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BACKGROUND: In Rio Grande do Sul, Brazil, a region with a subtropical climate, Rhipicephalus microplus is present in taurine cattle raised for beef and milk. In addition, ticks resistant to multiple acaricides are present in this region. Recently, fluralaner (isoxazoline) was launched on the market. Thus, there is a need to evaluate the effects of fluralaner for the control of R. microplus on taurine cattle. In addition, occurrence of myiasis by Cochliomyia hominivorax larvae after tick parasitism and weight gain of cattle during the experimental period were evaluated. METHODS: Thirty naturally infested cattle were divided into two experimental groups: T01, treated with fluralaner (2.5 mg/kg) pour-on; T02, control. T01 received fluralaner on Days 0 (early summer in January), 42 and 84 (early autumn), whereas T02, a control group, received palliative treatment with a spray formulation when the group mean was ≥ 30 ticks. Counts of R. microplus females and calculation of the efficacy of fluralaner were performed on Days 3, 7, 14, 28, 35, 42, 56, 70, 84, 98, 112 and 126. The occurrence of myiasis was assessed throughout the study period. In addition, the weight, weight gain and daily weight gain of the animals were evaluated. RESULTS: In the 12 evaluations performed, the parasitic load of T01 was near zero. Fluralaner showed 99.5% efficacy on the 3rd day after the first treatment and 100% efficacy from Day 7 to Day 126. Cochliomyia hominivorax larvae (n = 6; p = 0.0251) were found only in the control group (T02). At the end of the study, the animals subjected to treatments with fluralaner gained 32.8 kg more than the animals in the control group. CONCLUSIONS: Application of fluralaner in summer and autumn, with 42-day intervals between treatments, was effective to control R. microplus on taurine cattle, which also gained more weight than control cattle. Additionally, no cases of myasis were documented in animals treated with fluralaner.

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Transgenic insect strains with tetracycline repressible (Tet-Off) female-lethal genes provide significant advantages over traditional sterile insect techniques for insect population control, such as reduced diet and labor costs and more efficient population suppression. Tet-Off systems are suppressed by tetracycline-class antibiotics, most commonly tetracycline (Tc) or doxycycline (Dox), allowing for equal sex ratio colonies of transgenic insects when reared with Tc or Dox and male-only generations in their absence. Dox is a more stable molecule and has increased uptake than Tc, which could be advantageous in some insect mass-rearing systems. Here, we evaluated the suitability of Dox for rearing Tet-Off female-lethal strains of Australian sheep blowfly, Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), and New World screwworm, Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae), and the effects of dosage on strain performance. For both species, colonies were able to be maintained with mixed-sex ratios at much lower dosages of Dox than Tc. Biological yields of C. hominivorax on either antibiotic were not significantly different. Reduction of Dox dosages in C. hominivorax diet did not affect biological performance, though rearing with 10 or 25 µg/mL was more productive than 50 µg/mL. Additionally, C. hominivorax mating performance and longevity were equal on all Dox dosages. Overall, Dox was a suitable antibiotic for mass-rearing Tet-Off female-lethal L. cuprina and C. hominivorax and was functional at much lower dosages than Tc.

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Screwworm flies are mass-reared and released along the Panama-Colombia border to prevent reinfestation of Central and North America. The cost of the production facility, labor, and diet materials makes mass-rearing the most expensive component of the program. The mass-rearing diet has a large impact on the quality and quantity of insects produced, both of which are necessary for the successful implementation of the sterile insect technique. The diet currently used to rear screwworm flies in Panama contains dried bovine red blood cells, dried bovine plasma, egg powder, milk replacement powder, cellulose (thickening agent), formaldehyde (antimicrobial), and water. Here, we tested an alternative diet containing 2 chicken by-products, which cost less and are locally available, to replace the egg powder and milk replacement powder currently used in the diet. We used 2 screwworm colony strains in our test, the current production strain (Jamaica) and an early female-lethal strain. The chicken diet performed similarly to the production diet with the Jamaica strain, while further optimization will likely be needed for transgenic strain. Finally, nutritional analysis conducted on 7 diet ingredients will assist with diet optimization and the identification of alternative diet ingredients.

