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Physiology 13/10/2022

Cycle

The cycle of Trypanosoma cruzi within the vector insect

Patrícia Azambuja

Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/FiocruzEmail: azambuja@ioc.fiocruz.br

Eloi S. Garcia

Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/Fiocruz

Email: egarcia@fiocruz.br

Triatomines belonging to order Hemiptera, subfamilyTriatominae, family Reduviidae, with about 130 species, are hematophagous (Figure 1), and are considered potencial vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease.

Figure 1

Trypanosoma cruzi, a flagellated protozoa belonging to order Kinetoplastida and to the Trypanosomatidae family, infects, in natural conditions, more than 100 species of mammals of different orders, as reviewed by Devera and collaborators. The parasite exists in nature in different populations of vertebrate hosts, such as humans, wild animals and domestic animals, and of invertebrates, such as vector insects. T. cruzi has morphological and functional variations, alternating between stages during which they go through binary division and non-replicating and infecting forms. Replicating forms include epimastigotes present in the digestive tract of the vector insect and the amastigotes observed inside mammal cells. Non-replicant infecting forms, called metacyclic trypomastigotes, can be found in the feces and urine of the vector insect, while trypomastigotes circulate in the blood of mammals.

Recent investigations using analysis of isoenzymes, promotor rRNA activity, sequence of mini exon genes and micro satellite markers have shown that T. cruzi corresponds to 2 divergent genetic subgroups, called lineages 1 and 2, as described by Briones and collaborators. There is only a small difference in the biological behavior of natural populations ofT. cruzi belonging to genetic groups T. cruzi 1 and T. cruzi 2 isolated from different hosts (human, wild reservoirs, and vector insects). Enzymatic electrophoresis and amplified polymorphic DNA have shown that lineage 2 can be divided in 5 types, as described by Tibayrenc’s group.

During the phase of the cycle in the invertebrate host, T. cruzi differentiates into epimastigotes. Then, in the hindgut, these differentciate into metacyclic trypomastigotes (a process known as metacyclogenesis), which are eliminated through the feces and urine of the fector insect and can then infect the vertebrate host, as reviewed by Brener and Garcia and collaborators. The parasite cannot penetrate intact skin, and can only infect the host via mucosa or skin lesions. In these mammals, parasites develop within the cells and are released into the circulating blood after the host’s cells break, as reviewed by Garcia and collaborators.As the insect feeds, the trypomastigotes found in the blood of the infected vertebrate host are ingested by the insect. A few days after the insect’s blood meal, the parasites turn into epimastigotes and spheromastigotes. Once the infection is established, the epimastigotes within the stomach of the vector insect begin dividing repeatedly through binary division, and can adhere to the perimicrovillar membranes of the intestinal cells. In large numbers, the epimastigotes attach themselves to the rectal cuticle, differentiate themselves into metacyclic trypomastigotes and now both forms, differentiated or not, can be eliminated through feces and urine (Figure 2).

Figure 2

A drastic alteration occurs in the transformation from epimastigotes into trypomastigotes in the cycle developed in the vector insect, including the metacyclic trypomastigotes’ ability survive in mammals, unlike epimastigotes, which are destroyed by the complement system, as shown by Fernandez-Presas and collaborators. Studies by Ferguson and Previato and collaborators showed that on the surface of T. cruzi in the epimastigote form there are differences in the composition of carbohydrates, proteins and lipids, when compared with the trypomastigote form. These molecular alterations protect the trypomastigote from the complement system of the vertebrate host.

The susceptibility to natural infection of a vector species by a certain strain of T. cruzi is variable. It is also important to take into account the aspect of adaptation of the parasite strain to a certain species of vector insect for the success of its full development, as demonstrated by Garcia and Dvorak. The level of infectivity of strains of T. cruzi in the insect and in mammal hosts may diminish after several passages of the parasites in culture media, as described by Chiari. Perlowagora-Aszumlewics and collaborators have shown that a species of vector insect tends to be more susceptible to a strain of the parasite within the same area of geographical distribution in the different regions. It is interesting to observe that infection by T. cruzi does not significantly alter the vector’s behavior, as reviewed by Schaub.

Not only the kinetic of the multiplication of epimastigotes in a certain species of triatomines, but the metacyclogenesis process is also dependent on the strain and/or clone of the infecting parasites, as demonstrated by Garcia and Azambuja and by Kollien and Schaub. As proposed by the group of Tibayrenc and Lima and collaborators, as T. cruzi is an heterogeneous species, the effect of interaction and cooperation between different subpopulations of parasites in the intestinal environment of the vector insect should be taken into account.

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Interaction dynamics

Dynamics of the interaction of Trypanosoma cruzi with the vector insect

Patrícia Azambuja

Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/Fiocruz

Email: azambuja@ioc.fiocruz.br

Eloi S. Garcia

Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/Fiocruz

Email: egarcia@fiocruz.br

The establishment of infection by T. cruzi in the digestive tube of the vector insect is dependent on and regulated by physiological and biochemical factors of the vector insect, as proposed by Carlos Chagas and Emanuel Dias. Bio assays were used to evaluate a variety of factors that influence the development of the parasite, such as ultrastructural alterations of the epithelial cells of the midgut, litic factors, digestive enzymes and hormonal development physiology of the insect by the group of Garcia and Azambuja. After the ingestion of infected blood, in the anterior and posterior regions of the midgut (stomach and intestine, respectively), the parasites come across components that exist in these digestive compartments. These include hemolitic factors, peptides derived from the D-globin chain and lectins, which can modulate the dynamic of the multiplication and differentiation of T. cruzi in the digestive tube of the vector insect, illustrating the complexity of the mechanisms involved in the interaction with T. cruzi .

