Parasitol Res (2013) 112:415–425 DOI 10.1007/s00436-012-3153-x
ORIGINAL PAPER
Morphology of the midgut of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) adult ticks in different feeding stages R. N. Remedio & B. R. Sampieri & M. C. R. Vendramini & N. M. Souza & L. A. Anholeto & T. A. G. B. Denardo & M. I. Camargo-Mathias
Received: 3 September 2012 / Accepted: 26 September 2012 / Published online: 6 October 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract The intestinal epithelial cells of ticks are fundamental for their full feeding and reproductive success, besides being considered important sites for the development of pathogens. Rhipicephalus sanguineus ticks are known for their great medical and veterinary importance, and for this reason, the knowledge of their intestinal morphology may provide relevant subsidies for the control of these animals, either by direct acaricidal action over these cells or by the production of vaccines. Therefore, this study aimed to describe the midgut morphology of male and female R. sanguineus ticks in different feeding stages, by means of histological analysis. Significant differences were observed between the genders, and such alterations may refer mainly to the distinct demands for nutrients, much higher in females, which need to develop and carry out the egg-laying process. In general, the midgut is coated by a thin muscle layer and presents a pseudostratified epithelium, in which two basic types of cells can be observed, connected to a basal membrane—generative or stem and digestive cells. The latter was classified as follows: residual, deriving from the phase anterior to ecdysis; pinocytic, with vesicles containing liquid or pre-digested components of blood; phagocytic, with entire cells or remnants of nuclear material inside cytoplasmic vesicles; and mature, free in the lumen. Digestion is presumably intracellular and asynchronous and corresponds to a process which starts with the differentiation of generative cells into pinocytic digestive cells, which subsequently start to phagocytize intact blood R. N. Remedio : B. R. Sampieri : M. C. R. Vendramini : N. M. Souza : L. A. Anholeto : T. A. G. B. Denardo : M. I. Camargo-Mathias (*) Laboratório de Histologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Avenida 24-A, 1515, P.O. Box 199, Jardim Bela Vista, Rio Claro, São Paulo 13506-900, Brazil e-mail:
[email protected]
cells and finally detach from the epithelium, being eliminated with feces.
Introduction Ticks are arthropods capable of causing direct damage to their hosts or even act as vectors of relevant infectious agents because of their blood-feeding behavior (Dantas-Torres et al. 2006). Among them, the ixodid ticks (suborder Ixodida) constitute, currently, the most important group of pathogen vectors inside the phylum Arthropoda, being compared only to mosquitoes that belong to the family Culicidae (Dantas-Torres 2010). For this reason, many ixodid species have attracted the attention and interest of agencies that manage public health worldwide (Paz et al. 2008). Among the ixodid ticks, the brown dog tick, Rhipicephalus sanguineus, is considered the most frequent species worldwide and, undoubtedly, the most important species involved in the transmission of pathogens to dogs, as well as occasionally responsible for the transmission of diseases to humans (Dantas-Torres 2010). These facts confer great economical, medical, and veterinary importance to these animals, when the aim is to control their infestations and consequently prevent the transmission of diseases. Ehrlichia canis, transmitter of canine monocytic ehrlichiosis, and Babesia canis, causative agent of canine babesiosis (Blagburn and Dryden 2009), stand out as the main microorganisms transmitted by these ectoparasites. In addition, some studies have indicated that this species may be a potential vector of Leishmania chagasi, causative of canine visceral leishmaniasis (Coutinho et al. 2005); Leishmania infantum (Dantas-Torres et al. 2010), and Rickettsia rickettsii, which causes the Rocky Mountain spotted fever (Cunha et al. 2009).
