Amblyomma triste (Koch, 1844) (Acari: Ixodidae): morphological description of the ovary and of vitellogenesis

The present study presents the morphology, histology, and the dynamics of vitellogenesis in females of the tick Amblyomma triste. The ovary in this species is of the panoistic type, therefore it lacks nurse cells. It is composed of a layer of epithelial cells that outwardly form the wall of the ovary, but also originate the pedicel, the structure that attaches the oocytes to its external margin, as well the oocytes themselves. In Amblyomma triste, the oocytes develop in four synchronic stages, which differs from the process in other tick species. The classification of the stages of the oocytes was carried out based on the presence of four morphologic characteristics: cytoplasm appearance; site of the germ vesicle; presence, quantity, and constitution of the yolk granules and presence of chorium.     

Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the process of the vitellogenesis in oocytes of the tick Rhipicephalus sanguineus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a wall formed by small epithelial cells with rounded nuclei which delimit the lumen. The oocytes in the different developmental stages in this tick species were classified into five stages (I-V). They remain attached to the ovary during vitellogenesis by a cellular pedicel and afterwards the mature oocytes (stage V) are released into the ovary lumen.     

Antiviral effect of the egg wax of Amblyomma cajennense (Acari: Ixodidae)

The control of viral infections, especially those caused by influenza viruses, is of great interest in Public Health. Bio prospection has shown the presence of active principles in the hemolymph of arthropods, and in the salivary gland of ticks, and some of these are of interest for the development of new pharmacological drugs. Ticks lay their eggs in the environment, and to protect them from desiccation and microbial attack they involve the eggs in a waxy layer produced by an organ known as Gené’s Organ. In this study, the eggs wax from tick Amblyomma cajennense (Fabricius) was extracted using ice cold phosphate buffer. The antiviral activity was evaluated with picornavirus and influenza virus. In both cases egg wax was able to inhibit virus replication. For influenza virus, an amount as small as 12 μg/mL of crude egg wax suspension neutralized 128 UHA (hemaglutinant unit) of H₁N₁ influenza virus. With picornavirus, egg wax led to a 256-fold reduction in virus production by L929 cells. Egg wax was not cytotoxic to VERO, MDCK and L929 cell, being observed that the cell morphology was preserved with concentration as high as 2 mg/mL. In addition no genotoxic effect was observed for Vero cells, suggesting a very interesting potential antiviral activity.     

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females

Many studies have been conducted with plants whose extracts have the potential to be used for pest control. One of these plants is Azadirachta indica (neem), whose main active ingredient is azadirachtin, a compound shown to have acaricide and insecticide activity. Rhipicephalus sanguineus (brown dog tick) is currently considered to be an “urban pest,” because of its high levels of infestation and its ability to attack humans. In the present study partially and fully engorged R. sanguineus females were exposed to aqueous extracts of neem at concentrations of 10% and 20%, and to a control treatment. The results showed that differently from what was observed in the control, the pedicel cells of females exposed to neem at both concentrations lost their original shape. In the latter cases, the cytoplasm of the cells became fully vacuolated, especially near the germinal vesicle (oocyte nuclei) and in the oocyte pole, which is in contact with the cells of the pedicel. Oocytes in early stages of development (I and II) of ticks treated with both concentrations had irregular germinal vesicle, including the presence of two nucleoli as well as fragments of these. Oocytes in stages IV and V of exposed individuals showed full granular cytoplasm with bigger yolk granules when compared to the early stages. Chorion of mature oocytes was also altered, showing folds and deformations along their entire extension. The observed changes in cells of the reproductive system of R. sanguineus, especially in the oocytes, indicated the potential of neem as a new alternative method to control these ectoparasites.     

Effects of ricinoleic acid esters from castor oil of Ricinus communis on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

This study examines the effects of ricinoleic acid esters from Ricinus communis castor oil on the vitellogenesis of Rhipicephalus sanguineus ticks attached to hosts that were fed with commercial rabbit food containing these esters. The oocytes of ticks from the treatment group (TG) showed cytoplasmic changes that inhibited the development of oocytes I and II to the advanced stages (IV and V) in addition to preventing the maturation of oocytes V, resulting in small ones. In addition, sperm was not observed in ampoules. Our findings confirm the acaricide potential of ricinoleic acid esters.     

A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A₂ inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A₂ enzymatic activity.     

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Amblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates.     

