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.

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.

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.

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.

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.

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.

Scanning electron microscopy and morphometrics of nymph and larva of the tick Hyalomma impressum (Acari: Ixodidae).

Nymphs and larvae of ixodid ticks play an important role on the distribution of ticks and transmission of pathogens. They almost infest small mammals and birds which either move from place to place as rodents or migrate across different countries as migratory birds. The morphological descriptions of nymph and larva of the tick Hyalomma impressum were firstly studied in details by scanning electron microscopy and morphometric analysis. The distinguished characters of H. impressum nymph are as follows: dorsal idiosoma (excluding scutum) with 26-27 pairs of setae, posterior margin of scutum is narrowly rounded, posterolateral margins of scutum is straight, cervical grooves on the scutum extend to the midlength, coxa I with two large spurs, coxae (II-IV) with one small spur for each, spiracle is an egg shape with a numerous pores, palpus does not project beyond the hypostome, with nine setae dorsally and six setae ventrally, hypostome has cylindrical shape, dental formula 2/2, teeth number per file is eight in the outer file and seven in the inner file, basis capitulum has triangular shape without setae dorsally and tetragonal shape with three pairs of setae ventrally. The distinctive characters of H. impressum larva are as follows: idiosoma with 13 pairs of setae for each dorsal (including scutal setae) and ventral (excluding coxal setae), scutum with cervical grooves is narrow and shallow extending about one third of the scutal length, posterior margin of scutum is broadly rounded, posterolateral margins of scutum is straight, fold-like indistinctive spurs on coxae II and III, palpus with eight setae dorsally, three setae ventrally and one seta apically, hypostome with dental formula 2/2, teeth number per file (excluding small basal and apical teeth) is seven in the outer file and six in the inner file, basis capitulum without setae dorsally and with three pairs of setae ventrally.

Salivary glands of Amblyomma cajennense (Acari: Ixodidae): a histological and an ultrastructural overview.

The present study on the salivary glands of semi-engorged Amblyomma cajennense females has identified the various cell types present in this tissue and allowed its morphohistochemical characterization. Marking techniques were applied to detect polysaccharides (PAS), proteins (bromophenol blue), lipids (Nile blue) and calcium (von Kossa), as well as those of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results obtained by TEM showed and confirmed that these individuals' glands are also formed by round acini that are connected to the common excretory duct through acinar and intermediate ducts. Histological data as well as ultrastructural ones showed that the glands are formed by types I, II and III acini. In this study with salivary glands polysaccharides, proteins, lipids and calcium were observed in the cytoplasm and/or cell secretion granules-both free or forming complexes, as the intensity of the marking varied according to the cell as well as the type of acini analyzed, showing the structural and functional complexity of the tick salivary glands, characteristics that give the multifunctional character to this organ.

Redescription of the male and description of the female of Ixodes abrocomae Lahille, 1916 (Acari: Ixodidae).

The male of Ixodes abrocomae Lahille, 1916 (Acari: Ixodidae) is redescribed and the female described for the first time from specimens collected on the rodents Abrothrix longipilis (Waterhouse), A. olivaceous (Waterhouse) and Phyllotis xanthopygus (Waterhouse) at Coquimbo, Chile. The males of I. abrocomae are peculiar in having the combination of the following features: length and width less than 2 mm and 1 mm, respectively; hypostome notched with two rows of stout denticles and several small internal denticles; article II of the palpi with two conspicuous dorsal setae; coxa I with two subequal spurs; coxae II-IV with a single spur plus an indication of a second spur; and a scutum with long, scattered hairs except for the glabrous postero-median field which reaches to the marginal fold. The females of I. abrocomae are peculiar in possessing a combination of: a pointed hypostome, with a 3/3 dentition of flared denticles; a long, narrow scutum with few 'hairs' and with punctations which are especially numerous in the posterior region; a triangular basis capituli, with oval porose areas lacking definitive borders and separated by the width of one area, and a sinuous posterior margin with small cornuae; one spur on coxae I-IV; and conspicuous setae on the interno-dorsal face of palpal article II and the ventral face of article I. Sequences of 16S rDNA were identical for male and female I. abrocomae, but differ by 3.8% and 5.5% from sequences of their closest relatives, I. stilesi Neumann, 1911 and I. sigelos Keirans, Clifford & Corwin, 1976, respectively. Characters enabling the separation of I. abrocomae from Ixodes spp. distributed in the southwestern Neotropics are presented. Records of I. abrocomae in different climatic areas and on different, widely distributed rodent hosts indicate that this species may be present beyond its known Chilean territorial range (Regions III and IV).

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.

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.

Histopathology and ultrastructure features of the midgut of adult females of the tick Amblyomma cajennense Fabricius, 1787 (Acari: Ixodidae) in various feeding stages and submitted to three infestations.

