Introduction

Endosymbionts are usually classified as obligate (or primary) and facultative (or secondary) endosymbionts according to the extent of the mutual dependence between the host and the endosymbiont (Chaves et al. 2009). Primary endosymbionts, restricted to specialized tissues or cells, are usually obligate and essential for the survival of the host and their own vertical transmission. Secondary endosymbionts, not restricted to specific tissues, are usually facultative and nonessential for the host and transmitted either vertically or horizontally between the same or different species (Moran et al. 2008; Gosalbes et al. 2010; Santos-Garcia et al. 2015). Many insect species are usually simultaneously infected by multiple endosymbionts, including one primary endosymbiont and various secondary endosymbionts (Dale and Moran 2006; Distel et al. 2002), or two obligate mutual endosymbionts (McCutcheon and Moran 2007). These endosymbionts participate in host’s reproduction (Himler et al. 2011), development (Koropatnick et al. 2004), speciation (Stouthamer et al. 1999), defense against natural enemies, immunity (Haine 2008), environment stress (Brumin et al. 2011; Hendry et al. 2014), and offer nutrition for the host (Todd et al. 2015).

Hard ticks (Acari: Ixodidae) are blood-feeding ectoparasites (Munderloh et al. 2005). So far, several symbiotic bacteria had been identified in ticks, such as Coxiella-like (Klyachko et al. 2007), Francisella-like (Ivanov et al. 2011), Wolbachia-like (Zhang et al. 2011), Rickettsia-like (Gillespie et al. 2012), Arsenophonus-like (Dergousoff and Chilton 2010), “Candidatus Midichloria mitochondrii” (Sassera et al. 2006), and Rickettsia peacockii (Niebylski et al. 1997). However, unlike their counterparts in insects, the knowledge on the functions of tick endosymbionts remained limited. For instance, Coxiella-like endosymbiont might impact the reproduction of Amblyomma americanum (Zhong et al. 2007); Coxiella-like endosymbiont might provide vitamin for the lone star tick (Todd et al. 2015); and Candidatus Midichloria mitochondrii, the endosymbionts of Ixodes ricinus, is coupled with the process of engorgement and molt (Epis et al. 2013).

Haemaphysalis longicornis (Acari: Ixodidae), a three host tick, is extensively distributed in China (Chen et al. 2010), Korea, Japan, New Zealand, and Australia (Hoogstraal et al. 1968; Tenquisf and Charleston 2001). This species transmits a variety of pathogens (Yu et al. 2015), including Babesia microti (Guan et al. 2010), Ehrlichia chaffeensis (Sun et al. 2008), Anaplasma bovis (Lee and Chae 2010), A. phagocytophilum (Zou et al. 2011), C. burnetii (Lee et al. 2004), spotted fever group rickettsiae (Lee et al. 2003), Borrelia burgdorferi (Tian et al. 1998), and a new bunyavirus (Yu et al. 2011), all of which are able to cause damage to livestock production and to pose threat on human health. Our previous studies reported that three endosymbionts, Coxiella-like (CLS-Hl), Arsenophonus-like (ALS-Hl), and Rickettsia-like endosymbionts (RLS-Hl), coexist in this species (Liu et al. 2013). In addition, CLS-Hl appears to be the primary endosymbiont of H. longicornis, and specifically infects ovaries and Malpighian tubules with 100% prevalence. In addition, CLS-Hl has the characteristics of stable vertical transmission in the laboratory population (Liu et al. 2013). However, the functions of CLS-Hl have yet to be evaluated. In this study, we investigated the functions of CLS-Hl in ticks.

Materials and methods

Sample collection and rearing of ticks

Haemaphysalis longicornis samples were collected in Xiaowutai National Natural Reserve Area in China by flag dragging. Collected ticks were reared on the ears of rabbits as previously described (Liu et al. 2005). After detachment, ticks were collected and incubated at 26 ± 1 °C with a humidity of 75 ± 5% and a light: dark regime of 16:8 h. The protocol of all animal experiments was approved by the Institutional Animal Care and Use Committee of Hebei Normal University.

