Artificial-membrane feeding of the ixodid tick,Rhipicephalus appendiculatus,to repletion

An artificial feeding device was constructed with a Baudruche, membrane bearing olfactory and tactile stimuli, to induce voluntary attachment and feeding of the brown ear tick,Rhipicephalus appendiculatus. Preparation of the membrane and experimental conditions used are described. Unfed adults ofR. appendiculatus, 2–3 weeks after moulting, were confined on the membrane, which was treated with cattle ear-wash and other stimuli. They attached and commenced sucking blood within 12–48 h. Fully engorged ticks started detaching from the membrane on the 6th day. After a 5–6-day incubation period, they started laying eggs which hatched into normal larvae.     

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.     

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.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Purification and characterization of an anticoagulant from the salivary glands of the ixodid tick Rhipicephalus appendiculatus

An anticoagulant isolated from salivary gland extracts of the ixodid tick Rhipicephalus appendiculatus was purified by gel filtration on Sephadex G-100, ion exchange on DEAE-cellulose, aprotinin-Sepharose, and by high-pressure-liquid size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the anticoagulant activity was associated with a protein of an apparent Mr of 65 kDa. The purified molecule had a pI in the range of 8.0-8.5 on chromatofocusing and was stable over a wide pH range, but was heat labile and susceptible to inactivation by trypsin and reductive alkylation. The anticoagulant did not inhibit thrombin-initiated fibrin formation and had no detectable fibrino(geno)lytic or phospholipase-like activities. Although it inhibited factor Xa-induced clotting of bovine plasma, it did not affect the amidase activity of factor Xa toward a synthetic substrate, suggesting that the anticoagulant acts at a site distinct from the active site of factor Xa or on other components of the prothrombinase complex.     

Boophilus microplus: digestion of hemoglobins by the engorged female tick

When the cattle tick Boophilus microplus, after dropping from its host, was maintained at 27 C, the digestion of hemoglobins in the gut proceeded at a steady rate and was virtually complete by the 13th day. The rate was essentially the same whether the ticks were strains susceptible (Yeerongpilly) or resistant (Biarra) to organophosphorous compounds or whether the hosts were British breed, Brahman or banteng cattle, or buffalo. Ferrihemoglobin appeared in the gut contents and hematin was deposited. About 10% of the hematin released from the hemoglobin was transferred to the eggs and from them to the larvae. Translucent ticks, sometimes found on heavily infested hosts and referred to as “serum” ticks, had about half the normal hemoglobin content.     

HlLgm2, a member of asparaginyl endopeptidases/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion

Here we describe a cDNA encoding the second asparaginyl endopeptidase/legumain (HlLgm2) from the midgut of the ixodid tick Haemaphysalis longicornis. Endogenous HlLgm2 was expressed in all the developmental stages of the tick, localized mainly in the midgut epithelium and was up-regulated by the host blood-feeding process, as demonstrated by immunoblotting and immunohistochemistry. RT-PCR and real-time PCR showed that the HlLgm2 gene was expressed at a lower level during all phases of blood-feeding than our previously characterized legumain (HlLgm) gene from the same tick. More strikingly, there was no expression of HlLgm2 mRNA beyond 96 h of blood-feeding, while HlLgm mRNA expression continued until full engorgement. Escherichia coli-expressed recombinant HlLgm2 (rHlLgm2) efficiently hydrolysed the legumain-specific synthetic substrate. rHlLgm2 activity was inhibited by iodoacetamide and N-ethylmaleimide and also by Fe(2+), Cu(2+), Co(2+) and Ni(2+). rHlLgm2 digested bovine haemoglobin and exhibited strict specificity for the asparaginyl bonds on the carboxy-terminal side of a peptide, as demonstrated by internal amino acid sequence analysis of the cleaved bovine serum albumin products. Our results suggest that HlLgm2, together with HlLgm, plays a pivotal role in host blood-meal digestion process.     

