The extraction of high-molecular-mass DNA from hair shafts.

A simple and efficient method is presented for the extraction of DNA from hair shafts. DNA preparations obtained by this approach can be made amenable to restriction enzyme digestion, thereby allowing further molecular biological analysis.

Light- and electron microscopical observations on the caecilian spleen. A contribution to the evolution of lymphatic organs.

Red and white pulp were distinguished in the spleen of the caecilian species Ichthyophis paucisulcus and Afrocaecilia taitana. The red pulp was composed of endothelium-lined sinusoids and reticular connective tissue. Between the processes of the reticulum cells, accompanied by fine collagen fibrils, the following cell types were found: lymphocytes, macrophages (frequency containing fragments of erythrocytes), neutrophils, eosinophils, mast cells and/or basophils (metachromatic granules), thrombocytes, plasma cells, pigment cells as well as cells which presumably represent blast cells. Morphological evidence suggested the formation of thrombocytes in the red pulp. Besides sinusoids, ellipsoids and peculiar arteriolar vessels with a high endothelium and a loose layer of muscle cells were observed. Veins were concentrated in the splenic periphery. White pulp consisted of arterioles which were surrounded by a lymphocyte sheath. Follicles were not identified with certainty. Occasionally mitotic figures were associated with lymphocytes. On the basis of our findings, we suggest the following functions of the caecilian spleen: destruction of aged erythrocytes, formation of thrombo- and lymphocytes as well as of plasma cells and, to a marked lesser degree, of other blood cells.

Histological and histochemical observations in the stomach of the Senegal sole, Solea senegalensis.

An histological and histochemical study was conducted on the stomach of adult Senegal sole, Solea senegalensis specimens. The stomach was made up of four distinct layers: mucosa, lamina propria-submucosa-, muscularis and serosa. Surface epithelial, glandular and rodlet cells were present in the mucosa. Cells of the columnar epithelium contained a basal nucleus. Numerous mitochondria, granular endoplasmic reticulum and Golgi apparatus consisting of several parallel cisternae and vesicles were observed in the cytoplasm of these cells. The lysosomes were small, round and dense. The gastric glands were numerous in the pyloric and fundic regions but absent in the cardiac stomach. These glands were formed by two cell-types: light and dark cells. The light cells were characterised by numerous mitochondria, while dark cells had slightly fewer mitochondria and a tubulo-vesicular system. Rodlet cells similar to those observed in other teleostean fish were present among the epithelial cells. Although the epithelial cells of the mucosa contained a weak presence of neutral and acid mucopolysaccharides/mucosubstances, these substances were abundant in the lamina propria-submucosa. Proteins rich in arginine, lysine, cysteine and cystine were rarely present in the mucosa and lamina propria-submucosa of stomach, while proteins rich in tyrosine were abundant in these layers. Acid phosphatase, and ATP-ase (pH 7.2 and 9.4) activities were detected in the mucosa and lamina propria-submucosa. Alkaline phosphatase activity was not detected.

The female genital system of Ooperipatellus decoratus (Onychophora, Peripatopsidae): an ultrastructural study.

The female genital system of the oviparous peripatopsid Ooperipatellus decoratus consists of an ovary, oviducts equipped with receptacula seminis and additional pouches, uteri, and a vagina. It is examined using transmission and scanning electron microscopy. The ovary is made up of paired ovarian tubes united anteriorly and posteriorly and differentiated into a sterile dorsal part and a fertile ventral part with exogenous oocytes. Fertilization presumably occurs in the oviducts once the oocytes pass the receptaculum seminis. Although the receptacula seminis have been reported to occur in juvenile O. decoratus females only, the present study reveals that they are present in adult females as well. Their wall consists of a cuboidal epithelium covered with a thin collagen-muscle layer. The additional pouches are projections of the oviducts facing the receptacula seminis. They are distally closed to the haemocoel by a flattened epithelium and lack external muscle cells. A thin collagen layer is only found proximally. The uteri are characterized by a columnar epithelium with folded cell membranes allowing extension of the uteri, thus facilitating the passage of the large uterine eggs towards the vagina. Another dominating feature of the uteri is a distally increasing secretory production, which probably contributes to chorion development. Cilia occurring along the entire length of the uteri are considered to assist in the transport of eggs towards the vagina.

