Morphology,ultrastructure, and chemical compounds of the osmeterium of <Emphasis Type="Italic">Heraclides thoas</Emphasis> (Lepidoptera: Papilionidae)

The osmeterium, found in papilionoid larvae, is an eversible organ with an exocrine gland that produces substances in response to the mechanical disturbances caused by natural enemies. The anatomy, histology and ultrastructure of the osmeterium, and the chemical composition of its secretion in Heraclides thoas (Lepidoptera: Papilionidae) were studied. Heraclides thoas larvae have a Y-shaped osmeterium in the thorax. The surface of the osmeterium has a rough cuticle lining cells with papillae and irregular folds, whereas the cells that limited the gland pores are irregular, folded, and devoid of papillae. Two types of cells are found: (i) cuticular epidermal cells on the surface of the tubular arms of the osmeterium and (ii) secretory cells of the ellipsoid gland within the region of the glandular pore. Cuticular epidermal cells show a thick cuticle, with several layers divided into epicuticle and lamellar endocuticle. Secretory cells are polygonal, with extensive folds in the basal plasma membrane that formed extracellular channels. The cytoplasm has mitochondria, ribosomes, and numerous vacuoles, whereas the nucleus is irregular in shape with decondensed chromatin. The chemical composition of the osmeterial secretion comprised (Z)-α-bisabolene (25.4%), α-bisabol (20.6%), β-bisabolene (13.1%), (E)-α-bisabolene 8%), β-pinene (9.91%), longipinene epoxide (8.92%), (Z)-β-farnesene (6.96%), β-caryophyllene (2.05%), farnesol (1.86%), linalyl propionate (1.86%), and 1-octyn-4-ol (1.07%). The morphological features suggest that the cuticular epidermal cells play a major role in the maintenance and protection of the osmeterium, whereas secretory cells are responsible for production of osmeterial secretions.     

Effects of heat stress in the leaf mitotic cell cycle and chromosomes of four wine-producing grapevine varieties

Grapevine varieties respond differentially to heat stress (HS). HS ultimately reduces the photosynthesis and respiratory performance. However, the HS effects in the leaf nuclei and mitotic cells of grapevine are barely known. This work intends to evaluate the HS effects in the leaf mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato, and Viosinho. In vitro plants with 11 months were used in a stepwise acclimation and recovery (SAR) experimental setup comprising different phases: heat acclimation period (3 h—32 °C), extreme HS (1 h—42 °C), and two recovery periods (3 h—32 °C and 24 h—25 °C), and compared to control plants (maintained in vitro at 25 °C). At the end of each SAR phase, leaves were collected, fixed, and used for cell suspensions and chromosome preparations. Normal and abnormal interphase and mitotic cells were observed, scored, and statistically analyzed in all varieties and treatments (control and SAR phases). Different types of chromosomal anomalies in all mitotic phases, treatments, and varieties were found. In all varieties, the percentage of dividing cells with anomalies (%DCA) after extreme HS increased relative to control. TF and Viosinho were considered the most tolerant to HS. TF showed a gradual MI reduction from heat acclimation to HS and the lowest %DCA after HS and 24 h of recovery. Only Viosinho reached the control values after the long recovery period. Extrapolating these data to the field, we hypothesize that during consecutive hot summer days, the grapevine plants will not have time or capacity to recover from the mitotic anomalies caused by high temperatures.     

Floral nectary and osmophore of <Emphasis Type="Italic">Epipactis helleborine</Emphasis> (L.) Crantz (Orchidaceae)

The analysis of flowers collected at different stages of anthesis provides strong evidence to conclude that the shell-shaped hypochile and the knobs of epichile form a nectary. The scent comes from the aromatic constituents of nectar and the epichile tissue and the apices of all tepals (osmophores). The comparison between pollinated and unpollinated flowers revealed that the anthesis of unpollinated flowers lasted up to the 16th day. The nectariferous secretory cells formed single-layered epidermis and several layers of underlying parenchyma built by small, isodiametric cells with thin walls and dense cytoplasm, relatively large nuclei, supplied by collateral vascular bundles. During the floral lifespan, the residues of secreted material were higher on the hypochile cells. The lipoid-carbohydrate material and lipid globules in the cell walls and in the cytoplasm were localised. The abundance of starch grains was observed at the beginning of anthesis and their gradual reduction during the flower lifespan. At the end of anthesis in unpollinated flowers, the lipoid-carbohydrate-phenolic materials have been demonstrated. The phenolic material was the same as in plastoglobuli. The features such as irregular plasmalemma, the secretory vesicles that fuse with it, fully developed dictyosomes, numerous profiles of ER indicate vesicle-mediated process of secretion. The substances could be transported by vesicles to the periplasmic space via granulocrine secretion and then to the external surface. Both micro-channels and slightly developed periplasmic space were visible in the hypochile epidermis. This is the first time for anatomical survey of secretory tissue in pollinated and unpollinated flowers of E. helleborine.     

