Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Larvaterapia aplicada a heridas con poca carga de tejido necrótico y caracterización enzimática de la excreción, secreción y hemolinfa de larvas / Effect of maggot therapy on minimally necrotic tissues: characterization of larval enzymatic excretion/secretion

Introducción. Las úlceras crónicas son una afección con un impacto negativo importante en la calidad de vida de los pacientes y en el sistema de salud; la aparición de infecciones y su difícil manejo, así como la presencia de tejido necrótico, afectan el pronóstico de curación. La larvaterapia se presenta como una opción para el desbridamiento y el manejo de infecciones de úlceras crónicas. Objetivo. Evaluar la larvaterapia en heridas con poca carga de tejido necrótico y evaluar las excreciones, secreciones y la hemolinfa de las larvas, respecto a su contenido enzimático. Materiales y métodos. Se reporta una serie de tres casos clínicos con úlceras crónicas y poca carga de tejido necrótico, tratados con larvaterapia, y se evalúa su evolución por los índices PUSH (Pressure Ulcer Scale for Healing) y Wound Bed Score, así como el patrón electroforético y contenido enzimático por zimograma de las excreciones y secreciones, y de la hemolinfa de las larvas. Resultados. Con solo una aplicación de la larvaterapia se evidenció una mejoría del aspecto de la herida y en los puntajes evaluados; en el PUSH hubo una disminución de 2,3 puntos, en promedio, y con el Wound Bed Score, un incremento de 2,7, lo que demuestra una mejoría en ambas escalas. Conclusión. Se encontró una actividad enzimática diversa en su contenido de excreciones y secreciones, con predominio de actividad de la proteasa de tipo serina.

Introduction. Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. Objective. The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. Results. The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demontrated with major activity belonging to serine protease family. Conclusions. Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.

Impact of environmental temperatures on resistance to organophosphate insecticides in Aedes aegypti from Trinidad / Repercusión de las temperaturas ambientales sobre la resistencia de Aedes aegypti a los insecticidas organofosforados en Trinidad

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and β-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.

OBJETIVO: Examinar los efectos del aumento de las temperaturas de desarrollo larvario sobre el estado de resistencia a los insecticidas organofosforados de las poblaciones de Aedes aegypti en Trinidad. MÉTODOS: En 2007 y 2008 se llevaron a cabo ensayos biológicos y bioquímicos en larvas de A. aegypti recogidas en el 2006 de ocho áreas geográficamente separadas en Trinidad (Trinidad y Tabago). Las poblaciones larvarias se desarrollaron en cuatro temperaturas (28 ± 2 ºC, 32 ºC, 34 ºC y 36 ºC) antes de los ensayos biológicos con insecticidas organofosforados (fentión, malatión y temefós) y los análisis bioquímicos para las enzimas de esterasa. RESULTADOS: La mayoría de las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC fueron susceptibles al fentión (mortalidad > 98%) pero resistentes al malatión y al temefós (mortalidad < 80%). Se encontró una asociación positiva entre la resistencia a los insecticidas organofosforados y la mayor actividad de αy β-esterasas en las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC. Aunque las poblaciones larvarias que se desarrollaron a temperaturas mayores mostraron variaciones en la resistencia a los organofosforados, hubo un aumento general de la sensibilidad. Sin embargo, los aumentos o las disminuciones en los niveles de actividad de las enzimas no siempre se correspondieron con un aumento o disminución en la proporción de individuos resistentes desarrollados a las temperaturas más altas. CONCLUSIONES: Aunque el recalentamiento del planeta puede causar un aumento de la transmisión del dengue, según los resultados de este estudio el uso de insecticidas para la prevención y el control del dengue todavía puede ser eficaz si las temperaturas aumentan según lo proyectado.

