Appl. Entomol. Zool. 45 (1): 115–120 (2010) http://odokon.org/

Nardonella endosymbiont in the West Indian sweet potato postfasciatus (Coleoptera: )

Takahiro HOSOKAWA* and Takema FUKATSU National Institute of Advanced Industrial Science and Technology (AIST); Tsukuba, Ibaraki 305–8566, Japan (Received 15 July 2009; Accepted 16 August 2009)

Abstract The West Indian sweet potato weevil, , is a notorious pest of the sweet potato, Ipomoea batatas. We examined the potential presence of a bacterial endosymbiont in the pest weevil. The bacterial 16S rRNA gene and groEL gene were detected by PCR from the . Cloning, sequencing and molecular phylogenetic analyses of the bacterial genes demonstrated that E. postfasciatus is associated with a g-proteobacterial endosymbiont of the genus Nardonella. In situ hybridization detected the endosymbiont in the female ovaries, indicating its transovarial transmis- sion through host generations. This study is the first to identify Nardonella from the weevil subfamily Cryptorhynchi- nae. The potential relevance of the endosymbiont in the biology and management of E. postfasciatus is discussed.

Key words: Euscepes postfasciatus; Ipomoea batatas; bacterial endosymbiont; Nardonella

oryzae, for example, a large organ called the bacte- INTRODUCTION riome is found around the junction of the larval The West Indian sweet potato weevil, Euscepes foregut and midgut, wherein a specific g-pro- postfasciatus (Fairmaire) (Coleoptera: Curculion- teobacterial endosymbiont is harbored endocellu- idae: ), is a notorious pest of the larly and plays various biological roles for the host sweet potato, Iponema batatas (L.), in tropical and (Heddi and Nardon, 2005). It is conceivable subtropical countries worldwide (Raman and Al- that E. postfasciatus is also associated with such a leyne, 1991). In Japan, E. postfasciatus was first microorganism and, if so, the involvement of the found on Okinawa Island in the mid-20th century endosymbiont should be taken into account for and has rapidly spread throughout the Ryukyu management of the pest weevil. Archipelago (Kohama, 1990). The weevil seriously In this study, we characterized the bacterial en- damages sweet potatoes not only in the fields but dosymbiont of E. postfasciatus using molecular also during storage. Hidden inside potato tubers, phylogenetic and in situ hybridization analyses. eggs, larvae and pupae of E. postfasciatus are diffi- cult to control by insecticide spraying. Eradication MATERIALS AND METHODS programs on the basis of the sterile insect tech- nique and integrated pest management have been Insects. Sweet potatoes containing eggs and lar- conducted (Yasuda, 1998; Kuba et al., 2000), but it vae of E. postfasciatus were harvested from sweet is still worth exploring alternative approaches to potato fields in Yomitan, Okinawa in April 2008 controlling E. postfasciatus. and kept in plastic containers at room temperature. represent the most successful insect Adult insects emerging from the potatoes were group, embracing over 60,000 described fixed in acetone and used in this study. (Alonso-Zarazaga and Lyal, 1999). Many, if not all, Cloning and sequencing. DNA was extracted are associated with bacterial endosymbionts from the whole abdomen of female insects using (Buchner, 1965). In the rice weevil Sitophilus the NucleoSpin Tissue Kit (MACHEREY-NAGEL).