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The importance of this animal group has led to numerous studies on its biology, especially related to the control of its infestations. However, the structure and function of the digestive system of R. sanguineus, which have fundamental relevance for tick ecological success and for the development of pathogens (Agbede and Kemp 1985), are still unknown, although it was described for species belonging to the same genus, such as Rhipicephalus (Boophilus) microplus (Agbede and Kemp 1985, 1987; Kongsuwan et al. 2010) and Rhipicephalus appendiculatus (Walker and Fletcher 1987; Agyei and Runham 1995), during the feeding process, in different stages. Moreover, morphological descriptions of the intestinal tract of ixodid ticks from other species, such as Haemaphysalis longicornis (Koh et al. 1991; Matsuo et al. 2003) and Amblyomma cajennense (Caperucci et al. 2009, 2010), have also been carried out. According to Coons and Alberti (1999), the midgut of ticks is divided into an anterior and a post-ventricular region, lined by a simple pseudostratified epithelium composed of cells that have distinct classifications and functions. In general, six different cell types have been described in the anterior midgut of many tick species, namely, replacement (or stem cells), digestive, secretory, undifferentiated, endocrine, and vitelogenic cells. The intestinal cells correspond to the first site of contact between ticks and the immune responses of the hosts (Agbede and Kemp 1985). Recently and in accordance with the current needs, new methods of control of R. sanguineus have been proposed, such as the ministration of food enriched with acaricides to tick hosts, as performed by Arnosti et al. (2010a, b). The active ingredients of these products, which start to circulate in internal systems of ticks, would be possibly absorbed in large quantities in the intestinal epithelium. Thus, the knowledge of the morphology and physiology of tick’s intestinal cells would facilitate the evaluation of the effects of such substances. In addition, research regarding to morphological and functional aspects of tick intestine would be crucial for the discovery of potential antigenic sites for the production of vaccines against ticks (Caperucci et al. 2009). Based on the information exposed, this study aimed to describe the midgut morphology of R. sanguineus fed and unfed males and unfed, semi-engorged, and engorged female ticks, in order to characterize the cell types that constitute this organ in different feeding stages, providing information that will certainly contribute to the overall understanding of the biology of these animals, aiding in the control of infestations and future prevention of pathogen transmissions.
Methods Unfed and fed males and unfed, semi-engorged, and engorged female ticks of R. sanguineus (Latreille, 1806) (Acari: Ixodidae) species were used in this experiment. Unfed male and female
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ticks were obtained directly from the colony maintained in a biological oxygen demand (BOD) incubator under controlled conditions (28±1 °C and 80 % humidity, in a 12-h photoperiod), in the Biosciences Institute’s vivarium of São Paulo State University (UNESP), Rio Claro/SP, Brazil. In order to obtain ticks in different feeding stages, couples of R. sanguineus were released inside special feeding chambers set on the back of naïve New Zealand White female rabbits (without prior exposure to tick infestation), following the methodology described by Bechara et al. (1995). Then, semi-engorged females with approximately 5 days of feeding, fully engorged females, and fed males were collected and maintained under controlled conditions in a BOD incubator before dissection. The collected ticks were dissected under stereomicroscope in Petri dishes containing phosphate-buffered saline solution (7.5 g NaCl+2.38 g Na2HPO4 +2.72 g KH2PO4 +1,000 mL of distilled water) for withdrawal of midgut samples. The collected material was immediately fixed in Bouin’s aqueous solution for 72 h and, then, transferred to sodium phosphate buffer solution (pH7.2), for 24 h. The samples were subsequently subjected to dehydration in an ascending series of ethanol (70, 80, 90, and 95 %, for 20 min in each solution) and overnight infiltration in Leica historesin, followed by polymerization with Leica historesin plus a polymerizing agent. The resin blocks containing the material were sectioned on a Leica RM2255 microtome, and the sections were submitted to staining with hematoxylin and eosin (HE) technique for the observation and description of the general morphology of the tissue (Junqueira and Junqueira 1983). The microscopic slides obtained were mounted in Canada synthetic balsam, and the material was photographed in a Leica DM2000 light photomicroscope, equipped with a Leica DFC280 camera, by means of the Leica IM50 software. All experimental procedures performed in this study were approved by the Ethics Committee in Animal Use, CEUA, UNESP, Rio Claro/SP, Brazil, protocol number 3727, decision number 012/2010.