Ultrastructure of hepatocytes in golden ide (Leuciscus idus melanotus L.; Cyprinidae: Teleostei) during thermal adaptation.

The morphological alterations of hepatocytes of golden ide, Leuciscus idus melanotus, following adaptation to low and high temperatures (14 and 28 degrees C) were investigated by means of light and electron microscopy. The temperature-dependent behaviour of peroxisomes was visualized cytochemically with the alkaline diaminobenzidine medium; the morphological studies were supplemented by the biochemical determination of catalase activity. Cold adaptation of ide hepatocytes is manifested by proliferation and stacking of endoplasmic reticulum, an enhanced secretory activity of Golgi fields and a higher number of peroxisomes as compared with the warm-adapted animals. The latter organelles are characterized by a marked heterogeneity in size, shape and catalase activity, and by a more intimate association with mitochondria and endoplasmic reticulum. The occurrence of small peroxisomal profiles is restricted to lower temperature. Catalase activity can be shown both cytochemically and biochemically to increase during cold adaptation. Whereas the number of mitochondria seems to be unaffected by thermal adaptation, stacking of mitochondria as well as the formation of intramitochondrial membrane piles indicate cold-adaptive processes. A feature typical of warm-adaptation is the formation of membrane-glycogen complexes, which may represent the morphological expression of enhanced carbohydrate metabolism documented in a decreased storage of glycogen at 28 degrees C. At 28 degrees C lipid is the predominant storage product. These findings indicate that fish liver is well-suited to serve as a model for the analysis of the interaction of environmental temperature conditions and hepatic morphology.

Temperature induced alterations in the liver of wall lizard (Hemidactylus frenatus): morphological and biochemical parameters.

Wall lizards (Hemidactylus frenatus) were adapted to 20, 25, and 30 degrees C, and the liver was examined using standard transmission electron microscopy (TEM) and biochemical analysis. Peroxisomes were visualized after using the 3,3'-diaminobenzidine (DAB) technique. Catalase, uricase and protein content were determined biochemically. The hepatocytes of animals adapted to higher temperature displayed larger lipid inclusions than those of animals adapted to lower temperature. Rough endoplasmic reticulum was better developed in the animals kept at low temperature (20 and 25 degrees C) than in the animals held at 30 degrees C. Cytoplasmic crystalline structures were visualized, and better developed in the hepatocytes at 25 degrees C. Peroxisomes varied with the temperature, being more frequent in the animals kept at 20 degrees C, while the bigger ones prevailed in the animals kept at 30 degrees C. The higher catalase activity at higher temperature was correlated to an increase in staining intensity of DAB-incubated peroxisomes as visualized cytochemically in TEM. The biochemical results confirmed the cytochemical reaction observed by TEM. The hepatocytes of the animals at 30 degrees C showed a reduction in the number of peroxisomes, however, at this temperature the largest peroxisomes with a stronger reaction to DAB and a higher activity of catalase predominate. In contrast, the uricase activity showed no significant variation in relation to adaptation temperature. Overall, these data show the morphological and functional plasticity of hepatocytes to temperature adaptation of H. frenatus.

Histochemistry of lectin-binding sites in Halicryptus spinulosus (Priapulida).

