Bifidogenic effect of grain larvae extract on serum lipid, glucose and intestinal microflora in rats.

The main objective of this study was to investigate whether orally administered Korean grain larvae ethanol extract (GLE) had a bifidogenic effect in normal rats. Male Sprague-Dawley rats were divided into a negative control group (CO) and GLE orally administered (5.0, 7.0 and 9.0 mg/100 g body weight) groups. Thymus and spleen weights dosedependently increased by 128.58 percent and 128.58 percent, respectively, but abdominal fat decreased by 19.18 percent after GLE administration compared with that in the CO group (p less than 0.05). Serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, and glucose decreased by 30.26 percent, 7.33 percent, 27.20 percent, and 6.96 percent, respectively, whereas highdensity lipoprotein cholesterol increased by 129.93 percent in the GLE groups compared with those in the CO group (p less than 0.05). IgG, IgM, IgA in the GLE groups increased 203.68 percent, 181.41 percent, and 238.25 percent, respectively, compared to that in the CO group (p less than 0.05). Bifidobacteria and Lactobacillus increased by 115.74 percent and 144.28 percent, whereas Bacteroides, Clostridium, Escherichia, and Streptococcus decreased by 17.37 percent, 17.46 percent, 21.25 percent, and 19.16 percent, respectively, in the GLE groups compared with those in the CO group (p less than 0.05). Total organic acids, acetic acid, and propionic acid increased by 151.40 percent, 188.09 percent, and 150.17 percent, whereas butyric acid and valeric acid decreased by 40.65 percent and 49.24 percent, respectively, in the GLE groups as compared with those in the CO group (p less than 0.05). These results suggest that Korean GLE improves the bifidogenic effect by increasing cecal organic acids and modulating gut microflora via a selective increase in Bifidobacterium in normal rats.

Transfer of Escherichia coli O157:H7 to spinach by house flies, Musca domestica (Diptera: Muscidae).

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.

Effects of heavy metals on insect immunocompetent cells.

The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals.

Effects of zinc on programmed cell death of Musca domestica and Drosophila melanogaster blood cells.

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn(2+), an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn(2+), and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn(2+) increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn(2+) also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.

Circadian release of pigment-dispersing factor in the visual system of the housefly, Musca domestica.

In the present study we examined profiles of nerve fiber varicosities containing dense core vesicles (DCVs) in the distal medulla of the housefly's optic lobe using electron microscopic methods. These profiles are infrequent among other neuronal profiles and do not contain presynaptic specializations for the release of DCVs. Presynaptic elements surrounded by electron-translucent vesicles were only occasionally detected, whereas synaptic input sites to the profiles containing DCVs were never observed. Among the varicosities in the distal medulla, those immunoreactive to pigment-dispersing hormone (PDH) are most numerous. The DCVs of PDH-immunoreactive (PDH-ir) varicosities differ by size from DCVs of other profiles. Moreover, in the day/night cycle PDH-ir varicosities show differences in structure revealing the rhythmic accumulation and release of PDF. There were fewer PDH-ir DCV per varicosity profile in flies fixed 1 hour after lights-on than in flies fixed 1 hour after lights-off. Moreover, at the beginning of the day all DCVs harbored an electron-dense matrix, while at the beginning of the night numerous electron-lucent DCVs were observed. By applying a bath stimulation with a high potassium concentration we also showed that depolarizing events are involved in peptide release in the medulla. After potassium treatment immunolabeling with anti-PDH serum was weaker and PDH-ir varicosities were smaller and more distant from each other than in control animals.

Effects of locomotor stimulation and protein synthesis inhibition on circadian rhythms in size changes of L1 and L2 interneurons in the fly's visual system.

Axons of monopolar cell interneurons L1 and L2 in the first optic lobe (lamina) of the fly Musca domestica undergo cyclical changes in diameter. These axons swell during the day and shrink during the night. In addition, the axons' size depends on light conditions since they are largest in continuous light (LL), somewhat smaller under day/night (LD) conditions, and smallest under constant darkness (DD). In this study we found that sizes of both cells can further increase in free flying flies under LD conditions, while the visual stimulation alone does not have significant effect on the cross-sectional area of L1 and L2 axons. The stimulation of free flying had no effect on L1 and L2 sizes if it was performed at the beginning of subjective day in LL or DD. Our results indicate that a maximal increase in size of L1 and L2 is observed when stimulation of free flying is synchronized with a fly' daily peak of activity. We also found that protein synthesis is needed to increase size of monopolar cell axons during the day when they normally swell.

The segmentation and visualization of a neuron in the housefly's visual system.

