Biologia 67/5: 966—972, 2012 Section Zoology DOI: 10.2478/s11756-012-0086-x

Pathogens of bark (: 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, 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,

Abstract: The presence and diversity of 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 . 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, 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 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 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 967 N, 11.024 – – (%) ◦ n 47.965 ◦ Nematoda N., 23 E. .l., 41 – 13 (65) – 47 (100) – – 2 (12.5) – – 3 (10) (%) n 38.944 ◦ Chytr. typ. 39.893 ◦ 1100 m a.s .l., 42 sp. N, 24 – – 7– (3.2) 201 (91.8) – (%) n Chytr. 55.100 ◦ sp. E; 5 – Vitosha Mt., Zlatni mostove, 1400 – – (%) .l., 41 n 1 (2.3) – 1 (3.3) – 7.143 ◦ N, 24 – 3 (21.4) 3 (21.4) – 2 (14.3) – 4 (11.8) 11 (32.3) – 6 (17.6) – – – 4 (44.4) – – – (%) n 48.883 ◦ .l., 41 E; 7 – Pirin Mt., above Dobrinishte Vill., – – – – – – – (%) n 4.170 ◦ N, 23 – – – – (%) n E; 2 – Lyulin Mt., Monastery St. Cyril and Methodius, 1050 m a.s 4134.608 .l., E; 9 – Rhodope Mt., Byala Cherkva Resort, 1500 m a.s 48.467 (%) ◦ n N, 23 1000 m a.s 20.480 ◦ (%) 5.417 n ◦ N, 23 E; 4 – Rhodope Mt., Beglika Place, 1570 m a.s 9 – – N ItEPV B. bassiana B. brongniarti I. farinosa G. typographi Gregarina 47 – 2 (4.2) – 1 (2.1) 13 (27.6) – 34 – 2 (5.9) – 20 – 1 (5) 1 (5) 44 – – 30 – – 219 1 (0.4) 14 (6.4) 4 (1.8) – 52 (23.7) – .l., 42 39.835 40.542 ◦ ◦ L. 16 – 1 (6.2) – N, 23 Griseb. 14 – – (L.) H. sp. 2.098 ◦ Picea abies Pinus peuce Pinus sylvestris Pinus sylvestris Ulmus Picea abies Pinus sylvestris Karst. Picea abies Pinus nigra J.F.Arnold E; 6 – Maleshevska Mt., near Nikudin Vill, Site/Date Host plant 9/ 5.09.2009 7/29.05.2010 2/21.04.2009 5/ 2.09.2010; 1/ 6.10.2010; 6/ 5.10.2010; 2/ 22.10.2010 1/ 10.08.2010 6/ 15.10.2010 8/ 07.04.2009 4/ 10.08.2010 3/ 02.05.2010 14.181 4/ 15.06.2010, ◦ N., 23 (L., (Eichhoff, 36.817 E; 8 – Sofia, Borisova gradina Park, 580 m a.s.l., 42 ◦ (Gyllenhal, 1827) 33.712 typographus Ips sexdentatus (Borner, 1776 ) 1758) Ips amitinus 1871) Orthotomicus longicol- lis Pyrochroa coccinea (L., 1761) Tomicus piniperda (L., 1758) Hylurgops palliatus (Gyllenhal, 1813 ) Rhagium inquisitor (L., 1758) Beetle Host Acanthocinus aedilis (L., 1758) ◦ 23 m a.s.l., 42 Table 1. Infected bark beetles species and found pathogens. Collection sites in Bulgaria:E; 1 3 – Rila – Mt., Rila Kurtovo Mt., Place, above 1720 Yakoruda, m 970 a.s m a.s.l., 42 968 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 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. ty- five isolates of B. brongniartii (Saccardo) Petch, 1926 pographus and I. sexdentatus (Borner, 1776) and Grega- and two isolates of Isaria farinosa (Holmsk.) Fries, 1832 rina spp. were observed in the gut of four beetle species (formerly Paecilomyces farinosus (Holm ex S.F.Grey) (Table 1). The prevalence of gregarines was high in Brown & Smith, 1957 (Table 1). Identified fungal these host species: Rhagium inquisitor L., 1758 (44%), species from genera Beauveria and Isaria are anamorph Pathogens of forest beetles 969

Table 2. Morphometrical data of Gregarina spp. and G. typographi (in µm).

