Pathogens of the spruce bark beetles Ips typographus and Ips duplicatus

Cent. Eur. J. Biol. • 4(4) • 2009 • 567–573 DOI: 10.2478/s11535-009-0044-y

Central European Journal of Biology

Pathogens of the spruce bark beetles Ips typographus and Ips duplicatus Research Article

Jaroslav Holusa1*, Jaroslav Weiser1, Zdenek Zizka2

1 Department of Forest Protection and Game Management, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, CZ-16521 Praha 6-Suchdol, Czech Republic

Institute of Microbiology, Academy of Sciences, CZ-142 20 Praha 4, Czech Republic

2

Received 21 May 2009; Accepted 29 July 2009

Abstract: Pathogens of two important bark beetles, Ips typographus and Ips duplicatus, both in outbreaks connected with infestation of spruces by the fungus Armillaria ostoyae, were compared at four localities in the eastern Czech Republic. Low infestations of Chytridiopsis typographi, Nosema typographi, Menzbieria chalcographi, and Gregarina typographi were detected in I. typographus. In I. duplicatus, only C. typographi and G. typographi were found and with low infection levels. The microsporidium, Larssoniella duplicati, was not detected in I. typographus, but was detected in I. duplicatus at all localities in almost 80% of the samples (a sample consisted of 40-50 beetles collected at one locality in one period) and often with a very high infection level (up to 57% of the beetles infected in a sample). The infection level of L. duplicati did not differ between generations of I. duplicatus. I. duplicatus overwinters mainly in the adult stage, and no decrease in the number of infected overwintering I. duplicatus was observed. The relatively constant infection level of L. duplicati suggests that transmission is unlikely to be horizontal via oral ingestion. Keywords: Microsporidia • Gregarina • Ips typographus • Ips duplicatus • Outbreak by Armillaria ostoyae

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction Declining and dead spruce trees have occurred on thousands of hectares in the eastern Czech Republic [1], southern Poland [2], and northwestern Slovakia [3]. In accordance with Manion’s theory, the decline of spruce forests, is a result of predisposing factors and those factors that actually kill the tree. Predisposing factors include low soil pH, nutrient deficiency, water deficit, and mechanical damage by logging machines. Trees stressed by drought are further stressed by decreased water supply because roots break in the drying soil [1]. Once the trees have been stressed and their roots damaged, infection by the honey fungus (Armillaria ostoyae (H. Romagnesi) Herink) become obvious, and the trees are often then colonized and

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killed by bark beetles. The bark beetles Pityogenes chalcographus (Linnaeus, 1761) and Pityophthorus pityographus (Ratzeburg, 1837) are the most abundant in young forests while Ips duplicatus (Sahlberg, 1836), I. typographus (Linnaeus, 1758), I. amitinus (Eichhoff, 1871), and P. chalcographus are predominant in mature forests [1,2]. The pathogens of I. typographus have been studied by many authors [4-26]. On the other hand, only two reports on pathogens of I. duplicatus have been published [27-28]. The recently described microsporidium, Larssoniella duplicati Weiser, Holusa et Zizka, 2006 was observed in I. duplicatus (but not in I. typographus and I. amitinus) in the eastern Czech Republic as well as in its area of origin in northeastern Poland [27-28].

* E-mail: [email protected]

J. Holusa, J. Weiser, Z. Zizka

Both I. typographus and I. duplicatus aggregate on Norway spruce (Picea abies (L.) Karst.), feed on phloem [29], and thus may have similar pathogens. The objectives of the current study were (i) to identify and compare the pathogens of these two bark beetles in one area and (ii) to determine the infestation levels of pathogens over several generations of these bark beetles.

