Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus

The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa α-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the α-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an α-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin.This is the first ultrastructural study to show differences in the cell morphology of resistant larvae and further investigation is needed to understand the impact of the lack of expression of midgut enzymes on the physiology of resistant insects.     

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.     

Acute and subchronic exposure of diplopods to substrate containing sewage mud: Tissular responses of the midgut

Several discussions regarding the agricultural use of sewage mud have occurred; however, its use has been questioned due to the risks of soil contamination mainly by metals. Diplopods are saprophagous invertebrates, agile in colonizing several layers of soil. Due to the habit of this group, several researchers have proposed their use in ecotoxicological analyses. This study aimed to expose these invertebrates to substrate containing sewage mud of a STS (Sewage Treatment Station) from São Paulo State, Brazil and analyzed its toxic potential by morphological analysis of the midgut of the diplopod Rhinocricus padbergi, region of the digestive tube where digestion and nutrient absorption occur. The animals were exposed in mud concentrations at 1%, 10% and 50% mixed with soil from the collection site for periods of 7 (acute exposure), 15 (intermediary exposure) and 90 (subchronic exposure) days in each treatment. The animals from the control group did not present alterations at all the exposition times. As an acute response (7 days), it was observed in the animals exposed to mud at 1% and 10% an increase in the epithelial renovation and in the liberation of secretory vesicles of glycoprotein content. In the animals exposed to mud at 50%, the main alteration observed was the increase in the number of cytoplasmatic granules in the fat body cells; the epithelium seemed to be in reorganization process in all the observed individuals, suggesting an intense epithelial renovation. In the intermediary period of exposure (15 days), in the animals exposed to mud at 1% it was observed an increase in the number of haemocytes among the cells of the fat body, distributed both separately and grouped; the animals exposed to mud at 10% presented the same evidence of epithelial renovation observed in the animals exposed for 7 days at a concentration of 50% of mud; it was also observed an increase in the number of haemocytes, which reacted strongly to the PAS technique. Cytoplasmatic granules in the hepatic cells, fat body cells and haemocytes were also observed in great number, being that great part presented calcium in their constitution. In the animals exposed to mud at 50%, the alteration observed was the presence of numerous secretory vesicles of the apocrine type through all the epithelia of vacuolated aspect; hepatic cells with cytoplasmatic granules were also observed. As subchronic response (90 days), it was observed that all the fat body cells were completely taken by cytoplasmatic granules, into which it was, detected the presence of calcium; such granules were also observed in the apical region of the principal epithelial cells. These results suggest the existence of toxic agents in the analyzed mud sample, whose different concentrations trigger responses that occur in different manners, intensities and velocities that might prevent the entrance and action of toxic agents in the organism of the studied invertebrate.     

Transient changes in quartz resonators following exposure to pulse ionization

Abstract

This paper is a review of the transient effect of pulse ionization on the resonant frequency and Q of quartz resonators. A number of different samples of both natural and synthetic quartz crystals have been used. Fifth-overtone 5, 32, and 125 MHz thickness shear (AT-cut) crystal units were fabricated and studied using several different oscillator circuits, one of which was designed such that the resistance R_s (αQ ^?1) of the crystal unit at series resonance as well as frequency could be measured after pulse exposure. With the exception of one purposely altered crystal, all the resonators exhibited a negative-transient frequency offset after irradiation which thereafter anneals as a function of t ^?1/2 over nearly four decades of time. The pulse irradiation effect on Q ^?1 is most striking at 5 MHz in natural quartz units, which suffer a thirty-fold increase in acoustic absorption after exposure to 10⁴ rad. The transient increase in the acoustic absorption in natural quartz anneals within 10 seconds to a value somewhat larger than the preirradiation absorption. Theoretical models are described which account for these observations.     

AP threshold elevation in the guinea pig following exposure to a broadband noise

Sixty guinea pigs were exposed to a steady-state broadband noise with a falling frequency spectrum. The sound-pressure level was varied between 96 and 117 dB SPL, and the duration of the exposure was varied from 3 to 12 h. After 4-5 weeks, the auditory thresholds were determined by electrocochleography at 14 frequencies, and the results were compared with a control group. With increasing sound-pressure level, the thresholds became elevated at all frequencies. The maximum threshold elevation also exhibited a slight shift toward higher frequencies. With increasing exposure time, the threshold elevations increased and shifted into the high-frequency region, whereas the low-frequency region was less affected. Linear regression analysis showed that the average threshold elevation between 1 and 20 kHz did not deviate from that predicted by the equal-energy hypothesis. However, the high-frequency loss at 5-20 kHz was very dependent on the exposure time, whereas the 1- to 4-kHz loss was not. There was no sign of any critical intensity with sudden increments in threshold elevation.     