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BACKGROUND: The New World screwworm fly, Cochliomyia hominivorax, is widely distributed across South America. This parasitic insect is a significant cause of primary myiasis in animals, including dogs. There is an urgent need for a rapid and efficient treatment to improve the recovery of affected animals. In the present study we evaluated the potential of lotilaner for the treatment of myiasis caused by C. hominivorax larvae in naturally infested dogs. Lotilaner belongs to the isoxazoline class of chemical compounds and is marketed as Credelio™ for use against ticks and fleas in dogs and cats. METHODS: Eleven dogs with naturally acquired myiasis were enrolled in this study based on the severity of lesions and the number of identified larvae. All animals received a single oral administration of lotilaner at a minimum dose of 20.5 mg/kg body weight. After treatment, the number of expelled larvae, live or dead, was determined at 2, 6 and 24 h, and the larval expulsion rate, larvicidal effect and overall efficacy were calculated. After 24 h, the remaining larvae were removed, counted and identified. The lesions were cleaned, and palliative treatment was administered when necessary, according to the animal's health status. RESULTS: All larvae were identified as C. hominivorax. The larval expulsion rate was 80.5% and 93.0% at 2 and 6 h post-treatment, respectively. Lotilaner showed an overall efficacy of 100% at 24 h post-treatment. CONCLUSIONS: Lotilaner demonstrated a rapid onset of action and a high efficacy against C. hominivorax. We therefore recommend lotilaner for the effective treatment of myiasis in dogs.

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The New World screwworm, Cochliomyia hominivorax, causes myiasis in livestock, humans, and other warm-blooded animals in much of South America and the Caribbean. It has been eradicated from North and Central America using the sterile insect technique and a biological barrier is currently maintained at the Panama - Colombian border. However, C. hominivorax is still a threat to eradicated areas as outbreaks can and do occur. In order to identify the origin of a fly involved in an outbreak scenario, diagnostic tools would be beneficial. Recently, the geographic population structure of this species was identified using single nucleotide polymorphisms (SNPs). Here we characterize the three major regional clusters: South America, the Inner Caribbean, and the Outer Caribbean. The objective of this study was to develop a SNP (single nucleotide polymorphism) panel to distinguish between these three clusters. A panel was developed using two unique SNPs per region for a total of six SNPs. This diagnostic SNP assay will allow for rapid source determination of flies from future incursions in order to intercept introductory pathways and aid in the control of New World screwworm.

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The New World screwworm, Cochliomyia hominivorax, is a major parasite that causes myiasis in livestock, humans, and other warm-blooded animals in the western hemisphere. There is a permanent biological border that is maintained between Panama and Colombia, as it has been eradicated from North and Central America. However, it still exists in much of the Caribbean and South America causing an estimated annual loss of $3.6 billion dollars in South America alone. Less information is available for C. hominivorax in the Caribbean. Thus, here we examined its presence and genetic landscape in order to gain insights into this fly's distribution in this region. First, through sampling efforts, novel GPS (Global Positioning System) coordinates were collected. Second, the environmental correlates of those presence points were examined. Next, samples were sequenced in order to obtain a pairwise ΦIT genetic distance matrix. And lastly, this matrix was used to create a genetic landscape of divergence. The results of the genetic landscape show flies as more diverse in Trinidad and Tobago and less diverse in the Dominican Republic. This is perhaps due to the proximity of Trinidad to Venezuela and gene flow may be occurring between these two areas. This information will aid in screwworm surveillance and control programs by providing environmental correlates and a view into the distribution of these flies.

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BACKGROUND: Feral swine (Sus scrofa) are highly invasive and threaten animal and human health in the Americas. The screwworm (Cochliomyia hominivorax) is listed by the World Organization for Animal Health as a notifiable infestation because myiasis cases affect livestock, wildlife, and humans in endemic areas, and outbreaks can have major socioeconomic consequences in regions where the screwworm has been eradicated. However, a knowledge gap exists on screwworm infestation of feral swine in South America, where the screwworm is endemic. Here, we report screwworm infestation of feral swine harvested in Artigas Department (Uruguay), where the Republic of Uruguay shares borders with Brazil and Argentina. METHODS: Myiasis caused by the larvae of screwworm were identified in feral swine with the support and collaboration of members of a local feral swine hunting club over a 3-year period in the Department of Artigas. Harvested feral swine were examined for the presence of lesions where maggots causing the myiasis could be sampled and processed for taxonomic identification. The sites of myiasis on the body of infested feral swine and geospatial data for each case were recorded. The sex and relative size of each feral swine were also recorded. Temperature and precipitation profiles for the region were obtained from public sources. RESULTS: Myiases caused by screwworms were recorded in 27 of 618 the feral swine harvested. Cases detected in males weighing > 40 kg were associated with wounds that, due to their location, were likely caused by aggressive dominance behavior between adult males. The overall prevalence of screwworm infestation in the harvested feral swine was associated with ambient temperature, but not precipitation. Case numbers peaked in the warmer spring and summer months. CONCLUSIONS: This is the first report on myiasis in feral swine caused by screwworm in South America. In contrast to myiasis in cattle, which can reach deep into host tissues, screwworms in feral swine tended to cause superficial infestation. The presence of feral swine in screwworm endemic areas represents a challenge to screwworm management in those areas. Screwworm populations maintained by feral swine may contribute to human cases in rural areas of Uruguay, which highlights the importance of the One Health approach to the study of this invasive host species-ectoparasite interaction.

