bs_bs_banner Environmental Microbiology (2013) 15(7), 1956–1968 doi:10.1111/1462-2920.12001 Actinobacteria as essential symbionts in firebugs and cotton stainers (Hemiptera, Pyrrhocoridae) Hassan Salem,1 Elisabeth Kreutzer,2 Introduction Sailendharan Sudakaran1 and Martin Kaltenpoth1,2* Insects, the most abundant animal class on earth, engage 1Insect Symbiosis Research Group, Max Planck Institute in a remarkable diversity of symbiotic associations invol- for Chemical Ecology, Jena, Germany. ving microbial partners (Buchner, 1965). Many of these 2Department of Zoology, University of Regensburg, partnerships benefit the insect host by improvements Regensburg, Germany. to the metabolism, physiology and catabolic capacity through nutritional supplementation or the degradation Summary of complex dietary compounds (Douglas, 1998; 2009; Moran, 2002). Additionally, an increasing number of Actinobacteria engage in defensive symbioses defensive associations are being described where the with several insect taxa, but reports of nutritional symbionts protect their respective hosts and/or the host’s contributions to their hosts have been exceptionally food resources from parasites, pathogens or parasitoids rare. Cotton stainers (Dysdercus fasciatus) and (Currie et al., 1999; Kellner, 2002; Oliver et al., 2003; red firebugs (Pyrrhocoris apterus) (both Hemiptera, Kaltenpoth et al., 2005; Scarborough et al., 2005; Kroiss Pyrrhocoridae) harbour the actinobacterial symbionts et al., 2010). Coriobacterium glomerans and Gordonibacter sp. Members of the bacterial phylum Actinobacteria are as well as Firmicutes (Clostridium sp. and Lactococ- especially prevalent as defensive symbionts due to their cus sp.) and Proteobacteria (Klebsiella sp. and a ecological and physiological prerequisites, including the Rickettsiales bacterium) in the M3 region of their mid- ability to utilize a diverse range of nutritional resources gut. We combined experimental manipulation with and a remarkable versatility in producing secondary community-level analyses to elucidate the function metabolites with antibiotic properties (Kaltenpoth, 2009). of the gut symbionts in both pyrrhocorid species. In contrast, direct evidence for nutritional mutualisms Elimination of symbionts by egg-surface sterilization involving this bacterial group has been limited to vectors resulted in significantly higher mortality and reduced of the Chagas disease (Rhodnius prolixus) and their growth rates, indicating that the microbial community vitamin-supplementing Rhodococcus endosymbionts plays an important role for host nutrition. Fitness (Durvasula et al., 2008). Nonetheless, an increasing of symbiont-deprived bugs could be completely number of studies suggested Actinobacteria to be restored by re-infection with the original microbiota, involved in the nutrition of a range of invertebrates. In while reciprocal cross-infections of microbial com- scarab beetles of the genus Pachnoda, a number of munities across both pyrrhocorid species only par- bacterial strains with hemicellulolytic capabilities were iso- tially rescued fitness, demonstrating a high degree of lated from the hindgut, including Promicromonospora host–symbiont specificity. Community-level analyses pachnodae, an actinobacterial species capable of produc- by quantitative PCRs targeting the dominant bacterial ing a range of xylanases and endoglucanases – two strains allowed us to link the observed fitness effects enzyme families involved in (hemi)cellulose degradation to the abundance of the two actinobacterial symbi- (Cazemier et al., 1999; 2003; Andert et al., 2010). Addi- onts. The nutritional mutualism with Actinobacteria tional studies have also demonstrated the occurrence of may have enabled pyrrhocorid bugs to exploit Malva- Actinobacteria across different termite species. However, les seeds as a food source and thereby possibly evidence for their nutritional contributions alongside indi- allowed them to occupy and diversify in this ecologi- cations of species-specific associations remains to be cal niche. provided (Bignell et al., 1991; Shinzato et al., 2007). In this study, we present evidence for a highly specific part- nership involving members of the Pyrrhocoridae insect family and two actinobacterial symbionts. Received 21 May, 2012; revised 21 September, 2012; accepted 22 September, 2012. *For correspondence. E-mail mkaltenpoth@ Within the Pyrrhocoridae, an oligophagous family of ice.mpg.de; Tel. (+49) 3641 571800; Fax (+49) 3641 571810. bugs, the best known members are cotton stainers of the © 2012 John Wiley & Sons Ltd and Society for Applied Microbiology Actinobacteria as essential symbionts in firebugs and cotton stainers 1957 Fig. 1. Quantitative PCR analyses of the six dominant microbial strains across the four experimental treatments for D. fasciatus. Symbiont numbers represent estimated 16S rRNA gene copy numbers obtained from qPCR assays. Shading of boxes signifies the experimental treatment. Lines represent medians, boxes comprise the 25–75 percentiles, and whiskers denote the range. Grey bands represent the range of unspecific background amplification for the negative controls. Different letters above boxes indicate significant differences in copy numbers (repeated-measures ANOVA, P < 0.05). genus Dysdercus, which are serious pests of cotton, and pods (Kershaw and Kirkaldy, 1908; Ahmad and Schaefer, the red firebug (Pyrrhocoris apterus), an important model 1987; Kristenova et al., 2011). organism for endocrinology and physiology of hemime- Previous studies investigating the microbial community tabolous insects (Socha, 1993). Cotton stainers exhibit a of P. apterus revealed the presence of extracellular gut cosmopolitan distribution mirroring that of the cotton cul- symbionts (Coriobacterium glomerans) belonging to the tivars, with each continent having its own group of species actinobacterial family Coriobacteriacae within the diges- (Pearson, 1958; Ahmad and Schaefer, 1987). Dysdercus tive tract of the insects, particularly in the M3 section species harm cotton crops through the indelible staining (Haas and Konig, 1987; Kaltenpoth et al., 2009). Further of the cotton fibre resulting from the excrements of the characterization of the whole microbiota of P. apterus bug or the accidental processing of insect-bearing bolls, using bacterial 16S rRNA amplicon pyrosequencing as well as through the emanation of seed juices as a yielded an additional actinobacterial strain belonging to result of puncturing and feeding. Additionally, feeding by the Coriobacteriacae family (Gordonibacter sp.) alongside puncturing young cotton bolls usually results in the reduc- a range of other facultative and obligate anaerobes such tion of boll size (Pearson, 1958). Pyrrhocoris apterus also as Clostridium sp., an undescribed Rickettsiales bacte- specializes on seeds of the plant order Malvales, particu- rium, Klebsiella sp. and Lactococcus sp. (Sudakaran larly dry seeds of linden trees (Tilia cordata and T. platy- et al., 2012; Fig. 1). The consistency of the symbiotic phyllos) (Socha, 1993; Kristenova et al., 2011). However, microbial community across geographical localities and some studies also reported on the exploitation of seeds of different food sources in P. apterus suggests that the other plants groups within and – to a more limited extent complex microbiota might have co-evolved with the hosts – also beyond this plant order, as well as on the occa- over millions of years, and that they contribute signifi- sional feeding of firebugs on dead or weakened arthro- cantly to the fitness of the insect (Sudakaran et al., 2012). © 2012 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 1956–1968 1958 H. Salem, E. Kreutzer, S. Sudakaran and M. Kaltenpoth Across different insect lineages, the mode of transmis- and the D. fasciatus symbionts, especially because this sion of symbionts from one generation to another is highly bacterial group contains many species (and even genera) variable. The majority of insects harbouring intracellular with 16S rRNA similarities of more than 97% (our OTU primary symbionts rely on transovarial transmission clustering threshold similarity). modes where the infection originates inside of the female hosts during the early stages of oogenesis or embryogen- Success of symbiont manipulation procedure esis (Buchner, 1965; Schroder et al., 1996; Douglas, 1998; Sauer et al., 2002; Nardon, 2006). In Hemiptera, Egg surface sterilization successfully eliminated C. glom- however, post-hatch transmission is the most common erans in aposymbiotic treatments across both pyrrhocorid mechanism of transfer, with symbiont acquisition resulting species as confirmed by C. glomerans-specific diagnostic from ingestion of adult faecal droplets (Beard et al., 2002), PCRs (data not shown). In addition, the reinstitution of through probing of symbiont containing capsules depos- the microbial community using mid-gut suspensions from ited close to the egg clutch (Fukatsu and Hosokawa, conspecific as well as heterospecific individuals con- 2002; Hosokawa et al., 2005; 2006), or by egg-surface firmed that the nymphs readily accepted symbionts result- contamination and subsequent probing and uptake of the ing from the experimental smearing of their egg surfaces. symbionts by the nymphs (larvae) during the early devel- Symbiont abundance estimates from quantitative PCR opmental stages (Prado et al., 2006). The latter route of analyses of corresponding treatments for the two pyrrhoc- symbiont transfer has also