Morpho-physiological analysis of the insect fat body: A review

The insect fat body is the main organ of the intermediate metabolism of insects. The majority of proteins of the haemolymph is synthesized in this tissue, which also presents the functions of lipids, carbohydrates and proteins storage. This tissue is also responsible for the synthesis of vitellogenins, proteins with an important role in the reproduction of the insects, being incorporated into the oocytes during vitellogenesis. The fat body consists of thin layers or strings, generally one or two cells thick, or small nodules suspended in the hemocele through connective tissues and trachea. Within a species, the structure of this tissue is more or less constant, but can have considerable differences between insects of different orders. In this way, this article makes a review about the main morpho-physiological features of the fat body cells of the insects, as well as a phylogenetic study of the fat body between basal and derived species of the Attini tribe ants.     

Enzymatic studies in foetal brain and liver of mouse following in utero exposure to ultrasound

Lactate, malate and succinate dehydrogenases (LDH, MDH and SDH) were estimated in the foetal brain and liver following in utero exposure of mice to a continuous wave of unfocussed ultrasound. Enzymes in both tissues showed a significant increase (P greater than 0.001), indicating high energy demand due to depletion of oxygen. The increase was recorded even on the twentieth day of pregnancy.     

Recovery of embryos of Zizania palustris following exposure to liquid nitrogen

Factors affecting survival of embryos of Zizania palustris after exposure to liquid nitrogen were investigated. Moisture content was the most critical factor. Embryos desiccated to a moisture content between 0.36 and 0.56 g water per g dry weight had highest survival after liquid nitrogen exposure (56 +/- 4% and 52 +/- 9% respectively). The recovery of embryos in the light following liquid nitrogen exposure also influenced survival. Survival increased from 35 +/- 4% to 56 +/- 4% when embryos containing 0.36 g water per g dry weight were recovered in the dark compared to light. Embryos exposed to desiccation and freezing stresses were subject to increased levels of lipid peroxidation. Light exacerbated lipid peroxidation in recovering embryos. Furthermore, catalase and peroxidase, enzymes involved in reducing reactive oxygen species, increased in activity in response to stress, and were further up-regulated in tissues recovered in the light. This study suggests that there are a number of factors influencing the survival of tissues exposed to liquid nitrogen and recovery procedures that reduce oxidative stress should be employed.     

Vitellogenin is glycosylated in the fat body of the stick insect Carausius morosus and not further modified upon transfer to the ovarian follicle

Synthesis and secretion of vitellogenin (Vg) polypeptides were studied in egg-laying females of the stick insect Carausius morosus following in vivo exposure to [³⁵S]-methionine and acetyl-N-[³H]-glucosamine. The specificity of radioisotope incorporation was assessed by in vitro inhibition with tunicamycin and carbohydrate extraction with endo-glycosidase H. Vg polypeptides change in molecular weight during synthesis in the fat body and are not further modified upon transfer to the haemolymph or to the oocyte, suggesting that they are already fully glycosylated prior to secretion.

Radioactivity in the fat body was initially distributed over cisternae of the rough endoplasmic reticulum and gradually transferred to the Golgi apparatus. Within an hour of exposure, electron-dense granules budding from the trans-Golgi network became preferentially labeled. Radioactivity in the ovarian follicle was restricted to the yolk granules of the cortical ooplasm and to the amorphous material lying within the intercellular channels of the follicular epithelium. This amorphous material was also shown to react positively when tested with a monoclonal antibody raised specifically against a Vg polypeptide.     

Ferritin in iron containing granules from the fat body of the honeybees Apis mellifera and Scaptotrigona postica

It is already known that the behaviour of the honeybee Apis mellifera is influenced by the Earth's magnetic field. Recently it has been proposed that iron-rich granules found inside the fat body cells of this honeybee had small magnetite crystals that were responsible for this behaviour. In the present work, we studied the iron containing granules from queens of two species of honeybees (A. mellifera and Scaptotrigona postica) by electron microscopy methods in order to clarify this point. The granules were found inside rough endoplasmic reticulum cisternae. Energy dispersive X-ray analysis of granules from A. mellifera showed the presence of iron, phosphorus and calcium. The same analysis performed on the granules of S. postica also indicated the presence of these elements along with the additional element magnesium. The granules of A. mellifera were composed of apoferritin-like particles in the periphery while in the core, clusters of organised particles resembling holoferritin were seen. The larger and more mineralised granules of S. postica presented structures resembling ferritin cores in the periphery, and smaller electron dense particles inside the bulk. Electron spectroscopic images of the granules from A. mellifera showed that iron, oxygen and phosphorus were co-localised in the ferritin-like deposits. These results indicate that the iron-rich granules of these honeybees are formed by accumulation of ferritin and its degraded forms together with elements present inside the rough endoplasmic reticulum, such as phosphorus, calcium and magnesium. It is suggested that the high level of phosphate in the milieu would prevent the crystallisation of iron oxides in these structures, making very unlikely their participation in magnetoreception mechanisms. They are most probably involved in iron homeostasis.     

