Morphological identification and molecular characterization of economically important ticks (Acari: Ixodidae) from North and North-Western Egypt.

Ticks represent a major source of growing economic and public health concern, especially in the tropics and sub-tropics. Towards evidencing ticks' invasion in the North and North-Western parts of Egypt, the present study aimed to investigate the morpho-molecular aspects of those ectoparasites using stereomicroscopy, scanning electron microscopy (SEM) and sequencing of the mitochondrial 16S ribosomal DNA gene (rDNA) and nuclear second internal transcribed spacer (ITS2). Our analysis confirmed the presence and well-distribution of Rhipicephalus sanguineus s.l. infesting dogs and sheep (Alexandria governorate), Rhipicephalus annulatus infesting cattle (Beheira governorate), and Hyalomma dromedarii infesting camels (Marsa Matruh governorate) from North/North-Western Egypt. 16S rDNA and ITS2 sequences of the ticks were amplified using universal and gene-specific sets of primers, sequenced and analyzed. Lengths of amplified 16S rDNA sequences in all examined tick species were found to be similar in size (approximately 460 bp); however, they differed in base pair constitutions, whereas ITS2 lengths were 1,500 bp, 1,550 bp, and 1,800 bp for Rh. annulatus, Rh. sanguineus s.l., and Hy. dromedarii, respectively. Phylogenetically, based on the 16S rDNA results, Rh. sanguineus s.l. ticks clustered with the southeastern Europe lineage from Romania and Greece, Rh. annulatus ticks were similar to Turkish populations, and Hy. dromedarii were close to the isolates from Tunisia. Similarly, based on ITS2 sequences, Rh. sanguineus s.l. from dogs were showing 99% similarity to Nigerian populations; however, those collected from sheep were closer to Iranian populations with 4.1% nucleotide divergence between the two populations of different hosts. Rh. annulatus ticks were identical to a population from Romania, whereas Hy. dromedarii was close by 99.7% similarity to a population from Kenya. This is the first study reporting nucleotide sequences of 16S rDNA and ITS2 in integration with morphological identification of ticks from this part of Egypt.

Elemental Enrichment of the Exoskeleton in Three Species of Tick (Arachnida: Ixodidae).

Three species of adult hard tick (Ixodidae) were examined with scanning electron microscopy-energy dispersive X-ray spectroscopy to obtain elemental profiles of their exoskeletons and determine the presence of trace elements. The scutum, tarsal claws, chelicerae, and hypostome were examined on females and males of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. The only trace elements present included chlorine, calcium, and sodium. Chlorine was the most abundant trace element and occurred in all examined regions. The chelicerae generally possessed the highest weight percentages of Cl (up to 11.32 ± 1.36%) across all 3 species, although high weight percentages of Cl (up to 8.78 ± 2.77%) were also present in the hypostome teeth of most specimens. All 3 trace elements were present in the hypostome of A. americanum and I. scapularis, but Ca and Na appear to be absent from the teeth of D. variabilis. In general, there were few differences in the elemental profiles of the exoskeletons between the sexes of any species. This study confirms the presence of alkali metals (Na) and alkaline earth metals (Ca) in adult ticks, which are also common in other arachnids; however, the absence of transition metals such as zinc from the exoskeletons of ticks is uncommon and only shared with species of Ricinulei and Opiliones.

Enduring Challenge of Invasive Ticks: Introduction of Amblyomma oblongoguttatum (Acari: Ixodidae) into the United States on a Human Traveler Returning from Central America.

Introduction of exotic tick vectors of bacteria, protozoa, viruses, and filarial parasites into the United States has accelerated in recent years, primarily because of globalization, increased frequency of travel, and a rise in legal and illegal animal trades. We herein report introduction of a live specimen of Amblyomma oblongoguttatum on a human into the United States from Central America, and we review 4 previous similar incidents. This tick species occurs widely in the neotropics, from western and southern Mexico, southwards through Central America, to the northern half of South America. It is a potential vector of bacterial agents of spotted fever group rickettsioses, raising concern that if A. oblongoguttatum ticks become established in this country, they might also be able to carry pathogens of human and veterinary concern. Given the potential for exotic ticks as vectors of numerous pathogens, proper surveillance, interception, and identification of these ticks are vital to protecting human and veterinary health. Rigorous governmental inspections of imported livestock and pet animals at ports of entry and educating human travelers and medical practitioners about the risks should be part of an overall national tick program.

