Voltage gradients and microtubules both involved in intercellular protein and mitochondria transport in the telotrophic ovariole of Dysdercus intermedius

Summary In the telotrophic ovariole of Dysdercus intermedius the intercellular transport consists of different subsystems. Microinjection of FITC-labeled slowly diffusing proteins with opposite electrical net charges and of mitochondria was used to study the translocation of macromolecules and organelles. a) By intracellular measurements a voltage gradient of about 4 mV between the tropharium as the more negative side and the previtellogenic oocytes could be demonstrated. b) After injection into the tropharium negatively charged proteins migrated according to the electropotential gradient via the trophic cords into the oocytes. Positively charged proteins, however, were retained in the tropharium. c) After injection into previtellogenic oocytes both negatively and positively charged proteins moved into the trophic cords. Thus, the effectiveness of the electropotential gradient on the distribution of charged proteins is more pronounced from the tropharium side. d) Mitochondria microinjected into the trophic core were probably aligned along microtubules and translocated towards the trophic cords. — These results suggest that in the telotrophic bug ovariole a number of intercellular transport subsystems contribute to provide previtellogenic oocytes with nurse cells products. An electrophoretic transport mechanism for soluble proteins acting especially within the tropharium and a microtubule-associated transport for mitochondria could be evidenced.     

Membranes and calcium sequestration during spermiogenesis in the cotton seed bug (Dysdercus intermedius: Heteroptera)

Summary By use of osmium ferricyanide (OsFeCN) staining the fate of cytoplasmic membranes was followed during spermiogenesis in the cotton seed bug (Dysdercus intermedius). During early spermiogenesis interzonal lamellae of endoplasmic reticulum become aggregated as a stack of membranes traversing the entire cell body from the nucleus to the cytoplasmic bridge connecting neighbouring spermatids. Cisternae of endoplasmic reticulum ensheath the acroblast from which vesicles of different sizes are pinched off into the cytoplasm. The oxalate method was used to show that acroblast and associated vesicles are calcium-sequestering sites in spermatids. Membrane profiles with dense calcium oxalate precipitate derived from the acroblast form an uninterrupted membranous sheath at the apical side of the nucleus where the proacrosome will be attached. With further development of the spermatids, the vesicles derived from the acroblast also participate in forming a calciumsequestering sheath enveloping the axoneme and the mitochondrial nebenkern derivatives.     

Bioelectric aspects of the hemipteran telotrophic ovariole (Dysdercus intermedius)

Summary Two systems of steady extra-cellular currents were found along the surface of the telotrophicDysdercus ovarioles by means of a vibrating probe. The first covers the subgerminal tropharium and all the previtellogenic follicles. The current leaves the 3 or 4 small follicles of early euplasmic growth stages laterally and enters the syncytial tropharium. We presume that a similar intracellular current flows between the trophoplasm and the ooplasm which are interconnected by narrow nurse strands. Preliminary intracellular measurements indicate a potential gradient within this continuous cytoplasm, the ooplasm being electropositive to that of the tropharium. This current system fits into a model of polarized intracytoplasmic transport by electrophoresis. It is possible to explain the well known directed and selective flow of RNA from the tropharium via the nurse strands into the oocytes by means of such a model. The second current system occurs around every one of the 2 to 8 vitellogenic follicles. The pattern is completely different from that described for the first system. In the vitellogenic stages the current enters the follicle laterally all along the now much extended surface. It is balanced by a strong peak current which leaves the interfollicular region. As data on intracellular currents are not yet avialable, it is only a matter of speculation whether the circuit is closed through the ooplasm or only by a tangential loop through the follicle epithelium. The possible significance of this second current system for vitellogenin accumulation and uptake by the vitellogenic oocytes is also uncertain as yet.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

Heterologous gap junctions between oocytes and follicle cells of an insect,Dysdercus intermedius,and their potential role as ion current pathways

