Compatibility between Leptolegnia chapmanii and diflubenzuron and neem oil for the control of Aedes aegypti / Compatibilidad de Leptolegnia chapmanii con diflubenzurón y aceite de neem parael control de Aedes aegypti

Abstract Leptolegnia chapmanii is pathogenic to mosquito larvae. The compatibility betweenL. chapmanii and two insect growth regulators (IGR), diflubenzuron and neem oil, was evaluated. L. chapmanii was grown on culture media containing different concentrations of eachIGR. The mycelial growth was significantly reduced with the highest concentrations of IGR(F6,98= 268, p < 0.05). However, the production of zoospores (F6,56= 0.93, p > 0.05) and the lar-val mortality of Aedes aegypti (F6,56= 0.95, p > 0.05) were not significantly different amongtreatments. Furthermore, the percentage of adult emergence in the presence of different con-centrations of diflubenzuron or a neem formulation was determined, and the pathogenic activityof zoospores was evaluated at the concentrations that inhibit the emergence at 30, 50 and 90%.The pathogenicity of zoospores was not significantly different among treatments (F6,14= 0.54,p > 0.05), and the larval mortalities were above 90% in all cases.

Resumen Leptolegnia chapmanii es un patógeno de larvas de mosquitos. Se evaluó la com-patibilidad entre L. chapmanii y dos reguladores del crecimiento de insectos (IGR, por sus siglasen inglés), diflubenzurón y aceite de neem. L. chapmanii creció en medios de cultivo con diferentes concentraciones de estos IGR. El crecimiento micelial fue menor con las concentraciones más altas (F6,98= 268; p < 0,05). No hubo diferencias significativas en la producción de zoosporas (F6,56= 0,93; p > 0,05) ni en la mortalidad de las larvas de Aedes aegypti (F6,56= 0,95; p > 0,05).Se determinó el porcentaje de emergencia de adultos en presencia de diferentes concentra-ciones de diflubenzurón o de un formulado a base del aceite de neem. También se evaluó lapatogenicidad de las zoosporas a las concentraciones de los IGR que inhibieron la emergenciadel 30, 50 y 90% de adultos. No hubo diferencias significativas en la actividad de las zoosporasal comparar los tratamientos (F6,14= 054; p > 0,05). La mortalidad de las larvas fue superior al90%.

Compatibility between Leptolegnia chapmanii and diflubenzuron and neem oil for the control of Aedes aegypti.

Leptolegnia chapmanii is pathogenic to mosquito larvae. The compatibility between L. chapmanii and two insect growth regulators (IGR), diflubenzuron and neem oil, was evaluated. L. chapmanii was grown on culture media containing different concentrations of each IGR. The mycelial growth was significantly reduced with the highest concentrations of IGR (F6,98=268, p<0.05). However, the production of zoospores (F6,56=0.93, p>0.05) and the larval mortality of Aedes aegypti (F6,56=0.95, p>0.05) were not significantly different among treatments. Furthermore, the percentage of adult emergence in the presence of different concentrations of diflubenzuron or a neem formulation was determined, and the pathogenic activity of zoospores was evaluated at the concentrations that inhibit the emergence at 30, 50 and 90%. The pathogenicity of zoospores was not significantly different among treatments (F6,14=0.54, p>0.05), and the larval mortalities were above 90% in all cases.

[Praziquantel, levamisole and diflubenzuron in the control of Dolops carvalhoi (Crustacea: Branchiura) and Anacanthorus penilabiatus (Monogenea: Dactylogyridae) in Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae)]. / Praziquantel, levamisol e diflubenzuron on controle de Dolops carvalhoi (Crustacea: Branchiura) e Anacanthorus enilabiatus (Monogenea: Dactylogyridae) em Piaractus mseopotamicus Holmberg, 1887 (Osteichthyes: Characidae).

