Selenium-containing compounds exhibit diverse biological activities, such as antioxidative, anti-inflammatory, and cancer preventive effects. Using Caenorhabditis elegans as a model organism, we assessed the toxicity, neuroprotective, antioxidant properties, and impact on longevity of 11 selenoacetylenes. Their toxicity and bioactivities varied based on molecular structure. Selenoacetylenes with butyl substituents were toxic to Galleria mellonella larvae. The presence of but-3-in-2-ol radical increased antiprotozoal activity against Tetrahymena pyriformis. Compared to the positive control (Nimitz® EC), selenoacetylenes were less toxic to nematode worms and eggs. Selenoacetylenes significantly reduced amyloid beta (Aβ) paralysis in C. elegans CL4176 worms, increased longevity by 18 to 22%, along with improving survival after oxidative or thermal stress. Galantamine, showed inferior results. These findings enhance our understanding of selenoacetylenes on neuroprotection, antioxidant activity, and longevity in C. elegans. Future mammalian studies will further elucidate mechanisms and explore the potential therapeutic use of selenoacetylenes in treating Alzheimer’s disease and longevity. Seleniumcontaining Selenium containing activities antioxidative antiinflammatory, antiinflammatory anti inflammatory, inflammatory anti-inflammatory effects organism neuroprotective properties 1 structure larvae but3in2ol butinol but 3 2 ol pyriformis Nimitz® Nimitz (Nimitz EC, EC , EC) eggs Aβ (Aβ C CL CL417 22 22% stress Galantamine results neuroprotection Alzheimers Alzheimer s CL41 CL4

Abstract Cabbage larvae (Pieris brassicae, Lepidoptera: Pieridae) are an important pest in organic farming; therefore, biological pest control alternatives are being sought. The effect of Beauveria bassiana (Bb) combined with diatomaceous earth (DE) in a polyvinyl alcohol flask (Bbl), as well as the combination between them (Bb+DE, BbI+DE) on P. brassicae and Galleria mellonella larvae inoculated by aspersion or immersion was evaluated. Prior to the experiment, the viability of B. bassiana was assessed and one of three commercial brands of diatomaceous earth was selected based on the shape and damage caused to G. mellonella. Radial shapes with and without edges were more common in the diatomaceous earth types, but these did not define efficacy on cuticle damage. The result showed that the Bb+DE treatment had greater damage, followed by the Bbl+DE treatment applied by aspersion. The highest mortality in G.mellonella was 70.1 % with Bb at 120 h after sprinkled, while in P. brassicae, mortality reached 42.1 % with Bbl+DE; thus, it is important to highlight the importance of the inoculation method and larvae types used when effectiveness bio-trials are performed.

Resumen La larva de la col (Pieris brassicae, Lepidóptera: Pieridae), es una plaga importante en la agricultura orgánica, por lo que se buscan alternativas para su control. En este estudio, se evaluó el efecto de Beauveria bassiana sola (Bb) y combinada con tierra de diatomea (TD) en matrices de alcohol polivinílico (BbI), así como la combinación entre estos (Bb+TD, BbI+TD) en larvas de P. brassicae y Galleria mellonella inoculadas por aspersión y por inmersión. Previo al experimento, se evaluó la viabilidad de B. bassiana, y seleccionó una de tres marcas comerciales de tierra de acuerdo con su forma y daño provocado en G. mellonella, donde las formas radiales con y sin borde fueron mas frecuentes en los tipos de tierra, sin embargo, estas no definieron la eficacia en daño de cutícula. El resultado mostró que el tratamiento Bb+TD tuvo mayor daño, seguido del tratamiento BbI+TD aplicados por aspersión. La mortalidad más alta en G. mellonella fue 70.1 % con Bb a las 120 h después de aperjado, mientras que en P. brassicae la mortalidad alcanzó 42.1 % con BbI+TD, por lo que hay que destacar la importancia del método de inoculación y el tipo de larva utilizada al realizar bioensayos de efectividad.