* To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2010.115

115 116 T. HOSOKAWA and T. FUKATSU

Bacterial genes were amplified from the DNA sam- in acetone were dissected in 70% ethanol. Ovaries ples by PCR with AmpliTaq DNA Polymerase were dissected from the abdomen and fixed in (Applied Biosystems) and primers 16SA1 (5- Carnoy’s solution (chloroform : ethanol : acetic AGA GTT TGA TCM TGG CTC AG-3) and acid6 : 3 : 1) for a day, and then bleached with 6% 16SB1 (5 -TAC GGY TAC CTT GTT ACG ACT H2O2 in ethanol for a week (Koga et al., 2009). In T- 3 ) for the 16S rRNA gene, and groEL2F (5- situ hybridization targeting symbiont 16S rRNA ATG GGB GCT CAA ATG GTK AAA-3) and was conducted using an oligonucleotide probe la- groEL2R (5-CTC TTT CAT TTC AAC TTC NGT beled with AlexaFluor555, imozou99 (Al555-5C- BGC A-3) for the groEL gene, respectively. The CT TCA TTA GGC AGA TTC-3). The bleached temperature profile was 94°C for 4 min, followed ovaries were incubated in hybridization buffer (20 by 35 cycles of 94°C for 30 s, 55°C for 1 min, and mM Tris-HCl [pH 8.0], 0.9 M NaCl, 0.01% SDS, 72°C for 2 min. 30% formamide) containing 50 nM of the probe PCR products (1.5 kb for 16S rRNA gene and and 0.5 mM SYTOX Green (Invitrogen). The 0.9 kb for groEL gene) were cloned using the stained ovaries were observed under an epifluores- pT7Blue T-Vector (Takara) and Escherichia coli cent microscope (Axiophot; Carl-Zeiss) and a laser DH5a competent cells (Takara). The length of the scanning microscope (PASCAL5; Carl-Zeiss). inserted DNA fragment was checked by direct PCR Diagnostic PCR. DNA samples extracted from of white colonies using primers Univ19 (5-GTT the whole abdomen were subjected to diagnostic TTC CCA GTC ACG ACG T-3) and Rev20 (5- PCR detection of the symbiont 16S rRNA gene AGC TAT GAC CAT GAT TAC GC-3). Colonies with AmpliTaq Gold DNA Polymerase (Applied potentially containing target DNA fragments were Biosystems) and primers 16SA1 and imozou cultured in a liquid medium, from which inserted 16S729R (5-CGT GAA TAA GTG TCA GTC plasmids were extracted with QIAprep-Spin TTC A-3). The temperature profile was 95°C for Miniprep Kit (QIAGEN). 10 min, followed by 35 cycles of 94°C for 30 s, Cycle sequencing reactions were conducted 55°C for 1 min, and 72°C for 1 min, which specifi- using ABI PRISM BigDye Terminator v3.1 under cally amplified a 0.8-kb segment of the symbiont a temperature profile of 96°C for 1 min, followed 16S rRNA gene. by 26 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Products were analyzed with an RESULTS AND DISCUSSION ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). The gene sequences determined in Phylogenetic placement of bacterial 16S rRNA this study have been deposited in the DDBJ nu- gene sequence from E. postfasciatus cleotide sequence database under accession num- Only one bacterial 16S rRNA gene sequence bers AB506808 (16S rRNA gene) and AB506809 was obtained from the female abdomen of E. post- (groEL gene). fasciatus. The same sequence was reproducibly Phylogenetic analyses. Multiple alignments of identified from three adult females, exhibited a nucleotide sequences were generated using high AT-biased nucleotide composition of 58.8%, ClustalW (Thompson et al., 1994). All sites includ- belonged to the g-Proteobacteria, and showed ing gaps were excluded, and 1,184 sites for the 16S high sequence similarities to the sequences of en- rRNA gene and 923 sites for the groEL gene were dosymbionts of dryophthorid weevils (Nardonella subjected to phylogenetic analyses. Phylogenetic dryophthoridicola) and those of molytine weevils trees were constructed by the neighbor-joining, (Nardonella hylobii) (Lefevre et al., 2004; Conord maximum-parsimony, and maximum-likelihood et al., 2008). The highest BLAST hit for the se- methods using PAUP* 4.0b10 (Swofford, 2002) quence was the Nardonella endosymbiont of Hylo- and Modeltest 3.7 (Posada and Crandall, 1998). bius transversovittatus (91.5% [1,330/1,453] se- Bootstrap values were calculated with 1,000 repli- quence identity; accession number EF434872). cations in neighbor-joining analysis and 100 in Figure 1 shows the phylogenetic placement of the maximum-parsimony and maximum-likelihood 16S rRNA gene sequence in the g-Proteobacteria. analyses, respectively. The sequence fell into the clade of Nardonella In situ hybridization. Adult females preserved spp., forming a well-supported monophyletic group Endosymbiont of the West Indian Sweet Potato Weevil 117

Fig. 1. Phylogenetic position and AT content of the bacterial endosymbiont of E. postfasciatus on the basis of 16S rRNA gene sequences. The maximum-likelihood tree is shown, while neighbor-joining and maximum-parsimony methods estimated substan- tially the same topologies. Numbers attached to each node are bootstrap values (neighbor-joining/maximum-parsimony/maximum- likelihood). Asterisks indicate values lower than 70%. Host insects and sequence accession numbers are shown in parentheses and brackets, respectively. with Nardonella of molytine weevils. Diagnostic Ovarial localization of the bacterial 16S rRNA PCR detected the sequence from all 10 adult males in E. postfasciatus and 10 adult females examined. Fluorescent in situ hybridization detected the specific localization of bacterial 16S rRNA in the Phylogenetic placement of bacterial groEL gene female ovaries. Symbiont signals were concen- sequence from E. postfasciatus trated in the germalia at the tip of each ovariole In addition, a 923-bp bacterial groEL gene se- (Fig. 3), suggesting transovarial transmission of the quence was obtained from E. postfasciatus. The symbiont through the host generations. same sequence was reproducibly identified from three adult females. The sequence also exhibited a Identification of Nardonella endosymbiont in E. high AT-biased nucleotide composition of 71.0%, postfasciatus belonging to the g-Proteobacteria. Since no Nar- From these results, we concluded that E. postfas- donella groEL gene sequences have been deposited ciatus is associated with a g-proteobacterial en- in the DNA databases, no high BLAST hits for the dosymbiont of the genus Nardonella. This study is sequence were identified: the highest hit was the the first to report a bacterial endosymbiont from sequence of the endosymbiont of the monkey louse the West Indian sweet potato weevil. Pedicinus obtusus (79.5% [731/920] sequence identity; accession number AB478980). Figure 2 Implication of endosymbiont evolution in wee- shows the phylogenetic placement of the groEL vils gene sequence in the g-Proteobacteria, wherein Over 60,000 species of weevils have been de- the sequence represented a distinct lineage. scribed around the world (Alonso-Zarazaga and Lyal, 1999). Since the early 1900s, a number of 118 T. HOSOKAWA and T. FUKATSU

