Morphometric Study on Male Specimens of Hyalomma anatolicum (Acari: Ixodidae) in West of Iran.

BACKGROUND: Hyalomma anatolicum is the well-known hard tick, which is one of the most important livestock and human pathogens vector, wide range in host and distributed in all over the Hyalomma geographic fauna as well as in Iran. Taxonomy of the Hyalomma ssp. is debatable whereas their identification is a problematic work. The reasons for this claim is time consuming Delpy's researches in Iran also Schulze School, Feldman-Muhsam and the Russian tick workers. We would like to understand morphometric variation in the field collected H. anatolicum in Iran also validating some morphologic quantitative and qualitative characters.
METHODS: A total 247 field-collected tick specimens from different geographical regions in west of Iran includes Khuzestan and Lorestan Provinces were studied. The morphologic characters of the ticks were measured by the calibrated stereomicroscope armed scaled lens. The measurements were analyzed using SPSS for windows, version 16 on an IBM PC, so varied shapes of species in different geographic regions were drawn by the aid of a drawing tube connected to a light stereomicroscope.
RESULTS: One way ANOVA test revealed significant differences among the quantitative parameters in five zones (P < 0.001) also each zone to other zone by Post Hoc Tests e.g. LSD. No significant differences in the lateral grooves length/conscutum length ratio parameter were found.
CONCLUSION: Morphometric variation in Hyalomma spp is poorly studied. The variation in range and quantity of the morphometric parameters of H.anatolicum underlies that the correct recognition and key construction for Hyalomma species dependes on a complement morphometric study on the other species.

Introduction

The small Anatolian hard tick, Hyalomma anatolicum Koch, 1844 is a vector of the important pathogen agents, includes animals and human (Hoogstraal 1979, Burkot and Graves 2004). This species is the most abundant Hyalomma species in its geographical zone (Kaiser and Hoogstraal 1964). H. anatolicum formerly was includes of two subspecies anatolicum and excavatum, but recently reestablish as full species rank (Apanaskevich and Horak 2005). Identification of Hyalomma spp. is not simple (Pomerantzev 1950) and is very debatable because endless verity in faces which is due to exchange of environmental condition between different populations and genetic instability within their gene pool, hybridation and teratogenic abnormality (Delpy 1936b , Pervomaisky 1950, Hoogstraal 1956). Morphometric study of H. anatolicum in its geographical zone is not yet investigated, although some taxonomic characters on museum and field collected specimens was introduced but not measured (Mazlum 1968, Apanaskevich and Horak 2005). The medically and veterinary importance of H. anatolicum showed in Iran (Hashemi-Fesharki 1997, Telmadarraiy et al. 2010) and is widely distributed throughout in this country (Rahbari et al. 2008). Misidentification of H. anatolicum to closely related species e.g. Hyalomma asiaticum, H. excavatum, H. marginatum, generally the other Hyalomma species in Iran is a very common manner between tick workers whereas we have not an original and complete Hyalomma identification key for Iranian specimens. The traditional morphometric studies started by Delpy in Iran (Delpy 1936a, 1937a, 1937b), he believed the correct identification of Hyalomma species might be based on observation of the numerous specimens from any species.

The purpose of the present investigation was to identify valuable discriminating characters for male specimens of H. anatolicum also intraspecific variation study of the morphologic characters of this species in the five Iranian population in west of Iran based on Delpy’s conception (Delpy 1936a). The present study was designed to obtain information on morphometric variability on one of the most important Hyalomma species in the Iranian ixodid fauna. This study was performed on males of H. anatolicum since the morphologic characters of the females may be shallow and obscure when the tick engorged.

Materials and Methods

Material examined

A 247 field-collected tick specimens from different geographical regions in west of Iran includes Khuzestan and Lorestan Provinces were studied (Table 1). Ticks were examined for preliminary identification by several Hyalomma identification keys including Delpy 1936a, Pomerantzev 1950, Kaiser and Hoogstraal 1964, and other related keys. The morphologic characters of ticks were measured by the calibrated stereomicroscope armed scaled lens (Stemi SR-Zeiss Germany).

Table 1.

