. High variation was recorded in the expression of the four calcification stages, either between different locations along a single LSP or between different preparations observed at the same diurnal time. All four skeletogenesis stages took place during both day and night periods, indicating that an intrinsic process controls S. pistillata calcification. This study was supported by the Israel Science Foundation (206/01 to J.E.), by the BARD, US-Israel Bi-National Agricultural Research and Development, by INCO-DEV project (REEFRES), and by CORALZOO, EC Collective Research project.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

Growth retardants stimulate guggulsterone production in the presence of fungal elicitor in fed-batch cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the stimulatory effects of growth retardants [ALAR (N,N-dimethylaminosuccinamic acid) and CCC (chlormequat chloride)] and fungal elicitor on guggulsterone accumulation in cell cultures of C. wightii are reported. CCC at 1 mg l⁻¹ enhanced guggulsterone content (~123 μg l⁻¹) when added on the fifth day after inoculation, while ALAR at 2.5 mg l⁻¹ increased guggulsterone content (~116 μg l⁻¹) when added on the tenth day. In a two-stage fed-batch process, combined treatment with fungal elicitor and growth retardant caused a significant increase (~353 μg l⁻¹) in guggulsterone content in cell cultures after 17 days of growth. This represents an approximately fivefold increase over the guggulsterone contents in initial cultures of this plant.     

Acute toxic impacts of three heavy metals (copper,zinc, and cadmium) on <Emphasis Type="Italic">Diaphanosoma brachyurum</Emphasis> (Cladocera: Sididae)

Diaphanosoma brachyurum (Cladocera: Sididae) is a common limnetic species in summer-temperate and tropical water bodies. Few studies have investigated the sensitivity of D. brachyurum to toxic chemicals despite this species often being dominant in natural lakes and ponds. We performed acute toxicity tests of three heavy metals, copper (Cu), zinc (Zn), and cadmium (Cd), to D. brachyurum. For D. brachyurum, the lethal concentration (LC)₅₀ values of Cu (24-h LC₅₀ = 16.4 μg/L, 48-h LC₅₀ = 10.4 μg/L) and Zn (24-h LC₅₀ = 253.4 μg/L, 48-h LC₅₀ = 174.1 μg/L) were lower than those for D. magna, one of the most used test organisms for toxic chemicals. On the other hand, for D. brachyurum the 24-h LC₅₀ of Cd (166.4 μg/L) was much greater than that for D. magna, and the 48-h LC₅₀ of Cd (69.8 μg/L) was comparable. Our results indicate that D. brachyurum may be more strongly influenced by Zn and Cu than is D. magna. It is likely that the summer plankton community in which Diaphanosoma species is dominant is more sensitive to heavy metals than a community in which Daphnia species are dominant.     

Process of Carbonate Precipitation by Deleya halophila

Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the formation of carbonate crystals by Deleya halophila. The formation of calcium carbonate crystals (polymorphous aragonite) by D. halophila is a sequential process that commences with a nucleus formed by the aggregation of a few calcified bacterial cells and the subsequent accumulation of more calcified cells and carbonate, which acts to weld the bacteria together. The process leads to the formation of spherical bioliths measuring approximately 50 μm in diameter. The mechanism of carbonate precipitation by D. halophila under our working conditions represents a process of induced biomineralization.     

Epidermal ultrastructure of Seison nebaliae and Seison annulatus, and a comparison of epidermal structures within the Gnathifera

 The epidermis of both species of Seison is syncytial and has a characteristic internal layer divided into two distinct sublayers. Sublayer I is very thin (0.03 μm) and bounded to the outer cell membrane of the epidermis. Sublayer II is 0.5 μm thick and separated from sublayer I by a thin layer of cytoplasm. Intrusions of the outer cell membrane of the epidermis perforate the internal layer, before terminating within the cytoplasm. The intrusions of the cell membrane of S. annulatus are coated by an electron-dense material, the annulus, when passing through the internal layer. Bundles of filaments are present in the epidermis of S. nebaliae. A comparison of epidermal structures within the Gnathifera Ahlrichs, 1995, confirms phylogenetic relationships earlier proposed by the author. Accepted: 19 December 1996     

In vitro regeneration in <Emphasis Type="Italic">Dierama erectum</Emphasis> Hilliard

The genus Dierama comprises plants with a potential to be developed as ornamentals. D. erectum seeds were decontaminated and germinated on 1/10th strength Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) media without plant growth regulators or sucrose. In an experiment investigating the effects of 6-benzyladenine (BA), meta-Topolin (mT), kinetin (KIN) and zeatin (Z) with or without α-naphthaleneacetic acid (NAA), the highest shoot number per hypocotyl (4.20 ± 0.51) was obtained from MS medium supplemented with 1.0 μM Z after 8 weeks. This was followed by a combination of 2.0 μM KIN and 2.0 μM NAA with 3.67 ± 0.81 shoots per explant. BA treatments produced 3.20 ± 0.22 shoots per hypocotyl explant when 2.0 μM was combined with 1.0 μM NAA, while mT gave 3.09 ± 0.99 shoots per explant when 2.0 μM mT was combined with 2.0 μM NAA. Adventitious shoot regeneration was optimised when shoots were grown under a 16-h photoperiod at 100 μmol m⁻² s⁻¹ on MS medium supplemented with 1.0 μM BA. This resulted in an average of 12.73 ± 1.03 shoots per hypocotyl explant. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation of this species. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) and had an average root number of 2.73 ± 0.40. After 2 months of acclimatisation, plants had formed corms. The largest corms (of diameter 0.45 ± 0.03 cm) were produced in plants pre-treated with 0.5 μM IBA. The highest plant survival percentage of 73% was also associated with this treatment.     