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Cochliomyia hominivorax (Diptera: Calliphoridae) has been considered the most important cause of myiasis in animals in the South America with most cases being recorded in cattle. In Brazil, the risk of myiasis in swine population is underestimated, leading to difficulties in the implementation of preventive measures. The aim of this study was to report the occurrence of myiasis infesting a swine population over a one-year period and to evaluate the anatomical distribution of these larvae on the animal's body. From May 2019 to April 2020, a total of 2038 inspections were carried out on pigs reared in two farms located in the state of Pernambuco, Northeastern Brazil. Fly larvae retrieved were morphologically identified and the anatomical distribution of myiasis was graphically represented by a heat map indicating its frequency in swine based on the interpolation of data. Cochliomyia hominivorax larvae (n = 78) of second (n = 29) and third (n = 49) developmental stages were recovered in 13 animals (0.64%) out of 2038 inspections performed. The occurrence of myiasis was detected in eight anatomical sites, with higher concentration on the forehead region and on ears. Data herein obtained provide valuable information on the epidemiology and biological aspects of C. hominivorax affecting pigs. In addition, it indicates that the forehead and ears are important sites of parasitism most likely due the high frequency of skin lesions in these areas.

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Cochliomyia hominivorax larvae cause myiasis in animals and humans. To register a commercial product to control this dipteran is necessary to experiment on animals. The in vitro test was standardized to evaluate the larvicidal efficacy of commercial topical products. Five formulations were analysed in vitro and in vivo. For the in vitro test, a colony was formed and three replicates (n = 200) of each larval stage (L1, L2 and L3) were treated. The viability of the larvae was evaluated after 5 and 30 min, and at 1, 2, 6, 12, 24, 48, 60 and 72 h post-treatment (HPT). For the in vivo test, 30 bovines divided into six groups were castrated to achieve natural infestation with C. hominivorax. Animals in the treated groups received the product. Myiasis and efficacy were evaluated 12, 24, 36, 48, 60 and 72 HPT. Four formulations tested in the in vitro test achieved 100% efficacy at 24 HPT. In the in vivo experiment only one achieved 100% efficacy at 24 HPT. However, all products achieved the maximum efficacy by the end of study. The in vitro test developed here could be adopted to evaluate the efficacy of topical products for the control of C. hominivorax larvae.

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Abstract Cochliomyia hominivorax (Coquerel, 1858), the New World screwworm, causes primary myiasis in wild and domestic animals in tropical and subtropical regions of Brazil. Although this species is considered to occur throughout the country, organized information about its recorded distribution has not been available until now. This article aimed to provide a comprehensive review of the historical and current data published on both immature (myiasis) and adult stages of C. hominivorax in Brazil. A total of 174 articles were found; of these, 141 articles reported myiasis cases in cattle (146 records), humans (68 records), and other mammalian hosts (40 records), and captures of adult flies were reported in 33 articles. C. hominivorax is widespread in Brazil, having been recorded in 208 municipalities in all major biomes of the country.

Resumo Cochliomyia hominivorax (Coquerel, 1858), a mosca-da-bicheira, causa miíase primária em animais silvestres e domésticos em regiões tropicais e subtropicais do Brasil. Embora esta espécie seja considerada de ocorrência em todo o país, informações organizadas sobre sua distribuição com base em registros não estavam disponíveis até o momento. Este artigo teve como objetivo fornecer uma revisão abrangente dos dados históricos e atuais publicados sobre o registro de estágios imaturos (miíase) e adultos de C. hominivorax, no Brasil. De 174 artigos encontrados, 141 relataram casos de miíase em bovinos (146 relatos), humanos (68 relatos) e outros mamíferos hospedeiros (40 relatos) e capturas de adultos de C. hominivorax foram registradas em 33 artigos. C. hominivorax encontra-se amplamente distribuída no Brasil, tendo sido registrada em 208 municípios brasileiros em todos os principais biomas do país.

