Phagocytosis of latex beads and bacteria by hemocytes of the triatomine bug Rhodnius prolixus (Hemiptera: Reduvidae)

Insect circulating hemocytes are primarily responsible for the immune defense against parasites and pathogens. Here, we have analyzed phagocytosis of both biotic (bacteria) and abiotic (latex) particles by circulating hemocytes of 5th-instar nymphs of the triatomine bug Rhodnius prolixus. The following hemocyte types were identified: prohemocytes, plasmatocytes, granulocytes, oenocytoids and adipohemocytes. There was a considerable change in the relative percentage of plasmatocytes and prohemocytes in the hemolymph after challenge with both latex beads and bacteria. Granulocytes and oenocytoids also change their relative percentage in response to latex bead and Staphylococcus aureus, respectively. No significant change was observed in adipohemocytes at any time or treatment. Our data demonstrated that plasmatocytes were the only cell type involved in phagocytosis of foreign particles. As in mammal cells, phagocytosis by both zipper and trigger mechanisms were observed for the uptake of latex beads and bacteria. Neither melanization nor micro-aggregation was observed towards latex particles or Escherichia coli. On the other hand, R. prolixus produced a strong melanization reaction against S. aureus, thus showing that differences exist in the responses to E. coli and to S. aureus. Ultrastructural changes observed in plasmatocytes, adipohemocytes and oenocytoids suggest that these hemocyte types are directly involved in the immune defense of R. prolixus against foreign particles.     

Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera,Apidae, Meliponini): Cell types and their role in phagocytosis

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.5 μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris.     

Effect of sweet flag rhizome oil (Acorus calamus) on hemogram and ultrastructure of hemocytes of the tobacco armyworm, Spodoptera litura (Lepidoptera: Noctuidae)

Effect of the essential oil of Acorus calamus L. rhizomes, was studied on hemocytes of the tobacco armyworm, Spodoptera litura. The oil was administered in oral application at concentration of 500 and 1000 ppm to last instar larvae of S. litura and its effect on ultrastructure of hemocytes and hemogram was evaluated. The oil was administered in topical application at 250 microg dose to pupae to ascertain its effect on total and differential hemocyte counts. At both scanning (SEM) and transmission electron microscopic (TEM) levels, the major effect of oil treatment was observed on plasmatocytes (PLs) and granular hemocytes (GRs). SEM study revealed that the cytoplasmic projections of granular hemocytes were reduced, while the filopods of plasmatocytes remained unaffected. The vacuolization in the cytoplasm and degeneration of the organelles in both plasmatocytes and granular hemocytes was observed by TEM. However, no such deformities were observed in prohemocytes (PRs), spherulocytes (SPs), and oenocytoids (OEs). A concentration-dependent decrease has been observed in the larval body weight and hemolymph volume (HV), 24-72 h after treatment. In comparison to the controls, the maximum percentage growth inhibition (GI) was recorded to be 58.28 and 66.48, respectively at 500 and 1000 ppm after 72 h treatment. Similarly after 72 h treatment, the percentage reduction in hemolymph volume was 61.38 and 69.05, respectively at 500 and 1000 ppm. Total hemocyte count (THC), in larvae computed from five recorded hemocyte types viz. PRs, PLs, GRs, SPs and OEs, decreased only after 48-72 h of treatment. The maximum decrease in THC was recorded to be 29.15 and 49.05% at 500 and 1000 ppm, respectively, after 72 h of treatment. There was continuous decline in THC in pupae after 24-72 h treatment. DHC study revealed that both the concentrations of oil in 6th instar larvae of S. litura caused a decrease in PRs, PLs and SPs and increase in GRs and OEs after 24-72 h of treatment. Since A. calamus oil treatment causes the injury to both PLs and GRs and also affects the hemogram, it can be inferred that cellular defence reactions of S. litura are impaired.     

Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge

The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an important aquacultured decapod species as well as an invasive species in some European countries. In the current investigation we characterized the different classes of circulating blood cells in A. leptodactylus by means of light and electron microscopy analysis and we explored their reaction to different latex beads particles in vivo by total and differential cell counts at 0.5, 1, 2 and 4h after injections. We identified hemocytes by granule size morphometry as hyaline hemocytes with no or rare tiny granules, small granule hemocytes, unimodal medium diameter granule hemocytes and both small and large granule containing hemocytes. The latter granular hemocytes showed the strongest phenoloxidase l-DOPA reactivity both in granules and cytoplasm. A. leptodactylus respond to foreign particles with strong cellular immune responses. All treatments elicited a total hemocyte increase with a conspicuous recruitment of large granule containing hemocytes. All hemocyte types mounted some phagocytic response but the small granule hemocytes were the only ones involved in phagocytic response to all foreign particles with the highest percentages. These results (1) depict the variability in decapod hemocyte functional morphology; (2) identify the small granule hemocyte as the major phagocytic cell; (3) suggest that the rather rapid recruitment of large granule hemocyte in all treatments plays a relevant role by this hemocyte type in defense against foreign particles, probably in nodule formation.     

Cold resistance in the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

We have investigated cold resistance, measured by the supercooling point (SCP) temperature, in life stages of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), collected in Brittany poultry houses. Mean SCP values drastically increased during the insect ontogeny: egg (-26.1 C), first instar larvae (-21.6 C), last instar larvae (-15.5 C), pupae (-11.6 C), teneral adults (-12.0 C) and mature adults (-13.1 C). Nymphal metamorphosis and adult maturation did not promote substantial decrease of freezing resistance. The SCP values reflect the physiological states of the developmental stages especially the absence of ice nucleating agents: (i) lower SCP values in egg and unfed newly-emerged larvae I (i.e. -25.1 C), (ii) higher SCP values in fed larvae (i.e. -14.7 C), pupae and adults most likely due to the presence of ice nucleation sites in the gut. A tropical species, A. diaperinus, seems not to use its potential cold hardiness even in winter to remain in this warm habitat in temperate regions.     

Changes in the haemocytes of Agrotis ipsilon larvae (Lepidoptera: Noctuidae) in relation to dimilin and Bacillus thuringiensis infections

Five types of haemocytes are observed in the fourth larval instar of the black cutworm, Agrotis ipsilon: prohaemocytes (PRs), plasmatocytes (PLs), granulocytes (GRs), spherule cells (SPs) and adipohaemocytes (ADs). Infection of A. ipsilon fourth larval instar with Bacillus thuringiensis and dimilin resulted in a reduction of the total haemocyte count. Changes in the differential haemocyte population during bacterial and dimilin infections have been assessed. The PRs % decreased significantly while SPs, PLs, and GRs % increased significantly after the application of the two insecticides at 12 and 24 h. Ultrastructural alternations and malformations have been observed in circulating haemocytes of A. ipsilon larvae treated with dimilin and B. thuringiensis.     

Morphological characterization via light and electron microscopy of Atlantic jackknife clam (Ensis directus) hemocytes

The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim’s stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96 × 10⁶ cells mL⁻¹ while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function.     

Ultrastructural aspects of naturally occurring wound in the tunic of two ascidians: Ciona intestinalis and Styela plicata (Tunicata)

Efficient wound healing is essential for all animals from insects to mammals. Ciona intestinalis and Styela plicata are solitary ascidians belonging to urochordates, a subphylum that occupies a key phylogenetic position as it includes the closest relative to vertebrates. Urochordate first physical barrier against invaders is the tunic, an extracellular matrix that is constantly exposed to all kinds of insults. Thus, when damage occurs, an innate immune response is triggered to eliminate impaired tissue and potentially pathogenic microbes, and restore tissue functionality. Ultrastructural aspects of the tunic in the wound healing process of two ascidians are described. In the injured areas, we evidenced thinning of the tunic and areas of low fibre density, dense intratunic bacterial and protozoan population, and inflammatory aspects such as the increase in tunic cells, their aggregates, and phagocytosis. This is the first report on tunic physical wounding occurring in the natural habitat.     

Ultrastructural characterization of the hemocytes of Culex quinquefasciatus (DIPTERA: Culicidae)