2. Experimental Procedures The study area is situated in the eastern Czech Republic. The four study localities (Table 1) were located in intensively cultivated hills with forest coverage of 9-70% and a dominance of Norway spruce, which constitutes 30-50% of the trees [30]. All spruce forests in this area are planted and managed and have recently been stressed by drought and A. ostoyae [1]. The localities were located at a similar latitude, the distance between adjacent localities ranged from 18 to 28 km, and locality altitude ranged from 380 to 450 m above sea level. I. typographus and I. duplicatus emerge from the forest duff on warm spring days and fly to host trees that are stressed. These species use aggregation pheromone to attract more individuals of the same species to the tree for the purpose of killing the tree and for mating. The pheromone attracts both sexes. The attracted males join the attack and secure an area for mating and oviposition; this area consists of a hole and chamber beneath the bark called a “nuptial chamber”. The females construct a tunnel (“maternal gallery”) beneath the nuptial chamber in which to lay eggs. In all species of the genus Ips, several females join each male in his nuptial chamber [31-32]. Both beetle species have two or three generations per year with the main peaks of bark beetle emergence in April/May, July and August/September [31]. Mature beetles were collected from five trees (adjacent trees were about 100 m apart) in each of the four localities in each of six sample periods. Different trees were used for each sample period because beetle-infested trees are removed by forest managers. Samples periods were selected based on the times when new generations begin, however this is associated with weather and vegetation season. In 2002, the sampling periods were 25-27 May, 29-30 July, and 17-19 September. In 2003, the sampling periods were 16-27 May, 26-27 June, and 27-30 August. The beetles were collected from trees that had been recently invaded by beetles and that had signs of infestation by A. ostoyae (bulging trunk base, root decay, and syrrocium under bark). In spring (the first sampling

period), adult beetles of the parental generation (P) were collected as they emerged from their nuptial chambers in the tree trunks. In summer (the second sampling period), the beetles of the parental generation (P) were collected by removal from the maternal galleries and offspring adult beetles of F1 generation were also collected. Thus, in the summer beetles of two generations were collected from the same tree. Only mature beetles of the F1 generation at the end of maternal feeding were studied because beetle pathogens become easier to detect as the beetle mature [13]. At the end of summer (the third sampling period), we collected mature beetles of the F1 generation after they had mated and laid eggs and mature beetles of the F2 generation (Figure 1). The same collection methods were repeated in the second year of study. In total, five generations (P, F1, F2, F3, F4) of beetles were collected from the trees. For each locality and sample period, 40(-50) beetles of each species were collected. Sometimes it was problematic to find mature beetles that had already left galleries or were not mature, but this was mainly limited to the locality of Bystřice nad Olší. Beetles and associated bark were stored in small film boxes (volume of 30 cm3) at 4°C and were dissected within 10 days. Because the beetles were inactive at 4°C, transmission of diseases among beetles within one box was not possible. 2002 May

July

P

P F1

2003 September

May

June

F2

F2

August

F1 F2

F3

F3 F4

Figure 1.

The generations of adult bark beetles in six sampling periods in 2002 and 2003 (P = parental; F1 = offspring of P; F2 = offspring of F1 and overwintering generation for 2003, and so forth).

Given the specific interactions between L. duplicati and their host beetles [27], only beetles that were living when collected were dissected. Midguts of all beetles were removed with the last abdominal segment and were examined according to the method of Wegensteiner et al. (1996) [15]. The beetles were decapitated and dissected in a drop of water on a microscopic slide, and the whole gut together with the Malpighian tubules, the gonads, and parts of the adipose tissue were examined for the presence of pathogens using a light microscope (150x and 500x magnification). To estimate the abundance of bark beetles, the presence of bark beetles in 50 trees that had been infested by beetle, cut, and left on the forest floor were 568

Pathogens of the spruce bark beetles Ips typographus and Ips duplicatus

examined in each of the six sampling periods. For each of these trees, the beetle entry holes were counted on four 1 m sections; the sections were located in the middle of the crown, at the base of the crown, in the middle of trunk below the crown, and near the trunk base. The levels of pathogen infection between the two species of bark beetle were compared with a twosample t-test. Infection levels between generations were compared by Kruskal Wallis one-way analysis of variance. Regression and all other analyses used α=0.05 and were performed with the Statistica 8.0 [33]. In the analyses, the term infection level indicates the percentage of beetles infected in each sample of beetles, and the term constancy indicates the percentage of samples with at least one infected beetle.

3. Results During the two years of sampling (2002 and 2003), 715 and 653 specimens of I. typographus and 863 and 588 specimens of I. duplicatus were dissected

Beetle species

Locality

Latitude; Longitude

Number of beetles (P/F1/F2/F3/F4)

Pustá Polom

49°48-52'; 17°58'18°04'