This assay evaluated the control efficacy of diflubenzuron, praziquantel and levamisole added to the diet of pacu (Piaractus mesoptamicus) infected with Anacanthorus penilabiatus and Dolops carvalhoi. 19 water tanks of 300 L capacity were utilized with 28 fish in each one. The treatments were made by mixing the active principles in the diet. The experiment was evaluated in four harvests done 1 day before and 3, 7 and 15 days after the treatment. The medicated feeding was applied for 7 days. The results of efficacy suggest that the diflubenzuron alone or associated with levamisole and praziquantel was efficient against the crustacean D. carvalhoi and the efficacy in the 3, 7 and 15 days evaluations ranged from 96.2 to 100%. Against the monogenean the drugs did not present efficacy. The results suggest the use of diflubenzuron for the control of D. carvalhoi in captive fishes in special conditions.

Praziquantel, levamisol e diflubenzuron no controle de Dolops carvalhoi (Crustacea: Branchiura) e Anacanthorus penilabiatus (Monogenea: Dactylogyridae) em Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae) / Praziquantel, levamisole and diflubenzuron in the control of Dolops carvalhoi (Crustacea: Branchiura) and Anacanthorus penilabiatus (Monogenea: Dactylogyridae) in Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae)

Neste trabalho, avaliou-se a eficácia antiparasitária do praziquantel, levamisol e diflubenzuron administrados via oral, adicionados à ração, para pacus (Piaractus mesopotamicus) infectados por Anacanthorus penilabiatus e Dolops carvalhoi. Foram utilizadas 19 caixas d'água de 300 L de capacidade, comportando 28 peixes cada. Os tratamentos foram feitos misturando os princípios ativos nas dietas. A intensidade parasitária e eficácia foram avaliadas 1 dia antes e 3, 7 e 15 dias após o início da alimentação com ração contendo diflubenzuron, levamisol e praziquantel isolados ou associados em diferentes concentrações por 7 dias. Os resultados da eficácia terapêutica sugerem que, isoladamente ou associado com levamisol e praziquantel, o diflubenzuron é eficiente contra o crustáceo D. carvalhoi, demonstrando que a eficácia dos tratamentos nos dias 3, 7 e 15 variou de 96,2 a 100 por cento. Contra os monogenóides, as drogas não apresentaram eficácia satisfatória. Os resultados sugerem o uso do diflubenzuron para o controle de D. cavalhoi em peixes de cativeiro e em condições de quarentenário.

This assay evaluated the control efficacy of diflubenzuron, praziquantel and levamisole added to the diet of pacu (Piaractus mesoptamicus) infected with Anacanthorus penilabiatus and Dolops carvalhoi. 19 water tanks of 300 L capacity were utilized with 28 fish in each one. The treatments were made by mixing the active principles in the diet. The experiment was evaluated in four harvests done 1 day before and 3, 7 and 15 days after the treatment. The medicated feeding was applied for 7 days. The results of efficacy suggest that the diflubenzuron alone or associated with levamisole and praziquantel was efficient against the crustacean D. carvalhoi and the efficacy in the 3, 7 and 15 days evaluations ranged from 96,2 to 100 percent. Against the monogenean the drugs did not present efficacy. The results suggest the use of diflubenzuron for the control of D. carvalhoi in captive fishes in special conditions.

Susceptibility of Aedes aegypti (L) to the insect growth regulators diflubenzuron and methoprene in Uberlândia, State of Minas Gerais

Aedes aegypti (L) (Diptera: Culicidae) was reared in several concentrations of diflubenzuron and methoprene under laboratory conditions in Uberlândia, State of Minas Gerais, southeastern Brazil. Characteristics such as LC50 and LC95, the susceptibility of immature stages of different ages to these insect growth regulators and their residual effects were studied. The LC50 and LC95 of diflubenzuron and methoprene were 5.19 and 12.24 ppb; 19.95 and 72.08 ppb, respectively. While diflubenzuron caused great mortality in all larval instars, methoprene was more effective when the mosquito was exposed from the start of the fourth larval instar onwards. Commercial concentrations of these two insect growth regulators close to LC95 presented greater residual activity than did their respective technical formulations. The parameters were compared with those obtained elsewhere. The characteristics investigated here indicate that these insect growth regulators are effective alternatives for controlling the dengue vector in the Uberlândia region.