Pereira and collaborators reported the presence of lectins in the digestive tube and hemolymph of R. prolixus, suggesting that these molecules are involved in the development of T. cruzi in the vector insect. Mello and collaborators demonstrated differences in the development of three strains of T. cruzi in the intestine of R. prolixus and linked the infectivity of these parasites to the hability of the digestive tube extract to agglutinate the flagellates in vitro. Thus, lectins agglutinated clone Dm28c of T. cruzi , which presented the maximum level of infectivity, while, on the contrary, strain Y of the parasite, which was not agglutinated, was broken in the digestive tube of the vector insect, as described by Azambula and collaborators and by Mello and collaborators.

Frainderaich and collaborators demonstrated that the metacyclogenesis of T. cruzi was induced in vitro by peptides derived from α-D-globin. Garcia and collaborators studied, in R. prolixus in vivo, the effects of hemoglobin and synthetic peptides corresponding to the fragmented regions of the α-D-globin sequence on the development and transformation of epimastigotes of T. cruzi into metacyclic trypomastigotes. This differentiation in the digestive tube of the insect occurred when hemoglobin and the synthetic peptides corresponding to residues 30-49 and 35-73 of α-D-globin were added tto the plasma diet. However, synthetic peptide 41-73 did not induce the differentiation of epimastigotes into metacyclic trypomastigotes, even in the presence of the first two peptides. Hemoglobin proved to be an important blood component for the development of parasites. These results identified an uncommon molecular mechanism that modulates the dynamics of the transformation from epimastigotes into metacyclic trypomastigotes in the digestive tube of the vector insect.

Recently, Azambuja and collaborators isolated and identified, in the stomach of R. prolixus, Serratia marcescensbiotype A1a (called RPH), producer of the pigment called prodigiosin. This bacterium, or S. marcescens SM365, which also produces the pigment, or variant marcescens DB11, which does not produce prodigiosin, lysed erythrocytes, but only those that produce prodigiosin were able to destroy the Y strain of T. cruzi. The parasite lysis mechanism involves rapid bacterial adhesion to the parasite’s membrane, followed by disintegration of the parasite. It appears that mannose-dependent receptors are involved in the lysis mechanism. This sugar protects the parasite from lysis caused by S. marcescens, but does not interfere with the hemolysis caused by this bacterium. These authors suggested that the trypanolytic effect of the bacterium is different from the hemolytic activity, as this bacterium has fimbriae of mannose type 1 that mediates adherence and lysis of trypanosomes. Therefore, the study of bacteria in the digestive tube of the invertebrate host can provide new tools to block the development of the parasite in the vector insect.

As for digestive enzymes, Borges and collaborators demonstrated that insects infected by T. cruzi had increased activity of cathepsin D. However, feeding R. prolixus with SH-dependent proteinase inhibitor had no effect on the infection of the vector insect. Urisc-Bedoya and Lowerngerger revealed that the level of transcription of cathepsin B did not vary when comparing infected and non-infected vector insects.

One of the important processes of the physiological interaction between T. cruzi and its vector insect is parasite adherence to the surface of intestinal cells. Nogueira and collaborators, using video microscope analysis, showed that the epimastigote and trypomastigote forms of T. cruzi move in the direction of the surface of intestinal epithelial cells. While tryptomastigote forms do not adhere to the epithelium, epimastigotes adhere to the epithelium of the stomach and the intestine. This recognition process apparently involves molecules of glycoinositol-phospholipids (GIPLs), found in abundance on the surface of epimastigotes, as described by Colli and Alves. The inhibition of a gene involved in the synthesis of glycoproteins of T. cruzi resulted in the non-adherence of the parasite’s flagellum to the intestinal epithelium, diminishing the population of T. cruzi in the vector insect, as described by Ribeiro de Jesus and collaborators.Garcia and collaborators showed that the interaction T. cruzi – vector insect can be modulated by the azadirachtin compound(Figure 3), an inhibitor of insect growth obtained from the plant Azadirachta, that strongly interferes with the insect’s neuroendocrine regulation. This drug, administered orally before, during or even after infection by T. cruzi, not only interfers with the development of the insect, but also with the establishing of a parasite infection in the digestive tube of different triatomine species.

Figure 3

Garcia and collaborators showed that parasites in their epimastigote form did not adhere to the epithelium of the insect’s midgut when larvae of R. prolixus were beheaded or treated with azadirachtin. Treating the insect with ecdione and implanting normal heads into insects treated with azadirachtin reverted the adhesion process and the development of the insects. The insects that were given the drug or were decapitated showed a disorganization of the perimicrovillar membranes of intestinal epithelial cells, indicating that the pathways of the prothoracicotropic hormone-stimulator of ecdisone synthesis is involved in the development process of T. cruzi in its vector insect (Figure 4).

Figure 4

Therefore, highly specialized interactions between T. cruzi – vector insect are likely influenced by the biological effects of natural compounds obtained from plants over the physiological conditions of the insects due to their interference with hormonal homeostasis.

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Ecology 

Vector ecology

Liléia Diotaiuti

Group of Triatomines and Chagas disease Epidemiology of the René Rachou Institute/Fiocruz

Email: diotaiuti@minas.fiocruz.brThe occurrence of triatomines in nature is subject to the availability of food and hiding places. These insects associate to different food sources, preferably warm blood animals, such as birds and mammals, but depending on the ecotope, they can also feed on poikilotherms, such as amphibians and reptiles (Figure 1).