An experimental investigation into the effects of flooding on the ixodid tick Boophilus microplus (Canestrini)

Summary The effect of water immersion on the non-parasitic stages of Boophilus microplus were investigated both under laboratory and field conditions. Survival of engorged female ticks was influenced by their age and the temperature of the water. Eggs and larvae were more resistant to submerison than were engorged females and their survival was increased at low temperatures and in water with high oxygen content. The persistence of this tick in areas prone to flooding was attributed to the survival of parasitic stages and to larvae on the pasture. Heavy rains produce favourable pasture conditions for tick reproduction so that a large increase in population size may be expected to follow such rain.     

Morpho-functional changes in the midgut of ixodid ticks (Acari: Ixodidae) during the life cycle

Morpho-functional studies performed in five species of the genus Ixodes, including I. pacificus, I. pavlovskyi, I. persulcatus, I. ricinus, and I. scapularis demonstrated that the replacement of the midgut epithelium lags in each consecutive life stage and is not synchronized with basic processes of morphogenesis and organogenesis during molts. The midgut epithelium of each preceding stage is retained and functions during feeding of the subsequent life stage.     

COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities

We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96 h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48 h post-feeding.     

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.     

Molecular and reverse genetic characterization of serine proteinase-induced hemolysis in the midgut of the ixodid tick Haemaphysalis longicornis

Enzyme-induced hemolysis has been shown to occur in the midgut of ticks; however, little is known about the molecular basis for hemolytic activity. We report here the molecular and reverse genetic characterization of a hemolytic midgut serine proteinase, HlSP, recently identified from the ixodid tick Haemaphysalis longicornis. Endogenous HlSP was found in the midgut lumen and its contents, indicating that HlSP is extracellularly secreted. Recombinant H. longicornis serine proteinase (rHlSP) expressed in Escherichia coli showed dose-dependent hemolytic activity towards rabbit erythrocytes, with a maximum hemolysis of 94.5% within 1 h in vitro. Tests of pH dependency showed that rHlSP displayed optimal activity at pH 6.0. In binding assays, rHlSP showed high affinity to band 3, which shares the major erythrocyte membrane proteins. Disruption of HlSP-specific mRNA by RNA interference resulted in inhibition of the degradation of host erythrocyte membranes by endogenous HlSP in the knock-down ticks, indicating that HlSP plays a crucial role in the hemolysis in the midgut of haematophagous ticks. Our results suggest that HlSP may be essential for initiating the proteolytic cascade for the degradation of the host blood-meal.     

Serotonin-like immunoreactivity in the central nervous system of two ixodid tick species

Immunocytochemistry was used to describe the distribution of serotonin-like immunoreactive (5HT-IR) neurons and neuronal processes in the central nervous system (CNS), the synganglion, of two ixodid tick species; the winter tick, Dermacentor albipictus and the lone star tick, Amblyomma americanum. 5HT-IR neurons were identified in the synganglion of both tick species. D. albipictus had a significantly higher number of 5HT-IR neurons than A. americanum. The labeling pattern and number of 5HT-IR neurons were significantly different between sexes in D. albipictus, but were not significantly different between sexes in A. americanum. 5HT-IR neurons that were located in the cortex of the synganglion projected processes into the neuropils, invading neuromeres in the supraesophageal ganglion including the protocerebrum, postero-dorsal, antero-dorsal and cheliceral neuromeres. In the subesophageal ganglion, dense 5HT-IR neuronal processes were found in the olfactory lobes, pedal, and opisthosomal neuromeres. Double-labeling with neurobiotin backfilled from the first leg damaged at the Haller’s organ revealed serotoninergic neuronal processes surrounding the glomeruli in the olfactory lobes. The high number of the 5HT-IR neurons and the extensive neuronal processes present in various regions of the synganglion suggest that serotonin plays a significant role in tick physiology. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. The U.S. Government’s right to retain a non-exclusive, royalty free license in and to any copyright is acknowledged.     

Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24 h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity.     

Augmentation of host's naturally acquired immunity by solubilized membrane-bound midgut proteins of the tick Rhipicephalus appendiculatus.

Immune resistance of rabbits to the hard tick Rhipicephalus appendiculatus induced by combined tick infestations and immunization with solubilized midgut membrane proteins were compared with resistance due to 1-3 infestations or immunization alone. Results demonstrate that the level of exposure of rabbits to ticks used in the study does not significantly affect the immune expression resulting from immunization and that the latter augments the resistance due to infestation.     

