Occurrence of pathogens in Ips typographus

Biologia 69/1: 92—100, 2014 Section Zoology DOI: 10.2478/s11756-013-0286-z

Occurrence of pathogens in Ips typographus (Coleoptera: Curculionidae) and in other spruce bark beetles from the wilderness reserve D¨ urrenstein (Lower Austria) ¨ndel Rudolf Wegensteiner, Andrea Stradner & Uwe Ha University of Natural Resources and Life Sciences Vienna, Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection, Gregor Mendel Str. 33, A-1180 Vienna, Austria; e-mail: [email protected]

Abstract: The study presents new data on spatial distribution of bark beetle pathogens, on changes in frequency over several years and on their prevalence during different time periods within a year from several locations within the wilderness reserve D¨ urrenstein (Lower Austria). The occurrence of pathogens was investigated in Ips typographus (during five years), in Pityogenes chalcographus (during two years) and in Ips amitinus (in one year). In total, seven pathogen species could be detected in I. typographus. The most dominant pathogen species were the Ips typographus-Entomopoxvirus (ItEPV), the sporozoan species Gregarina typographi and the microsporidium Chytridiopsis typographi; the latter two pathogen species were recorded every year and at about similar high (G. typographi) or low (C. typographi) rates, the ItEPV in strongly varying rates. The neogregarine Mattesia cf. schwenkei and the two microsporidia Nosema typographi and Unikaryon montanum were found in I. typographus only sporadically and the rhizopodan species Malamoeba scolyti was found once. The number of infected males and females was relatively similar with almost all pathogen species in most of the years except U. montanum, which occurred exclusively in females. Three pathogen species were recorded in P. chalcographus which were Gregarina typographi, Mattesia cf. schwenkei and Chytridiopsis typographi. Two pathogen species were observed in I. amitinus, Gregarina typographi and Chytridiopsis typographi. Key words: Ips typographus; Ips amitinus; Pityogenes chalcographus; microorganisms; virus

Introduction The spruce bark beetles Ips typographus (L., 1758) and Pityogenes chalcographus (L., 1761) are throughout their distribution area in central and northern Europe the most important phloeophagous insect pests, especially in secondary Picea abies (L.) H. Karsten stands. After storm disasters, these two species can proliferate ideally under favourable weather conditions. Both species are known for a rapid increase of their populations, which are often initially established on windbroken or wind-thrown trees and spread further due to storm-damaged or to otherwise physiologically weakened trees. Ips amitinus (Eichhoff, 1871) is also known to occur on P. abies, preferring breeding material similar to both former species. Only relatively few publications focused on bark beetle pathogens (ref. in Wegensteiner 2004). Nevertheless, it was shown that a large number of different pathogens can occur especially in I. typographus (Wegensteiner & Weiser 1995, 1996a, b; Wegensteiner et al. 1996; Weiser et al. 1997, 1998; Keller et al. 2004). The

pathogen complex of associated living spruce bark beetles was investigated in a master thesis (Haidler 1998) and a PhD thesis (Händel 2001). The results of these studies provided surprising insights into the variety of pathogens within the guild of spruce bark beetles (Haidler et al. 2003; H¨ andel et al. 2003). There are very diverse theories on the prevalence and incidence of pathogens in bark beetles, and their importance as regulators in the population dynamics (ref. in Wegensteiner 2004). An essential prerequisite is on one hand, the temporal and spatial coincidence of hosts with their pathogens. On the other hand the continuous presence of both groups in numbers to ensure the permanent establishment of the pathogens was described by F¨ uhrer (1975, 1985) as critical minimum host density. There is currently hardly any knowledge about the function of pathogens in the different phases of population dynamics of bark beetles. However, it is assumed that pathogens involved in the natural limitation of bark beetle populations are relatively important (Stephen et al. 1993). Apart from the results of two

Dedicated in commemoration to our venerated teacher and promoter Prof. Dr. Jaroslav Weiser (Czech Republic).

Unauthenticated c 2013 Institute of Zoology, Slovak Academy of Sciences

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Pathogens in bark beetles

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Fig. 1. Skeleton map of the north-eastern part of Austria and magnified detail with sampling areas in the wilderness reserve D¨ urrenstein (the boundary line defines the wilderness area) in the conservation forest area (Rothwald), in the periphery of the conservation forest area (Edelwies) and in the orographically separated area (Hundsau).

