The structure of Mordacia mordax insulin supports the monophyly of the Petromyzontiformes and an ancient divergence of Mordaciidae and Geotriidae

The phylogenetic relationships between the two southern hemisphere lamprey families (Geotriidae and Mordaciidae) and their northern hemisphere counterparts (Petromyzontidae) are unresolved. Insulin was isolated from an extract of islet-containing intestinal tissue from ammocoetes of the Australian lamprey, Mordacia mordax. Its primary structure was established as A-chain: GIVEQCCHRK10CSIYDMENYC20N and B-chain: SALMGTGGTH10LCGSHLVEAL20YVVCGQRGFF30 YTP[SKG]. Although the residues in parentheses are only tentatively assigned, mass spectrometry supports the proposed sequence and demonstrates that Mordacia proinsulin, unlike proinsulin from Geotria australis, is fully processed to mature insulin. Insulins from M. mordax and G. australis and from the northern hemisphere lampreys Petromyzon marinus and Lampetra fluviatilis share a pentapeptide extension to N-terminus of the B-chain (Ser-Ala-Leu-Xaa-Gly) that has never been found in the insulins of any other vertebrate class. This observation provides support for the claim that the Petromyzontiformes constitute a monophyletic group. M. mordax insulin differs from that of G. australis by 18 amino acid residues but by only four residues from the common sequence of P. marinus and L. fluviatilis insulin. These data are consistent with the view that Geotriidae and Mordaciidae have been separated for a long period and suggest that G. australis insulin has undergone an accelerated rate of molecular evolution.     

Gold and Silver Trapping by Uncultured Magnetotactic Cocci

We studied the sites of gold and silver trapping by uncultured magnetotactic cocci from microcosms using transmission electron microscopy and energy-dispersive X-ray analysis. Two morphotypes were found to trap gold or silver. Morphotype 1 had large magnetite crystals frequently twinned in an unusual way and contained phosphorus-rich granules and electron-lucent inclusions probably composed of polyhydroxyalkanoates. Morphotype 2 presented smaller crystals with smaller width/length ratios and granules containing C, O, P, S, Cl, Na, Mg, Ca, and Fe, called phosphorus-sulfur-iron granules due to the presence of relatively large amounts of phosphorus, sulfur and iron. Gold was found in morphotype 2 bacteria, mainly in phosphorus-sulfur-iron granules. Additionally, the capsule presented small deposits that seemed to be composed of elemental gold. Silver was found in both spherical and rosette-shaped crystalline deposits also containing sulfur at the cell envelope of morphotype 1 bacteria. The rosette-shaped deposits had six subunits, suggesting that a homohexameric macromolecular assembly might be involved in their nucleation process. This seems to be an example of a highly organized structure mineralized incidentally by a biologically induced biomineralization process.     

Analysis of metal-containing granules in the barnacle Tetraclita squamosa

Metal-containing granules were isolated from specimens of the barnacle Tetraclita squamosa collected from a metal contaminated site in Hong Kong. The chemical composition, crystallographic and morphological properties of the deposits were analysed by X-ray powder diffraction, electron microscopy, energy dispersive analysis by X-rays and infrared spectroscopy. The most abundant metals were iron, calcium and zinc and the major anion was phosphate. Oxalates, carbonates and sulfur derivates were also found in small amounts. The deposits were amorphous to X-ray and electron diffraction and were approximately spherical in shape with diameters between 100 and 500 nm.     

