Avaliação do sistema de monitoramento computadorizado de digestão in vitro: 1. Testes preliminares

O objetivo deste trabalho foi testar o sistema de monitoramento computadorizado da produção de gás in vitro. Essa técnica facilita estudos da degradação das frações solúveis e insolúveis das forragens, quantificadas pela produção de gás (CO2 e CH4) oriunda do metabolismo microbiano e medida por sensor de pressão. Diversas quantidades de amostras (50 a 110 mg de feno de alfafa) e outros alimentos foram testadas. Também, a influência da quantidade de líquido ruminal (2,0 ou 3,0 mL), com ou sem barras magnéticas agitadoras nos frascos incubatórios, na digestibilidade de 100 mg de feno de alfafa, foi estudada. A quantidade de 100 mg de amostra, 2,0 mL de líquido ruminal e sem barra magnética agitadora, proporcionou os menores coeficientes de variação na produção de gás. em conclusão, 2,0 mL de líquido ruminal, sem barra e com 100 mg de amostra, apresentaram maior precisão na curva de digestão.

Comparação do sistema de monitoramento computadorizado de digestão in vitro com os métodos in vivo e in situ: 2. Uso do resíduo da matéria seca de forragens

RESUMO - O objetivo deste trabalho foi comparar o sistema de monitoramento computadorizado da produção de gás in vitro com os métodos in vivo e in situ. Nas comparações, foram utilizadas silagens de milho com alto/baixo teores de matéria seca, com/sem inoculante. A digestibilidade das silagens com alto teor de matéria seca (MS), com/sem inoculação, não apresentou diferenças entre os métodos analisados. Quando avaliadas separadamente do efeito do inóculo, essas silagens diferiram nos métodos in vitro/gás e in situ, mas não no in vivo. Entretanto, quando analisadas somente sob efeito do inoculante, apenas no método in situ houve diferença. Não foram encontradas diferenças na digestibilidade da fibra em detergente neutro (FDN) das silagens com alta ou baixa MS, inoculadas ou não, bem como em relação ao pH ao final da digestão. em conclusão, o desaparecimento da MS e FDN quantificada pelo resíduo no sistema in vitro/gás foi semelhante aos demais métodos avaliados.

Avaliação do sistema de monitoramento computadorizado de digestão in vitro: 3. Desaparecimento da matéria seca e/ou FDN pela produção de gás

RESUMO - Os objetivos deste trabalho foram avaliar o sistema de monitoramento computadorizado da produção de gás in vitro e compará-lo com os métodos in vivo e in situ. Nos métodos de digestibilidade (MS), foram utilizadas amostras de silagens de milho com alto/baixo teor de MS, com/sem inoculante. Avaliando a digestibilidade das silagens, pelo gás produzido na fermentação, os resultados da extensão da degradação (A+D) foram: 21,5; 22,6; 22,1; e 20,9 mL de gás/100 mg MS, sem diferença significativa. Os coeficientes de determinação obtidos na produção de gás total, em relação ao potencial de degradação obtido in situ, mostraram-se elevados para silagem de milho com alta MS inoculada (R²= 0,99), alta MS não-inoculada (R²= 0,98), baixa MS inoculada (R²= 0,94) e baixa MS não-inoculada (R²=0,93). O desaparecimento da MS e/ou fibra em detergente neutro (FDN), quantificada pelo gás produzido no sistema in vitro/gás, apresentou resultados semelhantes aos demais métodos avaliados.

Chitinolytic activities in the gut of Aedes aegypti (Diptera : Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut. (C) 2003 Elsevier B.V. All rights reserved.

X chromosome heterochromatic pattern and nucleolar synthesis in pupal ovaries of Aedes aegypti

C-banding and silver-staining techniques were used to examine pupal ovaries of Aedes aegypti from Sao Jose do Rio Preto (Brazil). Silver staining in ovary cystocytes showed two basic patterns relative to the nucleolar morphology: viz (1) a single, compact small body; and (2) multiple bodies encompassing large nuclear areas. These two types of cystocytes were present in the ratio of 7:1, which is the same as the number of nurse cells and oocytes, respectively, in each follicle. This suggests the possibility of eventually using such a nucleolar morphological difference to recognize both cell types in developmental stages before emergence. Silver nitrate staining in metaphase chromosomes revealed centromeric bands on all six chromosomes. The C-banding pattern in metaphase chromosomes showed an intercalary band in one of the X arms, as described previously in other populations. In ovary cystocytes (pachytene stage) this C-positive band seemed to consist of two chromomeres. Phase contrast microscopy showed that the nucleolus was associated with the distal chromomere of this intercalary C-band, indicating that the nucleolus organizer region was located in that part of the heterochromatic band.

Genetic variation along time in a brazilian population of Aedes aegypti (Diptera : Culicidae), detected by changes in the esterase patterns

Aedes aegypti from the Brazilian cities of Sao Jose do Rio Preto (SJ) and Goiania (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.