Table 1. WGS 84 coordinates (East and North), altitude (in m) and IUCN-category of the three sampling areas within the wilderness area D¨ urrenstein. WGS 84 coordinates

Rothwald Edelwies Hundsau

East

North

Altitude

IUCN-category

15◦ 5 38.9080 15◦ 3 22.6343 15◦ 2 35.2941

47◦ 46 55.3971 47◦ 45 31.5072 47◦ 46 46.8710

1000m 1100m 950m

Ia reserve Ia reserve Ib reserve

field studies on the effects and on transmission of Gregarina typographi (Fuchs, 1915) (Wegensteiner et al. 2010; Lukášová & Holuša 2011) most other investigations on the effects of pathogens (virus, bacteria, fungi, microsporidia and protozoa) in bark beetles are confined to laboratory experiments (ref. in Wegensteiner 2004). In the present study, we accepted the axiom that a large diversity of pathogen species guarantees a high regulatory potential in undisturbed ecosystems. Based on that, the following questions were addressed: (i) what pathogen species occur in the bark beetle species, I. typographus, P. chalcographus and I. amitinus from the wilderness reserve D¨ urrenstein, (ii) can the same pathogen species be found homogeneously in the beetles from various collection points within the wilderness area regardless if the sampling area is inside or outside the core area of the strict nature reserve, and (iii) can these pathogen species be detected continuously and at an approximately constant level or at a changing level in the course of several consecutive years.

Material and methods Study area Bark beetles were collected in the wilderness reserve D¨ urrenstein (Lower Austria), partly in the International Union for Conservation of Nature-(IUCN)-category Ia areas “Rothwald” and “Edelwies” and partly in the IUCNcategory Ib area “Hundsau”, from 2001 to 2005. As a consequence, this was not conducted earlier than late May or early June, in some few cases (long snow coverage) in late June or early July when snow allowed access to the study areas. The IUCN-category Ia strict reserve core area “Rothwald” was selected representing the ”undisturbed” development of a local bark beetle population for a long time. Another sampling site was a reference area outside that strict reserve core area with the local name “Edelwies” (forest regeneration area on a forest clearing caused by a storm in the year 1990). Beetles were also collected in a third area bordering west to the areas “Rothwald” and “Edelwies”, with the local name “Hundsau” (forest without significant management since 1997), orographically separated from the other areas by a mountain ridge (1500 m high) (Fig. 1, Table 1).

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R. Wegensteiner et al.

Beetle collection The number of bark beetle sampling sites varied depending on the local availability of trap trees or trap logs. In most cases infested wind-thrown or wind-broken trees were found from the previous autumn or winter at the same sampling place or next to that place (“Rothwald 1”). Presence of bark beetle infested material was ensured in that site in case we did not find an infested tree or log and (because tree felling was not allowed in the strict reserve core area) by manually entering two uninfested log sections (each 120 cm long) from 2002 to 2005. In addition, infested trees within the strict reserve core area (found by chance) were selected to get additional beetles (punctiform sampling sites: R-a to Rd). The distances between these sub-sampling places within the area Rothwald were 300–500 m (see Table 2: punct. and Table 4: a, b, c and d) in 2001 and 2003. Beetles were attracted to two uninfested log sections (each 120 cm long) at the sampling site “Edelwies” from 2001 to 2004. Beetles were collected from attacked (standing or lying) trees at the site “Hundsau” from 2001 to 2005. Beetles were collected either directly from the bark of trees or logs, or by removing colonized bark from the trees, or by cutting colonized log sections. Beetles that were collected out of the bark were transported to the laboratory and stored separately by location and species in an incubator at 15 ◦C (± 1.5 ◦C) until further processing. Colonized bark pieces or log sections were transferred into breeding cages in the institute’s insectary at 23 ◦C (± 2.0 ◦C) and incubated under long-day conditions (L:D = 16:8). Log sections which were stored over winter (2002 to 2003 and 2003 to 2004) in the garden of the institute were incubated after overwintering the same way starting in May. Emerging bark beetles were collected daily and stored together with fresh spruce bark chips in Petri dishes at 15 ◦C (± 1.5 ◦C) in an incubator until further processing (maximum seven days).

abolish the initial diagnosis. All pathogen species were documented photographically. Archival storage of smears was performed in slide boxes. No statistical analyses were conducted due to the high variation in number of dissected beetles and differences in the mode of sampling.

