Evidence for Wolbachia in Leafhoppers of the Genus Eupteryx with Intersexual Morphotypes
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Bulletin of Insectology 66 (1): 109-118, 2013 ISSN 1721-8861 Evidence for Wolbachia in leafhoppers of the genus Eupteryx with intersexual morphotypes 1 2 2 2 Catarina HENKE , Herbert NICKEL , Stefan SCHEU , Ina SCHAEFER 1Campus Klein-Altendorf, Rheinische Friedrich Wilhelms University Bonn, Germany 2J.F. Blumenbach Institute of Zoology and Anthropology, Georg August University Göttingen, Germany Abstract Leafhoppers (Hemiptera Cicadellidae Typhlocybinae) of the genus Eupteryx are important pests on medical and culinary herbs including sage (Salvia officinalis L., Lamiaceae), causing severe economic damage. Individuals of Eupteryx decemnotata Rey and Eupteryx melissae Curtis show a modified genital morphology at two geographically distant populations in Germany (Bonn and Göttingen). Typical female and male sexual characters are merged. In another species of Typhlocybinae a similar intersexual phe- notype, representing feminized males, was explained by Wolbachia infection. We investigated E. decemnotata and E. melissae from both locations for infection by a molecular screening study (PCR) with three Wolbachia specific genes (16S rRNA, ftsZ, wsp). The screening strongly supports Wolbachia infections in both host species in Göttingen and in E. melissae from Bonn. Phy- logenetic analyses of the ftsZ, wsp and the host-specific COI gene indicate a single infection in E. melissae, but infection with two different strains in E. decemnotata and host-mediated distribution of Wolbachia. Further, the data indicate horizontal Wolbachia transmission between these leafhopper species. This is the first study demonstrating the presence of Wolbachia in Eupteryx leaf- hoppers. Rapid spread of Wolbachia in Eupteryx populations can potentially threaten sage cultivations if morphologically modi- fied individuals represent feminized males, thereby increasing the reproductive potential of infected populations. We discuss pos- sible implications of Wolbachia infection inducing a feminoid phenotype for the population dynamics of leafhopper pests. Key words: feminization, reproductive parasites, horizontal transmission, molecular screening, plant pest, medical herbs, spice herbs, Wolbachia supergroup. Introduction tions in its host (Werren, 1997; Stouthamer et al., 1999), such as cytoplasmic incompatibility, parthenogenesis, Leafhoppers (Hemiptera Cicadellidae Typhlocybinae) male-killing and feminization of karyotypic males of the genus Eupteryx are important pests of medical (Hurst et al., 1999; Rousset, 2000; Hiroki et al., 2002; and culinary herbs. Originally native to Mediterranean Hunter et al., 2003). Wolbachia are transmitted mater- countries, some of these insects expanded their ranges nally and the modifications on the host enhance vertical rapidly in central Europe in the 20th century through transmission within host populations by increasing the commercial trade of their host plants. Species produce frequency of infected individuals (cytoplasmic incom- up to three generations per year and cause modest to se- patibility) or by inducing female-biased sex ratios vere damage to cultivated herbs (Dachler and Pelzmann, (Hurst et al., 1999; Stouthamer et al., 1999). Wolbachia 1999; Vidano and Arzone, 1976; Nusillard, 2001; strains are divided into eight supergroups (A-H) with a Nickel, 2003). Damage of plants by Eupteryx results wide range of host species including nematodes and ar- from piercing the leaf parenchyma which causes loss of thropods, but are particularly numerous in insects (Wer- assimilation tissue and leaf stippling (Pollard, 1968; ren et al., 1995; Zhou et al., 1998; Lo et al., 2007). 1969). Such damages have been reported from cultiva- Horizontal transmission has been increasingly detected tion sites in Portugal, Switzerland, Austria, Slovenia, in a variety of species but transmission routes are little Greece, UK, Germany and the northern USA (Nickel understood (Breeuwer and Jacobs, 1996; Werren and and Holzinger, 2006; Rung et al., 2009). Cultivation of Bartos, 2001). Established infections with Wolbachia sage (Salvia officinalis L.) is particularly affected since accompanied by altering karyotypic males into func- this plant is of high pharmaceutical value and therefore tional females (feminization) can strongly accelerate cultivation is expanding (Hoppe, 2005). So far available population growth due to higher frequency of reproduc- pest control strategies are insufficient in both organic tive individuals. For detection and characterization of and conventional agriculture (Röhricht, 2005; Jung, Wolbachia infection a common approach is to use 2009). multi-locus sequence typing (MLST) introduced by We investigated populations of both Eupteryx decem- Baldo