Those studying biological systems are often interested in the morphology of the various microscopic organelles. The three dimensional reconstruction and visualization of objects provide a powerful tool to understand the nature of each object, and its relationship to other objects. Segmentation is the key to 3D analysis and study of objects that have been recorded with a series of sectioned images, such as from a confocal laser scanning microscope (CLSM). Segmentation is the process of completely separating or isolating the individual objects in an image. A seed-based semi-automatic segmentation tool has been developed to aid in the process of 3D visualization of objects recorded with serial sectioned images, including a boundary creation method that maintains the separate identity of contacting objects. This segmentation tool also allows the user to retain background information as a separate object, providing important reference and landmark information for the object of interest. This paper summarizes the main parts of the segmentation algorithm and presents 3D reconstructions of visual neurons of the housefly, Musca domestica. These reconstructions are compared to typical 3D images produced from other widely used software packages, including standard CLSM imaging software and the popular ImageJ supported by National Institute of Health (NIH). Efforts are underway to develop a user-friendly graphical user interface (GUI) for the segmentation algorithm to entice broader used in research settings.

cDNA cloning of the housefly pigment-dispersing factor (PDF) precursor protein and its peptide comparison among the insect circadian neuropeptides.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean beta-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%-94%) and similarity (89%-100%) to insect PDFs and also to the crustacean beta-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs.

Embryonic and adult neurones of the housefly (Musca domestica) in culture.

This paper provides a detailed description of the dissociation and maintenance in culture of cells derived from embryonic tissue and from the central nervous system of adult housefly, Musca domestica. Dissociation of embryonic tissue produced several cell types of which muscle and neurone-like cells predominated. These cells were morphologically distinct once development had started in vitro with muscle cells often producing rhythmic contractions. Neurones had small cell bodies (less than 10 mum) and showed extensive outgrowth of neurites. Neuronal cell bodies readily sealed onto patch pipettes and with pipette solutions containing nicotinic agonists, single channel currents could be recorded. With cell-attached patches, these currents were inward at the cell resting potential and reversed with depolarization. Channels with two different conductances could be seen in the majority of patches.

Cobalt-coupled neurons of a giant fibre system in Diptera.

Certain intact nerve cells in flies can be filled with cobalt from presynaptic or postsynaptic neurons. This cobalt coupling is best demonstrated in giant fibre systems where the phenomenon was originally termed 'transsynaptic staining'. Fine structural analysis of silver-intensified, cobalt-coupled neurons indicates that the passage of cobalt ions occurs at gap junctions that are accompanied by conventional chemical synapses. Cobalt-coupled systems in dipterous insects are uniquely identifiable and can always be detected between the same kinds of neurons. The visualization of cobalt-coupled neurons allows the identification of functional pathways linking the brain to motor neuropils.

Cytology of cobalt-filled neurons in flies: cobalt deposits at presynaptic and postsynaptic sites, mitochondria and the cytoskeleton.

Combined light and electron microscopy of identified neurons requires an intracellular marker that is both photon opaque and has electron scattering properties. We describe results using cobalt chloride block intensified with silver as an intracellular label. The novelty of the method is its integration in tissue fixation, prior to dehydration, resulting in fine grain precipitates that resolve certain intracellular structures. Filled neurons are clearly distinguishable from unfilled profiles by cobalt-silver precipitates. Energy dispersive X-ray analysis confirms that silver is specifically deposited onto cobalt sulphide cores which are characteristically associated with microtubules, mitochondria, presynaptic and postsynaptic specializations and gap junction-like membrane appositions.

A pair of descending neurons with dendrites in the optic lobes projecting directly to thoracic ganglia of dipterous insects.

The lobula descending neuron (LDN) of dipterous insects is a unique nerve cell (one on each side of the brain) that projects directly from the lobula complex of the optic lobes to neuropil in thoracic ganglia. In the supraoesophageal ganglia the LDN has two prominent groups of branches of which at least one is dendritic in nature. Postsynaptic branches are distributed in the lobula and some branches, the synaptic relations of which are not yet known, extend to the lobular plate. A second group of branches is found among dendrites of the descending neurons proper, in the lateral midbrain. The arborization of LDN in the lobular (and lobula plate) map onto a retinotopic neuropil region subserving a posterior strip of the visual field of the compound eye. The arborizations in the lobula complex are extremely variable in size. The numbers of dendritic spines they possess vary greatly between left and right optic lobes of one animal, and between individual animals.

The housefly interfacetal hair: ultrastructure of a presumed mechanoreceptor.

The external and internal fine structure of the housefly interfacetal hair and its sensory dendrite was studied with the scanning and transmission (high and low voltage) electron microscopes. The hair shaft contains no dendrites, and is usually situated within a socket on the lens surface. Immediately beneath and directly connected to the base of each hair is a bipolar neuron whose dendrite tip is enveloped in a shealth cell which, in turn, is surrounded by a second sheath cell. Septate junctions are seen between all these cells and contiguous portions of a large pigment cell. At the hair base, the dendrite of the neuron terminates in a tubular body only 1.5 mum in diameter which is filled with about 400 microtubules in highly ordered (in parallel pentagonal and hexagonal) arrays and whose sides are fused to neurofilaments in parallel. Another filament (ca. 70 A diameter) is in the center of each microtubule-neurofilament polygon. Structures proximal to the tubular body are typical for a scolopoid sensillum, i.e., connecting cilium (9 times 2 + 0 microtubules) with rootlet and basal bodies, unmodified dendrite, perikaryon and axon. The axon has not been traced to its synapse. The high degree of internal organization and shortness of the tubular body, as well as its eccentric insertion into the hair shaft lead to the hypothesis that this hair may be a highly sensitive mechanoreceptor. On the basis of their single innervation, these hairs could monitor flight speed from the degree of hair deflection caused by wind in general or particular laminar air currents flowing past the eyes during flight.