Jorge E. Rabinovich

Exploration for Triatoma virus (TrV) infection in laboratory-reared triatomines of Latin America: a collaborative study*

Triatoma virus (TrV) is a small, non-enveloped virus that has a +ssRNA genome and is currently classified under the Cripavirus genus of the Dicistroviridae family. TrV infects haematophagous triatomine insects (Hemiptera: Reduviidae), which are vectors of American trypanosomosis (Chagas disease). TrV can be transmitted through the horizontal faecal-oral route, and causes either deleterious sublethal effects or even the death of laboratory insect colonies. Various species of triatomines from different regions of Latin America are currently being reared in research laboratories, with little or no awareness of the presence of TrV; therefore, any biological conclusion drawn from experiments on insects infected with this virus is inherently affected by the side effects of its infection. In this study, we developed a mathematical model to estimate the sample size required for detecting a TrV infection. We applied this model to screen the infection in the faeces of triatomines belonging to insectaries from 13 Latin American countries, carrying out the identification of TrV by using RT-PCR. TrV was detected in samples coming from Argentina, which is where the virus was first isolated from Triatoma infestans (Hemiptera: Reduviidae) several years ago. Interestingly, several colonies from Brazil were also found infected with the virus. This positive result widens the TrV’s host range to a total of 14 triatomine species. Our findings suggest that many triatomine species distributed over a large region of South America may be naturally infected with TrV.

Biological Control of the Chagas Disease Vector Triatoma infestans with the Entomopathogenic Fungus Beauveria bassiana Combined with an Aggregation Cue: Field, Laboratory and Mathematical Modeling Assessment

Background Current Chagas disease vector control strategies, based on chemical insecticide spraying, are growingly threatened by the emergence of pyrethroid-resistant Triatoma infestans populations in the Gran Chaco region of South America. Methodology and findings We have already shown that the entomopathogenic fungus Beauveria bassiana has the ability to breach the insect cuticle and is effective both against pyrethroid-susceptible and pyrethroid-resistant T. infestans, in laboratory as well as field assays. It is also known that T. infestans cuticle lipids play a major role as contact aggregation pheromones. We estimated the effectiveness of pheromone-based infection boxes containing B. bassiana spores to kill indoor bugs, and its effect on the vector population dynamics. Laboratory assays were performed to estimate the effect of fungal infection on female reproductive parameters. The effect of insect exuviae as an aggregation signal in the performance of the infection boxes was estimated both in the laboratory and in the field. We developed a stage-specific matrix model of T. infestans to describe the fungal infection effects on insect population dynamics, and to analyze the performance of the biopesticide device in vector biological control. Conclusions The pheromone-containing infective box is a promising new tool against indoor populations of this Chagas disease vector, with the number of boxes per house being the main driver of the reduction of the total domestic bug population. This ecologically safe approach is the first proven alternative to chemical insecticides in the control of T. infestans. The advantageous reduction in vector population by delayed-action fungal biopesticides in a contained environment is here shown supported by mathematical modeling.

The functional and numerical responses of Trissolcus basalis (Hymenoptera: Platygastridae) parasitizing Nezara viridula (Hemiptera: Pentatomidae) eggs in the field

Trissolcus basalis has been used as a biological control agent of its main host, Nezara viridula, in many countries. However, estimations of its functional and numerical responses in the field are lacking. We estimated the density of N. viridula eggs, the proportion of parasitized N. viridula eggs, and the number of T. basalis adults/trap in the field. We transformed relative parasitoid density to an absolute density, and estimated the parasitoids attack rate, a, and the mutual interference parameter, m, in two ways: following Arditi & Akçakaya (1990) and using the Holling-Hassell-Varley model with two iterative techniques. The attack rate estimated by both methods were a=1.097 and a=0.767, respectively. Parameter m varied less between methods: m=0.563 and m=0.586, respectively, and when used to calculate the number of parasitized N. viridula eggs per m2, differences with the observed values were not significant. The numerical response of T. basalis was affected by the sex allocation of their progeny and the proportion of adult parasitoids trapped decreased with field parasitoid population density. Theoretical models show that 0<m<1 is a stabilizing factor and previous re-analysis of field data showed a mean m value of 0.8. The Holling-Hassell-Varley model leads to a flexible description of the functional response allowing to predict acceptable weekly host parasitism. The pre-imaginal parasitoid survival and the change in sex ratio as a function of parasitoid density adequately describe the numerical functional response of the parasitoid.