Expression and Characterization of alpha-Methylacyl CoA Racemase from Anisakis simplex Larvae

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as alpha-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40degrees C) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Records of Chrysomya albiceps in Northern Italy: an ecological and forensic perspective

Knowledge of the carrion-breeding insects present at a local level is important and necessary for defining the post-mortem interval. Climate changes and globalisation are affecting species ranges and population dynamics. In this note, we report the incidence of Chrysomya albiceps (Diptera: Calliphoridae) on dead human bodies and carrion in Northern Italy. These data confirm the spread of this species in the Northern regions. The partial sequencing of a 583-bp region of the cytochrome oxidase subunit 1 gene of an Adriatic population did not reveal any difference compared to the same genomic region in the African and South American populations of this species.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (ì = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (ì = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Efeito de íons Cu2+ e Zn2+ em atividade Ca-ATPásica isolada de larvas de Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) / Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

As ATPases, um importante alvo de inseticidas, são enzimas que hidrolisam o ATP e utilizam a energia liberada no processo para realizar algum tipo de trabalho celular. A larva de Pachymerus nucleorum (Fabricius) possui uma ATPase que apresenta alta atividade Ca-ATPásica, mas não expressa atividade Mg-ATPásica. Nesse trabalho, foi testado o efeito de íons zinco e cobre na atividade Ca-ATPásica dessa enzima. Mais de 90 por cento da atividade Ca-ATPásica foi inibida em 0,5 mM de íons cobre ou 0,25 mM de íons zinco. Na presença de EDTA, mas não na sua ausência, a inibição por zinco foi revertida pelo aumento da concentração de cálcio. A inibição por íons cobre, não foi revertida nem na presença e nem na ausência de EDTA. O tratamento da fração ATPase com cobre, previamente ao ensaio de atividade ATPásica, não inibiu a atividade Ca-ATPásica sugerindo que o íon cobre não liga diretamente a enzima. Os resultados sugerem que íons zinco e cobre formam complexo com o ATP e se ligam à enzima inibindo sua atividade Ca-ATPásica.

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90 percent of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Coexistence of three different Drosophila species by rescheduling their life history traits in a natural population.

We present evidence for coexistence of three different Drosophila species by rescheduling their life history traits in a natural population using the same resource, at the same time and same place. D. ananassae has faster larval development time (DT) and faster DT(egg-fly) than other two species thus utilizing the resources at maximum at both larval and adult stages respectively. Therefore, D. ananassae skips the interspecific competition at pre-adult stage but suffers more from intraspecific competition. However, D. melanogaster and D. biarmipes have rescheduled their various life history traits to avoid interspecific competition. Differences of ranks tests for various life history traits suggest that except for DT(egg-pupa), the difference of ranks is highest for the combination of D. melanogaster and D. ananassae for all other life history traits. This difference is maintained by tradeoffs between larval development time and pupal period and between pupal period and DT(egg-pupa) in D. ananassae.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4°C and the supernatants were used in enzymatic assays at 30°C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50°C, a temperature that reduces enzyme stability to 80 and 60 percent of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

preliminary characterization of digestive proteease in the mid-gut of the third larval instar of gasterophilus intestinalis

Protinases contained in the mid-gut of the early third instar of Gasterophilus intestinals have been tentatively identified by midgut hydrolysis of synthetic substrates. Trypsin was identified by maximal hydrolysis of benzoyl-DL-arginine-p-nitroanilide [BApNA] at pH 8 and chymotrypsin by maximal hydrolysis of benzoyl-L-tyrosine ethyl ester [BTEE] at pH 9. Carboxypeptidase A and B were identified by their maximal hydrolysis of hippuryl-DL-phenyllactic acid and hippuryl-L-arginine at pH 9 and 8 respectively. Aminopeptidase was identified by maximal hydrolysis of leucine-p-nitroanilide at pH 9. The mid-gut also showed activity of aspartic proteinase and identified it as cathepsin D.A drug [Banmith 12.5% pyrantel tartarate] used for the routine control of helminthes parasites of horses and donkies in Egypt was used in vitro to investigate its effect on the optimal activity of studied enzymes. It was found that the drug has no effect on trypsin and carboxypeptidase A while it decreases the activity of chymotrypsin, aminopeptidase and acidic proteinase and was also found to increase the activity of carboxypeptidase B greatly

Characterization of glycosidase in the mid-gut of the early third larval instars in gasterophilus intestinalis

Mid-gut homogenates from the early third larval instars of Gasterophilus intestinalis contain at least four glycosidases. Enzyme activity was found alpha-galactosidase and beta-galactosidase. Also-alpha-amylase showed activity in midcythomogenates. All enzymes had acidic pH optima of 3.6-6.A drug [Banminth 12.5% pyrantel tartarate] used for the routine control of helminthes parasites of horses and donkies in Egypt was used in vitro to investigate its effect on the optimal activity of studied enzymes

Evaluation of the possible role of five enzymes in the metabolism of IBA in mustard aphid, Lipaphis erysimi (Kalt.).

