Coleoptera: Chrysomelidae) in North Turkey

Distribution and occurrence of microsporidian pathogens of the willow flea beetle, Crepidodera aurata (Coleoptera: Chrysomelidae) in North Turkey

Mustafa Yaman, Goniil Algl, Beyza Gonca Gfiner & Sabri Una]

Yaman, M., Alg1, G., Guner, B. G. & Unal, S. 2015: Distribution and occurrence ofmicrosporidian pathogens ofthe willow flea beetle, Crepidodera aurata (Co- leoptera: Chrysomelidae) in North Turkey. — Entomol. Fennica 26: 171—176.

In this study, microsporidian pathogens in Crepidodera aurata populations were investigated. Totally 1,728 C. aurata adults were examined for microsporidian pathogens and 78 of them were found to be infected. Two species of microsporidia; Microsporidium sp.1 and Microsporidium sp.2 were observed in the C. aurata populations from ten localities in North Turkey. They show considerable difference from each other in the spore morphology and dimension, infection rate and host locality. The spores ofMicrosporidium sp.1 were oval in shape and measured from 3.66 to 5.66 um in length and from 1.35 to 2.22 um in width (n=50). The spores of Microsporidium sp.2 were slightly curled and measured from 2.44 to 3.55 um in length and from 1.25 to 1.55 um in width (n=50). These microsporidia were recorded from C. aurata for the first time. Here we present occurrence and distribution oftwo microsporidia in C. aurata populations as natural potentially suppressing factors.

M. Yaman, G. Algi & B. G. Gu'ner, Department ofBiology, Faculty ofScience, Karadeniz Technical University, 61080, Trabzon, Turkey; Corresponding au— thor’s e—mail.‘ muyaman@lz0tmail. com S. Unal, Department ofForest Engineering, Faculty ofForestry, Kastamonu University, Kastamonu, Turkey Received 11 March 2015, accepted 2 June 2015

1. Introduction 2009, Urban 201 1). It is an important pest species of poplars and willows in different countries The family Chrysomelidae, one of the largest (Aslan et al. 1999, Mikhailov & Hayashi 2002, families of the order includes Czemiakowski 2005 Urban 201 Urban Coleoptera, phyto- , 1). (201 1) phagous insects. A member of this family, the mentioned that this species is the most important willow flea beetle Crepidodera aurata Marsham species of the genus in forestry. Alternative to is a widely distributed species (Aslan et al. 2004, pesticides, biological control strategies or sup- Urban 201 1). This species naturally occurs in Eu- pressing factors have not been yet searched to rope, northern Africa, Caucasus, Anatolia (Tur- find possible effective methods to struggle this key), Iran, Kazakhstan, Siberia, Mongolia, China pest. and Korea (Aslan et al. 1999, Aslan & War- The members of the family Chrysomelidae chalowski 2005, Baselga & Novoa 2007, Bukejs are frequently infected by pathogenic protists. 172

WAkyam * o u

Fig. 1. Localities m """" Where Crepidodera 360 km auraz‘a adults were col-— Booted in Turkey.

Pathogenic and parasitic organisms infecting this section. pins. ct smears were examined under a family have aroused interest as potentially sup-«- microscope for identification of microsporidian pressing factors of populations (To-n pathogens. an infection was observed9 the guebaye er a2 1988913011131” 19889 Theodorides slides were aha-dried and fixed with methanol for 19889 & Lipa 19959 Yaman 200239.b., 3 min. They were then washed with distilled wan 20049 20079 20089 Yaman 251:2... adek 20039 ter9 stained for approximately ten. hours in freshly 62‘ ad. 20049 er al. 20089 Yaman er at! prepared 5% solution of Giemsa stain, washed in 200839 2011a9b). Several new pathogen species running tap water and airmdried (Toguebaye er al. from different groups have been isolated and 1988). Giemsamstained preparations were then characterized from these insects recently (Yaman care Hy reexamined under a microscope. ’ Radek 2003 Yaman er a1. How- tected fresh and stained, were measured 9 2008b9 2010). spores even there is no record on the entomopathogenic protists infecting C. aurora. croscope with a DPn—25 digital camera and a 1n the present study we aimed at searching for S Soft imaging System. the first time microsporidian pathogens and docum- menting their occurrence and distribution. in the ' populations of C. aurora as natural potentially a BS “S suppressing factors of this important pest. During the study9 1W0 possibly new micro-w sporidian species were observed in the C. aurora ’ ES populations. These microsporidia showed considerable difference in the spore m0. 29L Insect I The first icrosporidium (Isolate 1) was obn- served in Vezirképrfi, Samsun provience. The Totally 19728 C. durum adults were collected spore of this microsporidium was oval in shape from a Wide geographical area (especially most and measured from 3.66 to 5.66 11min length and poplar breeding areas from a, piece of land of from 1.35 to 2.22 “mm Width (11:50). The second 7799000 with a high sampling ratio (10 10... microsporidian species (Isolate 2) was observed cahties and 26 samplings) in Turkey from April in the rest ofthe investigated localities. The spore to September during 2013 and 2014 (Fig. 1). of this microsporidium was slightly curled and measured from 2.44 to 3.55 pm in length and from 1.25 to 1.55 pm in Width (r1250).

