Abstract: In this study, Ips sexdentatus (Coleoptera: Curculionidae, Scolytinae) populations from Kastamonu (Turkey) were investigated for the occurrence of entomopathogens. Two species were encountered in the duration of this study and these were Gregarina typographi (Apicomplexa, Gregarinidae) and Metschnikowia typographi (Ascomycota, Metschnikowiaceae). Within the I. sexdentatus samples used in this study total infection rate of the pathogens G. typographi and M. typographi was calculated as 28.9%. Measurements of the Metschnikowia species identified from I. sexdentatus were as follows: mean length 16.86±2.2 μm and mean width 2.8±0.3 μm (n = 125). The ascospores of this pathogen were characteristically needle-shaped and therefore, easily distinguishable under the light microscope. The occurrence rate for the ascomycete pathogen (M. typographi) was 3.1%. The gregarine pathogen measured 108.1-203.5 μm and was identified as Gregarina typographi. The occurrence rate for the Gregarine pathogen was 25.8%. The first report for Turkey (Kastamonu) of an ascomycete fungus, Meschnikowia typographi from the pine bark beetle Ips sexdentatus (Boerner) is given in this study.

INTRODUCTION

The pine bark beetle Ips sexdentatus (Boerner) causes serious economic losses in spruce and pine forests in Europe and Turkey (Yuksel, 1998; Yuksel et al., 1999; Wegensteiner et al., 2005). Pheromone traps, mechanical and chemical control strategies have been used to control this pest for a long time in Turkey (Yuksel, 1998). Despite all the attempts, this pest still causes serious economic losses in oriental spruce and pine forests in various parts of Turkey including Kastamonu. Moreover, chemicals have a detrimental effect on the predators and parasites of these bark beetles and on the ecosystem. In an attempt to destroy the bark beetles, its predators and parasites are killed by chemicals conveniently enabling Ips sp. to thrive. On the other hand, natural enemies of Ips sp. are very interesting and certainly have advantages over chemicals as control agents. Although, there are several studies on the parasites and pathogens of Ips sp. from different parts of the world (Fuchs, 1915; Wegensteiner and Weiser, 1995; Wegensteiner et al., 1996; Weiser et al., 1998; Weiser et al., 2003; Wegensteiner and Weiser, 2004; Wegensteiner et al., 2005; Weiser et al., 2006), there are only two studies on I. sexdentatus in Turkey (Yaman, 2007; Yaman et al., 2008). Therefore, there is very little information on parasite and pathogen spectra of Ips sp. occurring in various parts of Turkey. The present study is significant because it reports on the occurrence of G. typographi and M. typographi in I. sexdentatus from Kastamonu region (Turkey) for the first time.

MATERIALS AND METHODS

In 2007, adults of I. sexdentatus were collected from pine forests from three localities in the Kastamonu district of Turkey, i.e., Saraycik, Ahlatcik and Subasi. During the collection, the specimens were randomly collected, without discriminating between male and female or mature adult and callow adult. Each beetle was dissected in a physiological solution (0.8% NaCl) and wet smears were examined under a light microscope at a magnification of 40-1000x. Several life stages of the detected pathogens were measured and photographed. Positive smears were fixed with methanol and stained with Giemsa’s dye according to Wegensteiner et al. (1996). Pathogens and parasites found throughout the period of observation were measured and recorded. Infection was also confirmed under TEM microscope (Yaman and Radek, 2005).

RESULTS AND DISCUSSION

During this study, two pathogens were observed in the population of Ips sexdentatus collected from Kastamonu. The pathogens observed are species of genera Metschnikowia and Gregarina. M. typographi belonging to the order Ascomycota is a spore forming scrounging yeast infecting the mid-gut lumen and surrounding cells. Asci of the fungus are also found free in the hemolymph in large numbers. G. typographi is a typical member of Apicomplexa, Gregarinidae, its gamonts are found in the mid-gut lumen and its trophozoites bind to the mid-gut epithelium.

The morphological features of the observed fungus include navicular asci having two needle-shaped ascospores attenuated at both ends and vegetative forms resembling typical round to tubular fungal cells. Measurements of asci are reported as mean length 17.6±2.4 μm and width 2.8±0.3 μm (11.5-24.1x1.4-4 μm) (n = 182) and mean sizes of ascospores are 16.88±1.4 μm in length and 1.54±0.3 μm (13.7-19.8x1.06x2.13) in width. This pathogenic fungus which forms two ascospores per ascus was observed with a light microscope (Fig. 1a). Of the 426 beetles examined, 13 were infected by the fungus M. typographi. Total pathogen infection rate of I. sexdentatus is 28.9%. Total rate of Metschnikowia infection is 2.81%. The rate of ascomycete infection from Saraycik is 3.5% and the infection rate from Subasi is 2.9%. We did not determine any Metschnikowia pathogen from Ahlatcik.

In this study, we also observed a gregarine pathogen from these beetles and observed several life stages of this pathogen, such as trophozoite with epimerite, gamont and cyst (Fig. 2) in anterior part of the intestinal lumen of the bark beetles. The measurements of gamonts of the observed gregarine parasite are 108-203 μm (Table 1). A 110 of the examined beetles were infected with G. typographi. Total gregarine infection rate is 25.8%. Gregarine infection rate is 33.3% at Ahlatcilik 31.7% at Saraycik and 16.6% at Subasi.

The pathogens observed in the present study were known species in Ips sp. And they were identified as Metschnikowia typographi and Gregarina typographi according to the study (Weiser et al., 2003; Wegensteiner et al., 2005; Yaman, 2007; Yaman and Radek, 2008).