Abstract One of the most commonly used fungi in pest control, which has a wide range of hosts, is Beauveria bassiana. This entomopathogen has adapted to different environments and can be found in several places, so in the present work, 60 soil samples were collected in 2020 in Acatlán de Pérez Figueroa, Oaxaca, by means of Galleria mellonella, used as bait for its isolation. Five isolates were obtained from this procedure, which were evaluated in larvae of Galleria mellonella at 24, 48, 72, 96, 120, 144, and 168 h in order to know their efficacy in mortality and mycosis. In the methodology, the DNA of the fungi was extracted and the ITS region of the 5.8S rRNA gene was amplified by PCR, the product was sequenced and the sequences were compared with others existing at the National Center for Biotechnological Information. The result was one isolate of Beauveria pseudobassiana and four of Beauveria bassiana, of which isolate 11 showed 100% mortality and mycosis at 96 h, unlike the commercial isolate and the control, which needed an additional 72 h to kill all larvae, in addition to showing 20% and 60% of mycosis, respectively. The time of 96 h was significantly different (p≤ 0.05) because during this time, most of the larvae died and mycosis occurred. Identifying entomopathogenic fungi and conducting studies of their effectiveness in larvae facilitates the start of new research experiments.

Resumen Uno de los hongos más usado en el control de plagas, el cual tienen un amplio rango de hospedantes es Beauveria bassiana. Este entomopatógenos se ha adaptado a distintos ambientes y se puede encontrar en varios lugares, por lo que en el presente trabajo se colectaron 60 muestras de suelo en Acatlán de Pérez Figueroa, Oaxaca, por medio de Galleria mellonella utilizado como cebo para su aislamiento, en el año 2020. De este procedimiento se obtuvieron cinco aislados los cuales se evaluaron en larvas de Galleria mellonella a 24, 48, 72, 96, 120,144 y 168 h con el fin de conocer su eficacia en mortalidad y micosis. En la metodología se extrajo el ADN de los hongos y la región ITS del gen 5.8S rRNA fue amplificada por PCR, el producto fue secuenciado y las secuencias fueron comparadas con otras existentes en el centro nacional para la información biotecnológica. El resultado fue un aislado de Beauveria pseudobassiana y cuatro de Beauveria bassiana de los cuales el 11 presentó 100% de mortalidad y micosis a las 96 h a diferencia del aislado comercial y el testigo los cuales necesitaron 72 h adicionales para matar la totalidad de larvas, además de presentar 20% y 60% de micosis respectivamente. El tiempo de 96 h fue significativamente diferente (p≤ 0.05) debido a que en éste murió la mayoría de las larvas y hubo micosis. Identificar hongos entomopatógenos y realizar estudios de su efectividad en larvas facilita el inicio de nuevos experimentos en la investigacion.

Resumo Inga cylindrica, uma árvore frutífera tropical da família Fabaceae (subfamília Mimosoideae), é nativa da América do Sul. As sementes desta família são uma fonte essencial de inibidores de tripsina, que apresentam bioatividade promissora para aumentar a defesa do hospedeiro contra patógenos. O objetivo do presente estudo foi caracterizar as atividades antimicrobiana e antibiofilme do inibidor de tripsina extraído de sementes de I. cylindrica, ICTI. O ICTI demonstrou atividade antifúngica com concentração inibitória mínima (CIM) de 32,11 μmol.L-1 e concentração fungicida mínima (CFM) de 32,1 μmol.L-1, contra Cryptococcus gattii, Candida albicans, Candida glabrata e Candida guilliermondii. A combinação de ICTI com Anfotericina B teve um efeito sinérgico significativo, reduzindo a concentração do antibiótico em 75% para C. albicans e 94% para C. gatti. O aumento significativo (16 x) na atividade observado com ergosterol (1,01 mol.L-1) para C. albicans e C. gatti, e a falta de atividade contra cepas bacterianas, sugerem que o ICTI interfere na integridade da membrana celular fúngica . O tratamento com ICTI a 10 x MIC resultou numa redução de 51% na formação de biofilme e numa diminuição de 56% nas colónias maduras de biofilme para C. albicans. Finalmente, o ICTI não apresentou toxicidade no modelo in vivo de Galleria mellonella. Dadas as suas propriedades antifúngicas e antibiofilme, o ICTI pode ser um candidato promissor para o desenvolvimento de novos protótipos de medicamentos antimicrobianos. cylindrica subfamília Mimosoideae, Mimosoideae , Mimosoideae) Sul patógenos I CIM (CIM 3211 32 11 μmol.L1 μmolL1 μmolL μmol.L 1 μmol L μmol.L- CFM (CFM 321 32, μmol.L1, 1, gattii guilliermondii 75 C 94 gatti 16 (1 1,01 101 01 (1,0 mol.L1 molL1 molL mol.L mol mol.L-1 bacterianas 51 56 Finalmente mellonella antimicrobianos 3 L1 7 9 ( 1,0 0 (1, mol.L- 5

