Effect of Temperature on Reproductive Fitness of the Engorged Tick, Haemaphysalis longicornis (Acari: Ixodidae)

All ticks used in this study were the second generation ticks originated from the adult H. longicornis collected from vegetation by flag -dragging in Xiaowutai National Natural Reserve Area (39°50´to 40°07´N, 114°47´ to 115°30´E) in Hebei Province, north China. They were allowed to feed on the ear of rabbits, and the non -parasitize stages were maintained in colony incubator under laboratory conditions (23 ±1°C, 75 ±5% RH with a light: dark regime of 6: 18 h). The engorged females were collected and used on the day detached freely.

After detachment, the engorged females were rinsed with water, dried with tissue paper, and weighed individually. Females that within the normal range of the engorged body weights (174.3–317.7 mg) were placed into individual glass tubes. A group of 3 females was maintained in colony incubator and served as control. The remaining groups (three females each) were exposed to 0°C and 40°C for 12 h, 24 h, 48 h and 72 h, and at 4°C and 37°C for 5 d, 10 d, 15 d and 20 d, respectively. Each single female was observed daily, and after above exposure they were transferred back to the colony incubator.

After stressed at different temperatures, the mortality of the engorged female H. longicornis was determined. Biological characteristics of the survived females including pre -ovipositon period (number of days from detachment to the beginning of oviposition), oviposition period (number of days from the beginning to the end of oviposition), egg mass laid and the incubation period of the eggs (number of days from the beginning of oviposition to the hatching of the first larva) were recorded, and then the reproductive efficiency index (REI) (number of eggs/weight of the engorged female) was calculated (Dantas -Torres and Otranto, 2011). Analysis of variance was employed to test for the statistical significance of group differences using Statistica V6.0 (StatSoft, USA).

The effects of temperature stresses on reproductive fitness of the engorged tick H. longicornis were listed in Table I. The engorged H. longicornis could survive all the time treatments at 0°C and 4°C, and both the temperature stress increased the pre -oviposition periods (P<0.05), except the group stressed at 4°C for 20 days failed to oviposit. When stressed at 37°C and 40°C, the prolonged exposure time (48 h and 72 h at 40°C, 15 days and 20 days at 37°C) resulted in 67% mortality of engorged H. longicornis. When stressed at 37°C, the 5 days and 10 days exposure groups showed shorter preoviposition periods (P<0.05). When stressed at 40°C, the 12 h and 24 h exposure prolonged the preoviposition periods (P <0.05) (Table I).

For the oviposition periods of H. longicornis, most temperature stresses showed no effects, except the groups exposed at 37°C for 5 days and 10 days, and 40°C for 24 h (P <0.05). As for REI of the engorged H. longicornis, most groups stressed at 0°C were elevated, and when compared to the control group, no differences were observed among the groups at 4°C and 37°C for 5 days, and 40°C for 12 h (P>0.05). With prolongation of time exposure at 4°C, 37°C and 40°C, the REI of H. longicornis declined progressively. The incubation periods were shortened when stressed at 37°C for 5 days and 40°C for 12 h, and at 0°C regardless of the exposure time (P<0.05). No egg hatchment was observed at 37°C for 10 days or longer, as well as at 40°C for 72 h and 4°C for 20 days. The hatachment rate of H. longicornis showed no differences among groups at 40°C for 12 h and 0°C regardless of time exposure (P>0.05), whereas other treatment groups all resulted in low hatchments (Table I).

The present study investigated the effects of temperature stress on fecundity of the engorged H. longicornis. Results indicated that the engorged H. longicornis showed high tolerance to cold and low tolerance to heat, as exposure at 37°C for 15 days and 20 days, and at 40°C for 48 h and 72 h resulted in 67% mortality, whereas no mortality was observed when stressed at 0°C and 4°C, though these stressed at 4°C for 20 days failed to oviposition. The high temperature stress (~40°C) resulting in death of H. longicornis has been described previously (Heath, 2016). The tick H. longicornis is found freeze susceptible, and the low temperature stress can enhance the cold hardiness of unfed ticks (Yu et al., 2014). In China, the unfed nymphal and adult H. longicornis are able to survive overwinter in the field, and the adults are active from April when the lowest teperature around 5°C to September with lowest temperature around 10°C, which offers possibilities to meet low temperatures for engorged females (Zheng et al., 2012). In New Zealand, all fed and unfed stages of H. longicornis as well as eggs can overwinter (Heath, 2016). All of these demostrated considerable level of cold tolerance of the engorged H. longicornis.

Means within a column with different superscripts differ significantly (P<0.05). NA, not applicable.

Pre -ovipositon periods of H. longicornis were prolonged after 0°C, 4°C and 40°C treatment, whereas it was shortened after 37°C stress (P<0.05), which indicated that the engorged H. longicornis could adapt a wide range of temperatures. This has been proved by the long active months from April to September in the field (Zheng et al., 2012). Correspondingly, the tick Rhipicephalus sanguineus has been reported to oviposit at 37°C (Jacobs et al., 2004), whereas oviposition did not occurr in H. leachi leachi (Adejinmi and Akinboade, 2011). Possible explanation may attribute to the different metabolic rates of theses ticks when facing extreme conditions (Adejinmi and Akinboade, 2011).

The oviposition periods of H. longicornis were varied among different temperature stress, and the 0°C and 4°C treatment showed no influences, whereas the prolonged exposure to high temperatures (37°C and 40°C) led to a shorter oviposition periods which subsequently resulted in a low REI. Additionally, the 4°C treatment for 10 days and 15 days also resulted in a low REI, which suggested that the low temperature treatments still cause some harmful effects on the tick H. longicornis though the exposure time was not long enough to stop oviposition. Similar phenomenon was also observed in R. sanguineus stressed at 8°C for 60 days which could also oviposit but without hatchement (Dantas -Torres and Otranto, 2011). Most incubation periods of eggs were shortened after temperature treatment on the engorged H. longicornis, and 37°C stressed for more than 10 days resulted in no hatchment. Treatments at 0°C on engorged ticks displayed no effects on egg viability, whereas 4°C, and pronlonged 37°C and 40°C treatments on engorged H. longicornis decreased the hatchment rate, and this may due to that the engorged females have started oviposition during temperature stress, and hence the eggs were also subjected to stress.

The results obtained in the current study suggested that the tick H. longicornis could adapt a wide range of climate conditions and showed high tolerance to cold and low tolerance to heat. These findings advanced our knowledge on the field adaptation of H. longicornis and provided new insights into the epidemiology of H. longicornis -borne diseases. However, further investigation remains required to explore the intriguing adaptation of this tick species under background of global climate changes.

This work was supported by National Natural Science Foundation of China (31400342), the Natural Science Foundation of Hebei Province (C2015205124) and Natural Science Research Programs of the Educational Department of Hebei Province (BJ2016032).

The authors have no competing interests.