Results The trees or logs were most densely infested by I. typographus every year; the other species (P. chalcographus and I. amitinus) were much rarer. Overall, during the 5-year study 8,620 adult I. typographus were examined, the number of analysed P. chalcographus in the years 2002 and 2003 (n = 855) and of analysed I. amitinus in 2002 (n = 178) was significantly smaller. Many of the well-known pathogens of the three bark beetle species were detected; however, this was not the case for all years, all sampling sites and all sub-samples within each year. Ips typographus From the conservation forest core area ”Rothwald 1” a total of 4,553 individuals were dissected from 2002 to 2005; from isolated “punctiform” collections (punct.) also in the conservation forest core area 1,001 beetles were dissected in 2001 and 2003; from the area “Edelwies” 1,909 individuals were inspected from 2001 to 2004 and from the area ”Hundsau” 1,157 I. typographus were dissected in the years 2001 to 2005 (Table 2). Diversity of pathogen species was variable in the different sampling sites. Gregarina typographi was detected in high infection rates in beetles from all sampling sites and from all samples. The Ips typographus Entomopoxvirus (Weiser & Wegensteiner, 1994), Chytridiopsis typographi (Weiser, 1954; 1970) and Unikaryon montanum (Weiser, Wegensteiner & Žižka, 1998) were also found in the beetles from all sampling sites, but only in much lower rates. Remarkably, the highest pathogen diversity (seven pathogen species) was found in beetles from the area “Edelwies” (Table 2).

Beetle dissection The number of examined beetles depended on colonization density in the bark. The focus of the microscopic analysis was on I. typographus, which were examined during the entire study period. In addition, other bark beetle species emerging from the log sections, P. chalcographus from two locations in the years 2002 and 2003 and Ips amitinus from one location in 2002 were dissected as well. Living adult beetles of all three species were dissected individually on microscopical slides under a stereo microscope (Wild M3C, at magnifications 16× to 25×); the pathogen diagnosis (except entomopathogenic bacteria and fungi) was conducted in a normal light microscope (Reichert Polyvar, at magnifications 40× to 400×). After first diagnosis fresh smears were fixed with methanol and stained with Giemsa’s dye solution (Wegensteiner & Weiser 1996a). In case of uncertain first diagnosis stained smears were re-inspected in the light microscope (at magnifications 400× to 1000×) to confirm or

Rothwald 1 (IUCN-Ia): Five different pathogen species were detected in beetles from the area Rothwald 1: these were the It EPV, the eugregarine, G. typographi, the neogregarine, Mattesia cf. schwenkei (Purrini, 1970), and the microsporidia, C. typographi and U. montanum. The species G. ty-

Table 2. Pathogen occurrence in I. typographus (in %) from four sampling in total from 2001 to 2005. Area R1 R punct. Ew Hu

N

ItEPV

M.s.

G.t.

Matt.

C.t.

N.t.

U.m.

4,553 1,001 1,909 1,157

4.0 0.4 1.8 1.2

– – 0.05 –

36.5 22.9 27.2 41.0

0.09 – 0.05 –

2.1 0.1 2.6 2.9

– – 0.2 –

0.07 0.50 0.05 0.70

Explanations: ItEPV – Ips typographus-Entomopoxvirus, M.s. – Malamoeba scolyti, G.t. – Gregarina typographi, Matt. – Mattesia cf. schwenkei, C.t. – Chytridiopsis typographi, N.t. – Nosema typographi, U.m. – Unikaryon montanum. Sites: R 1 – Rothwald 1, R punct. – Rothwald punctiform sampling, Ew – Edelwies, Hu – Hundsau. N – number of dissected beetles.