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The aim of this study was to evaluate the larval expulsion, larvicidal effect, retention rate of dead larvae and overall efficacy of sarolaner on the treatment of myiasis caused by New World screwworm Cochliomyia hominivorax in naturally infested dogs. Eight Beagle dogs received a single dose of saroalner 40 mg, with dosage ranging from 2.7 mg/kg to 3.7 mg/kg. Evaluations occurred every 15 min in the first hour, every hour for up to 6 h, and after 24 h of treatment. At 24 h post-treatment, total wound cleaning was performed, including removal and counting of remaining live and dead larvae. The animals received antibiotic, analgesic and wound cleaning support until complete wound healing. The average expulsion of the larvae was 50.9 % occurring mainly after 4 h of treatment. The larvicidal effect was 70.6 % at 6 h after treatment and 100 % at 24 h. The mean retention rate of dead larvae of sarolaner was 33.9 %, The overall efficacy was 100 %, thus making sarolaner an excellent treatment option in myiasis caused by C. hominivorax larvae in dogs.

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In southern South America, namely Argentina and Chile, Cochliomyia hominivorax (Coquerel) is the main myiasic agent on humans and domestic animals. The distribution pattern of the species is poorly known and the southern limit of its geographic distribution is unclear. The aims of this study are to elucidate the basic environmental factors associated with occurrence of this myiasic species, evaluation of models constructed on the basis of occurrence data based on adult specimen records to predict geographic occurrence of myiasis, evaluation of unsurveyed sites of high potential of occurrence of the species, and recognition and prioritization of areas that need medical control and specific prophylaxis practices related to this pest. The maximum entropy modeling system (Maxent) was used. Maps of potential distribution of C. hominivorax were produced using two different datasets, models obtained with all localities known for the species (combining medical data and taxonomic data) and only-taxonomic models (excluding medical data). The results obtained include an updated compilation of occurrence of the species in Argentina and Chile. Predictive models obtained in this work indicated that large areas of central-eastern territory of Argentina has the potential for C. hominivorax occurrence, probably reaching the parallel 42° S as is indicated by the complete datasets. Only-taxonomic models fail to predict those myiasic cases occurring in the nearer areas of the Andean chains. The main variables associated with the distribution of C. hominivorax were, alternatively, isothermality or minimum temperature of the coldest month. These results provide a new analytical resource of high potential for the prevention of myiasis and to supports further epidemiological studies.

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Pin-site myiasis is an underreported complication of surgical interventions. We present a case of myiasis caused by the New World screwworm fly (Cochliomyia hominivorax) in a pin site of a chronic nonhealed wound 12 years after the intervention. This infection apparently was the result of poor perfusion of the leg.

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Myasis is the infestation by fly larvae (Diptera) in live vertebrates including humans. Myasis has been reported most commonly in tropical and subtropical areas around the world with poor sanitation and presence of cattle. Neonatal umbilical myiasis is an important cause of death in bovines and produces major economic losses in the livestock industry. However, its presentation in humans is rare, with a few cases reported worldwide. Moreover, umbilical myasis can be life-treating due to the risk of larvae migration to deeper tissues of the abdomen, omphalitis, and sepsis. We describe the case of a 7-day-old infant admitted to the hospital due to umbilical cord myiasis. In total, 55 larvae were removed from the wound and identified as Cochliomyia hominivorax. The patient recovered satisfactorily after treatment with ivermectin and amoxicillin. A literature search was performed in Pubmed, Medline, Lilacs and Google Scholar, with 64 cases of myasis by C. hominivorax being reviewed. Oral cavity, wounds, scalp and natural orifices are the main affected anatomical areas. Risk factors include the extremes of age, male sex, poor hygiene, alcohol and drug use, cancer, and mental disability. Programs for human myiasis prevention and surveillance are needed in neotropical areas where living conditions make it difficult to implement control strategies.

