The effect of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: influence of group size

Developmental time and mortality in uninfected larvae of the reduviid bug Triatoma infestans and in those infected by feeding a mixture of blood and cysts of the homoxenous trypanosomatid Blastocrithidia triatomae were compared. Larvae were maintained isolated in 77-cm3 (area 9.6 cm2) beakers or in groups of 20, 30, 40, and 50 bugs per 1-liter beaker (area 722 cm2). In uninfected groups, only a minor proportion of isolated bugs showed delayed development, but in groups infected with B. triatomae, additionally, a retardation in groups of 50 larvae occurred. Infected bugs needed more time to complete development in fourth and fifth instar than did uninfected bugs. Mean mortality rates of about 10% in uninfected groups were unaffected by group size. Mortality rates in most groups of infected bugs were about 50%, but in groups consisting of 50 larvae a statistically significant higher mortality rate of 75% was observed. This indicates a subpathological overcrowding stress, increased by the synergistic action of the flagellate.     

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.     

Pathological effects of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bugs Triatoma sordida, T. pallidipennis and Dipetalogaster maxima after coprophagic infection

ABSTRACT. Developmental time and mortality in nymphs of the reduviid bugs Triatoma sordida (Stål), Triatoma pallidipennis (Stål) and Dipetalogaster maxima (Uhler) were studied in uninfected groups and in those infected with Blastocrithidia triatomae Cerisola et al. (Trypanosomatidae). In T. sordida and T. pallidipennis, major vectors of Chagas' disease in Brazil and Mexico respectively, infection with B. triatomae was associated with slight developmental retardations in the final instars, and increased mortality in the pre-adult instar. These effects were less evident in T. pallidipennis, but in this species a total infection rate of only 5–15% was achieved, compared with about 45% in T. sordida. In contrast, D. maxima was strongly affected by B. triatomae: nymphal development was retarded and complete mortality occurred in some groups exposed to infection, although not all bugs were infected.     

Developmental time and mortality of the reduviid bug Triatoma infestans with differential exposure to coprophagic infections with Blastocrithidia triatomae (Trypanosomatidae)

Developmental time and survival in larvae of the reduviid bug Triatoma infestans were studied in uninfected groups and in those exposed to a coprophagic infection with Blastocrithidia triatomae. Addition to young uninfected larvae of (a) different numbers of infected bugs, (b) infected and uninfected bugs, and (c) fresh or dry infectious feces were compared. Retardation of development was evident in groups given infected larvae or fresh feces. Mortality rates were correlated with infection rates and were higher in groups given more infected bugs, independent of the presence of uninfected bugs. Therefore, bugs did not discriminate between feces from infected and uninfected bugs, or they did not reject infectious feces. Dry feces had to be redissolved with fresh feces before infection was possible.     

Experimental Infection of Triatoma infestans and Rhodnius prolixus with Trypanosomatidae of the Genera Crithidia and Blastocrithidia*

SYNOPSIS. Dissections of Triatoma infestans and Rhodnius prolixus at various intervals after experimental infection with insect Trypanosomatidae of the genera Blastocrithidia and Crithidia (B. euschisti, C. luciliae, C. acanthocephali and C. mellificae) disclosed the protozoa to be surviving and in most instances undergoing reproduction in the experimental bugs for as long as 2 months following infection. Epimastigotes and amastigotes were seen in bugs infected with B. euschisti and choanomastigotes and amastigotes were observed in insects experimentally infected with the Crithidia. Since C. acanthocephali and C. luciliae were from clones, this is the 1st finding of these 2 structural types in this genus using cultures originating from a single choanomastigote. In view of the loose host specificity of the insect Trypanosomatidae as further emphasized by this work, it would appear that the necessity is increased for field workers in trypanosomiasis and leishmaniasis areas of the world to become well acquainted with the infective potentialities of the insect trypanosomatids of their areas for the vectors of Trypanosoma and Leishmania along with the possible association in nature of these vectors with insects which might harbor insect-limited Trypanosomatidae.     

Scanning Electron Microscopic Studies of Blastocrithidia triatomae (Trypanosomatidae) in the Rectum of Triatoma infestans (Reduviidae)12

ABSTRACT. The morphology of Blastocrithidia triatomae, when colonizing the rectum of Triatoma infestans, was studied by scanning electron microscopy. The posterior end of the epimastigote's body is tapered and often rolled up. The “straphangers” (cyst stages) are connected with the flagellum by “cytoplasmic bridges”; after “straphanger” detachment these bridges persist as remnants. A comparison of colonization densities of B. triatomae and Trypanosoma cruzi shows that both prefer to colonize the rectal gland, but minor density differences are apparent for B. triatomae in other rectal regions. The colonization density in established infections of B. triatomae is always greater than that of T. cruzi, especially near the midgut exit.     