Previously, Weiser et al. (2003) isolated and identified M. typographi from the bark beetles Ips typographus and Ips amitinus (Coleoptera: Curculionidae, Scolytinae). Wegensteiner et al. (2005) also found M. typographi from I. sexdentatus in Austria.

Morphological features obtained from the electron and light microscopical (Fig. 1a and b) observations of the determined fungus pathogen suggest that it is Meschnikowia typographi. Yaman and Radek (2008) described an ascomycete from Dendroctonus micans (Kugelann) from Turkey and identified it as Metschnikowia sp. and it measured 18.48±2.05 μm (14.7-22.3) in length and 2.1±0.4 μm in width. Weiser et al. (2003) measured asci of M. typographi as 13-17x1.5-2.5 μm from I. typographus and 17-22x2-2.5 μm from I. amitinus. Additionally I. typographus and I. sexdentantus are from the same beetle family (Coleoptera: Curculionidae) and they share the same terrestrial habitat therefore they also share pathogens. I. sexdentatus and D. micans are also pests that share the same habitat in Turkey.

Table 1:	The measurements of gregarine parasite from Ips sexdentatus
TL: Total Length; LP: Length of Protomerite; LD: Length of Deutomerite; WP: Width of Protomerite; WD: Width of Deutomerite; LP:TL: Ratio of the length of protomerite to total length; WP:WD: Ratio of the width of protomerite to the width of deutomerite

Fig. 1:	a) Differential interference contrast micrographs of M. typographi from I. sexdentatus; asci (A) and ascospores (a). Bar: 10 μm, b) Thin section of M. typographi showing the early stage in the formation of a bud. Bar: 500 nm

Table 2:	Comparison of measurements of Gregarina typographi from different studies
a: Max.-Min.: TL: Total Length; LP:TL: Ratio of the Length Of protomerite to Total Length; WP:WD: Ratio of the width of protomerite to the width of deutomerit

Fig. 2:	Life stages of Gregarina typographi: A-trophozoite, B-gamont, C-associative form, D- two gamonts in the process of rotation previous to cyst formation E-cyst still in rotation with thin cyst wall. (E: Epimerite; P: Protomerit; D: Deutomerite; Pr: Primate; St: Satellite). Bar: 50 μm

Although, I. sexdentatus and D. micans cannot be found in the same habitat elsewhere, in Turkey these bark beetles infest the same spruce stands together. Therefore, it would not be surprising to find the same pathogens in these bark beetles. The spores of Metschnikowia sp observed in I. sexdentatus in the present study and in D. micans by Yaman and Radek (2008) have the same physiological and morphological characters and ultrastructural studies prove that the Metschnikowia sp. determined from I. sexdentatus certainly is Metschnikowia typograpi.

According to these facts, Metschnikowia sp. has two different hosts which are D. micans and I. sexdentatus in Turkey. Besides, the occurrence and ultrastructure of M. typographi isolated from I. sexdentatus is reported for the first time from Turkey in the present study.

The rate of ascomycete infection from Saraycik with 3.5% is higher than the infection rate from Subasi with 2.9%. We did not determine any Metschnikowia pathogen from Ahlatcik. Wegensteiner et al. (2005) reported that the rates of M. typographi infection from I. sexdentatus varied between 0.9-51.4%. Weiser et al. (2003) reported that the rate of M. typographi infection was 1.8% from I. sexdentatus and 5.9% from I. amitinus.

On the other hand Yaman and Radek (2008) found Metschnikowia infection up to 50% in Dendroctonus micans populations at Black sea region of Turkey. Observed infection level in this study is not as high as that recorded from I. sexdentatus by Wegensteiner et al. (2005) and D. micans by Yaman and Radek (2008). It is similar with that recorded from I. sexdentatus and I. amitinus by Weiser et al. (2003).

There are several reports in the literature about the occurrence of G. typographi from two Ips species (I. sexdentatus and I. typographus) which are most economically important in Turkey. These studies include Purrini (1978) and Wegensteiner et al. (1996)’s report on G. typographi from I. typographus (L), Theodorides (1960)’s report on the same pathogen from I. sexdentatus, Takov et al. (2007) report from I. sexdentatus in Bulgaria, Kereselidze and Wegensteiner (2007)’s report from I. typographus in Georgia and Yaman (2007)’s report from I. sexdentatus in Turkey. G. typographi was also reported from Bulgaria and Georgia therefore, this shows that G. typographi is also present in adjacent countries indicating the distribution of this pathogen.

The measurements of gamonts of the observed gregarine parasite are similar with G. typographi reported by Yaman (2007) in Turkey measuring 80-275 μm. Morphological features and characters show that this gregarine pathogen is Gregarina typographi. The measurements of G. typographi are compared with the results of Lipa (1967), Takov et al. (2007) and Yaman (2007) in Table 2.

Rate of gregarine pathogen infection from Saraycik and Ahlatcik are higher than Subasi. Wegensteiner et al. (2005) reported that the rates of G. typographi infection from I. sexdentatus varied between 16.7-45.5%. Yaman (2007) found 16.1% gregarine infection in I. sexdentatus from Trabzon. Presented gregarine infection rate from Kastamonu is higher than the rate reported by Yaman (2007) but lower than the rate recorded by Wegensteiner et al. (2005).

CONCLUSION

Till now, one pathogen, G. typographi has been recorded from I. sexdentatus in Trabzon (Turkey). In this study, additional to this pathogen, M. typographi is reported in I. sexdentatus from Turkey for the first time confirming G. typographi in I. sexdentatus populations in Kastamonu.

ACKNOWLEDGEMENTS

This study was supported by The Research Foundation of Karadeniz Technical University (Project num.: 2007.111.004.4).