Results The midguts of females and males of the tick R. sanguineus were outlined for each feeding stage, as shown in Fig. 1. Moreover, morphologic characteristics of each feeding stage were summarized in Tables 1 and 2. Externally, midgut of R. sanguineus females is formed by a thin muscular layer consisting of thin fibers whose thickness varies according to the feeding stage, presenting flattened and strongly basophilic nucleus, intensely stained with hematoxylin thence (Fig. 2a–l). In males, the midgut is surrounded by a muscular layer with thickness equal to or somewhat thinner than in females, with cells containing flattened and intensely basophilic nucleus (Fig. 3a–f).
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Internally, the intestine of both genders is covered by a pseudostratified epithelium, where the following cell types were observed, depending on the feeding stage: intestinal stem cells or generative cells and digestive cells (residual, young, and mature). Secretory cells were not observed in any feeding stage. Diameter and luminal content showed different characteristics, depending on feeding stage. In unfed ticks, both males and females, the intestinal lumen was narrow and empty (Figs. 2a, b, and 3a–c), differently from observed in semi-engorged females, which presented intraluminal content apparently fluid and with no evident presence of blood cells (derived from the host) (Fig. 2c–h). In the final feeding stages, however, both males and females present a dilated lumen, with a large amount of erythrocytes inside (Figs. 2i–l and 3d, f). Digestion is preferentially intracellular and occurs through pinocytosis and phagocytosis of the host blood components. It is also asynchronic, as cells are in different physiologic stages during the digestive process. R. sanguineus female ticks In all feeding stages, generative cells were observed in small size and its form varying from cubic to prismatic. They present a cytoplasm weakly stained by eosin, with nonreactive regions, especially around the nucleus, except in engorged females, where those regions were found in the cell basal portion. These cells have a central nucleus in which chromatin is denser in unfed individuals and becomes more disperse as the feeding process progresses. In the nucleus’ interior, it was observed to have one or two nucleoli (Fig. 2b, f, and j). In unfed subjects, digestive cells with residual material were observed, probably from the nymph feeding stage, a developmental stage before molt to adult. Residual digestive cells presented a shape, varying from round to elongate towards the lumen, and are based upon the epithelium basal membrane. In its cytoplasm, round red endosomes were observed, with similar staining to erythrocytes found in the lumen. Dispersed chromatin inside the nucleus was noted. The majority of cells presented, around the nucleus, nonstained regions. Residual bodies were not observed in these cells cytoplasm (Fig. 2a, b). In semi-engorged females, digestive cells are significantly larger and were found over the basal membrane of the intestinal epithelium or free in the lumen. Already differentiated digestive cells adhered to the basal membrane of the epithelium and found between generative cells correspond to early stages, which are beginning the process of harvesting and digesting blood. As the digestive process progresses, those cells become elongated, until they detach from the epithelium and move to the intestinal lumen, with the cytoplasm harboring the residual bodies composed by digestion by-products. This characterizes them as mature digestive cells (Fig. 2c–h).