""" 22.2 mmscou emmafio observed microsporidial infections in seven ofthe ten investigated localities in the both Each of C. durum adults was placed individually years (Table 1). Isolate 1 showed infection from into a small drop of water on a microscopic slide 1.7 to 5% and isolate 2 from 0.8 to 25%. Total 111- and dissected in Ringer’s solution by using dis- fection was 5.4% in 2013 and 4.5% in 2014. The ENTOMOL. FENNICA Vol. 26 ° Microsporidia ofa chrysomelid beetle 173

Table 1. Occurrence of rnicrosporidian pathogens in populations of Crepidodera aurata (Coleoptera: Chrysomelidae) in Turkey.

Locality No. of examined Sampling No. of Infection beetles date infected rate (%) beefles

Samsun 1-Vezirkopr'u 118 25.4.2014 2 1.7 40 05.9.2014 2 5 2-lrmaks1rt1 30 03.6.2013 2 6.6 100 23.4.2014 9 9 84 01.6.2014 8 9.5 44 28.6.2014 11 25 14 28.6.2014 0 0 20 20.9.2014 0 0 3-K1z1lot 46 27.3.2013 3 6.5 87 01.6.2014 5 5.7 118 24.6.2014 6 5 39 27.6.2014 2 5.1 14 28.6.2014 0 0 12 06.9.2014 0 0 2 20.9.2014 0 0 4-Havaalan1 66 24.4.2014 6 9.1 109 01.6.2014 4 3.6 89 28.6.2014 6 6.7 Kastamonu 16 14.4.2013 0 0 113 26.4.2014 1 0.8 201 28.6.2014 4 2 Kocaeli 71 02.5.2014 0 0 Akya21 62 03.5.2014 0 0 Bilecik 65 19.5.2014 0 0 Bursa 65 17.5.2014 2 3.1 Bolu 103 16.5.2014 5 4.8

Total 1,728 78 4.51

infection rates for both pathogens were variable taxonomic character for microsporidia taxonomy between the localities (Table 1) and reached 25% (Sprague et al. 1992). It seems that at least one in some localities. We did not observe any infec- Microsporidium species infects C. aurata. On the tion in three (Kocaeli, Akyaz1 and Bilecik) ofthe other hand, spore morphology and size, host 10- investigated localities. More details on the occur- cality and infection rates are also important char- rence and distribution of the rnicrosporidian acteristics to discriminate microsporidia infecting pathogens are given in Table 1. insects (Larsson 1999, Yaman & Radek 2003, Yaman et al. 2008b). Two rnicrosporidian pathogens found in the C. aurata populations show 4. Discussion considerable differences from each other in the spore shape and size (Table 2) as well as in infec- In this study, two rnicrosporidian pathogens, Iso- tion capability (1.7 to 5% Isolate 1 and 0.8 to 25% late 1 and Isolate 2, are presented from C. aurata for Isolate 2) and locality (Isolate 1 in Vezirképrii populations for the first time. To date there is no and Isolate 2 in the rest ofthe investigated locali- rnicrosporidian report from C. aurata. Micro- ties) of the host population. Similarly, two new sporidia have been known relatively host specific microsporidia were identified from the popula- and the host affinity can be accepted as a valid tions of Chaetocnema tibialis (Illiger) (Coleop— 174 Yaman et al. ° ENTOMOL. FENNICA Vol. 26

Table 2. Microsporidian species first found in Chrysomelidae (Coleoptera).