Six hemocytes cell types from Culex quinquefasciatus were identified by light and transmission electron microscopy: They are prohemocytes (9.3%), spherulocytes (1.6%), adipohemocytes (0.8%), oenocytoids (4.6%), plasmatocytes (43.4%) and granulocytes (40.3%). The prohemocytes were the smallest hemocytes encountered in the hemolymph, displaying a large and centrally located nucleus, almost filling the whole cell. The spherulocytes, which were small hemocytes, presented small and numerous spherules with a lamellar pattern and an electron-dense core. Rare adipohemocytes were observed in the C. quinquefasciatus hemolymph, presenting large nucleus with an evident nucleolus, cytoplasm containing rough endoplasmic reticulum (RER), mitochondriae and lipid inclusions. C. quinquefasciatus oenocytoids showed homogeneous cytoplasm with several granules, completely or partially filled with amorphous material. These cells showed abundant smooth endoplasmic reticulum (SER) and dense mitochondriae. By light microscopy analysis we identified two morphological types of plasmatocytes, granular and agranular. However, ultrastructural investigation revealed that the granular cells contained lipid inclusion between RER membranes, instead of membrane-bounded granules. The granulocytes presented a fusiform or circular profile and displayed a unique and very complex process of granules formation, including organization of polysomes inside vesicles that protrude from the Golgi system, synthesis of a proteinaceous material, condensation of the granule matrix and recycling of endoplasmic membranes. Intense endocytic pathways were also observed in the granulocytes.     

Hemocytes ultrastructure of Aedes aegypti (Diptera: Culicidae)

Mosquitoes have an efficient defence system against infection. Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. We have identified by light and transmission electron microscopy six hemocytes cell types from the hemolymph of Aedes aegypti. They were: prohemocytes (20%), adipohemocytes (29%), granulocytes (16%), plasmatocytes (27%), oenocytoids (7%) and thrombocytoids (0.9%). The prohemocytes were the smallest hemocytes found in the hemolymph. Its cytoplasm occupies only a narrow area around the nucleus. The adipohemocytes were the most abundant cell type presented. These hemocytes exhibited a large lipid like vesicle and mitochondria. In electron micrographs, the granulocytes showed cytoplasm containing dilated rough endoplasmic reticulum (RER) and a round or elongated mitochondria. Electron-dense granules with a proteinaceous material were also present. The plasmatocytes were polymorphic and exhibited plasma membrane with irregular processes, philopodia and pseudopodia. Ultrastructural investigation revealed that the reticular cytoplasm showed a well-developed RER, a Golgi and vacuoles. Oenocytoids showed homogeneous cytoplasm with many mitochondria and ribosomes are scattered throughout the cytoplasm, abundant RER and a small smooth endoplasmic reticulum (SER) present at the cell poles. Thrombocytoids were very fragile and few in number. Similar characteristics were found in oenocytoids, possessing a homogeneous cytoplasm with poorly developed organelles, few mitochondria and granules.     

Distortion product otoacoustic emission (2f1-f2) amplitude growth in human adults and neonates

Distortion product otoacoustic emissions (DPOAEs) are thought to be by-products of an active amplification process in the cochlea and thus serve as a metric for evaluating the integrity of this process. Because the cochlear amplifier functions in a level-dependent fashion, DPOAEs recorded as a function of stimulus level (i.e., a DPOAE growth function) may provide important information about the range and operational characteristics of the cochlear amplifier. The DPOAE growth functions recorded in human adults and neonates may provide information about the maturation of these active cochlear processes. Two experiments were conducted. Experiment I included normal-hearing adults and term-born neonates. The 2f1-f2 DPOAE growth functions were recorded for both age groups at three f2 frequencies. Experiment II was an extension of the first experiment but added a subject group of premature neonates. The results of these studies indicate that DPOAE growth functions most often show amplitude saturation and nonmonotonic growth for all age groups. However, premature neonates show monotonic growth and the absence of amplitude saturation more often than adults. Those premature neonates who do show saturation also show an elevated threshold for amplitude saturation relative to adults. In contrast, term neonates are adultlike for most measures except that they show a larger percentage of nonsaturating growth functions than adults. These results may indicate immaturity in cochlear amplifier function prior to term birth in humans. Outer hair cell function and/or efferent regulation of outer hair cell function are hypothesized sources of this immaturity, although some contribution from the immature middle ear cannot be ruled out.     

Fat body of Prorhinotermes simplex (Isoptera: Rhinotermitidae): ultrastructure, inter-caste differences and lipid composition