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Morphologically, young digestive cells, still attached to the basal membrane of the epithelium, are prismatic and with a dilated apical portion. According to each digestive cell development stage, its cytoplasm can house both materials that are released by the lyse of red blood cells in the lumen, taken by pinocytosis, as well as intact blood cells of hosts, taken by phagocytosis. Young pinocytic digestive cells were found near the basal membrane and presented many homogeneous vacuoles moderately stained by eosin, varying in size. It was also frequent in these cells the presence of non-stained regions around the nucleus, which is central, presenting slightly dispersed chromatin and one to two nucleoli. As digestion progresses, there is an accumulation of granules with colors varying from yellow to brown in the cytoplasm, especially in the apical portion of the cell (Fig. 2e, g). Young phagocytic digestive cells were found in a distance from the basal membrane, while still maintaining contact with it. They are more elongated and present a cytoplasm filled by digestive vacuoles strongly stained by both hematoxylin and eosin, with varying shape and contents. Its nucleus is preferentially found mid-apically, with condensed chromatin, making the observation of nucleoli difficult. Along the digestive process, as in the pinocytic digestive cells, yellow granules accumulate in the cytoplasm, especially in the cell apex. Contact of these cells with the basal membrane is of difficult observation (Fig. 2e, h). In the intestinal lumen, mature digestive cells containing residual bodies and cytoplasmic granules in great quantities were observed. These cells are spheric and were found detached from the epithelial lining of the midgut, being excreted along with the animal feces (Fig. 2c, d). In completely engorged females, young digestive cells are possibly phagocytic because they were clearly encompassing blood erythrocytes and presented reddish endosomes. These cells are bigger and elongate towards the intestinal lumen. In its interior, there is a nucleus with similar characteristics to those of generative cells. Moreover, regions not reactive to HE were also observed to be dispersed through the cytoplasm. Digestive residues were not observed in this cell type (Fig. 2i, k). Mature digestive cells are spheric, free in the lumen, or even adhered to young digestive cells, with almost all the cytoplasm filled with vesicles with red content, similar to red blood cells. The cells have irregular shape and many prolongations. The nucleus shape accompanies that of the cell and is irregular, with possible apoptosis characteristics (Fig. 2i, l). R. sanguineus male ticks In the midgut of unfed and fed males, cells smaller than those of females were observed, forming a pseudostratified
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Fig. 1
Schematic drawing of R. sanguineus intestine cells for each feeding stage: a unfed female, b semi-engorged female, c engorged female, d unfed male, e fed male. Mature digestive cell (MDC), phagocytic digestive cell (PhDC), pinocytic digestive cell (PDC), residual digestive cell (RDC), generative cell (GC), basal membrane (bm), chromatin (chr), cytosol (cy), digestive vesicle (dv), endosome (e), hemosomes (he), lipid granules (lg), muscular layer (ml), nucleus (n), nucleolus (nu), pinosome (p), pinocytic digest cell in formation (pdcf), phagosome (ph), pycnotic nucleus (pn), vesicle (v). Bars a, d, and e 20 μ; b, c 60 μ
epithelium (Fig. 3a–f). In both feeding stages, generative cells are small and have similar characteristics to those of unfed females, with shape varying from cubic to prismatic and a cytoplasm with regions not reactive to the technique. The nucleus is found in the central region of the cell and has a condensed chromatin with one to two nucleoli, in the unfed stage. As the feeding process progresses, the chromatin is found more dispersed in the nucleus (Fig. 3b, c, e, and f). In unfed subjects, only one digestive cell type was observed, the residual digestive cells, possibly remaining of the previous developmental stage. These cells have large round vesicles in their interior, which did not stain by the employed technique, and are found in the cell apex. However, they do not present residual bodies and apparently are still connected to the basal membrane. In fed males, young digestive cells were observed with generative cells layer, also adhered to the intestine basal membrane, being prismatic and larger than the generative. In males, young digestive cells can be also classified in two types: pinocytic and phagocytic. Young pinocytic digestive cells have a cytoplasm completely filled by vacuoles with varying size and contents, some moderately stained by eosin and other non-stained. As in females, they are probably related to the digestion of components from cell lysis of the host blood. The nucleus have a disperse chromatin, and nucleoli are found in its interior. Around the nucleus, regions non-reactive to HE were observed (Fig. 3e, f). Young phagocytic digestive cells are more elongated and present most of the granules not reactive to HE. Digestive vesicles containing intact host cells, such as white blood cells and erythrocytes, were observed in the cytoplasm and may have arisen by the phagocytosis process, although in much less quantity than in females. They present a large nucleus with condensed chromatin (Fig. 3e, f). Mature digestive cells were always found free in the intestinal lumen. These cells are spheric and have many nonstained granules. However, almost no residual body or yellow granule was observed in these cells, unlike in females. In the lumen, besides the presence of the detached digestive cells, there are blood cells of the white lineage from the host and many erythrocytes, which will possibly be phagocytosed and digested by intestinal cells (Fig. 3e).
