Ultrastructural studies on the midgut epithelium and digestion in the female tickArgas (Persicargas) arboreus (Ixodoidea: Argasidae)

The ultrastructure of the midgut epithelium and digestion in the female tickArgas (Persicargas) arboreus are described before and after feeding, up to oviposition. The epithelium consists of secretory cells, digestive cells (DI and DII), and regenerative cells which may differentiate into any of the other cell types. In unfed ticks, the midgut wall consists mainly of type DII digestive cells retained from a previous feeding, and a few regenerative cells. Within 3 days after the tick feeding, haemolysis of the host blood components occurs in the midgut lumen. Secretory cells, the first differentiation of the regenerative cells, are presumed to produce a haemolysin and an anticoagulant which are released by merocrine and holocrine secretions. The DII cells seen in unfed ticks, and secretory cells which have completed their secretory cycle, start to have a specialized surface for endocytosis characteristic of type DI digestive cells. From 5 to 7 days after feeding up to the female oviposition, type DI cells which have completed their endocytosis are transformed into type DII digestive cells specialized for intracellular digestion and the storage of reserve nutrients required by the tick for long starvation. The various phases of the digestive cycle are considered according to ultrastructural changes of the midgut epithelium.     

Morphofunctional changes in the midgut of the Ixodes ricinus nymphs (Acari: Ixodidae) during developmental diapause

Changes in the midgut of the Ixodes ricinus nymphs at the stage of developmental diapause were studied. It is established, that the midgut of the tick nymphs undergoes the identical and synchronous changes at the development without diapause and in the state of diapause. In 7-8 months after feeding the midgut of the nymphs containes the digestive cells with gematine and food inclusions, as well as reserve cells, like the midgut of unengorged moulted female. But the midgut of diapausing nymphs retaines such condition during the whole period of diapause.     

Morphofunctional changes in the midgut of tick females of the genus Ixodes (Acari: Ixodidae) during and after feeding

Cyclic changes of the midgut epithelium were observed in females of 5 ticks species of the genus Ixodes during 7-10 days of feeding. The midgut epithelium of unfed females is represented by the digestive cells of nymphal phase and stem cells. The digestive cells of nymphal phase are functional during 1.5-2 days after attachment of the tick, and then, after the tearing away they go into the gut lumen. The secretory cells substitute the digestive cells of nymphal phase and finish their growth during the 4-4.5 days. Secretion of digestive enzymes is performed by the holocrine type with tearing away a whole cell. Intracellular digestion takes place in the digestive cells of four consequent generations. The secretory and digestive cells form a peritrophic matrix on their surface. The presence of peritrophic matrix gives an evidence the maturity and functional activity of the secretory and digestive cells. We suggest, that the peritrophic matrix takes part in intracellular digestion, namely in the process of micropinocytosis. The phagocytosis was not found in the ticks investigated. Digestion in the midgut lumen is performed by enzymes of the ruptured secretory and digestive cells, that is proved by the haemolysis of erythrocytes in the zone of their contact with these cells. The digestive cells of each generation functioned almost synchronously, with largest difference in starting about 12 hours.     

Morphofunctional changes in the midgut of virgin tick females of the genus ixodes (Acarina: Ixodidae) during and after feeding

The midgut epithelium of the virgin females in Ixodes ricinus, L. pacificus, and I. persulcatus is represented by the digestive cells of nymphal phase and stem cells. Digestion of feed is performed by the one generation of digestive cells of nymphal phase and two generations of digestive cells of adult females. The generation of secretory cells and two generations of digestive cells are absent. The feeding of virgin females is not completed but interrupted in the second phase during the period of slow feeding and preparation of the intestine for the third phase.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Ultrastructural changes of the midgut epithelial cells in feeding and moulting nymphs of the tick Haemaphysalis longicornis

The midgut epithelial cells in nymphs fed on laboratory rabbits were examined during feeding and after detachment. The midgut epithelium at the unfed stage consisted of digestive cells of lower activity, containing such nutritive substances as protein, lipid and glycogen. As feeding proceeded, the cells became active in intracellular digestion. At the middle of the feeding stage, the spent digestive cells derived from the active digestive cells began to be replaced by the new digestive cells of lower activity. After detachment, the pinocytotic activity of the above cells increased greatly, and the digestive activity increased to some extent. As a result, many large endosomes were formed by fusion of numerous pinosomes. Thereafter, endosomes decreased in size as digestion proceeded and there was an increase of haematin granules. On day 7 after detachment, the new digestive cells of lower activity, belonging to the 'nutritional reserve' type, appeared adjacent to the spent digestive cells which had almost exhausted all endosomes, and these new cells had completely replaced the spent cells by day 3 after moulting.     