Ultrastructure of the fat body was studied in following castes and developmental stages of Prorhinotermes simplex: larvae of the first and the second instar, pseudergates, presoldiers, soldiers, nymphs, imagoes and mature ergatoid neotenic reproductives of both sexes. Fat body always consists of two principal cell types: adipocytes and urocytes. Adipocytes are characterized by a presence of large amounts of storage substances, namely lipid droplets, glycogen rosettes and proteins in the form of either biocrystals or vacuoles. Proportion of these components strongly varies during ontogeny. Adipocytes are equipped by a large central vacuole in which lipid droplets may resolve. Cytoplasm of urocytes contain glycogen rosettes and spherical or irregular concretions, other organelles are rare. Only adipocytes change their inner structure in the course of ontogeny: amount of glycogen decreases during the postembryonic development, it is the major kind of inclusion in the larvae but lacks in nymphs and imagoes; opposite trend is performed by lipids. The changes in protein content are less obvious but are explained and discussed. The total amount of triacylglycerols (TAGs) was found to be roughly 100microg in a pseudergate, 250microg in a nymph, and 30microg in a soldier. The most abundant fatty acids in TAGs are oleic (O), stearic (S), palmitic (P) and linoleic (L) acid. TAGs form a complex mixture with OOO, OPO, OLO and OOS being the most abundant isomers. Only negligible differences exist among the castes.     

New calibration method for position detector for simultaneous measurements of force constants and local viscosity in optical tweezers

We present a new method to calibrate a quadrant photodiode used as position detector to monitor latex beads trapped in optical tweezers. The method combines the dragging Stoke’s force with the thermal noise analysis (power spectral density (PSD)) of the Brownian motion of the trapped bead. Position detector calibrations used in other similar methods normally utilise a bead attached to the coverslip: the voltage-position calibration factor is found by translating the bead across the waist of a laser beam. The so determined calibration factor is then assumed to be the same when beads are investigated in the optical trap. This procedure presents some drawbacks since attached beads can be affected by proximity effects due to the coverslip glass surface which alter the position sensor response itself. On the contrary, our method is able to provide, simultaneously, the calibration factor, the trap stiffness, and the local viscosity of the medium making use of a single trapped bead.     

Magnetic ground state and electronic structure of CeRu(2)Al(10)

We present a combined theoretical and experimental study of the electronic structure for CeRu(2)Al(10) based on ab initio band structure calculations and x-ray photoemission spectroscopy (XPS) data. Our calculations were performed for the base unit cell and for the hypothetical unit cell which enables antiferromagnetic ordering. The stability of the magnetic phase was investigated within fixed spin moment calculations. When additional 4f correlations are not included in the LSDA C U approach, CeRu(2)Al(10) exhibits an unstable magnetic configuration with the difference in total energy per unit cell between the weakly magnetic state and the non-magnetic one of the order ~0.3 meV. We found that Coulomb correlations among 4f electrons, when they are included in the LSDA C U approach, stabilize the magnetic structure. In the weakly correlated system (small U) an antiferromagnetic (AFM) ground state with the lowest total energy is preferred. The situation is, however, the opposite when the 4f correlations are strong. In this case the ferromagnetic (FM) ground state is preferred. By comparing our calculations with the experimental data we conclude that the 4f correlations in CeRu(2)Al(10) are weak. We also carried out a structural relaxation of atomic positions within the Cmcm unit cell and we found that the Al atoms exhibit noticeable displacement from their positions known from x-ray diffraction (XRD) analysis.     

Numerical design of a 3-D microsystem for bioparticle dielectrophoresis: The Pyramidal Microdevice

A device for the detection of bioparticles (latex beads, cells, proteins, DNA,…) present in a small volume of a liquid sample is described. Dielectrophoresis (DEP) is used to enhance the transport of bioparticles towards the reactive surface of the detector. A Pyramidal Microdevice (PM) is designed using numerical simulation. Unlike conventional coplanar electrode microchips, this original 3D configuration is able to collect particles on a surface for both positive and negative DEP. Numerical simulations show the location of the collection zones in the PM according to the DEP mode applied. PM performance is evaluated with the calculation of the collection kinetics. The influence of the voltage is studied. Experiments on PM with latex microparticles confirm calculations. Their replacement by DNA molecules attests to the feasibility of a PM collecting such extremely small particles using DEP.     

Type of the second hole subband on the Si(110) surface: Spin splitting anisotropy

Magnetoresistance measurements have been performed for the cases of one and two occupied size-quantization hole subbands in silicon field-effect transistors prepared on the Si (110) surface. In both cases, Shubnikov-de Haas oscillations exhibit very weak sensitivity to the in-plane component of the magnetic field. This indicates that both lowest subbands are formed by heavy holes. This conclusion disagrees with the wide-spread opinion that the second subband is the ground subband of light holes in the system under consideration.     

