Biological differences between two allopatric populations of <Emphasis Type="Italic">Amblyomma cajennense</Emphasis> (Acari: Ixodidae) in Argentina

The fertility of hybrids from two distinct populations of Amblyomma cajennense from different ecological regions of Northern Argentina was analyzed. Two colonies of A. cajennense from El Rey National Park (RNP), Salta Province (24o41′S, 64o36′W), and Copo National Park (CNP), Santiago del Estero Province (25o55′S, 61o43′W) were established infesting rabbits with adults collected from vegetation. Reproductive parameters of the first generation in laboratory of each colony and their crosses were evaluated considering engorged weight of females, engorged period of females, pre-oviposition period of females, minimum egg incubation period, reproductive efficiency index [REI = number of eggs laid/weight of the females in mg], and fertility efficiency index [FEI = number of hatched larvae/weight of the females in mg]. Infestations were made as follows: Group 1) RNP males and females; Group 2) CNP males and females; Group 3) males from CNP and females from RNP; Group 4) males from RNP and females from CNP. The engorgement weight of the females from CNP that mated with males from RNP was significantly lighter than those of the engorged females obtained in the other 3 crosses, and the engorged period of the females from CNP that mated with males from RNP was significantly longer that the engorged period of the females belonging to the remaining groups. The females from group 3 and 4 had a FEI extremely low in comparison with the FEI obtained from the engorged females originated from the groups 1 and 2. Biological implication of these findings is discussed.     

Salivary glands of <Emphasis Type="Italic">Amblyomma cajennense</Emphasis> (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.     

<Emphasis Type="Italic">Coxiella</Emphasis> Symbionts in the Cayenne Tick <Emphasis Type="Italic">Amblyomma cajennense</Emphasis>

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.     

Physiological effects upon <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae) infected with <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Ascomycota: Hypocreales)

Unfed adult Amblyomma americanum were exposed to the entomopathogenic fungus Beauveria bassiana. Ticks exposed to the fungus exhibited reduced survival and increased water loss as indicated by change in weight. Treated ticks survived 7.2 ± 0.22 days (mean ± SE) and controls survived 17.9 ± 0.73 days (P = 0.01; df = 57). At death, ticks exposed to the fungus had lost 25.2 ± 0.84% of their starting weight; control ticks had lost 14.1 ± 0.85% of their starting weight (P = 0.01; df = 96). Water loss was highest immediately following inoculation, although losses continued to be higher than in uninoculated ticks. This suggests that fungal penetration causes sufficient cuticle damage to cause desiccation, although other water-loss avenues exist, including increased time of spiracular opening. Additionally this study did not eliminate the possibility of a negative impact on water vapor uptake. This is the first study to investigate the effect of an entomopathogenic fungus on the water balance of a tick.     

Biology and life cycle of <Emphasis Type="Italic">Amblyomma incisum</Emphasis> (Acari: Ixodidae)

Amblyomma incisum Neumann is a major tick species in the Atlantic Forest of Brazil. Tapir is the main host for adult ticks and a high aggressiveness of nymphs to humans has been reported. In this work data on the biology and life cycle of this tick species is presented for the first time. It was shown that horse is a suitable host for A. incisum adults and rabbit for larvae and nymphs. It was also shown that A. incisum is a big tick species (mean engorged female weight of 1.96 g) with a long life cycle which lasts 262.3 days when maintained at 27°C and 85% RH. These laboratory conditions were, however, inappropriate and egg hatching rate (1.2%) was very low. Nevertheless egg hatching of ticks in a forest patch increased considerably (72.2%) indicating that this A. incisum population is highly dependent on a forest-like environment.     

In vitro efficacy of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> sensu lato against unfed <Emphasis Type="Italic">Amblyomma parvum</Emphasis> (Acari: Ixodidae)

Amblyomma parvum Aragão (Acari: Ixodidae) is a tick species found with wide distribution in the Neotropical region. Even though it is a wildlife-related tick, it is also a frequent parasite of domestic animals, is aggressive to human beings and may harbor pathogenic microorganisms. Therefore, it is a target species for control on domestic animals, particularly those at the rural–wildlife interface. Herein, the efficacy of two isolates (E9 and IBCB 425) of an entomopathogenic fungus, Metarhizium anisopliae sensu lato, already evaluated for ticks that parasitize domestic animals, was tested against unfed A. parvum adults. Both isolates displayed high acaricidal efficacy after immersion in fungal conidial suspensions for 5 min. Isolate E9 killed all ticks by the 7th day post-treatment, and isolate IBCB 425 did so by the 11th day. Tick mortality of 80 and 90% was achieved as early as the 3rd and 4th days, respectively, with both treatments. Thus, if a commercial M. anisopliae s.l. acaricide against domestic animal ticks is developed, it would also be effective against A. parvum.     