Histochemical changes in the midgut of two ixodid tick speciesBoophilus microplus andRhipicephalus appendiculatus during digestion of the blood meal

The changes in the midgut epithelia of two ixodid tick species,Boophilus microplus andRhipicephalus appendiculatus, have been studied using several histochemical techniques. It was revealed that there is an accumulation of RNA at the time of tick attachment to the host and prior to the arrival of the blood meal, indicating that the midgut digest cell is furnished with the machinery characteristic of a synthetic cell. There appears to be a synchrony in the appearance of granules with peroxidase activity and the uptake of haemoglobin into the midgut digest cells. Alkaline phosphatase activity was observed in the midgut epithelia of all ticks except in a few of the long-starved ticks, and was concentrated in the apical plasma membrane regions of those digest cells involved in absorption and the intracellular digestion of haemoglobin. The presence of these enzymes suggests that the midgut digest cell is a multifunctional cell capable of both secretory and digestive activities. The colloidal material in the midgut lumen was found to result from the accretion of several products both secreted and excreted by the midgut epithelial cells and exhibited different staining reactions depending on which component dominated. The nature of the material suggests that in addition to its digestive function it may serve as a sink to bind all the by-products of digestion and thereby facilitate their excretion.     

Ultrastructure of the midgut in Ornithodoros papillipes (Acarina, Argasidae) nymphs in the blood assimilation period

Three types of cells (reserve, digestive and secretory) differing in their ultrastructural characters were found to exist in the epithelium of the midgut of nymphs of O. papillipes during the blood assimilation. Reserve and secretory cells are dominant at the first stage of digestion while digestive cells are most abundant at the second one. Ultrafine structure of secretory and digestive cells points to the labile relationship between these types of cells. The same digestive cells assimilate blood both by means of phagocytosis of formed elements and by pinocytosis of its liquid components. The identity of ultrastructural peculiarities of endocytosis and intracellular blood digestion in ixodid and argasid ticks is shown.     

Morphofunctional changes in the midgut of tick nymphs of the genus Ixodes (Acarina: Ixodidae) during and after feeding

The midgut epithelium of feeding nymph is represented by the digestive cells of larval phase. Digestion of the main part of feed is performed by the one generation of digestive cells of nymphal phase after detachment, during moult. This period precedes the apolysis. The generation of secretory cells is absent on the nymphal phase. Secretory vacuoles are formed in the digestive cells of larval phase. All functioning cells form a peritrophic matrix on their apical surface. The replacement of the digestive cells of larval phase by the digestive cells of nymphal phase proceeds gradually, during the first 5-10 days after detachment. The beginning of the accumulation of digestive inclusions in the young digestive cells of nymphal phase takes place in the 10-15 days after detachment.     

Ultrastructure of the midgut and blood meal digestion in the adult tickDermacentor variabilis

Digestive cells in the midgut of male and femaleDermacentor variabilis (Say) took up the blood meal in coated vesicles and smooth flask-shaped vesicles, and deposited it in endosomes which were digested via heterophagy. Iron was concentrated in residual bodies.Digestion occurred in three distinct phases in mated females: (1) continuous digestion (initiated by feeding) occurred during slow engorgement; (2) reduced digestion (initiated by mating) occurred in mated females during the period of rapid engorgement; (3) a second continuous digestion phase (initiated by detachment from the host) occurred throughout the post-feeding periods of preoviposition and oviposition.It is proposed that the stem cells in the midguts of unfed females were progenitors of digestive, replacement, and presumed vitellogenic cells in midguts of mated feeding females. Digestive cells were present in all three digestion phases. Only during the first continuous digestion phase did digestive cells fill up with residual bodies, rupture and slough into the lumen, or did whole cells slough into the lumen. During the other two digestion phases no sloughing of digestive cells was observed. At the end of oviposition the digestive cells were filled with residual bodies. Replacement cells were present only during the first continuous-digestion phase. Presumed vitellogenic cells were present only during the reduced-digestion phase and during the second continuous-digestion phase. Stem cells in unfed males developed only into digestive cells in feeding males. Fed males and fed unmated females had only the first continuous-digestion phase. After being hand-detached from the host, unmated 13-day-fed females went through cellular changes associated with the reduced-digestion phase and second continuous-digestion phase of fed mated females, then began ovipositing. Maximum development of the basal labyrinth system and lateral spaces matched the known time of maximum water and ion movement across the midgut epithelia.Spectrophotometric analyses of lumen contents and midgut cells, sampled after detachment from the host, showed that concentrations of protein and hemoglobin at day 1 post-detachment decreased by one-half at the beginning of oviposition, while hematin increased about twofold by the end of oviposition. This supported the idea of the presence of a second continuous-digestion phase.     