Tick dissection

After tetracycline treatment, ovaries and Malpighian tubules were dissected from the 4th day of engorged females under a stereomicroscope at 10 × 23 magnification using a micro-clipper in sterile phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4) as previously described (Liu et al. 2013). Then tissues were collected in 1.5 ml sterile vials (Axygen, USA) and stored at − 80 °C until use.

DNA extraction

To assess the change of CLS-Hl densities in these collected tissues, DNA was extracted. Before DNA extraction, dissected tissues were washed in sterile phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4) for three times. Subsequently, genomic DNA was extracted according to the manual of DNeasy Blood & Tissue Kit (Qiagen, Germany). Then the genomic DNA was stored at − 20 °C until use. The DNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA).

Antibiotic treatments

Tetracycline HCl (Solarbio) were diluted with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4) at the concentration of 100 mg/ml. The doses of each antibiotic were 17 ng/mg weight of feeding females (Camille et al. 2015). Forty females on the 4th day of feeding were pulled from host rabbits and were injected with either PBS or tetracycline under a dissecting microscope with a Hamilton injector (1–10 µl). The back of ticks was sticked on double-sided adhesive adhered to a glass slide, and the Hamilton injector was tilted to a 45° angle to the microscope stage. The tip of the needle was inserted into the hemocoel through the space between the first and second legs. The needle was left place for at least 30 s and then slowly withdrawn after injection of 0.8–1 µl into females. Females that have been handled were put into incubator for 24 h to ease the pain of the wound after tetracycline or control treatment (26 ± 1 °C, humidity 75 ± 5% with a light: dark regime of 16:8 h). After injection, these females were fed on rabbits that had been treated with PBS and tetracycline respectively. Engorged females were weighed and other biological parameters were measured after dropping from the host rabbits.

Rabbit treatment was performed in two steps. Firstly, 10 mg/kg of antibiotic was injected to the rabbits every day until all ticks dropped. Secondly, 1.2% tetracycline (w/v) was administered to the rabbits through drinking water until all ticks dropped (Camille et al. 2015). The control group was treated using PBS with the same procedures.

Semi-quantitative polymerase chain reaction (PCR) assay

A housekeeping gene (usually actin) is used as a reference to quantify the change of expression of target genes after tetracycline treatment. To assess the change of CLS-Hl densities after tetracycline treatment, semi-quantitative PCR was performed with specific primers (Table 1). PCR reactions were carried out in 20 µl volume containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM of mixed dNTPs, 2.5 U Platinum Taq DNA polymerase (Invitrogen, USA), 0.5 mM of each primer and 2 µl of template DNA. PCR reaction was performed as: 1 cycle of 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 15 s, and 72 °C for 15 s, with a final extension at 72 °C for 5 min. Nuclease free water was used as a negative control during each set of PCR reactions. PCR products were electrophoresed in 1% agarose gels and visualized using AlphaImager HP (Alpha Innotech, USA). The IntDen of the target stripe was carried out using ImageJ (imagej.nih.gov/ij/).

Table 1 Oligonucleotide primers used for PCR amplification
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Statistical analysis

CLS-Hl densities in the ovaries and Malpighian tubules were estimated through the IntDen ratio between 16S rRNA of CLS-Hl and actin of tick tissues. The ratios of the IntDen were measured and analyzed with Statistica 6.0 (StatSoft, USA). T test was applied to analyze the densities of CLS-Hl between PBS treatment and tetracycline treatment.

Results

Reduction of CLS-Hl in ovaries and Malpighian tubules

The ovaries and Malpighian tubules were dissected from the 4th day of engorged females of tetracycline- and PBS-treated groups. The CLS-Hl densities were assessed using semi-quantitative PCR approach. The results showed significant reduction of CLS-Hl densities in ovaries (P < 0.05, Fig. 1) and Malpighian tubules (P < 0.05, Fig. 2) after tetracycline treatment comparing to PBS controls. The ratios of CLS-Hl and actin IntDen decreased from 3.62 to 0.32 in the ovaries and from 3.08 to 0.57 in the Malpighian tubules in females treated with tetracycline and PBS, respectively.

