DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM)

A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with root cells and their visualization under CLSM. DTAF (5-(4,6-dichlorotriazinyl) aminofluorescein) is a fluorescent dye that has been widely used for staining various biological samples for fluorescent microscopy. It reacts with polysaccharides and peptides at ordinary conditions. The possible application and efficiency of DTAF for CLSM studies were examined in various aspects of cyanobacterial-plant interactions. Seedlings of Pisum sativum, Vigna rediata and Triticum aestivum were co-cultivated and stained with DTAF as a fluorochrome. Extracellular and intracellular interactions of cyanobacteria and the plant root surface were observed by CLSM. Results were compared with staining by other commonly used lectins. Advantages of the use of DTAF over other stains are its penetration into root tissues and binding with polysaccharides, mainly the cellulose. The staining was smooth, which clearly showed minute details on the cell of surface and root hairs with higher resolution. The emission wavelength for DTAF is 517 nm, which is highly advantageous as cyanobacteria have auto-fluorescence at 665 nm, and both can be simultaneously used in CLSM by visualizing in different channels. This worked efficiently with all three plants used and with filamentous and unicellular cyanobacterial strains. Cyanobacterial presence was not only clearly observed on the root surface, but also inside the root tissue and epidermal cells. The easy protocol and absence of tissue processing make DTAF a useful probe for studies of cyanobacterial associations with plant roots by CLSM.     

Effect of Copper Ions on Early Developmental Stages of the Mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia)

The effect of exposure to copper in seawater (0.005, 0.01, and 0.02 mg/liter) on the development of Mytilus trossulus was examined in the stages of fertilization, blastula, early veliger, veliger, and veliconch. Copper in a concentration of 0.01 and 0.02 mg/liter inhibited the development and growth of the embryos and larvae. At 0.005 mg Cu/liter, the embryos and larvae were capable of adaptation. If M. trossulus embryos and larvae were maintained for 1–2 days in seawater containing 0.01 mg Cu/liter and then transferred to clean water, the consequences were largely dependent on the developmental stage at which the exposure to copper took place. The early developmental stages were more sensitive to the effect of copper than veliger larvae.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Yaroslavtseva, Sergeeva.     

Biological control of the zebra mussel Dreissena polymorpha and the snail Biomphalaria glabrata, using Gramicidin S and D and molluscicidal strains of Bacillus

Applications of Final Whole Culture (FWC) or primary powder material from strains of each of four Bacillus species (B. alvei, B. brevis, B. circulans, B. laterosporus) used singly, as well as the antibiotics Gramicidin S (GS) and Gramicidin D (GD) used singly, were found to be molluscicidal against several life cycle stages of the zebra mussel Dreissena polymorpha. Combinations of the bacterial material with either GS or GD were either additive (for GS) or antagonistic (for GD). The smaller the life cycle stage of the animal, the more sensitive it was to either the bacterial or antibiotic agent. The veliger stage was particularly sensitive to each agent, with the molluscicidal effect being more rapid in the veliger (5 h) than in the adult (6 days). The molluscicidal effects of these agents (at 1–100 μ g ml⁻¹) against the veliger stages of the zebra mussel were comparable to the activity of B. thuringiensis and B. sphaericus against their target organisms. These agents used singly were also active against small adult Biomphalaria glabrata, the snail vector of schistosomiasis (eg at tenths of μ g ml⁻¹ of GS). Received 09 July 1996/ Accepted in revised form 06 November 1996     

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

 Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla. Accepted: 10 February 1999     

Specific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples

An important issue in the management of zebra mussel (Dreissena polymorpha) populations is early, rapid, and accurate detection of the planktonic larvae (veliger) of the zebra mussel. The goal of this study was to explore the feasibility of developing a molecular approach for the detection of zebra mussel larvae in diverse environments. In this study a Dreissena polymorpha-specific 18S ribosomal RNA gene targeted oligonucleotide primer (ZEB-715a) and Polymerase Chain Reaction (PCR) assay was developed and compared with cross-polarized microscopy as a means to detect zebra mussel veligers in plankton samples. The design of the zebra mussel-specific primer was facilitated by sequencing nearly the complete 18S rRNA gene from the zebra mussel and three other closely related freshwater Veneroids including the quagga mussel (D. bugensis), the dark false mussel (Mytilopsis leucophaeata), and the Asian freshwater clam (Corbicula fluminea). The specificity of the primer for the zebra mussel was empirically tested by using the primer as a direct probe in a blot hybridization format. A single veliger in a plankton sample could be detected by PCR using this approach. Veliger detection sensitivity using the PCR approach was estimated to be over 300 times more sensitive than cross-polarized light microscopy based techniques. Cross-polarized light microscopy and the PCR technique were used to identify the presence of zebra mussel larvae in plankton samples that were collected from a variety of natural and industrial water sources. Detection results (presence or absence) were generally consistent between the two methods. Although additional studies will be required before routine application of molecular based veliger detection technology is available, a long-term goal of this work is the application of molecular technology to the development of a field device for the routine detection and quantification of zebra mussel veligers.     