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Table 3. Pathogen occurrence in I. typographus males (m) and females (f) (in %) from the sampling site “Rothwald 1” in the core area of the strict reserve from 2002 to 2005. N

ItEPV

M.s.

G.t.

Matt.

C.t.

N.t.

U.m.

Year 2002 2002/03* 2003 2003/04* 2004 2005

m

f

m

f

m

f

m

f

304 76 667 330 563 78

448 81 766 405 731 104

1.3 11.8 8.2 6.1 0.7 –

3.3 7.4 6.1 3.9 0.7 –

– – – – – –

– – – – – –

21.7 77.6 41.2 63.9 28.4 9.0

27.9 74.1 32.2 60.0 27.9 5.8

m

f

m

f

m

f

m

f

0.3 – – 0.3 – –

– 1.2 – 0.2 – –

0.7 1.3 4.9 2.4 1.2 –

0.9 1.2 2.7 2.0 1.0 2.9

– – – – – –

– – – – – –

– – – – – –

– – 0.3 – 0.1 –

Explanations: N – number of dissected beetles (for abbreviations Table 2). * After overwintering in the garden of the institute from 2002 to 2003 and from 2003 to 2004.

Table 4. Pathogen occurrence in I. typographus males (m) and females (f) (in %) from isolated punctiform sampling sites (R-a to R-d) from the core area of the strict reserve in 2001 and 2003. N Year

Sample

2001 2001 2001 2003

R-a R-b R-c R-d

ItEPV

M.s.

G.t.

Matt.

C.t.

N.t.

U.m.

m

f

m

f

m

f

m

w

m

f

m

f

m

f

m

f

16 58 349 57

29 72 358 62

6.3 – 0.3 –

3.4 – 0.3 –

– – – –

– – – –

37.5 8.6 27.5 24.6

31.0 6.9 24.3 11.3

– – – –

– – – –

– – – –

– – – 1.6

– – – –

– – – –

– – 0.3 –

– – 1.1 –

Explanations: N – number of dissected beetles (for abbreviations see Table 2).

Table 5. Pathogen occurrence in I. typographus males (m) and females (f) (in %) from the sampling site Edelwies from 2001 to 2004. N

ItEPV

M.s.

G.t.

Matt.

C.t.

N.t.

U.m.

Year 2001 2002 2002/03* 2003 2003/04* 2004

m

f

m

f

6 406 11 226 84 159

3 500 11 235 93 175

– 2.7 – 1.8 – –

– 3.6 – 0.9 – –

m – – – – –

f

m

– – – 0.4 – –

– 26.6 63.6 19.0 54.8 21.4

f – 30.4 63.6 13.2 63.4 18.3

m

f

m

f

m

f

m

f

– 0.2 – – – –

– – – – –

– 3.0 – 6.2 1.2 0.6

– 2.4 – 3.0 2.2 –

– – – – – 0.6

– – – 0.4 – 0.6

– – – – – –

– 0.2 – – – –

Explanations: N – number of dissected beetles (for abbreviations see Table 2). * After overwintering in the garden of the institute from 2002 to 2003 and from 2003 to 2004.

pographi, C. typographi and the It EPV (not in 2005), were found in beetles from all samples and years and in both sexes. M. cf. schwenkei was only sporadically recorded, in males and in females, whereas U. montanum was found only in two years in females (Table 3). The frequency of It EPV was variable in beetles of both sexes after hibernation in 2002/2003 and 2003/2004. The frequency of G. typographi was much higher in males and females after overwintering. The C. typographi infection rates were also variable comparing the two periods of beetles overwintering (Table 3). Rothwald “punctiform” collections (R punct.) (IUCN Ia): A very limited pathogen species spectrum was found in the “punctiform collected” I. typographus (“R-a” to “R-d”), especially noticeable is the absence of the microsporidium C. typographi (with one exception: “R-d”) (Table 4).