Priapulida represent one of the phylogenetically oldest multicellular animal groups. In multicellular animals (Metazoa) cell-to-cell and cell-to-matrix interactions are often mediated by carbohydrate residues of glycoconjugates. To analyze the carbohydrate composition of a phylogenetically old species, lectin histochemistry was employed on 5 specimens of the priapulid Halicryptus spinulosus. Many lectins bound to the chitin-containing cuticle, including those specific for carbohydrates other than N-acetylglucosamine, the principle building block of chitin. The connective tissue of the animals contained both N-acetylglucosamine and N-acetylgalactosamine. Mannose residues were widely distributed with the exception of the cuticle, but complex type carbohydrates were not present in the entire animal. Sialic acid residues were only detected in the cuticle and brush border of the intestinal epithelium, while fucose was limited to the cuticle. Thus, the lectin-binding pattern indicated that sugars typical for the linking region of both N- and O-glycoproteins in mammals are also present in H. spinulosus. Carbohydrate residues that are typical for the complex type of N-linked glycans in vertebrates are not present as are carbohydrate residues typical for the termination of O-linked carbohydrate chains. Hence, a truncated form of both N- and O-linked glycosylation is present in H. spinulosus indicating that more complex patterns of glycosylation developed later during evolution.

Impact of bacillus thuringiensis var. israelensis On larvae of chironomus thummi thummi and psectrocladius psilopterus (Diptera: chironomidae)

Chironomid larvae, especially species of the subfamily Chironominae, are known to be sensitive to the mosquitocidal bacterium Bacillus thuringiensis var. israelensis (B.t.i.). In this study, bioassays and electron microscopic investigations were carried out with third- and fourth-instar larvae of Chironomus thummi thummi Kieffer (subfamily Chironominae) and Psectrocladius psilopterus Kieffer (subfamily Orthocladiinae) in order to study the sensitivity of species belonging to different chironomid subfamilies. Both species showed susceptibility to increased B.t.i. concentrations, with LC50 values (24 h) ranging from about 40- to 60-fold the LC50 for Aedes aegypti (LC50 (24 h) is 0.77 mg/L for C. thummi thummi and 1.17 mg/L for P. psilopterus). C. thummi thummi was shown to be twice as sensitive as P. psilopterus. Ultrastructural investigations of the anterior midgut showed cellular alterations in larvae exposed to a high B.t.i. concentration (2.8 mg/L, about 50-fold the LC50 for A. aegypti), such as swelling of mitochondria, dilatation of intercellular spaces and basal labyrinth, fenestration or disorganization of the Golgi complex, concentric arrangement of rough endoplasmic reticulum, and an increase of lysosomes and myelin figures. Electron-lucent regions within the cell, cell protrusion, and, in some cases, swelling or lysis of cells were further effects observed in treated animals. Most effects were found in both species, though they seemed to be more severe in C. thummi thummi. The alterations coincide with those known from target organisms (Culicidae, Simuliidae). This study shows that there is a difference in sensitivity to B.t.i. between chironomid species from different subfamilies and that the susceptibility of chironomid larvae to the bacterial toxins is due to damage of the midgut epithelium as it is in target organisms. Copyright 1999 Academic Press.

A histological and histochemical study of the oesophagus and oesogaster of the Senegal sole, Solea senegalensis.