Heterochromatic banding pattern in two Brazilian populations of Aedes aegypti

We analysed samples of Aedes aegypti from Sao Jose do Rio Preto and Franca (Brazil) by C-banding and Ag-banding staining techniques. C-banding pattern of Ae. aegypti from Sao Jose do Rio Preto examined in metaphase cells differed from Franca. The chromosomes 2, 3 and X showed centromeric C-bands in both populations, but a slightly stained centromeric band in the Y chromosome was observed only in Sao Jose do Rio Preto. In addition, the X chromosome in both populations and the Y chromosome of all individuals from Sao Jose do Rio Preto showed an intercalary band on one of the arms that was absent in Franca. An intercalary, new band, lying on the secondary constriction of chromosome 3 was also present in mosquitoes of both populations. The comparison of the present data with data in the literature for Ae. aegypti from other regions of the world showed that they differ as to the banding pattern of sex chromosomes and the now described intercalary band in chromosome 3. The observations suggested that the heterochromatic regions of all chromosomes are associated to constitute a single C-banded body in interphase cells. Ag-banding technique stained the centromeric regions of all chromosomes (including the Y) and the intercalary C-band region of the X chromosome in both populations. As Ae. aegypti populations are widespread in a great part of the world, the banding pattern variations indicate environmental interactions and may reveal both the chromosome evolutionary patterns in this species and the variations that may interfere with its vector activity.

Monitoramento sanitário de um sistema integrado de tratamento de águas residuárias da suinocultura

Objective. To assess the potential for contamination of wastewaters from pig farming. Methods. Wastewaters from pig farming were stored in a tank. After 0, 30, 60, 90, and 120 days of hydraulic retention, they were added to lysimeters filled with argillaceous, sandy, or medium soil. Finally, these lysimeters were submitted to simulations of either a rainy season or a dry season. The number of colony-forming units (CFUs) of total coliforms, fecal coliforms, and fecal streptococci was measured in the effluents of the storage tank (for the various periods of hydraulic retention), in the percolate from the lysimeters, and in the three types of soil. The microbiological analyses were carried out using the membrane filter technique. The pH analyses were done potentiometrically. Results. For the three microorganisms, the largest decrease in bacterial counts in the storage tanks occurred with 90 or 120 days of retention. There was a marked decrease in the bacterial count in the percolates of the three soils. For the three soil types the greatest reduction in bacterial counts was found in medium soil, due to its acidity (pH < 7.0). Hydraulic retention was not sufficient to ensure the sanitary adequacy of the wastewaters and their use for irrigation, given that fecal coliform values were above 1 000 CFU per 100 mL. Therefore, adding the residues to the soil was considered a second stage of treatment. Conclusions. The retention of wastewaters followed by adding them to soil was effective in minimizing the contaminating effect of pig farming residues. The storage time for wastewaters from pig farming could be decreased from 120 to 90 days.

Variation of genetic expression during development, revealed by esterase patterns in Aedes aegypti (Diptera, Culicidae)

Polyacrylamide gel electrophoresis was used to analyze esterase patterns during development of Aedes aegypti from the cities of Marília and São José do Rio Preto (SJRP), Brazil. The zymograms showed a total of 23 esterase bands, 22 of which were in the specimens from Marília and 19 in those from SJRP. These esterase bands were considered to be the product of 23 alleles distributed tentatively in eight genetic loci. Most of the alleles were developmentally regulated. The larval stage expressed the greatest number of them (19 alleles, from the eight loci, in Marília; and 17 alleles, from seven loci, in SJRP). The pupal stage expressed 10 alleles from seven loci, in both populations, and the adult stage expressed 8 alleles from five and six loci in SJRP and Marília, respectively. Some alleles that were active in every stage were developmentally controlled at the level of expression (amount of product). A single allele was constitutively and highly expressed, in larvae, pupae, and adults, in both populations. Differences in esterase synthesis among stages are probably due to regulatory mechanisms acting in agreement with the requirements of a variable number of processes in which esterases are involved. The larval stage is the most active in developmental processes and shows very intense intake of food and very high mobility. These features may demand increased esterase production at that stage. Comparison of the two populations examined showed (besides the existence of alleles that they do not share) that they exhibit differences in the control of expression of other alleles. Such findings may reflect genetic differences between founders in each population, but the possibility of involvement of the intensive use of insecticides in SJRP is also discussed.

