The Diel Activity Pattern of Haemaphysalis longicornis and Its Relationship with Climatic Factors

Simple Summary Many tick species pose a threat to public health by transmitting diseases to humans and animals. Haemaphysalis longicornis (The Asian longhorned tick) is known to be the main vector that transmits severe fever with thrombocytopenia syndrome virus. In the United States, it was first identified in New Jersey during 2017 on domestic sheep. Even though H. longicornis is an important vector, research on its major activity period is lacking. Tick activity patterns may vary depending on climate factors, the host, and region. This study aimed to survey the patterns of diel activity in H. longicornis and the potential effects of climatic factors on tick activity. The highest activity occurred at 10:00 to 14:00. Avoiding the times of highest activity is considered the best way to reduce contact with ticks. Abstract Haemaphysalis longicornis is one of the most medically important carriers of various pathogens. Although H. longicornis is an important vector, only basic ecological and biological information has been obtained, primarily focusing on its abundance and distribution. This study determined the most active time and meteorological conditions for the diel activity of H. longicornis. The diel activity pattern of H. longicornis was the highest between 10:00 and 14:00, and the lowest between 22:00 and 02:00. The major activity temperature of H. longicornis was between 25 °C and 40 °C, with the highest activity at 35 °C. The relative humidity was between 30% and 70% during the active period. Temperature had the highest correlation with diel activity (R = 0.679), followed by humidity (R = −0.649) and light intensity (R = 0.572). Our results provide basic information for the development of tick-borne disease vector control programs and tick surveillance.


Introduction
Ticks transmit numerous viral, bacterial, and protozoan pathogens to humans and other animals.As the twenty-first century advances, tick-borne viruses and novel viruses will definitely emerge [1], such as severe fever with thrombocytopenia syndrome (SFTS), Heartland virus, Crimean Congo hemorrhagic fever virus (CCHFV), and Powassan virus.Additionally, Jingmen tick virus [2], Yezo virus [3], Toyo virus [4], and Songlin virus [5] have been identified in China and Japan.
Ticks are increasing in density and expanding their geographical distribution owing to climate change [6,7].Additionally, global warming causes increased tick activity and changes in tick development, reproduction, and survival rates, as well as changes in the development rates of pathogens that are carried by vectors [7,8].Ixodes ricinus is widely distributed in northwestern Europe, and climate change has expanded its range northward into Norway [9].The black-legged tick I. scapularis has been recorded over an expanded geographic range in the eastern-to-northern parts of the USA over the last half of the century [10].Additionally, an invasion of the red sheep tick (Haemaphysalis punctata) was first identified at Rhode Island, USA in 2010 [11].After the Asian long-horned tick (Haemaphysalis longicornis) was first reported in New Jersey [12], the distribution area has expanded to the southern area of the USA and was recently confirmed in Georgia [13].Modeling analysis using climate data shows that H. longicornis has a broad potential distribution in the United States and Mexico [14].Currently, there is great concern regarding the potential role of H. longicornis as a vector for transmitting pathogens to humans and domestic animals.
H. longicornis Neumann is distributed across eastern China, Japan, the Russian Far East, and the Republic of Korea (ROK).It was introduced to Australia over a century ago [15][16][17].H. longicornis was first discovered in the United States in 2017 [12] and has expanded to various regions [18][19][20][21].Because H. longicornis is a three-host Ixodid tick, each life stage (adult, nymph, and larva) must include feeding with blood for development into the next life stage [16].Furthermore, exotic populations of H. longicornis commonly reproduce asexually via parthenogenesis [16,22].Typically, in the summer, adult females lay 1000 to 4000 eggs in moist soil [23].Laboratory studies have shown that eggs hatch approximately 25 days after oviposition when stored at 25 • C [24].Larvae that hatch in the field immediately search tall grass or shrubs for a host [16,25,26].They can often be found in clusters browsing together at the tips of plants [26].After blood feeding, the larvae molt into nymphs and typically undergo diapause during the winter [16].Field surveys demonstrate that H. longicornis overwinters mainly at the nymphal stage in the topsoil layer [27].In late winter or early spring, depending on the temperature and photoperiod, the nymphs become active again and quest for new hosts [16], such as cattle, sheep, deer, or horses [25].Generally, one generation occurs over the course of a year as ticks undergo a diapause to increase their chances of survival [16,28].
The risk of tick-borne diseases in humans is influenced by many factors; however, the final issue is the questing activity time of unfed ticks.Therefore, studies have been conducted on the diel activity patterns of various species.Questing activities are influenced by climatic and environmental factors.The endogenous rhythm of the ticks and meteorological factors (particularly the temperature, humidity, and insolation) are considered to be the main parameters governing tick diel activity [46,47].Humidity is one of the most influential factors affecting questing activity and tick survival [48].Additionally, the activity and behavior of the host may be important [47].Studies on the activity patterns of ticks have been conducted in several species, and the activity patterns greatly differ for each species.For example, the adult stages of the Dermacentor reticulatus peak activity occurs at 18:00-18:25 in spring and 14:00-14:25 in autumn [49].Ixodes scapularis exhibited the highest activity in the early morning, whereas contrast Amblyomma americanum nymphs showed peak activity around noon [50].I. ricinus activity peaked between 23:00 and 03:00 [51].Although H. longicornis is an important vector, only basic ecological and biological information has been obtained, primarily focusing on its abundance and distribution.
This study aimed to survey the patterns of diel activity in H. longicornis and the potential effects of climatic factors on tick activity.Ultimately, we could identify the major activity times of ticks, provide guidelines to reduce the risk of contact between humans and ticks, and prevent the spread of tick-borne infectious diseases.