Differences in lipid granulation as the basis for a morphologic differentiation between third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum

Differences in the distribution of lipid granules between unstained third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum cultured at 15 C was found to be an effective means for differentiating these 2 species of canine hookworms. In contrast, larvae cultured at 22 C were less easily differentiated based on the distribution of lipid granules. After culturing at 15 C, third-stage larvae of U. stenocephala were motile and exhibited 32 well-demarcated intestinal cells which contained intracellular lipid granules. Intestinal cells were easily visualized due to the absence of extraintestinal lipid granulation. Ancylostoma caninum third-stage larvae cultured under similar conditions were significantly less motile and contained extraintestinal accumulations of lipid granules which obscured intestinal cells. Both species exhibited an overall decrease in lipid granulation during a 14-day observation period following culture at 15 C. Morphologic differentiation of these 2 species after 14 days was based on the absence of intra- and extra-intestinal lipid in U. stenocephala and the presence of some lipid granules in both these locations in A. caninum. The first- and second-stage larvae of both species cultured at 15 C exhibited dense accumulations of extraintestinal lipid granules and were morphologically indistinguishable. This suggests that the observed difference in lipid granulation between the third-stage larvae of U. stenocephala and A. caninum cultured at 15 C is due to differences in lipid utilization during the third stage rather than differences in lipid synthesis by the first- and second-stage larvae and is related to the adaptation of these parasites to their respective climatic regions.     

Occurrence and Structure of Iron Inclusions in Adipocytes of Larval Lampreys

The occurrence and structure of adipocytes in the larvae of two lamprey species, Geotria australis and Petromyzon marinus, were examined by electron microscopy. Adipocytes from both species possessed large electron-dense inclusions which histochemical and energy dispersal X-ray analyses show as containing iron. The greatest concentration of inclusions in adipocytes was found in the nephric fold of G. australis. While some iron is present in the cytoplasmic matrix as ferritin, the majority is seen in large ammocoetes in membrane-bound dense aggregations of haemosiderin. The wide variety of inclusion types seen in smaller larvae may reflect on the method of formation of these inclusions within the cell. Because of the high level of iron loading in the larval lamprey nephric fold, this readily accessible tissue may provide a valuable model for studies of iron metabolism in vertebrates.     

Exceptional iron concentrations in larval lampreys (Geotria australis) and the activities of superoxide radical detoxifying enzymes.

This study aimed to elucidate the way in which larvae of the lamprey Geotria australis counteract the potential problems of the very high concentrations of non-haem iron they contain and thereby avoid the deleterious effects associated with iron overload in other vertebrates. Particular attention has been paid to ascertaining whether increasing concentrations of iron are accompanied by (i) change to a less readily available form of iron and (ii) an increase in the activity of those detoxifying enzymes responsible for minimizing the production of harmful hydroxyl radicals via the Haber-Weiss reaction. The mean concentrations of haemosiderin and ferritin in larval G. australis were each far higher in the nephric fold than in either the liver or intestine, but all these concentrations were much greater than those in rat liver. Since haemosiderin releases iron far more slowly than ferritin, the iron it contains is much less readily available to catalyse the Haber-Weiss reaction. It is thus relevant that (i) non-haem iron in the nephric fold occurred to a greater extent as large dense haemosiderin granules than as ferritin molecules and (ii) the proportion of iron in the form of haemosiderin rose with increasing concentration of total non-haem iron. A strong correlation was also recorded between the activity of superoxide dismutase in the nephric fold and the concentrations of total non-haem iron and its haemosiderin and ferritin components. This demonstrates that enzyme detoxification of O2.- rises with increasing amounts of iron. The exceptional iron concentrations in the nephric fold were not reflected by a greater measured activity of superoxide dismutase than that found in other tissues. However, the nephric fold was shown to contain an augmentation factor which is presumed to enhance the activity of this enzyme in vivo. The activity of catalase and glutathione peroxidase, which catalyse the breakdown of H2O2 to O2 and water, were each significantly correlated with the concentration of ferritin.     

Electron cytochemistry of developing and mature eosinophils in the bone marrow of the fowl and the duck

Synopsis Enzyme cytochemical studies have been carried out on eosinophils in the fowl and the duck. Peroxidase was found in all regions of the Golgi apparatus, the rough endoplasmic reticulum and the perinuclear cisternae of the early cells. In fowl eosinophil granules irregular deposits of peroxidase and arylsulphatase final reaction product were found, but the acid phosphatase deposits were even. In the duck in contrast, peroxidase was demonstrated in the external part of the granule only. Acid phosphatase and arylsulphatase were found in both the interna and the externa of the duck eosinophil granules. An ammoniacal silver nitrate reaction for the presence of the histone arginine was also studied. Silver deposits were found occupying all regions of the granules of eosinophils from both species of bird.The presence of the hydrolytic enzymes acid phosphatase and arylsulphatase in avian eosinophil granules supports the theory that these structures are lysosomal in nature and that they correspond with mammalian eosinophils in this respect.     

