Design and Synthesis of Novel 3-Nitro-1H-1,2,4-triazole-1,2,3-triazole-1,4-disubstituted Analogs as Promising Antitrypanosomatid Agents: Evaluation of In Vitro Activity against Chagas Disease and Leishmaniasis.

A series of 28 compounds, 3-nitro-1H-1,2,4-triazole, were synthesized by click-chemistry with diverse substitution patterns using medicinal chemistry approaches, such as bioisosterism, Craig-plot, and the Topliss set with excellent yields. Overall, the analogs demonstrated relevant in vitro antitrypanosomatid activity. Analog 15g (R1 = 4-OCF3-Ph, IC50 = 0.09 µM, SI = >555.5) exhibited an outstanding antichagasic activity (Trypanosoma cruzi, Tulahuen LacZ strain) 68-fold more active than benznidazole (BZN, IC50 = 6.15 µM, SI = >8.13) with relevant selectivity index, and suitable LipE = 5.31. 15g was considered an appropriate substrate for the type I nitro reductases (TcNTR I), contributing to a likely potential mechanism of action for antichagasic activity. Finally, 15g showed nonmutagenic potential against Salmonella typhimurium strains (TA98, TA100, and TA102). Therefore, 3-nitro-1H-1,2,4-triazole 15g is a promising antitrypanosomatid candidate for in vivo studies.

Diagnosis of Cutaneous Leishmaniasis Using FTIR Spectroscopy and Machine Learning: An Animal Model Study.

Cutaneous leishmaniasis (CL) is a polymorphic and spectral skin disease caused by Leishmania spp. protozoan parasites. CL is difficult to diagnose because conventional methods are time-consuming, expensive, and low-sensitive. Fourier transform infrared spectroscopy (FTIR) with machine learning (ML) algorithms has been explored as an alternative to achieve fast and accurate results for many disease diagnoses. Besides the high accuracy demonstrated in numerous studies, the spectral variations between infected and noninfected groups are too subtle to be noticed. Since variability in sample set characteristics (such as sex, age, and diet) often leads to significant data variance and limits the comprehensive understanding of spectral characteristics and immune responses, we investigate a novel methodology for diagnosing CL in an animal model study. Blood serum, skin lesions, and draining popliteal lymph node samples were collected from Leishmania (Leishmania) amazonensis-infected BALB/C mice under experimental conditions. The FTIR method and ML algorithms accurately differentiated between infected (CL group) and noninfected (control group) samples. The best overall accuracy (∼72%) was obtained in an external validation test using principal component analysis and support vector machine algorithms in the 1800-700 cm-1 range for blood serum samples. The accuracy achieved in analyzing skin lesions and popliteal lymph node samples was satisfactory; however, notable disparities emerged in the validation tests compared to results obtained from blood samples. This discrepancy is likely attributed to the elevated sample variability resulting from molecular compositional differences. According to the findings, the successful functioning of prediction models is mainly related to data analysis rather than the differences in the molecular composition of the samples.

Design, synthesis and antitrypanosomatid activity of 2-nitroimidazole-3,5-disubstituted isoxazole compounds based on benznidazole.

Chagas disease and leishmaniasis are neglected diseases of high priority as a public health problem. Pharmacotherapy is based on the administration of a few drugs, which exhibit hazardous adverse effects and toxicity to the patients. Thus, the search for new antitrypanosomatid drugs is imperative to overcome the limitations of the treatments. In this work, 46 2-nitroimidazole 3,5-disubstituted isoxazole compounds were synthesized in good yields by [3 + 2] cycloaddition reaction between terminal acetylene (propargyl-2-nitroimidazole) and chloro-oximes. The compounds were non-toxic to LLC-MK2 cells. Compounds 30, 35, and 44 showed in vitro antichagasic activity, 15-fold, 12-fold, and 10-fold, respectively, more active than benznidazole (BZN). Compounds 30, 35, 44, 45, 53, and 61 acted as substrates for the TcNTR enzyme, indicating that this might be one of the mechanisms of action involved in their antiparasitic activity. Piperazine series and 4-monosubstituted compounds were potent against T. cruzi parasites. Besides the in vitro activity observed in compound 45, the in vivo assay showed that the compound only reduced the parasitemia levels by the seventh-day post-infection (77%, p > 0.001) compared to the control group. However, 45 significantly reduced the parasite load in cardiac tissue (p < 0.01) 11 days post-infection. Compounds 49, 52, and 54 showed antileishmanial activity against intracellular amastigotes of Leishmania (L.) amazonensis at the same range as amphotericin B. These findings highlight the antitrypanosomatid properties of 2-nitroimidazole 3,5-disubstituted isoxazole compounds and the possibility in using them as antitrypanosomatid agents in further studies.