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Discussion The morphological description of internal organs of ticks can be considered an important tool in Parasitology, providing the basis for further studies, particularly with respect to the development of effective control methods, such as the use of neem extracts (Denardi et al. 2010) and andiroba oil (Vendramini et al. 2012), for example. Structures like synganglion (Roma et al. 2012a, b) and ovaries (Oliveira et al. 2005) have already been described for R. sanguineus ticks, since they correspond to important acaricidal sites of action. The description of midgut structure, in turn, would be of great importance for the study of other application methods of acaricides, as well as the development of parasites. Although midgut morphology of some tick species has been described by researchers for the past 30 years, many differences have been observed concerning general characteristics, especially regarding the description of cell types, demonstrating a wide variety of achieved results even in tick species that belong to the same family. The difficulty in defining the morphological nature of epithelial cells of the ticks’ midgut contributed to the origin of many nomenclatures. In the present study, R. sanguineus (males and females), in different feeding stages, showed only two cell types: (1) stem cells, here called generative cells, following the nomenclature proposed by Caperucci et al. (2010), and (2) digestive cells classified as young and mature. The nomenclature here used for the description of young digestive cells was divided according to their endocytic activity (pinocytosis or phagocytosis), adapting the criteria proposed by Agbede and Kemp (1985) and Walker and Fletcher (1987). Unlike other descriptions, the hypothesis of Walker and Fletcher (1987) in which the phagocytic cells would be derived from pinocytic was maintained. However, the motility of these cells, as proposed by these same authors, was ruled out. In the present work, the pseudostratification, observed along the epithelium in all feeding stages of males and females, occurred mainly because the cells were found in different digestion stages, with observed cell morphologies varying from cubic to prismatic. The same epithelium was described by Caperucci et al. (2009) in the midgut of unfed female ticks of A. cajennense. However, unlikely in unfed subjects, semi-engorged and engorged females from this tick species showed a stratified epithelium. Digestion supposedly occurs intracellularly in R. sanguineus, through pinocytosis and phagocytosis in all feeding stages, confirming the data found for R. appendiculatus (Walker and Fletcher 1987), in which digestion occurred by activity of the lysosomal acid phosphatase system of digestive cells. However, some data showed that other sources of acid phosphatase in the ticks’ digestive tract, as well as the secretory cells of the gut, can pre-digest the ingested material in a way that a selective endocytosis can occur more efficiently (Walker and Fletcher 1987). The gut secretory cells would be
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Table 1 Summary of morphological results found in intestinal cells of R. sanguineus female ticks in different feeding stages Cell type
Generative cells
Residual digestive cells
Pinocytic digestive cells
Phagocytic digestive cells
Mature digestive cells
Characteristics Feeding stage
Presence/absence
Description
♀ unfed
Present
♀ semi-engorged
Present
Small cells, cubic or prismatic Cytoplasm with unstained regions Nucleus with condensed chromatin One to two nucleoli Similar to unfed individuals
♀ engorged
Present
♀ unfed
Present
♀ ♀ ♀ ♀
semi-engorged engorged unfed semi-engorged
Absent Absent Absent Present
♀ engorged ♀ unfed ♀ semi-engorged
Absent Absent Present
♀ engorged
Present
♀ unfed ♀ semi-engorged
Absent Present
♀ engorged
Present
mainly responsible for the lysis of the host blood cells, facilitating the uptake of blood constituents (Agbede and Kemp 1985). This can be particularly important during initial feeding stages, in a way that the tick can select the most favorable nutrients for its rapid growth, with subsequent excretion of residues (Walker and Fletcher 1987).