List of parameters used for characterization and zones of study

Quantitative Parameters

Basis capituli depression posteriodorsally

Cervical grooves length/conscutum length ratio

Conscutum length

Conscutum length/width ratio

Conscutum width

Lateral grooves length/conscutum length ratio

Parma length

Parma length/width ratio

Parma width

Qualitative Parameters

Arch

Caudal depression

Pigmented parma

Zones of Study

*I: Ramhormoz, Izeh, Baghmalek and Masjedsolyman

*II: Dashteazadegan, Shadegan and Shush

**III: Aleshtar and Taf

**IV: Kohdasht

**V: Mamolan

These zones are situated in Khuzestan Province

These zones are situated in Lorestan Province

The measurements were analyzed using SPSS for windows, version 16 on an IBM PC. Significances were measured at the 5% level. Afterwards, varied shapes of this species in different geographic regions were drawn by the aid of a drawing tube connected to a light stereomicroscope.

Description of used characters

The list of zones and characters for male specimens include nine quantitative and three qualitative parameters summarized in Table 1.

Basis capituli depression posterodorsally

The cornua are paired projections from the outer margins of the posterior dorsal surface of the basis capituli. The depression of between cornua is characteristic for identification.

Cervical grooves

These are paired depressions near the central anterior part of the conscutum; they go down from the raised intercervical field into the cervical fields (also known as mesial grooves).

Conscutum: The sclerotized (=hardened) plate which covers most of the dorsal surface of male ixodid ticks (so called scutum in female).

Lateral grooves

In the conscutum of males a groove may be present in the lateral area, starting most clearly near the position of the spiracles and possibly extending forward to the eyes (in some literature these are called marginal grooves or lines).

Parma: Festoons are a regular series of bulges in the posterior margin of many adult ixodid ticks. The central festoon may also be called the parma when it is developed as a distinct structure, separated from the surrounding festoons by grooves.

Arch: In Hyalomma males the pair of festoons next to the central one is paracentral and may be joined anteriorly to form an arch shape.

Caudal depression

In Hyalomma males the posterior of the conscutum may be broadly convex (caudal depression is absent) or it may be broadly concave (caudal depression is present).

Pigmented parma

The parma may be pale colored (unpigmented), or dark colored (pigmented).

Results

The result of relevant to the quantitative parameter is summarized in Table 2 to Table 4. One way ANOVA test revealed significant differences among the quantitative parameters in five zones (P< 0.001) also any zone to other zone by Post Hoc Tests eg LSD. No significant differences in the lateral grooves length/conscutum length ratio parameter were found (Fig. 4). Variation in some parameters (characters) is illustrated in Fig. 1, Fig. 2 and Fig. 3. The observation on qualitative parameters revealed the presence of three characters in under studying specimens which is summarized in Table 5.

Table 2.

Conscutum parameters of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces

Parameter	Zone	No	Size (mm)	*LSD
			Average	SD	Max	Min
Conscutum length	I	51	2.97	0.26	3.53	2.43	III,IV,V
	II	62	3.03	0.27	3.79	2.29	III,V
	III	45	3.48	0.31	4.04	2.78	I,II,IV,V
	IV	39	3.13	0.27	3.65	2.62	I,III
	V	50	3.14	0.24	3.61	2.52	I,II,III
Conscutum width	I	51	1.83	0.15	2.19	1.40	III
	II	62	1.87	0.18	2.29	1.46	III
	III	45	2.27	0.25	2.83	1.77	I,II,IV,V
	IV	39	1.88	0.15	2.21	1.44	III
	V	50	1.88	0.14	2.23	1.48	III
Conscutum length/width ratio	I	51	1.63	0.08	1.82	1.42	III,V
	II	62	1.63	0.08	1.85	1.44	III,IV,V
	III	45	1.55	0.07	1.69	1.40	I,II,IV,V
	IV	39	1.66	0.09	1.96	1.49	II,III
	V	50	1.67	0.07	1.82	1.39	I,II,III

Least Significance difference, P value in all parameters is similar (P< 0.001)

Table 4.

Other quantitative parameters of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces

Parameter	Zone	No	Size (mm)				LSD
			Average	SD	Max	Min
*Basis capituli depression	I	51	0.02	0.01	0.06	0.00	III
	II	62	0.03	0.01	0.08	0.00	IV,V
	III	45	0.03	0.01	0.06	0.02	I,IV,V
	IV	38	0.02	0.00	0.04	0.00	II,III
	V	50	0.02	0.00	0.06	0.00	II,III
**cervical grooves length/conscutum length ratio	I	49	0.33	0.06	0.51	0.25	III
	II	62	0.32	0.05	0.45	0.21	III
	III	45	0.42	0.10	0.80	0.26	I,II,IV,V
	IV	39	0.31	0.04	0.45	0.23	III,V
	V	50	0.34	0.07	0.57	0.23	III,IV

containing missing data in zone IV

containing missing data in zone I

P value in all parameters is similar (P< 0.001)

Fig. 4.

