Trypanosoma cruzi: quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans

This paper describes the development stages and numbers of flagellates of two strains of Trypanosoma cruzi living in the small intestine and rectum of the insect, Triatoma infestans, during the first 12 weeks postinfection (pi). Mainly epimastigotes and occasionally amastigotes and final trypomastigotes developed in the small intestine but after starvation periods of 3 or 4 weeks higher percentages of spheromastigotes including their transitional forms to/from epimastigotes were found. In the rectum, the percentage of final trypomastigotes increased in two steps; the second, but not the first, correlated with the development of intermediates originating from epimastigotes. For both strains the total number in the small intestine increased during the first 8 or 9 weeks, although there were reduced numbers when the bugs had starved for 3 or 4 weeks. In the rectum the numbers increased up to 10 weeks pi; only about 25% of these lived in the lumen, the others were located at the rectal wall. In small intestine and rectum the "Chile 5" strain of T. cruzi (zymodeme 1) nearly always reached higher population densities than the "Chile 7" strain (zymodeme 2).     

Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans

Population density and percentage of the different stages of an established infection of Trypanosoma cruzi were determined for two parts of the excretory system and for the rectum of fifth instars of Triatoma infestans unfed and 4 hr after feeding. These data were also evaluated for feces and urine of the fed bugs. In the first unfed group only small populations of the flagellate occurred in the Malpighian tubules and ampullae and not in all bugs. The three rectal populations (rectal lumen and anterior and posterior rectal wall) consisted of approximately equal numbers. About 10% were spheromastigotes and about 10% were stages intermediate to epimastigotes. Significantly fewer epimastigotes and more trypomastigotes were present on the rectal wall than in the lumen. Two intermediate forms leading to the trypomastigote stage occurred in similar numbers. In nearly all bugs the initial excretion (feces) contained the highest number of flagellates as compared to the following drops of urine. More flagellates were excreted through the urine than were contained in the excretory system of unfed bugs. The population in the feces reflected the percentage of forms present in the rectal lumen of unfed bugs, but in the urine the percentage of trypomastigotes increased up to 100%. Four hours after blood uptake, dissection of bugs still showed parasites in the Malpighian tubules and ampullae; the total number of parasites in the rectum was reduced by more than 50%. This reduction was more pronounced in the rectal lumen and on the posterior rectal wall. In stained smears from all three rectal populations there were rarely spheromastigotes but high percentages of epimastigotes. The intermediate stages leading to trypomastigotes mainly originated from short epimastigotes. Comparison of the T. cruzi populations before and after feeding demonstrates that the trypomastigotes in the urine should originate from the rectal wall, especially from the posterior part.     

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi in Triatoma infestans (Insecta, Hemiptera).

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi, which colonise the intestinal tract of the blood-sucking bug Triatoma infestans, was investigated. In established infections of controls without azadirachtin treatment, the small intestine of fifth-instar T. infestans contained up to 7 x l0(6) B. triatomae and the rectum 3 x 10(6). In comparison to this homoxenous flagellate, the population densities of T. cruzi in the respective regions were 99.3 and 76% lower. Treatment with azadirachtin (1 microg ml(-1)) via a blood meal and a concurrent infection with B. triatomae resulted in an increase of the population density (3 weeks p.i.), caused mainly by the mastigote stages in the rectum. In an established B. triatomae infection (12 weeks p.i.), feeding of azadirachtin did not affect the population density and composition. In an optimal T. cruzi-vector system, i.e. parasite and bug originate from the same locality, the treatment with azadirachtin at 20 weeks p.i. strongly reduced the population density in the small intestine of all bugs up to 100 days after treatment, but only in a minor percentage of the bugs in the rectum. Trypanosoma (cruzi incubated for up to 24 h in faeces of azadirachtin-treated bugs were not affected, indicating that the rectum of these bugs contained no toxic substances. The importance of these findings is that investigations of the mechanisms of action of azadirachtin offer a possibility to identify vector-derived compounds, which are necessary for the development of T. cruzi, thereby, giving us a possible new strategy to combat Chagas' disease.     

Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae

The effects of azadirachtin and ecdysone on the Trypanosoma cruzi population in the Rhodnius prolixus gut were investigated. T. cruzi were rarely found in the gut compartments of azadirachtin-treated larvae. High parasite numbers were observed in the stomach of the control and ecdysone groups until 10 days after treatment and in the small intestine and rectum until 25 days after treatment. High percentages of round forms developed in the stomachs of all groups, whereas azadirachtin blocked the development of protozoan intermediate forms. This effect was counteracted by ecdysone therapy. In the small intestine and rectum, epimastigotes predominated for all groups, but more of their intermediates developed in the control and ecdysone groups. Azadirachtin supported the development of round forms and their intermediates into trypomastigotes. In the rectum, trypomastigotes did not develop in the azadirachtin group and developed much later after ecdysone therapy. The parallel between the effects of azadirachtin and ecdysone on the host and parasite development is discussed on the basis of the present results because ecdysone appears to act directly or indirectly in determining the synchronic development of T. cruzi forms from round to epimastigotes, but not metacyclic trypomastigotes, in the invertebrate vector.     

Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids

In the intestinal tract of fifth instars of the hematophagous reduviid bugs Rhodnius prolixus and Triatoma infestans blood ingestion induced an initial decrease of the concentration of the respective symbiotic bacteria Rhodococcus rhodnii and Nocardia sp. and then within 10 days a 15- or 18-fold increase of the total population/bug to about 0.8 x 10(9) colony-forming units in R. prolixus and 1.8 x 10(9) colony-forming units in T. infestans. About 95-99% of the total populations of both symbionts developed in the anterior midgut regions, i.e., cardia and stomach. The passage from the blood-storing stomach to the digesting small intestine caused a considerable breakdown of symbiont populations, and only about 0.01% of the total population was present in the rectum. These were excreted mainly within 4 h after a blood meal. After infection with three species of trypanosomatids, R. rhodnii, the symbiont of R. prolixus, was affected by Trypanosoma rangeli, but not by Blastocrithidia triatomae or Trypanosoma cruzi. On the other hand, in T. infestans the concentration of Nocardia sp. was reduced after infection with B. triatomae, but not by T. rangeli nor T. cruzi. In long-term B. triatomae-infected T. infestans, this reduction and a reduced diuretic activity after feeding synergistically lowered the symbiont concentration in the singly deposited feces/urine drops drastically compared to uninfected controls. These data strongly support the theory of the mechanisms of pathogenicity of T. rangeli and B. triatomae for R. prolixus and T. infestans, respectively, that the primal point of attack is the host-specific symbiont, R. rhodnii and Nocardia sp., respectively.     

The Life Cycle of Trypanoplasma bullocki (Zoomastigophorea: Kinetoplastida)1

The leech Calliobdella vivida (Verrill) is the vector of Trypanoplasma bullocki. At 10°C, infective-stage flagellates were first present in the leech's proboscis sheath five days after feeding. At 5°C, infective-stage flagellates were not present in the leech's proboscis sheath until 10 days after feeding, but at 20°C, flagellates were located there as early as 24 h after feeding. Infected leeches retained flagellates through three subsequent feeds on uninfected fish. When flagellates were first observed in hogchoker, Trinectes maculatus (Bloch & Schneider), they were much larger than infective stages from the leech. Average flagellate length then decreased during early acute phase, but gradually increased thereafter. Peak parasitemia was greater in a hogchoker inoculated by only one leech but held at colder temperature than in a hogchoker inoculated by 45 leeches, suggesting that temperature may be more important than inoculum in determining peak parasitemia. Cell division in the fish host is described. SEM studies of fish blood flagellates revealed a pre-oral ridge and a cytostome.     

On the toxicokinetics and the metabolism of deoxynivalenol (don) in the pig

Eleven castrated male pigs weighing 88.1?±?3.9?kg on average were adapted to a diet containing DON (4.2?mg DON/kg) over a period of 7 days. Feed was given restrictively with 1.1?kg per meal (two meals per day). On the day of measurement, all pigs were slaughtered at different time intervals following the morning meal containing DON (1, 2, 3, 4, 5, 6, 8, 15, 18 and 24?h after feeding), with the exception of one pig which was slaughtered unfed. DON and de-epoxy-DON were analysed in serum and digesta from consecutive segments of the digestive tract (stomach, small intestine divided into three parts of a similar length, caecum, colon, rectum). DON was rapidly and nearly completely absorbed while passing through the stomach and the proximal small intestine. Maximum serum concentration appeared 4.1?h after the DON-containing meal and half of the systemically absorbed DON was eliminated after 5.8?h. De-epoxy-DON appeared in increasing proportions from the distal small intestine and reached approximately 80% of the sum of DON plus de-epoxy-DON in faeces collected from the rectum. It was concluded that de-epoxydation of DON, which primarily occurs in the hindgut, probably does not contribute much to a detoxification in the pig.     