Fig. 2. Phylogenetic position and AT content of the bacterial endosymbiont of E. postfasciatus on the basis of groEL gene se- quences. The maximum-likelihood tree is shown, while neighbor-joining and maximum-parsimony methods estimated substan- tially the same topologies. Numbers attached to each node are bootstrap values (neighbor-joining/maximum-parsimony/maximum- likelihood). Asterisks indicate values lower than 70%. Host insects and sequence accession numbers are shown in parentheses and brackets, respectively.

histological works have reported the presence of Future directions bacterial endosymbionts in diverse weevils (e.g., Thus far, the biological roles of Nardonella en- Pierantoni, 1927; Mansour, 1930; Scheinert, 1933; dosymbionts for their host weevils have not been Buchner, 1965; Nardon et al., 1985, 2002). Mean- studied. While Nardonella-weevil co-speciation while, the microbiological nature of these en- and the peculiar molecular features of Nardonella dosymbionts has been characterized for only a lim- genes are suggestive of an obligate mutualism ited number of weevil groups: Sodalis-allied en- (Lefevre et al., 2004; Conord et al., 2008), the bio- dosymbionts associated with dryophthorid grain logical basis of the association has been totally un- weevils of the genus Sitophilus, often referred to as known. Notably, E. postfasciatus can easily be SOPE (Sitophilus oryzae primary endosymbionts) maintained on sweet potatoes in the laboratory or other acronyms (Heddi and Nardon, 2005); and (Raman and Alleyne, 1991), making the insect an Nardonella endosymbionts identified from dryoph- ideal model for investigating the biological aspects thorid weevils of the genera Cosmopolites, Mata- of the Nardonella endosymbiont. Furthermore, an masius, Rhynchophorus, Scyphophorus, Spheno- artificial diet rearing technique has been estab- phorus and Yuccaborus (Lefevre et al., 2004) and lished for E. postfasciatus (Shimoji and Kohama, also from molytine weevils of the genus Hylobius 1996; Kuriwada et al., 2009; Ohno et al., 2009), (Conord et al., 2008). In this study, we first identi- which will enable physiological and nutritional fied a Nardonella endosymbiont from the cryp- studies on the Nardonella-weevil association. If the torhynchine weevil E. postfasciatus, broadening Nardonella endosymbiont is proven to be essential the range of weevils associated with Nardonella. for E. postfasciatus, the bacterial associate could The symbiont of E. postfasciatus and those of Hy- be a novel target for controlling this notorious pest lobius spp. certainly formed a monophyletic group insect of sweet potatoes. (Fig. 1), reflecting the host insect systematics and ACKNOWLEDGMENTS confirming that host-symbiont evolutionary coher- ence, as has been suggested previously (Lefevre et We thank Takashi Kuriwada and Dai Haraguchi at Okinawa al., 2004; Conord et al., 2008). Prefectural Plant Protection Center for providing samples and instruction in the biological aspects of E. postfasciatus. This work was financially supported by the Program for Promotion Endosymbiont of the West Indian Sweet Potato Weevil 119

