Resumen Los péptidos antimicrobianos (AMP) han tomado importancia en el desarrollo de nuevos antibióticos debido a su papel como inhibidores, no solo de bacterias sino también de virus, hongos y parásitos, entre otros. Desde el descubrimiento de los AMP, se han reportado miles, sin embargo, muchos de ellos no son adecuados para aplicaciones terapéuticas debido a sus largas secuencias de aminoácidos, baja potencia antimicrobiana y altos costos de producción. En este trabajo, proponemos utilizar redes neuronales recurrentes (RNN) con células LSTM para generar péptidos más potentes y económicos. Realizamos diferentes experimentos generando AMP sintéticos entre 12 y 20 aminoácidos. Los resultados muestran que podemos usar RNN y mejorar el proceso de generación en comparación con el método de plantillas manuales.

Abstract The antimicrobial peptides (AMPs) have taken importance in the development of new antibiotics because of their role as an inhibitor, not only of bacteria but also of viruses, fungi and parasites, among others. Since the discovery of AMPs, thousands have been reported, however, many of them are not suitable for therapeutic applications due to their long amino acid sequences, low antimicrobial potency and high production costs. In this work, we propose to use recurrent neural networks (RNN) with LSTM cells in order to generate more potent and economical peptides. We perform different experiments generating synthetic AMPs between 12 and 20 amino acids. The results show that we can use RNN and improve the generation process compared with the template method.

Abstract The increase in bacterial resistance to antibiotics available leads to the search for new compounds with antimicrobial potential, such as peptides and lipopeptides. In this work, eight short lipopeptides with the structural pattern Cn-X1 X2 X3-NH2 were de novo designed, synthesized by Fmoc solid phase and characterized by instrumental techniques. The results of the in vitro tests indicated that two of them, LIP 4 and LIP 12 display antibacterial activity against 4 pathogenic bacteria with minimum inhibitory concentrations (MIC) between 9.50 and 100 μM and between 8.50 and 10.0 μM, respectively; they did not displayed toxicity to human erythrocytes at concentrations between 3.13 and 50.0 μM. The antibacterial mechanism of action observed by scanning electron microscopy indicate that the cell membrane was the target, causing the formation of blisters and vesicles, with size ranging from 100 to 120 nm. The lipopeptide LIP 12, with higher activity, was stable to proteases of human blood serum.

Las lipasas son uno de los grupos de enzimas con mayor cantidad de aplicaciones industriales y biotecnológicas, en el procesamiento de alimentos lácteos y aceites, detergentes, cosméticos, cuero, productos farmacéuticos, papel, producción de surfactantes, biodiesel y recientemente como una alternativa promisoria para el tratamiento de aguas residuales contaminadas con lípidos. En este estudio se aislaron microorganismos a partir de muestras de aguas residuales con grasas a los que se les determinó la producción de lipasa extracelular utilizando p-nitrofenilpalmitato. De 149 aislados realizados, 37 mostraron actividad lipolítica y la cepa CCEI-1, identificada como Serratia marcescens, tuvo la mayor actividad a pH alcalino y a 30°C. el microorganismo degradó los lípidos y produjo pigmentación en medio de cultivo solido a pH 8,0 y 50°C. Los sobrenadantes de los cultivos de S. marcescens empleados para la determinación de la estabilidad térmica a 50°C, indicaron que la enzima es estable a ésta temperatura y en pH alcalino. La enzima fue purificada parcialmente y sometida a electroforesis (SDS-PAGE) donde se detectaron bandas entre 40 y 116 kDa. Los resultados obtenidos en este estudio indicaron que la cepa de S. marcescens CCEI-1 puede ser posible fuente de lipasas termoestables con futuras aplicaciones industriales y biotecnológicas.