Host species Acanthocinus Rhagium Pyrochroa Orthotomicus Ips Ips aedilis inquisitor coccinea longicollis typographus sexdentatus

Pathogen Gregarina spp. G. typographi

TL mean (min–max) 67 (64–70) 85 (72–98) 64.8 460 107.5 (32–198) 120 (55–260) LP mean (min–max) 9 (8–10) 19 (12–30) 19.2 80 37.5 (8.8–58.8) 25 (10–40) LD mean (min–max) 49 (48–50) 65 (44–80) 45.6 380 67.5 (22.5–137.5) 100 (41–231) WP mean (min–max) 11.5 (11–12) 25 (16–30) 18.4 80 41.2 (25–75) 40 (15–90) WD mean (min–max) 42 (40–44) 45 (30–72) 33.6 120 46.2 (20–85.5) 75 (30–110) LP/TL mean (min–max) 0.134 (0.125–0.142) 0.228 (0.166–0.405) 0.296 0.174 0.321 (0.140–0.490) 0.195 (0.086–0.380) WP/WD mean (min–max) 0.274 (0.275–0.273) 0.583 (0.250–0.823) 0.547 0.666 0.940 (0.630–1.13) 0.520 (0.250–1.8) LP/WP mean (min–max) 0.780 (0.727–0.833) 0.795 (0.533–1.66) 1.04 1 0.820 (0.421–1.53) 0.650 (0.227–1.150)