Nucleus with slightly condensed chromatin Also similar to unfed females Nucleus with highly dispersed chromatin Rounded or elongated, central nucleus Endosomes with reddish staining Unstained regions around the nucleus Without hemosomes – – – Elongated, with dilated apex Homogeneously reddish endosomes Unstained regions around the nucleus Central nucleus with dispersed chromatin Accumulation of hemosomes in the apex – – Elongated, distant from basal membrane Vesicles stained by both reactants Nucleus with medium apical localization Condensed chromatin Accumulation of hemosomes in the apex Large and elongated toward the lumen Unstained regions in the cytoplasm Vesicles with reddish staining Nucleus with dispersed chromatin Without hemosomes – Detached from basal membrane Large amount of hemosomes Irregular aspect, with projections Free in the lumen or adhered to other cells Vesicles with reddish staining Pycnotic irregular nucleus Without hemosomes
However, secretory cells were not found in R. sanguineus, suggesting that pre-digestion of the host blood cells and constituents is being done by other processes, like secretory activity of the salivary glands acini, for example. In H. longicornis nymphs, acini cells of the salivary glands would be responsible for the host blood cells lysis, which would already arrive pre-
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Table 2 Summary of morphological results found in intestinal cells of R. sanguineus male ticks in different feeding stages Cell type
Generative cells
Characteristics Feeding stage
Presence/absence
Description
♂ unfed
Present
Small cells, cubic or prismatic Cytoplasm with unstained regions Central nucleus with condensed chromatin One to two nucleoli Similar to unfed individuals
♂ fed
Present
Residual digestive cells
♂ unfed
Present
Pinocytic digestive cells
♂ fed ♂ unfed ♂ fed
Absent Absent Present
Phagocytic digestive cells
♂ unfed ♂ fed
Absent Present
Mature digestive cells
♂ unfed ♂ fed
Absent Present
digested to the intestinal lumen (Koh et al. 1991). As seen in Agyei and Runham (1995), the fact that secretory cells were not observed does not mean that they are not present. The intestine generative cells are found generally in the epithelium of each instar initial feeding stages, although more frequently observed in intermediate stages, during engorgement (Agbede and Kemp 1985; Walker and Fletcher 1987). In R. appendiculatus, referred cells were considered proliferative and pluripotent, as only one type was distinguished in this species intestine (Walker and Fletcher 1987). In R. sanguineus, only one type of generative cell was also observed, suggesting that these cells would be responsible by differentiation and proliferation of digestive cells. For Agyei and Runham (1995), enough evidence to reject the hypothesis that digestive cells are formed from the generative ones was not found. In R. appendiculatus, stem cells did not show secretory or digestive function. They showed, however, great accumulation of lipids in the cytoplasm (Walker and Fletcher 1987). This suggests that non-stained spaces in the generative cells of R. sanguineus may be energetic reserves (lipid droplets), generally dispersed on the cytoplasm of these cells in all feeding stages on males and females. In A. cajennense, generative cells of semi-engorged females also presented lipid droplets in great quantity in the cytoplasm,
Provenient from the phase anterior to ecdysis Elongated toward the lumen Rounded and unstained vesicles Large nucleus with condensed chromatin – – Cytoplasm with small vesicles Unstained vesicles dispersed in the cytosol Endosomes stained by eosin Nucleus with dispersed chromatin – More elongated, with most of vesicles not reactive to the technique used Vesicles with intact host cells, such as leucocytes and erythrocytes – Free in the lumen, detached from basal membrane Plenty of small and sustained vesicles
when analyzed ultrastructurally (Caperucci et al. 2010), thus corroborating the results obtained for R. sanguineus in light microscopy. Different nucleus morphology present in these cells indicated an alteration in cellular metabolism, during feeding of males and females. The nuclei presented dense chromatin during the fasting period, indicating that these cells had low metabolic activity during this stage. As the feeding process progressed, its mitotic activity raised, making possible generative cell differentiation, evidenced by lower condensation of nuclear material. As in other species, this change in the activity of intestine cells during the feeding stage suggests that the blood presence can stimulate both mechanically and chemically the development of digestive cells, due to mechanical stretching of the intestine or by contact of stem cells with blood (Agyei and Runham 1995). In unfed females and males of R. sanguineus, only one digestive cell type was observed, the residual cells, which received this denomination as they can be digestive cells that did not detach from the intestinal epithelium and are reminiscent of the feeding stage preceding molt. These cells showed large vesicles in its interior, which can be related to energy supply, probably used for surviving long periods without food, as suggested by Koh et al. (1991), whereby the fasting intestine would act as a nutrient reserve.