In Vivo Antileishmanial Effect of 3,5-Diaryl-isoxazole Analogues Based on Veraguensin, Grandisin, and Machilin G: A Glance at a Preclinical Study.

New treatment approaches targeting cutaneous leishmaniasis (CL) are required since conventional drugs exhibit limitations due to their several adverse effects and toxicity. In this study, we aimed to evaluate the in vivo intralesional treatment efficacy of five isoxazole derivatives previously synthesized and effective in vitro against intracellular amastigote forms of Leishmania (L.) amazonensis. Among the tested analogues, 7 exhibited relevant in vivo therapeutic effects. The in silico predictions provided interesting information about the toxicity, suggesting the safety of analogue 7. Experiments performed with Salmonella typhimurium strains (TA98, TA100, and TA102) showed a non-mutagenicity profile of 7. The treatment of Leishmania-infected BALB/c mice with isoxazole 7 showed remarkably smaller CL lesions and decreased the parasitism (by 98.4%) compared to the control group. Hence, analogue 7 is a promising drug candidate and alternative treatment for CL caused by L. amazonensis.

Assessment of equine piroplasmids in the Nhecolândia sub-region of Brazilian Pantanal wetland using serological, parasitological, molecular, and hematological approaches.

Brazilian Pantanal is the world´s largest wetland ecosystem, where cattle's ranching is the most important economic activity. The objective of this study was to compile some epidemiological features on equine piroplasmids from the Nhecolândia sub-region of Pantanal wetland through the evaluation of the patterns of T. equi and B. caballi infections in different groups of horses; identification of the tick species that infest horses; and to study phylogenetic relationships among Theileria equi 18S rRNA gene sequences. During October 2015, blood and serum samples were collected from 170 horses in four different categories. Ticks, after identification, had their hemolymph and eggs examined for the presence of piroplasmid sporokinets. Also we searched parasites in the peripheral blood smears of the investigated horses. The number of red blood cells (RBCs) and the packed cell volume (PCV) ​​were determined to test for anemia in the infected animals, and exposure to B. caballi and T. equi was evaluated by enzyme-linked immunosorbent assay. "Catch all primers" based on 18S rRNA gene were used in polymerase chain reactions (PCR) to detect equine piroplasmids, followed by three nested PCRs for the phylogenetic analysis. The serological results showed that 61.8% and 52.9% of the horses sampled were exposed to T. equi and B. caballi, respectively. Piroplasmid DNA was detected in 43.5% of the horses analyzed. Our sequencing revealed 98-100% identity with some sequences previously published in GenBank for T. equi, and microheterogeneity among others. We found that 51.2% of the animals sampled were infested with Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus (Boophilus) microplus, singly or co-infested. Since positive and negative animals presented similar RBC and PCV values, and no sporokinets were found on blood smears, hemolymph and eggs of the ticks collected, we suggest that infected equines can act as asymptomatic carriers for piroplasmosis in the studied region. Our results together showed the enzootic characteristic of equine piroplasmids in Pantanal region highlighting the importance of using different methods for detection these parasites. Moreover, breeding mares and foals should be monitored since they displayed the greatest occurrences for molecular test (59.0% and 86.1% respectively) and tick infestations (87.2% and 63.9% respectively).

Sugarcane stems as larval habitat for the stable fly (Stomoxys calcitrans) in sugarcane plantations / Colmos de cana-de-açúcar como habitat para o desenvolvimento de larvas de mosca-dos-estábulos (Stomoxys calcitrans) em plantações de cana-de-açúcar

Outbreaks of stable fly, Stomoxys calcitrans, cause losses for livestock producers located near sugarcane mills in Brazil, especially in southern Mato Grosso do Sul. The sugarcane mills are often pointed by local farmers as the primary source of these outbreaks; some mills also joined the farmers in combating the flies. Brazilian beef cattle production has great economic importance in similar level to bio-fuel production as ethanol. In this context, the wide-ranging knowledge on the biology and ecology of the stable fly, including larval habitats and their reproduction sites is extremely important for further development of control programs. This paper aims to report the occurrence and development of S. calcitrans larvae inside sugarcane stems in three municipalities of Mato Grosso do Sul. The sugarcane stems give protection against bad weather conditions and insecticide application. In this way, for sustainable sugarcane growth specific research concerning this situation should be conducted.

