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Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism's reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.

Insect storage proteins accumulate at high levels during larval development of holometabolous insects. During metamorphosis they are degraded, supplying energy and amino acids for the completion of adult development. The genome of Culex quinquefasciatus contains eleven storage protein-coding genes. Their transcripts are more abundant in larvae than in pupae and in adults. In fact, only four of these genes are transcribed in adults, two of which in blood-fed adult females but not in adult males. Transcripts corresponding to all Cx. quinquefasciatus storage proteins were detected by RT-PCR, while mass spectrometric analysis of larval and pupal proteins identified all storage proteins with the exception of one encoded by Cq LSP1.8. Our results indicate that the identified Cx. quinquefasciatus storage protein-coding genes are candidates for identifying regulatory sequences for the development of molecular tools for vector control.

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features., Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo., Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis., Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.

Wolbachia are α-proteobacteria that were first reported in Culex pipiens mosquitoes early in the twentieth century. Since then, the effect of Wolbachia on their host's reproduction has drawn attention and has been increasingly investigated. Given the extreme complexity of this interaction, new study cases are welcomed to enhance its understanding. The present work addressed the influence of Wolbachia on Cx. quinquefasciatus, the cosmopolitan member of the Cx. pipiens complex. Samples of a Cx. quinquefasciatus colony (wPip(+)) originated from individuals naturally infected by Wolbachiapipientis B strain, were cured with tetracycline, yielding a Wolbachia-free colony (wPip(-)). Both the presence of bacteria and the efficiency of bacterial elimination were checked by PCR of the wsp gene. Total reproductive unidirectional incompatibility occurred when wPip(-) females were crossed with wPip(+) males, whereas the other three types of reciprocal crosses were viable. Reproductive aspects were also comparatively evaluated between colonies. Concerning oviposition time during the first gonotrophic cycle, wPip(+) females developed and laid eggs earlier than did wPip(-) females. Reproductive fitness was higher among wPip(-) than wPip(+) females regarding the following parameters: fertility: egg rafts/fed females; fecundity: eggs/raft, and viability: larvae/eggs. Conversely, longevity of wPip(-) females was lower. Summarising, although the infected mosquitoes have the advantage of a higher longevity, they have lower reproductive fitness. Our results are partly distinct from all other reports on Aedes and Culex mosquitoes previously published., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24 h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity.

Gomesin is a cationic anti-microbial peptide of 18 amino acid residues isolated from the hemocytes of unchallenged tarantula spider Acanthoscurria gomesiana. This paper reports the first study of the processing and cellular location of an anti-microbial peptide (AMP) in spiders. Gomesin cDNA sequence analysis indicated that it is processed from a precursor containing a signal peptide (23 amino acid residues) and a negative C-terminal region (43 amino acid residues). The gomesin gene was constitutively transcribed in hemocytes and the gene product localized in hemocyte granules. The constitutive production of gomesin by a spider is discussed in the context of an ancient mechanism of AMP regulation and storage.

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm.

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.

A monoclonal antibody, 1E7, raised against tissue culture-derived trypomastigotes of Trypanosoma cruzi reacted with proteins located at the perinuclear region of the parasite as detected by immunofluorescence and immunogold electron microscopy. The antibody also recognized antigens in all trypanosomatids tested, including T. cruzi epimastigotes, as well as in many mammalian cells. Five principal antigens of 140-270 kDa soluble in 1 M NaCl were recognized by the monoclonal antibody, suggesting that the epitope may belong to more than one polypeptide since the same bands appeared even when the samples were treated with high concentrations of denaturing agents. The antibody reacted in Western blots with muscle myosin. Bacterial clones expressing fast skeletal muscle myosin head or tail cDNAs upon IPTG induction were used to demonstrate that 1E7 monoclonal antibody recognizes an epitope present in the tail region of the myosin heavy chain. This result adds to the on-going discussion related to the possible existence of an auto-immune component in the immunopathogenesis of Chagas' disease due to cross-reactive epitopes shared by the parasite and cardiac myosin.

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.

Leishmania braziliensis strain M2903 was adapted for growth and serially maintained as amastigotes at 34 degrees C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, amastigote growth took place in the absence of supplementary haemin and was unaffected when the initial medium pH was adjusted between 5.4 and 6.3. In contrast to promastigotes, which were elongated and exhibited very long free flagella endowed with the paraflagellar rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in axenic amastigotes was confirmed in Western blots and confocal immunofluorescence microscopy, by lack of reactivity with mAb 1B10. The antibody, which specifically labelled the paraflagellar structure, recognized a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes and two 70/74 kDa related proteins in L. braziliensis promastigotes. Surface 125I-labelling experiments identified promastigote-specific components (> 100, 74, 45/47 and 28 kDa) and at least 1, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respectively at 50 and 12.5 micrograms/ml, promastigotes were not agglutinated by PNA and agglutinated in the presence of LCA at concentrations of 100 micrograms/ml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerous organisms, able to proliferate at 34 degrees C in UM-54 medium, could be recovered 48 h later.