Lipases are one of the groups of enzymes with the highest number of industrial and biotechnological applications, processing of dairy foods and oils, detergents, cosmetics, leather, pharmaceuticals, paper, production of surfactants, biodiesel and recently as a promising alternative for the treatment of wastewater contaminated with lipids. In this study, microorganisms were isolated from wastewater samples containing fats and extracellular lipase production was determined using p-nitrophenylpalmitate. From the 149 isolates obtained, 37 showed lipolytic activity and strain CCEI-1, identified as Serratia marcescens, had the highest activity at alkaline pH and at 30°C. Also degraded lipids and produced pigmentation on solid medium at pH 8,0 and 50°C. The culture supernatants of S. marcescens used to determine thermal stability at 50°C indicate that the enzyme is stable at this temperature and at alkaline pH. The enzyme was partially purified and electrophoresed (SDS-PAGE) where bands between 40 and 116 kDa were detected. The results obtained in this study indicate that the strain CCEI-1 of S. marcescens can be a possible source of thermostable lipases with industrial and biotechnological applications.

Foram coletadas amostras de aguas residuais com graxas com a finalidade de realizar o isolamento de microrganismos nativos aos quais determinaram- se e quantificaram-se a produção da enzima extracelular lipase, utilizando um micro-método com p-nitrofenilpalmitato (p-NPP). No total, foram obtidos 149 isolamentos dos quais 37 mostraram atividade lipolítica sendo que o isolamento que apresentou a maior atividade foi selecionado para estudos de determinação das condições ótimas de pH e temperatura para a expressão da lipasa. O isolado CCEI-1, identificado como Serratia marcescens, apresentou a maior atividade a pH alcalino e 30°C, adicionalmente degradou lipídeos e teve produção de pigmentação em meio de cultura sólido a pH 8,0 e 50°C. Os supernadantes dos cultivos de S. marcescens empregados para a determinação da estabilidade térmica a 50°C mostraram resultados que indicam que a enzima é estável nessas condições pH alcalino. A enzima foi purificada parcialmente e sometida a eletroforese em gel SDS-PAGE onde foram detectadas bandas entre 40 y 116 kDa. Os resultados obtidos neste estudo indicam que a cepa S. marcescens CCEI-1 pode ser uma possível fonte de lipases termoestáveis com aplicações industriais e biotecnológicas.

Trichoderma es un género de hongos ampliamente conocido por la supresión de patógenos y la promoción del crecimiento en plantas, aumentando la disponibilidad y la absorción de nutrientes, incrementando concentraciones foliares de nutrientes en diferentes plantas. Con el objetivo de probar y prospectar el uso agrícola de aislamientos nativos colombianos, siete aislamientos previamente seleccionados en pruebas in vitro fueron probados para medir su efecto sobre los niveles de nutrientes y variables de crecimiento en fríjol bajo condiciones de invernadero. Para ello se sembraron las plantas en un andisol y en un ultisol como sustrato, luego estas fueron inoculadas con las diferentes cepas de Trichoderma. En estas plantas de fríjol se cuantificó el tiempo para alcanzar las diferentes etapas o estadios del cultivo. En floración se midieron área foliar, número de nudos y materia seca así como la concentración foliar de N, P, K, S, Mg, Ca, Fe, Zn, B, Cu y Mn. En cuanto a solubilización de nutrientes, en plantas establecidas en andisoles, la inoculación con algunos aislamientos de Trichoderma incrementa las concentraciones foliares de Cu, Fe, Ca, Mg y K con diferencias estadísticas significativas. En ultisoles las concentraciones foliares de P y Cu son mayores en plantas de fríjol inoculadas con Trichoderma, ratificando el efecto positivo del hongo en la concentración foliar de nutrientes. Demostrando que la solubilización de nutrientes por Trichoderma es un fenómeno influenciado por el sustrato o suelo agrícola entre otros factores.