On the toxicokinetics and the metabolism of deoxynivalenol (DON) in the pig

Eleven castrated male pigs weighing 88.1 +/- 3.9 kg on average were adapted to a diet containing DON (4.2 mg DON/kg) over a period of 7 days. Feed was given restrictively with 1.1 kg per meal (two meals per day). On the day of measurement, all pigs were slaughtered at different time intervals following the morning meal containing DON (1, 2, 3, 4, 5, 6, 8, 15, 18 and 24 h after feeding), with the exception of one pig which was slaughtered unfed. DON and de-epoxy-DON were analysed in serum and digesta from consecutive segments of the digestive tract (stomach, small intestine divided into three parts of a similar length, caecum, colon, rectum). DON was rapidly and nearly completely absorbed while passing through the stomach and the proximal small intestine. Maximum serum concentration appeared 4.1 h after the DON-containing meal and half of the systemically absorbed DON was eliminated after 5.8 h. De-epoxy-DON appeared in increasing proportions from the distal small intestine and reached approximately 80% of the sum of DON plus de-epoxy-DON in faeces collected from the rectum. It was concluded that de-epoxydation of DON, which primarily occurs in the hindgut, probably does not contribute much to a detoxification in the pig.     

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera-Reduviidae-Triatominae) IV: bionomic aspects on the vector capacity of nymphs

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.     

Transmission of Herpetomonas in Laboratory Populations of Drosophila melanogaster

Methods of transmission and the effects of temperature and mites on ageledeme development of Herpetomonas were examined in populations of Drosophila melanogaster maintained in the laboratory. Herpetomonas was observed in feces of infected adults taken from population cages and in the vomitus of clean flies shortly after feeding on a saline suspension of flagellates. Free-swimming flagellates were found in the moist areas of food cups. Adult D. melanogaster became infected when they fed on flagellates taken from the endoperitrophic space, the ectoperitrophic space or the Malpighian tubules. At 25°C the flagellates infected approximately 90% of the host population within 20 days. The high transmission rate was prematurely disrupted if host populations were subjected to changes in temperature. Free-swimming flagellates did not appear to be affected at these temperature changes. Food mites (Tyrophagus) established in the growth media of the fly nearly eliminated the Herpetomonas from Drosophila populations.     

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae)

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid–Schiff (PAS–Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS–Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.     

Trypanosoma cruzi: effects of infection on cathepsin D activity in the midgut of Rhodnius prolixus

Cathepsin D activity was estimated in midgut homogenates from Rhodnius prolixus, uninfected and experimentally infected with Trypanosoma cruzi, at different times after blood ingestion. No enzyme activity was found in the anterior midgut and rectum. In the posterior midgut, enzyme activity was found both in lumen and wall. In starved uninfected insects, in lumen and wall, cathepsin D activity was high, decreasing to a constant rate at 1-15 days after feeding. In insects infected with T. cruzi cathepsin D activity increased 1 and 3 days after blood meal. We suggest that these changes in cathepsin D activity in R. prolixus posterior midgut are due to the establishment of T. cruzi infection.     

Dynamics of feeding and defecation in Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) and its potential in the transmission of Trypanosoma cruzi

Adults of Triatoma vitticeps infected by flagellates similar to Trypanosoma cruzi are frequently captured by the inhabitants of rural areas in the Brazilian state of Espirito Santo. The dynamics of feeding and defecation were observed in three groups of adult triatomines, consisting of sylvatic T. vitticeps and laboratory-reared specimens of this species and T. infestans. Triatomines were observed from the moment they were presented with an immobilized chicken as a bloodmeal source until 240 min after feeding had ended. Mean times between the end of feeding and defecation for T. infestans, wild T. vitticeps and laboratory-reared specimens of the latter species were 1.2, 21.1, and 64 min respectively. All T. infestans defecated within 10 min of feeding, while only 29.9 of wild and 52.8% laboratory-reared specimens of T. vitticeps did so within this period. These results may explain the low efficiency of T. vitticeps in T. cruzi transmission to man. The shorter time between feeding and defecation in laboratory-reared T. vitticeps may indicate a change in behaviour of this species as a result of adaptation to an artificial environment.     