Cold preservation of endothelial cells in sucrose-based solution (SbS) and University of Wisconsin (UW) solutions: comparison of normoxic or hypoxic storage

Cold preservation of endothelial cells was studied, comparing primary endothelial cells (human umbilical vein endothelial cells - HUVECs) and a continuously growing cell line (ECV304 cells). Viability at the end of 24h cold preservation was measured by dye exclusion, whilst metabolism was assessed by Alamar blue conversion. Two preservation solutions were studied (UW solution) and sucrose-based (SbS) in both cell types. The response was similar in both cell types to preservation under normoxic conditions (with percentage dye exclusion maintained at about 80 percent in both preservation solutions) whereas under hypoxic conditions ECV304 were more sensitive to preservation in UW solution (dye exclusion reduced to 43.5+/-1.4 percent versus 73.6+/-14 percent (P<0.01). Metabolism assessed by Alamar blue conversion after cold preservation and rewarming was similar in both ECV304 and HUVECs after storage under normoxic conditions in UW solution, but in both cell types, metabolism was higher in SbS (P<0.05 and p<0.01) than in UW solution. Under hypoxic conditions, both cell types showed similar recovery of metabolism after storage in either UW or SbS. If the cells (in this case ECV304 under aerobic conditions) were stored for 24h and then allowed to rewarm in either of the respective preservation solutions (UW or SbS for 1h) before the Alamar blue test, metabolism was higher (p less than 0.01) in those exposed to SbS. UW solution and SbS provide similar protection for endothelial cells under hypoxic conditions, but SbS has some advantages under normoxic storage or if the cells experience variable temperatures in the presence of residual preservation solution at the end of cold preservation period.     

Antennal morphology and sensillar ultrastructure of Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae)

The parasitoid beetle Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an important parasite of longicorn beetles (Cerambycidae), and has been used in China for the biological control of the Asian longicorn beetle and the Japanese pine sawyer. In this study the antennal morphology and sensillar ultrastructure of D. helophoroides were observed using scanning electron microscopy and transmission electron microscopy. Two types of sensilla trichodea (Tr. 1 and Tr. 2), two types of sensilla basiconica (Ba. 1 and Ba. 2), three types of sensilla chaetica (Ch. 1, Ch. 2 and Ch. 3), and Bohm's bristles were identified according to the morphology and fine structure of each type of sensilla in both sexes. Ultrastructural studies revealed porous structures on the cuticle wall and dendritic branches in the inner lumen of Tr. 1, Tr. 2, Ba. 1, and Ba. 2, thereby suggesting chemoreceptor functions. No difference in shape, structure, sensilla distribution and typology was observed between the sexes. These structures likely have roles in the host locating and habitat searching behavior of adult D. helophoroides, and suggest future studies on the olfaction and host location behavior of D. helophoroides and other coleopteran parasitoids.     

Dielectrophoretic ratchets

We have experimentally applied some concepts of "force-free" motion to micron size particles (latex beads). The coupling of dissipation and local spatial asymmetry of the potential experienced by the beads can put them into motion. The potentials used in these experiments are of dielectrophoretic nature. To that end, electrodes of particular shapes were used in order to submit the considered suspensions to inhomogeneous ac electric fields. Two regimes were explored: i-the Brownian ratchet case in which a Brownian particle is successively trapped in a factory roof-like potential and left free to diffuse. ii-the shifted ratchets case in which two potentials exhibiting similar characteristics are applied successively, one of them being shifted by a fraction of their common period relatively to the other. In both cases, a good agreement with the theoretical predictions was observed. In particular, particles of different sizes were characterized by different macroscopic velocities leading to the prospect of promising separation techniques. (c) 1998 American Institute of Physics.     

Competition between charge- and spin-density-wave order and superconductivity in La(1.875)Ba(0.125-x)Sr(x)CuO(4)

We have performed a series of elastic neutron scattering measurements on 1/8-hole doped La(1.875)-Ba(0.125-x)Sr(x)CuO(4) single crystals with x = 0.05, 0.06, 0.075, and 0.085. Both charge-density-wave (CDW) and spin-density-wave orders are found to develop simultaneously below the structural transition temperature between the low-temperature orthorhombic (LTO) and low-temperature tetragonal (LTT) or low-temperature less-orthorhombic (LTLO) phases. In the ground state the CDW order is observed only in the LTT/LTLO phase and drastically degrades towards the LTO boundary. The x dependence of T(c) strongly suggests a direct effect of the CDW order on the suppression of superconductivity.