Biological aspects of <Emphasis Type="Italic">Amblyomma brasiliense</Emphasis> (Acari: Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20°C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.     

The life cycle of <Emphasis Type="Italic">Amblyomma auricularium</Emphasis> (Acari: Ixodidae) using rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) as experimental host

The life cycle of Amblyomma auricularium (Conil) is reported for the first time, using rabbits as experimental host. Developmental periods of free-living stages were observed in an incubator at 27 ± 1°C, 80 ± 10% RH and 24 h darkness. The complete life cycle, including pre-feeding periods for each parasitic stage, ranged from 97 to 162 days. The overall sex ratio was 1.16:1 (M:F). Feeding and premolt periods, molting success, and engorgement weight of nymphs were statistically different between males and females (P < 0.01), but because their ranges overlapped, they cannot be used to predict the sex with accuracy. The potential role of rabbits as experimental hosts for rearing A. auricularium in the laboratory is discussed.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Ascomycota: Hypocreales) as a management agent for free-living <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae) in Ohio

Unfed adult Amblyomma americanum were exposed to the entomopathogenic fungus Beauveria bassiana and placed in the field. Ticks exposed to the fungus experienced higher mortality than those in the control group (P = 0.001). It is recommended that for inclusion in a tick management program, the fungus be applied in the late evening during the months March through June as this is when ticks are most active and conditions suitable for fungal activity occur.     

Life-cycle and host preference of <Emphasis Type="Italic">Amblyomma ovale</Emphasis> (Acari: Ixodidae) under laboratory conditions

This study evaluated for the first time the life cycle of Amblyomma ovale in the laboratory. For this purpose, larvae and nymphs were exposed to Gallus gallus (chickens), Cavia porcellus (guinea pigs), Rattus norvegicus (wistar rats), Oryctolagus cuniculus (domestic rabbits), Calomys callosus (vesper mouse), and Didelphis albiventris (white-eared opossum). Nymphs were also exposed to Nectomys squamipes (South American water rat). Adult ticks were fed on dogs. The life-cycle of A. ovale in laboratory could be completed in an average period of ca. 190 days, considering prefeeding periods of 30 days for each of the parasitic stages. Vesper mice were the most suitable host for A. ovale larvae, whereas water rats were the most suitable host for A. ovale nymphs. Our results, coupled with literature data, strongly indicate that small rodents have an important role in the life history of A. ovale. Chickens (the only avian host used in the present study) showed to be moderately suitable hosts for subadult A. ovale ticks, indicating that wild birds might have a secondary role in the life history of A. ovale. Domestic dogs showed to be highly suitable for the adult stage of A. ovale, in agreement with literature data that indicate that the domestic dog is currently one of the most important hosts of A. ovale adult ticks in Latin America.     

Abundance estimation of <Emphasis Type="Italic">Ixodes</Emphasis> ticks (Acari: Ixodidae) on roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

Despite the importance of roe deer as a host for Ixodes ticks in central Europe, estimates of total tick burden on roe deer are not available to date. We aimed at providing (1) estimates of life stage and sex specific (larvae, nymphs, males and females, hereafter referred to as tick life stages) total Ixodes burden and (2) equations which can be used to predict the total life stage burden by counting the life stage on a selected body area. Within a period of 1½ years, we conducted whole body counts of ticks from 80 hunter-killed roe deer originating from a beech dominated forest area in central Germany. Averaged over the entire study period (winter 2007–summer 2009), the mean tick burden per roe deer was 64.5 (SE ± 10.6). Nymphs were the most numerous tick life stage per roe deer (23.9 ± 3.2), followed by females (21.4 ± 3.5), larvae (10.8 ± 4.2) and males (8.4 ± 1.5). The individual tick burden was highly aggregated (k = 0.46); levels of aggregation were highest in larvae (k = 0.08), followed by males (k = 0.40), females (k = 0.49) and nymphs (k = 0.71). To predict total life stage specific burdens based on counts on selected body parts, we provide linear equations. For estimating larvae abundance on the entire roe deer, counts can be restricted to the front legs. Tick counts restricted to the head are sufficient to estimate total nymph burden and counts on the neck are appropriate for estimating adult ticks (females and males). In order to estimate the combined tick burden, tick counts on the head can be used for extrapolation. The presented linear models are highly significant and explain 84.1, 77.3, 90.5, 91.3, and 65.3% (adjusted R ²) of the observed variance, respectively. Thus, these models offer a robust basis for rapid tick abundance assessment. This can be useful for studies aiming at estimating effects of abiotic and biotic factors on tick abundance, modelling tick population dynamics, modelling tick-borne pathogen transmission dynamics or assessing the efficacy of acaricides.     