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera-Reduviidae-Triatominae) III: patterns of feeding,defecation and resistance to starvation

Data from the Chagas Disease Control Program indicate a growing domiciliary and peridomiciliary invasion of Triatoma rubrovaria in the State of Rio Grande do Sul, where it has become the most frequent triatomine species captured there since the control of T. infestans. Bionomic characteristics that could influence the vectorial capacity of T. rubrovaria as vector of Trypanosoma cruzi were evaluated: patterns of (i) feeding, (ii) defecation, and (iii) resistance to starvation, using insects fed on mice. Fifty three percent of the females showed a defecation pattern conducive to chagasic transmission, defecating either on or near the bite site. The averages of the resistance to starvation varied from 48.1 to 179 days, for the first and fifth nymphal stages, respectively. Our study shows that with respect to the patterns of feeding, defecation and resistance to fasting, T. rubrovaria presented similar rates to the ones observed for other effective vector species, such as T. infestans. Thus, based on our studies we conclude that T. rubrovaria has biological characteristics that can positively influence its capacity to become infected and transmit T. cruzi, and also to keep residual populations after chemical control interventions.     

NPY和POMC基因在生长差异与饥饿再摄食鳙个体中的表达分析

为研究摄食促进基因和摄食抑制基因对鱼类生长调控和饥饿再摄食过程的影响,研究克隆了鳙神经肽Y(HynNPY)基因和前阿黑皮素原(HynPOMC)基因cDNA序列,采用qRT-PCR技术分析它们在生长显著差异鳙个体下丘脑、肠中的基因表达变化;设置对照组(连续投喂4周)、饥饿组、饥饿再摄食组,分析NPY和POMC在不同处理组鳙的下丘脑、肠中的基因表达变化。鳙NPY和POMC基因cDNA全长分别为839和799 bp,开放阅读框有291和657 bp,分别编码96和218个氨基酸。系统进化分析结果表明,鳙NPY和POCM基因具有高度保守性。鳙NPY在下丘脑的表达量最高,其次为肠和脑; POMC在肠道中的表达量最高,其次为下丘脑和肝脏。在相同环境下生长差异鳙个体的下丘脑和肠中, NPY在极大个体的表达量高于极小组个体, POMC在极小个体中的表达量高于极大个体。饥饿导致NPY在下丘脑表达上升,在肠表达量显著上升,恢复摄食后, NPY在下丘脑和肠中表达量下降; POMC在饥饿组下丘脑和肠中都表现为表达量呈显著上升,复投喂后POMC表达量逐步下降至接近对照组水平。肠组织学观察显示,极大个体的肠腔直径...     

Effect of fasting on the structure and function of the gastrointestinal tract of house sparrows (Passer domesticus)

Starvation is a condition that often affects animals in nature. The gastrointestinal tract is the organ system displaying the most rapid and dramatic changes in response to nutrient deprivation. To date, little is known about starvation phases and effects on the organ morphology and digestive function in small passerine birds. In this study, we determined the phases of starvation and examined the effect of final stage of starvation in the organ morphology and, intestinal histology and enzymatic function in the small intestine. Our results show the three phases of the classical model of fasting in a shorter period of time. The mass of heart, pancreas, stomach, small intestine and liver of long-term fasted birds was reduced between 20 and 47%. The mass decrease in small intestine was correlated with reduction in small intestinal histology: perimeter, mucosal thickness, villus height and width. In contrast, the enzyme activity of sucrase-isomaltase and aminopeptidase-N in enterocytes, all expressed per μg of protein, was higher in long-term fasted birds than fed animals. This suggest that, while autophagy of digestive organs is induced by starvation, consistent with phenotypic plasticity, the activity of sucrase-isomaltase and aminopeptidase-N remains high, probably as an anticipatory strategy to optimize digestion at re-feeding time.     

Growth of Trypanosoma cruzi in Cultures of Chick Embryo Cells, and Effects of Furazolidone and Tris (p-Aminophenyl)Carbonium Chloride