Peritrophic matrix in the midgut of tick females of the genus Ixodes (Acari: Ixodidae)

The formation of the peritrophic matrix in the midgut of females of 5 ixodid tick species (Ixodes pacificus, I. pavlovskyi, I. persulcatus, I. ricinus and I. scapularis) was studied by means of light and electron microscopy in different periods of the feeding and after detachment. The formation of the peritrophic matrix started when the first food portions came into the gut lumen, 9-12 hours after the attachment. Renovation of the peritrophic matrix took place during the whole feeding period; every new generation of midgut cells synthesized their own matrix. It was deposited on the apical surface of every midgut cell in the beginning of differentiation, and was functioning during the life of the cell. The peritrophic matrix separates spaces of the cavitary and cytozoic digestions.     

Macrophage migration inhibitory factor expression and protein localization in <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Ixodidae)

Amblyomma americanum (L.) ticks continue to emerge as disease vectors in many areas of the United States. Tick macrophage migration inhibitory factor (MIF) was first identified in A. americanum females and has been demonstrated to inhibit macrophage movement to the same extent as human MIF. This study was conducted to further characterize and elucidate the physiological role for MIF in tick feeding. A relative quantitative PCR assay was developed to determine the level of MIF gene expression during tick feeding. In addition, RNAi techniques were used to silence MIF prior to blood feeding. Physiological parameters of tick engorgement weight, length of feeding interval, and egg masses were observed to check for phenotypic manifestations of RNA silencing. Specific tick MIF antibody was used to localize MIF protein in frozen tick tissue sections. Tissue specific gene expression indicated that the midgut tissues were the most highly enriched for the MIF. Levels of gene expression did not parallel MIF protein pools seen in tissue sections. Of particular importance was the finding that unfed tick salivary glands appear to contain vesicles that are specific for MIF protein. This is the first demonstration of a pool of MIF that could be secreted during the first hours of tick feeding. While MIF silencing was demonstrated at the molecular level, no physiological phenotype was apparent. The MIF protein pools already available in the tissues may be sufficient to accomplish female tick feeding. Our studies show that the most prominent source of MIF during tick feeding is the midgut tissue. Future studies will address the role of MIF in blood feeding and nutrient digestion in the immature life stages of the tick.     

Haemaphysalis longicornis: molecular characterization of a homologue of the macrophage migration inhibitory factor from the partially fed ticks

The macrophage migration inhibitory factor (MIF) has been identified from some vertebrates and invertebrates. MIF is related to inflammation, tumor growth, and angiogenesis in vertebrates. Here, we report the molecular characterization of a homologue of MIF from partially fed Haemaphysalis longicornis. The sequence analysis of the H. longicornis MIF (HlMIF) indicated that its deduced amino acid sequence has an identity of 77% with the MIF of the tick Amblyomma americanum. Western blot analysis using the anti-His-HlMIF antibody showed that HlMIF was up-regulated during blood feeding. Immunohistochemistry showed that the endogenous HlMIF in partially fed ticks was localized to the midgut and epidermal cells. Moreover, the functional assay revealed that the GST-HlMIF inhibited the migration of human monocytes. In conclusion, we consider that HlMIF may facilitate blood feeding by inhibiting host macrophage migration to the feeding lesion or may participate in the proliferation and differentiation of cells in the tick body.     

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.     

The antioxidant role of xanthurenic acid in the Aedes aegypti midgut during digestion of a blood meal

In the midgut of the mosquito Aedes aegypti, a vector of dengue and yellow fever, an intense release of heme and iron takes place during the digestion of a blood meal. Here, we demonstrated via chromatography, light absorption and mass spectrometry that xanthurenic acid (XA), a product of the oxidative metabolism of tryptophan, is produced in the digestive apparatus after the ingestion of a blood meal and reaches milimolar levels after 24 h, the period of maximal digestive activity. XA formation does not occur in the White Eye (WE) strain, which lacks kynurenine hydroxylase and accumulates kynurenic acid. The formation of XA can be diminished by feeding the insect with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfonamide (Ro-61-8048), an inhibitor of XA biosynthesis. Moreover, XA inhibits the phospholipid oxidation induced by heme or iron. A major fraction of this antioxidant activity is due to the capacity of XA to bind both heme and iron, which occurs at a slightly alkaline pH (7.5-8.0), a condition found in the insect midgut. The midgut epithelial cells of the WE mosquito has a marked increase in occurrence of cell death, which is reversed to levels similar to the wild type mosquitoes by feeding the insects with blood supplemented with XA, confirming the protective role of this molecule. Collectively, these results suggest a new role for XA as a heme and iron chelator that provides protection as an antioxidant and may help these animals adapt to a blood feeding habit.     