A histological and histochemical study was performed in the buccal cavity and papillae, which were around the teeth, as well as in the oesophagus and oesogaster of the Senegal sole, Solea senegalensis adult specimens. The oesophagus and oesogaster were made up of four distinct layers: mucosa, submucosa, muscular and serous. Two morphological types of epithelial cells were distinguishable in the oesophageal mucosa: the more numerous type cells possessed an electron-dense cytoplasm, whereas the cytoplasm was electron-clear in the other cells. Mucus-secreting cells were the dominant feature of the epithelium throughout the oesophagus. These goblet cells were filled with numerous mucous droplets of low electron-density. The oesophagus was devoid of taste buds. In the oesogaster mucosa, three types of cells were distinguished: dark, rodlet and light epithelial cells. Dark epithelial cells showed different characteristics from that in the oesophagus: the nucleus was irregular with an electron-dense hyaloplasm, the cytoplasm had a scarce smooth and granular endoplasmic reticulum; a Golgi apparatus consisted of four parallel cisternae, dense granules without membrane, lysosomes and numerous mitochondria. The rodlet cells were elongated, contained rod-like structures and were surrounded by an electron-dense capsule-like structure. The bulk of the rodlet cell was composed of up to 20 extended rodlet units. Light epithelial cells of the oesogaster had the same characteristics as those observed in the oesophagus and contained numerous mitochondria with a dense matrix, abundant smooth endoplasmic reticulum and numerous vesicles. In the goblet cells of the papillae, sulfomucin was recognised, since they showed alcianophilia (alcian blue pH 1.0 and 0.5). These cells were negative to protein reaction (bromophenol blue) and contained -S-S- and SH groups. Enzymatic activities (alkaline phosphatase, acid phosphatase, ATPase (pH 7.2 and 9.4) and lipid reactions were negative in the goblet cells of the buccal cavity. Epithelial cells of oesophagus contained a weak presence of acid and neutral mucopolysaccharides. Oesophageal goblet cells contained carboxylated, sulphated (weakly and strongly ionised) mucosubstances and sialic acid. Most goblet cells did not contain proteins and presented disulphide (-S-S-) and sulphydril (-SH) groups. Proteins in general, and in particular those rich in lysine, tyrosine and arginine were present in the epithelium, lamina propria, submucosa and muscular layer of the oesophagus. Lipids in general and phospholipids were observed in the oesophageal epithelium while unsaturated, acid and neutral lipids were not observed. The lamina propria and submucosa contained a weak presence of phospholipids and unsaturated lipids. Acid phosphatase and ATPase (pH 7.2) activities were observed in the lamina propria, submucosa and muscular regions, while ATPase (pH 9.2) activity was weak in these areas. ATPase activity (pH 7.2 and 9.5) was very weak in the epithelium. Oesophageal goblet cells were negative to lipid and enzymatic reactions.

[Effect of starvation on the ultrastructure of hepatocytes of Hemidactylus frenatus (Lacertilia: Gekkonidae) with special emphasis on peroxisomes]. / Der Einfluss von Nahrungsentzug auf die Ultrastruktur der Hepatocyten von Hemidactylus frenatus (Lacertilia: Gekkonidae) mit besonderer Berücksichtigung der Peroxisomen.

The influence of starvation on hepatocyte ultrastructure of Hemidactylus frenatus (Lacertilia: Gekkonidae) was investigated with special emphasis on peroxisomes. Wall lizards (Hemidactylus frenatus) were sacrificed after different periods of starvation and their livers were processed for standard transmission electron microscopy. Peroxisomes were demonstrated by means of the 3,3'-diaminobenzidine (DAB) cytochemical technique. A control group consisted of individuals which were fed "ad libitum" with Tenebrio molitor larvae. After a 7-day period of starvation the ultrastructural observation of hepatocytes disclosed a marked reduction of glycogen and lipid inclusions associated with fragmentation of the endoplasmic reticulum (ER). In later stages of starvation (14 and 25 days) ER proliferation and partial reconstruction of glycogen aggregations were observed. Increasing numbers of peroxisomes were arranged either in clusters (14 days) or in close association with mitochondria, lipid droplets and elongated crystalloid structures (25 days). Particularly noteworthy is the increasing cytochemical response of these organelles to the DAB reaction, suggesting greater metabolic activity of catalase. These data suggest that morphological and functional plasticity of hepatocytes may contribute to adaptation of Hemidactylus frenatus to prolonged starvation.

Efficacy of Clostridium bifermentans serovar Malaysia on target and nontarget organisms.