Standardization of bioassays for monitoring resistance to insecticides in Aedes aegypti

Since the reintroduction of Aedes aegypti in Brazil in the 1980s, insecticide use for its control is routine. The chemical control efficacy is threatened by vectors developing resistance to insecticides. The World Health Organization, recognizing the impact of insecticide resistance in vector control programmes, proposed standardizing bioassays for detecting and monitoring resistance using a diagnostic dose method. As Brazil has a national programme for monitoring the resistance of Ae. aegypti populations to insecticides, this study was designed to compare diagnostic bioassays at WHO suggested concentrations and those estimated for local conditions. Populations were resistant to both temephos doses. But important differences were seen for fenitrothion and malathion, which could lead to under- or over-estimation of resistance respectively. These results and inclusion of a diagnostic dose bioassay standard for larvae are discussed.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): New perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Efficacy of topical permethrin as repellent against aedes aegypti's bites

Mosquitoes are the most important vectors of infectious diseases and their bites are related to several adverse skin reactions. Permethrin impregnated clothes are an efficient strategy against arthropods' bites; however, its topical efficacy as a repellent has not been well established. We studied the response to permethrin lotion 5 percent and N,N-Diethyl-meta-toluamide (DEET) spray 50 percent applied to the unprotected forearms of 10 volunteers. Each arm was exposed to 20 female mosquitoes of Aedes aegypti. We performed 71 bilateral comparative measurements evaluating the timing for the first bites. The average times for the arm without the product, with permethrin 5 percent, and with DEET 50 percent were: 7.9 seconds, 336.2 seconds and 7512.1 seconds. The results showed a significant difference between repellency times between either product and unprotected controls. In addition, there was a significant difference in time to first bite between permethrin and DEET treated arms (p<0.01). Permethrin affords some repellent activity against Aedes aegypti bites in this experimental setting. However, permethrin's profile of repellency was significantly inferior to that of DEET. © 2008 Dermatology Online Journal.

Uso do solo e monitoramento dos recursos hídricos no córrego do Ipê, Ilha Solteira, SP

Brazil has great potential of water resources, however, with deficiency in terms of qualitative and quantitative monitoring and knowledge of the influences of land use. So, this study had the purpose of presenting the qualitative and quantitative monitoring of water for irrigation and the influences caused by the use and occupation of land in the watershed of Ipê stream, in the municipality of Ilha Solteira, São Paulo. The qualitative monitoring was conducted from 2006 to 2011 and the quantitative monitoring from 2009 to 2011. In order to evaluate the influences, the Pearson correlation analysis was used. It was found that the watershed is mostly made up by the crop of sugar cane and the use in conflict with areas of permanent preservation are the fallow areas and pasture. The watershed is characterized with high concentrations of iron and coliforms, originating from degraded pastures and irregular use of urban and rural occupation. The small permanent preservation areas, bad soil conservation and expansion of urban areas imply in the deterioration of the quality and availability of water, generating socio-economic and environmental impact in the area.

Monitoramento temporal da pluma de contaminação no aterro de resíduos urbanos de Rio Claro (SP) por meio do método geofísico da eletrorresistividade

Several factors have contributed to the increase the world production of urban solid waste, resulting in difficulties to the correct management and the final environmentally appropriate disposal, in addition to causing serious socio-economic, environmental, and public health troubles. The soil and groundwater contamination by leachate in waste disposal sites is almost always present, leveraging the emergence and/or adaptation of methodologies for environmental assessment of these areas. The geophysical methods, especially the electrical resistivity method, are fast, reliable and low-cost alternative for this purpose. This paper reports the methodology adopted for the temporal monitoring of the plume of contamination of controlled landfill in the municipality of Rio Claro (SP). Electrical resistivity data were obtained in 1999 and 2008, by the resistivity method, using the techniques of vertical electrical sounding and electrical profile. In both years, similar tests were carried out in the same locations within the disposal area. The results indicated that the plume of contamination has resistivity values less than or equal to 50 ohm.m and moves guided by 2 flows: the main, to the southeast, and the secondary, to the west. Relatively to the first series of tests in 1999, the plume of contamination was higher and deeper in 2008, contrary to what was expected for a landfill deactivated for at least 10 years.

Avaliação de dois diferentes meios de cultura para o desenvolvimento de biopesticidas à base de Bacillus thuringiensis e sua aplicação em larvas de Aedes aegypti

The bacteria Bacillus thuringiensis var. israelensis (Bti) generates certain toxins with pesticide action, which can be used on the control of transmissible diseases by culicides, specially Aedes aegypti, the dengue vector. This biopesticide has been produced by submerged fermentation and, in Brazil, this production has been made by very little research centers and, more recently, by a unique small enterprise. For the implementation of a viable vectors control program through biopesticides, some studies about culture media are essential in order to join efficiency and low costs. In this way, agroindustrial wastes or by-products have been used as a nutrient source for the culture media production. In this study, corn steep liquor, a corn industrial processing by-product and tryptose, both with/without sugar addition, were compared as culture media. Cellular growth was evaluated by optical density at 620 nm, spore production by total viable cell count and LC50 by bioassays against 4th instar larvae. Among the four examined substrates, the medium composed by glucose plus corn steep liquor presented the best spore production and bioassay results.