Detection of triatomine infection by Triatoma virus and horizontal transmission: Protecting insectaries and prospects for biological control

Triatoma virus (TrV) is the only triatomine entomopathogenic virus identified so far. Propagation of TrV in insectaries depends on handling procedures and triatomine population dynamics. The effects of propagation can be devastating and entire colonies must often be sacrificed to prevent spread of the virus throughout the insectary. This study found that after 41.3 days from TrV ingestion of human blood with 0.04 mg of viral protein by 5th instar Triatomainfestans, viral particles could be detected by RT-PCR; in a second horizontal transmission experiment time to detection resulted in a mean of 42.5 days. These results should rise awareness of TrV dynamics in nature, help estimate the spread of this virus when TrV-infected field-collected insects are incorporated into an insectary, and provide a base for the consideration of TrV as an agent of biological control of some species of triatomines.

Life History Traits and Demographic Parameters of Triatoma infestans (Hemiptera: Reduviidae) Fed on Human Blood

ABSTRACT Triatoma infestans (Klug, 1834) (Hemiptera: Reduviidae), the main vector of Chagas disease in South America, feeds primarily on humans, but ethical reasons preclude carrying out demographical studies using people. Thus, most laboratory studies of T. infestans are conducted using bird or mammal live hosts that may result in different demographic parameters from those obtained on human blood. Therefore, it is of interest to determine whether the use of an artificial feeder with human blood would be operational to rear triatomines and estimate population growth rates. We estimated life history traits and demographic parameters using an artificial feeder with human blood and compared them with those obtained on live hens. Both groups of T. infestans were kept under constant conditions [28 ± 1°C, 40 ± 5% relative humidity, a photoperiod of 12:12 (L:D) h] and fed weekly. On the basis of age-specific survival and age-specific fecundity, we calculated the intrinsic rate of natural increase (r), the finite rate of population growth (k), the net reproductive rate (R o), and the mean generation time (T g). Our results show differences in life history traits between blood sources, resulting in smaller population growth rates on human blood than on live hens. Although demographic growth rate was smaller on human blood than on hens, it still remains positive, so the benefit/cost ratio of this feeding method seems relatively attractive. We discuss possibility of using the artificial feeder with human blood for both ecological and behavioral studies.

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients

BackgroundChagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake.MethodsIn this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015.ResultsOur results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts.ConclusionsWe presented the first experimental evidence of antibodies against TrV structural proteins in human sera.

Critical threshold meal size and molt initiation in Rhodnius prolixus

The molting process and body growth in Rhodnius prolixus (Hemiptera: Reduviidae) (Ståhl, 1859) are significantly influenced by the availability and quality of food. Based on the body weight of each stage, the present study provides estimates of a potential critical weight threshold required for molt initiation in R. prolixus. In addition, a new measure given by the area under the weight curve is proposed, which encapsulates both body weight and time. It is shown that this measure is consistent with the data, and allows the estimation of a pre‐refractory period (i.e. the time interval between the moment at which the critical weight threshold is reached and the moment when no further meals are accepted). The present analysis estimates the critical weight threshold as 1.6, 5.3, 12.9, 42.0 and 97.0 mg for stages 1–5, respectively, whereas the values of the area under the curve threshold as 5, 16, 31.2, 159.7 and 329.9 mg days for stages 1–5, respectively. The results of the present study confirm the existence of a weight‐dependent mechanism for the initiation of molting in R. prolixus.

Can Triatoma virus inhibit infection of Trypanosoma cruzi (Chagas, 1909) in Triatoma infestans (Klug)? A cross infection and co-infection study

Triatoma virus occurs infecting Triatominae in the wild (Argentina) and in insectaries (Brazil). Pathogenicity of Triatoma virus has been demonstrated in laboratory; accidental infections in insectaries produce high insect mortality. When more than one microorganism enters the same host, the biological interaction among them differs greatly depending on the nature and the infection order of the co-existing species of microorganisms. We studied the possible interactions between Triatoma virus (TrV) and Trypanosoma cruzi (the etiological agent of Chagas disease) in three different situations: (i) when Triatoma virus is inoculated into an insect host (Triatoma infestans) previously infected with T. cruzi, (ii) when T. cruzi is inoculated into T. infestans previously infected with TrV, and (iii) when TrV and T. cruzi are inoculated simultaneously into the same T. infestans individual. Trypanosoma cruzi infection was found in 57% of insects in the control group for T. cruzi, whereas 85% of insects with previous TrV infection were infected with T. cruzi. TrV infection was found in 78.7% of insects in the control group for TrV, whereas insects previously infected with T. cruzi showed 90% infection with TrV. A total of 67.9% of insects presented simultaneous infection with both types of microorganism. Our results suggest that TrV infection could increase adhesion of T. cruzi to the intestinal cells of triatomines, but presence of T. cruzi in intestinal cells would not increase the possibility of entry of TrV into cells. Although this study cannot explain the mechanism through which TrV facilitates the infection of triatomines with T. cruzi, we conclude that after TrV replication, changes at cellular level should occur that increase the adhesion of T. cruzi.