Graph illustrating variation in lateral grooves length/conscutum length ratio parameter of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces

Fig. 1.

Variation in cervical grooves length/conscutum length ratio parameter of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces (Original figure).

Fig. 2.

Variation in conscutum length parameter of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces (Original figure).

Fig. 3.

Variation in basis capituli depression parameter of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces (Original figure).

Table 5.

Qualitative parameters of H. anatolicum in two groups of specimen (with and without qualitative parameter)

[*]Group	Qualitative parameters
	Arch (n)	Pigmented parma (n)	Caudal depression (n)
I	21	30	57
II	225	216	188

I: specimens with qualitative parameter

II: specimens without qualitative parameter

containing missing data in group I and II in all qualitative parameters

Discussion

Morphometric analysis in Hyalomma spp. is poorly studied perhaps because morphologic characters in some species are obscure and unconfined. Delpy (1936a), after studying the genus Hyalomma, came to conclusion that diagnosis of Hyalomma species was almost impossible and suggested that the range of variations among laboratory bred offspring of individual females must be studied, for instance he reported high spectrum of qualitative variation in the adanal shields of Hyalomma dromedarii. Hoogstraal (1956) stated that the variation in the morphologic characters of Hyalomma species caused to nomination and presentation of the numerous Hyalomma species (about eighty species) by Schulze and his collaborators.

Delpy (1936a, 1937a) described all parasitic stages of the camel tick, H. dromedarii, he measured the length and width of the scutum, basis capituli, II and III palpi segments, spiracular plate and the many other characters in addition some of the comparative parameters. Hyalomma schulzei, (Olenev, 1931) the robust and rare Iranian Halomma species, also studied by Delpy (1937b), the measured characters include corps length and width, corps length to capituli length ratio and IV genua length ratio and the other characters (Delpy 1937b). Mazlum (1978) revealed the taxonomic status and presence of H asiaticum in the Iranian ixodid fauna and focused on scutum length as a taxonomic discriminating character for four closely related Hyalomma. Filippova and Musatov (1996) studied morphologic variability in eight population of prostriate tick Ixodes persulcatus (Schulze, 1930) to distance 8700 and 2900 km in the Western-Eastern and Northern-Southern populations, respectively, they have shown morphometric variation in the anal ring and gnathosoma length and width, hypostome, II and III palpi also I tarsi length.

Scutum size (body length in many literature) in the genus Hyalomma is a good interspecific discriminating character but no for closely related species as Apanaskevich and Horak (2005) reported 1.48–2 mm and 1.64–2.05 mm in conscutum size range of males of two similar and debatable species H. anatolicum and H. excavatum respectively. The statistical significance of size in H. anatolicum immature stages were studied by Arthur and Snow (1966), they reported a non-linear relationship between the length of the egg, and the emergent larva.

The parma (central festoon) is a suitable discriminating character for interspecific segregating of H. marginatum complex (Pomerantzev 1950) as parma is not seen in the representative specimens. Mazlum (1978) reported which is parma as a multishape character for identification of excavatum and asiaticum groups and showed oval, oval, subtriangular and subrectangular shapes in H. anatolicum, H. excavatum, H. asiaticum and H. dromedarii, respectively. Usually color of parma is dark in H. marginatum complex while sometime is pale and may be cause to misidentification of this species with H. anatolicum. Adler and Feldman-Muhsam (1948) used from pigmented and unpigmented parma phrase in the Palestin Hyalomma identification key, they believed the ticks of H. marginatum complex might be separated from the other species using this character (Adler and Feldman-Muhsam 1948). Pomerantzev (1950) remarked the basis capituli depression posteriodorsally as specific character for discriminating of H. asiaticum from H. anatolicum however stated more concave and slightly concave modal phrases for two mentioned species, respectively (Pomerantzev 1950). Kaiser and Hoogstraal (1964) used from depth of the basis capituli depression in addition to be angular in H. asiaticum and H. anatolicum, respectively. In the present study we observe several individuals from very deep to less deep range in H. anatolicum (0 to 0.08 mm), also its angularity were seen by our observation. Abdigoudarzi (2003) stated that the basis capituli depression posteriodorsally is a specific feature for differentiating of H. anatolicum from H. asiaticum specimens in the Iranian Hyalomma fauna (Abdigoudarzi 2003). The arch is a qualitative character which is debatable for identification of two closely related species H. anatolicum and H. excavatum as the former species this character not seen as connection of pair festoons 2 anteriorly and usually may be seen as wart like projection in the position of posteriodorsal groove to parma connection (Hoogstraal and Kaiser 1959b), or a weak junction similar to arch feature (Abdigoudarzi 2003) nevertheless in the future species the semicircle arch may be seen obviously. Adler and Feldman-Muhsam (1948) believed the arch is very important trait and may be causes to misidentification of the closely related Hyalomma species.

