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Coleoptera: Monotomidae) 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 Rhizophagus grandis (Coleoptera: Monotomidae) 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 beetle family Monotomidae with neogregarines and specifically the first finding of such a pathogen in the predatory beetle Rhizophagus grandis. 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 genus Mattesia (family Lipotrophidae) within the order Neogregarinida. Keywords: Bark beetles, Biological control, Dendroctonus micans, Neogregarine, Mattesia, Rhizophagus grandis. The great spruce bark beetle Dendroctonus micans 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 insect 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- Ophryocystidae 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, Lepidoptera 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.
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