Resumen Introducción. La creciente resistencia bacteriana a los antibióticos representa una amenaza mundial de salud pública. Las excreciones y secreciones larvarias derivadas de moscas necrófagas de la familia Calliphoridae podrían configurar una fuente promisoria para contrarrestar sus efectos. Objetivo. Comparar la actividad antimicrobiana de las excreciones y secreciones larvarias nativas, y de las mayores y menores de 10 kDa de Calliphora vicina y Sarconesiopsis magellanica (Diptera: Calliphoridae). Materiales y métodos. El bioensayo se hizo a partir de la técnica de turbidimetría y en el caso de las excreciones y secreciones menores de 10 kDa se determinó la concentración inhibitoria mínima (CIM). Resultados. Las excreciones y secreciones nativas y las menores de 10 kDa de C. vicina y S. magellanica, evidenciaron una potente actividad antibacteriana contra tres cepas de Staphylococcus aureus y cuatro bacterias Gram negativas, siendo las menores de 10 kDa más efectivas que las nativas en las dos especies de moscas evaluadas. Además, las menores de 10 kDa presentaron la misma efectividad, aunque en las pruebas de CIM se observó que las de S. magellanica fueron más potentes en todas las bacterias evaluadas, excepto contra la cepa de S. aureus ATCC 25923. Las mayores de 10 kDa no inhibieron el crecimiento bacteriano. Conclusión. Los resultados validaron, en general, que estas sustancias son fuente importante para el aislamiento y la caracterización de agentes antimicrobianos.

Abstract Introduction: The growing resistance to antibiotics worldwide represents a global threat to public health. The larval excretions and secretions derived from necrophagous flies from the Calliphoridae family could represent a promising source for counteracting their effects. Objective: To compare the antimicrobial activity of Calliphora vicina and Sarconesiopsis magellanica (Diptera: Calliphoridae) native excretions and secretions and those weighing more than 10 kDa and less. Materials and methods: We used the turbidimetry technique for the bioassay; we determined the minimum inhibitory concentration (MIC) for excretions and secretions weighing less than 10 kDa. Results: Calliphora vicina and S. magellanica native excretions and secretions and those weighing less than 10 kDa exhibited potent antibacterial activity against three Staphylococcus aureus strains and four Gram-negative bacteria; those weighing less than 10 kDa were more effective than the native ones in the two species of flies evaluated here. Furthermore, excretions and secretions weighing less than 10 kDa had the same effectiveness, except in the MIC trials where S. magellanica excretions and secretions weighing less than 10 kDa were more potent against all the bacteria evaluated, except for S. aureus ATCC 25923. Excretions and secretions weighing more than 10 kDa did not inhibit bacterial growth. Conclusions: These results potentially validate these substances as an important source for isolating and characterizing antimicrobial agents.

BACKGROUND The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs’ physico-chemical properties. Synthetic AMPs’ antibacterial activity was evaluated. FINDINGS Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules’ sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.

El objetivo principal del presente trabajo fue obtener cultivos celulares primarios derivados de tejido embrionario de Sarconesiopsis magellanica (Diptera: Calliphoridae), mosca importante por sus aplicaciones en el establecimiento del intervalo post-mortem. Se evaluaron siete medios de cultivo (Grace, Grace/L15, MM, VP12, MM/VP12, Eagle y Schneider), suplementados con 20% de suero fetal bovino. Se observó adhesión, crecimiento y proliferación celular en los medios L15, Schneider y Grace/L15, lográndose mejores resultados en los dos últimos. La obtención de la monocapa confluente, se presentó en un tiempo promedio de doce días, después de realizados los explantes. El patrón de crecimiento de los cultivos primarios mostró la presencia de vesículas con células adheridas a sus paredes, las cuales, ayudaron a la formación de la monocapa confluente. La morfología celular predominante en la monocapa correspondió a formas fibroblastoides y, en menor proporción, a epitelioides, mostrando las primeras una apariencia similar a células nerviosas. Adicionalmente, se registraron en los primeros días de los cultivos primarios, especialmente, en algunos conglomerados de células, movimientos contráctiles semejantes a los realizados por células musculares. Estos cultivos celulares primarios derivados de S. mangellanica representan, potencialmente, sustratos adecuados para realizar ensayos posteriores de susceptibilidad a infección con arbovirus y parásitos.

The present work was aimed at obtaining and characterizing cell cultures from Sarconesiopsis magellanica embryonic tissue (Diptera: Calliphoridae). This fly is important by its applications in the establishment of post-mortem interval. This study evaluated seven culture medium (Grace, Grace/L15, MM, VP12, MM/VP12, Eagle and Schneider), supplemented with 20% fetal bovine serum. The adhesion, cell growth and proliferation were observed in L15 medium, Schneider and Grace/L15, achieving better results in the last two. The confluent monolayer was present in an average of twelve days after that the explants were made. The growth pattern of the primary cultures showed the presence of vesicles with cells adhering to its walls, which helped to form a confluent monolayer. The predominant cell morphology in monolayer correspond to fibroblastoid a lesser extent to epithelioid, showing the first similar to nerve cells. Additionally, there was in the early days of primary cultures, especially in some clusters of cells, contractile movement, similar to those made by muscle cells. These primary cell cultures derived from S. magellanica represent potentially suitable substrates for further testing of susceptibility to infection with arboviruses and parasites.

