The ultrastructural studies in parasite-vectors interactions

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1 The ultrastructural studies in parasite-vectors interactions D. Feder 1, S. A. O. Gomes 2, S. C. Freitas 3, G. Santos-Machado 1,2 and J. R. Santos-Mallet 3 1 Laboratório de Biologia de Insetos, GBG, Universidade Federal Fluminense-UFF, Niterói, Rio de Janeiro, RJ, Brazil 2 Laboratório de Biodiversidade de Parasitas e Vetores, GBG, Universidade Federal Fluminense-UFF, Niterói, Rio de Janeiro, RJ, Brazil - CEP: Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, IOC-FIOCRUZ - Rio de Janeiro, RJ, Brazil, CEP: The Trypanosoma genera belongs to the Trypanosomatidae family and cause important human infection such as Chagas disease, also known as American trypanosomiasis, caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada and Europe. Trypanosoma rangeli is a protozoan that is nonpathogenic for humans and other mammals but causes pathology in triatomines from the genus Rhodnius. These trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Basically, bloodstream trypomastigotes are ingested by the insect vector and differentiate into epimastigotes that proliferate in the gut and eventually transform into metacyclic trypomastigotes able to infect vertebrate hosts. Colonizing different regions of the gut, T. cruzi and T. rangeli are confronted with several molecular factors, different epithelial and surface properties. These events are considered parasite-vector interactions and are also researched by transmission electronic microscopy (TEM) analysis. Keywords: Trypanosoma cruzi; Trypanosoma rangeli; Triatominae; Rhodnius prolixus; Quantun dots; parasite-vector interactions 1. Introduction The Kinetoplastida order are characterized by the presence of one or two flagella arising from a structure in the cell membrane, called, flagellar pocket. They have a single and long mitochondria that differentiates a typical, rich DNA organelle and that gives the name to the order: the kinetoplast. The cell of these microorganisms has a structure of microtubule stabilized by cross-links associated with the cell membrane, called subpellicular microtubules. Two suborders comprising the Kinetoplastidae are the Diplomonadina and Trypanosomatina. The latter with all representatives of the Trypanosomatidae family, is of great importance for Parasitology [1]. In this family are included twelve Genera flagellate protozoan parasites of a broad spectrum of organisms included in the Kingdom Protista, Animal and Plant [2]. Trypanosoma genus includes three species that infect humans: Trypanosoma cruzi and Trypanosoma rangeli in Latin America and Trypanosoma brucei in Africa [3]. The life cycle of these parasites involves passages through vertebrate and invertebrate hosts [4]. 2. Trypanosoma cruzi It is a flagellate protozoan agent of Chagas disease, also known as American trypanosomiasis. According to the World Health Organization (WHO) this disease is present in 21 countries in Central and South America, where there are 100 million people in risk areas [5] and about 7 to 8 million people affected [3]. It is a chronic, debilitating disease that a developments clinical presentation with very different characteristics and consequences in the vertebrates hosts, including the man. Once infected, the Trypanoma cruzi hosts may develop serious illnesses such as myocarditis, cardiomegaly, meningoencephalitis, megacolon, megaesophagous, among others. Doesn t exist immediate prospect of effective treatment for this disease [3], being the control of vector population inside and outside the home, one of the main strategies used to contain the spread of T. cruzi to human populations and animals in areas of epidemiological risk. Chagas disease is closely related to poor housing, as these favor the nesting bloodsucking insects of the Hemiptera order, Reduviidae family, Triatominae subfamily. The main route of transmission of T. cruzi to man occurs through contact of the feces of the insect vector, contaminated with metacyclic trypomastigotes forms of the parasite, during or shortly after blood feeding [6]. When they feed on blood of infected people or animals, triatomine bugs can ingest the trypomastigotes forms. These are converted into epimastigotes in the gut of triatomine. Epimastigotes reproduce by binary fission, and when they reach the terminal portion of the intestine (rectum) of triatomine, differentiate into highly mobile and infective metacyclic forms, which are eliminated in the feces of the insect vector, constituting the form infective to vertebrates. The main intracellular forms of Trypanosoma cruzi is amastigote phase that is present in the chronic phase of this disease [6]. 564

