Diversity and Phylogeny of Cardinium (Bacteroidetes) in Armored Scale Insects (Hemiptera: Diaspididae) Author(s): M. E. Gruwell, J. Wu, and B. B. Normark Source: Annals of the Entomological Society of America, 102(6):1050-1061. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/008.102.0613 URL: http://www.bioone.org/doi/full/10.1603/008.102.0613 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. SYSTEMATICS Diversity and Phylogeny of Cardinium (Bacteroidetes) in Armored Scale Insects (Hemiptera: Diaspididae) 1 2 2 M. E. GRUWELL, J. WU, AND B. B. NORMARK School of Science, Penn State Erie, Behrend College, 162 Nick Building, 4205 College Drive, Erie, PA 16563-0203 Ann. Entomol. Soc. Am. 102(6): 1050Ð1061 (2009) ABSTRACT Species of Cardinium are intracellular endosymbionts of arthropods. They are known to interfere with the mode of reproduction in some parasitoid wasp and mite hosts, and they are associated with parthenogenesis in the oleander scale, Aspidiotus nerii Bouche´ (Hemiptera: Diaspi- didae). Here, we survey A. nerii more broadly for presence of Cardinium species. In total, we tested 593 individuals in 301 populations, representing 132 identiÞed species of armored scale insects, by using polymerase chain reaction (PCR) and sequencing of 16S rDNA. We obtained positive PCR results in 67 individuals of 34 populations, representing 20 species and one unidentiÞed individual. Sequencing of these PCR products yields 20 new Cardinium haplotypes, raising the number of known insect-associated Cardinium haplotypes from 15 to 35. The new host-list elucidates the ecology of Cardinium, which in insects seems to be specialized on plant-feeding Hemiptera and associated parasitoid Hymenoptera. A phylogenetic analysis of all known insect-associated Cardinium 16S sequences reveals deep diversity in armored scale insects and implies horizontal transfer of Cardinium among armored scale insects and their parasitoids. KEY WORDS secondary endosymbiont, horizontal transfer, Encarsia, parthenogenesis The most widespread and notorious endosymbionts of Cardinium are known from a narrower range of insect insects are species of Wolbachia, a group of Alpha- hosts. Although fairly widespread in arachnids (Enigl proteobacteria that are well known for manipulating and Schausberger 2007, Duron et al. 2008, Groot and host reproduction (Werren and Windsor 2000). The Breeuwer 2008), so far in insects Cardinium has been four major host-manipulation phenotypes found in found only in the orders Hymenoptera and Hemiptera Wolbachia are typically categorized as follows: 1) cy- (Hunter and Zchori-Fein 2006, Duron et al. 2008, toplasmic incompatibility, in which sperm from in- Sacchi et al. 2008). Cardinium has been found to in- fected males are lethal to eggs from uninfected fe- duce cytoplasmic incompatibility and parthenogene- males; 2) male killing, in which infected males die sis in Encarsia wasps (Zchori-Fein et al. 2001, Hunter before maturity, usually at a very early embryonic et al. 2003, Zchori-Fein et al. 2004, Perlman et al. 2008) stage; 3) feminization, in which infected individuals and feminization in Brevipalpus mites (Weeks et al. that are genetically male develop as phenotypic fe- 2001). As with Wolbachia, a range of other more subtle males; and 4) parthenogenesis induction, in which effects on hosts also have been found, including en- infected individuals all develop as parthenogenetic hanced fecundity in Metaseiulus mites (Weeks and females (Werren 1997, Stouthamer et al. 1999). An- Stouthamer 2004) and broadened host range in En- other effect linked to Wolbachia in some cases is an carsia pergandiella Howard (Zchori-Fein et al. 2001). increase of fecundity and fertility (Stouthamer et al. Encarsia wasps, in which Cardinium is best charac- 1999). Wolbachia lineages are extremely widespread terized, attack whiteßies and armored scale insects, and occur in at least 12 insect orders (Werren et al. both of which are also known to harbor Cardinium. 