Figure 1

As Trypanosoma cruzi is a parasite of mammals only, it is necessary to differentiate sources of food from sources of infection. Wild sources of infection of triatomines are small mammals belonging to seven different orders: Marsupialia, Edentata, Chiroptera, Carnivora, Lagomorpha, Rodentia and Primates. From the epidemiological standpoint, marsupials are very relevant, having high rates of infection and high synanthropy, establishing a bridge between the wild and domiciliary cycles of the infection. The same applies to rodents, as some species are also synanthropic. In artificial environments, dogs and cats are the main reservoirs of T. cruzi. Dogs are considered sentinels in areas under control and can indicate the occurrence of the transmission cycle and the need to take steps to prevent the outbreak from expanding. In the peridomiciliary area, chickens are the main sources of food, but kissing bugs also feed on the blood of different animals in this environment. Small mammals, such as rodents, possums and dogs play an important role in the maintenance of the transmission cycle of T. cruzi in the artificial environment.Triatomines can be found in many different ecotopes, such as cracks between rocks, birds’ nests or mammals’ dens, in tree hollows or under the dried barks of trees etc (Figure 2). Depending on the characteristics of the hiding place, triatomine colonies can be associated to ecotopes considered stable or unstable.

Figure 2

In stable ecotopes, the presence of sources of food is more permanent, and micro climate characteristics suffer smaller variations regarding the external environment, therefore allowing for the development of colonies in higher densities. As an example, we can mention species of palm trees (Figure 3) whose tops have an architecture formed by the intertwining of leaves, creating an infinity of overlapping spaces. These are frequently colonized by triatomines, especially those of the genus Rhodnius. Many animals visit the tops of palm trees, such as birds, rodents, bats, marsupials, amphibians, and lizards: a variety of food sources for triatomines and for infection by T. cruzi. Another interesting example of stable ecotope is caves, colonized by large groups of bats, to which is associated the species Cavernicola pilosa.

Figure 3

In unstable ecotopes, the presence of food is sporadic, and temperature and humidity vary similarly to the external environment. This is how natural ecotopes of T. sordida are characterized: dried tree barks where food sources for triatomines are more restricted, depending on the nidification period of the species. In these conditions, triatomine colonies have their growth significantly restricted, and they usually consist of only a few insects (Figure 4).

Figure 4

Triatomines have many natural predators, beginning with other insect-eating Heteropta, spiders, birds, or even mammals such as rodents, marsupials and monkeys, which in this case results in an important infection pathway for the hosts. A special group of insects of the Scelionidae family draws attention for being egg parasites. The parasite relationshihp occurs through the development of parasitoid eggs within the triatomine’s eggs, whose larvae then devour the Heteroptera embryo (Figure 5). Some species of fungi (Beauverea bassianaMetarrhizium anisopliae) can also have a lethal action on triatomines, and are currently being studied in the search for natural alternatives to control triatomines.

Figure 5

Triatomines can disperse passively as they are taken from one place to another. Species that are associated to wood in the natural environment, such as T. sordida and T. pseudomaculata, are frequently introduced to the artificial environment when firewood is brought into the home for domestic use. Flying animals such as birds and bats are probably relevant for the dispersion of some species, such as T. sordida and C. pilosa. The main vector of Chagas disease in the Southern Cone countries, Triatoma infestans, was mainly dispersed through human migrations, as it was passively carried in travellers’ luggage and therefore reached a wide area of occurrence in a short period of time.Active dispersion, especially through the flight of adult specimens, has high epidemiological relevance, as it is related to the invasion of dwellings beginning with specimens coming from the wild cycle. Obeying a natural rhythm for each species, there is a trend of migration of adults to the artificial environment, i.e. the home and peridomiciliary hiding places, which may result in the formation of colonies (presence not only of adults, but also of immature forms) of these insects close to humans. The home (Figure 6 and Figure 7) is a highly stable environment, a “nest” that is inhabited throughout the year, and with a stabilized micro climate, as humans are very vulnerable to climatic changes and need a fairly controlled envionment, which benefits triatomines. The peridomiciliary area (Figure 8) also has characteristics of much stability: countless sources of food, such as chickens, dogs, cats, goats, pigs, rodents and others; constructions such as chicken coops, pigsties, barns, piles of rooftiles, wood and firewood etc. In these circumstances, triatomines that would only form small colonies in the wild can, in the artificial environment, be potentiated by the great availiability of food and hiding places, and therefore grow and form huge colonies, almost in a transgression of their own biology.

Figure 6
Figure 7
Figure 8

The vectorial transmission of Chagas disease is related to the populational density of vectors, i.e., the more kissing bugs, the more transmission. The species mostly associated to humans, such as T. infestans in countries of the Southern Cone and R. prolixus in some countries in northern South America and in Central America, frequently make up intradomiciliary colonies with average densities above 3,000 insects, exceptionally around 10,000, maintained by the food sources found within the homes, especially humans, dogs, cats, rodents and other animals that sleep indoors.

However, this ability to adapt to the artificial environment, more specifically to the intradomiciliary area, varies between species. T. infestans and R. prolixus are those that better develop in the intradomiciliary area, and they are also the ones to which the highest prevalence rates of Chagas disease are associated. Other species adapt very well to perodomiciliary conditions, but are less capable of forming colonies in the intradomiciliary area. The first are considered species of primary relevance for the domiciliary transmission of T. cruzi, while the others are considered secondary species. Triatomines considered to be of primary relevance are those that colonize homes in a permanent manner, with marked anthropophilia; these can be found beneath mattresses, in cracks in the walls, beneath loose stucco, in unused stoves or ovens, in boxes, behind pictures or anything else placed against walls, in birds’ nests or anywhere where dogs and cats sleep within the home. Secondary triatomines can produce small intradomiciliary colonies of a more temporary nature, especially in the absence of primary vectors, with varied degrees of anthropophilia, but they adapt well to artificial ecotopes, in particular those built with natural materials, such as chicken coops (walls, ceilings and nests), barns, poles or gateposts of pigsties or pens, pidgeon houses, piles of wood, bricks or tiles etc. Most triatomines, however, have exclusively wild habits, and even though some adult specimens can be found within dwellings, which they probably invade because they are attracted by light, they are unable to feed on the blood available in this environment. It is very likely that the ability to feed on the blood of different hosts that are not those to which triatomines are associated in the wild environment is a crucial factor for its adaptation to the artificial environment, qualifying species as ubiquist (which adapt to different environments, including the intradomiciliary area, making them more relevant in the epidemiology of Chagas disease) and specialist species (which live in restricted environments and are less capable of adapting to new opportunities).

