NORTH-WESTERN JOURNAL OF ZOOLOGY 8 (2): 353-357 ©NwjZ, Oradea, Romania, 2012 Article No.: 121203 http://biozoojournals.3x.ro/nwjz/index.html

A new neogregarine pathogen of grandis (Coleoptera: )

Mustafa YAMAN1*, Renate RADEK2 and Andreas LINDE3

1. Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey. 2. Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Str. 1-3, 14195 Berlin, Germany. 3. University of Applied Sciences Eberswalde, Applied Ecology and Zoology, Alfred-Möller Str. 1, 16225 Eberswalde, Germany. *Corresponding author, e-mail address: [email protected] (M. Yaman)

Received: 09. January 2012 / Accepted: 24. February 2012 / Available online: 27. May 2012 / Printed: December 2012

Abstract. Here we provide the first description of a natural infection of members of the family Monotomidae with neogregarines and specifically the first finding of such a pathogen in the predatory beetle . The fat body of the beetle is the site of infection, and the typical navicular oocysts are 11.87 ± 0.67 μm in length and 6.96 ± 0.43 μm in width (n = 60). Polar plugs are recognisable using light and electron microscopy. The oocyst wall is quite thick, measuring 400 to 500 nm. Oocysts are formed pairwise within a gamontocyst, and each oocyst has eight sporozoites. The described neogregarine pathogen in R. grandis has the typical characteristics of members of the (family ) within the order Neogregarinida.

Keywords: Bark , Biological control, micans, Neogregarine, Mattesia, Rhizophagus grandis.

The great tion in R. grandis. A new neogregarine pathogen of (Kugelann) (Coleoptera: Scolytinae) causes severe R. grandis is reported in this study. damage to spruce stands in the Black Sea area and After Yaman’s & Radek’s (2008) observations on the the Caucasus, resulting in significant economical neogregarine record in D. micans in 2005 and 2006, 226 losses. All efforts and resources dedicated to con- male and 301 female specimens of R. grandis were trolling this dangerous pest have been inadequate; obtained from the R. grandis rearing laboratory in Giresun it is still causing epidemics in the eastern Black Sea in July 2007. Samples from other laboratories could not be region of Turkey. The development of more effi- obtained in the same time period, because each laboratory cient, environmentally safe and sustainable meth- starts the mass-rearing procedures at different times of the year. However, all laboratories have the same specific ods for controlling this pest has thus become a conditions. Each was dissected in insect Ringer’s priority and necessity. As a result, studies in solution, and wet smears were prepared and examined search of means of biological control of this pest for presence of neogregarine cysts under a light were conducted. The predatory beetle Rhizophagus microscope at a magnification of 400–1000x. When an grandis is a proficient natural suppressing factor of infection was found, neogregarine oocysts were fixed D. micans. This very efficient and voracious hunter with methanol and dyed with Giemsa solution, is mass-reared using D. micans larvae as food. Un- measured, and then photographed using an Olympus BX51 microscope equipped with a DP-25 digital camera fortunately, the conditions during cultivation in and a DP2-BSW Soft Imaging System. The cysts were also the bark have been shown to allow a transmission studied with SEM and TEM microscopes, using a of pathogens from prey to predator (Yaman & previously reported technique (Yaman & Radek 2005, Radek 2007, Yaman 2008), which is an undesirable Yaman et al. 2008, 2010). The results were statistically situation in that R. grandis is the most important analysed using SPSS 11.0. factor in controlling D. micans populations. Patho- Only oocyst stages of the pathogen could be gens infecting the predator would certainly de- observed in adult R. grandis. Form and size of the crease the efficiency of the beetle as a biocontrol oocysts were very uniform, navicular in shape and agent. with plugs at the two poles (Figs. 1, 2, 3). Further- Yaman & Radek (2005) reported a pathogen more, oocyst size did not vary significantly (Fig. identified as Helicosporidium sp. in D. micans, and 3): within any given beetle as well as between dif- have confirmed the same infection in R. grandis ferent beetles, the oocysts (fixed in methanol and populations (Yaman & Radek 2007). Furthermore, stained with Giemsa) were 11.87 ± 0.67 (10.21- in 2008, Yaman and Radek recorded a neogre- 13.48) μm in length and 6.96 ± 0.43 (6.16-7.74) μm garine pathogen of D. micans in Turkey. This ob- in width (n = 60). They are formed in pairs within servation stimulated us to look for such an infec- a gamontocyst (Fig. 2). A polar plug heavily

