Histopathology of Rhipicephalus sanguineus (Acari: Ixodidae) ticks fed on resistant hosts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peripheral giant cell granuloma. An immunohistochemical and ultrastructural study.

OBJECTIVE: To study the nature of multinucleated and mononuclear cells from peripheral giant cell granuloma (PGCG). MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded sections of 40 cases of PGCG were immunohistochemically stained for vimentin, alpha I-antichymotrypsin, CD68, S-100 protein, lysozyme, leucocyte common antigen (LCA), factor VIII-related antigen and muscle cell actin. Six cases of PGCG were also studied by transmission electron microscopy. RESULTS: Vimentin, alpha I-antichymotrypsin and CD68 were expressed in both the mononuclear and multinucleated giant cells. Dendritic mononuclear cells, positive for S-100 protein, were noted in 67.5% of the lesions, whereas lysozyme and leucocyte common antigen were detected in occasional mononuclear cells. Ultrastructural examination showed mononuclear cells with signs of phagocytosis and sometimes interdigitations with similar cells. Others presented non-specific characteristics and the third type exhibited cytoplasmic processes and occasional Birbeck granules. Some multinucleated giant cells showed oval nuclei, abundant mitochondria and granular endoplasmic reticulum whereas others presented with irregular nuclei and a great number of cytoplasmic vacuoles. CONCLUSIONS: Immunohistochemical and ultrastructural results suggest that PGCGs of the jaws are composed mainly of cells of the mononuclear phagocyte system and that Langerhans cells are present in two thirds of the lesions.

Regression of the lateral oviducts during the larval-adult transformation of the reproductive system of Melipona quadrifasciata and Frieseomelitta varia

Toward the end of the larval phase (pre-pupa), the reproductive systems of Melipona quadrifasciata and Frieseomelitta varia workers are anatomically similar. Scanning electron microscopy showed that during this developmental phase the right and left ovaries are fused and form a heart-shaped structure located above the midgut. Each ovary is connected to the genital chamber by a long and slender lateral oviduct. During pupal development, the lateral oviducts of workers from both species become extremely reduced due to a drastic process of cell death, as shown by transmission electron microscopy. During the lateral oviduct shortening, their simple columnar epithelial cells show some signs of apoptosis in addition to necrosis. Cell death was characterized by cytoplasmic vesiculation, peculiar accumulation of glycogen, and dilation of cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum. The nuclei, at first irregularly contoured, became swollen, with chromatin flocculation and various areas of condensed chromatin next to the nuclear envelope. At the end of the pupal phase, deep recesses marked the nuclei. At emergence, worker and queen reproductive systems showed marked differences, although reduction in the lateral oviducts was an event occurring in both castes. However, in queens the ovarioles increased in length and the spermatheca was larger than that of workers. At the external anatomical level, the reproductive system of workers and queens could be distinguished in the white- and pink-eyed pupal phase. The metamorphic function of the death of lateral oviduct cells, with consequent oviduct shortening, is discussed in terms of the anatomical reorganization of the reproductive system and of the ventrolateral positioning of adult worker bee ovaries. (C) 2000 Wiley-Liss, Inc.

Feeding activity of Callinectes ornatus Ordway, 1863 and Callinectes danae Smith, 1869 (Crustacea, Brachyura, Portunidae) in Ubatuba, SP, Brazil

The feeding activity along the day cycle and the time consumed for extracellular digestion were evaluated in the portunids C. ornatus and C. danae. Swimming crabs were obtained from trawling in Ubatuba bay, São Paulo, Brazil, during both the rainy and dry seasons. In each season, daily scheduled samples were taken at dawn (±6 h), noon (±12 h), dusk (±18 h) and midnight (±24 h). All individuals were dissected and the degree of stomach replenishment was recorded. In order to estimate the time elapsed for extracellular digestion, crabs were fed, and groups were dissected at 30 min intervals to check the conditions of their stomachs. In general, both species show a higher feeding activity during periods of lower light intensity, as evidenced by an increased percentage of full stomachs in dusk and midnight samples. The obtained results support higher feeding activity at night in these species and indicate short time for extracellular digestion, not exceeding 8 h. Nevertheless, full stomachs were recorded in all sampling schedules. In this case, it should be considered that elimination of certain food items such as fish bones, mollusk shells and carapace fragments of crustaceans could take more time than other items. Additionally, some crab species could require a cycle of cell replacement in the midgut gland epithelium until they can take their next meal.