A new species of Amblyomma (Acari: Ixodidae) associated with monkeys and passerines of the Atlantic rainforest biome, Southeastern Brazil.

Recent studies have reported several larvae of an unidentified Amblyomma species on passerine birds in Atlantic rainforest fragments in southeastern Brazil. These larvae yielded a unique 16S rRNA haplotype designated as Amblyomma sp. haplotype Nazaré, which showed nucleotide identity levels of 91% to Amblyomma parkeri Fonseca & Aragão, 1952 and 88% to Amblyomma longirostre (Koch, 1844). Herein, we describe Amblyomma sp. haplotype Nazaré as a new species, Amblyomma romarioi n. sp. Martins, Luz & Labruna, through a formal description of the male and female adult stages. Amblyomma romarioi is morphologically and genetically most closely related to A. parkeri, A. longirostre and Amblyomma geayi Neumann, 1899. Among males, the rectangular basis capituli and rounded coxa I spurs separates A. romarioi from A. parkeri, A. longirostre, and A. geayi, which have basis capituli triangular or slightly hexagonal, and pointed coxa I spurs. Among females, the V-shaped genital aperture and coxa I rounded spurs of A. romarioi contrasts to the U-shaped genital aperture and coxa I pointed spurs in A. parkeri, A. longirostre, and A. geayi. Larvae of A. romarioi have been collected on 24 species of passerines. The few records of nymphs and adults were on the black-fronted titi monkey Callicebus nigrifrons (Spix, 1823). The current distribution of A. romarioi is restricted to the Brazilian Atlantic rainforest, southeastern Brazil, in areas with altitude between 363 and 1600 m, within the distribution of C. nigrifrons. We discuss ecological features of Amblyomma romarioi, comparatively to A. parkeri, A. longirostre and A. geayi. The present study increases the Brazilian tick fauna to 74 species.

Conidia and blastospores of Metarhizium spp. and Beauveria bassiana s.l.: Their development during the infection process and virulence against the tick Rhipicephalus microplus.

The current study compared the virulence of conidia and blastospores of Metarhizium robertsii (IP 146), M. anisopliae sensu lato (s.l.) (IP 363) and Beauveria bassiana s.l. (IP 361 and CG 307) against unfed larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). In addition, the development of fungal propagules on tick cuticle was investigated. Tick larvae were treated with fungal suspensions at 106, 107 or 108 propagules mL-1, and percent mortality was assessed every two days. Engorged females were immersed in fungal suspensions (1.0 × 107 propagules mL-1) for 1 min, and their biological parameters monitored daily. The virulence of conidia and blastospores against larvae varied considerably among the isolates tested. Only females treated with blastospores of IP 146 or IP 361 presented lower nutrient and egg production indices than the control group; the higher percent control was reached when females were treated with blastospores of IP 146 (98%), IP 363 (79%), or IP 361 (93%) in comparison to the groups treated with conidia, 71%, 59%, or 63%, respectively. Engorged females treated with conidia or blastospores of IP 146 or IP 361 were also examined by scanning electron microscopy (SEM). Germination of blastospores of IP 146 and IP 361 was observed on tick cuticle after 4 h incubation at 27 ±â€¯1 °C and RH > 90%, whereas germ tubes from conidia of both isolates were observed at 48 h. Appressoria in developing blastospores of B. bassiana IP 361 were observed after 4 h incubation, whereas no appressoria were seen in developing blastospores of M. robertsii IP 146. Blastospore penetrations by both fungal isolates through natural openings was also evidenced by SEM; fine sections of R. microplus engorged females treated with blastospores of IP 146 or IP 361 confirmed that these isolates penetrated through their cuticle and natural openings. Blastospores might be promising for use in biocontrol of ticks, since they are virulent against R. microplus and present rapid development on their cuticle.

Morphodifferentiation of Gené's organ in engorged Amblyomma sculptum Berlese, 1888 female ticks (Acari: Ixodidae).