The enzymatic activity of five enzymes viz. Glutathione S-transferases, Esterases, NADH dehydrogenase, NADH oxidase and Glutathione reductase were assessed under the influence of Indole butyric acid (IBA) (400 ppm) in the nymphs (48-52h old) of mustard aphid, Lipaphis erysimi fed on radish plants treated for 13, 25 and 37h. The activity of Glutathione S-transferases, Esterases and NADH dehydrogenase increased compared to that found in the control of the same age group of nymphs and it was concluded that these enzymes might be involved in the metabolism of IBA. The other two enzymes, NADH oxidase and Glutathione reductase showed no significant increase in their activity compared to that in the control of the same age group. It was hypothesized that the latter enzymes do not play any significant role in the metabolism of IBA.

Profiles of acid and alkaline phosphatases in the prawn Macrobrachium malcolmsonii exposed to endosulfan.

The present study was performed to determine the toxic effects of endosulfan on the quantitative and qualitative aspects of acid and alkaline phosphatases (ACP and ALP) in Macrobrachium malcolmsonii. Intermoult juvenile prawns were exposed to 32.0 ng/l of endosulfan for a period of 21 days. Samples were taken from the hemolymph, hepatopancreas, gills and muscle of test prawns on the 21st day. The content of ACP and ALP in the hepatopancreas of test prawns were found to be higher in comparison to respective controls. The levels of these enzymes in the hemolymph, gills and muscle of test prawns were found to be lower than in the same tissues of controls. In non-denaturing PAGE, phosphatases appeared as white bands. The intensity of white bands in the hepatopancreas of test prawns were found to be higher when compared to controls. In the hemolymph, gills and muscle of test prawns, the intensity of white bands were found to be lower in comparison to controls. The results of the present study suggest that endosulfan affects the quantity and quality of ACP and ALP in the tissues of M. malcolmsonii.

Performance of Spodoptera litura Fabricius on different host plants: influence of nitrogen and total phenolics of plants and mid-gut esterase activity of the insect.

Five host plants [castor, Ricinus communis (Carolus Linnaeus); cotton, Gossypium hirsutm (Carolus Linnaeus); tomato, Lycopersicum esculentum (Philip Miller); mint, Mentha arvensis (Carolus Linnaeus) and cabbage, Brassica oleracea (Carolus Linnaeus)] belonging to different families were used to study the performance of the Asian armyworm, Spodoptera litura larvae. Highest consumption of food and dry weight gain was observed in larvae fed on castor. Mint did not support optimum larval growth because of low digestibility and low efficiency of conversion of digested food to body matter. Dry weight gain ranged from 26.64 mg on mint to 86.80 mg in castor. These differences tend to be related to nitrogen and total phenolics content of the leaf tissues; however, the most clear-cut correlation is an inverse one between the host plant preference and the ratio of total phenolics to nitrogen in the leaf tissues. Mid-gut esterase activity in larvae showed an increasing trend with the increase in total phenolics: nitrogen ratio in the test plants and the order of mid-gut esterase activity in larvae was mint > cabbage > cotton > tomato > castor.

Cell nucleus activity during post-embryonic development of Apis mellifera L. (Hymenoptera: Apidae). Intranuclear acid phosphatase

We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues

Histochemical alterations of infective third-stage hookworm larvae (L3) in vaccinated mice.