The use of experimental chicken coops, installed in different situations regarding the natural or artificial environment, has made it possible to gather plenty of information on the development, variation and permanence of triatomines in artificial ecotopes, clarifying aspects related to the potential capacity for domiciliation of species as for environmental alterations.

In Brazil, species considered to be of primary relevance are T. infestans (already eliminated), P. megistus and T. brasiliensis. Secondary species are T. sordida, T. pseudomaculata, T. rubrovaria, R. neglectus, R. nasutus, P. lutzi, P. geniculatus, among others.

Some species can have different behaviors in different regions. Such is the case of Panstrongylus megistus, with great domiciliation capacity in the state of Minas Gerais, in the north of São Paulo and in some states of the North East region, while it is typically wild in the southern states of the country.

Characterization of triatomine populations has been using different tools, among them morphology, morphometry, isoenzyme electrophoresis pattern, genitalia morphology, cytogenetics, and countless molecular methods (RAPD, micro satellites, ITS etc). These markers make it easier to study populations dynamics, which determine the level of isolation between populations, or the gene flow between them. The dispersion model of T. infestans has been very well defined, based on the use of some of these techniques. Comparisons among populations of this triatomine obtained in the wild environment of Bolivia, in intra- and peri domiciliary areas in Uruguay, Argentina and, in Brazil, intradomicliary areas, prove the origin of this species in the inter Andean valleys of Cochabamba, where the triatomine has more genetic variability, which has been simplified along its area of occurrance in other countries. This also proves the passive dispersion of the species. Surprisingly, T. infestans in Brazil has a smaller quantity of autosomic DNA when compared with Bolivian populations. The exact meaning of this loss of genetic material is unknown, but this fact is likely related to a greater easiness to eliminate the species in Brazil by spraying dwellings with insecticides but also due to the appearance of populations resistant to pyrethroids in Bolivia. In Argentina and in Bolivia, peridomiciliary populations of T. infestans are a serious problem for the proposal of eliminating the species from Southern Cone countries. In the past few years, residual populations of T. infestans have been identified in the Brazilian states of Bahia and Rio Grande do Sul. A genetic study (micro satellite) has shown that these populations are very different from one another, that is, there is no gene flow between them. The study also included a reinfesting population of the state of São Paulo in the 1990’s. The analysis showed more intrapopulational variability only for the population from Bahia, suggesting (a) the existence of genetic exchange or (b) that this infestation has great insect density. This piece of information is very important, because it guides control actions mainly for the attempt of extending this outbreak or the existence of other infestation outbreaks in the region. In addition, tests of susceptibility to pyrethroids have shown that all populations were susceptible to the insecticide. The persistance of the infestation is therefore caused by an operational failure.

The high genetic variability of P. megistus has been demonstrated, but without any correlation with its epidemiological importance. However, a clear overlapping has been found between genetic patterns and the areas corresponding to landscape dominions of the caatinga, cerrado and Mata Atlantica, whose similarities/differences can be explained bio-geographically through the paleo processes of expansion and retraction of areas with higher aridity/humidity, which made it possible for P. megistus to disperse, always associated to more humid regions. This species has great epidemiological relevance and its evolutive cycle is longer, with only one generation being formed per year, but with high capacity of colonization of intradomiciliary areas: when it does colonize, it is predominantly found in bedrooms and beds. This is also a triatomine of historical importance, as it was examining specimens of P. megistus in the hinterland of the state of Minas Gerais that Carlos Chagas discovered and described the species Trypanosoma cruzi.

Triatoma brasiliensis is extremely common all over the North East region of Brazil, particularly associated to rocky formations in the caatinga, which are often located very close to human dwellings. Since the beginning of the Program for Control of Chagas Disease in the region, intradomiciliary populations have been much reduced. However, in the absence of insecticides, the populations present in the artificial environment are rapidly formed again, due to the great proximity and extension of their sources in the wild. It is interesting to note that even though there is gene flow between wild populations and the artificial environment, the populations can be very different from the genetic point of view, which shows that these populations are founded starting with a handful of specimens.

In 2006, the PAHO granted Brazil the certification of elimination of T. infestans from its territory. This was a herculean task that began in the late 1970’s, and which is worthy of much celebration: as previously mentioned, this species has always been associated to the most dramatic scenario of Chagas disease. However, there are other triatomine species that can play an important epidemiological role, some already well described, such as P. megistus and T. brasiliensis, and others still little known, but which in certain situations have been able to colonize the intradomiciliary area. Others are even more unknown but have already drawn attention because of the frequency at which they have been found. Apparently, Amazon species can transmit Chagas disease to humans without colonizing homes, in a process that still needs to be understood better. The perspective of populations of a same species having different behaviors and different epidemiological relevance opens up an important area for research and service. The definitive control of Chagas disease depends on a good understanding of the ecology of triatomines.