ascomycota (Hypocreales: Cordycipitaceae). Conidia most prevalent pathogens, followed by the fungi and and conidiophores of Aspergillus sp. (anamorph As- microsporidia. comycota, Eurotiales: Trichocomaceae) were observed ItEPV was found only in one individual of the in smears and durable specimens prepared from cadav- spruce bark beetle. Weiser & Wegensteiner (1994) first ers and from pure cultures of the isolates, obtained from reported the finding of ItEPV in the gut epithelium some adults of the following species: four of I. typogra- of I. typographus. Based on electron microscopical in- phus,twoofI. acuminatus, and one each of C. moraei, vestigations Zizka et al. (2001) reported that this virus T. piniperda and O. balcanicus, and nine of M. tonki- differed from all other entomopoxviruses of type A. Hai- neus. The record of B. bassiana in Tomicus piniperda, dler (1998) found ItEPV in I. typographus from Cen- O. longicollis, O. erosus, X. spinole, Taphrorychus vil- tral Austrian Alpes and H¨andel (2001) reported ItEPV lifrons and Phylobius sp. is the first report for Bul- not only in I. typographus, but also from I. amitinus garia. and the virus was also found in I. typographus in Bul- In the total number of investigated beetles are garia in 2003 (Takov et al. 2006). Low prevalences as also 13 bark beetle species included Magdalis frontalis in our study were also reported by Wegensteiner et (Gyllenhal, 1827) (18 ind.), Morimus asper funereus al. (1996) (0.1–3.6%) in and by Burjanadze & Mulsant, M¨uller, 1949–1953 (26 ind.), Rhagium bi- Goginashvili (2009) (0.9%) in Georgia. fasciatum F., 1775 (15 ind.), populi L., In our study, the infection rates of Ch. typographi 1758 (17 ind.), Chrysomela vigintipunctata Scopoli, in I. typographus were quite low, however the preva- 1763 (16 ind.), Curculio elephas (Gyllenhal, 1863) (85 lences of Chytridiopsis spp. in I. amitinus and O. longi- ind.), Otiorhynchus endroedi Angelov, 1964 (84 ind.), collis were considerably higher. Weiser (1954) described Otiorhynchus perdix (Olivier, 1807) (25 ind.), Phyllo- Ch. typographi from I. typographus and later this mi- bius oblongus (L., 1758) (11 ind.), Neocoenorrhinus ae- crosporidium was reported from 16 bark beetle species neovirens (Marsham, 1802) (23 ind.), Hylecoetus der- (Purrini 1978; Purrini & Weiser 1984, 1985; Wegen- mestoides (L., 1861) (35 ind.), Exomala hirtella (Brulle, steiner 1994; Laucius & Zolubas 1997; Haidler 1998; 1832) (20 ind.), Tomicus minor (Hartig, 1834) (10 ind.), Pernek et al. 2009; Holuša et al. 2007, 2009; Michalková in which no pathogens were observed. et al. 2012). Wegensteiner & Weiser (2004) published a Invasive larvae of nematodes were revealed in seven five year study on the prevalence of Ch. typographi in beetle species. Nematodes were not identified to species I. typographus. They reported infection rates between level because the observed larvae did not show a devel- 1.7 and 56.3%, but hypothesized that high infection oped reproductive system. The infections were found rates were probably due to the fact that they had col- in the gut and haemolymph of the beetles. The preva- lected beetles from infested log trees kept in the labo- lence was high and varied between 10% and 100% (Ta- ratory. In this situation, beetles which exit the log trees ble 1). do not have the option to leave and infest new trees but return to the old galleries, where the risk for a contact Discussion with the pathogens is high. The authors noticed that these data were not representative for natural popula- In this study several pathogen groups (ItEPV, G. ty- tions of the beetle. H¨andel et al. (2003) reported the pographi, Gregarina spp., Ch. typographi, Chytridio- presence of Ch. cf. typographi in I. amitinus. We sup- psis sp., B. bassiana, B. brongniartii, Isaria farinosa pose Chytridiopsis sp.wefoundinthesamehostmight and nematodes) were revealed in nine coleopteran be Ch. typographi however for the exact determination species. Four beetle species (Orthotomicus longicol- of the species molecular characterization is necessary. lis, Acanthocinus aedilis, Rhagium inquisitor and Py- Gregarina typographi was found in I. typographus rochroa coccinea) are new hosts for Gregarina spp., and I. sexdentatus in Bulgaria in 2003 (Takov et al. and Chytridiopsis sp. was reported for the first time 2006, 2007). This pathogen was described by Fuchs in O. longicollis. Gregarines and nematodes were the (1915) in I. typographus and reported in I. sexdenta- 970 D.I. Takov et al. tus by Theodorides (1960). Later G. typographi was pathogens found rarely in bark beetles (Landa et al. found in Germany, Austria and Czech Republic (Purini 2001; Jankevica, 2004; Keller et al. 2004; Sosnowska 1978; Wegensteiner 1994; Wegensteiner et al. 1996). et al. 2004; Wegensteiner 1992, 2004; Draganova et al. The information about the distribution of this pathogen 2010). The species B. caledonica Bissett et Widden, in I. typographus was complemented by Kereselidze & 1988 was found by Glare et al. (2008) as naturally oc- Wegensteiner (2007) and Burjanadze & Goginashvili curring pathogen of two forest beetle species belong- (2009) who found G. typographi in Georgia, and Ya- ing to the Curculionidae (Scolytinae). In our study man (2007) reported G. typographi from I. sexdentatus B. brongniartii was established only in two species, in Turkey. Additional morphometrical data about this I. typographus (in 5 dead adults) and H. palliatus (in gregarine were presented by Yaman (2007) and Takov 1 adult) and I. farinosa was found in I. sexdentatus (in et. al. (2011). 1 adult) and in I. acuminatus (in 1 adult). The eugregarines have life stages which are located In our study nematodes showed high infection rates in the gut lumen of the hosts, whereas microsporidia (between 11–100%). The reasons could be the synchro- and viruses are intracellular parasites. The infections nization of the nematodes life cycle with the one of the are spread by oral uptake of spheroids of ItEPV, oocysts bark beetle (Kaya 1984) and the fact that they actively of G. typographi and spores of Ch. typographi by the search for their hosts with hemoreceptors, which con- suitable hosts The mechanisms of infection are in di- tributes to a successful infection. rect relation with the biology of the hosts. A significant It should also be noticed that most of the patho- part of life cycle of bark beetles occurs under the bark gens we found in our study were established in bark bee- of trees and the pathogen infection occurs in the gal- tles. We suppose that the reason for this might be the leries where the hosts feed and release their excrements, peculiarities of their biology, namely the hidden mode which contain the infective stages of pathogen. The new of life in groups. In particular the process of mating, generation also feed in the galleries and therefore come where one male beetle is in contact with several female into contact with the pathogens. It is important to note beetles (Wegensteiner & Weiser 2004) and coprophagy that the contact between male and female beetles dur- (Wegensteiner & Weiser 1996a, b) which contributes to ing mating is also one of the possible ways for infec- the oral uptake of pathogens. tion and distribution of the pathogens (Wegensteiner & Weiser 2004; Lukášová & Holuša 2011). Fungal pathogens infect their hosts percutaneous- Acknowledgements ly. They produce extracellular enzymes, lysing the host cuticula, and penetrate through the integu- We are especially indebted to the National Science Fund of Bulgaria project DO-02-251/2008. ment with support of mechanical pressure of the grow- ing tubes of the germinating fungal conidia (St. Leger et al. 1986; Draganova 1988; Bidochka & Khachatourians References 1990; Gupta et al. 1992; St. Leger 1995). Bark beetles are susceptible to entomopathogenic fungi but their bi- Bidochka M.J. & Khachatourians G.G. 1990. 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