Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae)

Tribolium castaneum (Coleoptera: Tenebrionidae) is a common pest of stored grains and byproducts and is normally infected by Gregarina cuneata (Apicomplexa: Eugregarinidae). The life cycle of this parasite includes the sporozoite, trophozoite, gamont, gametocyte, and oocyst stages, which occur between the epithelium and lumen of the host's midgut. This study aims to describe the morphofunctional alterations in the midgut and determine the esterase patterns in T. castaneum when parasitized by gregarines. To achieve this purpose, midguts of adult insects were isolated, processed, and analysed using light and electron microscopy. We determined total protein content, amylase activity, and the expression and related activities of the esterases by using polyacrylamide gel electrophoresis (PAGE). The midgut of T. castaneum is formed by digestive, regenerative, and endocrine cells. The effects of parasitism on the digestive cells are severe, because the gregarines remain attached to these cells to absorb all the nutrients they need throughout their development. In these cells, the most common alterations observed include expansion and fragmentation of the rough endoplasmic reticulum, development of the smooth endoplasmic reticulum, changes in mitochondrial cristae, cytoplasmic vacuolization, formation of myelin structures, spherites, large intercellular spaces, autophagic vesicles, expansion of the basal labyrinth, and cytoplasmic protrusions. Deposits of glycogen granules were also observed. Amylase activity was reduced in parasitized insects. Regenerative cells were found in disorganized crypts and did not differentiate into new cells, thus, compromising the restoration of the damaged epithelium. Though few morphological alterations were observed in the endocrine cells, results suggest that the synthesis and/or release of hormones might be impaired. Nine esterases (EST-1 to 9) were identified in the midgut of T. castaneum and were expressed in varying levels in response to parasitism. Two additional isoforms of esterases were exclusively identified in the parasitized insects. The results of this study suggest that gregarines alter the morphology and physiology of the midgut. The changes may result in nutritional depletion and the impairment of other physiological processes, such as reproduction and development of the host. Thus, further studies are needed to uncover the possibility of utilizing gregarines as biological controllers of the insect pest population.     

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.     

The midgut of Cephalotes ants (Formicidae: Myrmicinae): Ultrastructure of the epithelium and symbiotic bacteria

The ultrastructural analysis of the midgut of Cephalotes atratus, C. clypeatus, and C. pusillus reveled that the midgut epithelium lays on a basal lamina and is composed basically of three cell types: digestive cells, regenerative cells, and goblet cells. In these ants, the rough endoplasmic reticulum, in addition to producing digestive enzymes, is involved in the formation of concretions and ion storage in specialized vacuoles present in the midgut. These concretions are spherocrystals and may contribute to stabilize the pH and to maintain symbiotic bacteria found between microvilli. The ultrastructure analysis of these bacteria revealed the presence of a double envelope typical of gram-negative bacteria. For the three species examined, the ultrastructure similarities are conspicuous, suggesting that this may be the pattern for the genus Cephalotes. Details of the relationship between bacteria and microvilli were examined.     

A Ru(II)-Based Nanoassembly Exhibiting Theranostic PACT Activity in NIR Region

Photoactivated chemotherapy (PACT) has appealing merits over traditional chemotherapy as well as photodynamic therapy (PDT) by virtue of its spatial and temporal control on drug activity and oxygen-independent mechanisms of action. However, the short photoactivation wavelengths, e.g., visible light–activated Ru(II)-based PACT agents, limit the clinical application severely. In this work, a facile construction of supramolecular nanoparticles from a poly(ethylene glycol) (PEG)-modified [Ru(dip)₂(py-SO₃)]⁺ (abbreviated as Ru-PEG, dip = 4,7-diphenyl-1,10-phenanthroline, py-SO₃ = pyridine-2-sulfonate) and 1,3-phenylenebis(pyren-1-ylmethanone) (BP) is shown. While Ru-PEG may undergo photoinduced ligand dissociation and release anticancer species of [Ru(dip)₂(H₂O)₂]²⁺, BP has extremely large two-photon absorption cross sections (δ₂) in the NIR region and intense fluorescence over the wavelengths where Ru-PEG has strong absorption. Thus, two-photon excitation of BP followed by an efficient Förster resonance energy transfer (FRET) from BP to Ru-PEG may lead to a potent inactivation against cisplatin-resistant cancer cells and 3D multicellular tumor spheroids (MCTSs). The residue fluorescence of BP also allows the cellular uptake of the particles to be visualized. This work provides a universal and convenient strategy to realize theranostic PACT in the ideal phototherapeutic window of 650–900 nm.     