354 Yaman, M. et al.

Figures 1-3. Fresh (Figs. 1 and 2) and Giemsa-stained oocysts (Fig. 3) of Mattesia sp. from R. gran- dis. Note oocyst pairs within a gamontocyst (Fig. 2) and heavily Giemsa-stained polar plugs at each cell pole of the oocyst (Fig. 3). Bar: 10 µm.

Figures 4-5. Oocysts of Mattesia sp., SEM. Note the navicular oocysts (Fig. 4) and the protrud- ing polar plug in some oocysts (Fig. 5). Bars: 5 µm (Fig. 4) and 2.5 µm (Fig. 5).

stained with Giemsa solution is present at each combined with electron microscopy revealed that cell pole of the oocyst (Fig. 3). The polar plugs are the neogregarine pathogen has the typical charac- also clearly discernable using electron microscopy teristics of the family Lipotrophidae within the or- (Figs. 4, 5, 8) and are 925 to 1120 nm thick (Fig. 8). der Neogregarinida. The characteristics of the Li- Some oocysts have protruded polar plugs (Fig. 5). potrophidae are navicular oocysts with pro- The spore wall is quite thick, measuring 400 to 500 nounced polar thickenings and containing eight nm (Figs. 6, 7, 8). Each oocyst includes eight sporozoites (see Figs 2, 7) (Perkins 2000). Members sporozoites (Fig. 6). of the other five families are distinct from the Other life cycle stages of the neogregarine neogregarine described here (Perkins 2000). In pathogen could not be observed in the wet or comparison, gamontocysts of the Gigaductidae are stained smear preparations. However, the de- enclosed in a thick gelatinous capsule that is not scribed results of light microscopic observations present in the neogregarine found in R. grandis. In

A new neogregarine pathogen of Rhizophagus grandis (Coleoptera: Monotomidae) 355 the Caulleryellidae, one or eight oocysts are tocyst/oocyst characteristics. There are five genera formed within a gamontocyst, while in the within this family: Farinocystis, Lipocystis, Lipotro- only a single one is found. The pha, Mattesia, and Menzbieria. Only the genus Mat- oocysts of the Syncystidae have three or four tesia is reported to generate as few as one or two spines extending from the poles and about 30-150 oocysts, while members of the other genera have oocysts are formed within a single gamontocyst. more than two oocysts in the gamontocyst. The With their spindle-shaped oocysts containing neogregarine from R. grandis we describe here, eight sporozoites, members of the Schizocystidae characterized by two spores with eight sporozoites resemble our pathogen. However, the genera within one gamontocyst (Figs 2, 7), thus un- characteristics are different from the neogregarine equivocally belongs to the genus Mattesia (Levine described here from R. grandis. The members of 1988, Kleespies et al. 1997, Perkins 2000). the included genus Schizocystis have no oocysts Levine (1988) listed nine described species in with prominent polar thickenings; the genus the genus Mattesia. These species have been de- Machadoella forms 3-6 or 24 oocysts (with polar scribed in the fat body tissue, Malpighian tubules thickenings), and the genus Lymphotropha pro- or intestine of the insect taxa Coleoptera, Hymen- duces more than two (4-16) oocysts per gamonto- optera, and Siphonaptera. Five spe- cyst. Therefore, only the family Lipotrophidae cies were recorded from the order Coleoptera: M. matches our pathogen in all details of the gamon- dispora, M. grandis, M. oryzaephili, M. schwenkei and

Figures 6-8. Mature oocysts of Mattesia sp., TEM. Longitudinal (Fig. 6) and cross (Fig. 7) sections of an oocyst including eight sporozoites. Thick polar plugs are seen (Fig. 8). Bars: 2 µm (Figs 6 and 7) 2.5 µm (Fig. 8).