Water Relations and Cell Wall Elasticity Quantities in Phaseolus vulgaris Leaves

The investigations were designed to test osmotic adjustment,cell wall bulk elastic modulus and stomatal behaviour duringand after water stress and rewatering in the primary and firsttrifoliolate leaf of Phaseolus vulgaris. Leaf water relationsquantities fully recovered after rewatering within a few hours;diffusion resistance to vapour flow, however, required 6 h.Leaf growth recovery was considerably delayed. Osmotic adjustmentwas absent during water stress in both the primary and the firsttrifoliolate leaf. The bulk elastic modulus (_v), however, waslower for the primary leaf (higher elasticity) than for thetrifoliolate leaves. These two types of leaves differed in theirdrought resistance in that the primary leaf exhibited wiltingat the end of the stress period (7 d) while the trifoliolateleaf remained relatively turgid. The bulk elastic modulus ofthe cell wall changed almost proportionally with the turgorpressure (_p). The structure coefficient (), an indicator forthe intensity of change of the bulk elastic modulus with turgorwas higher for the primary than for the first trifoliolate leaf.The leaf diffusion resistance (r), below the turgor loss point,changed proportionally with the solute potential with very similarregression lines for the relation of (r) versus RWC ¹. The datasuggest that greater drought resistance of the first trifoliolateleaf is related to a decreased bulk elastic modulus, but notto osmotic adjustment nor to differences in stomatal resistanceduring water stress. Key words: Phaseolus vulguris, Water stress, Recovery, Cell wall elasticity     

Pathogenicity and mucosal transmissibility of the R5-tropic simian/human immunodeficiency virus SHIV(AD8) in rhesus macaques: implications for use in vaccine studies

There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIV_AD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIV_AD8-CE8J, SHIV_AD8-CK15, and SHIV_AD8-CL98) exhibited robust replication in vivo and caused marked depletion of CD4⁺ T cells affecting both memory and naïve CD4⁺ T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIV_AD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections (Pneumocystis, Candida, and Mycobacterium). A single but unique founder virus, also present in the SHIV_AD8-CE8J swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIV_AD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4⁺ T lymphocytes in vivo, and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments.     

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled “Parthenon pillars.” In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.     

Early plant growth and biochemical responses induced by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 lipopolysaccharides in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seedlings are attenuated by procyanidin B2

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O₂ ^·?) and hydrogen peroxide (H₂O₂) levels increased in seedling roots treated with LPS (100 μg mL^?1). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 μg mL^?1), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.     

Structure of the style and pollen tube pathway in the Ziziphoid and Rhamnoid clades of Rhamnaceae

The ultrastructure of the style and pollen tube pathway before, during and after anthesis were studied in 13 species belonging to the tribes Pomaderreae, Paliureae, Colletieae and Gouanieae (Ziziphoid clade) and Rhamneae (Rhamnoid clade) using light microscopy and transmission electron microscopy. The aim of this study is to provide new morphological characters useful for phylogenetic analysis at suprageneric level in Rhamnaceae. The patterns of pollen tube growth and the ultrastructural changes undergone by cells of the style were also described. Species of Rhamneae (Scutia buxifolia and Condalia buxifolia) have a solid style, with the transmitting tissue forming three independent strands (S. buxifolia) or a central, single horseshoe-shaped strand as seen in transversal section (C. buxifolia) which could derive from the fusion of formerly independent strands. In contrast, Pomaderreae, Gouanieae and Paliureae showed semi-solid styles, while in Colletieae, as previously reported, the style is hollow with two or three stylar canals. The style anatomy and the ultrastructure of the pollen tube pathway show that there is a tendency towards a solid style with a single strand of transmitting tissue within the family. The three-canalled hollow style could be the plesiomorphic state of the character “type of style” in the family, the semi-solid style the synapomorphic state and the solid style with three strands of transmitting tissue the apomorphic state, with the solid style with a single strand of transmitting tissue as the most derived state. Therefore, Colletieae would be the most basal tribe of the Ziziphoid clade.     

Ring chromosomes: from formation to clinical potential

Ring chromosomes (RCs) are circular DNA molecules, which occur rarely in eukaryotic nuclear genomes. Lilian Vaughan Morgan first described them in the fruit fly. Human embryos very seldom have RCs, about 1:50,000. Carriers of RCs may have varying degrees of symptoms, from healthy phenotype to serious pathologies in physical and intellectual development. Many authors describe common symptoms of RC presence: short stature and some developmental delay that could be described as a “ring chromosome syndrome.” As a rule, RCs arise de novo through the end-joining of two DNA double-strand breaks, telomere-subtelomere junction, or inv dup del rearrangement in both meiosis and mitosis. There are family cases of RC inheritance. The presence of RCs causes numerous secondary chromosome rearrangements in vivo and in vitro. RCs can change their size, become lost, or increase their copy number and cause additional deletions, duplication, and translocations, affecting both RCs and other chromosomes. In this review, we examine RC inheritance, instability, mechanisms of formation, and potential clinical applications of artificially created RCs for large-scale chromosome rearrangement treatment.     

Post-embryonic development of the Malpighian tubules in <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera) workers: morphology,remodeling, apoptosis,and cell proliferation

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.