Genome Sequence of “Candidatus Walczuchella Monophlebidarum” the Flavobacterial Endosymbiont of Llaveia Axin Axin (Hemiptera: Coccoidea: Monophlebidae)
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GBE Genome Sequence of “Candidatus Walczuchella monophlebidarum” the Flavobacterial Endosymbiont of Llaveia axin axin (Hemiptera: Coccoidea: Monophlebidae) Tania Rosas-Pe´rez1,*, Mo´ nica Rosenblueth1,ReinerRinco´ n-Rosales2,JaimeMora1,and Esperanza Martı´nez-Romero1 1Centro de Ciencias Geno´ micas, Universidad Nacional Auto´ nomadeMe´xico, Cuernavaca, Morelos, Mexico 2Instituto Tecnolo´ gico de Tuxtla Gutie´rrez, Tuxtla Gutie´rrez, Chiapas, Mexico *Corresponding author: E-mail: [email protected]. Accepted: February 26, 2014 Data deposition: This project has been deposited at GenBank/DDJB/EMBL under the accession CP006873. Abstract Scale insects (Hemiptera: Coccoidae) constitute a very diverse group of sap-feeding insects with a large diversity of symbiotic asso- ciations with bacteria. Here, we present the complete genome sequence, metabolic reconstruction, and comparative genomics of the flavobacterial endosymbiont of the giant scale insect Llaveia axin axin. The gene repertoire of its 309,299 bp genome was similar to that of other flavobacterial insect endosymbionts though not syntenic. According to its genetic content, essential amino acid bio- synthesis is likely to be the flavobacterial endosymbiont’s principal contribution to the symbiotic association with its insect host. We also report the presence of a g-proteobacterial symbiont that may be involved in waste nitrogen recycling and also has amino acid biosynthetic capabilities that may provide metabolic precursors to the flavobacterial endosymbiont. We propose “Candidatus Walczuchella monophlebidarum” as the name of the flavobacterial endosymbiont of insects from the Monophlebidae family. Key words: scale insect, g-Proteobacteria, symbiosis, comparative genomics. Introduction endosymbionts of scale insects (Gruwell et al. 2007; Insects have specialized symbioses with certain bacteria that Rosenblueth et al. 2012). The genome of Uzinura diaspidicola provide diverse advantages to hosts. Generally, endosymbi- (endosymbiont of Diaspididae) was recently sequenced onts reside inside insect cells, sometimes in unique abdominal (Sabree et al. 2012). Flavobacteria from scale insects structures called bacteriomes (Wernegreen and Wheeler seem to have a single origin, except for Brownia rhizoecola 2009). Endosymbionts have reduced genomes and maintain (from Pseudococcidae: Phenacoccinae) tribe Rhizoecini functions that allow their hosts to live on nutrient-deficient (Rosenblueth et al. 2012). Some endosymbiotic diets such as plant sap or blood (Moya et al. 2008). Flavobacteria form a clade on the basis of 16S rRNA sequences Endosymbionts from different bacterial phyla have been stud- and have been proposed to cospeciate with their ied from various sap-sucking insects, mainly aphids and insects Monophlebidae hosts (Rosenblueth et al. 2012). Besides in the suborder Auchenorrhyncha (Moran 2007). scale insects, two other Flavobacteria have been reported as Flavobacteria have been found as endosymbionts of insect endosymbionts, Sulcia muelleri from Auchenorrhyncha several scale insects (Hemiptera: Coccoidea). Until now and Blattabacterium spp. from basal termites and cock- they have been reported within the families Diaspididae, roaches, and their genomes have been fully sequenced (Wu Lecanodiaspididae, Coccidae, Eriococcidae, Pseudococcidae, et al. 2006; McCutcheon and Moran 2007; Lo´ pez-Sa´nchez Ortheziidae, Coelostomidiidae, and Monophlebidae (Zchori- et al. 2009; McCutcheon et al. 2009; Sabree et al. 2009; Fein et al. 2005; Gruwell et al. 2007; Matsuura et al. 2009; McCutcheon and Moran 2010; Woyke et al. 2010; Neef Gruwell et al. 2010; Dhami et al. 2012; Rosenblueth et al. et al. 2011; Huang et al. 2012; Sabree et al. 2012; Patin˜ o- 2012). Flavobacteria have been considered to be the primary Navarrete et al. 2013; Tokuda et al. 2013). In scale insects, ß The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 714 Genome Biol. Evol. 6(3):714–726. doi:10.1093/gbe/evu049 Advance Access publication March 7, 2014 Genome Sequence of “Candidatus Walczuchella monophlebidarum” GBE Enterobacteriaceae endosymbionts have been frequently analysis with other flavobacterial endosymbionts, as well as an found to coexist with Flavobacteria, including insects from analysis of its metabolic complementarity with the enterobac- Diaspididae (Rosenblueth et al. 2012); although the genome terial endosymbiont. of U. diaspidicola has been sequenced from an insect that only has a single endosymbiont species (Sabree et al. 2012). Llaveia