Species Spore size and Infected organ Host shape (pm)

Nosema phyllotretae 4.2 X2—3 Adipose body Phy/lotreta atra Weiser, 1961 Phy/lotreta undulata Nosema gastroideae 3—4.8X2.5—3 Overall infestation Gastrophysa polygoni and Hostounsky & Weiser, 1973 several experimental hosts Nosema polygrammae 4.8 X2.05 Gut Polygramma Hostounsky & Weiser, 1975 undecem/ineata Nosema equestris 4—5><3 General infestation Gastrophysa viridu/a Hostounsky & Weiser, 1980 Leptinotarsa decem/ineata Nosema coui/Ioudi 3.4—4X1—1.5 Gut Nisotra sp. Toguebaye & Marchand, 1984 Nosema birgii 6.2><3.5 Eggs and general Mesop/atys cincta Toguebaye & Marchand, 1986 infestation, larvae and imago Nosema nisotrae 5.8X3.1 General infestation Nisotra sp. Toguebaye & Marchand, 1989 Nosema ga/eruce/Iae 4.95X2.89 Gut principally, Ga/eruce/Ia luteo/a Toguebaye & Bouix, 1989 adipose body, muscles, tracheae, malpighian tubules Nosema chaetocnemae 3.52X2.09 Gut, tracheae, Chaetocnema tibia/is Yaman & Radek, 2003 muscles, malpighian tubules Nosema tokati 3.82X1.3 Malpighian tubules Chaetocnema tibia/is Yaman et a/., 2008 Nosema leptinotarsae 2—5X1.9—3.3 Haemolymph Leptinotarsa decem/ineata Lipa, 1968 Nosema leptinotarsae 4.69><2.43 General infestation Leptinotarsa decem/ineata Yaman eta/., 2011 Unikaryon bouixi ovoid Gut and malpighian Euryope rubra Toguebaye & Marchand, 1983 1.6—2.5X1.5—1 .6 tubules Unikaryon matteii ovoid gut, malpighian Nisotra sp. Toguebaye & Marchand, 1984 3.72X1.96 tubules and muscles Unikaiyon nisotrae ovoid gut and adipose Nisotra sjoestedti Toguebaye & Marchand, 1986 2.33X1.66 tissue Unikaryon phy/Iotretae spherical to Malpighian tubules Phy/lotreta undulata Yaman eta/., 2010 ovoid, 3.80X1.90 Microsporidium sp.1 ovoid Haemolymph Crepidodera aurata (this study) 3.66—5.66X1.35—2.22 Microsporidium sp.2 Slightly curled Haemolymph Crepidodera aurata (this study) 2.44—3.55X1.25—1 .55

tera; Chrysomelidae) in different localities sporidia infecting the family Chrysomelidae in (Yaman & Radek 2003, Yaman et al. 2008b). spore size, type of infected tissue, infection rate, Yaman et al. (2008b) observed different infection host affinities and locality of the host population rates between Nosema chaetocnemae Yaman & (Table 2). The Isolate 2 from C. aurata clearly Radek and N. tokatz' Yaman, Radek & Togue- differs in spore size from microsporidian species baye, infecting C. tibialis populations in Turkey. infecting chrysomelids (Table 2). To identify There are different microsporidian species re- both isolates at the species level we need to com- corded from the members of the family Chryso- plete some morphological and ultrastructural melidae. The results show that the microsporidian studies on the different life stages and molecular pathogens presented here differ from micro- phylogeny on the microsporidian pathogens in- ENTOMOL. FENNICA Vol. 26 ° Microsporidia ofa chrysomelid beetle 175