The lateral groove length is a reliable character for interspecific identification of H. anatolicum as the value of this parameter in our study was not significant statistically. This means which the amplitude of variation in the mentioned character is limited. Our object from the studying of this important character was to obtain numerical value for its which is mentioned as qualitative character in many Hyalomma identification keys (Delpy 1936a, Hoogstraal 1956, Kaiser and Hoogstraal 1964, Apanaskevich and Horak 2005) for instance as lateral grooves not extending beyond the posterior third of the scutum (Hoogstraal 1956) or lateral grooves short, not reaching central third of scutum (Kaiser and Hoogstraal 1964), which are the vague definitions. Hoogstraal (1956) believed the lateral grooves length is an important character for discriminating of the genus Hyalomma, but Pomerantzev (1950) and Adler and Feldman-Muhsam (1948) was not mentioned this character in their Hyalomma identification keys. Abdigoudarzi (2003) after morphologic observation on the Iranian Hyalomma specimens reported that the lateral groove is a valuable character in some Hyalomma species and emphasis on the lateral grooves based on observation and description, no through statistics, therefore the present study may be complementary work.

The cervical grooves alike the lateral grooves which has never been measured, and described as cervical grooves short, not reaching mid-scutum (Kaiser and Hoogstraal 1964) or cervical grooves are seen as slight depression (Pomerantzev 1950) which are the vague descriptive statements. This study declare the length of the cervical grooves quantitatively, however the length of this character in under studied specimens of H. anatolicum was significance statistically, which means the trait from the cervical grooves which is important for discriminating of H. anatolicum from H. asiaticum may be its depth instead length of this character. Abdigoudarzi (2003) accepted our result on validity of the cervical grooves but stated that it may be a less useful and unstable character in him observed materials (Abdigoudarzi 2003).

Kaiser and Hoogstraal (1964) emphasize on elongation and are more depth of the cervical grooves of H. asiaticum from H. anatolicum while, Pomerantzev (1950) no stated about length of cervical grooves whereas considered the depth of this character as valuable taxonomic feature. Feldman-Muhsam (1962) shown that the cervical grooves are deep, wide and long in H. anatolicum of the Koch’s type collection. In the present study, we observed number of specimens of H. anatolicum with the deeper and longer cervical grooves. Identification of the Hyalomma species not is possible unless to observation of many specimens from species and measure of the taxonomic and morphologic traits, features or characters and data analyzed via morphometric and statistics methods. The variation in range and quantity of the morphometric parameters of H. anatolicum underlying that the correct identification and key construction for Hyalomma species depended on a complement morphometric study on the other species especially closely related species.

Table 3.

Parma parameters of H. anatolicum collected from different zones of Khuzestan and Lorestan Provinces

Parameter	Zone	No	Size (mm)				LSD
			Average	SD	Max	Min
*Parma length	I	51	0.15	0.03	0.21	0.10	III
	II	62	0.14	0.03	0.27	0.06	III,IV
	III	44	0.19	0.05	0.31	0.10	I,II,IV,V
	IV	39	0.16	0.02	0.21	0.12	II,III
	V	50	0.15	0.02	0.19	0.10	III
*Parma width	I	51	0.90	0.84	2.17	0.10	III
	II	62	0.17	0.04	0.27	0.10	III,IV
	III	44	0.21	0.05	0.39	0.12	I,II,IV,V
	IV	39	0.20	0.03	0.29	0.16	II,III
	V	50	0.19	0.03	0.29	0.12	III
*Parma length/width ratio	I	51	0.50	0.40	1.60	0.06	II,III,IV,V
	II	62	0.83	0.20	1.29	0.55	I,III
	III	44	0.95	0.28	1.67	0.50	I,II,IV,V
	IV	39	0.81	0.17	1.25	0.46	I,III
	V	50	0.78	0.20	1.33	0.46	I,III

Containing missing data in zone III

P value in all parameters is similar (P < 0.001)