Lucilia sericata (Diptera: Calliphoridae) es una mosca necrófaga de importancia médica y forense, utilizada en terapia larval y su desarrollo biológico es usado para establecer el intervalo postmortem. Para criar esta especie, generalmente, se recurre a tejidos animales que al descomponerse producen malos olores y contaminación. El objetivo de este estudio fue determinar las características del ciclo biológico de L. sericata empleando cuatro dietas diferentes a base de hígado de res (H), leche en polvo (LP), pescado (P) e hígado en polvo (HP). El estudio, se inició a partir de huevos tomados de una colonia de L. sericata, previamente establecida, bajo condiciones de laboratorio (24-27°C, 60% HR, fotoperiodo 12 horas luz). La dieta con la que se obtuvo la mayor duración en el ciclo de vida de L. sericata, desde huevo hasta pupa, fue LP y el menor tiempo de desarrollo de la especie, se estableció en la dieta HP. Por otro lado, la menor longevidad de las moscas, se consiguió en la dieta H; sin embargo, en este sustrato alimenticio, los insectos alcanzaron un tamaño mayor. De manera general, se registró un porcentaje mayor de hembras en las dietas LP y P. Potencialmente, las tres dietas sintéticas evaluadas presentaron características de sustratos alimenticios, óptimos para la cría de la mosca, en condiciones de laboratorio.

Lucilia sericata (Diptera: Calliphoridae) is a necrophagous fly of medical and forensic importance used in larval therapy, being its biological development important to establish the post postmortem interval. Animal tissues are generally employed to breed this specie, but these substrates, while decomposing produce bad odors and contamination. The aim of this study was to determine the characteristics of the life cycle of L. sericata using four different diets: beef liver (H), milk powder (LP), fish (P) and liver powder (HP). This study started from eggs taken from a colony of L. sericata previously established under laboratory conditions (24-27°C, 60% RH, photoperiod 12 hours light). The highest duration in the life cycle from egg to pupa was obtained on the diet LP, and the shortest developmental time was established on the diet HP. On the other hand the shortest longevity of adult flies was attained on the diet H. However, in this food substrate the flies were larger than on the others diets. In general, a higher percentage of females was registered with diets LP and P. Potentially, the three synthetic diets evaluated, revealed characteristics of optimal substrates for the breeding of the flies under laboratory conditions.

Introducción. Durante las últimas dos décadas, la terapia larval ha resurgido como una alternativa confiable y segura para la cura de úlceras cutáneas que no responden a los tratamientos convencionales. Objetivo. Evaluar el uso de las larvas de Lucilia sericata en el tratamiento de heridas infectadas con Pseudomonas aeruginosa en un modelo animal. Materiales y métodos. Se tomaron 12 conejos, los cuales fueron divididos al azar en 3 grupos homogéneos: al primero se le aplicó terapia larval, el segundo se trató con terapia antibiótica y el tercero fue establecido como control. A cada uno de los animales se les realizó una herida, luego se inoculó en ésta una suspensión de P. aeruginosa y, finalmente, al registrarse el desarrollo de la infección, se procedió, en los dos primeros grupos, a los tratamientos correspondientes. Para la evaluación macroscópica de las heridas, se tuvo en cuenta la presencia de edema y exudado, mal olor, inflamación alrededor de la herida y apariencia del tejido de granulación. Al proceso de cicatrización se le hizo seguimiento a través de una técnica dermohistológica. Resultados. Se registraron claras diferencias entre el grupo de animales tratados con terapia larval vs. el grupo tratado con terapia convencional de antibióticos, estableciéndose un periodo de 10 días para alcanzar la cicatrización en el grupo de terapia larval mientras que en el segundo grupo el proceso se cumplió en 20 días. Conclusiones. S e demostró la eficacia de las larvas de L. sericata en el tratamiento de heridas infectadas con P. aeruginosa.

Introduction. During the last two decades the larval therapy has reemerged as a safe and reliable alternative for the healing of cutaneous ulcers that do not respond to the conventional treatments. Objective. To evaluate the use of the larvae of Lucilia sericata as a treatment for infected wounds with Pseudomonas aeruginosa in an animal model. Materials and methods. Twelve rabbits were randomly distributed in 3 groups: the first group was treated with larval therapy; the second was treated with antibiotics therapy and to the third no treatment was applied, therefore was established as a control group. To each animal a wound was artificially induced, and then a suspension of P. aeruginosa was inoculated into the lesion. Finally, every rabbit was evaluated until the infection development was recognized and treatment was set up for the first two groups according with the protocols mentioned above. Macroscopic evaluation of the wounds was based on the presence of edema, exudates, bad odor, inflammation around the wound and the presence of granulation tissue. The healing process was evaluated by monitoring histological changes in the dermal tissue. Results. Differences in the time required for wound healing were observed between the first group treated with larval therapy (10 days) and the second group treated with conventional antibiotics therapy (20 days). Conclusion. The L. sericata larva is and efficient tool as a therapy for infected wounds with P. aeruginosa.