Fig. 1
figure 1

Reduction of CLS-Hl density in ovaries after tetracycline treatment. Bars represent standard errors of means and the asterisk indicates significant difference in CLS-Hl density between PBS treatment and tetracycline treatment at the same sampling time using t-test (P < 0.05)

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Fig. 2
figure 2

Reduction of CLS-Hl density in Malpighian tubules after tetracycline treatment. Bars represent standard errors of means and the asterisk indicates significant difference in CLS-Hl density between PBS treatment and tetracycline treatment at the same sampling time using t-test (P < 0.05)

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Temporal changes of CLS-Hl densities after tetracycline treatment

The CLS-Hl densities did not significant change after PBS treatment. However, the densities of CLS-Hl started to decrease slightly on day 4 after tetracycline treatment (Fig. 3, P > 0.05), continued to reduce and reached to a significant difference on day 11 after treatment (Fig. 3, P < 0.05). The ratios of CLS-Hl and actin IntDen decreased from 1.83 to 0.73.

Fig. 3
figure 3

Temporal changes of CLS-Hl densities after tetracycline treatment. The changes of CLS-Hl densities were detected at different time points of adult stage after tetracycline treatment. Bars represent standard errors of means. The difference on day 11 in the relative densities of CLS-Hl between PBS treatment and tetracycline treatment was significant using t-test (P < 0.05)

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The change of the biological parameters

The decrease of CLS-Hl densities resulted from tetracycline treatment had no significant effect on preoviposition period (P > 0.05, Table 2), but had significant effect on engorged weight, feeding time, number of eggs, oviposition period, and hatching period (P < 0.001, Table 2). Engorged weight decreased from 232.9 to 122.1 mg/♀; feeding time prolonged from 8.1 to 10.8 days; number of eggs decreased from 2390.5/♀ to 1147.5/♀; oviposition period prolonged from 19.9 to 24.9 days; and hatching period prolonged from 37.2 to 39.9 days.

Table 2 Change of biological parameters
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Morphological changes of engorged females and ovaries

Engorged females were measured after tetracycline treatment. The results suggested that the size of engorged females treated with tetracycline was significantly smaller than PBS and had lots of folds on the back (Fig. 4).

Fig. 4
figure 4

Morphological changes of engorged females after treatment. a Ticks injected with PBS, b ticks injected with tetracycline

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On the 4th day after engorgement, the ovaries were dissected. The number of ovarian follicles among the engorged females treated with tetracycline was less than that in case of those treated with PBS, indicating that the development of ovaries after tetracycline treatment was slower than that after PBS treatment (Fig. 5).

Fig. 5
figure 5

Morphological changes of ovaries after treatment. a Ticks injected with PBS, b ticks injected with tetracycline

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Discussion

In previous studies, we identified three types of endosymbionts from H. longicornis. These endosymbionts were named CLS-Hl, RLS-Hl, and ALS-Hl, respectively (Liu et al. 2013). CLS-Hl is a novel primary endosymbiont that only infects ovaries and Malpighian tubules and its prevalence in these tissues is 100%. CLS-Hl shows the characteristics of stable vertical transmission in the laboratory population (Liu et al. 2013). However, the functions of CLS-Hl have not been evaluated yet.

In this study, we treated the feeding females of H. longicornis with tetracycline. CLS-Hl was sensitive to tetracycline and had a significant reduction of its densities in ovaries and Malpighian tubules. The decrease of CLS-Hl densities led to remarkable changes in the host biological parameters including the decrease of engorged weight, lower number of eggs, prolonged time to feeding, and prolonged time to oviposition. We therefore concluded that CLS-Hl is essential for the reproduction and development of ticks. The average size of engorged females was smaller and engorged females shrinked after tetracycline treatment. The follicles developed slower and the number of follicles reduced in ovaries after tetracycline treatment, suggesting that CLS-Hl densities affect the amount of blood feeding of H. longicornis.

The knowledge of the functions of tick endosymbionts is limited. Treatment to engorged, mated females with tetracycline reduced reproductive fitness of the tick vector Amblyomma americanum (Zhong et al. 2007); treatment on the Ixodes ricinus with tetracycline affected metabolism of ticks (Camille et al. 2015); and Coxiella-like endosymbiont provided vitamin for A. americanum (Todd et al. 2015). In this investigation, CLS-Hl plays a role in the reproduction and development of H. longicornis. Therefore, the endosymbionts of ticks participate in tick’s reproduction, development, metabolism and offer nutrition for the tick.