Detection of Alpha and Gamma-Proteobacteria in <Emphasis Type="Italic">Amblyomma triste</Emphasis> (Acari: Ixodidae) from Uruguay

Amblyomma triste is the most prevalent tick species reported in human tick bites in Uruguay and has been found to be infected with Rickettsia parkeri, but no other microorganisms have been reported from this tick. A sample of 254 adults of A. triste was collected by flagging on vegetation in suburban areas in southern Uruguay. Pools of five ticks were assembled and a screening for the DNA from the resulting 51 pools was realized by PCR assays using primers for amplifying a fragment of 16S rRNA gene for members of Anaplasmataceae. Seventeen pools were positive (33%) and the sequenciation of the gene fragment amplified revealed the presence of a putative new Alpha-Proteobacterium (denominated Atri-uru). The phylogenetic analysis showed that this microorganism is closely related to the symbiont of I. ricinus denominated ‘Candidatus Midichloria mitochondrii’ and other associated organisms. This rickettsial symbiont of ticks is included in a recent new clade proposed for the Alpha subclass of the Proteobacteria. The discovery of this bacterium in A. triste is the first evidence of this group of Rickettsiales detected in the Genus Amblyomma, and the first record in South America. Also, in two of 17 positive samples a Gamma-Proteobacterium related to Francisella-like organisms was detected.     

Pathogenicity of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> (Deuteromycetes) and permethrin to <Emphasis Type="Italic">Ixodes scapularis</Emphasis> (Acari: Ixodidae) nymphs

Effectiveness of the entomopathogenic fungus Metarhizium anisopliae, for controlling nymphal Ixodes scapularis, was tested in laboratory and field trials. In the laboratory, M. anisopliae (Metschnikoff) Sorokin strain ESC1 was moderately pathogenic, with an LC₅₀ of 10⁷ spores/ml and induced 70% mortality at 10⁹ spores/ml. In a field study, however, 10⁹ spores/ml M. anisopliae did not effectively control questing I. scapularis nymphs, and significant differences were not detected in pre- and post-treatment densities. For nymphs collected and returned to the laboratory for observation, mortality was low in treatment groups, ranging from 20 to 36%. To assess whether a chemical acaricide would synergistically enhance pathogenicity of the fungus, we challenged unfed nymphal I. scapularis with combinations of M. anisopliae and permethrin, a relatively safe pyrethroid acaricide, in two separate bioassays. Significant interactions between M. anisopliae and permethrin were not observed, supporting neither synergism nor antagonism.     

Ecological implications on the aggregation of <Emphasis Type="Italic">Amblyomma fuscum</Emphasis> (Acari: Ixodidae) on <Emphasis Type="Italic">Thrichomys laurentius</Emphasis> (Rodentia: Echimyidae), in northeastern Brazil

We investigated the Amblyomma fuscum load on a pullulating wild rodent population and the environmental and biological factors influencing the tick load on the hosts. One hundred and three individuals of Thrichomys laurentius were caught in an Atlantic forest fragment in northeastern Brazil, as part of a longitudinal survey on ticks infesting non-volant small mammals. Ticks (n = 342) were found on 45 individuals and the overall mean intensity of infestation was 7.6 ticks per infested rodent. Ticks were highly aggregated in the host population and the negative binomial distribution model provides a statistically satisfactory fit. The aggregated distribution was influenced by sex and age of the host. The microhabitat preference by T. laurentius probably increases contact opportunities between hosts and aggregated infesting stages of the ticks and represents important clues about the habitat suitability for A. fuscum.     