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Fig. 2 a General view and b detail of the midgut epithelium of unfed R. sanguineus females. c, d General view and e detail of the midgut epithelium of semi-engorged R. sanguineus females. f Detail of the generative cells (gc) in semi-engorged females. g Detail of the pinocytic digestive cells (pdc) in semi-engorged females. h Detail of the phagocytic digestive cells (phdc) in semi-engorged females. i General view of the midgut epithelium of engorged R. sanguineus females. j Detail of the generative cells in engorged females. k Detail of the phagocytic digestive cells in engorged females. l Detail of the mature
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digestive cells (mdc) in engorged R. sanguineus females. Muscle layer (arrow), hemosomes/hematin (arrowhead), digestive vesicles (asterisk), cytoplasmic projections (cpr), cytosol (cy), endosomes (e), erythrocytes (er), lumen (L), lipid granules (lg), nucleus (N), nucleolus (Nu), pinosome (p), pseudostratified epithelium (pe), phagosome (ph), pycnotic nucleus (pN), residual digestive cell (rdc). Magnifications a, c, d, and i ×400; b, e–h, and j–l ×1,000. Bars a, c, d, and i 60 μm; b, e–h, and j–l 20 μm
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Fig. 3 a General view and b, c detail of the midgut epithelium of unfed R. sanguineus males. d General view and e, f detail of the midgut epithelium of fed R. sanguineus males. Muscle layer (arrow), digestive vesicle in formation (arrowhead), lipid granules (asterisk), pinocytic digestive cell in formation (double arrowhead), erythrocyte (er), generative cells (gc), lumen (L), mature digestive cells (mdc), nucleus (N), pinosome (p), pinocytic digestive cells (pdc), phagocytic digestive cells (phdc), pseudostratified epithelium (pe), phagocytosed erythrocyte (pher), phagocytosed leucocyte (phl), residual digestive cells (rdc), vesicles (v). Magnifications a, d ×400; b, c, e, and f ×1,000. Bars a, d 60 μm; b, c, e, and f 20 μm
Differences were observed, however, in the constitution of vesicles in residual cells of males and females of R. sanguineus. In unfed nymphs of H. longicornis, digestive cells presented endosomes positively stained by toluidine blue, indicating the presence of proteins (Koh et al. 1991). Digestive cells containing non-stained vacuoles, in turn, were observed in unfed females of R. (Boophilus) microplus (Agbede and Kemp 1985). In R. sanguineus, however, the used technique did not permit the identification of components present in the endosome of these cells. The staining differences may indicate that the feeding process is different between males and females before the passage to adult stage and sexual differentiation. During the feeding of the cattle tick, R. (Boophilus) microplus, two distinct young digestive cell types could be observed. One cell type showed many vacuoles containing hosts leukocytes and some diet soluble constituents, while the other, which develops later, did not show leukocytes (Agbede and Kemp 1985). This description differs from characteristics observed in semi-engorged females and fed males of R. sanguineus. It is believed that generative cells after differentiation in young digestive cells initially harvest, by pinocytosis processes, blood soluble constituents, from the extracellular pre-digestive
mechanisms. During digestion, these cells elongate towards the lumen, initiating phagocytosis of whole erythrocytes and leukocytes. Phagocytic cells of R. appendiculatus were easily recognized by the presence of leukocytes nuclei inside its endosomes (Walker and Fletcher 1987). In R. sanguineus, this fact was evidenced by the presence of endosomes heavily stained by hematoxylin, which may correspond to genetic material of leukocytes, since hematoxylin is an acidophilus dye. Two main digestive cell types can also be observed in R. appendiculatus during feeding process: (1) sessile, being mainly pinocytic, and (1) motile, which phagocyte whole erythrocytes and leukocytes, which originate from sessile cells and hold a large amount of residual bodies in the cytoplasm (Walker and Fletcher 1987). The midgut of R. sanguineus ticks showed very similar characteristics as those observed in R. appendiculatus. However, R. sanguineus digestive cells have no moving ability in the intestine. These cells’ contact with the basal membrane is unmade at the end of intracellular digestion, when phagocytic cells are detached and eliminated with feces. Either way, as in H. longicornis (Koh et al. 1991), a progression in intestine cell development was observed, initiating with the generative