Ultrastructural and ultracytochemical identification of the small granules in basophils from human and animals

The small granules in the basophils obtained from humans and animals were compared ultrastructurally and cytochemically. Cytochemically, there were no qualitative differences among the small granules in the species examined. The small granules in humans, guinea pigs and rabbits were approximately 0.16-0.22 micron, 0.15-0.17 micron, and 0.12-0.16 micron, in diameter, respectively. In all species small granules had a single unit membrane and contained some amorphous material. In immature cells many of the small granules were distributed near the Golgi apparatus, while in the mature cells many of them were found around the periphery of the cell. There were no morphological or cytochemical differences between the small granules of the immature cells and those of the mature cells. The negative reaction in the dialysed iron and high iron diamine methods showed that the small granules did not have acid mucopolysaccharides or sulfated glycoconjugates. The strong reaction of the small granules of all species to the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) test, which was especially prominent in rabbit, showed that the small granules have many periodate-reactive neutral glycoconjugates but no acidic glycoconjugates. Enzyme cytochemistry revealed that the small granules are negative for peroxidase and catalase but positive for acid phosphatase.     

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: bruchidae) and the induction of alpha-amylases in response to different diets

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.     

Occurrence of the autofluorescent pigment, lipofuscin, in polar crustaceans and its potential as an age marker

In crustaceans, the lack of reliable methods often prevents the determination of individual age. The quantification of the autofluorescent age pigment, lipofuscin, has revealed promising results in boreal and tropical species. We studied the presence of morphological lipofuscin and its possible application as an age marker in five Arctic and five Antarctic species, comprising decapods, amphipods and a euphausiid. Lipofuscin granules were located in the brain, using confocal fluorescence microscopy, and quantified from digital images. The pigment was found in 94 of 100 individuals and in all 10 species, and granules occurred in easily detectable amounts in 5 species. Two scavenging amphipod species, the Antarctic Waldeckia obesa and the Arctic Eurythenes gryllus, revealed the most conspicuous and numerous granules. There was a broad, though weak, correlation of lipofuscin concentration with individual body size within a species, but not with absolute body size of one species compared to another. In larvae of the decapod Chorismus antarcticus, lipofuscin accumulation was quantified over the 1st 4 months after larval release. Morphological lipofuscin is a potential index of age in those investigated species with a sufficient accumulation rate of the pigment.     

Cytochemical observations of hemocytes in decapod brachyuran crustacea (author's transl)]

Hemocytes from two species of crabs, Eriocheir sinensis and Carcinus maenas, have been submitted to different cytochemical reactions in order to bring out the nature of granules' content, either by specific coloration or be enzymatic digestion. Light as well as electron microscopy has been used. The granules appeared to be made essentially of basic proteins, rich in arginin and disulfide bridges but poor in tyrosin. These proteins are linked with nonglycogenic carbohydrates. Glycogen deposits of varied size are restricted to the cytoplasm in all the different cell-types but have never been detected inside the granules themselves. Lipids are poorly represented. The bulk of the granule material is made of neutral mucopolysaccharides although minute amounts of acid mucosubstances could be found in some instances.     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouse

The distribution of metals within the hepatopancreas of Oniscus asellus (Crustacea, Isopoda) from two uncontaminated sites, and two sites contaminated with zinc, cadmium and lead, has been studied by atomic absorption spectrophotometry, light microscopy, transmission and scanning electron microscopy and X-ray microanalysis. The hepatopancreas contains two types of intracellular granule. The first type, in the S cells, are spherical granules which contain copper, sulphur and calcium. In woodlice from contaminated sites, these ‘copper’ granules, also contain zinc, cadmium and lead. The second type, in the B cells, are flocculent deposits which contain iron. In woodlice from contaminated sites, these ‘iron’ granules also contain zinc and lead. Moribund woodlice from contaminated sites have large numbers of ‘copper’ and ‘iron’ granules in the hepatopancreas and a fine deposit of zinc and lead on the membranes of the cells. There are numerous microorganisms in close association with the microvillous border of the hepatopancreas of woodlice from all four sites. Within the microorganisms of Oniscus asellus from contaminated sites, there are deposits of material which contain zinc, lead, calcium and phosphorus ‘Copper’ and ‘iron’ granules could have evolved as storage sites for essential metals to be utilized when demand from the body exceeds uptake from the food. Woodlice in contaminated sites may be able to ‘detoxify’ potentially harmful amounts of essential and non-essential metals by storing them in a relatively insoluble form within these granules.     

Daily changes of neurosecretory type-II cell structure of Pieris larvae entrained by short and long days

The neurosecretory type-II cell (NS-II cell) group of each brain hemisphere consists of three kinds of cells: two small cells, six large ones, and two others having characteristic vacuolated endoplasmic reticulum (ER).Ultrastructural changes of large NS-II cells were observed through the fifth instar and diurnally when short-day and long-day larvae were compared. There were little differences between short-day and long-day larvae in cell structures on corresponding developmental days except for daily changes, but remarkable changes were observed every day through the instar. A secretory cycle through the instar was supposed being based on the ultrastructural changes in NS-II cells: reduced secretory activity on the first day, formation of organelles necessary for the synthesis of secretory materials throughout the instar on the second day, active synthesis and secretion of secretory material during the middle stage (third-fourth day), and reversion to a reduced level of cell activity after the cessation of feeding.In short-day larvae on the third to fourth day, NS-II cells contained large aggregates of secretory granules during the day except for the time of 13 hr after the onset of photophase when a decrease of secretory granules occurred. In long-day larvae, only a small amount of secretory granules was observed at 8 and 13 hr after the onset. Rough ER changed daily paralleling with the quantitative change of the secretory granules.Based on these differences of daily changes in NS-II cell activity between short-day and long-day larvae, it was concluded that photoperiodic time measurement of diapause induction depends on the daily secretory cycle entrained by the photoperiods during the larval stage.     

Oral Fimbriae and Papillae in Parasitic Lampreys (Petromyzontiformes)

There are 38 species of living lampreys, 18 parasitic and 20 nonparasitic. The parasitic species feed as adults, while the nonparasitic do not. The taxonomy and systematics of the living lampreys is based primarily on dentition characters. Since the number of oral fimbriae and oral papillae have never been systematically investigated in lampreys, we compared them in 17 of the 18 parasitic lampreys to assess their usefulness as taxonomic characters. Both showed little variation with total length and sex within a species, while exhibiting greater variation between species. Parasitic species belonging to the three lamprey families could be distinguished using the number of oral fimbriae: southern hemisphere Mordaciidae (0) and Geotriidae (55–65) and northern hemisphere Petromyzontidae (81–144). However, the taxonomic usefulness of the two characters at the species level was limited. Ten out of the 17 species of parasitic lampreys were placed into four distinct groups based on their numbers of oral fimbriae, and only in two of these was the character diagnostic. Twelve out of the 17 species were placed into two distinct groups based on their numbers of oral papillae and for none of these was the character diagnostic. Blood feeders and intermediate feeders (blood + flesh) were shown to have significantly higher numbers of oral fimbriae than flesh feeders. The higher numbers of oral fimbriae in the former two types of feeders were presumed to be linked to their greater need to create a good seal for feeding purposes. Blood feeders were also shown to have significantly higher numbers of oral papillae than either intermediates or flesh feeders. The higher numbers of oral papillae in the blood feeders were presumed to be linked to their greater need to find suitable attachment sites for feeding purposes.     