Aedes aegypti (L) (Diptera: Culicidae) foi criado em várias concentrações de diflubenzuron e methoprene sob condições de laboratório em Uberlândia, Minas Gerais, sudeste do Brasil. Foram estudados aspectos tais como, CL50 e CL95, suscetibilidade de estágios imaturos de diferentes idades a estes insect growth regulators e seu efeito residual. As CL50 e CL95 de diflubenzuron e methoprene foram: 5,19 e 12,24ppb; 19,95 e 72,08ppb, respectivamente. Enquanto diflubenzuron causou grande mortalidade em todos os estádios larvais, methoprene causou maior mortalidade quando o mosquito foi exposto a partir do início do quarto estádio larval. As concentrações comerciais dos dois insect growth regulators próximas às CL95 mostraram maior atividade residual que suas respectivas formulações técnicas. Os parâmetros são comparados com aqueles obtidos em outros locais. Os aspectos aqui investigados indicam estes insect growth regulators como alternativas efetivas para o controle do vetor da dengue na região de Uberlândia.

Significance of the sulfonylurea receptor (SUR) as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and Blattella germanica.

Diflubenzuron (DIMILIN) is a powerful insecticidal chemical which has been known for many years to inhibit chitin synthesis in vivo in insects and related arthropod species. However, its action mechanism has remained unresolved partly because of its inaction on any of the enzymes involved in chitin synthesis in vitro. Based on our previous work (Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L. Insect Biochem. Mol. Biol. 24 (1994) 1009) showing that diflubenzuron inhibits Ca2+ uptake by vesicles obtained from the integument of American cockroach, Periplaneta americana (L.), in vitro, we tested the hypothesis that the action site of diflubenzuron is an ABC (ATP binding cassette) transporter, probably a sulfonylurea-sensitive transporter. Glibenclamide, one of the most commonly used sulfonylureas for type II diabetes treatment, was the positive control. When given to immature insects, glibenclamide clearly caused toxicity, with symptoms indicating molting abnormality comparable to diflubenzuron. Its LD50 (0.472 microg/nymph) was approximately 2.8 times the value obtained for diflubenzuron (0.17 microg/nymph, topical) in German cockroach, Blattella germanica (L.). However, in terms of the inhibitory activities on chitin synthesis, in isolated integuments glibenclamide showed an identical potency to diflubenzuron in B. germanica nymphs. A competitive binding assay with [3H]-glibenclamide and unlabeled diflubenzuron clearly established that the latter is capable of competitively displacing the former radioligand. The KD values observed for vesicles prepared from fruit fly larvae, Drosophila melanogaster M., were 44.9 nM for glibenclamide and 65.0 nM for diflubenzuron, respectively. Furthermore, glibenclamide was found to affect Ca2+ uptake by isolated cuticular vesicles from B. germanica in a manner very similar to diflubenzuron. These results support our conclusion that the sulfonylurea receptor (SUR) is the target of diflubenzuron in inhibition of chitin synthesis in these two insect species.

Diflubenzuron, a benzoyl-urea insecticide, is a potent inhibitor of TCDD-induced CYP1A1 expression in HepG2 cells.