Clostridium bifermentans serovar malaysia (C.b.m.) is highly toxic to mosquito larvae. In this study, the following aquatic nontarget invertebrates were treated with high C.b.m. concentrations (up to 1,600-fold the toxic concentration for Anopheles stephensi) to study their susceptibility towards the bacterial toxin: Planorbis planorbis (Pulmonata); Asellus aquaticus (Isopoda); Daphnia pulex (Cladocera); Cloeon dipterum (Ephemeroptera); Plea leachi (Heteroptera); and Eristalis sp., Chaoborus crystallinus, Chironomus thummi, and Psychoda alternata (Diptera). In addition, bioassays were performed with mosquito larvae (Aedes aegypti, Anopheles stephensi, and Culex pipiens). Psychoda alternata larvae were very susceptible, with LC50/LC90 values comparable to those of mosquito larvae (about 10(3)-10(5) spores/ml). The tests with Chaoborus crystallinus larvae showed significant mortality rates at high concentrations, but generally not before 4 or 5 days after treatment. The remaining nontarget organisms did not show any susceptibility. The investigation confirms the specificity of C.b.m. to nematocerous Diptera.

Dispersal behavior of adult snow melt mosquitoes in the Upper Rhine Valley, Germany.

The dispersal behavior of female snow melt mosquitoes was studied in two forests in Rhineland-Palatinate, Germany, from April to August 1993. Both CDC-light-traps and human bait collections were used to collect mosquitoes. Sampling sites were chosen along a west-east and a north-south transect in treated and untreated parts of a forest with a village in its center. Around this settlement, breeding sites within a radius of 1.5 to 2.5 km were treated. It could be shown that this buffer zone is sufficient to prevent a nuisance caused by snow melt mosquitoes in the village. The results lead to the conclusion that snow melt mosquitoes do not regularly migrate over large distances but stay near their breeding sites. In a detailed study of the behavior of Aedes rusticus, it could be observed that these mosquitoes were resting in the interior of the forest during daytime and leaving it with increasing dusk up to 50 m from the forest edge. A comparison of landing rate counts near a row of trees and in the open field showed higher activity near the row of trees indicating visual orientation of the mosquitoes. Although the Ae. rusticus females left the forest regularly, no nuisance occurred in nearby villages. The treatment of breeding sites near settlements appeared to be sufficient to prevent a nuisance caused by the snow melt mosquitoes.

Studies on metacercariae from naiads in northeast Thailand.

One hundred and eleven naiads from six different dragonfly species were investigated for the presence of metacercariae. Sixty-nine animals (62.2%) were infected with intensities up to more than 150 cysts per animal. The metacercarial cysts were characterized by light and scanning electron microscopy. Two types of metacercariae could be differentiated by morphological features of cyst surface, cyst wall and size.

Studies on lophocercous cercariae from Bithynia siamensis goniomphalus (Prosobranchia: Bithyniidae).

Cercariae from Bithynia siamensis goniomphalus were studied in an area endemic for opisthorchiasis. Snails emitted different types of cercariae when shedding was induced by illumination. In addition to two lophocercous cercariae, a monostome cercaria (probably Notocotylus sp), two different furcocercariae and a xiphidiocercaria were found. Two similar types of lophocercous cercariae were distinguishable and one could be identified as Opisthorchis viverrini. To date the cercaria of O. viverrini has been regarded as the only lophocercous cercaria in the endemic area of opisthorchiasis. Therefore, differences between these cercariae were further characterized by scanning electron microscopy to enable us to avoid misinterpretations of O. viverrini in Bithynia.

Factors influencing the seasonal abundance of Aedes (Stegomyia) aegypti and the control strategy of dengue and dengue haemorrhagic fever in Thanlyin Township, Yangon City, Myanmar.