Trichoderma is a genus of fungi widely known for suppressing pathogens and promoting plant growth, enhancing the availability and absorption of nutrients and increasing foliar nutrient concentrations in different plants. With the goal of proving the prospect for agricultural use of Colombian native isolates, seven isolates that were previously selected in in vitro tests were tested to measure their effect on the nutrient levels and growth variables in beans grown under greenhouse conditions. The bean plants were sown in an Andisol and Ultisol, as the substrate, then they were inoculated with different strains of Trichoderma. The time these bean plants needed to reach the various stages of cultivation was assessed. For the flowering, the leaf area, number of nodes and dry matter and foliar concentrations of N, P, K, S, Mg, Ca, Fe, Zn, B, Cu and Mn were measured. In terms of nutrient solubilization in the plants established in Andisols, inoculation with the Trichoderma strains increased the foliar concentrations of Cu, Fe, Ca, Mg and K, with statistically significant differences. With the Ultisoles, the foliar concentrations of P and Cu were higher in the bean plants inoculated with Trichoderma, confirming the positive effect of the fungus on the leaf nutrient concentrations and demonstrating that nutrient solubilization by Trichoderma is a phenomenon influenced by the substrate or agricultural soil.

Las N-acíl homoserina lactonasas son una familia de metaloenzimas bacterianas que participan en la degradación de N-acil homoserina lactonas (AHLs) interrumpiendo el sistema de detección de quórum sensing de bacterias Gram negativas. A partir de una colección de cepas de Bacillus thuringiensis aisladas del filoplano de plantas colombianas que presentaron actividad tóxina contra insectos lepidópteros, 310 fueron probadas para determinar actividad lactonasa mediante el uso de sistemas de biosensores en presencia de N-hexanoilo-L-homoserina lactona (C6-HSL) y la N-octanoilo-L-homoserina lactona (C8-HSL) sintéticas. De estas cepas, el 251 mostraron actividad para ambas lactonas y de estas, el 57% mostró actividad a C6-HSL y el 43% a C8-HSL. Una cepa de B. thuringiensis- denominada 147-115-16- que mostró alta actividad para C6-HSL y C8-HSL, fue capaz de atenuar los síntomas de la pudrición blanda en rodajas de papa infectadas con Pectobacterium carotovorum. Esta cepa contiene un gen homólogo a aiiA, el cual este fue clonado, secuenciado y expresado en Escherichia coli DE3. La proteína recombinante AiiA147-11516 exhibe actividad para C6-HSL y C8-HSL, así como para N-(β-cetocaproil) (3-O-C6-HSL) y N-3-oxo-dodecanoil (3-O-C12-HSL). La cepa recombinante en presencia de P. caratovorum fue capaz de atenuar la infección, sugiriendo que interfiere con la acumulación o respuesta de las señales AHLs. Según estos datos y basados en el reporte previo sobre la actividad hidrolítica de la proteína recombinante AiiA147-11516, esta enzima posee un actividad contra un amplio número de sustratos lo cual sugiere su aplicación en la industria en el control de enfermedades, mediante la transformación de plantas.

The quorum-quenching N-acyl homoserine lactonases are a family of bacterial metalloenzymes that participate in degradation of N-acyl homoserine lactones (AHLs), disrupting the quorum sensing system of gram negative bacterial species. From a collection of Bacillus thuringiensis strains isolated in Colombia from plants and exhibiting toxic activity against lepidopteran insects, 310 bacterial isolates were tested to determine lactonase activity by using biosensor systems in presence of synthetic N-hexanoyl-L-homoserine lactone (C6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). From them, 251 strains showed degrading activity to both C6-HSL and C8-HSL, 57% exhibited degrading activity to C6-HSL and 43% to C8-HSL. One B. thuringiensis strain, denoted as 147-115-16, that exhibit high degrading activity to C6-HSL and C8-HSL, was able to attenuate soft rot symptoms in infected potato slices with Pectobacterium carotovorum. This strain contains an homologous of the aiiA gene that was cloned, sequenced and expressed in Esherichia coli DE3. The recombinant protein AiiA147-11516 display activity to C6-HSL, C8-HSL, N-(ß-ketocaproyl) (3-O-C6-HSL) and N-3-oxo-dodecanoyl (3-O-C12-HSL). The recombinant strain in the presence of P. caratovorum cultures was able to attenuate the infection, suggesting that it interferes either on the accumulation or response to the AHLs signals. Acording to this data and based on previous report from recombinant AiiA147-11516, this enzyme exhibit activity to wide range of catalytic substrates suggesting its industrial application in the disease control programs by plants transformation.