Diflubenzuron (DFB) belongs to a group of compounds called benzoyphenyl ureas acting as chitin synthesis inhibitors, which also inhibit growth of B16 murine melanomas. The present study was designed to investigate the effect of this insecticide, on CYP1A1 expression and induction in human hepatoma cells HepG2. Treatment of HepG2 cells over 72 h with noncytotoxic concentrations of DFB resulted in a strong dose-dependent decrease in constitutive ethoxyresorufin-O-deethylase activity. Moreover, DFB significantly decreased CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after 24 h exposure, as demonstrated by ethoxyresorufin-O-deethylase (EROD) activity and Northern blot analysis. Additional studies were performed both on parental HepG2 cells and HepG2-241c.1, which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene, cloned under the control of the human CYP1A1 promoter (-1140 to +59). Ribonuclease protection assays (RPA) analysis clearly demonstrated an inhibition of CYP1A1 transcription in both cell lines. Surprisingly, in corresponding experiments using 3-methylcholanthrene (3-MC) as a CYP1A1 inducer, DFB was less effective. Finally, in competitive binding studies using a 9S-enriched fraction of HepG2 cytosol, DFB was capable of displacing [(3)H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from its Ah receptor binding site. Taken together, these results support the involvement of a transcriptional mechanism in the inhibition of CYP1A1 expression in HepG2 cells by DFB, possibly via an Ah receptor antagonism.

Diflubenzuron stimulates phosphorylation of a 39 kDa integumental protein from newly molted American cockroach (Periplaneta americana).

We identified a 39 kilodalton (kDa) protein from newly molted American cockroaches (Periplaneta americana), isolated from a 100,000 g precipitate of a 1000 g supernatant from an integument homogenate, which exhibited increased phosphorylation following diflubenzuron (DFB) treatment. This 39 kDa phosphoprotein was partially purified from an intracellular membrane vesicle containing fractions obtained by discontinuous sucrose density centrifugation. Both the interfaces of the 30/40% and 40/50% sucrose density layers were found to be enriched in the 39 kDa phosphoprotein. Treatments with various chemicals such as the ionophores valinomycin and A23187, the protonophore carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and the vacuolar H(+)-ATPase inhibitor N-ethylmaleimide (NEM), like DFB, were also found to stimulate phosphorylation of 39 kDa protein. These results suggest that by disrupting the normal pH gradient occurring in certain acidic vacuolar-type membrane vesicles, phosphorylation of the 39 kDa protein will be increased. In addition, we found that by decreasing the external pH to 5.0 from about neutral, it was possible to stimulate the phosphorylation activity of this particular protein, and thus stimulate the action of DFB. We concluded the DFB's action is very probably related to its ability to disrupt the normal ionic balance of these vacuolar-type vesicles, leading to eventual disruption of the proton gradient within the vesicles.

Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L.

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10(-8) M). Such an inhibitory effect on DFB on Ca2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na+, and Li+ did not affect the calcium uptake. Among ionophores, agents disrupting H+ gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and gamma-S-GTP stimulated Ca2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.

Effects of diflubenzuron and clanfenur on mouse bone marrow cells.

Diflubenzuron (DFB) and Clanfenur (CFN) belong to a group of compounds called Benzoylphenyl Ureas (BPUs). Several BPUs regulate cell growth in insects and/or inhibit growth of B-16 murine melanomas. In view of potential clinical use for these compounds, DFB and CFN were selected as examples of BPUs and tested for effects on hematopoiesis in C57Bl/6 mice housed in a conventional environment. DFB and CFN exhibit anti-tumor activity in mice, cause little or no morbidity and mortality and rather than causing bone marrow suppression, which is usual for anti-cancer drugs, these agents stimulate hematopoiesis in vivo and in vitro. Stimulation in vivo was evidenced by increased (up to 112%) peripheral blood granulocytes 6 days after a single injection and enhanced granulopoiesis (approximately 25%) in bone marrow up to 18 days after treatment. That effects of DFB and CFN were on hematopoietic stem cells were indicated by 47% and 48%, respectively, increases in numbers of CFUs and 97% and 95%, respectively, increases in CFUgm. Further, bone marrow cells treated in vitro contained about twice the number of CFUs and CFUgm as control bone marrow cells. Almost all of the increase in number of spleen colonies, whether derived from donors treated in vivo or bone marrow cells treated in vitro, was accounted for by a corresponding increase in number of undifferentiated colonies. These data indicate that DFB and CFN treatment enhance numbers of pluripotential stem cells both in vivo and in vitro. The mechanism of enhancement, direct or indirect, remains to be determined.