2 3. Digestive tract of triatomine vectors Triatominae are hematophagous insects (figure 1a) and to ingest the blood of their hosts they use their piercing-sucking mouthparts. These are formed by the rostrum, composed of 3 segments, and when it is at rest, is adapted to the bottom of the insect head. Within it there is a set of stylets that consist of four structures, two mandibles and two maxillae, used for sucking blood and injecting substances produced by the salivary glands. The salivary glands have great diversification as to the number, size, shape and situation in different triatominae. They are generally located in the thoracic cavity, with the initial part of the digestive tract. There are three pairs, using the nomenclature - D1, D2 and D3 for each (figure 1c). The pair D1 corresponds to the major glands, D2 corresponds to the additional glands and D3 corresponds to the accessory glands. In all triatominae species studied, we have always found the three pairs of salivary glands, except for the genus Rhodnius which does not have the typical gland D3. In general, D1 and D2 glands have milky white or slightly yellowish color, as in P. megistus. However, in the genus Rhodnius, D1 and D2 glands are elongated and reddish in color [7]. The digestive system of the triatominae insects is divided into three parts: foregut, midgut and hindgut. The foregut is pharynx and esophagus, which are narrow structures that reach the entrance of the midgut that comprises the major portion of triatominae digestive tract formed by an anterior portion, the stomach, and a posterior portion, the guts (figure 1b-d). In this there is perimicrovillar membrane, also known as extracellular membrane that surrounds the microvilli of the epithelial cells of the midgut, extending to the lumen and ending in a blind bottom [8, 9]. The perimicrovillar membrane is synthesized by the epithelium of the total digestive tract at all stages of development. These structures are formed by glycerophospholipids, proteins, carbohydrates and hydrolytic enzymes, and they do not contain chitin. Moreover, this membrane that has selective permeability which allows digestive enzymes come to the bolus, and also that digestion products are absorbed from the intestinal epithelium [10](Peters, 1992). Finally, we found the hindgut, which makes up the final part of the digestive tract, consisting of a rectal bulb and the rectum (figure 1e), and has an important role in the reabsorption of water and minerals. The rectal bulb is a muscular sac with considerable deterrence capability, where the four Malpighian tubules flow into. During blood feeding, diuretic hormones are released, and water and ions are transported through the gut wall into the haemolymph to the Malpighian tubules and are then eliminated through the rectum [11]. Fig. 1 A- Adult of Rhodnius prolixus. B - End portion of the digestive system showing a rectal ampullae (RA) and the four Malpighian tubules (MT). C -Schematic localization of salivary glands of Triatoma infestans (adapted from [7]. D- General diagram of triatominae digestive system (Adapted from [12]). The regions between the lines represent the midgut. Arrows indicate food direction. E- Scheme of the hindgut of triatomine (adapted from [12]). E: esophagus, In: intestine, R: rectum, St: stomach, G1: 1 gland, G2: 2 gland, G3: 3 gland, LA: labium, Mi: midgut, Pv: proventiculum, SP: salivary pump, SD: salivary duct. 565

3 4. Quantum dots The semiconductor "quantum dot" (QDs) are synthesized from Cadmium Tellurium (CdTe) or Cadmium Selenium (CdSe) atoms, forming nanoparticles with fluorescent characteristics and superior luminescent properties [13]. QDs can bind with molecules such as proteins, peptides and nucleic acids [14] forming bioconjugates. These nanoparticles can also bind nonspecifically to the cell surface by electrostatic interaction or receptor [15] and many studies have shown that the uptake of QDs by cells occurs by endocytosis [16]. Quatum dots appear to enter the T. cruzi parasite cells by endocytosis through the cytostome and are delivered to reservosomes [17]. Moreover, an advantage of this nanoparticle is the resistance to chemical and metabolic degradation within the cells [16]. QDs toxicity is currently a much studied area in view of the medical and biological application [18]. The toxicity of QDs is associated with their physicochemical properties and the studied model [19]. CdTe citotocixity effects in epimastigotes of T. cruzi revealed changes in morphology followed by autophagy death [20, 21] (figure 2). Using monodansillcadaverine (MDC) as a fluorescent marker of autophagic vacuoles, were observed qualitative differences between the parasites control treated with 200μM of QDs (CdTe) (figure 2A) and group treated with quantum dots (200μM) plus 10mM of monodansylcadaverine (figure 2 B-C) confirming the process of autophagy in development. Vieira et al., (2011) by transmission electron microscopy showed that the use of 2μM CdTe did not alter the morphology of T. cruzi. However, several changes in parasites