Effect of periodic rest on hearing loss and cochlear damage following exposure to noise

Changes in hearing sensitivity and cochlear damage were determined in two groups of chinchillas exposed to an octave band of noise (OBN) centered at 0.5 kHz, 95 dB SPL on two different schedules: 6 h per day for 36 days, or 15 min/h for 144 days. Hearing sensitivity was measured behaviorally at 1/4-oct frequency intervals from 0.125 to 16.0 kHz before, during, and for a period of 1 to 2 months after the exposure, at which time the animals' cochleas were fixed and prepared for microscopic examination. Cochlear damage was determined by counts of missing sensory cells. Both exposures produced an initial shift of thresholds of 35-45 dB; however, after a few days of exposure, thresholds began to decline and eventually recovered to within 10-15 dB of original baseline values even though the exposure continued. Measures of recovery made after completion of the exposures indicated minimal permanent threshold shifts in all animals. The behavioral and anatomical data indicated that these intermittent exposures produced less temporary and permanent hearing loss and less cochlear damage than continuous exposures of equal energy.     

Systematic distortions of auditory space perception following prolonged exposure to broadband noise

Perceptual distortions referred to as aftereffects may arise following exposure to an adapting sensory stimulus. The study of aftereffects has a long and distinguished history [Kohler and Wallach, Proc. Am. Philos. Soc. 88, 269-359 (1944)] and a range of aftereffects have been well described in sensory modalities such as the visual system [Barlow, in Vision: Coding and Efficiency (Cambridge University Press, Cambridge, 1990)]. In the visual system these effects have been interpreted as evidence for a population of cells or channels specific for certain features of a stimulus. However there has been relatively little work examining auditory aftereffects, particularly in respect of spatial location. In this study we have examined the effects of a stationary adapting noise stimulus on the subsequent auditory localization in the vicinity of the adapting stimulus. All human subjects in this study were trained to localize short bursts of noise in a darkened anechoic environment. Adaptation was achieved by presenting 4 min of continuous noise at the start of each block of trials and was maintained by a further 15-s noise burst between each trial. The adapting stimulus was located either directly in front of the subject or 30 degrees to the right of the midline. Subjects were required to determine the location of noise burst stimuli (150 ms) in the proximity of the adapting stimulus following each interstimulus period of adaptation. Results demonstrated that following adaptation there was a general radial displacement of perceived sound sources away from the location of the adapting stimulus. These data are more consistent with a channel-based or place-based process of sound localization rather than a simple level-based adaptation model. A simple "distribution shift" model that assumes an array of overlapping spatial channels is advanced to explain the psychophysical data.     

Cytotoxicity and inflammation in human alveolar epithelial cells following exposure to occupational levels of gold and silver nanoparticles

This study explores the viability of rare earth-doped zirconia nanophosphors as probable candidates for white light emission. Undoped ZrO₂ and single- and double-doped ZrO₂:M (where M?=?Tb³⁺ and Eu³⁺) nanophosphors have been synthesized using a simple sonochemical process. The products were characterized using X-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDS), and photoluminescence spectrophotometry. The SEM micrographs show that resultant nanoparticles have dendritic shape. TEM and HRTEM studies showed that the size of the majority of the nanoparticles were around 28?±?5?nm. Characteristic blue and green emission from Tb³⁺ ions and red from Eu³⁺ dopant ions were observed. The CIE coordinates of the double-doped ZrO₂:Tb³⁺ (1.2?%):Eu³⁺ (0.8?%) nanophosphor lie in the white light region of the chromaticity diagram and show promise as good phosphor materials for new lighting devices.     

Ultrastructural study of first and second order neurons in the visual system of the crab Ucides cordatus following exposure to ultraviolet radiation

The visual system as an interface between the environment and the living organism can serve as a sensitive indicator especially in studies that deal with effects of radiation. The crab retina as the seat of the photoreceptors and the lamina ganglionaris as the place of second order neurons were the targets of our study. Conventional specimen preparation techniques for transmission electron microscopy (TEM) are adequate to preserve any modifications that may occur as a consequence of the experimental treatment. In this study we analyzed by TEM how retinal and lamina ganglionaris cells of the crab Ucides cordatus responded to a 30 min exposure to ultraviolet C (UV-C) and ultraviolet B (UV-B) radiation with doses of 7200 and 590 J/cm², respectively. The results show that damaged cells occurred in both retina and lamina ganglionaris, but that the retinal cells were affected to a greater extent. Morphological alterations of the pigment granules and an increase in the quantity of lipid droplets of the retinal cells were also observed. Additional changes include an increase in heterochromatin, nuclear karyolyses and karyorrhexes, distention of rough endoplasmic reticulum and mitochondrial disruptions. The observed morphological changes are indicative of apoptotic processes and show that an exposure to light of wavelengths of 254 and 312 nm may be injurious to the visual system of invertebrates.     