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cells activity increase, which start digesting blood elements, accumulating residual bodies, and protruding themselves to the intestinal lumen until detaching from the basal membrane and being replaced by new generative cells. H. longicornis ticks, however, showed no phagocytic cells. A great quantity of lipid droplets accumulates in the cytoplasm, particularly around the nucleus of digestive young cells in R. (Boophilus) microplus (Agbede and Kemp 1985) and R. appendiculatus (Walker and Fletcher 1987). During R. sanguineus engorgement, many vacuoles were observed in this region, suggesting that this lipid accumulation would also occur in this tick species. Many residual bodies also were present in this feeding stage. According to Agyei and Runham (1995), at the end of intracellular digestion, some digestive cells detach from the basal membrane, and its formation is preceded by the nucleus moving towards the apical portion of the cell. The detached cells are generally spherical and free, with vacuoles positively stained for proteins and many residual bodies, being called mature digestive cells. In R. (Boophilus) microplus, glycogen and lipid droplets accumulated in small deposits in mature cells. These cells also presented large quantities of granules dispersed in the cytoplasm, with colors ranging from yellow to dark brown (Agbede and Kemp 1985). Walker and Fletcher (1987) also observed accumulation of lipid droplets and residual bodies in mature intestinal cells of R. appendiculatus, detached from the basal membrane. In R. sanguineus, many yellow- or brown-colored granules were observed in males and females, corroborating the results obtained by these authors. Besides, in males, a great number of non-stained vesicles were noted. Such vesicles were not observed in mature digestive cells of semi-engorged and engorged females and may refer to lipid accumulations. These differences suggest that digestive processes may occur differently according to gender. During digestion in R. (Boophilus) microplus ticks, hemoglobin and other blood materials accumulate in vacuoles, which become larger as fusion with smaller ones occur (Agbede and Kemp 1985). As blood digestion occurs, byproducts are formed, such as hematin, which accumulates in the cytoplasm and is found in large quantities in mature digestive cells. Initially, they are yellow small granules, which then become larger and darker (Wigglesworth 1954; Agbede and Kemp 1985). Such granules could be observed in the apex of young digestive cells, as well as dispersed in the cytoplasm of mature digestive cells in R. sanguineus, and correspond to residual bodies originated from digestion of fluids and blood cellular components. Ticks ingest large volumes of blood, and therefore, large amounts of heme groups are produced inside the cell as a by-product of hemoglobin digestion. The heme group promotes the formation of free radicals, and for this reason, the majority of these molecules are accumulated inside
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a specialized organelle, limited by a membrane and present in digestive cells, called hemosome (Lara et al. 2003). In R. sanguineus fully engorged females, the presence of generative cells with distinct characteristics from previous stages was noted, as well as many cells that are apparently phagocytosing erythrocytes. At this stage, pinocytosis was not observed, which can indicate that the digestive process would change from one feeding stage to another. Such alterations may have happened due to changes in energy demand and blood volume. Within the lumen, a large amount of mature cells detached from the basal membrane of the intestinal epithelium was observed, showing plasma membrane prolongations. Midgut cells of fully engorged R. sanguineus females showed completely distinct characteristics when compared to other feeding stages, as when related to other tick species. Their characteristics completely differed from fully engorged R. appendiculatus females, which present phagocytic digestive cells with many leukocytes, as well as cells containing diet pre-digested components and basophilic cells (Agbede and Kemp 1985), and engorged R. (Boophilus) microplus females, in which phagocytic cells contained many hemosomes, few lipid droplets, and heterolysosomes with leukocytes from the host (Walker and Fletcher 1987). Based on previous reports and in results presented here, it is still not possible to suggest a reason for the presence of mature cells containing large amounts of phagocytosed vesicles and showing such plasma membrane projections. In general, the main digestion process in males and females seem to be intracellular and asynchronous, occurring mainly by pinocytosis and phagocytosis, with great differences specially according to feeding stage and gender. The data obtained will provide an important basis for further work, contributing to studies related to functional activities of this organ or even for the control of infestations of this tick species. Acknowledgments The authors are thankful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support. Conflict of interest None.
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