Biological Control 58 (2011) 182–191 Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon DNA barcoding of endoparasitoid wasps in the genus Anicetus reveals high levels of host specificity (Hymenoptera: Encyrtidae) ⇑ Yan-Zhou Zhang a, , Sheng-li Si b, Jin-Tu Zheng c, Hong-Liang Li b, Yu Fang a, Chao-Dong Zhu a, Alfried P. Vogler d,e a Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China b Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai 200031, PR China c Ningbo Technology Extension Center for Forestry and Specialty Forest Products, Ningbo 315012, PR China d Department of Entomology, Natural History Museum, Cromwell Road, London SW7 5BD, UK e Imperial College London, Division of Biology, Silwood Park Campus, Ascot SL5 7PY, UK article info abstract Article history: The genus Anicetus includes economically important biocontrol agents that are introduced for control of Received 20 June 2010 soft and wax scale insect agricultural pests (Ceroplastes spp.). Understanding of host–parasitoid associa- Accepted 10 May 2011 tions is critical to the successful outcome of their utilization in biological control projects. However, iden- Available online 15 May 2011 tification of these parasitoids is often difficult because of their small size and generally similar morphological features, and hence, studies on the host–parasitoid associations. Here, nucleotide Keywords: sequence data were generated from the mitochondrial COI gene and the D2 region of 28S rRNA to assess Hymenoptera genetic variation within and between species of Anicetus occurring in China. The results of this study sup- Encyrtidae port the use of the COI and the D2 region of 28S rRNA gene as useful markers in separating species of Anicetus ceroplastis species group Anicetus, even in cases where morphological differences are subtle. On the other hand, the COI gene is also DNA barcoding useful in recognizing species with much variation in morphology. DNA barcoding reveals high levels of mtDNA host specificity of endoparasitoids wasps in the genus Anicetus. Our results indicate that each Anicetus 28S rRNA species is adapted to a limited set of host species, or even are monospecific in their host choice. DNA sequences Ó 2011 Elsevier Inc. All rights reserved. Taxonomy Host specificity Ceroplastes Coccidae 1. Introduction Trjapitzin, 2010). Among them, Anicetus beneficus Ishii and Yasumatsu is one of the most successful biological control agents The genus Anicetus (Hymenoptera: Encyrtidae) includes about 50 (Yasumatsu, 1958, 1969; Smith, 1986), which continues to be used currently recognized species (Noyes, 2010; Trjapitzin, 2010). Spe- in biocontrol programmes (Krull and Basedow, 2005). Anicetus ben- cies of Anicetus are primary endoparasites of soft scales (Homoptera, eficus Ishii and Yasumatsu was discovered by Yasumatsu in Kyu- Coccidae), particularly the wax scale species belonging to Ceroplas- shu, Japan, in 1946 (Yasumatsu, 1958). Then it was introduced tes (Subba Rao, 1965; Annecke, 1967; Trjapitzin, 2010). Some of into the islands of Honshu and Shikoku and entirely suppressed these wax scale species, such as Ceroplastes rubens Maskell,Ceroplas- there its host, a dangerous pest of citrus plants (Yasumatsu, tes floridensis Comstock and Ceroplastes japonicus (Green), are 1958). The introduction of A. beneficus from Japan into South Korea polyphagous and are significant economic pests of important agri- and Australia for use against wax soft scale was successful (Kim cultural plants such as Citrus spp., the coffee tree, mango and guava et al., 1979, 1994; Smith, 1986), but its introduction into the Black around the world (Yasumatsu, 1958; Gimpel et al., 1974; Smith, Sea coast of Georgia and Israel was ineffectual because of the ab- 1986; Ben-Dov, 1993; Wang, 2001). sence of its host there (Trjapitzin, 2010). Other than Anicetus ben- Anicetus is well known in economic entomology since many eficus, Anicetus ceroplastis, Anicetus communis, Anicetus dodonia, species have been used in the biological control of wax and soft Anicetus nyasicus and Anicetus parvus have been also used for bio- scales (Homoptera, Coccidae) (Subba Rao, 1965; Annecke, 1967; control purposes (Noyes and Hayat, 1994; Trjapitzin, 2010). Since many species of Anicetus are considered effective natural ⇑ Corresponding author. enemies of agricultural pests, the taxonomy of the Anicetus has E-mail address: [email protected] (Y.