Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae)

Morphology and cytochemistry of Aedes aegypti’s cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 ºC, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 µm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84±2.54 µm in length and 5.31±1.26 µm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04±4,00 µm in length and 13.96±3.70 µm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08 % of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90 %) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week. Rev. Biol. Trop. 56 (2): 447-458. Epub 2008 June 30.

La primera línea celular de Aedes aegypti fue establecida por Grace en 1966 y desde entonces se han utilizado para el estudio de virus, bacterias y parásitos. En el presente trabajo se describen, por primera vez, algunas características citoquímicas de los cultivos celulares de A. aegypti, infectados con la cepa (MHOM/CO/87CL412) de Leishmania panamensis. También se realizó un estudio morfológico de las células del cultivo. Se observaron 30 células pequeñas con apariencia fibrolastoide de 10.84±2.54 µm de largo y 5.31±1.26 µm de ancho; otras 30 presentaron apariencia epitelioide con 23.04±4.00 µm de largo y 13.96±3.70 µm de ancho; éstas últimas predominaron sobre las de apariencia fibroblastoide. De 113 células, un 7.08%, presentaron abundantes gránulos citoplasmáticos positivos con la coloración de PAS, indicando presencia de polisacáridos. La prueba de peroxidasa dio un resultado negativo. El mayor porcentaje de infección (18.90%), de un total de 101 células, se presentó el día 6. Ultraestructuralmente, las células presentaron un citoplasma con aspecto vacuolado; algunas contenían parásitos, otras material fibrilar y otras estaban vacías. Los resultados indican que los cultivos celulares de A. aegypti pueden ser infectados por L. panamensis y mantener dicho proceso por aproximadamente una semana.

Activation and germination of spores of Bacillus thuringiensis var israelensis by alkaline pH and larval (Aedes aegypti) gut fluid.

Alkaline activation of the spores of crystalliferous (Cry+) and acrystalliferous (Cry-) strains of Bacillus thuringiensis var israelensis; wild type B. cereus and its transcipient crystalliferous derivatives and wild type B. subtilis was studied. Also the effect of larval (Aedes aegypti) gut fluid on the activation of spores of these strains was studied. Only the spores of the crystal forming strains were found to be activated by 0.1 M K2CO3 (pH 10) and by the larval gut fluid. The process of alkaline activation was independent of whether crystals were present with the spores in the activation solution. This indicates that protoxin in the spore coat is responsible for the alkaline activation process and may have ecological implications for the organism.

Total salivary gland proteins of female culex pipiens and aedes caspius [diptera: culicidae] and their fractionaation during adult development and after blood sucking

Salivary glands of Culex pipiens and Aedes caspius were analyzed to determine total protein content and its fractionation during adult female development and after blood sucking. In both species, the molecular weight of proteins ranged between 26.000 and 84.000 Daltons. These proteins were not identical in the two species. In Cx. pipiens, the total protein level increased during the first three days of adult development from 4.94 +/- 0.84 to 6.6 +/- 0.37 mug/gland. During this period, the salivary gland proteins were separated into 35, 34 and 37 fractions, respectively. Cx. pipiens was released in the human host 64% of the total proteins while taking a blood meal compared to unfed females. This decrease in protein level was proportional to protein fractions. Over the next six days, the protein level increased again to attain values comparable to those obtained prior to blood sucking. In Ae. caspius, the total protein level of the salivary glands did not change during the first 4 days of adult development [range between 3.13 +/- 0.27 and 3.19 +/- 0.36 mug gland]; but on the 5th day, 2-fold increase was observed