SYNOPSIS. Monolayers of cells of coverslips were produced by culturing known numbers of trypsinized chick cells in growth medium (solution 199 plus 20% calf serum) at 37 C for 2 days. The fluid was then replaced with maintenance medium (solution 199 plus 5% calf serum) containing various known numbers of T. cruzi and the preparations were incubated at 33 C for 5 days; fresh maintenance medium was substituted on the 2nd or 3rd day. The inocula of parasites were obtained from T. cruzi -cell cultures, supplemented with 2% sterile NNN overlay, or from NNN cultures.
The numbers of extracellular parasites, proportions of infected cells, and percentage distribution of infected cells relative to the number of intracellular leishmanial bodies were determined on days 2 or 3 and 5 of parasite cultivation in many experiments. Analyses of the data gave the following results. Extracellular parasites increased 2- to 14-fold during the first 2 or 3 days, depending upon the source and size of the inocula, and 10- to 20-fold during the last 2 or 3 days. Cell infection continued throughout incubation at daily rates of 1.4-4.5%; 8-22% of the cells became infected during the 5 days of incubation. Intracellular growth was reflected most clearly by increases in the proportion of cells having >10 leishmanial bodies. This increase was about 5% daily during the last 2 or 3 days of incubation.
A useful test procedure for assessing the antiparasitic action and chick embryo cell toxicity of drugs is illustrated by data obtained with furazolidone and tris ( p -aminophenyl)carbonium chloride.     

Resource allocation and time budgeting in adults of the cockroach,Periplaneta americana: The interaction of behaviour and metabolic reserves

Activity (to food, water and shelter) and resource allocation (mass budget, and size of various body components) were examined in populations of adult Periplaneta americana subjected to periods of starvation or sugar feeding. Following 13 days of starvation, roaches ate 5 times their normal daily ration and feeding required about 20 days to return to pre-starvation levels. When sucrose was substituted for the usual dog-food diet for 13 days, there was also a large increase in feeding initially. When the dog food was re-established, however, there was very little feeding for about one week. Although reproduction was markedly curtailed by starvation, females fed sucrose continued reproducing for at least 59 days. The results suggested that the roaches were mainly energy limited. Although the consumption of roaches was strongly affected by reserve depletion, eating was a small component of the time budget, and overall activity was relatively unchanged by starvation or sugar-feeding. There was a slight decrease in activity during starvation, but the circadian pattern remained unchanged. Females carrying oöthecae were highly active and were apparently attracted to food (even though they ate little). Thus the activity of the population was dominated by circadian rhythmicity and the reproductive cycle.     

禁食对东方蝾螈胃肠道嗜银细胞形态 和分布密度的影响

以改良的龙桂开浸银法,比较了喂食和禁食15 d后的东方蝾螈(Cynops orientalis)胃肠道的长度与重量、嗜银细胞的形态及分布密度的变化。结果表明,禁食后动物的肠鲜重下降,胃长度缩短。禁食处理对嗜银细胞的形态无明显影响,呈圆形、椭圆形、楔形、锥体形、毛笔头状和蝌蚪形等。喂食组的分布密度高峰为幽门,低谷为十二指肠;禁食组的分布密度高峰为贲门,低谷为十二指肠、回肠和直肠。除幽门和十二指肠外,禁食组其余胃肠道各部位的密度都高于喂食组。禁食使东方蝾螈胃肠道前、后端嗜银细胞的密度增加,这可能与东方蝾螈的摄食习性和能较长时间耐受饥饿的生理特性有关。     

Effects of food availability on larval development in the slipper limpet Crepidula onyx (Sowerby)

Effects of food availability on the larval survival and development of Crepidula onyx were studied in four experiments by feeding the larvae with different concentrations of the chrysophyte Isochrysis galbana and by starving the larvae for different periods of time. Food concentration had a clear impact on the survival, growth and development time of C. onyx veligers. Larval development occurred only at 10⁴ cells ml⁻¹ and higher algal concentrations. No shell increment was detected in the veligers cultured for 12 days at 10² cells ml⁻¹I. galbana or the blank control. At 10³ cells ml⁻¹, there was only a slight increase in shell length over 12 days. At 10⁴ cells ml⁻¹, about 40% of the larvae became competent in 18 days. At 10⁵ and 10⁶ cells ml⁻¹, more than 90% of the larvae reached competence in 7 days. Initial starvation negatively affected the larval development, but the sensitivity differed among parameters measured on day 5: lower survivorship was detected only for larvae that had suffered 3 days or longer initial starvation, whereas one-day initial starvation caused shorter shells and lower percentage of competent larvae. Three days of continuous feeding was required for 50% of the larvae to reach competence. After feeding for 3 days, most larvae could become competent to metamorphose even under starvation. The time of starvation was also critical: larvae that suffered 1-day food deprivation in the first 2 days of larval release had shorter shells and lowered percent competent larvae than those that suffered the same length of food deprivation in later stages of development. Our study thus indicates that both food concentration and short-term starvation have detrimental effects on the larval development of this species, and that once the larva has consumed certain amount of food, starvation may induce metamorphosis.