-Z. Zhang). been studied thoroughly (Tachikawa, 1955, 1958, 1963; Subba 1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2011.05.006 Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 183 Rao, 1965; Annecke, 1967; Hayat et al., 1975; Hayat, 1999; found that each Anicetus species oviposited on a species of Ceropl- Trjapitzin, 2010). Due to their small size and generally similar mor- astes other than the natural host under laboratory conditions. phological features and thus lack of distinct morphological charac- All species known so far from China belong the ceroplastes spe- ters, they are difficult to characterize on morphological data alone. cies group except for Anicetus annulatus Timberlake, Anicetus chin- In fact, many species have been typologically described on the ba- ensis Girault and Anicetus deltoideus Annecke (Table 1). However, sis of subtle differences in morphological features. This is true in their natural pattern of parasitism in the field is not clear. Specifi- the ceroplastes species group. cally, it remains unclear to what extent host associated differenti- In order to separate A. beneficus, A. ceroplastis, and Anicetus ation and subsequent speciation in this group of economically ohgushii, Tachikawa (1958, 1963) used some characters of the an- important parasitoids. As Loch (1997) stated, Anicetus has been tenna (length of the upper margin of the clava and the length of studied extensively, but the biology requires further study and tax- the funicle, as well as the size or the width of funicular segments), onomy of the genus appears to require revision. setae arrangement of forewing base cell, ovipositor and even the Many studies have shown that short fragments of mitochon- bristles on the scutellum. Several other nominal species, for in- drial DNA, in particular the ‘barcode’ marker COI, are effective in stance, Anicetus aligarhensis and Anicetus angustus (Hayat et al., diagnosing species (Hebert et al., 2004a, 2004b; Barrett and Hebert, 1975), Anicetus rarisetus and Anicetus rubensi (Xu and He, 1997), 2005; Ward et al., 2005; Clare et al., 2006; Smith et al., 2006, 2007, have been proposed on the basis of the above subtle differences. 2008). However, ‘barcoding’ of species faces the problem of intra- Unfortunately, these characters are not always reliable and often specific variation and incongruence of mitochondrial markers with difficult to assess for a non-specialist. Problems were experienced morphologically defined species that may limit the use of diagnos- by Loch (1997) in identifying females of A. beneficus; variation in tic markers. Hence, debates over the validity of the approach re- taxonomic characters of this species made identifications difficult main (Meier et al., 2006; Whitworth et al., 2007; Wiemers and using the keys of Ishii and Yasumatsu (1954), Tachikawa (1955, Fiedler, 2007; Hunter et al., 2008; Moura et al., 2008; Dasmahapat- 1958), Subba Rao (1965) and Annecke (1967). ra et al., 2010). Moreover, owning to interspecific mitochondrial As a result of technical difficulties in species identification, and Wolbachia transfers did take place, a very high error rate can studies of the host–parasitoid associations are difficult (Noyes, be achieved for both aspects of barcoding, specimen identification 1994; Shaw, 1994; Quicke, 1997; Santos et al., 2011). Most species and species discovery (Whitworth et al., 2007). In practice, DNA- of Anicetus are known as parasitoids of Ceroplastes spp. and some based identification is already well-established in the literature, have been recored for a broad host range. For example, A. beneficus and has become the standard for identification within many taxo- has been reported to use more than six species of Ceroplastes as nomic groups, for host specificity test (Smith et al., 2006; Li et al., hosts (see Table 1). On the other hand, a single Ceroplastes species, 2010) and for study of the geographical variability of host–parasit- such as C. rubens can host as many as 6 Anicetus species. Even in oid interactions (Santos et al., 2011). China, at least 5 Anicetus species have been reported using C. rubens The high species diversity and broad host use make Anicetus an as their host (Xu et al., 2003). In Japan, A. beneficus, A. ceroplastis excellent potential model system for the investigation of speciation and A. ohgushii are reported to have their own natural host in the patterns and host specificity. Here, the barcode COI fragment of genus Ceroplastes (Tachikawa, 1963). However, Ohgushi (1958) Anicetus species reared from Ceroplastes in mainland China was Table 1 Host–parasitoid index of ceroplastis species group from China. Species Host Citations Anicetus aligarhensis Hayat, Alam & Agarwal Ceroplastes sp. Hayat et al. (1975) and Hayat (1986) Ceroplastes japonicus Xu (1985) Anicetus beneficus Ishii and Yasumatsu Ceroplastes sp. Hayat (1986); Trjapitzin, 1989 Ceroplastes actiniformis Hayat et al. (1975) and Hayat (1986) Ceroplastes