Myogenesis in Aplysia californica (Cooper, 1863) (Mollusca,Gastropoda, Opisthobranchia) with special focus on muscular remodeling during metamorphosis

To date only few comparative approaches tried to reconstruct the ontogeny of the musculature in invertebrates. This may be due to the difficulties involved in reconstructing three dimensionally arranged muscle systems by means of classical histological techniques combined with light or transmission electron microscopy. Within the scope of the present study we investigated the myogenesis of premetamorphic, metamorphic, and juvenile developmental stages of the anaspidean opisthobranch Aplysia californica using fluorescence F‐actin‐labeling in conjunction with modern confocal laser scanning microscopy. We categorized muscles with respect to their differentiation and degeneration and found three true larval muscles that differentiate during the embryonic and veliger phase and degenerate during or slightly after metamorphosis. These are the larval retractor, the accessory larval retractor, and the metapodial retractor muscle. While the pedal retractor muscle, some transversal mantle fibers and major portions of the cephalopedal musculature are continued and elaborated during juvenile and adult life, the buccal musculature and the anterior retractor muscle constitute juvenile/adult muscles which differentiate during or after metamorphosis. The metapodial retractor muscle has never been reported for any other gastropod taxon. Our findings indicate that the late veliger larva of A. californica shares some common traits with veligers of other gastropods, such as a larval retractor muscle. However, the postmetamorphic stages exhibit only few congruencies with other gastropod taxa investigated to date, which is probably due to common larval but different adult life styles within gastropods. Accordingly, this study provides further evidence for morphological plasticity in gastropod myogenesis and stresses the importance of ontogenetic approaches to understand adult conditions and life history patterns. J. Morphol., 2008. © 2007 Wiley‐Liss, Inc.     

Characterization of the bacterial microbiota of Biomphalaria glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil

Roughly 200 000 000 people in 74 countries infected with schistosomes all share the fact that they came in contact freshwater harbouring infected snails. The aim of the study is to characterize the microbiota of wild and laboratory‐reared snails of Biomphalaria glabrata from Pernambuco, Brazil. The microbiota of these molluscs was identified biochemically by the VITEK 2 automated microbiological system. Antimicrobial susceptibility testing was carried out by the disc diffusion method with ß‐lactam antibiotics, aminoglycosides, quinolones, folate pathway inhibitors, fenicols and tetracyclines. The results showed that all bacteria identified were gram‐negative, including 11 bacterial genera: Aeromonas, Citrobacter, Enterobacter, Cupriavidus, Rhizobium, Stenotrophomonas, Pseudomonas, Klebsiella, Acinetobacter, Vibrio and Sphingomonas. Regarding the antimicrobial susceptibility, all the isolates exhibited resistance to amoxicillin and sensitivity to meropenem (beta‐lactam antimicrobials). The microbiota of the wild snails consisted predominantly of Enterobacter cloacae, while the laboratory‐reared snails predominantly showed Citrobacter freundii and Aeromonas sobria.

Significance and Impact of the Study

Biomphalaria glabrata is a Brazilian freshwater Planorbidae of great medical relevance as an intermediate host of Schistosoma mansoni. About a month after being infected by one or more miracidia larvae of a compatible schistosome, B. glabrata sheds thousands of cercariae into the water where they seek human skin and, if successful, penetrate to establish infection, eventually taking up residence and maturing in blood vessels of the small intestine. Results obtained from this study aim at targeting novel biological control strategies for schistosomiasis such as paratransgenesis. This is the first study on the microbiota of B. glabrata from Brazil.     

Investigation of oxidation in freeze-dried membranes using the fluorescent probe C11-BODIPY

Incorporation of the fluorescent probe C11-BODIPY^581/591 in two dried membrane systems, soy bean phosphatidylcholine liposomes freeze-dried in a carbohydrate/protein matrix and Lactobacillus acidophilus (La-5) freeze-dried in a carbohydrate matrix, was successful and could be visualised by Confocal Laser Scanning Microscopy (CLSM). The C11-BODIPY^581/591 probe is a lipid oxidation reporter molecule, which is known to associate with the lipids of biological membranes and exhibit a fluorescence shift from the red range to the green range of the visible spectrum when it is oxidised together with the lipids. The present study is the first to demonstrate that the C11-BODIPY^581/591 probe can be used in dried membrane systems, and that a detection of oxidation is possible by CLSM analysis directly on the dried samples.     