Simultaneous ultrastructural demonstration of heavy metals (silver, mercury) and acid phosphatase

A histochemical technique which permits the simultaneous visualization of heavy metals and acid phosphatase at the ultrastructural level is described. The technique was applied to the anterior pituitary gland, the spinal cord and the liver. In all of the tested organs, both mercury and silver were found to accumulate primarily in the lysosomes, although small amounts of both metals could be observed in other organelles including endocytotic vesicles. In the anterior pituitary, few mercury deposits were found in the secretory granules.     

Chemical defence in chrysomelid eggs and neonate larvae

ABSTRACT. Eggs and neonate larvae of chrysomelid beetles (sub-tribes Chrysomelina and Phyllodectina) were investigated for the presence of defensive substances.
The two isoxazolinone glucosides (compounds 1 and 2), characteristic of the adult defence secretion, were detected in the eggs of all studied species. Compound 2, containing a nitropropionate, is always present in concentrations (above 10^-2 M), which are highly deterrent to the ant Myrmica rubra. This compound is not at all or only slightly toxic to ants at 10^-2 M. Compound 1, devoid of nitropropionate, is a minor constituent, and is neither deterrent nor toxic to ants.
The five Chrysomela species studied and Phratora vitellinae also sequester salicin in their eggs in amounts highly deterrent and toxic to ants. A single Chrysomela egg often contains enough salicin to kill an ant. While the isoxazolinones are discarded with the egg shells, salicin is used by neonate larvae as a precursor for the production of salicylaldehyde in the thoracic defence glands, already functional at hatching. No salicin could be detected in the eggs of those species whose larvae produce cyclopentanoid monoterpenes, even if they feed on Salicaceae. No larva of any species seems to be able to produce detectable amounts of monoterpenes at birth. A very early defence, possible only in those species using salicin as the precursor for their defensive secretion, could be highly advantageous in protecting the clustered larvae during the long process of hatching and in avoiding cannibalism between siblings.
Only trace amounts of oleic acid were found in the eggs of Gastrophysa viridula , in contrast to previous reports on its presence in large quantities in the American G. cyanea.     

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Synopsis Coelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and -glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.     

The morphology and histology of the endocrine pancreas of the southern hemisphere lamprey, Geotria australis gray

The location and arrangement of the pancreatic endocrine tissue in larval and adult Geotria australis (Geotriidae) differ markedly from those exhibited by the comparable stages of Northern Hemisphere lampreys (Petromyzontidae). In larval Geotria australis, the main zones of islet proliferation are located laterally between the oesophagus and the inner edge of the two large intestinal diverticula unique to this species rather than dorsal and ventral to the oesophagus. In adult Geotria australis, the islet follicles are closely packed into a single discrete capsule which could be easily removed surgically, rather than into cranial, intermediate, and caudal cords. The differences in the adult can be related to a lack of involvement of the bile duct in islet formation during metamorphosis. While B cells were found in both larval and adult islet follicles, the PI acidophilic cells and argyrophilic cells, which appeared respectively at stages 3 and 4 in metamorphosis, were present in all adult stages.     

The fine structure of akinete formation and germination in Cylindrospermum

Summary Akinete formation and germination were studied in a species of Cylindrospermum using the electron microscope. The differentiation of a vegetative cell into an akinete is characterized by cell enlargement, sheath condensation, deposition of several spore envelope layers, including a dense fibrillar layer and deposition of large cyanophycin granules. The mature akinete in addition to the multilayered envelope retains internally a large number of cyanophycin granules, a photosynthetic thylakoid system, polyhedral bodies, lipid deposits and nucleoplasmic regions. Germination of the akinete can take place in several modes differing in detail. Most frequently the spore envelope remains intact and the germling which may or may not have divided emerges through a pore at one end of the envelope. The photosynthetic thylakoid system appears to increase by the fusion of small vesicles found in the cytoplasm. Alpha-granules are numerous and cyanophycin is nearly absent in the germling.