Summary In telotrophic insect ovaries, the oocytes develop in association with two kinds of supporting cells. Each ovary contains five to seven ovarioles. An ovariole consists of a single strand of several oocytes. At the apex of each ovariole is a syncytium of nurse cells (the tropharium), which connects by strands of cytoplasm (the trophic cords) to four or more previtellogenic oocytes. In addition, each oocyte is surrounded by an epithelium of follicle cells, with which it may form gap junctions. To study the temporal and spatial patterns of these associations, Lucifer yellow was microinjected into ovaries of the red cotton bug, Dysdercus intermedius. Freeze-fracture replicas were examined to analyze the distribution of gap junctions between the oocyte and the follicle cells. Dye-coupling between oocytes and follicle cells was detectable early in previtellogenesis and was maintained through late vitellogenesis. It was restricted to the lateral follicle cells. The anterior and posterior follicle cells were not dye-coupled. Freeze-fracture analysis showed microvilli formed by the oocyte during mid-previtellogenesis, and the gap junctions became located at the tips of these. As the microvilli continued to elongate until late vitellogenesis, gap junction particles between them and follicle cell membranes became arranged in long arrays. The morphological findings raise questions about pathways for the intrafollicular phase of the ion currents known to surround the previtellogenic and vitellogenic growth zones of the ovariole.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Effects of water from Nakhodka Bay (Peter the Great Bay, Sea of Japan) on the early development of the Sea UrchinStrongylocentrotus intermedius

Our studies show that seawater from different sites in Nakhodka Bay has a deleterious effect on the development of the sea urchinStrongylocentrotus intermedius. As development proceeded to the pluteus I stage, the percentage of abnormal larvae maintained in water from Nakhodka, Novitskogo, and Vrangelya Bights increased markedly (66.7±2.2, 67.1±2.6, and 54.6±1.8%, respectively). These larvae developed more slowly, were smaller, and differed from those in the control in the intensity of color of their echinochrome granules. In water from Nakhodka and Novitskogo bights, larval survival rates were lower than in the control. This suggests that the water of Nakhodka Bay is heavily polluted, especially in its harbor areas.     

The absence of cytoplasm ribosomes on the envelopes of chloroplasts and mitochondria in plants: Implications for the mechanism of transport of proteins into these organelles

Summary Chloroplasts and mitochondria ofChlamydomonas were examined by electron microscopy to determine if cytoplasmic ribosomes were associated with the envelopes of these organelles. Cells were treated with cycloheximide to prevent polypeptide chain completion, and resultant dissociation of envelope-ribosome associations. No extensive association of cytoplasm ribosomes with envelopes of chloroplasts, or mitochondria was detected in intact cells or in damaged cells in which cytoplasm was partly removed. Our results indicate that association of cytoplasm ribosomes with envelopes of chloroplasts or mitochondria is not an essential requirement for transport of polypeptides from cytoplasm to organelle.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Gibberellin synthesis inhibitors affect electron transport in plant mitochondria

NADH oxidation and cytochrome c reduction rates in the electrontransport chain were determined for mitochondria isolated from leaves of matureEuropean black alder (Alnus glutinosa L.) and exposed to arange of concentrations of the growth retardants flurprimidol andpaclobutrazol.NADH oxidation and cytochrome c reduction were enhanced by low concentrationsofboth compounds whereas higher concentrations reduced electron transport. Thisisthe first report of gibberellin synthesis inhibitors affecting electrontransport in plant mitochondria and provides evidence for another potentialmodeof action of this type of growth retardant.     

Absence of phytosterol dealkylation and identification of the major ecdysteroid as makisterone A in Dysdercus fasciatus (Heteroptera,Pyrrhocoridae)

The absence of phytosterol dealkylation in the cotton stainer bug, Dysdercus fasciatus, has been established and the major ecdysteroid in the fifth-stage larvae identified. The demonstration that the free and esterified sterols in D fasciatus consisted of 95–96% sitosterol and 4–5% campesterol, a similar composition to the cottonseed diet, together with the lack of conversion of [¹⁴C]sitosterol into cholesterol, establishes that phytosterol dealkylation does not occur in this insect species. The ecdysteroid titer determined by radioimmunoassay in the fifth instar of D fasciatus shows a distinct peak at day 6, the instar lasting for 7 days. Makisterone A was purified by HPLC from insects at a time of high ecdysteroid titer and identified as a major component by both fast atom bombardment and electron impact mass spectrometry. Gas-liquid chromatography/mass spectrometry (selected ion monitoring) confirmed the occurrence of makisterone A and revealed the presence of two unidentified compounds. One of these occurs in a similar amount to makisterone A and may be 26-hydroxymakisterone A, whereas only a minute amount of the other compound, which may be 20-deoxymakisterone A, was present; further identification of the latter compounds is necessary. C₂₇ ecdysteroids (eg, ecdysone and 20-hydroxyecdysone) and C₂₉ ecdysteroids (eg, podecdysone A) were undetectable. The specificity of the enzymes of ecdysteroid biosynthesis is discussed.     