Effects of periodic rest on physiological measures of auditory sensitivity following exposure to noise

Whole nerve action potential (AP) and single auditory-nerve fiber thresholds were measured in chinchillas exposed to noise. The exposure stimulus was a 500-Hz octave band of noise presented at 95 dB SPL for 15 min/h, for 4 or 40 days. The AP thresholds were elevated by about 40 dB on day 4, between 0.5 kHz and approximately 8 kHz. On day 40, AP thresholds at the same frequencies were lower by 10-25 dB, even though the noise exposure had continued. Single fiber threshold tuning curves exhibited pathologies similar to those previously observed following noise exposure. Tuning curves measured on day 40 were more normal in appearance. These results confirm that similar recovery of threshold observed in psychophysical experiments [Clark et al., J. Acoust. Soc. Am. 82, 1253-1264 (1987)] can be understood in terms of the sensitivity of the peripheral auditory system.     

Ultrastructural alterations in the ventricular myocardium of the adult Italian newt (Lissotriton italicus) following exposure to nonylphenol ethoxylate

Nonylphenols ethoxylates (NPEs) are surface active agents (surfactants) commonly used in cleaning products, in industrial processes, agricultural formulations and paints. They are found in sewage, municipal wastewaters and industrial effluents, and as contaminants in water bodies. Accumulating data suggest that exposure to NPEs can adversely affects functional properties of the neurologic, reproductive, immune, and endocrine systems. In order to examine whether NPEs exert similar damaging effects on the cardiovascular system, we used an amphibian model to examine the ultrastructural alterations of the ventricular myocardium following exposure to NPEs. Adult Lissotriton italicus in the aquatic phase were exposed to NPE10 (100 μg/L, ppb) for 96 h. Heart specimens were collected from a total of 10 individuals and processed for scanning and transmission electron microscopy. Our ultrastructural examinations demonstrated that amphibian ventricle is susceptible to the effects of NPEs. The most pronounced alterations were observed in the membrane compartments of both myocardial and endothelial cells as demonstrated by the presence of swollen mitochondria with disrupted cristae and dilated rough endoplasmic reticulum. We suggest that destabilization of the lipid milieu within membranes might represent one of the potential mechanisms by which NPEs exert their toxic effects on amphibian heart.     

A structural study of the retinal photoreceptor, plexiform and ganglion cell layers following exposure to UV-B and UV-C radiation in the albino rat

Over the last two decades, ultraviolet radiation levels (UV), reaching the Earth's surface, have been increasing at a rate of 1.5% per each 1% loss of the ozone layer. Moreover, artificial UV-sources have also proliferated and contributed to the rising UV-stress that many organisms have to face. To assess how the vertebrate retina responds to an exposure of short wavelength UV, we focused our attention on the rat retina, observing photoreceptor (containing outer and inner segments of rods and cones), inner plexiform, and ganglion cell layers by light and transmission electron microscopy using conventional and cytochemical techniques. We analyzed how cells of the layers in question responded to a 30 min exposure to UV-C and UV-B radiation with doses of 7200 and 590 J/cm², respectively. The results show that there are significant changes in the nuclei and cytoplasmic organelles of the exposed retinae when compared with those of the unexposed controls. The changes include an increase in heterochromatin, distension of rough endoplasmic reticulum, mitochondrial disruptions, and increases in the number of myelin bodies. The recorded morphological changes, especially those of the ganglion cells, are suggestive of apoptotic processes and show that the exposure of vertebrate retina to wavelengths ranging from 254 to 312 nm can produce alterations that are likely to impact negatively on the retina's proper functioning.     

The inner ears of Northern Canadian freshwater fishes following exposure to seismic air gun sounds

An earlier study examined the effects of exposure to seismic air guns on the hearing of three species of fish from the Mackenzie River Delta in Northern Canada [Popper et al. (2005). "Effects of exposure to seismic airgun use on hearing of three fish species," J. Acoust. Soc. Am. 117, 3958-3971]. The sound pressure levels to which the fishes were exposed were a mean received level of 205-209 dB re 1 microPa (peak) per shot and an approximate received mean SEL of 176-180 dB re 1 microPa(2) s per shot. In this report, the same animals were examined to determine whether there were effects on the sensory cells of the inner ear as a result of the seismic exposure. No damage was found to the ears of the fishes exposed to seismic sounds despite the fact that two of the species, adult northern pike and lake chub, had shown a temporary threshold shift in hearing studies.