Edelwies (IUCN Ia): In addition to the pathogen species found in the strict core area “Rothwald” two more pathogen species were identified in I. typographus from the area “Edelwies”. The rhizopodan species Malamoeba scolyti (Purrini, 1978; 1980) and the microsporidian species, Nosema typographi (Weiser, 1955) were found only in beetles from this sampling site (Table 5). The number of examined beetles is extremely small in 2001 and of the beetles from the overwintered log sections 2002/2003 and 2003/2004 as well. The frequencies of the individual pathogen species however, were similar to those as observed in the beetles from the area “Rothwald 1” (Table 5; compare Table 3). Comparing the infection rates before and after the winter and then again in the following summer, there were no consistent changes. Similar to the beetles of “Rothwald 1” G. typographi infection rates in males and females from the area “Edelwies” were always higher “after overwintering” compared to those from the pre-


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Table 6. Pathogen occurrence in I. typographus males (m) and females (f) (in %) from the sampling site Hundsau from 2001 to 2005. N

ItEPV

M.s.

G.t.

Matt.

C.t.

N.t.

U.m.

Year 2001 2002 2003 2004 2005

m

f

m

5 170 144 150 54

6 260 166 146 56

– – 2.1 – 14.8

f

m

f

m

– – – – –

– – – – –

– 50.6 63.9 16.7 18.5

16.7 – – – 3.6

f 50.0 61.2 44.6 8.9 21.4

m

f

m

f

m

f

m

f

– – – – –

– – – – –

– – 6.3 1.3 13.0

– 0.4 4.2 1.4 8.9

– – – – –

– – – – –

– – – – –

– 2.7 – 0.7 –

Explanations: N – number of dissected beetles (for abbreviations see Table 2).

Table 7. Pathogen occurrence in P. chalcographus males (m) and females (f) (in %) from the core area of the strict reserve “Rothwald 1” and from “Edelwies” in 2002 and 2003. N Area

G.t.

Matt.

C.t.

Year m

f

m

f

m

f

m

f

Rothwald 1

2002 2003

– 85

2 83

– 9.4

– 2.4

– –

– –

– –

– –

Edelwies

2002 2003

139 154

219 173

13.7 8.4

11.4 5.2

0.7 –

0.5 –

2.9 1.9

3.6 –

Explanations: G.t. – G. typographi, Matt. – M. cf. schwenkei, C.t. – C. typographi. N – number of dissected beetles.

ceding summer and those of the subsequent summer. It EPV infections were not recorded in beetles from the area “Edelwies” “after overwintering”. C. typographi was either not found immediately “after overwintering” (2002/2003) or in a much smaller number than in the previous summer (2003/2004); but C. typographi was diagnosed only in one male beetle in summer 2004 (Table 5). Hundsau (IUCN Ib): Beetles from the area Hundsau showed a pathogen species spectrum (apart from the absence of M. cf. schwenkei) similar to that of beetles from Rothwald 1. G. typographi was found in beetles from Hundsau in all years, C. typographi occurred only in the years 2002 to 2005, and U. montanum only in 2002 and 2004 (the latter species was found only in female beetles). The frequency of pathogens was variable in the five years period, and also in the number of infected female and male beetles (Table 6). Pityogenes chalcographus Pityogenes chalcographus infested trap trees and trap logs were found (together with I. typographus) only in two study areas, and could be analysed in the course of two consecutive years (2002 and 2003). Significantly fewer beetles were analysed from the site “Rothwald 1” compared to the site “Edelwies” because of lower beetle infestation density of the logs (Table 7). Rothwald 1 (IUCN Ia): Only G. typographi was detected in P. chalcographus from the site “Rothwald 1” and only in beetles from the year 2003, with more males than females being infected. Only two female beetles emerged in 2002 without any infection (Table 7).

Edelwies (IUCN Ia): The pathogen species spectrum in P. chalcographus from the site “Edelwies” was clearly more diverse, because in addition to the eugregarine G. typographi also the neogregarine, M. cf. schwenkei, was detected (but only in 2002), and furthermore C. typographi. Both males and females were infected with the three pathogen species, infection rates were higher in 2002 than in 2003 (Table 7). Ips amitinus Ips amitinus was found only in log sections from the area “Edelwies” (IUCN Ia) in one year (2002). The pathogens of this beetle species (71 males and 107 females) were limited to G. typographi (5.6% males) and C. typographi (1.4% males). Discussion The investigated bark beetle species, I. typographus, P. chalcographus and I. amitinus, were expected to represent the majority of attracted beetles checking relatively fresh spruce trees or logs; I. typographus was actually the dominant bark beetle species. Sp¨ ork (1992) also identified I. typographus as the most common bark beetle species in his investigations on the area Edelwies and in the conservation forest (Rothwald). I. amitinus was found in accordance with the results of another study in the conservation forest Rothwald (H¨ andel & Wegensteiner 2003) only in very small numbers. The small observed numbers of P. chalcographus and I. amitinus could be due to a low incidence of these two species or to an interspecific brood competition. On the other hand, already Wild (1953) and Mosbach & B¨ ohler (1983) described the flight of I. typographus preferentially on sunny stock rims. Such open area was given at the lo-