Study Site
This study was conducted in Iin-myeon, GongJu-si, Chungcheongnamd-do, Republic of Korea (36 • 24 ′ 45 ′′ N, 127 • 04 ′ 05 ′′ E).This area was selected as a suitable environment for hosts because there are resting places and water supply sources around it (Figure 1).Additionally, several ticks were collected from a previous study.The collection site was covered with grass and shrubs, with an area of approximately 0.045 km 2 .A 50 × 70 m experimental section was established at the center.
Insects 2024, 15, x 3 of 12 This study aimed to survey the patterns of diel activity in H. longicornis and the potential effects of climatic factors on tick activity.Ultimately, we could identify the major activity times of ticks, provide guidelines to reduce the risk of contact between humans and ticks, and prevent the spread of tick-borne infectious diseases.

Study Site
This study was conducted in Iin-myeon, GongJu-si, Chungcheongnamd-do, Republic of Korea (36°24′45′′ N, 127°04′05′′ E).This area was selected as a suitable environment for hosts because there are resting places and water supply sources around it (Figure 1).Additionally, several ticks were collected from a previous study.The collection site was covered with grass and shrubs, with an area of approximately 0.045 km 2 .A 50 × 70 m experimental section was established at the center.

Sampling Methods and Identification
The ticks were collected using a dry-ice bait trap.The main body of the trap was a white tarpaulin cylinder with an open top (36 cm diameter × 40 cm height).Additionally, a cylindrical ice chest (10 cm diameter × 30 cm height) containing 3 pieces of dry ice (ca, 2.5 kg total) was placed inside the trap to attract ticks [52].
The study was conducted on the third week of every month from April to September at 4 h intervals for 24 h in 2022.The experiment was performed randomly on sunny days.If it rained, we changed the collection period.In August, tick collection could not be conducted owing to rain and wet ground from the rainy season.The attracted ticks (only adults and nymphs) were counted and released 5 m away from the trap to minimize the effects of ticks being removed during collection.The distance between the traps was set to 20 m because the attraction distance of the ticks to dry ice was up to 8 m [17].To eliminate site bias, the traps were moved to different sites for each time period (Figure 2A).Each trap had a pole with reflective tape to easily find a trap at night (Figure 2B).

Sampling Methods and Identification
The ticks were collected using a dry-ice bait trap.The main body of the trap was a white tarpaulin cylinder with an open top (36 cm diameter × 40 cm height).Additionally, a cylindrical ice chest (10 cm diameter × 30 cm height) containing 3 pieces of dry ice (ca, 2.5 kg total) was placed inside the trap to attract ticks [52].
The study was conducted on the third week of every month from April to September at 4 h intervals for 24 h in 2022.The experiment was performed randomly on sunny days.If it rained, we changed the collection period.In August, tick collection could not be conducted owing to rain and wet ground from the rainy season.The attracted ticks (only adults and nymphs) were counted and released 5 m away from the trap to minimize the effects of ticks being removed during collection.The distance between the traps was set to 20 m because the attraction distance of the ticks to dry ice was up to 8 m [17].To eliminate site bias, the traps were moved to different sites for each time period (Figure 2A).Each trap had a pole with reflective tape to easily find a trap at night (Figure 2B).
For identification, the ticks collected during the last period were brought to the laboratory and identified to the species and life stage levels using a stereomicroscope (Olympus, Tokyo, Japan) and morphological keys [53].For identification, the ticks collected during the last period were brought to the laboratory and identified to the species and life stage levels using a stereomicroscope (Olympus, Tokyo, Japan) and morphological keys [53].

Environmental Measurements
The temperature (°C) and relative humidity (RH) (%) were measured at knee height (trap height) near the dry-ice trap using CEM DT-172 (CEM, Shenzhen, China) at three points at the collection site.The light intensity was measured using a CEM DT-8808 (CEM, Shenzhen, China) at the same location.The temperature, humidity, and light intensity were also measured when ticks were counted from the trap.

Statistical Analysis
The collection results were analyzed at 4 h intervals, and the collected ticks at each time period were compared using an analysis of variance (ANOVA).The mean of each group was used in this test.The relationships between the climatic variables were examined using Pearson's correlation coefficients.We used multiple linear regression to examine the relationships between climatic variables and the number of ticks collected.

Result
A total of 412 ticks were collected during the last period, including 53 adults and 376 nymphs.A total of 396 individuals were H. logicornis (48 adults and 365 nymphs) and 16 individuals were H. flava (five adults and 11 nymphs).From April to July, all ticks were H. longicornis.In September, 16 of the 64 ticks collected were H. flava and the remaining 48 were H. longicornis.

Environmental Measurements
The temperature ( • C) and relative humidity (RH) (%) were measured at knee height (trap height) near the dry-ice trap using CEM DT-172 (CEM, Shenzhen, China) at three points at the collection site.The light intensity was measured using a CEM DT-8808 (CEM, Shenzhen, China) at the same location.The temperature, humidity, and light intensity were also measured when ticks were counted from the trap.

Statistical Analysis
The collection results were analyzed at 4 h intervals, and the collected ticks at each time period were compared using an analysis of variance (ANOVA).The mean of each group was used in this test.The relationships between the climatic variables were examined using Pearson's correlation coefficients.We used multiple linear regression to examine the relationships between climatic variables and the number of ticks collected.

Result
A total of 412 ticks were collected during the last period, including 53 adults and 376 nymphs.A total of 396 individuals were H. logicornis (48 adults and 365 nymphs) and 16 individuals were H. flava (five adults and 11 nymphs).From April to July, all ticks were H. longicornis.In September, 16 of the 64 ticks collected were H. flava and the remaining 48 were H. longicornis.
Adults and nymphs showed the highest activity between 10:00 and 14:00.At this time, the average temperature was 36.0 • C and the average relative humidity was 34.4% (Figure 4).The higher the temperature, the greater the tick activity.Tick activity was the lowest between 02:00 and 06:00, except for in June.The tick activity during this period was statistically significant compared to April, May, and September.Adults and nymphs showed the highest activity between 10:00 and 14:00.At this time, the average temperature was 36.0 °C and the average relative humidity was 34.4% (Figure 4).The higher the temperature, the greater the tick activity.Tick activity was the lowest between 02:00 and 06:00, except for in June.The tick activity during this period was statistically significant compared to April, May, and September.Adults and nymphs showed the highest activity between 10:00 and 14:00.At this time, the average temperature was 36.0 °C and the average relative humidity was 34.4% (Figure 4).The higher the temperature, the greater the tick activity.Tick activity was the lowest between 02:00 and 06:00, except for in June.The tick activity during this period was statistically significant compared to April, May, and September.The activity patterns of ticks highly correlated with temperature (r = 0.679, p < 0.05), light intensity (r = 0.572, p < 0.05), and relative humidity (r = −0.361,p < 0.05) (Table 3).