Phylogenetic relationships and taxonomy of Altiplano populations of Biomphalaria (Gastropoda: Planorbidae): inference from a multilocus approach

Isolated closed basins provide a natural laboratory to study the differentiation among wild populations. Here we examined the phylogenetic relationships of the Southern Altiplano populations of Biomphalaria, a genus with medical importance, using nuclear (ITS1, ITS2) and mitochondrial (16S) ribosomal gene markers and a species of Helisoma as outgroup. Phylogenetic trees based on separate and combined analyses show that these populations form a particular lineage within Biomphalaria along with Biomphalaria peregrina (d'Orbigny, 1835) and Biomphalaria oligoza Paraense, 1974. The origin of this clade was estimated to have occurred in the middle Pleistocene. Molecular analyses showed that the nominal species Biomphalaria crequii ( Courty, 1907 ) from the Salar de Ascotán and Biomphalaria costata (Biese, 1951) from the Salar de Carcote, previously synonymized with Biomphalaria andecola (d'Orbigny, 1835) and B. peregrina, respectively, are distinct taxa. Molecular data did not resolve the relationship of Biomphalaria aymara Valdovinos & Stuardo, 1991 from the Isluga swamps to other Biomphalaria species, but confirm that the populations from the Lauca and Huasco basins may represent a distinct undescribed species of Biomphalaria from the Southern Altiplano. Snails examined for trematodes were found to be positive in some Altiplano localities. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 795–808.     

Spatial associations between actin filaments, endoplasmic reticula, mitochondria and fungal hyphae in symbiotic cells of orchid protocorms

The relationships between endoplasmic reticula (ER), mitochondria, and actin filaments (Afs) were observed in uncolonized and colonized cells of symbiotic protocorms ofSpiranthes sinensis (Orchidaceae) germinated in the presence of the fungus,Ceratobasidium cornigerum. Mitochondria and ER were observed by transmission electron microscopy, and with the fluorescent probe DiOC₆ (3) (3,3′-dihexyloxacarbocyanine) combined with confocal laser scanning microscopy (CLSM). An indirect immunofluorescence method using CLSM and an indirect, pre-embedding immunogold method at the ultrastructural level were used for observation of Afs. In uncolonized cells, cortical ER showed a polygonal pattern and ER formed a network throughout the cytoplasm. In the cortex, a smooth face of ER contacted the plasma membrane. Mitochondria were associated with ER. Afs were in close proximity to ER, mitochondria and amyloplasts. Colonized cells retained cortical ER, and a smooth face of ER was also closely associated with the perifungal membrane. ER and mitochondria were present in the cytoplasmic channels bridging between the central peloton and the peripheral cytoplasm. This distribution of ER and mitochondria during fungal colonization and senescence coincided with that of Afs. The changes in the arrays of Afs accompanying symbiotic fungal colonization and senescence occurred concomitantly with the changes in ER.     

Contribution à l'étude de l'organogenèse des Hétéropodes (Mollusca,Prosobranchia)

The histological larval anatomy of Oxygyrus keraudreni Lesueur, Carinaria lamarcki Péron and Lesueur, Pterotrachea coronata Forskal and Firoloida desmaresti Lesuour relating to the veliger organisation of Atlanta is described at the stages close to the metamorphosis.

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Contribution n° 50 du Groupe Scientifique du Centre Océanologique de Bretagne.     

Dual-rhodamine urea derivative, a novel chemidosimeter for Hg(II) and its application in imaging Hg(II) in living cells

Abstract  The synthesis and spectral properties of a chemidosimeter 1,4-di[2-(6-ethylamino-3-ethylimino-2,7-dimethyl-3H-xanthen-9-yl) benzoic acid (aminomethyl)-3-phenylthiourea] benzene (1) for Hg(II) ions are reported, and it has been demonstrated that 1 can be used as a fluorescent probe for monitoring Hg(II) ions in living cells. Graphical abstract  A highly sensitive fluorescent probe (1) was developed as a fluorescent and colorimetric chemodosimeter in dimethyl sulfoxide/methanol solution with a broad pH range (pH 5–10) and high selectivity toward Hg²⁺ ions but no significant response toward other competitive cations. Furthermore, by means of confocal laser scanning microscopy experiments, it is demonstrated that 1 can be used as a fluorescent probe for monitoring Hg²⁺ in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Survival,growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Abstract

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17°C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