Analysis of 3D structural differences in the IgG-binding domains based on the interresidue average-distance statistics

It is well-known that the IgG-binding domain from staphylococcal protein A folds into a 3α helix bundle structure, while the IgG-binding domain of streptococcal protein G forms an (α + β) structure. Recently, He et al. (Biochemistry 44:14055–14061, 2005) made mutants of these proteins from the wild types of protein A and protein G strains. These mutants are referred to as protein A219 and protein G311, and it was showed that these two mutants have different 3D structures, i.e., the 3α helix bundle structure and the (α + β) structure, respectively, despite the high sequence identity (59%). The purpose of our study was to clarify how such 3D structural differences are coded in the sequences with high homology. To address this problem, we introduce a predicted contact map constructed based on the interresidue average-distance statistics for prediction of folding properties of a protein. We refer to this map as an average distance map (ADM). Furthermore, the statistics of interresidue distances can be converted to an effective interresidue potential. We calculated the contact frequency of each residue of a protein in random conformations with this effective interresidue potential, and then we obtained values similar to ϕ values. We refer to this contact frequency of each residue as a p(μ) value. The comparison of the p(μ) values to the ϕ values for a protein suggests that p(μ) values reveal the information on the folding initiation site. Using these techniques, we try to extract the information on the difference in the 3D structures of protein A219 and protein G311 coded in their amino acid sequences in the present work. The results show that the ADM analyses and the p(μ) value analyses predict the information of folding initiation sites, which can be used to detect the 3D difference in both proteins.     

Functional morphology of Tethya species (Porifera): 1. Quantitative 3D-analysis of Tethya wilhelma by synchrotron radiation based X-ray microtomography

Rhythmic body contraction is a phenomenon in the Porifera, which is only partly understood. As a foundation for the understanding of the functional morphology of the highly contractile Tethya wilhelma, we performed a qualitative and quantitative volumetric 3D-analysis of the morphology of a complete non-contracted specimen at resolutions of 5.2 and 6.9 μm, using synchrotron radiation based X-ray computed microtomography (SR-μCT). For the first time, we were able to visualize all three major body structures of a complete poriferan without dissection of the shock-frozen, fixed and contrasted specimen in a near-to-life confirmation: poriferan tissue, mineral skeleton and aquiferous system. Applying a ‘virtual cast’ technique allowed us to analyze the structural details of the complete canal structure. Our results imply an extensive re-circulation of water inside the poriferan due to well-developed by-pass-canals, connecting excurrent and incurrent system. Nevertheless, the oscule region is strictly separated from the incurrent system. Based on our data, we developed a hypothetical flow regime for T. wilhelma, which explains the necessity of by-pass canals to minimize pressure boosts in the canal system during contraction. Additionally, re-circulation optimizes nutrient uptake, within small-sized poriferans, like T. wilhelma. Quantitative analysis allowed us to measure volumes and surfaces, displaying remarkable organizational differences between choanosome and cortex, by means of distribution of morphological elements. The surface-to-volume ratio proved to be very high, underlining the importance of the poriferan pinacoderm. We support a pinacoderm-contraction hypothesis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Dedicated to Prof. Dr. Michele Sarà (Genova, Italy), in honour of his 80th birthday in 2006.     

Multiple shoot formation and plant regeneration from stem nodal explants of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Summary Stem nodal explants of Paphiopedilum philippinense hybrids (hybrid PH59 and PH60) directly formed shoots when cultured on a modified half-strength Murashige and Skoog (1962) basal medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D: 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45 and 4.54 μM) for 6 mo. On hormone-free basal medium, the percentages of explants with shoots were 33.3% and 0% and the shoot numbers per explant were 1 and 0 in hybrid PH59 and hybrid PH60, respectively. In hybrid PH59, 4.52 μM 2,4-D plus 0.45μM TDZ induced a higher percentage of explants with shoots and shoot number per explant than did the hormone-free treatment. In hybrid PH60, although 4.52 μM 2,4-D and 0.45 μM TDZ promoted shoot formation, the highest shoot number was found with 4.52 μM 2,4-D alone. Plantlets, each having several roots, were obtained from regenerated shoots after transferring onto hormone-free basal medium for 3 mo. The plantlets were potted in sphagnum moss, and acclimatized well in a greenhouse.     

Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.