Abstract Inga cylindrica, a tropical fruit tree of the Fabaceae family (subfamily Mimosoideae), is native to South America. The seeds from this family are an essential source of trypsin inhibitors, which display promising bioactivity for increasing host defense against pathogens. The aim of the present study was to characterize the antimicrobial and antibiofilm activities of the trypsin inhibitor extracted from I. cylindrica seeds, ICTI. ICTI demonstrated antifungal activity with a minimum inhibitory concentration (MIC) of 32.11 μmol.L-1, and a minimum fungicidal concentration (MFC) of 32.1 μmol.L-1, against Cryptococcus gattii, Candida albicans, Candida glabrata and Candida guilliermondii. Combining ICTI with Amphotericin B had a significant synergistic effect, reducing the concentration of the antibiotic by 75% for C. albicans and 94% for C. gatti. The significant increase (16 x) in activity observed with ergosterol (1.01 mol.L-1) for C. albicans and C. gatti, and the lack of activity against bacterial strains, suggests that ICTI interferes with the integrity of the fungal cell membrane. Treatment with ICTI at 10 x MIC resulted in a 51% reduction in biofilm formation and a 56% decrease in mature biofilm colonies for C. albicans. Finally, ICTI displayed no toxicity in the in vivo Galleria mellonella model. Given its antifungal and antibiofilm properties, ICTI could be a promising candidate for the development of new antimicrobial drug prototypes. subfamily Mimosoideae, Mimosoideae , Mimosoideae) America inhibitors pathogens I (MIC 3211 32 11 μmol.L1, μmolL1 μmolL μmol.L 1, μmol L 1 μmol.L-1 MFC (MFC 321 32. gattii guilliermondii effect 75 C 94 gatti 16 (1 1.01 101 01 (1.0 mol.L1 molL1 molL mol.L mol mol.L-1 strains membrane 51 56 Finally model properties prototypes 3 L1 μmol.L1 μmol.L- 7 9 ( 1.0 0 (1. mol.L- 5 1.

Resumo Nematoides entomopatogênicos (NEP) das famílias Heterorhabditidae e Steinernematidae são amplamente utilizados no controle de insetos-pragas. No Brasil, os estudos relacionados a caracterização e identificação destes entomopatógenos são recentes e escassos. Nesse sentido, o objetivo deste estudo foi isolar NEP de amostras de solos coletadas em diferentes áreas no estado do Paraná, Brasil. Um isolado Heterorhabditis (UEL 08) detectado em amostra de solo em área de pastagem cultivada com braquiária, localizada em Londrina, Paraná, Brasil (23º34´311´´S, 050º58´298´´W), utilizando o método de “inseto-isca” com lagartas de Galleria mellonella. Para a identificação foram realizados estudos de morfometria e identificação molecular a partir da amplificação da região ITS. Algumas diferenças foram encontradas em termos de morfometria em comparação com a descrição original, entretanto, os dados moleculares demonstraram que a sequência obtida para o isolado UEL 08 é idêntica à sequência de referência de H. amazonensis (DQ665222), com a qual apresentou 100% de similaridade. Os estudos das características biológicas de H. amazonensis (UEL 08) revelaram que o isolado tem dois ciclos de vida distintos, um curto (216 h) e outro longo (288 h), sendo que ocorrem duas gerações em ambos os ciclos. O isolado UEL 08 H. amazonensis foi patogénico e virulento sobre os três hospedeiros avaliados. Notadamente, as larvas de G. mellonella e Alphitobius diaperinus foram consideradas mais susceptíveis do que os adultos do percevejo Dichelops (Diacereus) melacanthus, com percentagens de mortalidade de 100%, 85% e 46% de mortalidade, respectivamente. O ensaio de produção in vivo revelou um rendimento médio diário de 3,4 × 103 juvenis infectantes/g de larva hospedeira.