En el presente trabajo se evaluó la actividad tóxica de extractos de Eupatorium microphyllum L.F. sobre larvas de IV estadio del mosquito Aedes aegypti (Linneaus), bajo condiciones de laboratorio. Se utilizaron extractos acuosos en concentraciones del 500 mg L-1, 1.500 mg L - 1 y 2.500 mg L-1 y acetónicos en concentraciones de 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 y 50 mg L-1. Los bioensayos se realizaron por triplicado, cada uno con 20 larvas, expuestas durante 24 horas a 150 mL de solución. En todos los ensayos biológicos se emplearon grupos control. En la evaluación de los extractos acetónicos, se empleó un control negativo para evitar que la mortalidad de las larvas ocurriera a causa del solvente. Los extractos acuosos mostraron acción moderadamente baja en la mortalidad de larvas, menor del 20%. Por el contrario, la acción de los extractos acetónicos se observó a 10 y 20 mg L-1, con 15% de mortalidad, mientras que a 30 y 40 mg L-1 se registraron 22 al 38% de mortalidad, en tanto que a 50 mg L-1 la mortalidad fue del 95,4% con resultados estadísticos altamente significativos. Las concentraciones de los extractos acetónicos mostraron ser las más eficientes para el control de los mosquitos seleccionados. Ambos tipos de extractos mostraron efecto tóxico en larvas de A. aegypti ; sin embargo, se observó mayor efecto en los extractos acetónicos en relación con los extractos acuosos de E. microphyllum, lo cual constituye una alternativa viable en la búsqueda de nuevos larvicidas a partir de compuestos naturales.

In the present work the toxic activity of extracts of Eupatorium microphyllum L.F. was evaluated on 4 th instar larvae of the mosquito Aedes aegypti (Linneaus), under laboratory conditions. Aqueous extracts were utilized in concentrations of 500 mg L-1, 1,500 mg L-1 and 2,500 mg L-1 and acetone in concentrations of 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 and 50 mg L-1. The bioassays were carried out for triplicate each one with 20 larvae, exposed for 24 hours to 150 mL of solution. In all the bioassays were employed control groups. In the evaluation of the acetone extracts, a negative control was employed to avoid that the mortality of the larvae to occur on account of the solvent. The Aqueous extracts showed low moderate action in the mortality of larvae, less than 20%. On the contrary, the action of the acetone extracts was observed to 10 and 20 mg L-1 with 15% of mortality, while to 30 and 40 mg L-1 were registered 22 to 38% of mortality. However, to 50 mg L-1 the mortality was of 95.4% with highly significant statistical results. The concentrations of the acetone extracts showed to be the most efficient for the control of the mosquitoes selected. Both types of extracts showed toxic effect in larvae of A. aegypti, nevertheless, greater effect in the acetone extracts was observed relating to the aqueous extracts of E. microphyllum, which constitutes a viable alternative in the search of new larvicides from composed natural.

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Lutzomyia spinicrassa is a vector of Leishmania braziliensis in Colombia. This sand fly has a broad geographical distribution in Colombia and Venezuela and it is found mainly in coffee plantations. Baseline biological growth data of L. spinicrassa were obtained under experimental laboratory conditions. The development time from egg to adult ranged from 59 to 121 days, with 12.74 weeks in average. Based on cohorts of 100 females, horizontal life table was constructed. The following predictive parameters were obtained: net rate of reproduction (8.4 females per cohort female), generation time (12.74 weeks), intrinsic rate of population increase (0.17), and finite rate of population increment (1.18). The reproductive value for each class age of the cohort females was calculated. Vertical life tables were elaborated and mortality was described for the generation obtained of the field cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

The brain cell karyotype of New World sand fly Lutzomyia shannoni was described. This species has four pairs of chromosomes, 2N=8, with one pair of heteromorphic chromosomes.

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

A new cell line designated LSB-AA695BB, was established from embryos of the mosquito Anopheles albimanus. The primary culture was initiated in April, 1995, and the first passage was made 48 days later. Serial subcultures of the cells have been carried through 90 passages from Abril 1995 to February 1996. The cells were grown at 28°C in MK/VP12 medium, supplemented with 20% fetal bovine serum; the pH tolerance ranged between 6.8 to 7.0. The cells have also been adapted to MM/VP12 medium under the same pH, temperature and serum concentration. The majority of the cells were a fibroblast-type. Isozyme characterization showed a pattern similar to that of An. albimanus pupae and adults but distinct from Ae. taeniorhynchus and Ae. albopictus (C6/36) mosquito cell lines. The culture was shown to be free of mycoplasma, bacteria and fungi. Microsporidia contamination of transovarial transmission was controlled with 6.0 mg/ml of albendazole