Oriented responses of the triatomine bugs Rhodnius prolixus and Triatoma infestans to vertebrate odours on a servosphere

Oriented responses of both R. prolixus and T. infestans adults were recorded on a servosphere to mouse-odour, one of its components (CO₂), and to rabbit urine-odour. The volatiles were delivered in an air-stream under controlled conditions which excluded other sensory modalities. In stimulus-free air the triatomines walked preferentially downwind in straight bouts interrupted by stops or periods at relatively low speeds, all of variable duration. In odour-laden air, bugs maintained their typical walking habit but switched from negative to positive anemotaxis. The characteristic response to odour onset was to stop, sample the air with the antennae, turn upwind in situ, and then walk off in the direction of the source for at least a few seconds, i.e., odour mediated anemotaxis. Mouse-odour caused T. infestans to increase its speed to 5.3 cms^-1. Both species continued with the upwind response for some time after odour delivery ceased, but the crosswind component of the tracks was more prominent during this period — an effort, we presume, by the bugs to re-contact an odour plume. This investigation provides unequivocal evidence for host finding in triatomines by olfactory cues alone.     

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.     

Novel adipokinetic hormones in the kissing bugs Rhodnius prolixus,Triatoma infestans,Dipetalogaster maxima and Panstrongylus megistus

Peptides of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family were isolated and sequenced from the retrocerebral corpora cardiaca of four kissing bugs which are all vectors of the protozoan Trypanosoma cruzi responsible for Chagas’ disease. The sequence of three novel AKHs were deduced from the multiple MS^N electrospray mass data: the octapeptide pGlu-Leu-Thr-Phe-Ser-Thr-Asp-Trp amide (denoted Rhopr-AKH) in Rhodnius prolixus and Panstrongylus megistus, the nonapeptide pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly amide (denoted Triin-AKH) in Triatoma infestans and the decapeptide pGlu-Leu-Thr-Phe-Ser-Asp-Gly-Trp-Gly-Asn amide (denoted Dipma-AKH) in Dipetalogaster maxima. The sequences were confirmed by identical behavior of natural and synthetic forms in reversed-phase HPLC and by CID-MS mass spectra. Conspecific injections of a dose of 10 pmol of the respective synthetic peptides resulted in a small but significant increase of the lipid concentration in the hemolymph. These experiments suggest that AKHs in kissing bugs act to regulate lipid metabolism, possibly during dispersal flights which is one of the mechanisms whereby the insects reach new outbreak areas.     

Shelter selection in the kissing bugs Triatoma infestans and Rhodnius prolixus: Intra- and inter-specific interactions and quality evaluation

Triatomines display most of their activities during the night. Before sunrise, they search, select and occupy adequate shelters to stay during the photophase, avoiding exposure to diurnal predators. In this work, we first explored the interactions between individuals of the same or different species in the shelter selection process of Triatoma infestans (Klug 1834) and Rhodnius prolixus (Stål 1859). When two groups of insects (either of different nutritional status, nymphal instar or species) were released together over an experimental arena containing two identical shelters, all nymphs were distributed randomly, suggesting the absence of intra- and inters-pecific interactions. Secondly, we analysed their preferences for particular features of shelters by releasing one group of insects (either T. infestans or R. prolixus) over an arena containing two different refuges. Nymphs exhibited preferences for darker shelters with a vertical orientation of its substrate and elevated from the ground, highlighting the importance of such features in a shelter selection context. We conclude that these species disregard the presence of other individuals but evaluate certain characteristics of the shelters to choose them. This information may contribute to understanding the colonization/recolonization dynamic processes of these Chagas disease vectors.     

Lethal and sublethal effects of eucalyptol on Triatoma infestans and Rhodnius prolixus,vectors of Chagas disease

Eucalyptol is the common name for a cyclic ether monoterpene found in essential oils from Eucalyptus species and other plants. Several reports showed its insecticidal activity. In this work, visible symptoms of intoxication, effect on locomotor activity, knock‐down, and repellence produced by eucalyptol were evaluated on nymphs of Triatoma infestans Klug and Rhodnius prolixus Ståhl (both Hemiptera: Reduviidae). Both insects are among the main vectors of Chagas disease in Latin America. Visible symptoms of intoxication were similar to those observed for neurotoxic insecticides. A video tracking technique was used to evaluate locomotor activity and repellence by exposing the nymphs to impregnated papers. Hyperactivity (a non‐directional increase in locomotor activity) is a symptom of intoxication that is used to detect triatomines in rural houses, because it causes the insects to leave their refuges. Eucalyptol produced hyperactivity only in T. infestans at a concentration 1 000× higher than the positive control, deltamethrin [(S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐(2,2‐dibromoethenyl)‐2,2‐dimethylcyclopropanecarboxylate]. It also produced repellence on both species at a concentration 10× higher than the positive control, DEET (N,N‐diethyl‐3‐methylbenzamide). Knock‐down effect was evaluated by exposing the nymphs to impregnated papers in closed containers (contact and fumigation simultaneously). Values of knock‐down time for 50% of exposed nymphs (KT₅₀) were calculated for various concentrations of eucalyptol. The onset of knock‐down occurred more rapidly as the concentration increased. In the best cases, eucalyptol was 12–15× less toxic than the positive control dichlorvos (2,2‐dichlorovinyl dimethyl phosphate). After these results, eucalyptol seems discouraged as a hyperactivant agent for monitoring insects in rural houses. Nevertheless, its knock‐down and repellence effect on vectors of Chagas disease deserve further investigation.     