Abstract Entomopathogenic nematodes (EPNs) from Heterorhabditidae and Steinernematidae families are extensively used to control insect pests. In Brazil, however, relatively few studies have identified and characterized these entomopathogens. The objective of this study was to identify and characterize an EPN isolate obtained from soil samples collected in the state of Paraná, Brazil. An isolate (UEL 08) of Heterorhabditis was detected in a soil sample collected from a pasture area cultivated with Brachiaria grass in Londrina, state of Paraná, Brazil (23°34ʹ311ʹʹS, 050°58ʹ298ʹʹW), using the insect-baiting technique with Galleria mellonella larvae as hosts. The nematode was identified through morphometric studies and molecular analyses based on amplification of the rDNA ITS region. Although we identified certain morphometric differences compared with the original description, the molecular data indicated that the ITS sequence obtained for the UEL 08 isolate is identical to the reference sequence of H. amazonensis (DQ665222) and presented 100% similarity. Thus, the findings of our morphological and molecular studies confirmed that the isolated nematode is H. amazonensis, which is the first time this species has been registered in Paraná. Study of the biological characteristics of H. amazonensis (UEL 08) revealed that the isolate has two distinct life cycles – one short (216 h) and the other long (288 h) – and produces two generations in both cycles. We observed that H. amazonensis (UEL 8) was pathogenic and virulent to the three evaluated hosts, although with different virulence against these hosts. The larvae of G. mellonella and Alphitobius diaperinus were more susceptible than adult Dichelops (Diacereus) melacanthus, with 100%, 85%, and 46% mortality, respectively. Furthermore, an in vivo production assay revealed a mean daily yield of 3.4 × 103 infective juveniles/g host larvae.

Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae), woolly apple aphid (WAA) is an important pest on apples globally and a key pest of apple production in South Africa. The aphid has developed some level of resistance against several chemical insecticides. Entomopathogenic fungi (EPF) have been identified as promising biological control agents against a wide array of insect pests. The main aim of this study was to conduct a survey in local apple orchards in the Western Cape province for EPF and to use isolates to test the susceptibility of WAA under optimum laboratory conditions. Soil samples were collected from apple orchards and baited using larvae of Galleria mellonella and Tenebrio molitor to isolate EPF. Six EPF species: Beauveria bassiana, Cordyceps fumosorosea, Metarhizium brunneum, M. pinghaense, M. robertsii and Purpureocillium lilacinum were identified from the soil samples. The results from bioassays showed that Metarhizium robertsii and M. pinghaense isolates were the most effective with an average percentage mortality of > 90%. Metarhizium brunneum also proved to be effective when used against the insect with an average percentage mortality of > 80%, while B. bassiana and C. fumosorosea were the least effective with average percentage mortality of 52% and 48%, respectively. The lethal time required to kill 50% and 90% of the colony at a concentration of 107 conidia/ml over five days, LT50 and LT90, of M. robertsii (2.12 and 4.19 days, respectively) and M. pinghaense (2.05 and 4.45 days, respectively) showed to require similar mortality time in days of E. lanigerum. The results obtained in the study have provided an insight into the diversity of EPF species across apple orchards of the Western Cape and shown the efficacy of the Metarhizium isolates as potential biological control agents of the E. lanigerum.

Resumen Objetivos: Aislar nematodos entomopatógenos (NEP) nativos y evaluar la patogenicidad de la mezcla de Steinernema sp. y Heterorhabditis sp. sobre Eurhizococcus colombianus en condiciones de laboratorio. Alcance: La mezcla de los nematodos entomopatógenos nativos de los géneros Steinernema y Heterorhabditis controlan eficientemente a E. colombianus. Metodología: Se aislaron NEP nativos utilizando larvas de Galleria mellonella como insecto trampa, se mezclaron ambos nematodos y se evaluó su patogenicidad sobre los estados de desarrollo de E. colombianus. Resultados: Se aislaron y mezclaron NEP nativos de los géneros Steinernema y Heterorhabditis. Veinticuatro h después de la infestación sobre hembras de E. colombianus se presentó mortalidad del 95% en la dosis de 100JI/ 25μl, mientras que a las 192 h la mortalidad fue del 100% en las dosis de 10 y 100 JI/ 25μl. Cuando los NEP se aplicaron sobre los quistes de E. colombianus en dosis de 10, 100 y 1000 JI/ 25 μl, el rompimiento de estos fue del 43%, 21% y 24%, respectivamente. Conclusiones: La mezcla de los NEP aislados controlan eficientemente los estados de desarrollo de E. colombianus en las dosis evaluadas.