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cation “Edelwies” being a forest regeneration area on an almost open forest clearing. Such aspects are mentioned to be important for the occurrence of bark beetles (Lausch et al. 2011), of many Coleoptera species (M¨ uller et al. 2010) and of insect species in general (Lehnert et al. 2013). Therefore, it can be assumed that different climatic conditions, topographic features, various vegetation and canopy cover played an important role in the colonization of the trees and logs in the wilderness area. In addition, it cannot be estimated if the beetles in the trap trees or trap logs came from the immediate vicinity or from some distance. The migration potential of bark beetles is often underestimated, although already Botterweg (1982) indicated that I. typographus is able to migrate very long distances (up to 8 km) depending on their physiological status. In addition, flight of I. typographus and P. chalcographus was detected frequently also outside forest areas (Duelli et al. 1986). However, the infestation patterns of bark beetles are not completely understood in general as shown in a study in the Bavarian National Park (Lausch et al. 2011). The pathogen species spectrum in different spruce bark beetle species is unclear for the moment. Some pathogen species are considered to be “generalists” as they can occur not only in one but in several bark beetle species. For example, G. typographi is described in I. typographus and P. chalcographus (ref. in Wegensteiner 2004) whereas Dryocoetes autographus (Ratzeburg, 1837) and Ips sexdentatus (Börner,1767) are also indicated as potential hosts (Geus 1969). Similarly, even though C. typographi is found to be occurring in several bark beetle species (Zitterer 2002; Händel et al. 2003; Lukášová et al. 2013); but till now in no case these morphological findings were verified by molecular techniques. However, the transmission of pathogens is also likely to be of great importance. For example G. typographi in any case, has a phase of maturation outside the host organism and thus only after a certain time is available for infection of other individuals, which was confirmed by Lukášová & Holuša (2011). Most probably, it can be assumed that beetles are eating food in their galleries contaminated either with G. typographi or C. typographi or they are coprophagous as suspected by Wegensteiner & Weiser (1996a, b); in the latter case it would be possible for I. typographus to eat faeces loaded with micro-organisms triggered by some nutrition-recyclable material in the faeces. However, these theories can be assumed as very probable, as the gamont-cysts of G. typographi, the pansporoblasts of C. typographi and the occlusion bodies of It EPV are released together with the pathogen loaded faeces and only after an oral uptake of the pathogens by conspecifics become active again. In case this mode of horizontal transmission occurs, it looks to be possible that these pathogen species are causing diseases also nonspecific in associated living bark beetle species because of increased risk of contact in case of high beetle density. This would explain why the same pathogen species can be observed in different bark beetle species living

