Biologia 67/5: 966—972, 2012 Section Zoology DOI: 10.2478/s11756-012-0086-x Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria Danail Ilchev Takov1, Danail Dimitrov Doychev2, Andreas Linde3, Slavimira Atanasova Draganova4 & Daniela Kirilova Pilarska1,5 1Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1, Tsar Osvoboditel Blvd., Sofia 1000, Bulgaria; e-mail: [email protected] 2University of Forestry, 10, Kl. Ohridski Blvd., Sofia 1756,Bulgaria 3University of Applied Sciences Eberswalde, Alfred-M¨oller-Straße 1, 16225 Eberswalde, Germany 4Institute of Soil Science, Agrotechnologies and Plant Protection, 7 Shosse Bankya Str., 1080 Sofia, Bulgaria 5Faculty of Forestry and Wood Science, Czech University of Life Sciences, 129 Kamýcká str, CZ-16521 Prague 6, Suchdol, Czech Republic Abstract: The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis. Key words: beetle pathogens; Gregarina typographi; Chytridiopsis typographi; ItEPV; Beauveria bassiana; Isaria farinosa; Bulgaria Introduction The study of diversity and role of insect pest pathogens is related to the development of environmen- Forests are very valuable biological resources and their tally friendly methods to control the pest mass out- conservation and sustainable development is the basis breaks. The aim is to maintain a low density of pest in for their long term use. Each year insects cause ma- forests and to reduce the damages, respectively. There- jor damage to forestry. The role of beetles is very im- fore, the control of their density contributes to the qual- portant in this respect, as they are a part of the for- itative forest management. est ecosystems and this way connected with particular In the present work we obtain new data about tree species through feeding preferences. Bark beetles pathogens of bark beetles and other forest Coleoptera (Curculionidae: Scolytinae) are insect pests with high from different trees species, in particular on their diver- economical importance; they can cause mass drying of sity, occurrence and infection levels. coniferous forest trees. Therefore their pathogen com- plex is intensively investigated during the last years. More than 30 species of bark beetles were investigated Material and methods and 35 protozoan and microsporidian species (Weiser Nine forest stands from different regions of Bulgaria have 1954, 1977; Purrini 1978; Wegensteiner et al. 1996; been investigated from April 2009 to October 2010. A to- H¨andel et al. 2003; Wegensteiner & Weiser 2004; Kere- tal of 818 individuals of beetles, belonging to 22 species selidze et al. 2010; Lukášová & Holuša 2011; Holuša et were collected from different parts of spruce trees (stem, leaves, seeds, under bark). All collected insects were trans- al. 2012; Michalková et al. 2012), seven fungal species ◦ (Landa et al. 2001; Jankevica 2004; Keller et al. 2004; ferred to the laboratory and kept refrigerated at 1–4 Cto Sosnowska et al. 2004; Wegensteiner 1992, 2004; Glare reduce movement and prevent horizontal transmission of any pathogens. After the dissections, fresh preparations of et al. 2008; Draganova et al. 2010) as well as many ne- the gonads, Malpighian tubules, fat body, and the entire matode species (R¨uhm 1956; Massey 1974; Poinar 1975; gut from the imago hosts were examined for the presence Nedelchev et al. 2008) have been detected. The infor- of pathogens under light microscopy (×160–400) according mation about pathogens of other forest beetle species to Wegensteiner et al. (1996). When pathogens were ob- is also scarce. served, Giemsa stained smears were made of the infected c 2012 Institute of Zoology, Slovak Academy of Sciences Pathogens of forest beetles Table 1. Infected bark beetles species and found pathogens. Beetle Host Site/Date Host plant N ItEPV B. bassiana B. brongniarti I. farinosa G. typographi Gregarina sp. Chytr. sp. Chytr. typ. Nematoda n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Acanthocinus aedilis 9/ 5.09.2009 Pinus nigra 30 – – – – – 1 (3.3) – – 3 (10) (L., 1758) J.F.Arnold Rhagium inquisitor 2/21.04.2009 Picea abies (L.) H. 9 – – – – – 4 (44.4) – – – (L., 1758) Karst. Tomicus piniperda 3/ 02.05.2010 Pinus sylvestris L. 16 – 1 (6.2) – – – – – – 2 (12.5) (L., 1758) Hylurgops palliatus 4/ 15.06.2010, Picea abies 20 – 1 (5) 1 (5) – – – – – 13 (65) (Gyllenhal, 1813 ) 4/ 10.08.2010 Ips amitinus (Eichhoff, 1/ 10.08.2010 Pinus peuce Griseb. 