Semi-artificial mouse skin membrane feeding technique for adult tick, Haemaphysalis longicornis
© Hatta et al.; licensee BioMed Central Ltd. 2012
Received: 26 June 2012
Accepted: 11 October 2012
Published: 15 November 2012
An in vitro artificial feeding technique for hard ticks is quite useful for studying the tick-pathogen interactions. Here, we report a novel semi-artificial feeding technique for the adult parthenogenetic tick, Haemaphysalis longicornis, using mouse skin membrane.
Skin with attached adult ticks was removed from the mouse body at 4 to 5 days post-infestation for the construction of the feeding system. This system supplied with rabbit blood was kept in >95% relative humidity at 30°C during the feeding, and ticks were fully engorged (artificially engorged, AE) within 12 to 48 h. For comparison, ticks were fed to engorgement solely on rabbit or mouse for 5 days as controls (naturally engorged on rabbit, NEr, or mouse, NEm). Blood digestion-related gene expression in the midgut and reproductive fitness were compared. Body weight, egg mass weight, egg conversion ratio, and hatchability of eggs did not show any significant differences. We analyzed transcription profiles of selected genes assayed by quantitative RT-PCR and revealed similar patterns of expression between NEr and AE but some differences between NEm and AE or NEm and NEr.
Our results demonstrate that this semi-artificial feeding technique mimics natural feeding processes of ticks and can be utilized as a standardized method to inoculate pathogens, especially Babesia protozoa, into H. longicornis and possibly other tick species as well.
KeywordsHaemaphysalis longicornis Mouse skin membrane system Semi-artificial feeding technique Tick
As vectors of pathogens, ticks transmit viruses, rickettsia and protozoan parasites to both animals and humans. Artificial feeding systems are attractive tools for investigating the mechanisms of pathogen transmission as well as for studying the tick-pathogen interactions. First, artificial feeding systems can reduce variation within a given treatment group because the blood meal is supplied from the same donor, which reduces the variation that arises from individual host-tick relationships . Second, an animal experimental model is assumed to have a potent difficulty to control the infection in attached ticks with known numbers of pathogens, because pathogen load in ticks might be affected by the immune system of the hosts targeting tick molecules, such as protective antigen, subolesin . In this model using artificial feeding, the effects of the host’s immune responses against ticks are removed, and pathogens can be introduced into vectors in a controlled manner. So far, artificial feeding techniques have been used to feed a number of tick species of the family Ixodidae, including Rhipicephalus spp., Dermacentor spp., Amblyomma spp., Hyalomma spp., and Ixodes spp. using capillary tubes or membranes (briefly reviewed in ). Recently, Kröber and Guerin [1, 4, 5] established a method using a silicone membrane to engorge Ixodes ricinus. Tajeri and Razmi  also attempted to use this membrane for Hy. anatolicum anatolicum and Hy. dromedarii. These tick species have a long hypostome and fine palps with a wide range of motion and can reasonably be expected to completely penetrate the artificial membrane.
Naturally engorged ticks
The parthenogenetic Okayama strain of the tick H. longicornis has been maintained by feeding on rabbits in our laboratory since 1976 . In this study, NEr ticks were prepared according to the usual method described previously . Briefly, 20 adult ticks were placed on the ears of a rabbit to feed. At the beginning of the engorgement period, 9 ticks, which spontaneously detached from the rabbit after 5 days were collected. Of those, 6 randomly selected ticks were subjected to phenotypic analysis, and the remaining 3 ticks were used for transcription analysis. NEm ticks were also prepared according to the method described previously . Briefly, 5 mice (BALB/c, 3 weeks old) were infested with 10 adult ticks (2 ticks per mouse). Six randomly selected NEm ticks were subjected to phenotypic analysis, and the remaining 4 ticks were used for transcription analysis. Ethical approvals of conducting all animal experiments were provided by the Animal Care and Use Committee, National Institute of Animal Health (NIAH, Approval nos. 441 and 578).