Surtos de mosca dos estábulos, Stomoxys calcitrans, vêm causando prejuízos em fazendas de pecuaria localizadas próximas as usinas de cana no Brasil, especialmente no sul do Mato Grosso do Sul. As usinas são frequentemente apontadas pelos pecuarístas locais, como a principal fonte desses surtos e algumas destas usinas também se uniram aos agricultores no combate a este parasita. No Brasil, a produção de gado de corte tem grande importância econômica em nível semelhante ao de bio-combustíveis como o etanol. Neste contexto, o conhecimento amplo sobre a bioecologia, incluindo os habitats das larvas e os seus locais de reprodução, é extremamente importante para o desenvolvimento de programas de controle para a mosca dos estábulos. Este trabalho teve como objetivo registrar que larvas de S. calcitrans podem desenvolver dentro do colmo da cana. Larvas foram encontradas dentro de colmos de cana de açúcar em três oportunidades em diferentes cidades. O colmo da cana pode dar proteção para as larvas em condições climáticas desfavoráveis e aplicação de inseticidas. Desta forma, para o crescimento sustentável destas atividades, mais pesquisas específicas focadas nestas relações devem ser realizadas.

Experimental infection of Rhipicephalus sanguineus ticks with the bacterium Rickettsia rickettsii, using experimentally infected dogs.

We evaluated if Rickettsia rickettsii-experimentally infected dogs could serve as amplifier hosts for hipicephalus sanguineus ticks. In addition, we checked if Rh. sanguineus ticks that acquired Ri. rickettsii from dogs could transmit the bacterium to susceptible hosts (vector competence), and if these ticks could maintain the bacterium by transstadial and transovarial transmissions. Uninfected larvae, nymphs, and adults of Rh. sanguineus were allowed to feed upon three groups of dogs: groups 1 (G1) and 2 (G2) composed of Ri. rickettsii-infected dogs, infected intraperitoneally and via tick bites, respectively, and group 3 composed of uninfected dogs. After larval and nymphal feeding on rickettsemic dogs, 7.1-15.2% and 35.8-37.9% of the molted nymphs and adults, respectively, were shown by polymerase chain reaction (PCR) to be infected by Ri. rickettsii, confirming that both G1 and G2 dogs were efficient sources of rickettsial infection (amplifier host), resulting in transstadial transmission of the agent. These infected nymphs and adults successfully transmitted Ri. rickettsii to guinea pigs, confirming vector competence after acquisition of the infection from rickettsemic dogs. Transovarial transmission of Ri. rickettsii was observed in engorged females that had been infected as nymphs by feeding on both G1 and G2 dogs, but not in engorged females that acquired the infection during adult feeding on these same dogs. In the first case, filial infection rates were generally <50%. No tick exposed to G3 dogs was infected by rickettsiae in this study. No substantial mortality difference was observed between Ri. rickettsii-infected tick groups (G1 and G2) and uninfected tick group (G3). Our results indicate that dogs can be amplifier hosts of Ri. rickettsii for Rh. sanguineus, although only a minority of immature ticks (<45%) should become infected. It appears that Rh. sanguineus, in the absence of horizontal transmission, would not maintain Ri. rickettsii through successive generations, possibly because of low filial infection rates.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings.

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

The effect of temperature and fasting period on the viability of free-living females of Rhipicephalus sanguineus (Acari: Ixodidae) under laboratory conditions.

Little is known about the effect of temperature on viability of free-living phases of the life cycle of Rhipicephalus sanguineus (Latreille, 1806) despite of its importance as vector of several pathogens. Knowledge of the effect of abiotic factors on the capacity of a given tick species to infest new hosts is important for routine experimental activities under laboratory conditions, and may be relevant to understand the transmission of pathogens. The study evaluates the viability of R. sanguineus females held at 18 +/- 1, 27 +/- 1 and 32 +/- 1 degrees C and 80 +/- 5% RH (saturation deficits of 3.0, 5.3 and 7.2 mmHg, respectively) for three fasting periods (3 and 20 days and the day when female mortality reached approximately 50% after ecdysis), under laboratory conditions. In general, the best result on viability was obtained when rabbits were infested with unfed female ticks after three or 20 fasting days at both 27 +/- 1 and 32 +/- 1 degrees C and 80 +/- 5% RH.

Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO(2) tick trap.