Underwater survival in the dog tick Dermacentor variabilis (Acari:Ixodidae)

Ticks are blood-feeding arthropods known for their long survivability off the host. Although ticks are terrestrial, they can survive extended periods of time submerged underwater. A plastron is an alternative respiration system that can absorb oxygen from water via a thin layer of air trapped by hydrophobic hairs or other cuticular projections. The complex spiracular plate of ticks has been postulated to serve as a plastron but that function has not been verified. This study provides evidence of plastron respiration in the American dog tick, Dermacentor variabilis, and for the first time confirmed the existence of plastron respiration in Ixodidae. Longer survival rates in oxygenated water indicate that underwater respiration requires oxygen. Wetting the spiracular plate with alcohol debilitates any potential plastron function and lowers the survival rate. Survival underwater may also be enhanced by metabolic depression and possibly anaerobic respiration. This study describes the first example of plastron respiration in the Ixodidae.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

Amblyomma cajennense (Acari: Ixodidae): salivary gland cells of partially engorged females ticks and the production of lipid by their mitochondria

Morphologically, the salivary glands of ticks are paired structures consisting of a secretory and an excretory portion, lacking a reservoir for the storage of the secretion. The secretory portion is composed in females by cells that form acini classified into the types I, II, and III. The excretory possess a major duct, from which arise several intermediate ducts that then subdivide to form the canaliculi or acinal tubules, which end at the acini from where they collect the secretion. The present study describes the ultrastructural changes that occur in the mitochondria of cells of the acini I, II, and III in the salivary glands of partially engorged females of the Cayenne tick Amblyomma cajennense. The results show that this organelle exhibits completely disarrayed crests due to the presence of lipidic material inside the matrix and between the crests, thus demonstrating their participation in the production of the lipids that would be used structurally by the cells. These organelles with ultrastructural changes were denominated derived mitochondria.     

Determination of LC50 of permethrin acaricide in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Currently, the most efficient and widely used method of tick control still is the treatment with acaricides, especially permethrin (active ingredient of the Advantage^® Max3, Bayer), a pyrethroid with neurotoxic action. Due to the wide use of this acaricide in the control of the tick Rhipicephalus sanguineus, this study carried out laboratorial procedures to determine the LC₅₀ (lethal concentration fifty) of permethrin in semi-engorged females of R. sanguineus. Based on the result of 14 dilutions of permethrin in distilled water and later Probit analysis, the LC₅₀ of permethrin for R. sanguineus was 2062 ppm (1549-2675 ppm). This work can be used as a protocol with other chemicals, to determinate the LC₅₀, basic procedure for studies of control, resistance and behavior of ticks treated with acaricides, especially the brown dog tick R. sanguineus. Also, the knowledge of the LC₅₀ provides information on the potency of chemicals, the sensitivity of Arthropods to them and even estimates on pest control.     

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

Toxicity effect of the acaricide fipronil in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): preliminary determination of the minimum lethal concentration and LC(50)

Chemical acaricides, especially fipronil (active ingredient of Frontline®), are still the most effective method to control tick populations. In this study, the effectiveness of fipronil was assessed in semi-engorged females of the tick Rhipicephalus sanguineus. A protocol for an in vitro bioassay (AIT) was developed, and the LC₅₀ (lethal concentration 50%) and 95% confidence interval were determined. Ticks were immersed in Petri dishes with different concentrations of fipronil or distilled water for 2 min, dried, and placed in an incubator for 7 days. Dead R. sanguineus females treated with the 14 concentrations of fipronil were counted daily. Mortality results were compared with the Probit analysis, and the LC₅₀ and 95% confidence interval were calculated, g (95): LC₅₀ = 9.647 (4.711 to 13.470). This study was aimed at developing a more appropriate and updated protocol for an in vitro bioassay (AIT – adult immersion test), and providing information on the toxic potential of fipronil (elimination of ectoparasites with lower concentrations) and sensitivity of ticks, especially R. sanguineus, a pest of great interest, due to its occurrence in urban environments.     

Salivary gland degeneration and vitellogenesis in the ixodid tick Amblyomma hebraeum: Surpassing a critical weight is the prerequisite and detachment from the host is the trigger

The normal engorged body weight of female ixodid ticks (Acari: Ixodidae) is about 100× the unfed weight. Virgin female Amblyomma hebraeum normally do not feed beyond 10× the unfed weight. However, about 10–20% of a population of virgins will feed to perhaps 20× the unfed weight, but not much beyond that. In A. hebraeum, when females surpass about 10× the unfed weight, the following changes in physiology occur if they are removed from the host: (a) they will not reattach if given the opportunity, (b) their salivary glands (SGs) will undergo autolysis within 4 days if they are mated or 8 days if they are virgin, and (c) egg maturation and oviposition will occur in due course. Mated or virgin female ticks removed from the host below about 10× the unfed weight do not experience the latter changes (Kaufman, W.R., Lomas, L., 1996. ‘Male Factors’ in ticks: their role in feeding and egg development. Invertebrate Reproduction and Development 30, 191–198). In 1984 we named this transitional weight, the ‘critical weight’ (CW). Its absolute value is probably a species-specific characteristic (Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? Journal of Insect Physiology 53, 264–273). Although mated females tend to engorge within a day of surpassing the CW, virgin females surpassing the CW can remain attached to the host for at least several weeks more. It is not known whether the physiological changes in the SGs and ovaries listed above occur in those large virgins that remain attached, although we suppose that this would be maladaptive. Instead, we hypothesize in this study that surpassing the CW is only a prerequisite for inducing these changes, and that detachment is the actual trigger. We support our hypothesis by demonstrating that large virgins, remaining attached to a host for 8 days, did not undergo SG degeneration nor complete egg maturation during the attachment period. Those changes occurred only within 8 days following detachment. So some type of sensory information associated with attachment to the host, and still undefined, inhibits expression of the physiological changes hitherto associated merely with surpassing the CW.     