The digestive tube of the tick Amblyomma cajennense is responsible for the digestion during feeding on the host. This study analyzed the midgut of unfed, partially engorged, and fully engorged fed females as well as three infestations in rabbits. In A. cajennense, the digestive tube is long and from the midgut, two pairs of diverticula ramify and lead to a blind end. In some midgut regions were observed for the first time in ticks, structures termed here "nodules." The midgut of unfed females possesses a pseudostratified epithelium composed of digestive and generative cells. In partially engorged and engorged females at 1st infestation and partially engorged at 2nd infestation, the epithelium becomes stratified. In partially engorged females at 2nd infestation, the epithelium exhibits a third cell type: secretory cell. So the intestinal epithelium undergoes several changes during the feeding process in ticks at subsequent infestations. As infestations progress in the same host, the latter becomes more resistant and female ticks require more days to complete their feeding cycle, which in A. cajennense is 25 days.

Redescription of the female, description of the male, and several new records of Amblyomma parkeri (Acari: Ixodidae), a South American tick species.

The tick Amblyomma parkeri Fonseca and Aragão was described in 1952, based on female and immature ticks collected in the states of São Paulo and Santa Catarina, Brazil. Thereafter, there has been no further report of A. parkeri, and the male has remained unknown. Herein, we examined ticks collected on porcupines from a locality in the state of São Paulo. Some of the ticks were identified as Amblyomma longirostre (Koch, 1844), whereas others as A. parkeri, including male specimens, for which we provide the first description. We also provide additional reports of A. parkeri after examining collections of A. longirostre and Amblyomma geayi Neumann, 1899 from different tick collections. Morphological evidence to support the original description of A. parkeri is presented, supported by molecular analyses of portions of the 16S rRNA and 12S rRNA mitochondrial genes. Morphological particularities to separate A. parkeri, A. longirostre, and A. geayi are provided.

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.

The ornithodoros (Alectorobius) talaje species group (Acari: Ixodida: Argasidae): description of Ornithodoros (Alectorobius) rioplatensis n. sp. from southern South America.

A new species of argasid tick, Ornithodoros (Alectorobius) rioplatensis Venzal, Estrada-Peña, & Mangold, is described from larval and adult specimens collected in Uruguay. Further specimens from Argentina, Paraguay, and Chile are ascribed to the new species. Key distinguishing characters of larvae of the species are the number of pairs of dorsal setae (19-20 but typically 20 in O. rioplatensis, compared with 16-17 but typically 17 in O. puertoricensis and O. talaje), and the length of the hypostome, which is clearly longer in O. rioplatensis than in O. talaje. Morphological details of the adults of these species are inadequate for taxonomic purposes. The genetic distance between O. puertoricensis and O. rioplatensis, based on 16S rDNA sequencing, is approximately 12.7%. Variability in some morphological features, using multivariate morphometric discrimination to assess congruence between populations, was examined among larval specimens of O. talaje and O. puertoricensis collected from the United States, and Central and South America. Larval specimens of O. talaje from Guatemala (near the type locality of the species) were morphologically different from Mexican and southern U.S. populations originally described as O. talaje. In the absence of DNA data for these populations, and because of inadequate statistical discrimination among body characters, we prefer to refer to the Mexican and U.S. material as O. nr. talaje until further analysis of population variability is conducted using an adequate sample size.

New morphological data on fat bodies of semi-engorged females of Amblyomma cajennense (Acari: Ixodidae).

The present ultramorphological, histological and ultrastructural study on the fat body of semi-engorged females of Amblyomma cajennense revealed that this tissue is diffuse and consists of strands of cells surrounding the tracheal trunks. Morphometric analysis showed that the cellular and nuclear areas of round-shaped trophocytes are larger than those of cuboidal trophocytes, indicating that the arrangement of the former provides more contact area with the haemolymph. In this species, the fat body is found right underneath the integument and around organs. It consists of two cell types that despite distinct morphological characteristics and locations in the tissue, present the same histological features. In this study, these cells were termed cuboidal trophocytes when arranged as strands of cells and present in larger numbers, and round-shaped trophocytes when lying on these strands and observed in fewer numbers. Histological observations revealed that both types of trophocytes have one nucleus in their cytoplasm and also exhibit numerous vacuoles of different sizes and contents. Ultrastructural examination revealed that the organelles more frequently observed were the vesicular and lamellar rough endoplasmic reticulum, and mitochondria with tubular crests, indicating that they might be involved in lipid synthesis. Smooth endoplasmic reticulum was not observed. Cuboidal trophocytes arranged in strands, despite being closely associated, do not exhibit fused plasma membranes. Rather, the fusion of basal lamina of two neighboring cells is occasionally observed, acting as a selective permeability barrier. Here, a new terminology for tick fat body is proposed. It is based on fat body location (parietal, when located right underneath the integument instead of peripheral; and perivisceral, when located around organs instead of central) terminologies previously suggest by Obenchain and Oliver and for the cells constituting them, cuboidal trophocytes when arranged as strands, and round-shaped trophocytes when lying on these strands. Nephrocytes were not observed in semi-engorged females of A. cajennense.

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.