The was fecting chrysomelids. For now, we consider our Acknowledgements. study financially supported as a research the Scientific and organisms, tentatively classified in the collective project by Technological Council ofTurkey (1120807). Authors are thankfiil to the to be two distinct, undes- group Microsporidium, reviewers for their kind efforts to improve the manuscript. cribed species and call Isolate 1 as Micro— sporidium sp.1 and Isolate 2 as Microsporidium sp. 2. Further studies, however, are needed to fi- References nally resolve the taxonomic status of the new Aslan, I., Gruev, B. & Czbek, H. 1999: A preliminary re- from C. aurata. Here we are fo- microsporidia view of the subfamily Alticinae (Coleoptera, Chryso— cused on the occurrence and distribution of melidae) in Turkey. 7 Turkish Journal ofZoology 23: Microsporidium sp.1 andMicrosporidium sp.2 in 3737414. the C. aurata populations from several localities Aslan, 1., Czbek, H. & Warchalowski, A. 2004: Five new records, new localities and new host plants for the Tur— in a wide geographical area in Turkey. kish flea—beetle fauna (Coleoptera: Chrysomelidae: infections were observed in Microsporidial Alticinae). 7 Entomologica Fennica 15: 1387141. the seven often localities in the both investigated Aslan, I. & Warchalowski, A. 2005: Longitarsus ozbeki sp years (Table 1). Yaman (2007) observed Nosema n., a new species from Asia Minor (Coleoptera : Chry— meligetlzi infection in only two of the seventeen somelidae: Alticinae). 7 Entomologica Fennica 16: 2217224. Meligetlzes aeneus (Fabricius) (Coleoptera: Niti- Ayd1n, C., Yaman, M. & Tosun, O. 2009: Distribution of in In dulidae) populations investigated Turkey. Nosema phyllotretae (Microspora, Nosematidae) another Yaman found Nosema in- study, (2008) Weiser, 1961 in populations ofPhyllotreta arm (Cole— fection in Chaetocnema tibialis (Coleoptera: optera, Chrysomelidae) in Turkey. 7 Tiirkiye Parazi- Chrysomelidae) adults from two ofthe ten locali- toloji Dergisi 32: 1657168. A. & Novoa, F. 2007: of ties. Further, Ayd1n et al. (2009) observed No— Baselga, Diversity Chrysomeli— dae (Coleoptera) at a mountain range in the limit ofthe Weiser infection in semaphyllotretae Phyllotreta Eurosiberian region, northwest Spain: species rich— arm 7 (Fabricius) (Coleoptera: Chrysomelidae) ness and beta diversity. Entomologica Fennica 18: populations from only one of the five localities. 65773. The results confirm that the microsporidian Bukejs, A. 2009: To the knowledge of flea beetles (Cole— optera: Chrysomelidae: Alticinae) of the fauna of in the present have an extensive pathogens study 7 Latvia. Acta Zoologica Lituanica 19: 1097119. distribution in the of C. aurata. How- populations Czerniakowski, Z. W. 2005: Noxious insects in energy ever, we did not observe any infection in C. willow coppice nurseries. 7 Progress in Plant Protec- aurata populations from the three localities tion 45: 19724. M. & I. 2008: Effects of four (Kocaeli, Akyaz1 and Bilecik) which are close to Ertiirk, 0., Yaman, Aslan, Bacillus on the Colorado each other geographically (Fig. 1). soil-originated spp. potato beetle, Leptinotarsa decemlineata (Say). 7 Entomo— Total infection rate for both microsporidia logical Research 38: 1357138. was 4.51%, varying among the localities from 0.8 Hokkanen, H. M. T., Lipa J. J. 1995: Occurrence and dyna— to 25% (Table 1). Potentially high infection is mics of Nosema meligethi (Microsporida) in popula— promising for biological pest control. Crepz'do— tions of Meligethes aeneus (Coleoptera, Nitidulidae) in Finland. 7 Entomologica Fennica 6: 11718. dera aurata has one generation per year. Beetles Hostounskl, Z. & Weiser, J. 1973: Nosema gastroideae sp. overwinter as imago and lay eggs in spring. Lar- n. (Nosematidae, Microsporidia) infecting Gastrophy- vae summer and in the end ofAu- develop during sa polygoni and Leptinotarsa decemlineata (Coleop— gust, and the new generation appears (Aslan et al. tera: Chrysomelidae). 7 Acta Entomologica Bohe- 1999, Urban 2011). Therefore, the beetles of C. moslovaca 70: 3457350. Hostounskl, Z. & Weiser, J. 1975: Nosema polygrammae aurata occur on poplars and willows from April sp. n. and Plistophora fidelis sp. n. (Microsporidia, to October and skeletonize leaves of these plants. Nosematidae) infecting Polygramma undecimlineata During this developmental period, any disease (Coleoptera: Chrysomelidae) in Cuba. 7 Vistnik Fes— factor affecting population density is of great im- koslovenské Spoleenosti Zoologicke’ 39: 1047110. portant. The microsporidia presented here are the Hostounskl, Z. & Weiser, J. 1980: A microsporidian infection in Curcu— first recorded from C. aurata. This Otiorrhynchus equestris (Coleoptera,

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