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process.     

秀丽隐杆线虫脂肪沉积研究进展

脂肪的过度沉积会引发多种疾病,如心脏病、高血压、高甘油三酯血症、Ⅱ型糖尿病等。小白鼠(Mus musculus)和猪(Sus domesticus)是常用的研究脂肪沉积的模式动物,近年来随着研究的深入,发现脂肪代谢调控网络错综复杂,调控因子相互作用。秀丽隐杆线虫(Caenorhabditis elegans)具有结构简单、身体透明、便于观察、繁殖周期短、易于人工培养等特征,因此使得秀丽隐杆线虫进行脂肪调控的研究成为了可能。本文通过总结国内外线虫脂肪沉积方面的研究,综述秀丽隐杆线虫研究脂肪沉积的进展。     

The natural hosts for larvae and nymphs of Amblyomma neumanni and Amblyomma parvum (Acari: Ixodidae)

Based on the hypothesis that birds and rodents are important hosts for subadults of the Neotropical Amblyomma neumanni and Amblyomma parvum ticks, a survey of these type of hosts was carried out from July 2004 to March 2006, in Quilino (A. parvum) and Dean Funes (A. neumanni), Córdoba province, Argentina. Additionally, monthly tick counts were performed on cattle and goats with occasional tick search in other domestic hosts. Records of questing height of subadult ticks on vegetation were also carried out monthly. Rodents (n = 123) and birds (n = 122) captured in Dean Funes showed no infestation with A. neumanni. Apart of few nymphs found on horses, all larvae and nymphs of A. neumanni were on cattle with a larval prevalence and mean number of 22.2%, and 7.7 ± 22.52, respectively, and a prevalence of nymphs of 47.8% with a mean of 7.9 ± 18.49. The average questing height of larvae and nymphs of A. neumanni was 23.5 ± 17.1 cm and 30.7 ± 26.7 cm, respectively. A total of 138 rodents and 130 birds were captured in Quilino but the Caviidae rodent Galea musteloides carried 99.3% of larvae and 99.8% of nymphs of A. parvum, and no immature stages were detected on cattle, goat or vegetation. Tick counts on G. musteloides (n = 74) showed a prevalence of 42% and a mean number of 9.9 ± 24.83 for larvae, while nymphal infestation had a prevalence of 56.5% and a mean of 8.7 ± 11.31. Cattle appear to be suitable hosts to sustain the complete cycle of A. neumanni in nature (adult ticks infest cattle too) and questing height of subadults indicates that they are expecting to feed on medium and large-sized mammals, such as cattle and other ungulates. At least in the study site, G. musteloides is the principal host for the survival strategy of A. parvum subadults; adult ticks are common on cattle and goats. These hosts are introduced in the Neotropics but A. neumanni was able to develop a surrogate cycle independent of native hosts while A. parvum still depends on probably primeval hosts to sustain their larvae and nymphs.     

Apoptosis in the fat body tissue of the beetle Tenebrio molitor parasitised by Hymenolepis diminuta

Many insects experience a decrease in reproductive output when parasitised. We are investigating mechanisms underlying this fecundity reduction using the rat tapeworm, Hymenolepis diminuta infection of Tenebrio molitor beetles. These include an increase in the resorption of developing ovarian follicles and a decrease in fat body synthesis of vitellogenin. The latter is the direct effect of a molecule produced by the parasite. Here we report a study to determine whether vitellogenin synthesis and follicle resorption are the result of parasite-induced apoptosis in the respective tissues and whether the parasite molecule acts directly on the fat body by inducing apoptosis. In vivo, the number of fat body cell nuclei with chromatin condensation are significantly elevated in parasitised females at all days examined and peaked at day 7 post-infection. A TUNEL assay to detect DNA fragmentation confirmed these observations of apoptosis. However, when fat body from uninfected females was co-cultured with live metacestodes they did not cause cells to die by apoptosis, showing that the induction signal does not come directly from the parasite. The follicle resorption observed in the ovaries of infected beetles was not associated with apoptosis of the epithelial cells. The possibility of several mechanisms underlying fecundity reduction is discussed.