Paternal inheritance of mitochondria in rapeseed (Brassica napus)

Summary Transfer of a mitochondrially associated plasmid following sexual crosses in Brassica napus rapeseed suggested that paternal mitochondria were being transferred to the cytoplasm of the egg. To examine this possibility further, plants carrying the chloroplast (cp) marker of triazine resistance, but which had lost the plasmid associated with the mitochondria of this cytoplasm, were crossed as females to males carrying the polima cytoplasm. The males carried a nuclear fertility restorer gene on an extra chromosome to overcome the male sterility marker conferred by the mitochondria of this cytoplasm. Approximately 10% of the F₁ progeny displayed the male sterility and flower morphology of the male parent. Mitochondrial (mt) DNA from the progeny showed the combined restriction patterns of both parents, but this rut heterogeneity did not continue into subsequent generations. All progeny retained the cp DNA restriction patterns of the maternal plant as well as resistance to the herbicide atrazine. To date, sexually mediated cybrid plants have shown no morphological abnormalities and have maintained their unique combination of cp and mt traits through several sexual generations.     

Changes in axonal transport in neurones of Asterias vulgaris and Asterias forbesei produced by colchicine and dimethyl sulfoxide

Summary Gas flow analysis, autoradiography, and electron microscopy were performed on radial nerve cords of sea stars injected intragastrically with [¹⁴C] leucine tracer, a precursor of axonal proteins. Neurotubules, neurofilaments, and axonal transport were demonstrated for the first time in sea stars. This transport occurred at two rates (fast phase: 240–480 mm/da, slow phase: 20–40 mm/da) moving in both the cellulifugal and centripetal directions. The rate in the centripetal direction was slightly slower. Displacement of peaks of radioactivity appeared cyclic.Transneuronal transport apparently occurred between several groups of neurones suggesting the possibility of metabolic communication, perhaps across the synapse, between groups of neurones or even areas of the nervous system. Regenerating nerve cords demonstrated an increase in the rate (400–600 mm/da) and quantity (6–11 times as much) of label transported to cord termini.The relationship between colchicine and the disruption of neurotubular integrity, resulting in disturbances in fast phase transport, was confirmed. More significantly, the association between nerve injury and site or concentration of treatment is emphasized. Treatment of nerve cords with dimethyl sulfoxide (DMSO) produced more subtle effects than did colchicine, reversible 4–6 hours following the local treatment of the involved nerves. Axon bundles swelled and the organization of axons within the bundles was distorted. Stereochemical structure was suggested from this work as playing a key role in effecting fast phase transport.The authors are indebted to Dr. B. Kiefer, Department of Biology, Wesleyan University, Dr. L. Ross, Department of Anatomy, Medical College of Pennsylvania, and Dr. M. Shelanski, Department of Molecular Biology, Harvard University for their help and constructive criticism.     

Effect of diet and mating status on ovarian development and oviposition in the polyphagous predator Macrolophus caliginosus (Heteroptera: Miridae)