356 Yaman, M. et al.

Table 1. Mattesia species described in the order Coleoptera.

Mattesia species Spore size Infected Host References organ Mattesia dispora 12.2 x 6.7 µm unknown Laemophloeus ferrugineus, Finlayson 1950 L. minutes Mattesia grandis 9-10.9 x 4.9-11.3 µm Fat body Anthonomus grandis McLaughlin 1965 Mattesia schwenkei 17.5-20.4 x 7.5-9.2 µm (native) Fat body Dryocoetes autographus Purrini 1977 15.5-18.5 x 6.2-7.8 µm (stained) Mattesia schwenkei 16.3-21.2 x 6.3-10.5 µm (native), Fat body Hylurgops glabratus Purrini 1978 15.5-18.5 x 6.2-7.8 µm (stained) Mattesia trogoderma -- Fat body Trogoderma granaria Canning 1964 Mattesia oryzaephili 12 x 7 µm (native), Fat body Oryzaephilus surinamensis Ormières 1971 10 x 6 µm (stained) Mattesia sp. 14 x 5, tubular type, Fat body Ips typographus Žižka et al. 1997; 13-13.5 x 7, navicular type Händel et al. 2003 Mattesia sp. 20-22 x 8.5-10 µm Fat body Pityogenes chalcographus Händel et al. 2003 Mattesia sp. 11 x 6 μm (stained) Fat body Dendroctonus micans Yaman & Radek 2008 Mattesia sp. 11.87 x 6.96 (stained) Fat body Rhizophagus grandis This study

M. trogodermae (Table 1). The only named species ing these as nutrition for parent R. grandis and found to infect bark beetles is Mattesia schwenkei their new progeny. Under such conditions, a (15.5-18.5 x 6.2-7.5 μm in size) from Dryocoetes transmission of neogregarines from D. micans to autographus (Purrini 1977). Źižka et al. (1997) the predator, R. grandis could be possible. Such a found two yet unnamed types of Mattesia in the transmission was described for Helicosporidium sp. bark beetle Ips typographus; the tubular type meas- from D. micans to R. grandis by Yaman & Radek ures 14 x 5 μm and the navicular type 13-13.5 x 7 (2007). To prevent a disease transmission from μm. Händel et al. (2003) found a similar infection prey to predator and thus to improve the produc- in Ips typographus and Pityogenes chalcographus. Re- tion of this biological control agent, we recom- cently, Yaman and Radek (2008) recorded a Matte- mend that cultures of D. micans, serving as prey sia sp. (11 x 6 μm) infection in Dendroctonus micans, beetles, are free of neogregarine infection. There- the prey of Rhizophagus grandis (Table 1). As seen fore, D. micans larvae destined for use as food for in Table 1, the neogregarine pathogen in R. grandis R. grandis should be only collected from areas in has a slightly larger spore size than Mattesia sp. in which no infections of the prey by either Helico- D. micans. Until now, the only recorded pathogen sporidium sp. or Mattesia sp. have been detected. from R. grandis is the entomopathogenic alga Heli- cosporidium sp., described by Yaman and Radek

(2007). The pathogen described here is the first Acknowledgements. The study was financially supported neogregarine reported from R. grandis. It only in- as a research project by the Scientific and Technical fects the fat body of its host. Merogony and Council of Turkey (107T166). Dr. Mustafa Yaman was sporogony of many Mattesia species are known to awarded with a grant by the DAAD (German Academic take place in the fat body, whereby the tissue is Exchange Service) (316- A/09/03438). The authors wish lysed (Kleespies et al. 1997, Perkins 2000). Thus an to express their thanks to Prof. Dr. Klaus Hausmann, infection with Mattesia is pathogenic and may re- Berlin for making this study possible. duce the lifespan and reproduction of its host, leading to a decreased potential as a biological control agent. References As mentioned above, Yaman & Radek (2008) recorded a neogregarine infection (Mattesia sp.) Canning, E. U. (1964): Observations on the life history of Mattesia from D. micans, the prey of R. grandis. This preda- trogodermae sp. n., a schizogregarine parasite of the fat body of the Khapra beetle, Trogoderma graniarium Everts. Journal of tory beetle is mass-reared in laboratories for the Insect Patholology 6: 305-317. biological control of D. micans in several countries Finlayson, L. H. (1950): Mortality of Laemophloeus (Col., Cucujidae) where this insect is a threat to spruce forests. The infected with Mattesia dispora Naville (Protoz., Schizogregarinaria). Parasitology 40: 271-264. mass-rearing procedure includes collecting D. Händel, U., Wegensteiner, R., Weiser, J., Žižka, Z. (2003): micans larvae from infested spruce stands and us- Occurrence of pathogens in associated living bark beetles (Col.,

