View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by AIR Universita degli studi di Milano Journal of Insect Science RESEARCH Presence of Wolbachia in Three Hymenopteran Species: Diprion pini (Hymenoptera: Diprionidae), Neodiprion sertifer (Hymenoptera: Diprionidae), and Dahlbominus fuscipennis (Hymenoptera: Eulophidae) Dario Pistone,1 Alessandro Bione,2 Sara Epis,3 Massimo Pajoro,3 Stefano Gaiarsa,3 Claudio Bandi,3 and Davide Sassera3,4,5 1Natural History Collections, University Museum of Bergen, P.O. Box 7800, 5020 Bergen, Norway 2Dipartimento di Scienze Agrarie, Universita`degli Studi di Bologna, Viale Fanin 44, 40127 Bologna, Italy 3Dipartimento di Scienze Veterinarie e Sanita`Pubblica, Facolta`di Medicina Veterinaria, Universita`degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy 4Current address: Dipartimento di Biologia e Biotecnologie, Universita`degli Studi di Pavia, via Ferrata 9, 27100, Pavia, Italy 5Corresponding author, e-mail: [email protected], [email protected] Subject Editor: Sara Goodacre J. Insect Sci. 14(147): 2014; DOI: 10.1093/jisesa/ieu009 ABSTRACT. Sawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Downloaded from Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria (Wolbachia and Cardinium) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the pres- ence of Cardinium, 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single http://jinsectscience.oxfordjournals.org/ Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini, both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults. Key Words: endosymbiont, gynandromorphism, molecular screening, diprionid wasp Phytophagous insects represent a major threat for worldwide forests by can develop in one cocoon. The winter is spent as a full-grown larva or causing direct damage, consuming leaves or other parts of the host, and pupa within the host cocoon. also by vectoring micro-organisms that can trigger diseases in plants Haplodiploid sex determination, in which females develop from fertil- (Ayres and Lombardero 2000, Hulcr and Dunn 2011). Adults of ized diploid eggs and males develop from unfertilized haploid eggs, is Diprion pini (L.) (Hymenoptera: Diprionidae) feed preferentially on the most frequent and presumably ancestral mechanism of sex determina- leaves of plants of the genus Pinus. The areal of distribution of this spe- tion in the insect order Hymenoptera (Heimpel and de Boer 2008). Such by guest on December 3, 2015 cies covers the majority of continental Europe, in all areas where the is the case of Diprionidae, including D. pini and N. sertifer, where sexual host plants are present. In Italy, D. pini is frequently encountered in al- chromosomes are absent and sex is determined by ploidy of the newborn pine environments (Pornbacher 1992), but it has also been reported in (Rousselet et al. 1998). Males are aploid (e.g., n ¼ 14 in D. pini)while the Etna area in Sicily (Turrisi and Bella 1999). The species Neodiprion females are diploid (e.g., 2n ¼ 28 in D. pini). Gynandromorphism is an sertifer (Geoffroy) (Hymenoptera: Diprionidae) also occurs in a variety anomaly affecting sexual and somatic characters in different tissues of an of habitats, on pine host plants from northern and central Europe, as individual. An abnormal chimeric organism presenting a mixture of male well as in Asia and North America (Olofsson 1987). The larvae of both and female physical features is defined a gynandromorph (Narita et al. sawfly species are massive defoliators that can cause important damage 2010). The occurrence of gynandromorphic individuals was previously to the plants they feed on, also affecting the esthetic value in ornamental reported in D. pini (Beaudoin et al. 1994). tree plantations. In both species, the adult females lay eggs inside the Micro-organisms of the genus Wolbachia are gram-negative needles of host plants, and the damage is determined by trophic activity a-proteobacteria belonging to the order Rickettsiales, family of the larva consuming the leaves (Augustaitis 2007). Besides, as the Anaplasmataceae. Wolbachia is an obligated intracellular bacterium in- larvae mature, their feeding becomes more extensive and needles are fecting a wide range of hosts, from arthropods to filarial nematodes stripped. Complete defoliation of the trees may occur, affecting the (Bandi et al. 2001). Vertical maternal inheritance is considered the pri- growth of the parasitized plants. The larvae, however, do not feed on mary route of Wolbachia transmission