This study evaluates the impact of cattle-raising on ticks associated with wildlife in the Pantanal region of Brazil, by trapping free-living ticks using a CO(2) tick trap. The traps were equally distributed in two areas: a 600-ha protected area (PA) and the Nhumirim Ranch (NR). Both areas are contiguous and located at the sub-region of Nhecolândia (18 degrees 59'S; 56 degrees 39'W), Pantanal, Brazil. In each area, four habitat types were selected: cerrado (tropical savanna), forest patches, lakes and soda lakes. The last two are temporary lagoons of fresh and salted water, respectively. A total of 980 nymphs, 613 adults and 13 larvae of ticks Amblyomma were collected in 256 h of collection. In a pen of calves infested by Ornithodoros rostratus 114 specimens were collected, including larvae, nymphs and adults. Adults of A. cajennense and A. parvum were more abundant in the savanna at NR, but not differed among habitat types at PA. There was a tendency of having more nymphs in NR than in PA. The higher number of ticks found in the NR in comparison to the PA is not due to the presence of cattle itself but probably due to introduced dogs and horses, habitat alteration.

Influência do comportamento gregário nos processos de muda de Rhipicephalus sanguineus (Latreille, 1806) (acari: ixodidae), em condições laboratoriais / Influence of aggregation on the molting process of Rhipicephalus sanguineus, (Latreille, 1806) (acari: ixodidae) under laboratory conditions

Objetivou-se com o experimento verificar a influência de diferentes agregações sobre os parâmetros relacionados ao processo de ecdise ninfal e de adulto de Rhipicephalus sanguineus (Latreille, 1806). Foram utilizados como hospedeiros 10 coelhos domésticos (Oryctolagus cuniculus) primos-infestados. Foram analisados os períodos de pré-ecdise, de ecdise e de muda, o percentual e o ritmo de ecdise para todos os estágios estudados. Durante toda a fase não-parasitária, os carrapatos foram mantidos em condições controladas de umidade (80±5 por cento) e temperatura (27±1ºC). Um total de 1560 larvas ingurgitadas e de 1560 ninfas ingurgitadas foi utilizado. Os carrapatos foram divididos em seis grupos contendo um, cinco, dez, 20, 40 e 80 espécimes cada. Para cada um desses grupos foram realizadas dez repetições. Os processos de ecdise ninfal e de adulto foram acompanhados diariamente. Após a análise dos resultados, verificou-se que não houve relação entre as diferentes agregações e o percentual de ecdise em nenhuma etapa experimental. Em todos os outros parâmetros biológicos estudados, verificaram-se diferenças significativas (p<0,05). Os resultados obtidos sugerem que em elevadas agregações o processo de ecdise tende a ser mais rápido.

The objective of this experiment was to evaluate the influence of different aggregations on the molting process of Rhipicephalus sanguineus (Latreille, 1806). Tick-naïve domestic rabbits (Oryctolagus cuniculus) were used as experimental hosts for the parasitic phase. A total of 10 rabbits were used on the experiment. All tick free-living phases were kept in incubator at 27±1ºC, 80±5 percent relative humidity and scotophase. The periods of pre-molt, molt, percentage of ecdisis and molt rhythm of larvae and nymphs were studied. A total of 1560 engorged larvae and 1560 nymphs of R. sanguineus were divided in six groups of aggregations: 1, 5, 10, 20, 40 and 80 ticks with ten repetitions each one. The ecdises of nymphs and adults were daily observed. The percentage of ecdysis was the only parameter with no aggregations effects. All others parameters were statistically different (p<0,05). The results suggested that highest aggregations accelerate the molting process.

[Influence of aggregation on the molting process of Haemaphysalis leporispalustris (Acari: Ixodidae) under laboratory conditions]. / Influência do comportamento gregário nos processos de muda de Haemaphysalis leporispalustris (Acari: Ixodidae) em condições laboratoriais.

The aim of the experiment was to evaluate the influence of different aggregations on the molting process of the nidicoulus ticks, Haemaphysalis leporispalustris. Tick-naïve domestic rabbits (Oryctolagus cuniculus) were used as experimental hosts for the parasitic phase. All free-living phases were kept into 4.5 mL glass vials in incubators at 27+/-1 degrees C, 80+/-15% relative humidity and scotophase. The periods of pre-molt, molt, percentage of ecdisis and molting pattern of larvae and nymphs were studied. A total of 1560 engorged larvae and nymphs were divided in six groups of aggregations: 1, 5, 10, 20, 40 and 80 ticks with ten repetitions. The molting of nymphs and adults were observed daily. The percentage of ecdysis was the only parameter with no statistical difference among the groups (p>0.05). All others parameters were statisticaly different (p<0.05). The results suggested that highest aggregations accelerate the molting process of H. leporispalustris. We recommended the following aggregations: 4.4 ticks/ml for scientific research and less than 7.7 tick/ml for ticks maintenance.

[Influence of aggregation on the molting process of Rhipicephalus sanguineus, (Latreille, 1806) (Acari: Ixodidae) under laboratory conditions]. / Influência do comportamento gregário nos processos de muda de Rhipicephalus sanguineus (latreille, 1806) (acari: ixodidae), em condições laboratoriais.