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Behavior

The behavior of Chagas disease vectors

Marcelo Lorenzo 

marcelo@minas.fiocruz.br

Alessandra Guarneri 

guarneri@minas.fiocruz.br

Group of Vector Behavior and Interaction with Pathogens

Almost 100 years ago, Carlos Chagas presented to the world his brilliant discovery of the cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease, and its transmission by triatomines, hematophagous insects popularly known as kissing bugs. Today, and despite the many studies published since his discovery, there are still important unknown aspects of the behavior of vector insects of this parasite that affects the health of millions of people in the American continent.

Behavior can be defined as motion of parts of the body or of the individual as a whole, or, still, of groups of individuals, and the physiological basis for this motion. Like all animals that can move around, triatomines have a varied behavioral repertoire, fundamentally related to finding food, shelter and mating partners. Several papers published over the last 110 years have analyzed aspects of these insects’ behavior. Most of these works focused on the study of Triatoma infestans and Rhodnius prolixus, the two main transmitters of T. cruzi. However, for the remaining more than 130 species of triatomines that can also transmit the disease, very little has been studied.

Looking for food is a core part of the behavior of these animals, as food is crucial for survival and reproduction. Triatomines are hematophagous insects, that is, they feed on blood. These insects look for food preferably in warm-blooded animals, such as mammals and birds, but they may occassionally (or, depending on the species, habitually) feed on the blood of reptiles and amphibians, and even on the hemolymph of insects (hemolymph is a fluid present in invertebrates, with functions similar to those of blood). Immature stages need blood, a source of proteins and other nutrients, in order to develop. Females also use blood to produce eggs; in males, blood is fundamental for the production of spermatozoa. To find their sources of blood, the so-called hosts, triatomines chase “evidence” such as heat (body warmth)CO2, the smells and humidity that the bodies of these animals release into the environment. Some of these signals, such as smells, can attract the insects from several meters away. Others, such as body warmth, are only perceived when the triatomines are close to the host. It is possible that adults, which have wings and can fly, are capable of finding their way in much longer distances, using the host’s scents transported by air currents as guides.

Once in contact with the host, the insect needs to bite it. This means they need to find a blood vessel beneath the host’s skin and introduce the extremities of their mouth parts into the vessel in order to suck the blood. To detect the small variations in temperature present in regions of the skin located over blood vessels, triatoimines use their antennae, equipped with heat receptors on their surface. Once the vessel is detected for being warmer than the tissues surrounding it, these insects extend their proboscis and perforate the host’s skin, producing a small lesion. If this action were to be noticed by the host before the insect started feeding, the bug would probably die. As an adaptation, these insects inject saliva, containing anesthetic and vasodilating substances, while they bite. Other substances injected with the saliva inhibit blood clotting. As a result, the blood meal is quick for the insect and painless for the host. Triatomines suck large volumes of blood, up to 10 times their own body weight. And all this in less then 30 minutes!

When a triatomine gets to its adult stage, another process takes priority in addition to feeding: adult males and females begin looking for mating partners. This is crucial to produce descendants, the only goal of sexual reproduction. The sexual behavior of male and female triatomines differs significantly. Males show an active behavior, attempting to copulate by mounting other insects of the species that cross his path. Meanwhile, females, which do not show specific search behaviors, can either accept or reject copulation attempts from males, who, in many cases, may have to go away without achieving their goal. But why should a male be turned away? It is now known for sure yet, but it is likely that females evaluate information related to the quality of the males and can therefore estimate the quality of the descendants the pair will produce. These interactions are mediated by scents called sexual pheromones, produced by insects to facilitate the meeting and recognizing of partners of the same species.

When they feed on infected mammals, triatomines may ingest T. cruzi, which will then develop in the gut of these insects and is later eliminated with their feces. The parasite can be transmitted to new mammal hosts when the insect defecates on the skin of a host, during or immediately after the blood meal, or when the insect is ingested purposefully or accidentally. When this happens, the mammal may be infected and the parasite can begin a new phase of its cycle. Chagas disease can develop when this host is a human being. The parasite can also have negative effects on the triatomines themselves, especially if they fast for a long period of time. In addition, infection by T. cruzi can may alter the insect’s behavior. A recent paper showed that infected individuals have their mobility reduced during the early hours of the night, exactly the period during which these insects usually leave their shelter in search of hosts.

And how do triatomines meet humans? In some situations, the encountar is incidental, as most known species live in wild environments. But in most cases, what happens is a typical situation that repeats itself in many countries in Latin America, from Mexico to Argentina. Only a few species of triatomines have the ability to enter human dwellings and develop in those environments. This happens almost exclusively in rural araes, where there are houses made of mud walls, precariously maintained. In these dwellings, these nocturnal insects find plenty of hiding places they can use during the day. The more eclectic species – those that feed on the blood of different kinds of hosts – seem to find it easier to develop in human environments. In these places they can find plenty of sources of food, including humans, dogs, cats, chickens, and pigs. This variety of hosts and hiding places offers ideal conditions for these insects. But abundant food is not all it takes for triatomines to settle in a place. Most species cannot develop in or near human dwellings, for various reasons. One important factor seems to be the behavior called negative phototaxis. This simply means that triatomines avoid light, and this characteristic apparently plays an important role in their settling in human dwellings. Most Chagas disease vectors avoid at all costs leaving their hiding places during daytime. These hiding places offer them protection and a suitable micro climate, as triatomines have very defined preferences for areas with mild temperature and relative humidity, with little variation. It seems that each species has its own preferences. So only when darkness fall do triatomines leave their hiding places and look for hosts to feed on. Their hosts, in the normal conditions of many mudwall homes in different rural areas in Latin America, cannot see the triatomines because of poor lighting. From cracks in the mudwalls can come out countless triatomines, if they belong to the species that efficiently colonize human environments. A long time ago, ecologists that specialize in the study of these interactions indicated that only this way is it possible for such high numbers of Chagas disease cases to exist in Latin America. Only through permanent co-living with large numbers of triatomines does transmission of T. cruzi through insects’ feces becomes frequent and can have a significant epidemiological impact. The solution to avoid transmisson would therefore be to eliminate these insects from human environments. Even better: ideally, no humans should have to live in such precarious conditions.