from one host generation to an- new grown leaves, so the trees usually survive (Kurkela et al. 2005). other. The role of Wolbachia in the host is also variable; in fact, these Periodically, sawfly populations may increase to outbreak proportion, bacteria can behave as mutualists, pathogens, or even reproductive par- and reduction in plant increment and tree mortality can be registered asites. Wolbachia bacteria can exploit all known mechanisms of repro- over widespread areas (Coppel and Benjamin 1965). Dahlbominus fus- ductive parasitism, manipulating the host reproduction with different cipennis (Zetterstedt) (Hymenoptera: Eulophidae) is a parasitoid of strategies, including parthenogenesis, male killing, cytoplasmic incom- many sawfly species, including D. pini and N. sertifer, and it is largely patibility, and feminization of genetic males (Werren et al. 2008). In reared and released for pest control purposes. Females of Da. fuscipen- general, Wolbachia bacteria are strictly associated with the gonads but nis lay eggs in the larvae of diprionid cocoons. Several eggs are laid might also be present in different somatic tissues (Dobson et al. 1999). with a single insertion of the ovipositor, and up to 40 parasitoid larvae Cardinium, a recently described bacterial genus of maternally acquired VC The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 JOURNAL OF INSECT SCIENCE VOLUME 14 Table 1. Number of male, female, and gynandromorph D. pini individuals for each of the three laboratory colonies for each generation Colony Laboratory line 1 (Dpini1) Laboratory line 2 (Dipini2) Laboratory line 3 (1Â2) (Dpini3) Sex M F Gyn M F Gyn M F Gyn Generation 0 168 (2) 111 (4) 21 (2) 19 20 3 4 (2) 11 (2) 1 Generation I 143 10 14 68 (2) 35 (4) 12 (2) 1 1 1 Generation II 13 15 3 20 9 1 1 1 4 Generation III 11 9 1 7 10 10 Generation IV 44 (4) 52 (2) 7 (2) 19 (2) 84 (2) 8 Generation V 18 45 5 31 36 51 Generation VI 14 4 2 1 2 0 Generation VII 52 (4) 10 (4) 7 (2) 83 (4) 33 (2) 26 (2) Total Gyn/total individual 60/779 16/187 101/426 Gyn, gynandromorph; F, female; M, male. endosymbionts, widespread in spiders, arachnids, and other arthropod velopment. Two heat treatment experiments were performed each on groups (Martin and Goodacre 2009), is associated with reproductive 15 D. pini by increasing the rearing temperature. The first experiment manipulations such as cytoplasmic incompatibility, parthenogenesis, consisted of a stable increase in the rearing temperature to 25C, while Downloaded from and feminization in different arthropod species (Wu and Hoy 2012). the second consisted of an increase to 30C for three intervals of 24 h Investigations on the presence of endosymbionts in pests represent separated by 48 h at 20C. The hymenopteran parasitoid Da. fuscipen- the first step in developing a potential strategy for biocontrol purposes. nis was reared only on N. sertifer, at the conditions described earlier. For example, a study focused on the agent of Pierce disease in grape- In total, 30 individuals of N. sertifer (17 males and 13 females), 30 vines, the glassy-winged sharpshooter, showed that the spread of the of Da. fuscipennis (11 males and 19 females), and 50 individuals of disease could be controlled by an antagonistic bacterium that resides in D. pini (20 females, 20 males, and 10 gynandromorphs) were subjected http://jinsectscience.oxfordjournals.org/ the same insect vector (Bextine et al. 2004, Newman et al. 2004). to molecular screening for the presence of symbionts (see Table 1 for On the other hand, the discovery of Wolbachia in filarial nematodes led details). All individuals were stored in absolute ethanol, then washed in to the development of antisymbiotic therapy to cure filarial diseases sterile water, homogenized with a sterile pestle, and processed for DNA (Taylor et al. 2000). In general, considering that the wide interactions extraction using a commercial kit (DNeasy Blood & Tissue kit, Qiagen, between the host and the associated microbiota are a fundamental as- Hilden, Germany) following manufacturer’s instructions. A molecular pect of insect biology (Weiss et al. 2012), investigations on the role of screening was performed on the extracted DNAs to investigate the pres- bacterial symbionts are allowing the development of novel strategies ence of endosymbiotic bacteria belonging to the genera Wolbachia and to control pest and parasitic arthropods and to reduce their vector Cardinium. Three specific Polymerase Chain Reaction (PCR) proto- competence.