Invariance of demographic parameters using total or viable eggs

Recently Mou et al. (J. Appl. Entomol., 139, 2015 and 00) recommended that in population studies in which hatch rates vary with maternal age, demographic parameters should be calculated excluding unhatched eggs. A mathematical proof was provided to support this. In this note, we expose a flaw in their proof and show that the demographic parameters do not differ by considering either all eggs or only viable ones, as long as the beginning and end of a generation are defined accordingly.

Response to Chi, Mou, Lee and Smith

In our article, ‘Invariance of demographic parameters using total or viable eggs’ by C. Hernandez-Suarez, P. Medone and J. E. Rabinovich (this issue), we provide three different mathematical proofs that Ro will yield the same result using total or viable eggs, as long as the participating stages are defined accordingly. Thus, Ro can be defined as the ‘number of eggs that will replace each initial egg of a cohort in the period of one generation’, the ‘number of viable eggs that will replace each viable egg in the period of one generation’, the ‘number of adults that will replace each adult in the period of one generation’ and so on. Regrettably, Chi, Mou, Lee and Smith (CMLS)’s response to our article is merely tautological. In the last paragraph they mention: ‘Based on the above mathematical proof. . .’, however, there is no mathematical proof in their arguments, which even lacks of a single equation. They merely refer to equations in their original article (Mou et al. 2015), some of which have been criticized in our article, so no new formal mathematical evidence has been provided. Additionally, CMLS mention: ‘. . .misinterpretation and errors in their proof’, but again, there is no single exhibition of such errors in our work; in fact, there is no reference to any of our equations identifying them in our work, which discards any mathematical refutation to our proof. In their response, the authors used hypothetical and simplified data (tables 1–5 in CMLS) to substantiate their arguments, and sound scientific procedures indicate that no data set can be valued against mathematical proofs as the ones we have provided. Chi, Mou, Lee and Smith confirm our impression that there was a flaw in the experimental design, when they say ‘When the egg hatch rate of a particular species varies with maternal age, it is impossible to collect eggs representative of the entire population unless a prohibitively large number of eggs are used’. So, if the sample of the original number of eggs for initiation of the cohort study was not statistically representative of the population as a whole (at least in terms of hatching rates, something that we have confirmed from the article on H. dimidiata), then increasing the number of eggs should have been a methodological sound option, and the argument of excessive work should not override scientific procedures. Furthermore, CMLS make an unsustainable statement that because the hatch rates of female daily fecundity were based on a much large sample size and thus are more representative than the parent cohort, then the lx, mx and hx calculated using only viable eggs are more representative than those based on total eggs. There is no demonstration anywhere in the original CMLS article, nor in their rebuttal to our article, in the direction that the hatch rates of female daily fecundity can be considered more representative of the population as a whole. The sole fact that the number of eggs from the female’s daily fecundity that was analysed to estimate the cohort’s hatch rate was really high (23,309 eggs) cannot be used as an argument for representativeness; this would be so if all other conditions could be guaranteed as being identical. Furthermore, in the Discussion section of the original CMLS article, it was mentioned that Gillani et al. (2007) reported 96% hatching rate, even higher than the original batch of eggs used for the initiation of the cohort, and so further deviant from the average hatch rate of the female’s daily fecundity that CMLS consider ‘more representative’. In relation to mean generation time, the authors have focused their critique on a simple semantic concern (i.e. stage versus age). Our argument still holds if we replace stage by age. Chi, Mou, Lee and Smith also claim that as a theoretical proof must be inclusive and that our discussion regarding r and k is based on the approximate (or simplified method) of Birch (1948) that this needs no more refutation. Our mathematical proofs involving r and k are completely general and not restricted to any particular estimation method. Many years ago, there was a discussion on whether to calculate Ro by considering only females (because these are the ones laying eggs) or all individuals. Biologists then had to count how many eggs gave birth to