Drop off rhythm and survival periods of <Emphasis Type="Italic">Amblyomma lepidum</Emphasis> (Acari: Ixodidae) under field conditions

In this study, engorged Amblyomma lepidum ticks were found to drop off in two peaks, one in the morning and one in the evening. Most larvae and females engorged during the morning hours between 06.00 h and 10.00 h with a peak around 08.00 h, whereas the majority of the nymphs dropped in the evening between 18.00 h and 24.00 h with the peak around 22.00 h. Although the effect of time on drop-off patterns of the ticks was statistically significant (p≤ 0.001), there were no significant seasonal influences. Survival of unfed stages of A. lepidum was also studied and was found to increase from larvae to adult ticks. The longest survival periods of 10, 11 and 14 weeks were recorded during the wet season for larvae, nymphs and adults, respectively. It is concluded that environmental conditions required for survival of A. lepidum are optimal only during the wet season and that during other seasons the tick depends primarily on prevailing micro-climatic conditions for its survival.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Detection of a questing <Emphasis Type="Italic">Hyalomma marginatum</Emphasis><Emphasis Type="Italic">marginatum</Emphasis> adult female (Acari,Ixodidae) in southern Germany

The first finding of a questing Hyalomma marginatum marginatum female tick in Germany is described. The tick was found in May 2006 on the clothing of a person who had spent the preceding day in rural surroundings in southern Germany. As the infested person had also been visiting Spain where H. m. marginatum is known to occur some weeks prior to finding the tick, it is not clear whether the tick had been imported by him as a female or by another host in a preimaginal stage and succeeded to develop to an adult in Germany. H. m. marginatum is a thermophilic tick species usually occurring in relatively dry and warm regions of southern Europe, northern Africa and some parts of Asia. It is a vector of several disease agents of human relevance including Crimean-Congo hemorrhagic fever virus. Although, by PCR examination, the female was found neither infected with this virus nor with Rickettsia aeschlimannii, another human pathogen which has been found in Spanish Hyalomma ticks, its mere finding should be taken seriously and draw further attention to the increasing problem of the import and spread of putatively tropical vectors of disease to central Europe.     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

Biology of <Emphasis Type="Italic">Dermacentor silvarum</Emphasis> (Acari: Ixodidae) under laboratory conditions

The minimum life cycle of Dermacentor silvarum Olenev had a mean duration of 87.5 days (range 74–102 days) under laboratory conditions [(27±1 °C), 70% RH, 6 L: 18 D]. The mean time in (days) for the different stages of its cycle was as follows: incubation period of eggs was 15.3 days; prefeeding, feeding and premoulting periods of larvae and nymphs averaged 5.5, 4.0 and 7.3 days, and 5.2, 5.0 and 14.6 days, respectively; prefeeding, feeding, preoviposition and oviposition periods of female adults lasted for 7.8, 4.5, 4.3 and 14.0 days, respectively. There existed a highly significant correlation between engorged body weight of females and egg masses laid (r = 0.9877, p<0.001). The reproductive efficiency index (REI) and reproductive fitness index (RFI) in females were 11.09 and 9.58, respectively. No relationship between nymphal engorged body weight and resultant sexes was observed. Delayed feeding and non-oviposition (in June and July) existed in females, and low temperature (−10 °C) treatment for 45 days could terminate oviposition diapause. However, the egg masses laid by post-diapause females were significantly smaller than those laid by females engorged in March, April and May.     

A comparison of the cuticular properties of the female ticks <Emphasis Type="Italic">Ixodes pacificus</Emphasis> and <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) throughout the feeding period

The mechanical properties of the cuticle of Ixodes pacificus (Ip) are compared to those of Amblyomma hebraeum (Ah) from our earlier work. The 10-fold size difference between the species is expected to lead to significant differences in mechanical properties, because cuticular stretch depends on high internal hydrostatic pressure during the rapid phase of engorgement. We demonstrate here: (1) The cuticle of partially fed Ip is less stiff and viscous than that of Ah. (2) A stretch-recoil cycle in both ticks consists of recoverable deformation (ESv) and permanent deformation (ESp); ESp is higher in Ip, and increases sharply during the slow phase of engorgement, but not in Ah. (3) Injected dopamine (DA) increases ESp and reduces all measures of stiffness and viscosity, suggesting that a catecholaminergic neurotransmitter plays a fundamental role in modulating mechanical properties of the cuticle. However, unlike Ah, DA’s effect was not different from that of the control (1.2% NaCl). Mere insertion of the needle may have punctured the gut, causing the release of perhaps a catecholamine that increases ESp, an hypothesis supported by the fact that inserting a needle without any injection also caused an increase in ESp. (4) Stretch reduces ESp, but subjecting loops to pH 6.5 in vitro restores it. (5) Despite the smaller size of Ip, later onset of the rapid phase of engorgement, a thinner cuticle and different mechanical properties all reduce the internal pressure needed for stretch.