SNFYMPL荧光探针和共聚焦显微内镜在胃癌诊断中的应用

目的:探讨荧光探针SNFYMPLGGGSK-FITC与胃癌组织的结合能力,预测其在胃癌诊断中的应用价值。方法:收集我院2015年6月至10月收治的48例胃癌患者手术切除的肿瘤和癌旁组织,使用异硫氰酸荧光素FITC标记的线性七肽SNFYMPL及无关序列肽探针对其冰冻切片进行荧光染色,通过共聚焦激光扫描显微镜CLSM检测其结合能力,并与组织病理结果对比。结果:SNFYMPLGGGSK-FITC对胃癌组织荧光染色的阳性率为81.25%(39/48),明显优于无关序列肽探针的14.58%(7/48),差异有统计学意义(P0.001)。SNFYMPLGGGSK-FITC对癌旁组织荧光染色的阳性率为27.08%(13/48),明显低于对胃癌组织荧光染色阳性率,差异有统计学意义(P0.001)。结论:FITC标记的SNFYMPL短肽探针与胃癌组织较强结合能力,图像组织分辨率好,可以作为共聚焦激光显微内镜在胃癌诊断的潜在靶向分子进一步研究。     

Nucleoprotein cytochemistry during oogenesis in a unisexual fish, Poecilia formosa

Summary Cytochemical methods and electron microscopy were used to study changes in the chemical composition of nuclear, nucleolar and perinuclear bodies during the early stages of oocyte development inPoecilia formosa, an apomictic species of fish that produces only female offspring. Prominent accumulations of ribonucleoprotein (RNP) occur in nucleoli and appear on either side of the nuclear envelope during diplotene. In certain planes of section, RNP material seems to be in transit across this interface.En bloc acid extractions or RNAse treatment abolished basophilia and markedly reduced the electron density of both nucleoli and cytoplasmic nucleolar-like bodies. DNA-specific fluorescent probes such as mithramycin failed to reveal nucleolar cores in poeciliid oocytes, although the same procedures showed unequivocal localization of GC-rich DNA cores within multiple nucleoli of diplotene oocytes fromXenopus laevis or the rainbow trout,Salmo gairdneri. Also, cytological hybridization studies, utilizing [³H]rRNA as a probe for nucleolar oocytes. Feulgen-stained pachytene oocytes ofP. formosa have twice the number of chromosome strands seen in similar stages of oocytes from two, related bisexual species,P. mexicana andP. latipinna. Although the bivalent nature of these chromosomes could not be resolved with the light microscope, configurations resembling, but not identical to, synaptonemal complexes were identified by electron microscopy.     

Identification of schistosome hybrids and larval parasites using rRNA probes

Observations have been made on the ribosomal RNA (rRNA) gene units of hybrid progeny produced by experimental crosses of S. haematobium × S. mattheei, S. mattheei × S. bovis and S. haematobium × S. intercalatum. Hybridisation of DNA probe pSM 889 to restriction endonuclease digested DNA extracted from adult worms showed that each parental form could be differentiated by differences in the rRNA gene unit. In each experimental cross the F1 hybrid generation produced a composite major banding pattern of the two parental species. No differences associated with the stage of development were detected in the major bands of hybridisation when DNA extracted from various life-cycle stages of S. mansoni and S. margrebowiei was digested with EcoR1 and hybridised with probe pSM 889. Prepatent infections of S. mansoni in Biomphalaria glabrata were detected 16 days post-infection utilising probe pSM 389 and dot blot analysis. Small numbers of intact cercariae dotted onto nitrocellulose were detected using probe pSM 389, 10 cercariae being the minimum number required for accurate determination.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Life history strategy of Leptinaria unilamellata (d'Orbigny, 1835) (Mollusca,Pulmonata, Subulinidae)

Abstract

Life history theory predicts that the patterns of resource allocation in animals are associated with different strategies, selected in the course of evolution. In the present study, the life history of Leptinaria unilamellata was characterized under laboratory conditions. We determined the growth, reproduction, and longevity patterns of this species and elucidated the strategy related to the development of embryos, through direct observations and examination of the morphology of the gravid uterus. Furthermore, we attempted to analyze the glycogen and galactogen contents of the albumen gland, digestive gland and cephalopedal mass in order to understand energy allocation to life history traits, for three life stages. Leptinaria unilamellata's life history is characterized by great longevity, a short juvenile phase, early sexual maturity, and repeated reproductive events, with little reproductive effort at each event and some mortality shortly after the first reproduction. In the terraria, we found juveniles but no eggs. However, the results of the anatomical study showed no morphological connection between the embryos and the parental organism. Thus, this species should be described as ovoviviparous rather than viviparous. Egg retention in the parent organism is the primary cause of the release of juveniles, instead of eggs, enabling the offspring to withstand environmental stress. The higher quantity of galactogen found in the adults' albumen gland, as compared to juveniles and senescent individuals, as well as the ratio of glycogen to galactogen, reveal the allocation of energy to reproduction rather than to growth. The remaining energy is directed to the maintenance of omeostasis. Such pattern was confirmed by the low levels of glycogen and galactogen observed in the senescent stage, compared to the juvenile and adult stages. In the life strategy of L. unilamellata, the distribution of the reproductive effort among many events associated with ovoviviparity indicates a long-term investment in reproductive success.