A new neogregarine pathogen of Rhizophagus grandis (Coleoptera: Monotomidae) 357

Scolytidae) from different spruce stands in Austria. Journal of , Scolytinae). North-Western Journal of Zoology 4: Pest Science 76: 22-32. 99-107. Kleespies, R.G., Huger, A.M., Buschinger, A., Nähring, S., Yaman, M., Radek, R. (2005): Helicosporidium infection of the great Schumann, R.D. (1997): Studies on the life history of a European spruce bark beetle, Dendroctonus micans (Coleoptera: neogregarine parasite found in Leptothorax ants from North Scolytidae). European Journal Protistolology 41: 203-207. America. Biocontrol Science and Technolology 7: 117-129. Yaman, M., Radek, R. (2007): Infection of the predator beetle Levine, N.D. (1988): The Protozoan . 2 vol.. Rhizophagus grandis Gyll. (Coleoptera, Rhizophagidae) with the CRC Press, Boca Raton, FL. insect pathogenic alga Helicosporidium sp. (Chlorophyta: McLaughlin, R.E. (1965): Mattesia grandis n. sp., a sporozoan Trebouxiophyceae). Biological Control 41: 384-388. pathogen of the boll , Anthonomus grandis Boheman. Yaman, M., Radek, R. (2008): Pathogens and parasites of adults of Journal of Protozoolology 12: 405-413. the great spruce bark beetle, Dendroctonus micans (Kugelann) Ormières, R., Louis, C., Kuhl, G. (1971): Mattesia oryzaephili n. sp. (Coleoptera: Curculionidae, Scolytinae) from Turkey. Journal of Néogregarine parasite d'Oryzaephilus surinamensis L. (Col., Pest Science 81: 91-97. Cucujidae). Cycle et action pathogéne. Bulletin de la Société Yaman, M., Tosun, O., Aslan, I. (2008): On the occurrence of Zoologique de France 96: 547-556. Gregarine Parasite from Psylloides cupreus Koch 1803 Perkins, F.O. (2002): Order Grassé, 1953. In: Lee, (Coleoptera: Chrysomelidae) of Turkey. North-Western Journal J.J., Leedale, G.F., Bradbury, P. (eds.), An Illustrated Guide to of Zoology 4: 167-172. the Protozoa, 2nd ed. Society of Protozoologists, Lawrence, USA, Yaman, M., Tosun, O., Lipa, J.J., Aslan, İ. (2010): The first records of pp. 288-298. a gregarine pathogen and a mermithid parasite from Chrysolina Purrini, K. (1977): Über eine neue Schizogregarinen-Krankheit der fastuosa (Scopoli 1763) (Coleoptera: Chrysomelidae). North- Gattung Mattesia Naville (Sporoz., Dischizae) des Zottigen Western Journal of Zoology 7: 105-111. Fichtenborkenkäfers, Dryocoetes autographus Ratz. (Coleoptera, Žižka, Z., Weiser, J., Wegensteiner, R. (1997): Ultrastructures of Scolytidae). Anz. Schädlingskde., Pflanzenschutz, oocysts of Mattesia sp. in Ips typographus. Journal of Eukaryotic Umweltschutz 50: 132-35. Microbiolology 44: 25A, 98. Purrini, K. (1978): Protozoen als Krankheitserreger bei einigen Borkenkäferarten (Col., Scolytidae) im Königssee-Gebiet, Oberbayern. Anz. Schädlingskde., Pflanzenschutz, Umweltschutz 51: 171-175. Yaman, M. (2008): First results on distribution and occurrence of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in the populations of the great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: