Astheno-association: hard tick species new association type identied during the summer season 2019 on cattle in Jijel area, Algeria

To this date, ticks remain the major problem in the livestock sector because of their role as vectors for serious agents of disease propagation. Given the scarcity of studies on ticks in Algeria, associations of species were mentioned in only one document and were not well detailed in another. This paper is a synoptic review of seven tick species associations related to host biotic factors in Algeria. The aim is to better understand the behaviour and biology of ticks to propose a new control method based on the association index. A total of 53 infested cattle were detected and 1,214 ticks were manually collected and identied based on their morphology during the summer of 2019 (June to August). Seven tick species were identied: viz. Rhipicephalus (Boophilus) annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus, Hyalomma marginatum, Hyalomma lusitanicum, Hyalomma scupense, and Hyalomma anatolicum. The Principal Component Analysis (PCA) was used as an analytical tool to visualize similarities and correlations between cattle and females, males of tick species, according to three biotic factors (age, sex and breed of cattle). The results show that there is a relationship between the number of associated species and biotic factors (age of livestock). In addition, the degree of association is inuenced by the host and other tick species and a new type of "astheno-association" is emerging that limits the number of associated species to two or three.


Introduction
Ticks are veterinary and medically important arthropods because of their vectorial capacity. They are known as vectors for human and animal pathogens (protozoa, bacteria, viruses) and transmit a wider variety of infectious organisms (Nicholson et al., 2009). Host preferences and tick distribution are both important in understanding the ecology and epidemiology of tick-borne diseases (Mihalca et al., 2012). Given the paucity of tick studies in Algeria (Yous -Monod and Aeschlimann, 1986;Boulkaboul, 2003;Elfegoun et al., 2007;2013;2018;2019), only two studies are available in Algeria on tick species association. In addition, the only comprehensive study on tick associations in Algeria was published more than 34 years ago.
The rst study was carried out in 1986 by Yous -Monod and Aeschlimann. The authors found that the association degree vary between two to four species in the twelve species identi ed in 2086 cattle during two years (1981 to 1983). Furthermore, they identi ed two types of associations under abiotic factors, both types being related to the breeding methods and the geographical origin of the cattle. They reported that competition-exclusion was not observed. The same authors recommended statistical analysis of species associations, but no studies have been conducted so far.
The second study, carried out by Boulkaboul (2003), only underlined that "several species can parasitize the same animal. The minimum association of two species was observed in January and the maximum association ( ve species) in June. This of course conditions the parasitic load of an animal at a given time". In addition, most of the data on species associations are rather rare and old and are only available in Japan (Takashi 2007), thus not easily accessible to researchers for comparative studies. In addition, similar synoptic papers have not been published recently.
This study aims to address the following questions: (1) Which biotic factor is primarily chosen by ticks? (2) Is the degree of association in uenced by the host or by other tick species? (3) Where are associations of less than four species most frequent? (4) Is there or is there not a new type of association based on biotic factors? PCR analysis was used to test for similarities and correlation between species with dissimilar host biotic factors.

Study area
This study was carried out in the province of Jijel, in northeastern Algeria ( Fig. 1), in June, July and August 2019. Jijel is located between 36°48′N and 5°46′E, covers 2,577 km 2 . The climate is warm temperate with a typical hot and relatively dry season between June and August and a wet season from November to April dominate the region. Annual rainfall varies and the monthly temperature is between 800 and 900 mm/year, or 11.3 and 26 °C respectively. The hottest and driest months in Jijel are August and July (Source: climate-data.org).
An extensive system is used for cattle breeding in the study area. Cattle are kept on pasture 24 hours a day and cows are only brought in for calving. Farmers apply anti-tick control acaricides randomly and irregularly. For this study, ticks were manually removed from the skin surface of infested cattle; 1,214 ticks were collected and stored in vials containing 70% ethanol. Data on the age, breed and sex of the cattle were meticulously recorded, and the ticks were then identi ed in the laboratory of biosystematics and ecology of arthropods under a stereomicroscope according to the key of Walker et al. (2003).

Statistical analysis
All data was exported to an Excel® spreadsheet for Windows®. Principal Component Analysis (PCA) was performed using PAST® for Window®. It was used to visualize relationships between variables (female and male tick species) and similarities between observations (age, sex, and breed of cattle), understand the relationship between variables when analysing many variables, discover patterns and reduce a large amount of data to a subset of interesting relationships, and describe the strength of a relationship between two variables. For the PCA, 14 variables (seven species; females, males) were considered, to assess correlations between tick species and biotic factors (cattle age, breed, and sex), while comparisons of infestation rate (prevalence and intensity) and association degree were made using the ANOVA test. All statistical tests performed were considered at a 5% cut-off value (Schwartz, 1993).
Cattle were scored on 53 factors. The scores of each cattle were combined into 3 biotic factors, using the seven species (females and males) as factors, then the scores of each factor were combined into an overall score and ranking for each cattle to create the scatter plot and correlation matrix. Intensity varied from 1 tick on female Breton Black Pied cattle under 5 years of age to 124 ticks on female Breton Black Pied cattle under 6 years of age with an average of 22 ticks/cattle (Table 1 and table 3). The most or least infested cattle (intensity > 40 ticks) belong to Breton Black Pied cattle (8/53) and two Red Pied Lowland (2/53). The least infested cattle is the Breton Black Pied (intensity = 1 tick).

Similarities between cattle
There is a signi cant relationship between the number of ticks of each species and the number of species associations (p = 7.78E-20). There is no statistically signi cant difference in the prevalence and intensity of infestation according to the degree of species association (p > 0.05) ( Table 5).
Figures 2 show the two-dimensional approximation of the initial multidimensional space, which represents 70% of the initial variation in the data; each point in the plot represents a cattle and each axis represents a tick species, the distance between cattle represents the similarity between them; close points are cattle with similar pro les and distant points have dissimilar pro les. Any point in the plot can be projected orthogonally on the axes to show the approximate value of this variable.

Discussion
This study was carried out in June, July and August 2019 in the region of Jijel (humid climate) to consider the seasonal activity of the main vector tick species in north-eastern Algeria. 1,139 ticks were collected from 53 examined cattle. Seven species of ixodid ticks were collected from cattle, namely Rhipicephalus (Boophilus) annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus, Hyalomma marginatum, Hyalomma scupense, Hyalomma lusitanicum, and Hyalomma anatolicum.
In the Jijel region of Algeria, male and female hard tick species are associated, and these associations vary widely depending on biotic factors, including the sex, breed and age of the cattle.
This study aims to address the following questions: (1) Which biotic factor is primarily chosen by ticks? : The major factor that affect the infestation rate is primordialy cattle sex. (2) Is the degree of association in uenced by the host or by other tick species?: the degree of associations affect greatly the infestation rate, since these rate decreased when association degree increased (3) Where are associations of less than four species most frequent?: it is frequent in cattle age cattle age between ve and seven years categories (4) Is there or is there not a new type of association based on biotic factors? Yes there is a new type of association was observed during the study and we chose to refer to it as astheno-association (from the Greek asthen (ēś) "frail, weak, sickly"). This new type is clearly de ned as "an exposure of 2 or 3 species depending on the biotic factors of the host, without exclusion or competition". Yous -Monod and Aeschlimann (1986) described two types of associations, the mode of reproduction and the geographical origin of the livestock. The same authors observed associations of two to four species, which differs from our results.
In this study, negative correlations were found between females and males of some species. The latter nding is evident since Hyalomma males are more mobile than females and stay longer on livestock. This result was also observed in dogs infested with the brown tick R. sanguineus (Little et al., 2007). Micrometeorological and microhabitat factors on livestock are fundamental factors in tick survival and foraging behaviour (Daniel and Dusbabek, 1994).
These factors are dispersed for ticks, and the age of the livestock largely affects the number of females and males. The same observations were made with regard to sex and breed. Arthur (1962) reported that the degree of tick aggregation is highest in larvae but lowest in adults. A similar nding was made, with the number of infested cattle decreasing when the species associations changed to six species. In addition, spatial niche overlap was greater than temporal niche overlap in some tick species and there were interactions between tick species when at least two tick species occurred in the same season (Tsunoda, 2007). Host biotic factors determine the number of species due to coexistence, as has been observed with Rhipicephalus (Boophilus) annulatus and Rhipicephalus bursa. In addition, it can be advantageous to cluster in an optimal locality, as this can increase the chances of success (Tsunoda, 2007). The larger group offers a greater advantage to ticks even if the group includes other species (Tsunoda, 2007), which may explain why the association of more than three species is observed during the study period.

Conclusion
This paper aimed to investigate the behaviour and biology of ticks on cattle in Jijel area, in northeastern Algeria, to propose a new control method based on the association index. A total of 53 infested cattle were detected and 1,136 ticks were manually collected and identi ed on the basis of their morphology during the summer of 2019 (June, July and August). Seven species of ticks were identi ed: Rhipicephalus (Boophilus) annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus, Hyalomma marginatum, Hyalomma lusitanicum, Hyalomma scupense and Hyalomma anatolicum. Principal Component Analysis (PCA) was used as an analytical tool to visualize the similarities and correlations between cattle and female, male ticks species, according to three biotic factors (age, sex and breed of cattle). Based on the results, we can conclude that there is a relationship between the number of associated species and biotic factors. The association degree is in uenced by the host and other tick species, and the appearance of a new type of "astheno-association" is limited to two or three associated species. Elfegoun MCB, Gharbi M, Merzekani Z, Kohil K. 2018     Correlation bi-plot for Principal component analysis of females and males hard tick species on cattle according to cattle age, breed, and sex during summer 2019 in Jijel province, Algeria.