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

La Polilla Guatemalteca de la Papa, Tecia solanivora (Lepidoptera: Gelechiidae), es una de las plagas más importantes del cultivo de la papa en el norte de Sur América, causando pérdidas que oscilan entre el 50 y el 100% de la cosecha. En este trabajo aislamos una cepa nativa de Bacillus thuringiensis (146-15801) con alta actividad contra esta polilla y caracterizamos sus genes Cry. Esta cepa fue la más activa de once cepas evaluadas contra T. solanivora en bioensayos en el laboratorio. Técnicas de PCR específica y caminado genómico permitieron el aislamiento de los genes Cry mostrando un 99 y 95% de similitud con los genes reportados para Cry1Ac y Cry2, respectivemente. Experimentos de inmunolocalización demostraron que Cry1Ac y Cry2 se unen a las células epiteliales del intestino medio del insecto, sugiriendo que estas proteínas están involucradas en la actividad insecticida contra T. solanivora. Los resultados obtenidos en este trabajo son relevantes debido a que las cepas nativas constituyen una fuente de componentes biológicos para el desarrollo de nuevos bioinsecticidas, así como nuevos genes Cry que puedan ser usados para el control biológico.

The Guatemalan potato moth, Tecia solanivora (Lepidoptera: Gelechiidae), is one of the most important pests affecting potatoes in northern South America, causing crop losses ranging from 50 to 100%. In this work, we isolated a native strain of Bacillus thuringiensis (146-15801) with high activity against this moth, and we characterized its Cry genes. This strain was the most active out of eleven strains tested against T. solanivora in laboratory bioassays. Specific PCR and genome walk allowed the isolation of Cry genes showing 99 and 95% similarity to the sequences reported in Cry1Ac and Cry2 genes, respectively. Immunolocation experiments demonstrated that Cry1Ac and Cry2 bind to the midgut epithelial cells, suggest that these two proteins are involved in the insecticidal activity against T. solanivora. Results obtained in this work are relevant because native strains comprise a source of biological components for the development of new bioinsecticides, as well as new Cry genes that can be used for biological control.

Se evaluó la capacidad biocontroladora de ocho cepas Metarhizium anisopliae sobre la garrapata Boophilus microplus en condiciones de laboratorio y campo. La cepa 137bm causó el efecto más fuerte sobre la capacidad reproductiva de la garrapata, disminuyendo la oviposición de la garrapatas del 90 al 96% cuando las garrapatas adultas fueron tratadas in vitro con 1x108 y 1x109 conidias/ml. También redujo la viabilidad de los huevos de este artrópodo en más del 98% en las mismas dosis. En ensayos de campo se logró reducir la infestación de garrapatas en un 75% en vacas Holstein x Cebú, cuando se aplicó M. anisopliae 137bm a una concentración de 1x108 conidias/ml. Además, se observó que la fecundidad de las garrapatas provenientes de vacas tratadas con M. anisopliae fue tres veces inferior que las del grupo control. Por otra parte, la cepa 137bm aplicada en la pradera a una concentración de 5x1012 conidias/ha, causó una disminución en la población de larvas en cerca del 86% una semana después de una aspersión con conidias de M. anisopliae.

The biocontrol potential of eight strains of Metarhizium anisopliae was evaluated for the cattle tick Boophilus microplus under laboratory and field conditions. The strain 137bm caused the highest effect on the reproductive capacity of the tick, decreasing tick oviposition by 90 and 96% when adult ticks were treated in vitro with 1x108 and 1x109 conidia/ ml. It also reduced egg viability by more than 98% in the same doses. In the field trials it was possible to reduce tick infestation by 75% in Holstein x Cebú cows when M. anisopliae 137bm was applied at a concentration of 1x108 conidias/ ml. Also, the fecundity of the ticks coming from cows treated with M. anisopliae was three times lower than that of the control group. On the other hand, strain 137bm applied to the meadow at a concentration of 5x1012 conidia/ha concentration caused a decrease in the larval tick population close to 86% one week after an application with conidia of M. anisopliae.

