Swimming patterns of the quadriflagellate Tetraflagellochloris mauritanica (Chlamydomonadales, Chlorophyceae).

Chlamydomonadales are elective subjects for the investigation of the problems related to locomotion and transport in biological fluid dynamics, whose resolution could enhance searching efficiency and assist in the avoidance of dangerous environments. In this paper, we elucidate the swimming behavior of Tetraflagellochloris mauritanica, a unicellular-multicellular alga belonging to the order Chlamydomonadales. This quadriflagellate alga has a complex swimming motion consisting of alternating swimming phases connected by in-place random reorientations and resting phases. It is capable of both forward and backward swimming, both being normal modes of swimming. The complex swimming behavior resembles the run-and-tumble motion of peritrichous bacteria, with in-place reorientation taking the place of tumbles. In the forward swimming, T. mauritanica shows a very efficient flagellar beat, with undulatory retrograde waves that run along the flagella to their tip. In the backward swimming, the flagella show a nonstereotypical synchronization mode, with a pattern that does not fit any of the modes present in the other Chlamydomonadales so far investigated.

Water monitoring: automated and real time identification and classification of algae using digital microscopy.

Microalgae are unicellular photoautotrophs that grow in any habitat from fresh and saline water bodies, to hot springs and ice. Microalgae can be used as indicators to monitor water ecosystem conditions. These organisms react quickly and predictably to a broad range of environmental stressors, thus providing early signals of a changing environment. When grown extensively, microalgae may produce harmful effects on marine or freshwater ecology and fishery resources. Rapid and accurate recognition and classification of microalgae is one of the most important issues in water resource management. In this paper, a methodology for automatic and real time identification and enumeration of microalgae by means of image analysis is presented. The methodology is based on segmentation, shape feature extraction, pigment signature determination and neural network grouping; it attained 98.6% accuracy from a set of 53,869 images of 23 different microalgae representing the major algal phyla. In our opinion this methodology partly overcomes the lack of automated identification systems and is on the forefront of developing a computer-based image processing technique to automatically detect, recognize, identify and enumerate microalgae genera and species from all the divisions. This methodology could be useful for an appropriate and effective water resource management.

A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

Automatic and real time recognition of microalgae by means of pigment signature and shape.

Microalgae are unicellular photoautotrophic organisms that grow in any habitat such as fresh and salt water bodies, hot springs, ice, air, and in or on other organisms and substrates. Massive growth of microalgae may produce harmful effects on the marine and freshwater ecological environment and fishery resources. Therefore, rapid and accurate recognition and classification of microalgae is one of the most important issues in water resource management. In this paper, a new methodology for automatic and real time identification of microalgae by means of microscopy image analysis is presented. This methodology is based on segmentation, shape features extraction, and characteristic colour (i.e. pigment signature) determination. A classifier algorithm based on the minimum distance criterion was used for microalgae grouping according to the measured features. 96.6% accuracy from a set of 3423 images of 24 different microalgae representing the major algal phyla was achieved by this methodology.

Tetraflagellochloris mauritanica gen. et sp. nov. (Chlorophyceae), a New Flagellated Alga from the Mauritanian Desert: Morphology, Ultrastructure, and Phylogenetic Framing.

Morphological, ultrastructural, and molecular-sequence data were used to assess the phylogenetic position of a tetraflagellate green alga isolated from soil samples of a saline dry basin near F'derick, Mauritania. This alga can grow as individual cells or form non-coenobial colonies of up to 12 individuals. It has a parietal chloroplast with an embedded pyrenoid covered by a starch sheath and traversed by single parallel thylakoids, and an eyespot located in a parietal position opposite to the flagellar insertion. Lipid vacuoles are present in the cytoplasm. Microspectroscopy indicated the presence of chlorophylls a and b, with lutein as the major carotenoid in the chloroplast, while the eyespot spectrum has a shape typical of green-algal eyespots. The cell has four flagella, two of them long and two considerably shorter. Sequence data from the 18S rRNA gene and ITS2 were obtained and compared with published sequences for green algae. Results from morphological and ultrastructural examinations and sequence analysis support the placement of this alga in the Chlorophyceae, as Tetraflagellochloris mauritanica L. Barsanti et A. Barsanti, gen. et sp. nov.

Fundamental questions and concepts about photoreception and the case of Euglena gracilis.