Artificially engorged ticks
To prepare the mouse skin membrane, 5 to 7 adult ticks were allowed to feed on the shaven back of each of four tick-naïve SPF mice (BALB/c, 3 weeks old) following the described method . After 4 to 5 days (at the beginning of the expansion period ), a rectangular section (~9 cm2) of the mouse skin with the ticks attached was carefully removed from the mouse’s body. Figure 2A shows the Hematoxylin and Eosin (HE)-stained lesion of mouse skin membrane used for the feeding system. Hypodermal layers around the feeding lesion were carefully removed from the skin with sterile tweezers as much as possible. The body of the feeding system (Figure 2B) was constructed using the upper part of a Falcon tube (#352059, Becton, Dickinson and Company, Franklin Lakes, NJ) by cutting the tube at ~3 cm from the top. Then, the roof of the cap of the Falcon tube was removed and the skin membrane with ticks was placed on the mouth of the tube keeping the ticks outside and held tightly in place by applying the cap. Only two ticks were selected and allowed to feed within the area of the skin membrane in this system, and the ticks in excess of two were removed by tweezers and weighed. The mean body weight of the removed ticks (24.9 ± 5.2 mg) was subtracted from the weight of ticks after semi-artificial feeding to estimate weight gain. After construction, the inside of the membrane was washed with sterilized phosphate-buffered saline (PBS) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin (Life Technologies Corporation, Carlsbad, CA). Then, pre-warmed (30°C) rabbit blood containing 300 μl washed red blood cells (RBC) and 700 μl sterile serum (filtered with a syringe filter; #4652, 0.2 μm, Acrodisc Syringe Filters, Pall Co., Cornwall, UK.) was poured into the device (Figure 2C). To secure a sufficient volume of blood for the duration of tick feeding, we collected blood from a tick-naïve female SPF Japanese white rabbit (3- to 5-months-old). The open end of the tube was covered with a piece of parafilm. All procedures including system construction and blood exchange were performed inside a biosafety cabinet. The system was kept in a humidified chamber with >95% relative humidity at 30°C, and the rabbit blood was changed at every 12 h. When partially fed ticks of the expansion period (4–5 days post-infestation, DPI) were used, fully engorged ticks dropped off within 12 to 48 h of the onset of artificial feeding (Figure 2D-F). In contrast, ticks in the late-growth phase (3 DPI) required feeding on blood for more than 48 h to become engorged (data not shown). These feeding patterns were quite similar to the on-host feeding patterns of ticks described previously , suggesting that our in vitro feeding device effectively supports the expansion process of ticks and provides a sufficient amount of blood.
Primers for quantitative RT-PCR
Gene name and primer ID
Primer sequences (5′ to 3′)
HlSP (serine protease)
Longepsin (aspartic protease)
Longipain (cysteine protease)
HlSCP1 (serine carboxypeptidase)
Hlgut-defensin (anti-microbial peptide)
Even though a biosafety cabinet was used throughout the study during construction and manipulation of the feeding device, the possibility of microbial contamination in the blood cannot be completely neglected since we did not use antibiotics in the supplied blood. Nevertheless, similar expression patterns of the Hlgut-defensin gene among AE, NEr, and NEm ticks indicate that the feeding system was quite free from bacterial contamination. However, to increase the certainty of avoiding bacterial contamination, it would be better to apply antibiotics to this feeding system for other applications.
In conclusion, our findings suggest that the semi-artificial feeding technique for H. longicornis is very effective and may be used for all tick species, especially those with short hypostomes that do not penetrate deeply in the dermal layer of the host, such as Rhipicephalus ticks . Additionally, the technique is quite simple and cost effective since it does not require thinner artificial membrane , odorant (cow hair extracts), and/or feeding stimuli (adenosine triphosphate, ATP) [1, 4, 5] to enhance tick attachment. Although it is necessary to sacrifice mice in order to construct the device, the technique bears great promise for conducting in vitro assays, including the inoculation of pathogens, especially Babesia protozoa, based on results reported by Callow  that Rhipicephalus (Boophilus) microplus tick infection with one of the tick-borne bovine pathogens, Babesia bigemina, takes place during the rapid phase of feeding corresponding to the final stage (last 24 h) of the blood feeding. We expect that this technique will be useful in studies of tick physiology, tick-pathogen interactions, and tick-host interactions regarding novel tick genes that respond to the host defense mechanisms such as coagulation, inflammation, and immune responses.
Hematoxylin and eosin
H. longicornis serine proteinase
H. longicornis serine carboxypeptidase 1
H. longicornis legumain
H. longicornis leucine aminopeptidase
National Institute of Animal Health
Red blood cells
Reverse transcription polymerase chain reaction.
This work was supported by the Grant-in-Aids (to TH, NT and KF) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported by a grant (to NT and KF) for Promotion of Basic Research Activities for Innovative Biosciences from the Bio-oriented Technology Research Advancement Institution. We thank M. Shimada and M. Kobayashi for their generous help in preparing histological sections. We also thank Forte Science Communications (L. Gudex) for language editing of the manuscript.
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