Se evaluó la eficacia biológica de ocho aislamientos de Trichoderma spp. provenientes de suelos de Colombia para el control de los agentes causales de volcamiento, Sclerotium rolfsii y Rhizoctonia solani, bajo condiciones in vitro y de invernadero. Los análisis in vitro mostraron la capacidad antagónica de todos los aislamientos evaluados. En condiciones de invernadero, cuatro aislamientos fueron altamente eficaces contra S. rolfsii en plantas de fríjol en semillero (>90% de reducción de la enfermedad) y dos aislamientos fueron eficaces contra R. solani en plantas de algodón en semillero (58 y 61% de reducción de la enfermedad). El análisis UP-PCR y DS-PCR permitió determinar tres grupos de aislamientos; dentro de estas asociaciones formadas no se encontró ninguna relación evidente entre la posición en el dendrograma y la actividad antagónica, pero sí permitió separar las especies de Trichoderma por grupos, e incluso encontrar diferencias dentro de aislamientos de una misma especie. Los resultados muestran que el comportamiento micoparasítico de los aislamientos de Trichoderma spp. varía según el hongo fitopatógeno, evidenciando una amplia especificidad del antagonista por su sustrato, es decir por el hongo atacado; por lo tanto es necesario realizar cuidadosas selecciones del aislamiento de Trichoderma que se utilice en programas de control de fitopatógenos.

The biological effectiveness of eight isolates of Trichoderma spp. coming from Colombian soils was evaluated under in vitro and greenhouse conditions for the controlled causal agents of damping-off, Sclerotium rolfsii and Rhizoctonia solani. In vitro analysis showed the antagonistic capacity of all evaluated isolates. Under greenhouse conditions, four isolates were highly effective against S. rolfsii in bean nursery plants (>90% of disease reduction) and two isolates were effective against R. solani in cotton nursery plants (58 and 61% of disease reduction). The analysis UP-PCR and DS-PCR permitted the determination of three groups of isolates. Within these associations no clear link between the position in the dendrogram and antagonistic activity was found, but it allowed separation of species of Trichoderma by groups and even within isolates of the same species. The results indicate that the mycoparasitic capacity of Trichoderma spp. varies according to the phytopathogen fungi, showing a broad specificity of the antagonist for its substrate that is attacked by the fungus. Therefore it is necessary to make careful selections of the isolates of Trichoderma to be used in pathogens control programs.

A través de tablas de vida se analizan los estados de desarrollo que componen el ciclo de vida de Phyllophaga obsoleta Blanchard, una especie plaga del denominado “complejo chisa” de Colombia. Consecuientemente, se reconoce su importancia económica en varias regiones de Colombia. Además se discuten algunos aspectos relacionados con su fenología y distribución en el oriente del departamento de Antioquia (Colombia) en donde se halló asociada a varios cultivos agrícolas y ornamentales.

By means of life tables, the development stages that composed the life cycle of Phyllophaga obsoleta Blanchard were analyzed, which is part of the white grubs complex in Colombia. Consequently, its importance as an economic activity in Colombia recognized. Additionally, some aspects related to its phenology and distribution in the eastern part of the Antioquia department (Colombia) were analyzed, where it was found to be associated to agricultural and ornamental crops as well.

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this <FONT FACE="Symbol">¶</FONT>-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

Bacillus thuringiensis produces d-endotoxins that require proteolytic processing to become active. The activation of the B. thuringiensis subsp. medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin of B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity. Amino terminal sequence of the C. quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys31 and Asp32, with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu29 and Arg30, and for the chymotrypsin-resistant fragment between Arg30 and Lys31.

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot