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.     

Cannibalism and coprophagy are modes of transmission of Blastocrithidia triatomae (Trypanosomatidae) between triatomines

Transovarial transmission was not detectable among Blastocrithidia triatomae-infected Triatoma infestans. Rather, B. triatomae was transmitted directly between triatomines by cannibalism and coprophagy. Cannibalism conditions that excluded coprophagy always resulted in an infection of Dipetalogaster maxima. The efficiency of transmission was not influenced by the blood source--mice or chickens--fed to the infected donor bugs although chicken blood lyses the epimastigotes of the stomach population. Triatoma infestans was infected by coprophagy only if fed, not if unfed. Blastocrithidia triatomae in dry feces was taken up only if the feces were redissolved in fresh feces. Infections also appeared in groups of bugs fed on chickens previously used for feeding infected bugs.     

Cannibalism and Coprophagy Are Modes of Transmission of Blastocrithidia triatomae (Trypanosomatidae) between Triatomines

Transovarial transmission was not detectable among Blastocrithidia triatomae- infected Triatoma infestans . Rather, B. triatomae was transmitted directiy between triatomines by cannibalism and coprophagy. Cannibalism conditions that excluded coprophagy always resulted in an infection of Dipetalogaster maxima . The efficiency of transmission was not influenced by the blood source—mice or chickens—fed to the infected donor bugs although chicken blood lyses the epimastigotes of the stomach population. Triatoma infestans was infected by coprophagy only if fed, not if unfed. Blastocrithidia triatomae in dry feces was taken up only if the feces were redissolved in fresh feces. Infections also appeared in groups of bugs fed on chickens previously used for feeding infected bugs.     

Are dead Triatoma infestans a competent vector of Trypanosoma cruzi?

After Triatoma infestans death, Trypanosoma cruzi survived several days, maintaining the ability to infect a vertebrate host. Dead bugs from an endemic area collected during an official spraying campaign showed mobile rectal trypanosomes up to 14 days after vector death. Two days after vector death 2,760 trypomastigotes were found alive in its rectal material. However, the number of mobile trypomastigotes decreased significantly from the 5th day after death. Laboratory proofs with third and fifth nymphal stage showed similar results. Living trypanosomes were found in their rectal material at 10 days in third stage and even at 30 days in fifth nymphal stage. The mean number of trypomastigotes had no changes up to 10 days in third nymphal stage and increased significantly from 1 to 10 days in the fifth stage. Conjunctival instillation as well as intraperitoneal inoculation to mice, of metacyclic forms from dead T. infestans produced infection in the vertebrate host. Present results show that human contact with dead vector is highly probable in summer and living and infective T. cruzi are available for transmission in the vector.     

Ion concentrations in oviduct and uterine fluid and blood serum during the estrous cycle in the bovine

In the bovine up to 40% of embryos die before implantation but despite the importance of ions in oviduct and uterine fluid formation and in gamete, zygote and early embryo development there is very little published information on the ion concentrations of oviduct or uterine fluid. The free anions chloride, phosphate and sulphate and the free cations sodium, calcium, magnesium and potassium were measured in oviduct fluid on days 0, 2, 4 and 6 and in uterine fluid on days 6, 8 and 14 and in corresponding blood samples. Oviduct and uterine fluids were collected in situ. Sodium was 25-fold higher than potassium and 80-fold higher than the other ions and chloride was 10-fold higher than potassium and 40-fold higher than the other ions in oviduct and uterine fluid. Phosphate, sulphate, magnesium, potassium and calcium were at lower concentrations in all fluids. Oviduct calcium and sodium were higher on day 0 than other days. The most striking uterine differences were the higher potassium and lower chloride, sodium and magnesium on day 14 than other days. There were significant positive associations between oviduct and blood chloride, sulphate, magnesium and calcium while only uterine sulphate was positively related to its blood concentration. There was no relationship between fluid secretion rate and no association between the concentrations of systemic progesterone or oestradiol and any ion in oviduct or uterine fluid. The different concentrations and associations between ions in the oviduct, uterus and blood suggest a differential regulation of ion secretion by the oviduct and uterine epithelia.     

Inorganic ions modulate the path of phloem loading of sucrose in Ricinus communis L. seedlings

The impact of inorganic ions on sucrose fluxes in the cotyledons and on the pathway of phloem loading was studied in Ricinus communis L. seedlings. The cotyledons were incubated in defined solutions which contained either potassium, sodium, magnesium or calcium as chloride salts, or the sodium salts of sulphate or phosphate. Sucrose uptake from the medium into the cotyledons was only slightly affected by the salts. Sucrose efflux to the medium was increased by phosphate and sulphate and to a lesser extent by sodium and potassium. Phloem loading from the apoplasm and the symplasm was analysed by addition of labelled sucrose to the medium, determination of the specific radioactivity of sucrose in sieve-tube exudate and quantification of export into the seedling axis. Potassium and sodium stimulated the apoplasmic route of phloem loading of sucrose, mostly at the expense of loading from cotyledon sucrose pools. In contrast, sulphate and phosphate strongly inhibited the apoplasmic route whereas the (small) symplasmic flux from the cotyledon sucrose pools was less affected. Magnesium ions inhibited phloem loading by both pathways. The potential of ions in modulating the pathways of sucrose export in day to day operation of plants is discussed.     