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The New World screwworm (Cochliomyia hominivorax) is an obligate parasite that affects warm-blooded animals. It causes myiasis in livestock and humans, which is a problem for animal production and public health. The health and economic burden of myiasis on livestock production is largely unknown in Ecuador. We investigated the presence of the screwworm and analysed the epidemiology and spatial and temporal trends of myiasis in cattle farms of San Miguel de Los Bancos county. In total, epidemiological questionnaires were conducted in 110 farms, which were subsequently monitored for 12 months. The findings show that the initial and final prevalences in farms were 70% and 61.81%, respectively, and the average monthly prevalence was 15.08%. The initial and final prevalences in animals were 3.87% and 4.60% for bovines and 2.91% and 3.36% for all animals examined. The average percentage of new cases reported per month was 17.68% with a minimum of 10 and a maximum of 28 cases in October and May 2015, respectively. The cumulative incidence estimated that the risk for non-infested farms to become infested could reach 100% in approximately 6 months. The incidence rate is 168 per 1,000 farms at risk-monthly. The annual incidence was 459 per 10,000 for bovines at risk-annually. An analysis of hotspots based on the Getis-Ord Gi* index revealed no temporally stable hot spot, but one temporally stable cold spot, suggesting that most of the study area is generally favourable to infestation, except one cluster of farms.

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Cochliomyia hominivorax (Coquerel, 1858), the New World screwworm, causes primary myiasis in wild and domestic animals in tropical and subtropical regions of Brazil. Although this species is considered to occur throughout the country, organized information about its recorded distribution has not been available until now. This article aimed to provide a comprehensive review of the historical and current data published on both immature (myiasis) and adult stages of C. hominivorax in Brazil. A total of 174 articles were found; of these, 141 articles reported myiasis cases in cattle (146 records), humans (68 records), and other mammalian hosts (40 records), and captures of adult flies were reported in 33 articles. C. hominivorax is widespread in Brazil, having been recorded in 208 municipalities in all major biomes of the country.(AU)

Cochliomyia hominivorax (Coquerel, 1858), a mosca-da-bicheira, causa miíase primária em animais silvestres e domésticos em regiões tropicais e subtropicais do Brasil. Embora esta espécie seja considerada de ocorrência em todo o país, informações organizadas sobre sua distribuição com base em registros não estavam disponíveis até o momento. Este artigo teve como objetivo fornecer uma revisão abrangente dos dados históricos e atuais publicados sobre o registro de estágios imaturos (miíase) e adultos de C. hominivorax, no Brasil. De 174 artigos encontrados, 141 relataram casos de miíase em bovinos (146 relatos), humanos (68 relatos) e outros mamíferos hospedeiros (40 relatos) e capturas de adultos de C. hominivorax foram registradas em 33 artigos. C. hominivorax encontra-se amplamente distribuída no Brasil, tendo sido registrada em 208 municípios brasileiros em todos os principais biomas do país.(AU)

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Larvae (maggots) of Cochliomyia hominivorax, the New World Screwworm fly, are voracious consumers of living flesh that have a negative economic impact by decreasing productivity, predisposing to other pathogens, and, in severe cases, causing death of domestic livestock. Screwworm caused extensive financial losses to the livestock industry in North America prior to its eradication. Sterile insect technique (SIT) was used to eradicate screwworm throughout North and Central America and continues to be the main tool to control it in eastern Panama. The goal of this study was to evaluate the temporal and spatial trends of screwworm myiasis cases reported in the Province of Darien and Comarca Embera (border with Colombia), Panama, from 2001 to 2011. We hypothesized that screwworm cases would vary seasonally and be spatially clustered near Colombia as a result of effective eradication strategies in Panama and the presence of an autochthonous population of flies in western Colombia. Temporal and spatial data were retrieved from COPEG-USDA records (Panama) and analysed by anova, Ripley's K function, discrete Poisson spatial statistic scan and Getis-Ord Gi*. No significant temporal trend was found, but cases were spatially distributed in four clusters. One cluster of cases occurred from 2001 to 2003 and was considered a focal temporal and spatial cluster. One cluster occurred in 2001 and 2007 indicating more rare outbreaks in an area with fewer cattle. The two remaining clusters contained cases from 2004 to 2011 and 2001 to 2011 suggesting regular breaks in the control barrier due to occasional failures of the SIT programme, difficulties implementing border quarantine strategies, livestock smuggling or the movement of infested wildlife.

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MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies.

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The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30μg, 40μg and 50μg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30μg, 40μg e 50μg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases [...] (AU)

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RESUMO

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

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