Pathogen-targeted hydroxyl radical generation during melanization in insect hemolymph: EPR study of a probable cytotoxicity mechanism

The main component of the insect immune system is melanotic encapsulation of pathogenic organisms. Molecular mechanisms of destruction of an encapsulated pathogen are poorly understood. Reactive oxygen species (ROS) are considered as probable cytotoxic agents responsible for destruction of pathogenic organisms in insect hemolymph. In the present work the formation of H₂O₂ during melanization in Galleria mellonella hemolymph in the presence of catalase inhibitor NaN₃ was detected. Enhanced rates of H₂O₂ generation were observed in the hemolymph of insects activated by injection of bacterial cells. Using spin trapping technique in combination with electron paramagnetic resonance spectroscopy we demonstrated that production of H₂O₂ in the hemolymph causes the formation of highly toxic reactive oxygen species, hydroxyl radical. However, neither H₂O₂ nor hydroxyl radical were detected in the absence of NaN₃ in agreement with the high catalase activity in the hemolymph. These observations allow us to propose a unique mechanism of pathogen-targeted cytotoxicity based on localized hydroxyl radical generation within a melanotic capsule.     

Study of melanin localization in the mature male Calopteryx haemorrhoidalis damselfly wings

Damselflies Calopteryx haemorrhoidalis exhibiting black wings are found in the western Mediterranean, Algeria, France, Italy, Spain and Monaco. Wing pigmentation is caused by the presence of melanin, which is involved in physiological processes including defence reactions, wound healing and sclerotization of the insect. Despite the important physiological roles of melanin, the presence and colour variation among males and females of the C. haemorrhoidalis species and the localization of the pigment within the wing membrane remain poorly understood. In this study, infrared (IR) microspectroscopy, coupled with the highly collimated synchrotron IR beam, was employed in order to identify the distribution of the pigments in the wings at a high spatial resolution. It was found that the melanin is localized in the procuticle of the C. haemorrhoidalis damselfly wings, distributed homogeneously within this layer, and not associated with the lipids of the epicuticle.     

Structure of p-xylene from its P.M.R. spectrum in the nematic phase

It has been observed experimentally that circular dichroism bands can appear at absorption frequencies of achiral molecules when chiral species (which do not associate with the achiral molecules) are added to the solution. This paper treats those contributions to the induced activity in the achiral species which are the results of van der Waals coupling to the chiral species. Use of perturbation theory leads to the expression of the induced rotatory strength as a series in 1/r, r being used in a general sense as the effective achiral-chiral molecule separation. Averaging over all orientations of each molecule yields non-vanishing terms falling off as 1/r ⁶. The results suggest that the effect may be important even in completely dissociated systems.     

Stability of benzoporphyrin photosensitizers in water/ethanol mixtures: pKa determination and self‐aggregation processes

Benzoporphyrin monoacid derivatives, here named B3A and B3B, are promising new drugs for photodynamic therapy. Although both isomers show interesting characteristics as photosensitizing compounds, they have some distinct physicochemical properties such as the tendency to self‐aggregate in water‐rich media. Because pH drives the presence of each species, the pK_a of these compounds assumes strategic importance. However, traditional micro‐titration methods and UV–Vis absorption techniques fail to give reliable pK_a values due to the characteristics of this highly complex system, such as the precipitation of hydrophobic species, close pK_a values, and high absorption band superposition. In the present work, chemometric tools are employed to evaluate pK_a, and the kinetic tendency of monomers to undergo self‐aggregation is investigated. In solvent mixtures at low water percentage in ethanol, both B3A and B3B are stabilized in a monomeric state. However, in mixtures with a high water content, self‐aggregation takes place, mainly under a mild pH acid condition (3 < pH < 6), in which the prevalent protolytic species of both isomers is the neutral charged form, compounds with carboxylic and porphyrin free‐base groups. It is demonstrated that both isomers can undergo aggregation following a self‐catalytic mechanism, which is 2000 times slower to B3A than B3B. For B3A, the aggregation is manifested by a decrease in the monomer band with the aggregation band probably superposed to that of the monomer. For B3B, together with the decrease in the monomer band, a new band related to self‐aggregates is observed. Copyright © 2010 John Wiley & Sons, Ltd.     