et al. (2006). notata Rey and Eupteryx melissae Curtis with a high In this study, we tested two populations of E. melissae proportion of individuals with a novel malformation of (from Bonn and Göttingen, Germany) and one popula- the female ovipositor. In Italy Negri et al. (2006) found tion of E. decemnotata (from Göttingen, Germany) that a similar malformation in the leafhopper Zyginidia positive for Wolbachia infection by polymerase chain pullula (Boheman) was induced by the reproductive reaction (PCR) screening, using three Wolbachia parasite Wolbachia. housekeeping gene loci i.e., 16S rRNA, ftsZ and wsp. At Wolbachia is a group of intracellular inherited bacteria both locations individuals with modified genital charac- and well known as agents of various reproductive altera- ters had been observed since 2008. In the following we shall refer to these individuals as “feminoid” since their amplified gene 1-2 µl BSA (3%); H2O was added to a karyology has not yet been studied. Additionally, the final volume of 25 µl. All PCR conditions included an host-specific COI gene was sequenced from all investi- initial activation step at 94 °C for 15 min and a final gated individuals. With phylogenetic analyses of all four elongation step at 72 °C for 10 min. genes we ask if infection at the two locations was based PCR conditions for ftsZ were 34 cycles with 94 °C for on single or multiple events. The consequences of a 1 min (denaturation), 61.5 °C for 1 min (annealing) and Wolbachia infection for modified phenotypes in leaf- 72 °C for 2 min (elongation) and yielded a 737 bp frag- hopper populations with respect to population dynamics ment using the primers ftsZunif (5‘-GG(CT) AA(AG) and the suspected relevance of Wolbachia for the pest GGT GC(AG) GCA GAA GA-3‘) and ftsZunir (5’-ATC management in agriculture will be discussed. (AG)AT (AG)CC AGT TGC AAG-3‘; Lo et al., 2002). For wsp conditions were 35 cycles with 94 °C for 1 min (denaturation), 55 °C for 1 min (annealing) and 72 °C Materials and methods for 1 min (elongation), amplifying a 610 bp fragment using the primers wsp_81F (5’-TGG TCC AAT AAG Sample collection TGA TGA AGA AAC-3‘) and wsp_691R (5’-AAA In summer 2009 leafhoppers were collected from two AAT TAA ACG CTA CTC CA-3‘; Braig et al., 1998). different populations in Germany, Bonn, [Klein- For 16S rRNA, 30 cycles with 94 °C for 1 min (denatu- Altendorf, competence centre for horticulture (Kompe- ration), 52 °C for 1 min (annealing) and 72 °C for 2 min tenzzentrum Gartenbau, KoGa)] and Göttingen (histori- (elongation) generated a 900 bp fragment using the cal Botanical Garden); sampling locations are 300 kilo- primers 16S_F_V1 (5‘-TTG TAG CCT GCT ATG meters apart. Insects were collected at three different GTA TAA CT-3‘) and 16S_R_V6 (5‘-GAA TAG GTA dates during the vegetation period by sweep-netting. TGA TTT TCA TGT-3‘; O’Neill et al., 1992). Moving forward with constant speed, fifteen catches The amplification of the 658 bp fragment of COI con- were made per sample, using forehand and backhand sisted of 35 cycles with 94 °C for 30 s (denaturation), strokes. Intoxication with ethyl acetate followed imme- 51 °C for 1 min (annealing) and 72 °C for 1 min (elon- diately, insects were transported to the laboratory and gation) using the primers HCO2189 (5‘-TAA ACT stored in 95% ethanol at 4 °C. Eupteryx specimens were TCA GGG TGA CCA AAA AAT CA-3‘) and determined after Ribaut (1936) under a stereomicro- LCO1490 (5‘-GGT CAA CAA ATC ATA AAG ATA scope. For quantitative analysis individuals were sexed, TTG G-3‘; Folmer et al., 1994). counted, abundance of E. melissae and E. decemnotata All PCR runs included a positive control for Wolba- was determined and genital morphology was examined. chia (infected individuals of Bryobia sp.). All products were run on 1% agarose gels and visualized by ethidium DNA extraction bromide staining. Positive samples were purified with Total genomic DNA was extracted from single indi- the QIAquick PCR Purification Kit (Qiagen, Hilden, viduals using the DNeasy® Blood & Tissue Kit Germany) following the manufacturer’s protocol and (Qiagen, Hilden, Germany) according to the manufac- sent for sequencing. Purified PCR products were se- turer’s protocol: ‘Purification of Total DNA from Ani- quences at the Department of Experimental Phycology mal Tissues (Spin Column Protocol)’. Each insect was and Culture Collection of Algae (Georg August Univer- homogenized with a pipette tip prior to lysis in 180 µl sity Göttingen, Germany). buffer ATL and 20 µl Proteinase K (600 mAU/ml) at In total, 157 leafhoppers were screened for Wolbachia 56 °C for 2-3 hours. After lysis, DNA was washed and and for the host COI gene, 51 individuals of E. melissae eluted in 50 µl buffer AE. sampled in Bonn (11 females, 9 males, 31 feminoids), The concentration of extracted DNA was quantified