14 – – – – – 3 (21.4) 3 (21.4) – 2 (14.3) 1871) Ips typographus (L., 5/ 2.09.2010; Picea abies 219 1 (0.4) 14 (6.4) 4 (1.8) – 52 (23.7) – – 7 (3.2) 201 (91.8) 1758) 1/ 6.10.2010; 2/ 22.10.2010 Ips sexdentatus 6/ 5.10.2010; Pinus sylvestris 47 – 2 (4.2) – 1 (2.1) 13 (27.6) – – – 47 (100) (Borner, 1776 ) 7/29.05.2010 Orthotomicus longicol- 6/ 15.10.2010 Pinus sylvestris 34 – 2 (5.9) – – – 4 (11.8) 11 (32.3) – 6 (17.6) lis (Gyllenhal, 1827) Pyrochroa coccinea 8/ 07.04.2009 Ulmus sp. 44 – – – – – 1 (2.3) – – – (L., 1761) Collection sites in Bulgaria: 1 – Rila Mt., Kurtovo Place, 1720 m a.s.l., 42◦5.417 N, 23◦48.467 E; 2 – Lyulin Mt., Monastery St. Cyril and Methodius, 1050 m a.s.l., 42◦38.944 N., 23◦11.024 E; 3 – Rila Mt., above Yakoruda, 970 m a.s.l., 42◦2.098 N, 23◦40.542 E; 4 – Rhodope Mt., Beglika Place, 1570 m a.s.l., 41◦48.883 N, 24◦7.143 E; 5 – Vitosha Mt., Zlatni mostove, 1400 m a.s.l., 42◦36.817 N., 23◦14.181 E; 6 – Maleshevska Mt., near Nikudin Vill, 1000 m a.s.l., 4134.608 N, 23◦4.170 E; 7 – Pirin Mt., above Dobrinishte Vill., 1100 m a.s.l., 41◦47.965 N, 23◦33.712 E; 8 – Sofia, Borisova gradina Park, 580 m a.s.l., 42◦39.835 N, 23◦20.480 E; 9 – Rhodope Mt., Byala Cherkva Resort, 1500 m a.s.l., 41◦55.100 N, 24◦39.893 E. 967 968 D.I. Takov et al. Fig. 1. A – Spheroids of ItEPV in the gut of Ips typographus; B – Pansporoblasts of Chytridiopsis sp. in Orthotomicus longicollis;C – Gamonts of Gregarina sp. in Orthotomicus longicollis; D – Gamonts of Gregarina sp. in Rhagium inquisitor.Scales20µm(A,B), 200 µm(C),50µm(D). tissues (Weiser 1977). Sizes of spores and gregarine tropho- I. sexdentatus (28%), I. typographus (24%). With the zoites were measured with an ocular micrometer (Carl Zeiss exception of I. sexdentatus and I. typographus, the ga- micrometer) at ×160 and 400 magnifications. Cadavers of mont number in the other hosts in which Gregarina insects with symptoms of mycosis were placed in a hu- spp. were observed was low and thus the species iden- mid chamber for the colonization of cadavers by fungal tification was difficult. Since this is the first record of pathogens, which were then isolated into pure cultures on gregarines in these bark beetle species, we present in- SDAY (Sabouraud dextrose agar with yeast extract) and identified according to morphological characteristics (Sam- formation on gamont size (Table 2). son et al. 1988; Humber 1997). Conidia and conidiogenous Microsporidian infections (Chytridiopsis typogra- cells were observed in smears stained with methylen blue phi Weiser, 1954 and Chytridiopsis sp. were revealed in and in durable specimens with lactophenol and aniline blue, the gut of three bark beetle species (Table 1). Chytri- using a transmission interference microscope (BX60 DIC diopsis typographi was detected in I. typographus and Olympus), equipped with a digital camera and a Cell B Chytridiopsis sp. in I. amitinus (Eichhoff, 1871) and O. image capture software. longicollis (Gyllenhal, 1827). The size of alive cysts Statistical analysis was performed using the computer of Chytridiopsis sp. in I. amitinus varied from 9.6 to program STATISTICA, version 7.0 (StatSoft Inc. 1999). 12.8 µm(n = 20), and in O. longicollis from 6.4 to 9.6 µm(n = 20). The prevalence of Chytridiopsis sp. Results in I. amitinus and O. longicollis was high and reached 21.4% and 32.3%, respectively (Table 1). O. longicollis Of all 22 investigated coleopteran species, pathogens is new host for Chytridiopsis sp. (Fig. 1). were found in 9 species (Table 1). The virus ItEPV Fifty five individuals from the collected dead in- Weiser & Wegensteiner was established only in a single sects belonging to 15 coleopteran species showed symp- individual of Ips typographus (L., 1758). Large numbers toms of mycoses and 38 fungal isolates were obtained of spheroids, filling the whole gut of the beetle, were from them in pure cultures on SDAY plates. Thirty observed (Fig. 1). one isolates found in 11 insect species were identified The protozoa Gregarina typographi Fuchs, 1915 as Beauveria bassiana (Bals. – Criv.) Vuillemin, 1912. and Gregarina spp. were observed in six beetle species. Two different bark beetle species were initial hosts of G. typographi was detected in the gut lumen of I.