The Gené's organ (GO) secretes a waxy substance on eggs that reduces water loss and has antimicrobial properties. The current study evaluated morphological and histochemical aspects of GO in Amblyomma sculptum from the period of post-feeding - when ticks detach from the host - to the stage just before oviposition. In this species, GO is composed of a corpus and two pairs of glands, namely, cranial and caudal. Glandular cells are joined laterally by a system of interdigitating membranes with junctional complexes. Histochemistry showed that lipid droplets became more evident as GO developed, while glycogen gradually disappeared, and proteins were detected only near the onset of oviposition. The ultrastructural results revealed a marked distension of the cuticle filled with an amorphous material. Glandular cells showed poor endoplasmatic reticulum, many mitochondria mainly in the basal cell poles and a very developed basal labyrinth. We concluded that the development of GO in A. sculptum ticks was continuous and progressive, and it started after detachment from the host. Additionally, the ultrastructure study suggests that gland cells have an important absorption ability and a low synthetic activity, which indicates that the majority of wax precursors are derived from haemolymph.

An Exotic Hitchhiker: A Case Report of Importation into Connecticut from Africa of the Human Parasitizing Tick, Hyalomma truncatum (Acari: Ixodidae).

We report the importation into Connecticut, U.S.A., of an exotic tick, Hyalomma truncatum (Koch) (Acari: Ixodidae), on a human with recent travel history to Africa. The tick was identified using key morphological characters and through DNA sequencing. This case report highlights continuing risk associated with the importation of exotic tick vectors of medical and veterinary significance on international travelers returning to the United States from abroad.

Morphometric Analysis of Chemoreception Organ in Male and Female Ticks (Acari: Ixodidae).

The Haller's organ plays a crucial role in a tick's ability to detect hosts. Even though this sensory organ is vital to tick survival, the morphology of this organ is not well understood. The objective of this study was to characterize variation in the morphological components of the Haller's organ of three medically important tick species using quantitative methods. The Haller's organs of Ixodes scapularis Say (Ixodida: Ixodidae) (black-legged tick), Amblyomma americanum (L.) (Ixodida: Ixodidae) (lone star tick), and Dermacentor variabilis (Say) (Ixodida: Ixodidae) (American dog tick) were morphologically analyzed using environmental scanning electron microscopy and geometric morphometrics, and the results were statistically interpreted using canonical variate analysis. Our data reveal significant, quantitative differences in the morphology of the Haller's organ among all three tick species and that in D. variabilis the sensory structure is sexually dimorphic. Studies like this can serve as a quantitative basis for further studies on sensor physiology, behavior, and tick species life history, potentially leading to novel methods for the prevention of tick-borne disease.

Keys for the morphological identification of the Australian paralysis ticks (Acari: Ixodidae), with scanning electron micrographs.

The Australian paralysis ticks (Ixodes: Sternalixodes) are of considerable medical and veterinary importance within Australia. This is because of their ability to cause paralysis but also their capacity to transmit pathogens and induce allergic reactions. The available keys for the identification of the group (Ixodes holocyclus Neumann, Ixodes cornuatus Roberts, and Ixodes hirsti Hassall) are ambiguous and contain errors, making accurate identification of the group difficult. Illustrations of varying quality and accuracy of these species have been published in the past; however, they have never been collectively treated using scanning electron microscopy (SEM). Female Australian paralysis ticks are here examined using SEM, and the first pictorial key for this group is presented along with an accurate text key.

Redescription of Amblyomma integrum adults and immature stages.

Amblyomma integrum Karsch, 1879 (Acari: Ixodidae) is one of four Amblyomma Koch, 1844 species with eyes found in southern India and Sri Lanka. The immature stages of this species were poorly described. Therefore, accurate identification is difficult. Here we re-describe the male, female, nymph and larva of A. integrum and illustrate all the stages in greater detail for the first time. A set of diagnostic morphological characters is defined to distinguish this species from other sympatric species of eyed Amblyomma in any parasitic stage of development. Adults of A. integrum parasitize mostly various larger mammals whereas nymphs and larvae use mostly larger and medium mammals. Amblyomma integrum is recorded from India (Andhra Pradesh, Goa, Karnataka, Orissa and Tamil Nadu States) and throughout Sri Lanka.

Sample holder for axial rotation of specimens in 3D microscopy.

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

Oocyte maturation in the sloth's giant tick Amblyomma varium (Acari: Ixodidae) in an ecological context.

The sloth's giant tick Amblyomma varium Koch, which is a neotropical species that inhabits tropical rainforests, is the largest tick reported to date. The adult stage of this tick parasitizes mammals from the families Bradypodidae and Magalonychidae (Xenarthra) nearly exclusively. This study aimed to describe morphological and histological features of the reproductive system and the oocyte maturation process of this tick species. The ovary of A. varium is a long single tubular organ that is horseshoe-shaped, winding and arranged in the posterior part of the body. Two oviducts are connected to the ovary on each side; these thicken at certain region forming the uterus (common oviduct), followed by a muscular connecting tube, vagina and genital aperture. A large number of oocytes at different stages of development are attached to the ovary wall by the pedicel, as they reach maturity they are released into the ovary lumen and from there to the genital aperture. These oocytes develop simultaneously and asynchronically along the ovary. Amblyomma varium oocytes were classified into five development stages (i.e., I-V), and specific characteristics were observed; the processes of yolk and chorion deposition begin early in oocytes stage II, and oocytes V exhibit a very thick chorion and eggs of a large size. These characteristics are likely adaptations that enhance the survival and the reproductive success of this extremely host-specific tick, which is limited to a particular environment.

Scanning electron microscopy of all parasitic stages of Haemaphysalis qinghaiensis Teng, 1980 (Acari: Ixodidae).

Haemaphysalis qinghaiensis Teng (Acta Zootaxon Sin 5:144-149, 1980) is an endemic species in China. This tick species was first described based on engorged or semi-engorged specimens, and the drawings and description in words of morphological characteristics were poor. Therefore, the present study aims to redescribe morphological characteristics of all active stages of this tick species in detail by scanning electron microscopy. Additionally, a comparison between H. qinghaiensis and other sympatric Haemaphysalis species was also analyzed. Males of H. qinghaiensis can be distinguished from sympatric Haemaphysalis species by the following characters: palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; the tips of palpal segment III not so strongly recurved inward to become "pincerlike" and lacking dorsal spur; dental formula 5/5; lateral grooves enclose first festoon; coxa IV with a short, broadly triangular spur; tarsi somewhat humped; and spiracular plates long comma-shaped. Females of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; segment III of palpi lacking dorsal spur; dental formula 4/4; scutum subcircula; and tarsi somewhat humped. Nymphs of H. qinghaiensis can be distinguished from those of other species by palpi less salient laterally and curved in contour; dental formula 2/2; basis capituli rectangular, with distinct dorsal cornua, without ventral cornua; and spiracular plates with short and narrow dorsal prolongation. Larvae of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; basis capituli rectangular, without distinct cornua.

Morphological characteristics and developmental changes of the ovary in the tick Haemaphysalis longicornis Neumann.

Haemaphysalis longicornis (Ixodida: Ixodidae) is an important vector of transovarially transmitted parasites of the genus Babesia (Piroplasmida: Babesiidae). In the present study, we investigated the morphological characteristics and developmental changes of the ovary of H. longicornis. We show that the ovary of H. longicornis has a single tubular structure and is surrounded by a tunica propria. There is a longitudinal groove along one side of the ovary. During feeding and after engorgement, great changes can be observed in the ovary of H. longicornis and two rapid growth phases can be detected. The number of major protein bands of the ovary is significantly increased from day 3 of feeding and reaches a maximum on the day of engorgement. Therefore, the great diversity of proteins in the ovaries of H. longicornis can facilitate the identification of new targets for vaccine development.

Description of the larva of Amblyomma calcaratum Neumann, 1899 (Acari: Ixodidae) by light and scanning electron microscopy.

