The effects of aposymbiosis and of an infection with Blastocrithidia triatomae (Trypanosomatidae) on the tracheal system of the reduviid bugs Rhodnius prolixus and Triatoma infestans

We have investigated the effects of the absence of symbionts and of infection with a trypanosome on the tracheal supply to different organs in larvae of the blood-sucking bugs, Rhodnius prolixus and Triatoma infestans. In bugs grown without symbionts there were extensive reductions in the tracheal supply to all the internal organs examined. These bugs also excreted less fluid after blood meals, perhaps because of a reduction in the oxygen supply to the Malpighian tubules. Inclusion of vitamin B in the diet of these affected insects reversed both the adverse effects on diuresis and on the extent of the respiratory supply to the different internal organs. The results of these studies suggest that vitamin B may play a key role in the development and maintenance of an adequate tracheal supply to the tissues in these insects. Infection with the trypanosome Blastocrithidia triatomae also greatly reduced the density of tracheoles supplying the rectum, small intestine and Malpighian tubules in infected T. infestans, but not in R. prolixus. It is possible, therefore, that the parasite exerts at least part of its pathogenic effect by causing a vitamin B deficiency, that in turn affects oxygen supply to the tissues.     

Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus

Resistance levels to insecticides used in control of Chagas Disease vectors were assessed in two species of bugs (Hemiptera: Reduviidae): Triatoma infestans (Klug) from Brazil and Rhodnius prolixus Stål from Venezuela. The resistance ratios, compared to susceptible laboratory strains, were determined by topical application bioassays. The T. infestans PA strain exhibited resistance ratios of 7× to deltamethrin, 3.6× to β‐cyfluthrin and 3.3× to cypermethrin, but was susceptible to β‐cypermethrin and lambda‐cyhalothrin. Rhodnius prolixus CA strain showed resistance to all the pyrethroids evaluated, the resistance ratios ranging between 4.5× to lambda‐cyhalothrin and 12.4× to cypermethrin. Deltamethrin resistance in both strains was decreased by piperonyl butoxide, suggesting oxidative metabolism as cause of resistance.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph.

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrate erythrocytes and cultured epimastigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titers of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus.     

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.     

Ultrastructure of Symbiotic Bacteria in Normal and Antibiotic-Treated Blastocrithidia culicis and Crithidia oncopelti*

SYNOPSIS. An electron microscope study of diplosomes in Blastocrithidia culicis and bipolar bodies in Crithidia oncopelti has shown that both entities appear to be intracellular symbiotes and have a similar fine structure. They are enclosed by 2 unit membranes which are separated by a large space of very low density. The outer membrane is derived probably from the host cell. The matrix of the symbiotes is composed of dense ribosome-like particles and of areas of low density containing fine fibrillae. The particles are of the same size as ribosomes in bacteria and the fibrils have the characteristics of bacterial DNA. Thus, the lucid areas with fibrillae correspond to the nucleoids in bacteria. These observations suggest that the symbiotes are bacteria. The effect of chloramphenicol (CAP) and penicillin G (PCL) on these symbiotic bacteria was studied by culturing the host flagellates in media containing the antibiotics. The effect was analyzed at different intervals after the treatment by electron microscopy. After single treatment in the blood broth containing 0.08% (w/v) CAP, symbiotes appeared to have enlarged nucleoids, became deformed and eventually degenerated. In Grace's medium (supplemented with 10% fetal bovine serum) containing 0.6 or 2.4% (w/v) PCL, symbiotes of C. oncopelti remained unaltered, whereas some symbiotes of B. culicis became pleomorphic. Symbiotes of both species persisted after repeated transfers in PCL media and reverted to normal forms when transferred to PCL-free media. Sensitivity of symbiotes to CAP provides further evidence of their bacterial nature. The effect of PCL on the symbiotes of B. culicis suggests the presence in their cell envelopes of mucopeptide, which probably provides rigidity for maintaining the bacterial shape of the symbiotes.     

Impact of residual spraying on Rhodnius prolixus and Triatoma dimidiata in the department of Zacapa in Guatemala

As a vector control program to control Chagas disease in Guatemala, residual spraying of Rhodnius prolixus and Triatoma dimidiata was performed, and its impact was measured in the department of Zacapa. In order to identify infested villages and determine the degree of infestation, a baseline entomological survey to identify municipalities infested with vectors followed by an additional vector survey in areas known to be infested was conducted. Residual spraying using pyrethroid insecticides was performed at all the villages identified as being infested with the vectors. The residual spraying was shown to be highly effective against both vectors by the decrease in infestation indices after spraying. Analysis of the cost-effectiveness of the spraying showed that the average cost of insecticides per house is high when compared with that in Southern Cone countries.     

Reduced Growth of Blastocrithidia culicis and Crithidia oncopelti Freed of Intracellular Symbiotes by Chloramphenicol*

The intracellular symbiotes of Blastocrithidia culicis and Crithidia oncopelti can be eliminated from cultures of the flagellates by a single chloramphenicol (CAP) treatment. Effective dosages were determined to be 0.01–0.08% (w/v) CAP after a treatment for 2 weeks or more for B. culicis and 0.08% (w/v) after 1 month for C. oncopelti in most cases. Ineffective dosages only lowered the numbers of symbiote-bearing flagellates. Growth of both species of flagellates in the presence of CAP was reduced in proportion to the drug concentration. Repeated subcultures at effective dosages yielded symbiote-free flagellates, which maintained a low level of growth rate. After repeated subcultures at ineffective dosages, the growth rate rose and the symbiote-bearing cells, initially very few, increased in number. The lowest effective dosages proved to be marginal, often producing symbiote-free cultures, but occasionally cultures with a few symbiote-bearing cells. After repeated subcultures at these drug concentrations, symbiote-containing cultures grew faster than the symbiote-free cultures. Hence, the symbiotic bacteria benefit the growth of their hosts, perhaps by supplying essential factors that are inadequate even in a rich blood medium.