Cytoskeletal preparations of Trypanosoma cruzi trypomastigotes and epimastigotes contain a protein recognized by a monoclonal antibody (2G4) which is connected to the flagellar attachment zone of both stages of the parasite. Western blot analysis revealed that the antibody was able to recognize protein bands of molecular masses higher than 700 kDa up to 2500 kDa. These giant proteins do not seem to share sequences with beta-connectin since an anti-beta-connectin antibody did not recognize the T. cruzi proteins nor did the 2G4 monoclonal antibody recognize authentic beta-connectin. Immunofluorescence and immunogold electron microscopy provided evidence that this protein is located inside the cell body of the parasite, closely related to a corset of four microtubules known as subpellicular microtubule quartet. Immunogold labeling shows that the protein accompanies the flagellar attachment zone as long as the flagellum adheres to the cell body. It is proposed that these microtubule-associated proteins recognized by the 2G4 monoclonal antibody exist only in trypanosomatid forms having a junctional complex between the flagellum and the cell body and may act as transmembrane elements connecting the subpellicular microtubular quartet with the flagellum at the desmosome region.

Proteinase activities and megasomes were examined in axenically cultivated amastigote-like forms, freshly isolated lesion amastigotes, and promastigotes. Megasomes were absent in promastigotes and present in both amastigote stages, but they seemed to be less numerous and more homogeneous in cultured amastigote-like forms. Contrasting with the poor detection of proteinase activities in promastigote lysates, both types of amastigotes shared multiple proteinases, which were classified in two groups: (a) 60 to > 100 kDa, o-phenanthroline-sensitive activities; and (b) 23- to 40-kDa cysteine proteinases, of which those resolving as 35- to 40-kDa bands in gelatin gels were more clearly visualized in lysates of cultured amastigote-like forms. Incubation of both kinds of amastigotes with 0.25 to 1.0 microM of either Z-Phe-AlaCHN2 or Z-Tyr-AlaCHN2 selectively inactivated cysteine proteinases, but not the 35- to 40-kDa activities, which, again, were detected with higher intensity in cultured amastigote-like forms. The expression of the 35- to 40-kDa proteinases progressively increased when promastigotes were allowed to transform into amastigote-like forms or when lesion amastigotes were incubated at 34 degrees C for different time periods prior to exposure to Z-Phe-AlaCHN2; activities comparable to those of amastigote-like forms were attained within 24 to 48 hr. The activities resistant to Z-Phe-AlaCHN2 in vivo were fully inhibited by E-64 or Z-Phe-AlaCHN2 during gelatin digestion, suggesting that the 35- to 40-kDa proteinases were mainly inactive before cell lysis. The presence of cycloheximide (at 10, 50, and 100 micrograms/ml) during the pulse with Z-Phe-AlaCHN2 abolished the 35- to 40-kDa activities of lesion amastigotes and significantly reduced gelatin digestion by the similar enzymes of cultured amastigote-like forms. In the latter, the 35- to 40-kDa proteinases were no more detected when cycloheximide was given 60 min prior to Z-Phe-AlaCHN2. The results indicate higher rates of synthesis of the 35- to 40-kDa enzymes, and the existence of a more representative pool of inactive enzyme precursors, in cultured amastigote-like forms.

An ultrastructural cytochemical study of acid phosphatase activity performed in mouse endometrium on the second day of pregnancy showed that stromal cells which were heavily labeled by the cytochemical reaction had disarranged organelles. On the other hand, the cytoplasm of several stromal cells had collagen-containing phagosomes that were also labeled, indicating that the collagen fibrils were being digested by lysosomal enzymes. It is suggested that cell death and phagocytosis of collagen are events of the remodeling of the mouse endometrium that occur prior to decidualization.

The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.

The rapid disappearance of infective forms of Trypanosoma cruzi from the site of inoculation as well as the initial phase of infection produced by the parasite are not yet fully understood. To investigate this problem we used the hamster as an animal model considering the existence of the cheek pouch--a peculiar region devoid of lymphatic vessels. T. cruzi trypomastigotes were inoculated into the cheek pouch or into the footpad of animals previously infected or not with the same parasite. The results were followed from 3 up to 21 days postinoculation, by histological examination. In the cheek pouch of normal animals a large number of parasites could be seen up to 15 days post-inoculation and the inflammatory infiltrate had a focal distribution. Conversely, in the footpad the infiltrate was diffuse and no parasites could be detected. These observations indicate that the lymphatic system is the main route of T. cruzi dissemination from the site or inoculation. When hamsters were first inoculated in the footpad and 7 days later in the pouch, the inflammatory infiltrate at this point was less aggressive and no parasites could be detected.

Collagen fibrils were present within membrane-bound vacuoles in the cytoplasm of mouse decidual cells on the 7th day of pregnancy. The space between the vacuole membranes and the fibrils was narrow and frequently filled with a granular electron-dense material. The loss of banding of the collagen fibrils, their association with lysosomelike bodies, and the demonstration of acid phosphatase activity in the vacuoles indicate that the fibrils were internalized by the decidual cells and were being digested. It is suggested that phagocytosis of collagen is a mechanism of remodeling of the mouse decidua.

The initial interaction between infective forms of Trypanosoma cruzi and host cells in vivo was studied at the ultrastructural level. In order to follow these events, T. cruzi bloodstream forms were inoculated into the cheek-pouch of hamsters--a peculiar region devoid of lymphatic vessels. This region was chosen as injection site because, unlike other regions, trypanosomes remained there and multiplied locally up to 15 days after inoculation. Parasites were detected initially outside cells or inside neutrophils. Only after the first week following inoculation were developing and multiplying trypanosomes seen inside macrophages or other resident cells. Parasites persisted until 15-20 days after inoculation, but by about the 28th day they were no longer seen.