The larval stage of Amblyomma calcaratum Neumann is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. calcaratum originating from engorged females collected on Tamandua tetradactyla in the Jaraguá Mountain (23°40'S, 45°44'W), São Paulo County, Brazil. Eleven larvae were prepared and mounted on slides and observed under a light microscope equipped with a drawing tube. Three specimens were prepared for SEM. Several morphological characters are described, including the chaetotaxy of the idiosoma, palpi, and Haller's organ, as well as morphological features of the idiosoma, gnathosoma, and legs of A. calcaratum larvae. In addition, topographical and numerical patterns of integumentary structures on the larval idiosoma are described using a recently proposed nomenclature. On the idiosoma, setaes, lyrifissures, small glands, and large wax glands were found. These structures were observed isolated or associated over the entire idiosoma, except on the scutum, which lacks large wax glands. The topographical and numerical patterns of integumentary structures of the A. calcaratum larva showed only minor differences when compared with patterns of other Amblyomma larvae; however, a few key features can be used to differentiate A. calcaratum from other members of this genus.

First description of the immature stages and redescription of the adults of Cosmiomma hippopotamensis (Acari: Ixodidae) with notes on its bionomics.

Cosmiomma hippopotamensis (Denny, 1843) is one of the most unusual, beautiful, and rare tick species known to the world. All stages of this species possess a unique morphology, on the one hand making them easy to identify, while on the other they exhibit similarities to certain species of Amblyomma Koch, 1844, Dermacentor Koch, 1844, and Hyalomma Koch, 1844. Adults of C. hippopotamensis have been collected on only two occasions from their hosts, namely Hippopotamus amphibius L. and Diceros bicornis (L.), and have been recorded from only a few widely separated localities in East and southern Africa. Here, the larva and nymph are described and illustrated for the first time, while the male and female are illustrated and redescribed. Data on hosts, geographic distribution, and life cycle of C. hippopotamensis are also provided.

Description of larva of Amblyomma romitii (Acari: Ixodidae) by optical and scanning electron microscopy, including porotaxy and phylogenetic analysis.

The description of the larva of Amblyomma romitii Tonelli-Rondelli is based on optical and scanning electron microscopy. Larvae were obtained under laboratory conditions from an engorged female collected on capybara from Rurópolis municipality, State of Pará, Northern Brazil. Several characters are presented including the chaetotaxy of idiosoma, palpi and Haller's organ. The larval porotaxy (topographical and numerical patterns of integumentary structures) was presented and compared to that of the other Amblyomma spp. larvae. The mitochondrial 16S rDNA partial sequence of A. romitii generated in the present study was aligned with sequences previously determined for other Amblyomma species available in Genbank and with some species presently sequenced. The larval morphology of A. romitii and other Neotropical Amblyomma spp. larvae is discussed as well as the DNA sequence and its phylogenetic position among other species of the genus.

Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines.

As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

Morphological, biological and molecular characteristics of bisexual and parthenogenetic Haemaphysalis longicornis.

The reproductive mechanism of Haemaphysalis longicornis is quite different from many other animal species. In this article, several characteristics of parthenogenetic and bisexual populations of H. longicornis were analyzed, including some important micro-structures, synchronized life cycle feature and sequences of mitochondrial 16S rRNA gene. The results suggested even though many observations of the two populations were similar to each other, some important differences also existed. The genital apron of parthenogenetic females was wider than that of bisexual females. Parthenogenetic individuals (except engorged females) were significantly larger in weight than bisexual individuals (p<0.01; unfed nymph p<0.05); the difference of nymphal premoulting, female feeding and preoviposition, and egg incubation periods between the two populations were significant (p<0.01); hatch percentage of parthenogenesis was lower than that of bisexual population (69% and 73%, respectively); parthenogenetic individuals had a slightly longer development cycle than bisexual individuals (134 and 129 days, respectively). Hybridization attempts failed between them. Comparing to the two strains of bisexual H. longicornis, parthenogenetic strain of H. longicornis inserted two nucleotides of thymine, though the genetic distance of 16S rDNA between the bisexual and parthenogenetic populations was 0. In order to clarify the relationships of the two reproductive populations of H. longicornis, the sequences of 16S rDNA of four strains of two other species were also analyzed. The divergence between Hebei and Xinjiang strains of Rhipicephalus sanguineus was 0, and the sequences were totally identical between them. The divergence between the two subspecies of Hyalomma asiaticum was 0.01. The results indicated that the relationship between bisexual and parthenogenetic H. longicornis was certainly closer than that between subspecies, but farther than that between the same reproductive populations of conspecies.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae).

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-III and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption.