A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

ABSTRACTA current bottleneck in the functional analysis of the emerging parasitic model plant Cuscuta and the exploitation of its recently sequenced genomes is the lack of efficient transformation tools. Here, we describe the development of a novel highly efficient Agrobacterium-mediated transformation protocol for Cuscuta reflexa based on the parasitic structure referred to as adhesive disk. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Co-transformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a possibility for development of transformed cells into callus.ONE SENTENCE SUMMARYA protocol that yields high numbers of transformed cells in the adhesive disks of Cuscuta reflexa upon exposure to agrobacteria brings closer the vision of generating genetically modified Cuscuta.

Type of shelter and first description of the echolocation call of disk-winged bat (Thyroptera devivoi)

Thyropteridae is a family of bats endemic to the Neotropical region, and Thyroptera devivoi is the only species in the family that occurs exclusively in forest patches within savannas of northern South America and north of the Brazilian Cerrado. Primary data on the species are still scarce. Therefore, in this study our objective was to fill knowledge gaps on geographical distribution, roost-use, and echolocation for the species. We observed a T. devivoi colony of 15 individuals living under a dead palm leaf. The bats used the leaf as a roost for at least four days. After capturing one individual, we confirmed the species identification via skull size and the oblong shape of the adhesive disk. The new record reinforces the association of this species with non-forested formations, and its occurrence mainly in savannas. Echolocation calls of T. devivoi are consistent with those known for the genus, with multiharmonic, low intensity and high frequency pulses. Despite these new data, more studies are certainly needed to enhance distribution data for the species, as well as to clarify the biological and ecological requirements of the species.

New Adhesion Mechanism in Giardia: Role of the Ventrolateral Flange in the Attachment of Trophozoites to Rough and Porous Surfaces

The protozoan parasite Giardia, an intestinal flagellate, has evolved an unique attachment organelle called the ventral adhesive disk (VAD). This attachment organelle mediates attachment of the trophozbite to the microviUous border (MVB) of intestinal absorptive cells, and production of lesions via attachment are thought to lead to diarrheal disorder characteristic of giardiasis. The VAD has contractile proteins arranged around it's circumference and it has been suggested that contraction of this area might function like a purse-string suture. The resemblance of the VAD to a suction cup has led to the hypothesis that suction or a negative pressure produced under the VAD by a grasping action might produce the adhesive force regulating attachment. To test whether or not the generation of a negative pressure is necessary for attachment, experiments were designed in which the substratum was fabricated to have an uneven surface, or the substratum was composed of different arrangement of pores.

Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip

Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3′-dihexyloxacarbocyanine iodide. The definitive identification of endoplasmic reticulum required the development of a molecular label. We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein. In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs. By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy. Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level. In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope. Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions. In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions. Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk. The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells.

Development and ultrastructure of the haustorium of Viscum minimum. I. The adhesive disk

The adhesive disk of the haustorium of Viscum minimum Harvey develops an epithelium of uni- or bi-cellular secretory trichomes. Before contact with the host is obtained, the trichomes secrete adhesive cutinaceous material. This secretion contains fragments of the lamellar cuticle proper, the reticulate cuticular layer, and the noncutinized part of the cuticularized wall, in additions to pockets of granular material. The volume of the adhesive product per trichome may be several times the volume of the secretory cell itself except in front of the future intrusive organ where secretion is reduced. Formation of cutin cystoliths was observed in sections of wall of different ages within single trichomes. The trichomes are rich in mitochondria, plastids with starch, spherosomes, ribosomes, and ER that are associated with the strongly convoluted plasmalemma. A special type of tubular ER is described. Zones of collapsed cells are formed in the cortex as the apex of the wedge-shaped intrusive organ emerges. The cuticularized wall of the host then disintegrates, and the intrusive organ forms a fissure within the host. This becomes lined, to the depth of a few cells, partly with cuticularized and finally compressed cells originating from the adhesive epithelium, and partly with new cells produced by the intrusive organ. Key words: Viscum minimum, adhesive disk, adhesive epithelium, cuticle ultrastructure, cutin cystolith, haustorium, secretion.

Structure and development of Lobatostoma manteri sp.nov. (Trematoda: Aspidogastrea) from the Great Barrier Reef, Australia

Lobatostoma manteri sp.nov. is described. It differs from other species of this genus in the number of marginal alveoli (usually 56–62), the location of the testis near the posterior end and the large size of the cirrus pouch. Mature worms occur in the intestine of the fish Trachinotus blochi. Eggs containing fully developed larvae are laid. The eggs are eaten by snails and hatch in the stomach. Larvae have an oral sucker, pharynx, simple caecum, ventro-terminal acetabulum, two dorsal excretory bladder cells in front of the acetabulum, and a caudal appendage. They migrate into the digestive gland and differentiate to pre-adults with fully developed genital organs and the full number of alveoli on the adhesive disk; young spermatozoa and egg cells develop but do not mature. Pre-adults have a minimum number of 8500 sensory papillae on the surface. The worms are usually found in a cavity formed by enlargement of the main duct and one or more (?) side ducts of the digestive gland near the stomach in Cerithium moniliferum, or in the stomach and main ducts of the digestive gland of Peristernia australiensis. They may creep from the ducts into the stomach and back into the ducts. Fish become infected by eating snails. Worms from fish die soon after transfer into sea water but can be kept alive for up to 13 days in frog's Ringer solution or dilute sea water (1:5), in which they lay eggs containing larvae infective to snails. Worms from snails remain alive in sea water, dilute sea water, frog's Ringer or Tyrode solution. Eggs of worms from single infections have the haploid chromosome number of 7; there is normally no self-fertilization and development does not reach the blastula stage. The life-cycle of Lobatostoma manteri is the simplest two-host cycle of trematodes known. Reasons are given why it must be considered the most primitive one, of a type from which digenean life-cycles have evolved.