The objective of this experiment was to evaluate the influence of different aggregations on the molting process of Rhipicephalus sanguineus (Latreille, 1806). Tick-naïve domestic rabbits (Oryctolagus cuniculus) were used as experimental hosts for the parasitic phase. A total of 10 rabbits were used on the experiment. All tick free-living phases were kept in incubator at 27+/-1 degrees C, 80+/-5% relative humidity and scotophase. The periods of pre-molt, molt, percentage of ecdisis and molt rhythm of larvae and nymphs were studied. A total of 1560 engorged larvae and 1560 nymphs of R. sanguineus were divided in six groups of aggregations: 1, 5, 10, 20, 40 and 80 ticks with ten repetitions each one. The ecdises of nymphs and adults were daily observed. The percentage of ecdysis was the only parameter with no aggregations effects. All others parameters were statistically different (p<0,05). The results suggested that highest aggregations accelerate the molting process.

[The effect of immersion in distilled water on the engorged larvae and nymphs of Amblyomma dubitatum Neumann, 1899 (Acari: Ixodidae)]. / Efeito da imersão de larvas e ninfas ingurgitadas de Amblyomma dubitatum Neumann, 1899 (Acari: Ixodidae) em água destilada.

To evaluate the effect of the immersion of engorged larvae and nymphs of Amblyomma dubitatum experimentally fed on rabbits, the ticks were collected and formed control groups, without immersion, and groups witch were immersed by 24, 48, 72, 96 hours and, for engorged larvaes, by eight weeks, in test tubes containing distilled water. During all experiment ticks were kept at 27 +/- 1 degrees C, 80 +/- 10% RU and scotophase The parameters evaluated were: pre-ecdysis, ecdysis and moulting periods; and ecdysis and mortality percentages. When compared with the control group, the effect (p<0,05) of immersion varied intra and inter stages. Apparently, immersion was less deleterious to A. dubitatum in comparison with A. cajennense and R. sanguineus from neotropical region, thus reflecting a long host relations with the capybara, its primary host.

[Infestation with Linognathus africanus (Kellogg e Paine, 1911) (Linognathidae) and Bovicola caprae (Ewing, 1936) (Trichodectidae) in goat flock of the State of Rio de Janeiro, Brazil]. / Infestações por Linognathus africanus (Kellogg e Paine, 1911) (Linognathidae) e Bovicola caprae (Ewing, 1936) (Trichodectidae) em rebanho caprino no Estado do Rio de Janeiro.

Mixed infestations with Linognathus africanus and Bovicola caprae were diagnosed in a lot of 20 goats from a flock of 40, belonging to a farm from the Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ (22 degrees 44'38''S and 43 degrees 42'27''W). Animals were of both sexes, varied ages and different breeds (half blood Saanen/Boer, Saanen, cross breeds), were examined in April of 2004. With an exception of one goat, parasitized only by L. africanus, the remainders 19 (95%) were infested by the two species lice. Of 69 (71%) specimens of L. africanus and 28 (28.9%) of B. caprae collected the ratio female:male for L. africanus were 4.6 (23:5), while for B. caprae were 4.8 (57:12). The absence of L. africanus in recent surveys carried out in the semi-arid regions of the Northeast Brazil where more the 60% of examined goats were parasitized by B. caprae suggests that the adaptation to the climatic variation might be different for the two species.

[The effect of immersion in distilled water on the engorged larvae and nymphs of Amblyomma cajennense (Fabricius,1787)(Acari: Ixodidae)]. / Efeito da imersão de larvas e ninfas ingurgitadas de Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) em água destilada.

To evaluate the effect of the immersion of engorged larvae and nymphs of Amblyomma cajennense, these stages were immersed by 24, 48, 72 and 96 hours in test tubes containing distilled water. The period of premolt of the nymphs increased proportionally to the time of immersion (p<0.05). The ecdisis period, the longevity and the mortality (days) were similar in all of treatments (p>0.05). The percentage of ecdisis in nymphs was reduced after 72 hours of immersion of the larvae (p<0.05). None of the engorged nymphs survived 96 hours of immersion. The period of the adult premolt increased after 48 hours of immersion (p<0.05). The ecdisis period was similar in the treatments of 24 and 48 hours of immersion (p>0.05). The percentage of ecdisis decreased starting from 24 hours of immersion (p<0.05). There was not statistical difference (p<0.05) between the control group and the 24 hours group of immersion for the parameters longevity and mortality. These findings suggest that the engorged nymphs were more sensitive to the immersion that the engorged larvae.