Pathological effects of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans after infection by membrane feeding and long-term starvation

Developmental time and mortality in larvae of the reduviid bug Triatoma infestans were studied in uninfected groups and in those infected with Blastocrithidia triatomnae by in vitro feeding with 10(4) cyst stages/cm(3) blood. Bugs were subsequently subjected to two different starvation periods. In bugs fed at weekly intervals infection with B. triatomae was associated with developmental retardation, increasing in the final instars, and with increased mortality in the third and preadult instar. Compared to these groups, long-term starvation caused slightly lower mortality rates of uninfected groups and in infected bugs a remarkably higher mortality rate in the preadult instar. Total mortality rates of infected groups were nearly unaffected by starvation.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

The Development of Trypanosoma cruzi (Trypanosomatidae) in the Reduviid Bug Triatoma infestans (Insecta): Influence of Starvation

ABSTRACT Fifth instars of Triatoma infestans with established Trypanosoma cruzi infections were dissected after different periods of starvation to determine the population density and the percentage of different developmental stages of T. cruzi in the small intestine and rectum of the bugs. After a short starvation period of 20 days, the population density in the small intestine was 20% (about 60,000) of the rectal population. The population in the small intestine was strongly reduced after an additional ten days of starvation, and no flagellates could be found there 60, 90 and 120 days after the last feeding. In the rectum, this reduction went down to 1% of the initial population, but a total elimination never occurred. Usually the remaining population contained more live than dead flagellates. Starvation also resulted in an increase in the rectum in the number and percentage of drop-like forms, intermediates between sphere- and epi- or trypomastigotes, from 1% initially to about 10% after 90 days of starvation. The percentage of spheromastigotes increased from 2% at 20 days after the last feeding to about 20% after an additional 40 and 70 days. Therefore, the spheromastigotes of T. cruzi seem to be induced by stress conditions.     

Temporal change of the aggregation response in Triatoma infestans

Adults and larvae of Triatoma infestans spend daylight hours assembled in shaded places. An assembling factor has been demonstrated in the excrement of this species. We analysed different aspects of the dynamics of the response of bugs. Recently fed insects do not aggregate around faeces. They start to show a significant assembling response from the 8th hour after feeding onwards. Just deposited faeces do not evoke assembling, but a significant rejection instead. This reaction switches 3 h after deposition, when the faeces become attractive to the insects. The attractiveness of faeces persists for about 10 days and can be recovered after this time by rehydration. These findings are discussed in relation to the biological role of faeces and the dynamics of the use of refuges by T. infestans.     

The postembryonic development of the ocellar system of Triatoma infestans Klug (Heteroptera: Reduviidae)

Simple eyes or ocelli coexist with compound eyes in many adult insects. The change in the morphology of the ocelli along the five larval instars of Triatoma infestans was studied by light and scanning electron microscopy. Our analysis showed that the development of the ocelli of these bugs occurs gradually along the larval life. The photoreceptor layer is present from the second-instar onwards. The cornea appears first at the imaginal stage and grows up to the 18-20th day after the last ecdysis, associated to an increase in the retinal mass. Findings are discussed in a comparative fashion and in relation to the functionality of the ocellar system in T. infestans.     

Sensitivity of the sodium and potassium channels of Myxicola giant axons to changes in external pH

Myxicola giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of GNa was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K+ channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis.     

The role of water vapour in the orientation behaviour of the blood-sucking bug Triatoma infestans (Hemiptera,Reduviidae)

The behavioural response to water vapour of the haematophagous bug Triatoma infestans was analysed. Dry or humid discrete sources at different temperatures were used as stimuli for insects walking on a locomotion compensator. Humidity significantly increased the tendency of these bugs to orientate towards thermal sources. Furthermore, humid sources at room temperature were attractive to T. infestans, but this effect was limited to short-range distances. On the other hand, dynamic sources, i.e. airstreams carrying different water vapour contents did not affect the spontaneous anemotactic behaviour of this species, neither in sign (positive) nor in intensity. The anemotactic behaviour was also not influenced by the physiological water balance state of the bugs. Results are discussed in relation to the cues released by living hosts of triatomine bugs and in relation to their responses to air-currents.     

Scanning electron microscopic studies of Trypanosoma cruzi in the rectum of its vector Triatoma infestans

The cuticular surface of the rectum of Triatoma infestans and its colonization by a Trypanosoma cruzi strain originating from the same locality as the bugs were studied by scanning electron microscopy at different weeks post infectionem. On the basis of the cuticular folding, the rectum can be subdivided into five regions. The rectal gland has the finest structure and the region anterior to the anus the deepest folds. Try. cruzi always prefers to colonize the rectal gland, while the other regions are colonized in varying densities. Most of the flagellates are epimastigotes (long and short), except in the region at the entrance of the midgut, where trypomastigotes predominate.     