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Transcriptome

Transcriptome project

Pedro Lagerblad Oliveira

Biochemistry Laboratory of Hematophagous Arthropodes, Medical Biochemistry Institute, UFRJ

Email: pedro@bioqmed.ufrj.br

Under construction

Vector control

Controlling Chagas disease vectors

Alfredo Martins de Oliveira Filho

Biology Laboratory, Natural Products Research Hub, Federal University of Rio de Janeiro

Email: alfredomof@uol.com.br

About a century after its discovery, Chagas disease is still one of the most important parasitic diseases affecting humans in Latin America. In spite of the resources spent in research and control, the presence of vector triatomines in dwellings in most of the endemic areas can still be observed, and the risk of transmission of this trypanosome-caused disease within the human population remains. The fight against some of the most relevant vector species, such as Triatoma infestans, has been tireless, practically leading to its erradication in wide areas of Southern Cone countries. However, in most cases, species of vectors previously considered secondary have taken up their place, keeping the population under the same risk.

There is currently no vaccine and no perspective of a large-scale immunization process in a nearby future. There are no effective drugs against the disease either. The same drugs have been under use for more than 30 years and their prescription is limited, as they are only able to cure the disease in acute cases, and even then with several unwanted side effects. Controlling vectors and blood transfusions is therefore the most important tool to avoid transmission and dissemination of Chagas disease into new areas.

Many types of agents that control insects have been investigated, but results have not always provided a clear indication that they could be used in control campaigns. Here we can mention juvenile hormone analogs, most of which are produced at the Natural Products Research Hub of the Federal University of Rio de Janeiro (NPPN-UFRJ), which cause severe alterations in their life cycle and induce the appearance of sterile juvenilized adultoids, as described by Pinchin and collaborators, Oliveira Filho and collaborators, and Jurberg and ocllaborators, the precocens that have antagonic effects to those of juvenile hormone, inducing the appearance of precocious adultoids that are also sterile, and kitin inhibitors that affect the formation of the exoskeleton, not allowing for normal molting between the different stages of development of the insect. The main control effect identified is the sterilization of adultoids, but this does not prevent transmission of T. cruzi by the insects of the generation that received the treatment. One advantage is the fact that they are extremely safe for mammals. The disadvantages amount to them only working on some parts of the life cycle, as they are chemically unstable compounds and are slow to act. Another characteristic, the specificity of the target, may become a disadvantage, as there is limited interest in producing a compound for an extremely restricted market.

Biological control through the use of Microhymenoptera, insects that parasite on triatomine eggs, such as Telenomus fariai, described by Barret, have already proven to be inefficient in field conditions. Insect pathogens, such as Metarrhizium, have very limited use due to the environmental conditions necessary for the survival and spreading of this fungus. The use of traps with attractives (feromones) or without them (just hiding places for nymphs and adults) has already been proven to be inefficient, being useful only for the monitoring of infestations when vestiges are also used, such as exuviae, eggs or feces from these insects. Genetic control, as is used for certain pests through the release of infertile males, is not acceptable, as this is the vector of a disease that is very hard to treat.

Building or renewing dwellings, such as lining the walls and replacing thatched roofs with tiles are effective up to a point. However, if a simultaneous improvement in the socio-economic conditions of the population does not occur, together with education for health, these measures will not result in long-lasting results. This happens because dwellers soon begin adding new rooms or animal coops, built in a precarious fashion, resulting in cracks in the walls and ceilings that are soon colonized by the vectors present in the surrounding areas. Accumulated boxes, bags of food and other objects at home also create opportunities for shelter, therefore maintaining vector populations large and failing to prevent transmission. Improvements made to dwellings must be effective and persistent, preferably done by home owners themselves once their living conditions have also improved. Education for health should also take place as the result of a wish to participate in a community, in surveillance contexts, with the goal of preventing infestations from appearing again. At the moment, infested areas are usually very poor, and there is no evidence that these changes will take place any time soon.

Considering what was presented above, it is easy to realize that the only tool of practical use that health authorities have at their disposal to reduce transmission in affected areas is the use of insecticides. They provide a quick response to reduce the population of kissing bugs and, as a consequence, to reduce transmission. The method is also reasonably cheap and easy to implement, when compared to the other methods described above.

Humanity keeps on growing in numbers and so does our dependence on synthetic chemicals for our survival. In this context, insecticides play a prominent role in the protection of human health, directly by eliminating vectors of diseases or indirectly, as a consequence of the control of pests that affect plants, animals, and their products. This issue is frequently treated from an emotional standpoint, as the result of the various accidents that have occurred in different parts of the world, often causing irreversible changes to the environment. Pressure by ecologists against the improper use of these and other hazardous chemicals resulted in a review of the legislation in many countries, in the suppression of the production of various of these products, and also in a search for safer and more active molecules, as well as more efficient formulations that are specific for certain uses. Biological studies to evaluate these products have also been promoted, in an attempt to use lower doses for control and better application schemes, at a low cost and taking into account the reduction of environmental contamination.