Abstract Objectives: To isolate native entomopathogenic nematodes (NEP) and evaluate the pathogenicity of the mixture of Steinernema sp. and Heterorhabditis sp. on Eurhizococcus colombianus under laboratory conditions. Scope: The mixture of native entomopathogenic nematodes of the Steinernema and Heterorhabditis genera efficiently control E. colombianus. Methodology: Native NEPs were isolated using Galleria mellonella larvae as a trap insect, both nematodes were mixed and their pathogenicity was evaluated on the development stages of E. colombianus. Results: Native NEPs of the Steinernema and Heterorhabditis genera were isolated and mixed. Twenty-four h after the E. colombianus infestation on females, mortality of 95% was presented at the dose of 100 JI / 25μl, while at 192 h mortality was 100% in the doses of 10 and 100 JI / 25μl. When NEPs were applied to E. colombianus cysts in doses of 10, 100 and 1000 JI / 25 μl, their rupture was 43%, 21% and 24%, respectively. Conclusions: The mixture of the isolated NEPs efficiently control the development stages of E. colombianus at the doses evaluated.

Abstract Schinus terebinthifolia Raddi green fruits essential oil (EO) was evaluated regarding its phytochemical profile, antimicrobial and cytotoxic activities, and toxicity. Gas chromatography with mass spectrometry was applied to identify its constituents, thereafter the minimum inhibitory concentration, minimum bactericidal and fungicidal concentrations, and its antibiofilm activity were evaluated. The EO cytotoxicity was assessed in tumor and non-tumor human cells, and in vivo toxicity was evaluated in a Galleria mellonella model. The major constituents of S. terebinthifolia EO were alpha-phellandrene and beta-phellandrene. The EO had a weak activity against all strains of Candida albicans (MIC 1000μg/mL) and had no activity against non-albicans strains, bacteria, and C. albicans biofilm. Cytostatic activity against all tumor cell lines was shown. Additionally, cell viability remained at EO concentrations up to 62.5 μg/mL. At 16 mg/mL, 50% hemolysis was observed, and it had low toxicity in vivo. Overall, the S. terebinthifolia EO was characterized by low antimicrobial and antibiofilm activities, with no evidence of toxicity to human tumor and non-tumor cells.

Resumen Los nematodos entomopatógenos son parásitos obligados de insectos que habitan en el suelo, tienen gran potencial como organismos de control biológico por su amplio rango de huéspedes de plagas de importancia económica. En este estudio se determinó la presencia de nematodos nativos en suelos provenientes de la granja Tesorito de la Universidad de Caldas, Colombia, utilizándose la técnica de insecto trampa con larvas de Galleria mellonella. Se evaluó la mortalidad que causan los nematodos sobre larvas de Premnotrypes vorax. Se encontraron aislamientos pertenecientes al género Steinernema, se seleccionaron los más virulentos sobre G. mellonella y se inocularon sobre larvas de último instar de P. vorax, mostrando mortalidades entre el 33,3% y el 91,7%, 72 horas después de la infestación. El alto porcentaje de mortalidad que presentó P. vorax deja en evidencia que estos organismos se podrían utilizar como componente importante del manejo integrado de P. vorax en cultivos de papa.

Abstract Entomopathogenic nematodes are compulsory parasites of soil-dwelling insects. They have great potential as biological control organisms due to their wide range of economically important pest hosts. In this study, the presence of native nematodes was determined in soils from the Tesorito farm of Universdad de Caldas, Colombia, using the insect trap technique with Galleria mellonella larvae. The mortality caused by nematodes on Premnotrypes vorax larvae was evaluated. Isolates belonging to the genus Steinernema were found, the most virulent ones on G. mellonella were selected and inoculated on last instar larvae of P. vorax, showing mortalities between 33.3% and 91.7%, 72 hours after infestation. The high mortality rate of P. vorax showed that these organisms could be used as an important component of integrated management of P. vorax in potato crops.

Abstract Entomopathogenic nematodes (EPNs) can control pests due to their mutual association with bacteria. The use of these biological control agents is increasing worldwide due to advances in research about its control efficiency, range of action and mass production. The identification of EPNs adapted to specific environmental and climatic conditions is important for sustainable pest suppression in integrated management (IPM) programs. The objective is to report, for the first time, the occurrence of the Steinernema diaprepesi in Brazil. Steel mesh traps with Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae were buried in red latosol cultivated with Eucalyptus. Infective juveniles (IJs) were isolated from dead larvae and multiplied in healthy ones of this host to confirm its pathogenicity and to start a laboratory population from the strain found in the field. The DNA of the IJs was extracted and amplified using PCR technique with the universal primers D2A and D3B. The detection of S. diaprepesi is the first report of this nematode in Brazil, increasing the knowledge about its distribution in the world and the diversity of EPNs that must be considered as agents of biological pest control in the country.