Increased expression of ATG genes during nonfeeding periods in the tick Haemaphysalis longicornis

Ticks are long-lived hematophagous arthropods and have tolerance to starvation. They can survive without food during the host-seeking period for several months to years. To understand how ticks obtain energy over a long period of non-feeding (starvation), we focused on autophagy, a crucial proteolysis system via the lysosomes for various cellular processes that is induced during starvation in eukaryotes. In the present study, EST databases for several organs of the tick Haemaphysalis longicornis led to the identification of HlATG3, HlATG4 and HlATG8, homologues of 3 autophagy-related (ATG) genes, ATG3, ATG4 and ATG8/LC3/GABARAP, respectively, which are essential for the Atg8 conjugation system in model animals. Real-time PCR results revealed that the expression of HlATG3, HlATG4 and HlATG8 in the tick showed higher levels during the non-feeding period than the feeding period, suggesting that the Atg8 conjugation system is at work in unfed ticks. Notably, their expression levels were higher in the midgut, a digestive organ, of unfed than fed adults. Histological analysis demonstrated that lipids and glycogen accumulated within the epithelial cells of the midgut in unfed ticks, implying that the midgut of unfed ticks serves as storage of those components as nutrients during non-feeding. Furthermore, autophagic organelles were found in the midgut undifferentiated cells of unfed ticks. The starved condition appears to be associated with the increased expression of HlATG genes in the midgut of unfed ticks. Tick autophagy might help compensate for the loss of nutrients derived from host blood components during the non-feeding period.     

中华硬蜱叮咬不同免疫力新西兰兔后中肠上皮组织的形态动态变化

通过光镜和电镜观察了中华硬蜱Ixodes sinensis叮咬初次和再次感染宿主新西兰兔后不同时间（叮咬后24 h、48 h、72 h以及第 5天、第8天）中肠上皮组织的形态学动态变化。结果显示： 中华硬蜱叮咬前中肠上皮主要由替代细胞和少量体积较大的消化细胞构成;替代细胞数量多、体积小、呈圆形、胞质染色浅 。中华硬蜱叮咬初次感染宿主后,消化细胞随叮咬时间延长而增多增大,微绒毛较密集,排列整齐,胞质内细胞器丰富,各单位膜结构清晰,并出现顶端小管、小泡、大量脂滴和高铁血红素颗粒;近基膜的细胞膜内褶形成发达的基底迷路系统。中华硬蜱叮咬再次感染宿主后,中肠可发生一系列明显的病理变化,中肠基膜出现变薄、松散和断裂现象,消化细胞破裂、空泡化,消化细胞数量减少;消化细胞微绒毛减少、变短、排列不整,线粒体肿大,体嵴减少、变短甚至髓样变,粗面内质网扩张,脂粒及高铁血红素颗粒减少,细胞膜吞饮、吞噬现象减弱,消化细胞内结构紊乱和破坏。该研究结果提示初次叮咬导致了宿主的免疫抗性,再次叮咬后蜱中肠是宿主免疫力的主要作用部位。     

Digestive cells in the midgut of Triatoma vitticeps (Stal, 1859) in different starvation periods

Triatoma vitticeps (Stal, 1859) is a hematophagous Hemiptera that, although being considered wild, can be found in households, being a potential Chagas’ disease vector. This work describes the histology and ultrastructure of the midgut of T. vitticeps under different starvation periods. Fifteen adults of both sexes starved for 3, 7, 20 and 25 days were studied. In general, digestive cells had apical microvilli, basal plasma membrane infoldings and central nucleus. The perimicrovillar membrane was found in all insects examined. Digestive cells of anterior midgut had lipid droplets, glycogen granules, developed basal labyrinth associated with mitochondria suggesting their role in nutrient storage and in fluid and ion transport. The cells of median and posterior regions of the midgut were rich in rough endoplasmic reticulum, lysosomes, vesicles and granules with different electron-densities. Moreover, cells of the posterior portion of the midgut had hemozoyn granules and mitochondria in the apical cytoplasm close to microvilli, suggesting their role in blood digestion and active nutrient absorption. The midgut of T. vitticeps showed differences in digestive cells associated with the time after feeding, and the increase of vesicles amount in long starvation periods, which suggests enzyme storage, which is readily used after a blood meal.