DNA Barcoding of Endoparasitoid Wasps in the Genus Anicetus

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

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 floridensis Trjapitzin, 1989 Ceroplastes japonicus Herting (1972) Xu (1985) Jiang and Gu (1988) Yang and Ren (1999) Ceroplastes pseudoceriferus Herting (1972) Ceroplastes rubens Ishii and Yasumatsu (1954) Anicetus ceroplastis Ishii Ceroplastes ceriferus Trjapitzin (1989), Tachikawa (1963), Herting (1972), Yang and Ren (1999) and Xu and Huang (2004) Ceroplastes floridensis Trjapitzin (1989), Thompson (1954), and Xu and Huang (2004) Ceroplastes japonicus Herting (1972), Yang and Ren (1999) and Xu and Huang (2004) Ceroplastes pseudoceriferus Trjapitzin (1989), Tachikawa (1963), Kajita (1964), Herting (1972) and Xu and Huang (2004) Ceroplastes rubens Herting (1972) Anicetus howardi Hayat et al. (1975) Ceroplastes sp. Hayat et al. (1975) and Hayat (1986) Ceroplastes floridensis Xu and Huang (2004) Ceroplastes japonicus Xu (1985) and Xu and Huang (2004) Ceroplastes rubens Xu and Huang (2004) Anicetus ohgushii Tachikawa Ceroplastes floridensis Xu and Huang (2004) Ceroplastes japonicus Herting (1972), Liu et al. (1983), Xu (1985), Liao et al. (1987) and Trjapitzin (1989) Ceroplastes rubens Liao et al. (1987) Ceroplastes centroroseus Liao et al. (1987) and Trjapitzin (1989) Anicetus rarisetus Xu and He (1997) Ceroplastes ceriferus Xu and He (1997) and Xu and Huang (2004) Ceroplastes japonicus Xu and He (1997) and Xu and Huang (2004) Ceroplastes rubens Xu and He (1997) and Xu and Huang (2004) Anicetus rubensi Xu & He Ceroplastes rubens Xu and He (1997) and Xu and Huang (2004) Anicetus zhejiangensis Xu and Li (1991) Ceroplastes ceriferus Xu and Li (1991) and Xu and Huang (2004) Ceroplastes japonicus Xu and Li (1991) and Xu and Huang (2004) 184 Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 sequenced and analyzed to investigate inter/intraspecific genetic 3. Result variation of closely related species of Anicetus. The D2 region of 28S rRNA was further used to explore whether one or more species 3.1. Morphological identification were present. For many Chalcidoidea and other Hymenoptera 28S- D2 has been shown to be a good species marker (Campbell et al., More than 400 Anicetus specimens were reared from 6000 wild- 1993; Campbell et al., 2000; Babcock and Heraty, 2000; Triapitsyn caught individuals of adult or late-stage nymph wax scale insects et al., 2007). which were morphologically separated into 3 species, namely Through a combined morphological and molecular approach, Ceroplastes ceriferus, C. japanicus and C. rubens. Samples reared we aimed to ascertain if patterns of sequence variation at COI are from C. rubens showed marked morphological variation (see Figs. congruent with one another and with morphology in revealing of the samples, A797a, A835, etc., reared from C. rubens in Plates species boundaries. In addition, we employ molecular evidence 1 and 2). Some of these samples could be identified as A. beneficus to test if Anicetus from different host species represent a single spe- using the previous keys (Tachikawa, 1958, 1963; Annecke, 1967; cies, providing a test of host specificity. Hayat et al., 1975; Hayat, 2006) but other morphs were very difficult to delineate. For example, some females have setae proximal of linea calva, under marginal vein forming two rows in one fore- 2. Materials and methods wing, in the other one forming three rows or being irregular (see Figs. of A797a and A835 in Plate 1). Still other females have setae 2.1. Specimen sampling proximal of linea calva, under marginal vein forming one row in one forewing, in the other one forming two rows (see Figs. of All samples of Anicetus species were reared from hosts collected A8321 in Plate 1). On the other hand, even in the same batch of in the field. Twigs and/or leaves infested by live individuals of adult A. beneficus, we found a few specimens having broad frontovertex, or late-stage nymph wax scale insects (Ceroplastes spp.) vulnerable morphologically quite different from the others (see Figs. of A8301 to parasitisation were collected in the fields and brought back to in Plates 1 and 3). Some specimens reared from C. rubens are very the laboratory as quickly as possible. If two or more Ceroplastes probably A. rubens (Xu and He, 1997). However, A. rubens is very species presented on the same twig or leaf, they were carefully iso- similar to A. ohgushii and A. angustus (Hayat et al., 1975) in appear- lated and each kept in glass vials for at least 2 months to allow par- ance. Moreover, we found number of bristles on the scutellum to asitoids to emerge. As A. beneficus is a special case from both a vary a lot. For instance, in a series of specimens from Shanghai, taxonomic and a molecular perspective, its host was investigated the number of bristles on the scutellum may range from 15 to in more detail. All species were identified by