associated in P. abies (H¨ andel et al. 2003; Lukášová et al. 2013). The pathogen set in the analysed I. typographus from the wilderness area was very diverse with seven species compared to former studies in Austria and in the Czech Republic (H¨ andel et al. 2003; Lukášová et al. 2012); from the well-known species only Menzbieria chalcographi (Weiser, 1955) originally described only in P. chalcographus, later in I. typographus (Wegensteiner & Weiser 2004) was not found in our study. Within previous surveys on pathogen occurrence in I. typographus from punctiform collections in the conservation forest area in the early 1990s, only G. typographi (in relatively high infection rates) and C. typographi (in low infection rates or absent) were recorded (Wegensteiner 1994; Wegensteiner et al. 1996). In a followup study already six pathogen species were detected in I. typographus from two places in the conservation forest Rothwald (H¨ andel 2001). In the present study, three pathogen species were found in P. chalcographus and two in I. amitinus. Händel (2001) found in P. chalcographus four pathogen species (including one not accurately described microsporidium of the genus Unikaryon), and no pathogens were found in I. amitinus most probably due to the low number of investigated beetles. On the other hand Lukášová et al. (2013) found a much more diverse pathogen species spectrum (five species) in I. amitinus from the Czech Republic. Surprisingly the highest pathogen species diversity was observed in I. typographus from the area Edelwies (also in P. chalcographus), which was an open forest regeneration area where phyto-sanitary measures were performed in the past if necessary (till 1991). This raises the question from where the beetles immigrated to the logs on the area Edelwies, with two conceivable possibilities: from the immediately adjacent managed areas and/or from the forest conservation area. Studies on the pathogen occurrence in I. typographus-infested areas in managed and not-managed secondary spruce forests in Switzerland generally showed a much smaller range of pathogen species, and also that salvage logging operations do not lead to a depletion of the local pathogen species spectrum (Wegensteiner et al. 2007). Lukášová et al. (2012) reported three pathogen species only in I. typographus from the Šumava Mountains. Climatic conditions in different areas may favour or interfere with pathogen development, although almost nothing is known about the requirements of the bark beetle pathogens apart from entomopathogenic fungi (e.g., Mietkiewski et al. 1994; in Butt et al. 2001; in Roy et al. 2010). Generally it can be assumed that the risk of infection is most probably influenced by some abiotic factors, particularly by temperature, UV radiation or relative humidity. Such conditions can be even more important to beetles and pathogens in trap trees or trap logs in open space, compared to such material under a protective canopy. Unfortunately it was not possible to record the climatic conditions at the sites; but it looks plausible that the climatic conditions were more favourable for pathogens and more appropriate for their


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viability in cooler and shady locations in the wilderness core area Rothwald. This is in contrast to our results where the highest pathogen diversity was found in beetles from an open and sun-exposed area (Edelwies). The pathogen species composition and abundance of individual pathogen species varied not only in beetles from different locations, but also when comparing beetles from different years. Furthermore, variation was found comparing “summer emerged beetles” with such that emerged immediately “after the subsequent winter” in spring, and compared with those emerging “in the following summer”. These differences were not identical at all locations and not identical for all pathogen species. The reason for the differences in G. typographi and in It EPV can be explained by different development cycles and by a presumed different virulence. The It EPV develops completely intracellular (in cells of the midgut epithelium) in the host and is suspected to be relatively virulent. In contrast, the development of G. typographi and of other Gregarina spp. starts initially in the host (primarily developing intracellular, later sucking cell content of midgut cells) and completes the developmental cycle outside the host organism and is suspected to have low virulence (Tanada & Kaya 1993). No such dynamics were observed for C. typographi although some fluctuations occurred, generally at lower infection levels. Another explanation could be that a decrease in infection rates, may originate in large numerical increase of bark beetle abundance under favourable conditions within a short time period (more rapidly than the development of pathogens). An alternative could be that the total beetle infestation density in the bark might also play a certain role, with an increased chance of contact and an increased risk for infection at higher density. The results of our study are partly in contrast to the results of a previous study on the pathogen occurrence in beetles “before” and “after” overwintering; H¨ andel (2001) described that the It EPV occurred slightly more often “after hibernation” as “before the winter” and G. typographi slightly more often “before winter” than afterwards. This incongruence can be linked to partly low numbers of inspected beetles and to unknown differences in duration of beetles’ stay in their galleries. H¨ andel (2001) also describes a variable occurrence of individual pathogen species and in varying frequencies depending on the season of beetle collections in the conservation forest Rothwald. Significantly higher C. typographi infection rates occurred in an I. typographus laboratory stock compared to the infection rates in beetles from the field, but also varying within the year; however, phloem quality was suspected to have an important influence for seasonal differences in infection rates (Wegensteiner & Weiser 1996b). To some extent, differences in the number of infected males and females were found. This also corresponds to the observations of Wegensteiner & Weiser (1996a) in I. typographus from a conservation area near Freiburg in Germany. Changes in infection rates over the course of several years, however, proceeded with a few exceptions in both sexes always very similar also de-