1995, Arakaki et al. 2001, Noda et al. 2001, Dittmar and The other wasps in which Cardinium has been found Whiting 2004, Peng and JinJun 2004, Bordenstein and belong to the genera Aphytis and Plagiomerus, which Rosengaus 2005, Covacin and Barker 2007). are parasitoids of armored scale insects, and the genus Another lineage of endosymbiotic bacteria, the ge- Marietta, which is a hyperparasitoid of scale insects. nus Cardinium from the Bacteroidetes, has some of the Thus, in insects, Cardinium is known primarily from same host manipulation phenotypes as Wolbachia, but species that have a close ecological association with armored scale insects. In a survey of Cardinium and related bacteria across 24 species of scale insects in six 1 Corresponding author, e-mail: [email protected]. families, Cardinium was found in two species, both of 2 Department of Plant, Soil, and Insect Sciences and Graduate Program in Organismic and Evolutionary Biology, Fernald Hall, Uni- which were armored scales (Zchori-Fein et al. 2005). versity of Massachusetts, Amherst, MA 01003. Here, we survey Ͼ130 species of armored scale insects 0013-8746/09/1050Ð1061$04.00/0 ᭧ 2009 Entomological Society of America November 2009 GRUWELL ET AL.: DIVERSITY OF Cardinium BACTERIA IN ARMORED SCALES 1051 by using Cardinium-speciÞc 16S primers. We Þnd a trols, based on samples from Provencher et al. (2005), greater diversity of Cardinium haplotypes from ar- to ensure accuracy and avoid contamination. mored scale insects than are known from all other PCR reactions were visualized using 1.5% agarose insects combined. The phylogeny of insect-associated gel electrophoresis and cleaned using Exo SAP-IT Cardinium lineages is consistent with frequent hori- enzymatic digestion (USB, Cleveland, OH). Cleaned zontal transfer among armored scale insects and their products were cycle sequenced and analyzed on a parasitoids. 3730xl DNA analyzer (Applied Biosystems, Foster City, CA) at Brigham Young University (Provo, UT). Com- pleted sequences were edited and compiled using Materials and Methods Sequencher 4.2 (Gene Codes Corporation, Ann Ar- Cardinium Survey Across Diaspidid Scale Insects. bor, MI) and imported into a BLAST (Altschul et al. We sampled 593 individuals from 301 populations in 1997) search on GenBank (Benson et al. 2004) to 132 identiÞed species of armored scale insects. Of the verify identity as Cardinium. 301 populations, 263 were identiÞed to species, 20 Cardinium in Males. Female scale insects were used were identiÞed as undescribed new species in an iden- for the above-described survey, and for most other tiÞed genus and 18 were unidentiÞed. For 87 species, systematic work on scale insects, because they are we used the same genomic DNA extractions that were much more readily collected than male scale insects used for a previously published phylogenetic study of and because adult females are the only life stage that the armored scale insects (Morse and Normark 2006). can be reliably identiÞed to species. This stems from Two species from that study, Selenaspidus articulatus the fact that male scale insects are much shorter-lived Morgan and Dactylaspis sp. undesc. #3, were no than female scale insects, with a brief, winged adult longer available. To this sample we have added 45 phase. Thus sampling (generally by searching plant species and 38 undescribed or unidentiÞed popula- surfaces for sessile life stages) is biased toward fe- tions. Each new sample was tested for quality by poly- males. Nonetheless, most of our stored samples in- merase chain reaction (PCR) and sequencing of the clude many individuals, and subadult males are often 28S gene from the host scale insect. For each popu- present. lation we tested a minimum of two individuals and up In Aspidiotus nerii Bouche´ (Hemiptera: Diaspidi- to eight when the material was available (Table 1). dae), Cardinium is found only in the parthenogenetic Our sample includes a broad diversity of the armored lineage and is absent from sexual populations (Prov- scale insects, including multiple representatives from encher et al. 2005). This implies that Cardinium in A. the Þve major tribes and Ϸ6% of described species. nerii has a parthenogenesis-inducing phenotype. To The sample also includes 17 of the 36 species in which assess whether Cardinium always has such a pheno- parthenogenesis has been reported previously type in armored scale insects, all Cardinium-positive (Brown 1965, Danzig 1986, Gerson 1990, Nur 1990, samples were examined for the presence of males. If Kosztarab 1996, Gill 1997). See Table 1 for a list of one or more males was found, DNA was prepared from all species tested and