axin axin (Monophlebidae) harbors both endosymbionts pre- Materials and Methods viously found in scale insects: a flavobacterium and an entero- Nomenclature bacterium (Rosenblueth et al. 2012). In this article we did not use the word “Candidatus”aspartof Llaveia axin axin (Llave) (called “niij” by native people) is a the name for symbiotic bacteria, and we italicized the genus giant scale mainly restricted to tropical lowland regions of the and species (e.g., S. muelleri instead of Candidatus Sulcia states of Michoaca´n, Guerrero, and Chiapas in Mexico, and in muelleri). Guatemala, although there are reports previous to 1995 of its presence in the Mexican states of Guanajuato, Veracruz, and Yucata´n(Ben-Dov 2005). It is characterized by its use in the DNA Extraction, PCR, and Cloning manufacture of native traditional crafts that provide an eco- Seven L. axin axin female adults were collected from each of nomic benefit to the local people. A yellow fat that is obtained the following host plants: Acaciella angustissima (Ejido Flores from the female insect is used to prepare a lacquer to coat Mago´ n, Mpo. Venustiano Carranza), Jatropha curcas,and traditional art crafts making them resistant to heat, water, and Spondias purpurea (Chiapa de Corzo City) from the state of decay. The lacquer can be mixed with other natural products Chiapas, Mexico. Total DNA from the freshly collected insects to obtain different colors. It has been used on eating utensils was extracted as reported earlier (Rosenblueth et al. 2012). without toxic effects and also as a medicine unguent for ex- PCR was performed with bacterial universal 16S rRNA primers ternal wounds or pain (Williams and MacVean 1995; Martı´nez fD1 (50-AGAGTTTGATCCTGGCTCAG-30)andrD1(50-AAGGA 2006). GGTGATCCAGCC-30), which amplify products of about 1,500 During its early stages of development, the niij establishes bases (Weisburg et al. 1991). The PCR products were cloned on the young leaves and stems of its host plants, specially and 60 individual plasmid clones were sequenced by Acacia cochliacantha, A. angustissima (reclassified as Macrogen Inc. (Korea). Sequences were compared with the Acaciella angustissima), Spondias sp., and Jatropha curcas nt database of NCBI using the BlastN algorithm. (Rinco´ n-Rosales and Gutie´rrez-Miceli 2008; Suazo-Ortun˜ o et al. 2013) where it sucks the plant sap. As the insect Fluorescent In Situ Hybridization of the Bacteriome grows, it keeps moving on the plant until it becomes an adult and reaches the principal trunk. Three-year-old plants Fluorescent in situ hybridization (FISH) was performed as can have around 300–400 insects but only during a short time described by Koga et al. (2009) with some modifications. in the year (from July to October). Locals collect the females at Twenty-day-old freshly collected L. axin axin first instar the end of the rainy season, rendering most of them to obtain nymphs (fig. 1a) were dehydrated with a 30–100% ethanol the lacquer, but preserving a few to obtain eggs (around 500 series, fixed overnight in Carnoy’s solution, washed with eth- eggs per female). Locals place these eggs at the crown of the anol, and treated for a few days in 6% hydrogen peroxide in plants when the rainy season begins and the eggs hatch a few 80% ethanol. The samples were washed several times with hours later (MacVean 1999). The populations of L. axin axin absolute ethanol, then with xylene, and embedded in paraffin. have declined due to its overexploitation, as well as overgraz- They were cut into 10-mm sections with a rotary microtome ing, forest fires, and deforestation of the host plants. As they and mounted on silane-coated glass slides. Sections were are considered pests of some commercial crops, such as dewaxed through several washes with xylene and ethanol. Spondias purpurea, they have been eliminated (Rinco´ n- Hybridization buffer with 100 nM of the probe was added Rosales and Gutie´rrez-Miceli 2008; Suazo-Ortun˜ oetal. to the samples and was incubated at 28 C overnight in a 2013). Host plant species of L. axin axin have in common humidified chamber. The oligonucleotide probe used was the production of tannins (Rinco´ n-Rosales and Gutie´rrez- Cy5_DcFlv1450 (50-Cy5-ATACCTCCGACTTCCAGGA-30), Miceli 2008; Islam et al. 2011; de Sousa Arau´ jo et al. 2012) which targets 16S of Flavobacteria of Drosicha sp. which could be toxic compounds for insects. Endosymbiotic (Matsuura et al. 2009)andL. axin axin. After washing with bacteria could have a role in the insect detoxification as in the PBS, the samples were stained with 2 mg/ml of DAPI and they case of pesticide detoxyfication in stinkbugs by their were mounted with citifluor antifade solution. In order to Burkholderia