The ability to sense light can be considered the most fundamental and presumably the most ancient property of visual systems. This ability is the basis of phototaxis, one of the most striking behavioral responses of motile photosynthetic microorganisms (i.e. microalgae) to light stimuli, which allows them to move toward or away directional light. In order to fully exploit the information content of light (intensity, direction, distribution) microorganisms need proper perceiving devices, termed photoreceptors, which must act as sensors, to perceive wavelength and direction of light, as transducers, to convert the light signal into chemical and/or electrical information, but also as amplifiers and eventually as transmitters. This review describes the universal structural, behavioral and physiological features necessary for the proper functioning of these devices in algae, and how these features have been investigated by means of different analytical techniques such as for example microspectroscopy, digital fluorescence microscopy, two photons FLIM. The insight of the photoreceptive response mechanism is explained using the unicellular alga Euglena gracilis, in which the different structural, behavioral and physiological features combine to achieve a concerted, efficient response to light stimuli.

Chemistry, physico-chemistry and applications linked to biological activities of ß-glucans.

ß-Glucans is the common name given to a group of chemically heterogeneous polysaccharides. They are long- or short-chain polymers of (1-->3)-ß-linked glucose moieties which may be branched, with the branching chains linked to the backbone by a (1-->6)-ß linkage. ß-(1-->3)-Glucans are widely distributed in bacteria, algae, fungi and plants, where they are involved in cell wall structure and other biological function. ß-Glucans have been shown to provide a remarkable range of health benefits, and are especially important against the two most common conventional causes of death in industrialized countries, i.e. cardiovascular diseases (where they promote healthy cholesterol and blood glucose levels) and cancer (where they enhance immune system functions). This Highlight provides a comprehensive and up-to-date commentary on ß-glucans, their chemistry, physico-chemistry, functional role in immunological responses, and possible applications as therapeutic tools. In addition, we discuss the mechanism behind their health benefits, which are not yet fully understood.

A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization.

Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle.

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.

MICROSPECTROPHOTOMETRY AS A METHOD TO IDENTIFY KLEPTOPLASTIDS IN THE NAKED FRESHWATER DINOFLAGELLATE GYMNODINIUM ACIDOTUM(1).

A relatively small number of freshwater dinoflagellates are involved in symbiotic association with cryptophytes. The chloroplasts of the cryptophytes are retained by the dinoflagellate and give it the characteristic phycobilin pigmentation, either phycoerythrin or phycocyanin. The pigment characterization of the retained chloroplasts can give precise and accurate information about the type of cryptophyte preyed upon by the dinoflagellate. For this purpose, we performed microspectrophotometric evaluation of the pigments of Gymnodinium acidotum Nygaard and three different cryptophytes present in samples collected from a tributary of the river Arno, in Tuscany (Italy). The comparison of the different spectroscopic data allowed us to discriminate effectively among the cryptophytes preyed upon by the dinoflagellate.

Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor.

This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90A). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor.

Absorption microspectroscopy, theory and applications in the case of the photosynthetic compartment.

We performed microspectroscopic evaluation of the pigment composition of the photosynthetic compartments of both algae and higher plants. The feasibility of microspectroscopy for discriminating among species and/or phylogenetic groups was tested on laboratory cultures. Gaussian bands decompositions, and a fitting algorithm, together with fourth-derivative transformation of absorbance spectra, provided a reliable discrimination among chlorophylls, phycobiliproteins and carotenoids. Comparative analysis of absorption spectra highlighted the evolutionary grouping of the algae into three main lineages in accordance with the most recent endosymbiotic theories.

Microspectroscopy of the photosynthetic compartment of algae.

We performed microspectroscopic evaluation of the pigment composition of the photosynthetic compartments of algae belonging to different taxonomic divisions and higher plants. The feasibility of microspectroscopy for discriminating among species and/or phylogenetic groups was tested on laboratory cultures. Gaussian bands decompositions and a fitting algorithm, together with fourth-derivative transformation of absorbance spectra, provided a reliable discrimination among chlorophylls a, b and c, phycobiliproteins and carotenoids. Comparative analysis of absorption spectra highlighted the evolutionary grouping of the algae into three main lineages in accordance with the most recent endosymbiotic theories.

A short flagella mutant of Dunaliella sallina (Chlorophyta, Cholorophyceae).

Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 + 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed.

Fluorescence behavior of Euglena photoreceptor.

To provide new information on the series of structural changes that Euglena photoreceptive proteins undergo inside the photoreceptor in response to light, we measured in vivo emission fluorescence spectra in the stable intermediates of its photocycle. Our emission spectra give a certain indication that fluorescent proteins are present in the Euglena photoreceptor and that they undergo a photocycle. On the basis of our data, we suggested that at least two stable intermediates, one of which is fluorescent, can be discriminated at room temperature and with our time resolution.