Glucose metabolism during embryogenesis of the hard tick Boophilus microplus

Glucose metabolism plays an essential role in the physiology and development of almost all living organisms. In the present study we investigated glucose metabolism during the embryogenesis of the hard tick Boophilus microplus. An increase in glucose and glycogen content during the embryonic development of B. microplus was detected and shown to be due to the high enzyme activity of both gluconeogenesis and glycolytic pathways. Glucose 6-phosphate (G-6P), formed by hexokinase, is driven mainly to pentose-phosphate pathway, producing fundamental substrates for cellular biosynthesis. We detected an increase in glucose 6-phosphate dehydrogenase and pyruvate kinase activities after embryo cellularization. Accumulation of key metabolites such as glycogen and glucose was monitored and revealed that glycogen content decreases from day 1 up to day 6, as the early events of embryogenesis take place, and increases after the formation of embryo cellular blastoderm on day 6. Glucose and guanine (a sub-product of amino acids degradation in arachnids) accumulate almost concomitantly. The activity of phosphoenolpyruvate carboxykinase was increased after embryo cellularization. Taken together these data indicate that glycogen and glucose, formed during B. microplus embryogenesis after blastoderm formation, are produced by intense gluconeogenesis.     

Boophilus microplus (ixodid tick): Fine structure of the gut basophilic cell in relation to water and ion transport

Basophilic cells in the guts of female ticks are derived from the basal remnants of type 2 secretory cells. As viewed by electron microscopy, these cells have microvilli uniformly distributed on the luminal surface, but they lack the abundant pinocytotic vesicles and lysosomes characteristic of digest cells. The cytoplasm is filled with well organized rough endoplasmic reticulum, Golgi complexes and secretory granules. Infoldings of a basal labyrinth extend the contact of the cell with the underlying haemolymph, and there are many mitochondria in the cell processes between folds. This morphology appears to fit the cell for functioning in active water transport across the gut wall. Subsequent to a final rapid phase of engorgement, the basophilic cell reorganizes its cisternae of rough endoplasmic reticulum into whorls and parallel arrays and resumes a secretory role.     

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.     

Adenosine triphosphatases in the cattle tick Boophilus microplus

The nature and distribution of the magnesium dependent ATPases of Boophilus microplus have been studied in partially engorged, adult female ticks. The tissues examined, namely salivary glands, ovaries and gut, all contain at least three enzymes: an ouabain sensitive, Na,K-ATPase; an enzyme inhibited by azide and dicyclohexyl carbodiimide and stimulated by bicarbonate and sulphite; an enzyme inhibited by neither azide nor ouabain but stimulated slightly by several monovalent cations. These activities are compared with those of well-characterized ATPases from other sources.     

Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis

We characterize here a cDNA from the ixodid tick Haemaphysalis longicornis, which encodes an asparaginyl endopeptidase, legumain (HlLgm), that was present as a functional molecule in the midgut of this tick. Endogenous HlLgm was detected as a 38-kDa antigen in H. longicornis extracts and was seen throughout all developmental stages. Endogenous HlLgm was mainly localized in the midgut epithelium by immunohistochemistry, and was shown to be up-regulated by the host blood-feeding process. Recombinant HlLgm (rHlLgm) produced in Escherichia coli was shown to hydrolyze the synthetic substrate Z-Ala-Ala-Asn-MCA at the rate of 6.42x10(-4)mumol/min/mg protein. Its activity was inhibited by the thiol blocking reagents iodoacetamide and N-ethylmaleimide. The enzyme was shown to possess a unique feature of having an autocatalyzed cleavage at asparagines(364-365) at the C-terminus of both endogenous HlLgm and rHlLgm. rHlLgm degraded bovine hemoglobin and bovine serum albumin (BSA) showing its strict specificity for hydrolysis of the peptide on the carboxyl side of the asparagines, as demonstrated by internal amino acid sequence analysis of proteolytic product of BSA cleavage. These results suggest that HlLgm plays an important role in host blood-meal digestion and may be critical for the final process of digestion of blood components.