Some laboratories linked to universities such as UFRJ or research institutions like Fiocruz are involved in research and development of means to control Chagas disease vectors in Brazil and in Latin America. BHC (benzene hexachloride) was the first choice insecticide to control triatomines in national campaigns in Brazil and many other countries. Although it is efficient, the product had a series of inconveniences, as it was a very stable organochloride that accumulated in living tissues. In addition, it is also biomagnified, i.e the higher it was in the food chain, the higher its accumulated values were. Laboratory and field assays were carried out by Pinchin and collaborators, Mariconi and collaborators, and Oliveira Filho, in the search for alternative insecticides, formulations and application techniques to control the main vector species in Brazil, having tested organophosphates such as chlorpyrifos ethyl and methyl, malathion, fenithrotion, pirimiphos-methyl and cholrfhoxim. Other substances that were tested were the carbamates propoxur and bendiocarb, as well as synthetic pyrethroids deltamethrin, permethrin, bifenthrin, fenpropathrin, esfenvalerate, cyphenothrin, prallethrin, pynamin, neo-pynamin, alpha-cypermethrin, cyfluthrin, and lambda-cyhalothrin. As a result of these assays, pyrethroids were selected for use in the campaings carried out so far, following the recommendations of doses, formulations and need for reapplication according to the residual effect observed for these products on T. infestans, T. sordida, T. brasiliensis, T. pseudomaculata, Panstrongylus megistus andRhodnius nasutus. These results were also supported by various works describing capture techniques and entomological evaluation, described by Pinchin and collaborators, Oliveira Filho, Briceño-Leon and collaborators, in addition to large-scale field assays regarding what happened within the homes and in the peridomiciliary area and the evicting action of insecticides, as described by Pinchin and collaborators and Oliveira Filho and collaborators. The consequences of the use of insecticides for the near environment and the planet as a whole were also studied by Oliveira Filho.

The analysis of vector control methods using insecticides takes into account the recovery of kissing bug population some time after application. Are these insects the descendents of those that survived the application or are they the result of the migration of non-treated local specimens? If reinfestation is due to survivors, more frequent reapplications will be necessary, or the application of products with more residual power. If the issue is caused by migrants, then it is advisable to extend the geographical coverage of the treatment, as proposed by Schofield. However, if the treatment uses a product or formulation with sufficient residual effect, both survival and migration can be avoided. In the first case, survivors will be eliminated once they leave their hiding places or when they emerge from laid eggs, looking for food. In the latter, recolonization of homes or annex structures is not expected during the period of insecticide activity. The biggest problem was finding insecticides with this level of persistence on porous and alkaline surfaces, usually made of clay, and that were still safe for intra-domiciliary use.

Studies by Castleton and collaborators, Pinchin and collaborators, and Oliveira Filho looking for slow-release formulations that could avoid constant reapplications thanks to increased persistence resulted in the invention of a formulation based on polyvnyl acetate and co-polymers. This technology was transferred to a Brazilian industry and its expressive production was used in the North East region of the country to control T. brasiliensis and T. pseudomaculata and in Argentina, Chile, Paraguay, Honduras and Nicaragua to control other species, obtaining good contro levels.

Laboratory and field assays on the susceptibility of various species of triatomines to organochlorines, organophosphates, carbamates and pyrethroids showed sharp differences between species. As pyrethroids represent the products most commonly used in control campaigns, biological assays were made to determine the intrinsic activity of some of the main compounds used against five of the main vector species. When comparing pesticides, lambda-cyalothrin proved to be more efficient than the three other pyrethroids, taking into account the lethal doses (LD) for 50 and 99% of the population tested. Three of the four products tested showed little effect on P. megistus and because of these parameters should not be indicated to control individuals of this species (Table 1).

Table 1. 50 and 99 lethal doses for five species of triatomines of four technical-grade pyrethroids applied topically, diluted in an acetone solution. Values expressed in µg i,a,/5th-stage nymph. Average weight of nymphs: T. infestans 143 mg, T. brasiliensis 118 mg, T. sordida 56 mg, P. megistus141 mg and R. prolixus 73 mg (Oliveira Filho, 1994).

These performance differences between technical (non formulated) products cannot be transferred directly to field situations in which formulated products interact with insects for long periods. However, these intrinsic toxicity differences for these insects will certainly result in the recommendation of different doses for use in the field.

The elimination of vector transmission of Chagas disease was taken as a priority by most Latin American governments. The WHO/TDR Southern Cone Initiative, involving Argentina, Bolivia, Brazil, Chile, Paraguay, Peru and Uruguay, where there are about 11 million infected people and 50 million people exposed to the risk of acquiring the disease, proposed the erradication of T. infestans from its territory (except for Bolivia, where this species is also found in wild environments and its erradication is not expected). This commitment has led to actions that produced a significant reduction in the incidence of this disease, according to the WHO and as described by Moncayo.

More recently, two other blocs of countries interested in controlling the transmission were created. One is the Andean Countries Initiative, involving Colombia, Ecuador, Peru and Venezuela, where there are about 6 million infected people and about 25 million people are exposed to the risk of infection. Another bloc was organized by Central American countries, and includes Costa Rica, El Salvador, Guatemala, Honduras, Mexico, Nicaragua and Panama. The main vector in these countries is Rhodnius prolixus, as demonstrated by Guhl and Vallejo and Ponce. In Brazil, the erradication of T. infestans was successful, in practical terms. Increasing attention has been dedicated to other species that had been considered secondary until not longo ago, such as T. sordidaT. brasiliensisT. pseudomaculata and, in some areas, P. megistus.

Nelson showed that kissing bugs’ resistance to insecticides has been a rare occurrence and in most cases does not affect control activities, as the doses used in the field are many times higher than the diagnostic doses recommended for the identification of resistant insects. Picollo suggests that in Bolivia, however, the failure of treatment with pyrethroids (whose mechanism of action targets sodium canals in neurone membranes) to control T. infestans, is due to the high level of resistance it has reached – in some villages, 640 more than susceptible ones. In an attempt to solve the problem, an exchange was suggested, for another insecticide with a different mechanism of action, a carbamate, which is a cholinesterase inhibitor.