Václavovice

and inspected. In total, five species of pathogens were diagnosed (Table 1). The microsporidium Chytridiopsis typographi (Weiser, 1954) and the gregarine Gregarina typographi (Fuchs, 1915) were found in both bark beetles, but the microsporidium Nosema typographi (Weiser, 1954) and the gregarine Menzbieria chalcographi (Weiser, 1955) were found only in I. typographus. The microsporidium L. duplicati was detected only in I. duplicatus. Mixed infections were also observed in the studied hosts. In I. typographus, the infection levels of the pathogen species differed slightly between sampling localities (Table 1). N. typographi and M. chalcographi infection was very low and rare: in both cases, they were found in only two beetles (≤5% of the beetles sampled). In 2003, no case of infection with N. typographi or M. chalcographi was found. Infection by C. typographi was found in 17.5% of the samples although its infection level was rather low in most cases (≤5%) and never exceeded 25%. C. typographi was even missing in some localities. G. typographi was present in 35% of the samples, and its mean infection level was lower than 5%. Occasionally it reached 20% (Table 1). Pathogens and infection levels (%) Chytridiopsis typographi

Nosema typographi

Menzbieria chalcographi

Larssoniella duplicati

Gregarina typographi

455 (80/80/145/100/50)

2.6

0.0

0.003

0.0

4.3

49°44'; 18°21'

420 (80/80/120/100/40)

0.0

0.2

0.0

0.0

0.7

Jánské Koupele

49°44'; 17°43'

375 (80/85/150/40/40)

0.3

0.3

0.0

0.0

0.5

Bystřice nad Olší

49°36´; 18°43'

118 (40/40/0/0/38)

0.0

0.0

0.0

0.0

1.6

Constancy

17.5

5.0

5.0

0.0

35.0

Ips typographus

Ips duplicatus Pustá Polom

49°48-52'; 17°58'18°04'

422 (80/80/120/92/50)

10.6

0.0

0.0

25.0

1.4

Václavovice

49°44'; 18°21'

370 (50/80/120/80/40)

0.0

0.0

0.0

21.8

1.6

Jánské Koupele

49°44'; 17°43'

416 (90/86/120/80/40)

0.9

0.0

0.0

12.6

0.0

Bystřice nad Olší

49°36´; 18°43'

243 (40/40/83/40/40)

0.0

0.0

0.0

3.4

0.0

Constancy

10.0

0.0

0.0

78.0

28.0

Table 1. Infection of adult bark beetles Ips typographus and Ips duplicatus by five pathogens in four localities. Infection levels are the percentage of

beetles infected and the total number of beetles collected over six sample periods in 2002 and 2003. Constancy indicates the percentage of samples with at least one infected beetle.

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In I. duplicatus, C. typographi was found in only 10% of the samples and in only two localities; these samples had very low infection levels. G. typographi was also present in only 10% of the samples and with low mean infection levels (≤5%); its highest infection level in a sample was 30% (Table 1). Another microsporidium, L. duplicati, was present in I. duplicatus in all localities in almost 80% of the samples and often with a very high infection level (as high as 57%; Table 1). Infection levels of C. typographi were equal in both bark beetles species (t=1.21; P>0.10), but L. duplicati was found only in I. duplicatus (t=-6.55; P0.05; I. duplicatus: C. typographi: r=0.06; P>0.10; L. duplicati: r=0.68; P>0.05; G. typographi: r=0.24; P>0.10). Infection levels of particular generations in both species were very low for most pathogens and varied between 0 and 6% for C. typographi and G. typographi. Differences in infection levels among generations were not significant (C. typographi in I. typographus: χ2=5.13, P>0.10; G. typographi in I. typographus: χ2=12.82, P>0.10; C. typographi in I. duplicatus: χ2=13.71, P>0.10; G. typographi in I. duplicatus: χ2=6.08, P>0.10).

Infection levels of L. duplicati among generations varied between 5 and 35% (Figure 2), but differences among generations were not significant (χ2=13.59, P>0.05). Post hoc tests did not show differences between generations (Kruskal-Wallis test: H (8, n=30)=11.98; P>0.10.

4. Discussion Fewer pathogens of I. typographus were detected in this study and in Holusa et al. (2007) [28] than in some former studies. Two pathogens appearing in I. typographus in the Sumava Mountains [34] and in Austria [10,15,21-23] were absent in our samples: Entomopoxvirus typographi Wegensteiner and Weiser, 1995 and Malamoeba scolyti Purini, 1980. The microsporidium Unikaryon montanum (Weiser, Wegensteiner & Zizka, 1998), known from the foci in Austria and Germany [3,19], also was not found in our study. Infection rates of C. typographi and G. typographi varied little among samples. They were very similar to those recorded by some authors [14,15,21,23]. Although both pathogens often occur together with high infection levels [35], a high infection level is also possible when only one pathogen is present, e.g., in G. typographi [24]. Low prevalence of N. typographi is common [6,14,22,34]. Infection by M. chalcographi is rare but can be very high in some cases [23,34].