The mirid bug Macrolophus caliginosus is commercially reared on eggs of Ephestia kuehniella, constituting an effective but expensive factitious food. Artificial diets can decrease the rearing costs of this natural enemy, but developing and evaluating an artificial diet is a very time-consuming activity. In the current study, development and reproduction of M. caliginosus on two artificial diets based on egg yolk were investigated. The artificial diets resulted in longer development and lower adult weights, but survival was comparable with that of control insects fed E. kuehniella eggs. Reproductive potential of the predator reared on factitious and artificial foods was assessed using a dissection method. The influence of nymphal food on fecundity was less important than that of adult food. Adults fed E. kuehniella eggs had a preoviposition period of about 4 days, whereas adults offered only plant material started laying eggs about 7 days after emergence. Ovarian scores at day 7 were higher for females fed E. kuehniella eggs than for those given access only to a tobacco leaf. Ovarian scores were not significantly affected by mating status. In a final test, a parallel comparison of two methods for assessing reproductive response to diet was made. Here, adult couples were offered one of four diets: E. kuehniella eggs, one of two artificial diets or no food. Half of the females were dissected and the other half was held for determining lifetime oviposition. Females fed E. kuehniella eggs had superior ovarian scores and laid more eggs than those fed either artificial diet or those given no extra food. A good correlation (r = 0.97) was obtained between ovarian scores and oviposition data, indicating that dissecting females after 1 week provides a reliable estimate of fecundity as affected by diet quality. Rapid reproductive assessments as used in the current study will help to increase the rate of development of artificial diets and may contribute to more cost effective production methods for augmentative biological control agents.     

Wing Movement Behavior in Long- and Short-Winged Morphs of the Flightless Bug Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae)

The aim of the present paper was to study, with the use of video-recordering and computerized methods, the flight and wing movement behavior in two wing morphs of the flightless bug Pyrrhocoris apterus during the first 14 days of their adult life. We demonstrated the capability of macropterous males and females of this heteropteran to flap their fore- and hindwings for the first time. Flight was not observed in any of the tested males and females. We revealed that wing flapping does not occur earlier than on the fourth and fifth day after adult emergence in males and females, respectively. Time-series analysis of the obtained data showed that the maximum activity of wing flaps is limited to the light phase of the 12-h light/12-h dark photocycle, with the peak occurring in the morning, i.e., about 3 h after turning the light on. No substantial differences in the peaks of wing flapping activities in males versus females were found. The present paper is the first experimental demonstration of wing flap activity in a representative species with a nonfunctional alary polymorphism.     

Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Sperm transfer in the antCarebara vidua F. Smith (Hymenoptera: Formicidae)

Summary Sperm transfer in the myrmicine antCarebara vidua was studied by making histological sections of newly mated queens. Queens are multiply inseminated, and longitudinal sections through the sperm- filled vagina show that the bundle of sperm from each male is enclosed in a layer of accessory gland secretion terminating in a plug of secretion in the female's vaginal orifice. This spermatophore breaks down soon after mating. The huge quantity of sperm packed into the spermatheca (ca. 16 mm³) suggests that the queen lays a large number of eggs in her lifetime, confirmed by the average of 1672 ovarioles in the two ovaries.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

Contributions of Robert (Bob) J. O’Neil to the field of predaceous Heteroptera

Predaceous Heteroptera are important components of natural and managed habitats. The extent of predatory habits among taxa ranges from those that are totally (or nearly totally) predaceous to those that are nearly totally phytophagous, but occasionally feed on arthropod prey. Because of that variability in predatory habit, the contributions of heteropteran taxa to management of pest species will vary greatly. The late Robert (Bob) J. O’Neil (1955–2008) added significantly to understanding the roles of and benefits from facultatively predaceous Heteroptera. His focus on behavioral and physiological attributes that permitted facultatively predaceous species to persist in agricultural habitats demonstrated that facultative predators could help prevent pest outbreaks, but were less effective at reducing numbers of species already considered above economic thresholds.     

An autoradiographic study of calcium transport in spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The route of calcium transport to the sites of spicule formation in the gorgonian Leptogorgia virgulata has been examined by the use of ⁴⁵Ca as a tracer in light- and electron-microscopic autoradiography. From 1 to 15 min after the 15-min incubation the tracer accumulates in the axis. After 15 min there is a movement of label out of the axis largely to the peripheral region of the axis, the axial epithelium, and the mesoglea. By 60 min much of the label is in the spicules reaching its maximum level at 120 min. When calcium enters the scleroblast from the mesoglea, it appears to be transported to the spicule by electron-dense bodies. There does not appear to be a simultaneous release of all ionic calcium from the axis, but rather a continuously increasing efflux which levels off at 60–120 min. Not all of the calcium reaching the axis will traverse it en route to spicules; instead, a portion of it apparently precipitates as an amorphous compound.Contribution No. 550; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208 USA