Resumen Se estudió la macromorfología de cepas de Beauveria bassiana (B1), Metarhizium anisopliae (M1) e Isaria javanica (HPI-210) y se determinó su crecimiento radial a temperaturas de 25, 28, 30, 35 y 40 °C en medio PDA suplementado con polvos de Diatraea considerata, Spodoptera frugiperda y Galleria mellonella. Los hongos se inocularon adicionando 2 µl de una suspensión con 1 x 107 esporas/ml. Después de 15 días se midió el crecimiento radial de las cepas en mm/d. Para determinar la interacción entre las tres cepas con el polvo de los tres insectos más el control PDA y las cinco temperaturas, se usó un análisis factorial 3 x 4 x 5; los datos del crecimiento de los hongos fueron analizados mediante un ANOVA y una prueba de Tukey. Se encontraron diferencias estadísticas significativas en el crecimiento a diferente temperatura, respecto al control; el crecimiento óptimo en los tres hongos fue a 28 °C, la cepa B1 suplementada con polvo de D. considerata creció 2,76 ± 0,06 mm/d, M1 con G. mellonella 2,77 ± 0,02 mm/d, y HPI-210 con D. considerata 3,27 ± 0,06 mm/d. La cepa B1 creció 0,45 ± 0,03 mm/d, M1 1,22 ± 0,0 mm/d a 30 °C, mientras que HPI-210 no creció después de 28 °C. Estos resultados sugieren que las tres cepas fueron inducidas a crecer mejor por efecto de los medios suplementados con los insectos; a 28 °C, HPI-210 presentó el mayor crecimiento/d (F = 25,24, E8, P = 0,0001) seguido de M1 y B1, mientras que M1 con suplemento de S. frugiperda creció mejor a 30 °C (F = 38,33, E8, P = 0,0001). Estos resultados de crecimiento radial, desarrollo macromorfológico y tolerancia a la temperatura permiten seleccionar a las mejores cepas para evaluar su virulencia en insectos plaga.

Abstract The macromorphology of Beauveria bassiana (B1), Metarhizium anisopliae (M1) and Isaria javanica (HPI-210) strains was studied, as well as its radial growth at temperatures of 25, 28, 30, 35 and 40 °C in PDA medium supplemented with powder of Diatraea considerata, Spodoptera frugiperda and Galleria mellonella. The fungi were inoculated by adding 2 µl of a suspension with 1 x 107 spores/ml. Afterwards the radial strains growth in mm/d was measured during 15 d. To determine the interaction between the three strains, the three lepidopteran powders, control PDA and the five temperatures, a factorial analysis 3 x 4 x 5 was used; fungi growth data was analyzed by ANOVA and Tukey test. The macromorphological characteristics of each species were studied, finding significant statistical differences in the growth at different temperatures, with respect to the control. The optimal growth for the three fungi was at 28 °C. Strain B1 supplemented with D. considerata powder grew 2.76 ± 0.06 mm/d, M1, G. mellonella 2.77 ± 0.02 mm/d and M1 HPI-210 D. considerata 3.27 ± 0.06 mm/d. Strains B1 grew 0.45 ± 0.03 mm/d and M1 1.22 ± 0.03 at 30 °C, while HPI-210 did not grow after 28 °C. These results suggested that the strains were induced by medium contained insect supplementation; at 28 °C, HPI-210 obtained the best growth/d (F = 25.24, E8, P = 0.0001), followed by M1 and B1, while M1 supplemented with S. frugiperda grew best at 30 °C (F = 38.33, E8, P = 0.0001). These results of radial growth, macromorphology and tolerance of strains at temperature, allow the selection of the best strains to assess its virulence in insect pests.

Abstracts Escherichia coli is an environmental bacterium frequently implicated in bovine mammary infections. Although specific virulence factors are unknown, biofilm-forming bacteria are associated with persistent infections, and motile bacteria exhibit increased virulence. An analysis was done of the genetic relationship, biofilm formation capacity and motility of bovine mastitis-associated E. coli, as well as the in vivo virulence of representative bacterial isolates. Of the 34 isolates, 67.7 % belonged to phylogenetic group A, 17.6 % to group B1 and 14.7 % to group D. Genetic relationship analysis done with (GTG)5-PCR indicated that the analyzed bacteria are diverse, with only two isolates exhibiting 100 % similarity, and the remaining 32 being grouped into seven main clusters with more than 70 % similarity. Biofilm formation capacity ranged from strong to moderate and weak in 76.5 % of the isolates. The csgA and fimA genes were detected in 52.9 % of the biofilm-forming isolates. Most (70.6 %) of the isolates were motile. In vivo infection assays using Galleria mellonella showed the biofilm-forming bacteria to be more pathogenic than the non-biofilm-forming ones. The studied bovine mastitis-associated E. coli were genetically diverse. Biofilm formation capacity and motility were variable among the isolates, but the biofilm-forming bacteria were more pathogenic than the non-biofilm-forming ones.