incubated with 200μM CdTe were shown as mitochondrial swelling; many vacuoles; double membranes containing intense vacuolization and concentric structures, which also features a cell death by autophagy (figure 3B). These data contribute to the understanding of cellular dynamics and kinetics of the QDs (including uptake mechanisms and intracellular distribution), providing basic information for biomedical and biological applications of QDs as well as the manipulation of QDs less toxic to cells. Fig. 2 A- T. cruzi treated with 200μM of QDs (CdTe) and B-C T. cruzi treated with 200μM of QDs + 10mM of monodansylcadaverine.the arrows shows autophagosomes expressing the cadaverine protein labeled. Fig. 3 Transmission electron microscopy analysis of T. cruzi epimastigotes labeled with CdTe QDs. A- Control epimastigotes presenting typical morphology. B- The incubation with 200 µm QDs induced morphological alterations in the parasites led to intense cytosolic vacuolization (V) and plasma membrane alterations such as blebbing (black arrows) formation. N, nucleus; F, flagellum; K, kinetoplast. 5. Triatominae/ Trypanosoma cruzi Interaction While the insects feed on blood from an infected vertebrate host, its can acquire trypomastigotes forms of the parasites, passing through the mouthparts, thus reaching the midgut (small intestine) (figure 1b-d) [22]. In the stomach, most of the ingested bloodstream trypomastigotes differentiate into epimastigotes, the main replicative stage responsible for the maintenance phase of infection in the vector. The midgut is divided into 2 parts of different diameters and functions: a 566

4 secretory anterior and posterior absorptive [23]. Hemolytic activity and proteolytic enzymes of triatomine midgut are responsable by an environment hostile to the development of parasites [24]. After passage into the midgut and rectum the epimastigotes divide repeatedly and adhere to the perimicrovillar membranes of the intestinal cells (Figure 4 A-B-C- D) [25, 26, 27]. The proctodeo comprising the rectal ampulla and the rectum, the pyriform is a structure which is the last part of the digestive tract, playing an important role in the absorption of water and mineral recovery [28]. In the proctodeo the differentiation of epimastigotes into metacyclic trypomastigotes occurs. Following attachment to the waxlayer of the rectal cuticle by hydrophobic interactions, epimastigotes transform into non-replicative but infective metacyclic trypomastigotes [29]. The metacyclic trypomastigotes accumulate in proctodeo and are shed in the feces and are able to infect the mammalian host [6]. Fig. 4 A- Midgut cells imaging- Intestinal Epithelium of Rhodnius prolixus: Laser transmission; B- minor autofluorescence present in midgut cells, C- Intestinal Epithelium of Rhodnius prolixus shows an In vitro incubation of Trypanosoma cruzi cells D- Confocal analysis shows an In vitro incubation of Trypanosoma cruzi cells of alive QDs-labeled T. cruzi epimastigotes with yellow emitting CdSe quantum dots (After Feder et al., 2009[15]). 6. Trypanosoma rangeli Trypanosoma rangeli Tejera, 1920 is outher hemoflagellate heteroxenic parasites, that infects humans and domestic and wild animals, usually have insects as vectors that infect mammals and is present in several countries of Central America and South America, so living in sympatry with T. cruzi [30]. T. rangeli was discovered in Venezuela, later being found in Colombia, Guatemala, Guyana, Chile and Argentina [31]. This parasite is not pathogenic to vertebrate hosts, however causes damage to invertebrate host [32]. T. rangeli belongs to the Trypanosomatidae family and has morphological characteristic of the subgenus Herpetosoma, but is distinguished by its biological cycle in the insect vector. Currently, the taxonomic position of T. rangeli is still controversial, whether it belongs to salivate and / or stercoraria section. [33]. The natural development of T. rangeli in the invertebrate host begins with the ingestion of blood of the vertebrate host containing trypanosomes. In the insects, these parasites multiply in the midgut, penetrate in the hemocoel (bathed by the hemolymph cavity) and migrate to the salivary glands where they differentiate into infective forms that are injected with the saliva through the bite in the vertebrate host, thus continuing the cycle [34]. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. After ingested as trypomastigotes by its vectors multiplies as epimastigotes in the midgut and invade the hemocoel. In a few days after infection, short epimastigotes (figure 5 A-C) appear in the hemocoel of the insect and soon, they disappear to be replaced by a massive colonization by long epimastigotes (Figure 5 B-D) [35, 36]. The long epimastigotes survive in the hemolymph and/or inside the hemocytes and migrate to and complete their development in the salivary glands [37]. Consequently, the cellular differentiation process is a crucial event during the life cycle of T. rangeli. Although not pathogenic to its vertebrate hosts, the overlap of the geographical distribution of T. rangeli and T. cruzi has been a complicating agent for health programs. The epidemiological impact of T. rangeli is associated with the presence of surface antigens similar to T. cruzi, indicating a cross serology, ie, false-positive for Chagas disease. In addition, there are records of about 2500 people with mixed infections in several countries in Latin America [38]. Fig. 5 Trypanosoma rangeli: A-short epimastigote and B- long epimastigote by optic microscopy; C-D - short and long epimastigote by transmission electron microscopy. N- nucleus; c- cinetoplast; F flagellum. 567

5 6.1 Triatominae / Trypanosoma rangeli Interaction In invertebrates, T. rangeli has as main ability to invade the hemolymph of the triatomine vector following pathway to the salivary gland of the insect, which complete their life cycle by inoculating infective forms in the act of biting and blood ingestion, for the vertebrate host [32]. Moreover, T. rangeli, after the invasion of the hemocoel, can develop intracellularly in all organs of the insect vector, R. prolixus [39]. These parasites need to survive and escape of the mechanisms to defense processes triggered in the invertebrate host as cellular and humoral immune response of these insects. T. rangeli to reach the hemocoel of R. prolixus, seem to escape of humoral and cellular defense present in the hemolymph [40] and invade the salivary glands of insect vectors (Figure 6 A-B and C), and thus, are introduced in the vertebrate host, during salivation, early blood meal [41]. Fig. 6 A-B Salivary gland (SG) of Rhodnius prolixus and C- Confocal analysis shows an in vitro incubation of alive Trypanosoma rangeli QDs-labeled with salivary gland of Rhodnius prolixus. The parasite detects the target cell, salivary gland (T rangeli) and moves towards it. Nucleus (N), parasites (arrows). Acknowledgements We are gratefully to the Electron Microscope Plataform of Instituto Oswaldo Cruz FIOCRUZ, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), PROPPI-UFF and CEPOF UNICAMP. References [1] Rey L.Parasitologia o Ed. Rio de Janeiro. Guanabara Koogan, 856p [2] Souto-Padrón, S. The surface charge of Trypanosomatids. An Acd. Bras. Cienc. 2002; 4: [3] World Health Organization (WHO), Chagas disease (American trypanosomiasis). Available: http;// Acessed: may/2014. [4] Stevens JR., Noyes HA., Schofield, CJ., Gibson, W. The molecular evolution of Tripanosomatidae. Adv. Parasitol. 2001; 48:1-56. [5] Organização Pan-Americana de Saúde (OPAS), Chagas disease (American Trypanosomiasis). Available:http;//new.paho.org/hq/index.php?option=com_content&view=article&id=2382&itemid=392&lang=fr. Acessed: may/2014. [6] Garcia ES., Genta, FA, Azambuja, P. and Schaub, GA. Interactions between intestinal compounds of triatomines and Trypanosoma cruzi. Trends in Parasitol. 2010; 26: [7] Barth R Estudos anatômicos e histológicos sobre a Subfamília Triatominae. IV. O complexo das glândulas salivares de Triatoma infestans. Memórias do instituto Oswaldo Cruz 52: [8] Terra WR. Evolution of Digestive System of Insects. Annual Review Entomology 1990; 35: [9] Albuquerque-Cunha JM, Mello CB, Garcia ES, Azambuja P, Souza W de, Gonzalez MS, Nogueira NFS, Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus. Mem. Inst. Osw. Cruz 99: [10] Peters W. Peritrophic membranes. Ed. Springer & Verlag, New York, USA. 1992; 238. [11] Kollien AH, Schaub GA. The development of Trypanosoma cruzi in Triatominae. Parasitology Today 2000; 16: [12] Lacombe, D. Estudos anatômicos e histológicos sôbre a subfamília Triatominae (Heteroptera, Reduviidae). VII. estudo anatômico do ducto intestinal do Triatoma infestans. Mem. Inst. Osw. Cruz; 55(1):69-111, maio [13] Alivisatos AP, Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science 1996; 271: [14] Weng J, Song X, Li L, Qian H, Chen K, Xu X, Cao C, Ren J. Highly luminescent CdTe quantum dots prepared in aqueous phase as an alternative fluorescent probe for cell imaging. Talanta 2006; 70(2): [15] Parak WJ, Pellegrino T, Plank C Labelling of cells with quantum dots. Nanotechnol 2005; 16:R9-R21. [16] Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol 2003; 21: [17] Feder D, Gomes SAO, de Thomaz AA, Almeida DB, Faustino WM, Fontes A, Stahl CV, Santos-Mallet JR and Cesar C L. In vitro and in vivo Documentation of Quantum Dots Labeled Trypanosoma cruziand Rhodnius prolixus Interaction using Confocal Microscopy. Parasitol Res 2009; 106: [18] Marquis BJ, Love SA, Braun KL, Haynes CL. Analytical methods to assess nanoparticle toxicity. Analyst 2009; 134: Pelley JL, Daar AS, Saner MA. State of academic knowledge on toxicity and biological fate of quantum dots. Toxicol Sci 2009;112:

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