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.     

Epidemiology of Chagas disease in Guatemala: infection rate of Triatoma dimidiata,Triatoma nitida and Rhodnius prolixus (Hemiptera,Reduviidae) with Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida,Trypanosomatidae)

A five-year domiciliary collection in the 22 departments of Guatemala showed that out of 4,128 triatomines collected, 1,675 were Triatoma dimidiata (Latreille, 1811), 2,344 were Rhodnius prolixus Stal 1859, and only 109 were T. nitida Usinger 1939. The Chagas disease parasite, Trypanosoma cruzi, was found in all three species. Their natural infection rates were similar in the first two species (20.6%; 19.1%) and slightly lower in T. nitida(13.8%). However there was no significant difference in the infection rates in the three species (p = 0.131). T. dimidiata males have higher infection rates than females (p = 0.030), whereas for R. prolixus there is no difference in infection rates between males and females (p = 0.114). The sex ratios for all three species were significantly skewed. More males than females were found inside houses for T. dimidiata (p < 0.0001) and T. nitida (p = 0.011); a different pattern was seen for R. prolixus (p = 0.037) where more females were found. Sex ratio is proposed as an index to show the mobility of T. dimidiata in different populations. T. dimidiata is widely distributed in the country, and is also the main vector in at least ten departments, but R. prolixus with higher vectorial capacity is an important vector in at least two departments.     

Biological effect of 1-dodecanol in teneral and post-teneral Rhodnius prolixus and Triatoma infestans (Hemiptera: Reduviidae)

Topical application of 1-dodecanol was significantly more toxic against teneral first nymphs (1-3 h old) than post-teneral first nymphs (24 h old). The lethal dose ratios were 711,500 for Rhodnius prolixus and 3613 for Triatoma infestans. No significative difference between LD50 was found when 1-dodecanol was injected in recently hatched adult R. prolixus (1-4 h old) nor in older adults (24 h old). These values were similar to those calculated for deltamethrin (an effective triatomicide), showing that 1-dodecanol had no insecticidal properties when it was applied by injection. Topical application of high dose of 1-dodecanol (1 microg/i) on teneral first nymphs of R. prolixus, produced an interruption of the darkening process of the cuticle, and probably in the development of its physiological properties.     

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.     

Isolation and separation of cysts and epimastigotes of Blastocrithidia triatomae Cerisola et al., 1971 (Trypanosomatidae)

Developmental stages of the flagellate Blastocrithidia triatomae were isolated according to differences in density or in surface charge from dry feces or the intestinal content of reduviid hosts or from in vitro cultures which contained host cells. Isopycnic ultracentrifugation on isoosmotic Percoll gradients separated cysts (ϱ = 1.145 g/cm³) from epimastigotes (ϱ = 1.075 g/cm³), representing the two major morphological stages of B. triatomae. Moreover, a continuous shift in buoyant density of encystating and/or excystating stages from the level of mature cysts to lower densities displayed by epimastigotes was observed. This characteristic excluded the isolation of pure epimastigotes by density gradients. Nevertheless, approximately 1 × 10⁸ mature cysts per gradient were obtained by this procedure. Employing DEAE-ion exchange chromatography for cyst isolation, approximately 2 × 10¹⁰ cysts could be purified per column. In addition, DEAE-chromatography was used for the isolation of pure epimastigote fractions. The succesful isolation of B. triatomae-stages from three completely different sources suggests that the methods derived in our study may be easily adapted for the isolation of other insect trypanosomatids.     

Comparative kinetics of bloodmeal intake by Triatoma infestans and Rhodnius prolixus, the two principal vectors of Chagas disease

Chagas disease vector insects Triatoma infestans and Rhodnius prolixus (fifteen stage III nymphs per 4 litre cage) were allowed to feed on anaesthetized mice for 1 h (control group), or on active non-anaesthetized mice (NAM) for 2, 4 or 8 h exposure. The bloodmeal size (weight increase) for both species was proportional to the duration of contact with NAM, due to ingestion of multiple small bloodmeals, up to 142% of control weight for T. infestans with 8 h exposure to NAM. The mean weight increase of T. infestans nymphs after 4 h contact with NAM was similar to that of the control group, whereas for R. prolixus , 8 h contact with NAM gave only 64% of the control value. For both species of insect, within 4 h of feeding, >20% of the bloodmeal weight was lost by defaecation and diuresis. The proportions of unfed nymphs and mortality during 2 h contact with NAM were significantly higher for R. prolixus , demonstrating better exploitation of the host blood source by T. infestans , apparently because during blood-feeding the latter insect species caused less irritation to the host.