Purification and properties of AMP-deaminase from human uterine smooth muscle

AMP-deaminase from human uterine smooth muscle has been isolated, and properties of the enzyme were characterized. At pH 7.0, and in the presence of 100 mM potassium chloride the enzyme manifests a distinctly sigmoidal type of kinetics, with S0.5 parameter value about 12 mM. 1 mM ATP strongly activates the enzyme, and diminishes the value of S0.5 to 1.2 mM. In contrast to that 2.5 mM orthophosphate slightly inhibits the activity of AMP-deaminase studied and increases the S0.5 to about 14 mM. Similarly to ATP, orthophosphate does not influence the maximum velocity of the reaction. Electrophoresis in the presence of sodium dodecyl sulphate revealed that the molecular weight of human smooth muscle AMP-deaminase subunit is close to 37 kDa.     

Chronobiological basis of thermopreference in the haematophagous bug Triatoma infestans

The chronobiological basis of the daily dynamics of thermopreference was tested in adults of Triatoma infestans, continuously registering the preferred temperature of insects released over a temperature gradient for 13 days. We found that the thermopreference in T. infestans is a dynamic process that depends on the time of the day and the post-feeding time. When submitted to a 12:12 h light/darkness cycle (L/D), the preferred temperature reached the highest and the lowest values at the end of the light and dark phases, respectively. This daily rhythm persisted under constant conditions of illumination (D/D and L/L), suggesting the existence of an internal oscillator controlling this behaviour. Statistical analysis revealed that the thermopreference of insects kept under L/D exhibited a ca. 24 h periodicity, while insects kept in D/D and L/L showed free-running periods of tau((D/D))=23.35 and tau((L/L))=27.35 h, respectively. The persistence of a cyclic pattern of thermopreference under constant conditions, and free-running periods, close to, but different from 24 h, demonstrate the existence of an endogenous control of the thermopreference in this species. The biological relevance of these results is discussed in the light of the hypothesis that both the length of time elapsed since feeding and the time of the day modify thermopreference in these bugs. The gradual decline in preferred temperature following feeding may be associated with energy conservation during starvation. The rhythmic modulation of thermopreference may be associated with the daily rhythm of locomotion activity shown by these bugs.     

Growth inhibition ofChlorella pyrenoidosa produced by sodium dihydrogen phosphate and its reversal by calcium

Summary The growth ofChlorella pyrenoidosa in a special medium based on the critical concentrations of nutrients for autotrophic growth has been shown to be stimulated more by chloride with the sodium salt than with the potassium salt, more by sulphate in the presence of sodium than in the presence of potassium, and to be inhibited by sodium dihydrogen phosphate and not by potassium dihydrogen phosphate. Furthermore, it has been found that calcium reversed the growth inhibition produced by sodium dihydrogen phosphate, and that strontium only partially substituted for calcium.     

Mineral uptake and transport in xylem and phloem of the proteaceous tree,Banksia prionotes

Xylem sap of sinker (tap) root, cluster feeding roots, lateral roots and from an age series of main stem extensions of 6-year trees of Banksia prionotes was collected and analyzed for principal organic and inorganic solutes. During the phase of root uptake activity in winter and spring, cluster roots were principal xylem donors of malate, phosphate, chloride, sodium, potassium and amino acid N whereas other parts of the root served as major sources to the shoot of other cations, nitrate and sulphate. Sinker root xylem sap was at all times less concentrated in solutes than that of lateral roots into which cluster roots were voiding exported solutes. Phosphate was abstracted from xylem by stem tissue during winter and it and a range of other solutes released back to xylem immediately prior to extension growth of the shoot in summer. Phloem sap collected from mid regions of stems was unusually low in potassium and phosphate relative to chloride and sulphate in comparison with phloem sap of other species, and its low potassium: sodium ratio relative to xylem indicated poor discrimination against sodium during phloem loading. Data are discussed in relation to the asynchronous seasonal cycles of nutrient uptake and shoot growth.     

Statistical optimization of culture media for growth and lipid production of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> LB572

Botryococcus braunii has an outstanding ability to produce lipid; however, it is a slow-growing green microalgae. Statistical optimization of growth media was performed to faster growth and to increase lipid concentration. The effect of media composition on the growth of B. braunii LB572 was examined using fractional factorial design and central composite design. The media components examined include sodium carbonate, potassium phosphate, calcium chloride, magnesium sulfate, ferric citrate, and sodium nitrate. The results indicated that potassium phosphate and magnesium sulfate were major impact factors. The optimum concentrations of potassium phosphate and magnesium sulphate were found to be 0.058 and 0.09 g/L, respectively, for growth and 0.083 and 0.1 g/L, respectively, for lipid production. These values were validated using bubble column photobioreactors. Lipid productivity increased to 0.19 g/L/day in lipid-optimized media, with an average biomass productivity of 0.296 g/L/day and 64.96% w/w. In growth-optimized media, lipid productivity was 0.18 g/L/day, with an average biomass productivity of 0.304 g/L/day and 59.56% w/w.