Although in the past few years no intensive studies were made regarding new insecticides that could result in new tools to control Chagas disease vectors in infested domiciliary and peridomiciliary areas, there have also been no news of significant cases of resistance to insecticides that had been recommended previously and which may compromise their use. In this manner, the situation presented to this day has not suffered great changes that could invalidate the efforts of previous research that indicated various synthetic pyrethroid as insecticides effective in the control of the triatomine species that were most present in domiciliary and peridomiciliary infestations in the main endemic areas in Brazil and in the remaining Latin America. It could be possible to get around exceptional and localized casas of bubbles of resistance to pyrethroids by using organophosphates or carbamates that have already been studied as well.

The significant change occurred mainly in the profile of the disease. Chronic cases are currently more prevalent. These cases result from vector infection that took place in the past decades, as shown in a study by the Secretary’s Office of Health Surveillance on the historical series of Chagas disease data between 2000 and 2013(*1). In the last national survey, between 2000 and 2008, considering children below 5 in the rural area, the prevalence of the infection was 0.03%. However, as for 0.02% of these children there was a concomitant maternal infection, suggesting vertical transmission, there remained only 0.01% of cases of positivity exclusively in the child, indicating vectorial transmission. These figures point, without a doubt, to the success of vector transmission control campaigns that were made in the country for many years and which led to the interruption of the transmission via Triatoma infestans, as attested by the Pan-American Health Organization in 2006(*2). Other species that were also important for transmission in the national territory, such as Panstrongylus megistus, Triatoma sórdida, T. brasiliensis, T. pseudomaculata, and several species of Rhodnius no longer play a relevant role, although their presence can still be detected.

In a work by the Superintendence of Control of Endemics in São Paulo(*3) between 2010 and 2012, 3,867 notifications of insects were received, of which 72% turned out to be triatomines (2,785). Examination of the cases showed that 746 households were infested and more than 15,000 specimens of triatomines were found, of which 3.4% were infected with Trypanosoma cruzi. The prevalent species collected was T. sordida, in the peridomiciliary area. No inhabitant was found to be infected, which points to an interruption of vectorial transmission in the state.

Between 2007 and 2011(*1), more than 770,000 triatomines of 62 species were collected in Brazil. T. vitticeps, R. robustus and P. lutzi showed the highest rates of natural infection: 52%, 33.3% and 29.4%, respectively. Rare cases of transmission point to the involvement of species of wild habits with high rates of natural infection, occurring even in municipalities that were considered non endemic for the disease.

On the other hand, cases of oral transmission, which were previously quite uncommon, have been detected lately, indicating changes in the epidemiological pattern. Between 2000 and 2013, 1,570 acute cases were detected in the country, of which 68.9% were transmitted orally and only 6.4% by vector (*1). Of this total of acute cases, 91.1% occurred in the North Region, mainly in the state of Pará (74.7%). Outbreaks of oral transmission began to be investigated seriously in 2005 in the state of Santa Catarina. These were likely caused by ingesting sugarcane juice contaminated with T. cruzi. Since then, up to 2013 there had been 112 outbreaks reported in the country, involving 35 municipalities of the Amazon region, due to the probable ingestion of contaminated foods, especially açaí berries, bacaba, jaci, and sugarcane juice. In the state of Pará, where about 75% of Brazilian cases were reported, more than 50% of the acute cases notified between 2007 and 2013 had first symptoms between August and November, the period of açaí berry harvest.

Today the reappearance of Chagas disease no longer depends on the classic transmission model by T. infestans, or by other species that colonize the intra or peridomiciliary area, in a close relation with humans. Entomological surveillance remains important, associated to actions of education in health, with the goal of detecting cases of acute disease as early as possible. It is also important to have an active interface with Health Surveillance for the adoption of measures to reduce and prevent new cases of oral transmission. Data records must also be improved, as 23% of acute cases reported between 2000 and 2013 had means of transmission unknown, as can be seen in the records of the Secretary’s Office of Health Surveillance (*1).

New research lines are recommended, like those that attempt to determine the factors that make a wild population of triatomines invade and colonize the domiciliary or peridomiciliary environment or to not settle and fail to form relevant colonies. Its relevance is due to the fact that eliminating domestic populations remains possible by applying residual insecticides, while in the second case the role of vector control is limited, as invasion attempts will always be expected (*4).

Unless punctual situations occur in which triatomines once again effectively occupy domiciliary and peridomiciliary areas, there will be no reason to apply insecticides the way they were traditionally used in the past. Entomological surveillance therefore remains important to detect cases that are worthy of intervention.

Bibliography

  1. Boletim Epidemiológico, Doença de Chagas Aguda 2000-2013. Secret. Vigilância Saúde-MS. Vol. 46(21):1-9, 2015.
  2. Ferreira, I.L.M., Silva, T.P.T., Eliminação da transmissão da doença de Chagas pelo Triatoma infestans no Brasil: um fato histórico. Carta ao editor. Rev. Soc. Bras. Med. Trop., 39(5), set-out 2006.
  3. Silva, R.A., Barbosa, G.L.&Rodrigues, V.L.C.C. Vigilância epidemiológica da doença de Chagas no estado de São Paulo no período de 2010 a 2012. Epidemiol. Serv. Saúde, Brasília, 23(2):259-267, abr-jun 2014.
  4. Research Priorities for Chagas Disease, Human African Trypanosomiasis and Leishmaniasis. Technical Report Series 975, 99p, 2012.

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