evolutionary stability of bacterial endosymbiosis in Cur- culionoidea: addiotional evidence of symbiont replace- ment in the Dryophthoridae family. Mol. Biol. Evol. 25: 859–868. Heddi, A. and P. Nardon (2005) Sitophilus oryzae L.: A model for intracellular symbiosis in the Dryophthoridae weevils (Coleoptera). Symbiosis 39: 1–11. Koga, R., T. Tsuchida and T. Fukatsu (2009) Quenching aut- ofluorescence of insect tissues for in situ detection of en- dosymbionts. Appl. Entomol. Zool. 44: 281–291. Kohama, T. (1990) Invasion and colonization of the sweet- potato weevils in Okinawa and current problems for their control. Plant Prot. 44: 115–117 (in Japanese). Kuba, H., T. Teruya and M. Sakakibara (2000) Eradication of weevils by sterile-insect-release method (9) Experi- mental eradication project of sweet potato weevils in Kume island. Plant Prot. 54: 483–486 (in Japanese). Kuriwada, T., N. Kumano, K. Shiromoto and D. Haraguchi (2009) High population density and egg cannibalism re- duces the efficiency of mass-rearing in Euscepes postfas- ciatus (Coleoptera: Curculionidae). Fla. Entomol. 92: 221–228. Lefevre, C., H. Charles, A. Vallier, B. Delobel, B. Farrell and A. Heddi (2004) Endosymbiont phylogenesis in the Dryophthoridae weevils: evidence for bacterial replace- ment. Mol. Biol. Evol. 21: 965–973. Mansour, K. (1930) Preliminary studies on the bacterial cell mass (accessory cell mass) of Calandra oryzae: the rice weevil. Q. J. Microsc. Sci. 73: 421–436. Nardon, P., C. Louis, G. Nicolas and A. Kermarrec (1985) Discovery and study of symbiotic bacteria in 2 species of banana pests: the weevils Cosmopolites sordidus (Ger- mar) and Metamasius hemipterus (L.) (Coleoptera: Cur- culionidae). Ann. Soc. Entomol. Fr. 21: 245–258. Nardon, P., C. Lefévre, B. Delobel, H. Charle and A. Heddi (2002) Occurrence of endosymbiosis in Dryophthoridae weevils: cytological insights into bacterial symbiotic structures. Symbiosis 33: 227–241. Ohno, S., T. Sasaki, K. Urasaki and T. Kohama (2009) Im- Fig. 3. Localization of the bacterial endosymbiont in the provement of survival of the West Indian sweet potato ovary of E. postfasciatus. (A) A whole ovary observed under weevil, Euscepes postfasciatus (Coleoptera: Curculion- an epifluorescent microscope. (B) A germalium of an ovariole idae), by placing absorbent paper on the artificial diet observed under a laser scanning microscope. Red and green after egg seeding and ensuring air permeability of the signals indicate the symbiotic bacteria and host nuclei, respec- rearing tray. Appl. Entomol. Zool. 44: 13–22. tively. Pierantoni, U. (1927) L’organo simbiotico nello sviluppo di Calandra oryzae. Rend. Reale Acad. Sci. Fis. Mat. Napoli 35: 244–250. of Basic Research Activities for Innovative Biosciences (PRO- Posada, D. and K. A. Crandall (1998) Modeltest: testing the BRAIN). model of DNA substitution. Bioinformatics 14: 817– REFERENCES 818. Raman, K. V. and E. H. Alleyne (1991) Biology and man- Alonso-Zarazaga, M. A. and C. H. C Lyal (1999) A World agement of the West Indian sweet potato weevil, Eu- Catalogue of Families and Genera of Curculionoidea (In- scepes postfasciatus. In Sweet Potato Pest Management secta: Coleoptera) Excluding (Scolytidae and Platypodi- (R. K. Jansson and K. V. B. Raman, eds). Westview dae). Entomopraxis, Barcelona. 315 pp. Press, Boulder, pp. 263–281. Buchner, P. (1965) Endosymbiosis of with Plant Mi- Scheinert, W. (1933) Symbiose und Embryonalentwicklung croorganisms. Interscience, New York. 909 pp. bei Rüsselkäfern. Z. Morphol. Ökol. Tiere. 27: 76–128. Conord, C., L. Despres, A. Vallier, S. Balmand, C. Miquel, S. Shimoji, Y. and T. Kohama (1996) An artificial larval diet Zundel, G. Lemperiere and A. Heddi (2008) Long-term for the West Indian sweet potato weevil, Euscepes 120 T. HOSOKAWA and T. FUKATSU

postfasciatus (Fairmaire) (Coleoptera: Curculionidae). cific gap penalties, and weight matrix choice. Nucleic Appl. Entomol. Zool. 31: 152–154. Acids Res. 22: 4673–4680. Swofford, D. L. (2002) PAUP* Version 4.0b10 [computer Yasuda, K. (1998) Studies on integrated pest management of program]. Sinauer, Sunderland, Massachusetts. West Indian sweet potato weevil Euscepes postfasciatus Thompson, J. D., D. G. Higgins and J. J. Gibson (1994) (Fairmaire) and sweet potato weevil Cylas formicarius ClustalW: Improving the sensitivity of progressive multi- (Fabricius). Bull. Okinawa Agric. Exp. Sta. 21: 1–80 (in ple alignment through sequence weighting, position-spe- Japanese with English summary).