In Situ Monitoring the Photolysis of Fluoranthene Adsorbed on Mangrove Leaves Using Fiber-Optic Fluorimetry

A newly established fiber-optic fluorimetry was used to determine the fluoranthene (Fla) adsorbed on the leaf surfaces of three mangrove species, such as Excoecarla agallocha (Ea), Kandelia candel (Kc) and Aegiceras corniculatum (Ac). With the method, the photolysis of Fla adsorbed on Ea, Kc and Ac leaf surfaces was studied. Under the laboratory conditions, using a high pressure mercury lamp as light source, photolysis was found to be the main transformation pathway for the Fla adsorbed on the three mangrove leaves, whereas disappearance of the adsorbed Fla as a result of volatilization and absorption could be negligible. The photolysis of Fla adsorbed on the three mangrove leaf surfaces followed first-order kinetics with photolysis rates of Fla in the order of Ac > Ea > Kc. The photolysis half lives of Fla adsorbed on the leaves were from 14.32 min to 69.31 min. The different initial concentrations of Fla adsorbed on the selected mangrove leaves did not affect the photolysis rates of Fla. The absorption characteristics of leaf-wax played an important role to influence the different photolysis rates of Fla adsorbed on these three mangrove species.     

Immunolocalization of the short neuropeptide F receptor in queen brains and ovaries of the red imported fire ant (Solenopsis invicta Buren)

Background

Insect neuropeptides are involved in diverse physiological functions and can be released as neurotransmitters or neuromodulators acting within the central nervous system, and as circulating neurohormones in insect hemolymph. The insect short neuropeptide F (sNPF) peptides, related to the vertebrate neuropeptide Y (NPY) peptides, have been implicated in the regulation of food intake and body size, and play a gonadotropic role in the ovaries of some insect species. Recently the sNPF peptides were localized in the brain of larval and adult Drosophila. However, the location of the sNPF receptor, a G protein-coupled receptor (GPCR), has not yet been investigated in brains of any adult insect. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in queens of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera), an invasive social insect.

Results

In the queen brains and subesophageal ganglion about 164 cells distributed in distinctive cell clusters (C1-C9 and C12) or as individual cells (C10, C11) were immuno-positive for the sNPF receptor. Most of these neurons are located in or near important sensory neuropils including the mushroom bodies, the antennal lobes, the central complex, and in different parts of the protocerebrum, as well as in the subesophageal ganglion. The localization of the sNPF receptor broadly links the receptor signaling pathway with circuits regulating learning and feeding behaviors. In ovaries from mated queens, the detection of sNPF receptor signal at the posterior end of oocytes in mid-oogenesis stage suggests that the sNPF signaling pathway may regulate processes at the oocyte pole.

Conclusions

The analysis of sNPF receptor immunolocalization shows that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). To our knowledge, this is the first report of the cellular localization of a sNPF receptor on the brain and ovaries of adult insects.     

Comparison of Two Tricarbocyanine-Based Dyes for Fluorescence Optical Imaging

Optical technologies are evolving in many biomedical areas including the biomedical imaging disciplines. Regarding the absorption properties of physiological molecules in living tissue, the optical window ranging from 700 to 900 nm allows to use fluorescent dyes for novel diagnostic solutions. Here we investigate the potential of two different carbocyanine-based dyes fluorescent in the near infrared as contrast agents for in vivo imaging of subcutaneously grown tumours in laboratory animals. The primary aim was to modify the physicochemical properties of the previously synthesized dye SIDAG to investigate the effect on the in vivo imaging properties.     

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.     

Hund’s Case (a)-case (b) transition in the singlet-triplet absorption spectrum of pyrazine in a supersonic jet

An analytic expression is derived for calculating the intensities of individual spin-rovibronic lines in the fully resolved gas phase electronic spectrum of a polyatomic molecule, in which one of the zero-order electronic states is a triplet state. The expression is employed to calculate the effect of fine structure splitting on the singlet-triplet absorption spectrum of pyrazine using the parameters available from experiment. A transition from Hund’s coupling Case (a) to Case (b) on going from low J to high J rotational levels is predicted to occur at a moderate resolution of a few hundred MHz. The effect is more pronounced in pyrazine-d ₄ and the pyrazine-argon van der Waals complex owing to their larger mass. Zh. éksp. Teor. Fiz. 114, 63–90 (July 1998) Published in English in the original Russian journal. Reproduced here with stylistic changes by the Translation Editor.     

Steric effect in alkylation reactions by N‐alkyl‐N‐nitrosoureas: a kinetic approach

The alkylation reactions of 4‐(p‐nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to DNA bases, by five N‐alkyl‐N‐nitrosoureas (methyl‐, ethyl‐, propyl‐, butyl‐, and allylnitrosourea) were investigated in 7:3 (v/v) water/dioxane medium in the 5.0–6.5 pH range. Decomposition of alkylnitrosoureas (ANU) gives rise to alkyldiazonium ions that yield NBP‐R adducts directly or through carbocations in certain instances. The NBP alkylation rate constants by these species were determined. The following sequence of alkylating potential was found: methyl‐ > ethyl‐ > allyl‐ > propyl‐ > butyl group. Application of Ingold–Taft correlation analysis to the kinetic results revealed that the NBP alkylation reactions occur mainly through steric control. The values of the molar absorption coefficients of the NBP‐R adducts also reveal the determinant influence of a steric effect in the formation of alkylation adducts. The kinetic results are consistent with the biological activity of ANU. Copyright © 2008 John Wiley & Sons, Ltd.     

Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri

Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the safe and compatible integrated use of biopesticides.     

High spin state of Mn in an ideal monolayer on Ag(001)

We studied the magnetic properties of ultra-thin Mn films deposited on Ag (001) held at 80 K with soft X-ray absorption and magnetic circular dichroism. The observed shape and branching ratio of the Mn 2p absorption edge as a function of Mn coverage demonstrate that, up to , the Mn adopts a stable high spin state similar to the Mn atom Hund's rule ⁶ S _5/2 ground state. Above this coverage a rapid transition from localized high spin to itinerant low spin behavior of the Mn 3d electrons is evidenced. Magnetic circular dichroism shows no sign of long range ferromagnetic order in these films at 80 K. The data, first confirm the large atomic-like local magnetic moment, and second are in line with the in-plane antiferromagnetic order, reported recently (Phys. Rev. B 57, 1141 (1998)), for Mn in the nearly ideal on-top Mn monolayer formed by 0.9 ML deposited at 80 K. Received: 4 May 1998     

Reduction of Se(VI) to Se(-II) by zerovalent iron nanoparticle suspensions

The reaction of selenate (Se(VI)) with zerovalent iron nanoparticles (nano Fe⁰) was studied using both conventional batch equilibrium and X-ray spectroscopic techniques. Nano Fe⁰ has a high uptake capacity for removal of dissolved Se(VI) reaching concentrations as high as 0.10 Se:Fe molar ratio in the solid product mixture. Kinetic studies of the Se(VI) uptake reaction in batch experiments showed an initial reaction rate (0–30 min) of 0.0364 min^?1 which was four times greater than conventional Fe⁰ powder. Analysis of the oxidation state of Se in the solid products by X-ray absorption near edge structure (XANES) spectroscopy showed evidence for the reduction of Se(VI) to insoluble selenide (Se(-II)) species. Structural analysis of the product by extended X-ray absorption fine structure (EXAFS) spectroscopy suggested that Se(-II) was associated with nano Fe⁰ oxidation products as a poorly ordered iron selenide (FeSe) compound. The fitted first shell Se–Fe interatomic distance of 2.402 (±0.004) Å matched closely with previous studies of the products of Se(IV)-treated Fe(II)-clays and zero-valent iron/iron carbide (Fe/Fe₃C). The poorly ordered FeSe product was associated with Fe⁰ corrosion product phases such as crystalline magnetite (Fe₃O₄) and Fe(III) oxyhydroxide. The results of this investigation suggest that nano Fe⁰ is a strong reducing agent capable of efficient reduction of soluble Se oxyanions to insoluble Se(-II).     

Species-Related Difference to Noise Reduction Between Trees in Urban Forest: The Abidar Forest Park (Case Study)

Sound pollution is one of the most important urban problems which endangers mental and physical health of the residents. This study was aimed to assess the influence of different tree species, including Fraxinus rotundifolia, Robinia pseudoacacia, Platanus orientalis, Platycladus orientalis, and Pinus eldarica, in reducing noise pollution in the Abidar Forest Park. A further objective was to identify the contaminated areas of Sanandaj city and to propose suitable noise absorbent tree species in consistent conditions. For each tree stands the noise measurements were performed during intervals at frequencies of 250, 500 and 1000 Hz, besides an open area with the same topography. With regards to the second purpose, a total of 50 stations with residential, commercial, residentialcommercial, and green space applications were selected across the city. Equivalent Continuous Sound Pressure Level (Leq) was determined in five replicates for 30 min. The measurements were performed under stable weather conditions and low wind velocity at 17:00 (traffic peak) in summer and fall. All of the Leq values were above the threshold noise level. The highest noise reduction was recorded in summer (i.e., green season); Platanus and Platycladus species demonstrated the highest and lowest noise absorption (31.43 dB and 22.28 dB, respectively). Furthermore, a meaningful difference was observed between Leq values of commercial, residential, commercial-residential, and green space urban applications, and the central parts of the city showed noticeable noise pollution. Taken together, due to being exposed to higher than the acceptable threshold noise level, the residents of Sanandaj will be endangered to health problems in the near future; thus consideration should be given to the noise pollution sources.