Resumen Escherichia coli es una de las bacterias ambientales que frecuentemente se ha visto implicada en infecciones de la glándula mamaria bovina. Aunque se desconocen los factores de virulencia específicos involucrados en dicho proceso, se ha observado que las bacterias formadoras de biopelículas se asocian con infecciones persistentes. De igual forma se ha planteado que las bacterias móviles pueden ser más patógenas. El propósito del trabajo fue analizar la relación genética, capacidad de formación de biopelículas y movilidad de E. coli aislada de casos de mastitis bovina, así como la virulencia in vivo de aislados representativos. El 67.7 % de las bacterias pertenece al grupo filogenético A, el 17.6 % al grupo B1 y el 14.7 % al D. El estudio de la relación genética mediante (GTG)5 reveló que solo dos bacterias son idénticas genéticamente, el resto se organizaron en siete grupos distintos con 70 % de similitud. El 76.5 % de las bacterias fue capaz de formar biopelículas de forma fuerte, moderada o débil. Además, los genes csgA y fimA se detectaron en el 52.9 % de las bacterias formadoras de biopelículas. El análisis de movilidad mostró que el 70.6 % fue móvil. Mientras que, el análisis de patogenicidad en larvas de Galleria mellonella reveló que bacterias formadoras de películas y móviles fueron capaces de matar un mayor número de larvas a las 24 h que las no formadoras. Los resultados indican que las E. coli causantes de mastitis bovina son muy diversas y pertenecen principalmente al grupo filogenético A y que las formadoras de biopelículas y móviles son más patógenas.

BACKGROUND Essential oils (EO) extracted from Cinnamomum verum has been used as an antimicrobial agents for centuries. The effects of C. verum leaf oil against virulence of microorganisms is not well studied yet. OBJECTIVES This study evaluates the effect of C. verum leaf oil against three virulence factors of Candida albicans, C. tropicalis and C. dubliniensis and its in-vivo toxicity. METHODS Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing Candida cells to adhere to the polystyrene surface for 2 h. Biofilm formation of Candida in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. In-vivo toxicity of EO was determined using Galleria mellonella larvae. Chlorhexidine digluconate: positive control. RESULTS Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by C. albicans, C. tropicalis and C. dubliniensis with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC50 for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of Candida. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with in-vivo experiment model at any concentration tested. CONCLUSION C. verum leaf oil acts against virulence factors of Candida and does not show any toxicity.

ABSTRACT The objective of this study was to determine the influence of selected abiotic factors on the viability and pathogenicity of H. amazonensis strain HC1, as initial studies to obtain a commercial formulation. Three trials were developed, where infective juveniles (IJ) were maintained under the effect of each factor for several weeks. In the first test, it was evaluated the effect of different types of water (tap water, boiled, distilled and deionized) on JI at the concentrations of 5x104 and 105 IJ per plate (15 cm diameter). The data related to the viability of IJ were processed through the two-factor analysis of variance and the Duncan´s multiple range test (95 % significance level). Using the water type selected in the first test, three solutions at pH 5, 7, and 9 were prepared and evaluated at the temperatures of 22±1°C, 25±2°C, and 27°C. The values were processed by Wald's test by correction, and CompaProWin 2.0 software was used. The viability and infectivity of IJ in the temperature and pH tests were determined by using the one-in-one test with larvae of Galleria mellonella L. The highest survival values of IJ were obtained with distilled and boiled water, pH 5 and 7, and at temperatures of 22±1° C.