scribed by H¨ andel (2001). In polygamous living species (e.g., I. typographus, P. chalcographus or I. amitinus) the infection of the males or females can be very significant in case of horizontal pathogen transmission (to or from females or males). At least for G. typographi this kind of horizontal transmission in the nuptial chamber is assumed in I. typographus (Lukášová & Holuša 2011). Concerning sampling method it is evident that removing beetles directly from their mother galleries, if so a statement can be made about the infection status of the parental beetles; a too small number of offspring beetles studied (n < 30) can provide data from an individual breeding system, but cannot be considered representative for an entire local bark beetle population. Taking into account the reproductive potential of an I. typographus or P. chalcographus female and the polygamous breeding characteristics, at least dissecting numbers of > 100 to 200 individuals should be aspired in order to obtain data reliable for a local bark beetle population. Collecting parental bark beetles by cutting them out of the bark is very time-consuming and often connected with mechanical damage of the beetles and is not easy at all for offspring beetles before they emerge. Another method for collecting beetles for infection analysis is the incubation of field-attacked log sections in the laboratory or under semi-field conditions allowing autonomous beetles emergence; this method provides data on infections in parental beetles and in filial beetles separately. However, such data cannot stand for the same emergence data as in the field (e.g., different temperature and moisture regime in the field). To get a sufficient high sample size the mode of beetle sampling must be considered to be important for the interpretation of infection data. This consideration is possibly underscored, e.g., by the absence of C. typographi in beetles of our study from non-recurring punctiform collections in Rothwald 2001 to be insignificant. Another collection method, using pheromone traps for bark beetle collections through several emptying dates (if possible through the entire swarming period of beetles), can give infection data of swarming beetles in a certain locality in a certain period. The exact spatial allocation of beetles from a trap is also not possible, and moribund, non-swarming but infected beetles will be disregarded even though they still may act as disease transmitters. Till now the impact of all the pathogens on bark beetles is unknown. The virulence of a pathogen and the disease progress must play a major role in many activities and on the life span of the host (Tanada & Kaya 1993). These facts together can result in positive or negative effects on the migration capacity of beetles, as shown with G. typographi causing intensified migration in I. typographus (Wegensteiner et al. 2010). How such behavioural changes are triggered is not yet clarified, for wide dissemination of a pathogen it seems certainly favourable. Furthermore, it is not known if any pathogen infection influences the orientation of bark beetles to pheromones, alleviated or intensified. For instance the effect of a microsporidium, Nosema locustae (Canning, 1953) was identified interrupting the



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aggregation pheromone response in Locusta migratoria manilensis (Meyen, 1835) (Shi & Njagi 2004). We achieved the aim to investigate the pathogen species spectrum, which was variable in all three beetle species (I. typographus, P. chalcographus and I. amitinus) from different places within the wilderness area D¨ urrenstein. Especially in I. typographus we found a very diverse set of pathogen species. Ambiguous were the results concerning the presence of some pathogen species which were detected continuously, but at a changing level in the course of several consecutive years; other pathogens were observed sporadically or only once. Despite our results further investigations are necessary to elucidate precisely the life cycles of the pathogens and their requirements. Another focus must be on the effects of pathogens on the bark beetles and on possible interactions between different pathogen species. Understanding all the relationships and the regulative effects in undisturbed forests will help to develop new microbial control measures or control strategies against bark beetles.

Acknowledgements The investigations were financially supported by the Austrian Ministry for Education, Science and Arts within the multidisciplinary project “Dynamics in mountain forests – natural disturbances and regulatory mechanisms” (project number GZ 30.940/1-VI/A/4a/2002). Furthermore, we thank Dipl.-Ing. Dr. Christoph Leditznig, Reinhard Pekny and Hans Zehetner from the wilderness reserve D¨ urrenstein management, and Dipl.-Ing. Johannes Doppler and Thomas Fritz from the der Rothschild’s forest administration in Langau for their support in the project. Thanks to M.Sc. Josef Pennerstorfer (BOKU University Vienna) for creating the maps shown in figure one and to Dr. Bernard Papierok (Paris) for valuable comments on the manuscript.

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