Discussion
In the ROK, the peak developmental stage is summer for adults, spring for nymphs, and fall for larvae [52,54].This study obtained consistent results, showing that adults were most frequently confirmed from June to July and nymphs from May to June.Additionally, 96.1% (396/412) of the identified ticks were H. logicornis, and a small amount of H. flava was collected only in September.These results are consistent with those of the other reports stating that the dominant species was H. longicornis [55,56].
H. longicornis is present in ten countries of the world, feeds on a variety of animal hosts, and is related to 30 human pathogens, including various species of Rickettsia, Anaplasma, Borrelia, Babesia, and viruses [17,29,34].The prediction model revealed that H. longicornis could potentially spread to more extensive regions, which is important for public health and veterinary care.Therefore, ecological research on H. longiconis in field environments is important [57].In this study, we confirmed the activity patterns of H. longicornis in the field.
The dial activity patterns of Ixodes ricinus, I. scapularis, I. persulcatus, and Amblyomma americanum were reported, which are vectors for Lyme disease [50,51,58].Additionally, Dermacentor variablis (which is a vector for Rocky Mountain spotted fever and tularemia) has been studied [49].In this study, H. longicornis in the ROK showed the highest activity between 10:00 and 14:00, which is similar to the activity patterns of I. persulcatus [58].These different diel activity times are influenced by species, environmental factors, the climate of the region, and microhabitats [59].
Our study shows that H. longicornis preferred temperature ranges between 18 and 35 • C, and the survivable temperature is known to be 12-40 • C [16,19].If temperatures exceed 40 • C, heat stress can cause death.When the temperature falls below 12 • C, hostseeking activities are slowed down or the ticks do not move [16].In this study, the temperature of the research period was investigated in a wider range, between 5.7 and 42.1 • C. The temperature between 10:00 and 14:00 (the most active period) was mainly 35-40 • C.This result is consistent with A. americanum, A. maculatum, D. andersoni, D. variablis, I. scapularis, and Rhipicephalus sangineus, which have activity patterns at temperatures higher than their preferred temperatures [60].A low activity was seen between 10:00 and 14:00 in June, because of the temperature exceeding 40 • C. Similarly, a low activity pattern appeared at temperatures below 12 • C during the nighttime in April and May.However, high activity was confirmed in June and July at 02:00-06:00, when the temperatures were approximately 20 • C.
Relative humidity is an important microclimatic factor for the survival and activity of H. longicornis, A. americanum, I. scapularis, I. ricinus, and D. reticulatus [49][50][51][61][62][63].However, there was a weak correlation between relative humidity and the number of ticks collected in this study.The critical equilibrium of relative humidity for H. longicornis is approximately 85% [57].When the humidity falls below this threshold, the tick continuously loses water [64], which has a negative effect on survival [65].In this study, the humidity at the time of the highest activity was approximately 50%, and it was judged that they showed considerable activity in replenishing soil moisture before the moisture evaporated.Therefore, it seems that high relative humidity did not have a significant effect on activity patterns.Additionally, water droplets formed on the collection trap because the humidity was close to 100% at night and dawn.Occasionally, the tick was locked in water droplets.It is thought that tick activity is inhibited even when it rains or humidity is very high.
Light intensity and temperature (R = 0.802) were highly correlated.However, light intensity and activity patterns showed a low correlation (R = 0.572).Climatic factors related to light are more strongly associated with diapause than with activity.In particular, the photoperiod has a greater influence than light intensity on activity [16,66].A long-day photoperiod triggers host-seeking behavior in H. longicornis in the laboratory [66].In this study, the overall tick activity pattern was the highest in June, when the day length was the longest.
Climatic stress decreases tick survival, but increases the rate of host-seeking activity [67].A variety of local field conditions can affect questing activity, including temperature and relative humidity [46,50], precipitation [68], time of day [69], seasonality [70,71], solar radiation [72], forest structure [73], and understory structure and invasive shrubs [74].Additionally, several intrinsic factors (such as geographic origin and local adaptation) may influence tick-questing activity [75,76], and infected pathogens boost tick mobility [77].For example, it is necessary to study the ecology of ticks through the analysis of various aspects as well as climatic factors.
Also, host preferences influence tick distribution, and the survival of tick populations depends on the presence of suitable hosts [78].H. longicornis is known to prefer large mammals (e.g., cattle, sheep, deer) over small ones [16].In ROK, H. longicornis was identified in medium-and large-sized mammals such as cattle, goats, wild boars, roe deer, raccoons, badgers, and migratory birds [79][80][81], but not in small mammals (rodents) [82].However, in the United State, H. longicornis have been found on medium-and large-sized mammals (coyotes, rabbits, raccoons, and deer), as well as small mammals (possums and marmots) [83].Tick distribution can be influenced by several factors, including local host populations and species [84].Although this study provided general guidelines for the highest activity of H. longicornis, climate changes may alter the activity of the species.The study is limited to a particular region.It is unknown if other environmental, climatic conditions and the effects of the host have a definite effect on their activity.
Ticks are associated with the transmission of various pathogens of medical and veterinary importance.They are considered one of the most important arthropod vectors after mosquitoes [85].Our study determined the period of major daily activity of H. longicornis.These results will help inform the public about the risk of exposure to ticks during daytime outdoor activities.It also provides basic information for the development of tick-borne disease vector control programs.

Figure 1 .
Figure 1.Map of the study site where the traps were installed.(A) The location of the site shown broadly and the white box is the actual survey location (image source by Korea Statistical Information Service).(B) Panoramic view of the study site.

Figure 1 .
Figure 1.Map of the study site where the traps were installed.(A) The location of the site shown broadly and the white box is the actual survey location (image source by Korea Statistical Information Service).(B) Panoramic view of the study site.

Figure 3 .
Figure 3.Total tick (adult + nymph) activity patterns by month (indicated by averaging the collected tick in each time period by month).

Figure 3 .
Figure 3.Total tick (adult + nymph) activity patterns by month (indicated by averaging the collected tick in each time period by month).

Figure 3 .
Figure 3.Total tick (adult + nymph) activity patterns by month (indicated by averaging the collected tick in each time period by month).

Figure 4 .
Figure 4. Tick activity patterns, temperature, and relative humidity by time period.Trap indexes with the same letter are not significantly different at the 5% level of probability (Duncan's multiple range test).RH = relative

Table 1 .
Number of Haemaphysalis longicornis sampled (mean ± S.D) during the daytime and nighttime at 5 months.

Table 2 .
Mean ± S.D. of the total tick (adult + nymph) and climate factors (temperature and relative humidity range).

Table 3 .
Pearson's correlation coefficients comparing three climate factors and the collected ticks.