From June 2006 to May 2007, mosquito surveys were conducted in Thanlyin Township, Yangon City, Myanmar, to determine factors influencing the abundance of Aedes aegypti (Stegomyia aegypti) during the rainy season. Both the biological and environmental factors were included in this study. Increase in the hatchability of egg, larval survival rate, the shortened larval life-span and increased pupation rates supplemented by rainfall (i.e. continuous flooding of the containers, stimulate the continuous hatching of eggs) were observed for correlation with the increase in population density of Ae. aegypti during the rainy season in the study area. Control strategy of Ae. aegypti to analyze the infestation in the community (study area) with larval Ae. aegypti, integrated management measures including health education, attitudes and practices regarding dengue and dengue haemorrhagic fever, transmission of the disease and possible preventive measures, reduction of breeding sites and testing the efficacy of Bacillus thuringiensis israelensis (B.t.i.) with respect to the reduction level of Ae. aegypti larvae in breeding sources, were taken into consideration.

[Ultrastructure of hepatocytes is teleost fishes following food deprivation]. / Zur Ultrastruktur der Hepatocyten von Teleosteern bei Nahrungsentzug.

Liver tissue of nine teleost species has been investigated electron microscopically after periods of fasting. Ultrastructural changes tn the hepatocytes are described mainly from Haplochromis burtoni. 70 days of food deprivation have the following effects: the intercellular spaces are widened; the size of the hepatocytes is reduced; lysosomes are enlarged and reach the diameter of the nucleus and have increased in number; the amount glycogen is reduced; the endoplasmatic reticulum bears less ribosomes. All alterations are reversible. The comparison of different species mainly shows differences conceming to the amount of lipid, which may be reduced or enlarged.

[Electron microscopic studies of the gill epithelium of the amphibian teleost Periophthalmus vulgaris]. / Elektronenmikroskopische Beobachtungen am Kiemenepithel des amphibischen Teleosteers Periophthalmus vulgaris.

The gill epithelium of the airdwelling fish Periophthalmus vulgaris has been studied with the electron microscope. The following celltypes can be distinguished: flat covering epithelial cells, chloride cells, mucous cells, basal cells, various leucocytes as well as a specific granule containing cell which is possibly an epithelial cell. The covering epithelial cells exhibit a relatively smooth apical surface and contain in their apical half densely packed microfilaments, pinocytotic vesicles are rare. These characteristics are not to be found in water dwelling fish and possibly represent adaptations to the air containing surroundings. In the chloride cells are numerous, especially in the basal halves of the secondary lamellae. The distal parts of the secondary lamellae the barrier for the respiratory gases measures about 0,9 micrometer. The basal cells are ribosome rich replacement cells. Two types of mucous cells occur. Individual intraepithelial nerve fibres have been observed.

Septate junctions in the cephalic epidermis of Turbellarians (bipalium).

In the epidermis of turbellarians septate junctions of the pleated sheet type have been demonstrated in conventional thin sections and freeze fractured preparations. The structure of these junctions entirely agrees with that found in molluscs and arthropods.

Fine structure of the sensilla of Peripatopsis moseleyi (Onychophora).

Three types of sensilla occurring on the lips and on the antennae of Peripatopsis moseleyi have been investigated by scanning and transmission electron microscopy. On the lips sensory spines can be found which contain numerous cilia originating from bipolar receptor cells. They reach the tip of the spine where the cuticle is modified. The perikarya of the sensory cells, a large supporting cell with a complicated surface and a second type of receptor, form a bud-like structure and are surrounded by a layer of collagen fibrils. The second receptor cell bears apical stereocilia as well as a kinocilium which are directed towards the centre of the animal -- thus the cell appears to be turned upside down. The sensilla of the antennae are 1) sensory bristles containing two or three kinds of receptor cells, one of which bears an apical cilium and one kind of supportive cell and 2) sensory bulbs located within furrows consisting of receptor cells with branched cilia and two kinds of supportive cells which are covered by a modified thin cuticle. According to the electron microscopical findings the sensory spines on the lips are presumably chemoreceptors. The sensory bristles on the antennae can be regarded as mechanoreceptors and the sensory bulbs as chemoreceptors.