To study the histochemical alterations of hookworm L3 administered in a challenge dose to mice vaccinated previously with the larvae. Male Kunming strain mice vaccinated subcutaneously with 500 living Ancylostoma caninum L3 once every 2 weeks for a total of three immunizations before a final challenge with 500 L3 one week after the final immunization. The abdominal skin with underlying subcutaneous tissue and muscle were removed from the site of percutaneous challenge entry (from 2-3 mice), and fixed in absolute alcohol, cold acetone and 10% neutralized formalin. The tissue sections containing the L3 from the challenge dose were then stained histochemically of glycogen, RNA, DNA alkaline protein, acid mucopolysaccharide, collagen, reticulin, alkaline phosphatase (AKP) and adenosine triphosphatase (ATPase). Skin samples from non-immunized mice that were also subcutaneously inoculated with the L3 served as negative control. The L3 identified in cutaneous sections from vaccinated mice at 6-72 hours post-challenge exhibited reductions in parasite glycogen, alkaline protein, RNA and DNA, as well as reductions in acid mucopolysaccharide, collagen and reticulin contents in the parasite cuticle. There were also reduced enzyme AKP and ATPase activities. In contrast L3, identified in sections from non-immunized mice exhibited a normal histochemical appearance, as did some L3 who survived in vaccinated mice at 7-14 days post-challenge. Vaccination results in hookworm L3 damage which is manifested by reduced histochemical staining for the challenge inoculum of parasites. There is also reduced hydrolytic enzyme activity. The observed changes could reflect either host-mediated parasite structural damage and disintegration or possibly anti-metabolic properties of the host immune response.

Determinación de la resistencia a insecticidas y sus mecanismos bioquímicos en 2 cepas de Culex quinquefasciatus procedentes de Santiago de Cuba / Determination of the resistance to insecticides and their biochemical mechanisms in 2 strain of culex quinquefasciatus fron Santiago de Cuba

Se analizó el comportamiento de la resistencia a 3 insecticidas organofosforados (malation, clorpirifos y pirimifos metil), 3 piretroides (deltametrina, lambdacialotrina y cipermetrina) y 1 carbamato (propoxur) en poblaciones de Culex quinquefasciatus provenientes de 2 municipios de la provincia de Santiago de Cuba. Los valores del factor de resistencia determinaron que existe resistencia para malatión y clorpirifos. Sin embargo, a pesar de la existencia de una alta frecuencia en los mecanismos de esterasas elevadas y acetilcolinesterasa alterada, no se observó resistencia a pirimifos metil, lo cual corroboró la no afectación de este insecticida por estos mecanismos seleccionados en nuestras poblaciones de Culex quinquefasciatus. Se observó resistencia a los insecticidas piretroides deltametrina y lambdacialotrina en Santiago de Cuba, y moderada para cipermetrina en Santiago y San Luis; también se encontró en San Luis resistencia a deltametrina, pero moderada a lambdacialotrina. Los resultados obtenidos a partir del uso de los sinergistas S,S,S, tributil fosfotritiado (DEF) y piperonil butóxido (PB) indicaron que los mecanismos de resistencia de esterasas inespecíficas y las oxidasas de función múltiple están involucradas en la resistencia a piretroides en ambas cepas provenientes de Santiago de Cuba y San Luis. Se determinó, mediante las pruebas bioquímicas, que existió una alta frecuencia de los mecanismos de esterasas y acetilcolinesterasa alterada. Los resultados de las electroforesis en gel de poliacrilamida (PAGE), mostraron que la esterasa B1 aparece con mayor frecuencia asociada con las esterasas A6 y B6. Se infirió que esta asociación pudiera estar vinculada con la resistencia a piretroides

Caracterizaçäo isoenzimática da hexoquinase no desenvolvimento de Plebeia droryana (Hymenoptera, Apidae) e sua relaçäo com a atividade de vôo / Isoenzymatic characterization of hexokinase in the development of Plebeia droryana (Hymenoptera, Apidae) and its relation with flight activity

A enzima Hexoquinase foi estudada em P. droryana por meio de eletroforese em gel de amido agarose. Três regiöes anódicas com atividade enzimática foram observadas durante o desenvolvimento. A hexoquinase-1 (HK-1), cuja intensidade de coloraçäo aumenta de larva até adulto, provavelmente relacionada ao fornecimento de energia para os músculos torácicos de vôo e, conseqüentemente, com a atividade de vôo nesta espécie. A hexoquinase-2 (HK-2), que alcança intensidade máxima em pupa de olho marrom claro e hexoquinase-3 (HK-3), que alcança seu pico máximo de intensidade em imago e näo é observada na fase adulta. Esta isoenzima deve ter funçäo importante na metamorfose desta espécie.