RESUMEN El objetivo de este estudio fue determinar la influencia de tres factores abióticos sobre la viabilidad y patogenicidad de Heterorhabditis amazonensis Anadaló et al., cepa HC1, como estudios iniciales para la obtención de una formulación comercial. Se desarrollaron tres ensayos para determinar el efecto de tres tipos de agua diferentes (potable, hervida, destilada y desionizada), pH (5; 7 y 9) y rangos de temperaturas (22±1°C; 25±2°C; 27°C) sobre la viabilidad e infectividad de juveniles infectivos a los siete y 35 días. En el ensayo con los tipos de agua se utilizaron dos concentraciones de juveniles infectivos (JI): 5x104 y 105 JI por placa; los resultados se evaluaron a través de Análisis de Varianza bifactorial y prueba de rangos múltiples de Duncan (95 %). En los experimentos de pH y temperatura se presentan los resultados a los siete y 35 días, cuando se determinó viabilidad (%) e infectividad, medida a través del porcentaje de mortalidad de larvas de Galleria mellonella L. Los datos se procesaron mediante Prueba de Wald por corrección y se usó software CompaProWin 2.0. En ambas concentraciones, los mayores valores de viabilidad se obtuvieron cuando se empleó agua destilada. Entre 60 y 100 % de los JI sobrevivieron a los siete días en pH entre 5 y 9, con los mayores valores en pH 7; mientras que, a los 35 días, fue de 40 y 60 %. La temperatura fue el factor abiótico que más efecto negativo tuvo en la viabilidad e infectividad de la cepa. Este estudio evidenció que H. amazonensis cepa HC1, puede ser almacenada en formulaciones acuosas, en bandejas y esponjas, utilizando agua destilada con pH neutro y a temperaturas frescas de unos 22±1°C.

ABSTRACT It was evaluated the adequacy of an olfactometer, to which a glass cylinder and a plastic net were introduced to avoid displacement of the target insect larvae, allowing determining displacement and infection capacity of infective juveniles (IJ) of entomopathogenic nematodes in laboratories trials. For these assays, recently emerged IJ with 100 % viability of the strain HC1 of Heterorhabditis amazonensis Andaló et al. were used. For the assembly of the two branches device, some parts of a six arms olfactometer, glass cylinders, plastic meshes, and, as substrate, sterile silica sand with 10 % of humidity were used. The last instar larvae of Galleria mellonella (L.) use as the target insect came from a laboratory rearing and weighed about 200 mg. A 2000 IJ suspension was applied to the substrate on the end opposing to the PVC tube in which the G. mellonella larvae were kept in a glass cylinder protected with a plastic net to avoid their movement. Three replicas were kept at 27°C. Larval mortality was recorded at 24, 48, and 72 hours after nematode application. With the olfactometer assay, e it was checked mobility of IJ, which moved along 19.5 cm in 60 hours. Mortality of G. mellonella larvae reached 84.7 %. The two-branch olfactometer with the introduction of a glass cylinder and a plastic net, was useful for laboratory studies related to the effect of abiotic factor on entomopathogenic nematodes.

RESUMEN Se evaluó la adecuación de un olfatómetro, al que se le introdujeron un cilindro de vidrio y mallas plásticas para evitar el desplazamiento de las larvas del insecto diana a evaluar, permitiendo determinar la capacidad de desplazamiento e infección de los juveniles infectivos (JI) de nematodos entomopatógenos en experimentos en laboratorios. Para estos ensayos se utilizaron JI de la cepa HC1 de Heterorhabditis amazonensis Andaló et al., recién emergidos y con 100 % de viabilidad. Para el montaje del dispositivo de dos cámaras, se utilizaron partes de un olfatómetro de seis brazos, cilindros de vidrio, mallas plásticas y arena sílice esterilizada al vapor como sustrato con 10 % de humedad. Las larvas de último instar de Galleria mellonella (L.), utilizadas como insectos diana, procedían de una cría de laboratorio y tenían 200 mg de peso, aproximadamente. Al sustrato se le aplicó una suspensión que contenía 2000 JI en el extremo opuesto al tubo de PVC donde se situaron las larvas de G. mellonella; estas se mantuvieron en cilindros protegidos con malla plástica para evitar su desplazamiento. Se prepararon tres réplicas que se mantuvieron a 27°C. Se realizaron observaciones a las 24, 48 y 72 horas posteriores a la aplicación de los nematodos y se registró la mortalidad de las larvas. Con el ensayo del olfatómetro se comprobó la movilidad de los JI, los cuales recorrieron 19,5 cm en 60 horas. La mortalidad de las larvas de G. mellonella fue de 84,7 %. El olfatómetro de dos cámaras, con la introducción de cilindro de vidrio y malla plástica, resultó útil para los estudios del efecto de factores abióticos sobre el comportamiento de los nematodos entomopatógenos en el laboratorio.