Cell nucleus activity during post-embryonic development of Apis melliferaL. (Hymenoptera: Apidae). Intranuclear acid phosphatase

We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues.

Two Digestive Trypsins Occur in Three Species of Neotropical Anophelines

Trypsin activity increases in the midgut of Anopheles aquasalis, Anopheles albitarsis, and Anopheles darlingi after a bloodmeal. The activity returns to basal levels at the time the blood is completely digested. Affinity chromatography, reversed-phase high performance liquid chromatography (HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to sequentially purify the mosquito trypsins found in the midguts at 24 h after feeding. Amino-terminal sequencing of the purified trypsins showed the occurrence of two distinct trypsins in the midgut of each of the mosquitoes studied. The sequences obtained are similar to those of the trypsins of other hematophagous insects.

The ultrastructure of the intestinal epithelium in Oreochromis niloticus on a diet with different concentrations of zinc

The effects of diets with variable zinc levels on the midgut epithelial cells were studied in Oreochromis niloticus L. One hundred and twenty fry of tilapia were apportioned into 4 experimental groups (I, II, III and IV groups), with 30 fish in each treatment, 5 replicate aquaria per treatment containing 6 fish each. The animals of the 4 groups were fed with isonitrogenous (30% crude protein) and isoenergetic (3000 Kcal/Kg of digestible energy) diets with increasing quantities of zinc (44.59; 149.17; 309.93; 599.67 mg Zn/kg of diet), twice a day, for 93 days. Three fish from each group were sacrificed at 36, 66 and 93 days and samples of midgut were removed for ultrastructural analysis. After 93 days of treatment, 3 animals of each experimental group were used for the analysis of zinc concentration by atomic absorption spectrophotometry. The comparative relative index (CRI) revealed that the animals in groups II, III and IV contained, respectively, 1.99%, 34.67% and 22.78% more zinc than the mean concentration in animals from group I. The ultrastructural analysis showed enterocytes with swelling of smooth surfaced endoplasmic reticulum and dilated mitochondria with variable matrix rarefaction and cristae number reduction in the fish exposed to 599.67 mg Zn/Kg of diet at 66 and 93 days of treatment.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Intestino médio de larvas de Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae): estudo das células epiteliais ao microscópio de luz e eletrônico

The morphology of the midgut epithelium cells of Anticarsia gemmatalis (Hübner) larvae is described by light and transmission electron microscopy. The midgut of A. gemmatalis is the largest portion of the digestive tract, with three distinct regions: proximal, media and distal. Its wall is formed by pseudostratified columnar epithelial tissue having four cell types: columnar, goblet, regenerative, and endocrine cells. The columnar cells are numerous and long, with the apical portion showing many lengthy microvilli and the basal portion invaginations forming a basal labyrinth. The goblet cells have a large goblet-shaped central cavity delimited by cytoplasmic projections filled with mitochondria. The regenerative cells present electron-dense cytoplasm and few organelles. The endocrine cells are characterized by electron-dense secretory granules, usually concentrated in the cytoplasm basal region.

Susceptibility/resistance of Anticarsia gemmatalis larvae to its nucleopolyhedrovirus (AgMNPV): Structural study of the peritrophic membrane

This investigation compares the peritrophic membrane (PM) morphology along the midgut of susceptible (SL) and resistant (RL) Anticarsia gemmatalis larvae to the AgMNPV. The PM increased the thickness from the anterior to the posterior midgut region in both insects strain; however, the intensity of FITC-WGA reaction of the PM in the RL were greater than in SL. The PM in RL was ultrastructurally constituted by several layers of fibrous/vesicular materials in comparison with the few ones in SL. Our results showed that the structure of PM in the RL could be one of the resistance barriers to AgMNPV. © 2007.

Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: An evolutionary history

Background: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. Methodology/Principal Findings: Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. Conclusions/Significance: Herein the Hz formation is shown to be associated to an a-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance. © 2009 Mury et al.

Toxicological and histopathological effects of boric acid on atta sexdens rubropilosa (Hymenoptera: Formicidae) workers

The current study compared the toxicity of different concentrations of boric acid in adult workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae), with toxicological bioassays, and examining the dose-dependent and time-dependent histopathological changes, of the midgut, Malpighian tubules, and postpharyngeal glands. Our results revealed the importance of conducting toxicological bioassays combined with morphological analyses of the organs of ants chronically exposed to insecticides used in commercial ant baits. In vitro bioassays showed that boric acid significantly decreases the survivorship of workers regardless of concentration, whereas the morphological data suggested progressive dose-dependent and time-dependent changes in the organs examined, which were evident in the midgut. The midgut is the first organ to be affected, followed by the postpharyngeal gland and Malpighian tubules. This sequence is in agreement with the absorption pathway of this chemical compound in the midgut, its transference to the hemolymph, possibly reaching the postpharyngeal glands, and excretion by the Malpighian tubules. These progressive changes might be due to the cumulative and delayed effect of boric acid. Our findings provide important information for the understanding of the action of boric acid in ant baits in direct and indirect target organs. © 2010 Entomological Society of America.

Biosolid soil application: Toxicity tests under laboratory conditions

A large volume of generated sewage sludge makes its disposal a problem. The usage of sludge in agriculture is highlighted by a number of advantages. However, heavy metals and other toxic compounds may exercise harmful effects to soil organisms. This study evaluated the possible toxic effects of a biosolid sample, under laboratory conditions, for 30 days, using diplopods Rhinocricus padbergi and plants Allium cepa (onion) as test organisms. The data obtained demonstrated that the biosolid raw sample had genotoxic potential for Allium cepa root tip cells. In the diplopods exposed to biosolid sample, epithelium disorganization in the midgut and a reduction of the volume of the hepatic cells were observed after 7 days of exposure. After 30 days, the animals still showed a reduction of the volume of the hepatic cells, but in minor intensity. Allium cepa analysis showed genotoxicity, but this effect was reduced after 30 days of bioprocessing by diplopods. This study was important to know the effects as well as to determine how this waste could be applied concerning the soil living organisms and plants. © 2012 Cintya Ap. Christofoletti et al.

Side-effects of thiamethoxam on the brain andmidgut of the africanized honeybee Apis mellifera (Hymenopptera: Apidae)

The development of agricultural activities coincides with the increased use of pesticides to control pests, which can also be harmful to nontarget insects such as bees. Thus, the goal of this work was assess the toxic effects of thiamethoxam on newly emerged worker bees of Apis mellifera (africanized honeybee-AHB). Initially, we determined that the lethal concentration 50 (LC50) of thiamethoxam was 4.28 ng a.i./μL of diet. To determine the lethal time 50 (LT50), a survival assay was conducted using diets containing sublethal doses of thiamethoxam equal to 1/10 and 1/100 of the LC50. The group of bees exposed to 1/10 of the LC50 had a 41.2% reduction of lifespan. When AHB samples were analyzed by morphological technique we found the presence of condensed cells in the mushroom bodies and optical lobes in exposed honeybees. Through Xylidine Ponceau technique, we found cells which stained more intensely in groups exposed to thiamethoxam. The digestive and regenerative cells of the midgut from exposed bees also showed morphological and histochemical alterations, like cytoplasm vacuolization, increased apocrine secretion and increased cell elimination. Thus, intoxication with a sublethal doses of thiamethoxam can cause impairment in the brain and midgut of AHB and contribute to the honeybee lifespan reduction. © 2013 Wiley Periodicals, Inc.