Figure 2. Larssoniella duplicati infection levels in five consecutive generations of Ips duplicatus in the eastern Czech Republic. The inner square, the mean; the rectangle, ± SE; the bars, ± SD).

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Although infection levels should depend on the population density of bark beetles, infection levels in this study were not related to the abundance of bark beetles as represented by the volume of infested trees in the different localities. As noted by Wegensteiner and Weiser (1996) [21], however, the different levels of pathogen infection in an area may not correlate with different numbers of beetles when the beetles in that area represent one, highly mobile population. The potential mobility of beetles can prevent the development of beetle subpopulations with different pathogen sets [22]. Because the localities in the present study were only separated by about 20 to 30 km, the bark beetles could have readily moved from one locality to another; beetles are known to migrate regularly more than 1 km [32,36]. It follows that the bark beetles present in the entire study area (encompassing all four localities) represented one population for each species, and each of the two bark beetle populations in this study had a relatively constant pathogen infection level among the different localities. As discussed below, patches or subpopulations with very large numbers of beetles can occur when groups of beetle-infested trees are left in the forest, which was not the case in this study. Except for L. duplicati, pathogens were not abundant and infection levels were generally low in our study. This could be due to the low number of beetles in sampled at Bystřice nad Olsi. We suspect, however, that this arises from forest management and to the distribution of stressed trees. How a forest is managed likely affects the buildup of bark beetles and the pathogens of bark beetles. In unmanaged or poorly managed forests, trees infested by bark beetles usually occur in groups which generate hot spots of high beetle and pathogen numbers. The repeated development of new generations of bark beetles is common in these groups of infested trees and beetle numbers can remain very high over many generations. When their numbers are very high, young beetles often cannot find enough space for their maturation feeding, and are forced to continue feeding by crossing other nearby galleries. Such movement increases the chance of pathogen transmission and therefore results in an increase in pathogens [13]. Consequently, groups of infested trees generate hot spots of high beetle and pathogen numbers. This is the case in the Sumava Mountains (Czech Republic) and Austria localities, where C. typographi and Entomopoxvirus typographi can be abundant pathogens of bark beetles [15,28,34]. In contrast, beetle infestations in well-managed forests, like the forests in our study, are more patchy because foresters rapidly remove infested trees. Removal of trees heavily infested by bark beetles prevents the kind 571

of pathogen buildup that occurs in unmanaged forests. This has led to a substantial decrease in pathogens of bark beetles and even the disappearance of N. typographi and M. chalcographi in 2003 (this study) and 2004 [28]. Another reason why groups of trees with bark beetles were observed only occasionally during this study was that beetles usually infested individual spruce that were scattered in forests and were stressed and dying from drought and infection by honey fungus [1,37]. In some periods the key mortality factors are still drought [38] and infection by the honey fungus [1] rather than bark beetles. Given the removal of beetle-infested trees by foresters and the patchy nature of the infestation (as related to stress by drought and the honey fungus), it is perhaps not surprising that pathogens (except L. duplicati, see further) were not abundant and infection levels were generally low. Differences in the infection levels of C. typographi and G. typographi among generations in both bark beetle species were not significant. This could result from a low infestation level, but even when infection levels were higher, the numbers of infected beetles did not differ between generations [13]. L. duplicati is definitely specific to I. duplicatus [27-28]. L. duplicati can achieve a high infection level in I. duplicatus (present study, [28]) and the high infection level frequently occurs throughout the beetle’s range [28]. Infection levels did not show any trend over time in our study and has also been reported previously in one locality [28]. The lack of difference in the infection level of L. duplicati between bark beetle generations was confirmed by our study. I. duplicatus overwinters mainly in the adult stage [31,39], leading one to expect a decrease in the percentage of overwintering infected adults because of death resulting from infection. In an earlier study, high winter mortality resulted in relatively low numbers of living infected beetles in spring [13]. A relatively constant infection level of L. duplicati is consistent with the hypotheses that transmission is not horizontal or the result of oral ingestion of pathogen propagules [28]. In summary, C. typographis and G. typographi were detected in both I. typographus and I. duplicatus. These pathogens are known to overlap in host range and have been observed in various beetle species [22,26,35]. The microsporidium L. duplicati was very common in I. duplicates. The high infection level occured throughout the beetle’s range and did not show any trend over time [28]. N. typographi and M. chalcographi were found only in a few specimens of I. typographus at the beginning of our study. Their disappearance could be a result of very intensive forest management and elimination of bark beetle hot spots.


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