Evaluación del diflubenzuron-urea, regulador del crecimiento de los insectos en condiciones naturales simuladas en culex (c) quinquefasciatus say, 1823 (Diptera: Culicidae) / Evaluation of diflybenzurone-urea, growth regulator of insects under simulated natural conditions in culex (c) quinquefasciatus say, 1823 (Diptera: culicidae)

Se realizaron pruebas en condiciones naturales simuladas con 2 dosis de diflubenzuron-urea (dimilin o TH-6040) en larvas de Culex (C.) quinquefasciatus Say, 1823 (Díptera: Culicidae) de tercer estadío avanzado o principio de cuarto, procedentes de la Provincia de La Habana, las cuales fueron cultivadas en el laboratorio. Las pruebas se efectuaron en el período de marzo de 1983 a marzo de 1984, según la metodología de la OMS, 1975, a temperatura ambiente. Los resultados nos muestran que con una dosis de 0,448 kg IA/ha se obtuvo una efectividad residual de 9 días

Effects of chitin synthesis inhibitors on incorporation of nucleosides into DNA and RNA in a cell line from Manduca sexta (L).

Five putative chitin synthesis inhibitors (CSI) were tested to determine if they inhibited nucleoside incorporation into acid precipitable material in a cell line from Manduca sexta (L.). The results varied. Diflubenzuron (DFB) (100 micron) inhibited cytidine incorporation by 38%; EL-494 (100 micron) inhibited adenosine incorporation by 43%; Bay Sir 8514 (100 micron) inhibited uridine incorporation by 24%. Superdiflubenzuron (100 micron) was the worst inhibitor overall (18-22%) for the benzoylphenyl urea CSI. The triazine CSI, CGA 19255, was the best inhibitor tested with 60% inhibition for cytidine and 49% for adenosine incorporation into DNA and RNA. Examination of cells incubated with diflubenzuron by scanning electron microscopy revealed distinct external morphological changes. Transmission electron microscopy showed that crystalline structures accumulated in the cytoplasm of cells treated with DFB. The crystalline structures were assumed to be diflubenzuron and they persisted even after diflubenzuron was removed from the medium.

Inhibition of the uptake of nucleosides in cultured Harding-Passey melanoma cells by diflubenzuron.

Diflubenzuron (DFB) significantly inhibited the uptake of uridine, adenosine, and cytidine but not thymidine, in cultured Harding-Passey melanoma cells. Inhibition of nucleoside uptake was rapid (i.e., less than or equal to 5 min) and could not be reversed by washing. These results suggest that DFB may affect membrane properties and - as shown by in vivo tests - growth of melanoma cells.

Inhibition of melanogenesis in B16-F1 melanoma cells after exposure to diflubenzuron.

The insect growth regulator diflubenzuron was found to be a potent inhibitor of melanosome synthesis and release in mouse melanoma cell cultures, and after three to five successive passages of melanoma cells in growth medium containing this compound, these cells were unable to produce monolayers in untreated medium and were incapable of inducing tumor formation in mice. This is the first time that an insect growth regulator has been shown to have a deleterious effect on malignant cells of animals.

Mutagenicity tests of diflubenzuron in the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation assay, and the Ames Salmonella reverse mutation test.

Diflubenzuron, one of a new class of pesticides believed to act via inhibition of chitin synthesis in the developing insect cuticle, was tested for possible mutagenic activity using the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation test at the thymidine kinase locus, and the Ames Salmonella/microsome reverse mutation test. No mutagenic effect was found.