author ZYZ and the 42. These specimens were labelled with an alphanumerical code Ceroplastes hosts were identified by Professor San-An Wu, an expe- until their species identity is confirmed. rienced taxonomist of scale insects in China. Voucher specimens In general, specimens reared from Ceroplastes ceriferus are mor- were deposited in the Institute of Zoology, Chinese Academy of Sci- phologically uniform. After careful examination and comparison ences. Details of the species studied and their associated Ceroplas- with identified specimens by Tachikawa, they were identified as tes hosts are listed in Table 2. A. ceroplastis (see Figs. of A8031 in Plate 2). Anicetus specimens reared from C. japonicus can only be separated from A. ceroplastis by a minor difference in antenna shape, 2.2. DNA extraction, amplification and sequencing suggesting that they might be a different species (see Figs. of 071b in Plate 2). They are very similar to A. aligarhensis. Total genomic DNA was extracted from each individual using DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s protocols. Universal primers LCO1490 and HCO2198 (Folmer 3.2. DNA sequences analyses et al. 1994) were used to amplify 658 bp of the 50 end of mitochondrial cytochrome oxidase I (COI). Reactions (50 ll) consisted of 5 ll We obtained a sequence fragment of ca. 650-bp from all indi- of template DNA (not quantified), 5 ll of reaction buffer, 4 llof viduals, but deleted the terminal ambiguous part of the aligned dNTP mixture (2.5 mM each), 0.5 mM of each primer and 1.25 unit data, to result in a matrix of 616 nucleotides. The frequency of ade- of ExTaq polymerase. Reactions were conducted on an Eppendorf nine (A) and thymine (T) was high (A = 28.4%, C = 12.3%, G = 15.6%, Mastercycler gradient S thermocycler with the following profile: T = 43.8%). COI sequences revealed that the different Anicetus mor- 94 °C for 1 min followed by 35 cycles of 94 °C for 30 s, 47 °C for phs were split into four haplogroups in the neighbor-joining (NJ) 45 s, 72 °C for 1 min, and final extension in 72 °C for 3 min. Prod- tree (Plate 4). Specimens reared from C. rubens are divided into ucts were visualized on a 2% agarose E-GelH 96-well system (Invit- two groups. One clade includes those specimens which are very rogen) and samples containing clean single bands were sequenced probably A. rubensi mentioned above. The remaining Anicetus mor- using BigDye v3.1 on an ABI 3730xl DNA Analyzer (Applied Biosys- phs reared from C. rubens are clustered together, including those tems). The forward and reverse primers were used for amplifying we identified as A. beneficus. Specimens reared from Ceroplastes the D2 region of 28S rRNA gene: [F] 50-CGT GTT GCT TGA TAG ceriferus (identified as A. ceroplastis) form a clade. Specimens reared TGC AGC-30 and [R] 50-TTG GTC CGT GTT TCA AGA CGG G-30 (Camp- from C. japonicus, which probably are A. aligarhensis, form another bell et al., 1993). The sequencing procedure of PCR products is clade. Across these haplogroups, the COI region showed 11.9–15.6% same as in Zhang et al. (2008). Contigs were made using Sequen- mean sequence divergence (or a mean of 73.3–96.1 nucleotide cher v4.0.5 (Gene Codes) and were subsequently aligned by eye substitutions over 616 bp) between groups. However, intra- in Bioedit (Hall, 1999). Sequence divergences were calculated using haplogroup polymorphism in the COI region was lower, with the the K2P distance model (Kimura, 1980) and a NJ tree of distances number of segregating (polymorphic) sites and number of haplo- (Saitou and Nei, 1987) was created to provide a graphic represen- types per group ranging from 0 to 12 and 1 to 7, respectively. tation of the patterning among-species divergences using MEGA4 The majority of nucleotide substitutions in the COI region consti- (Tamura et al., 2007). tuted synonymous changes. For example, in the most diverse We verified our database coverage for Anicetus by using BLASTn group based on the nucleotide sequence (here referred to as A. ben- 2.2.16 (Altschul et al. 1997) to find the top 100 sequences matching eficus haplotype group), only 2 of 12 codon changes were non- one newly sequenced individual. synonymous. Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 185

Table 2 Specimens of Anicetus used in the study (collectors’ names are abbreviated as follows: HDY = Dun-Yuan Huang; LHL = Hong-Liang Li; LJ = Jie Li; YF = Feng Yuan; ZTX = Tong-Xin Zhang; ZYZ = Yan-Zhou Zhang); DNA vouchers are in IZCAS and ZYZ identiﬁed the species).

Species Voucher code Host Location Date Collector Anicetus beneficus 7951 Ceroplastes rubens Jiangxi, Yichun 15.vi.2007 HDY Anicetus beneficus 7952 Ceroplastes rubens Jiangxi, Yichun 15.vi.2007 HDY Anicetus 797a 797a Ceroplastes rubens Shanghai (Minhang park) 11.v.2008 LHL Anicetus 797b 797b Ceroplastes rubens Shanghai (Minhang park) 11.v.2008 LHL Anicetus 797c 797c Ceroplastes rubens Shanghai (Minhang park) 13.v.2008 LHL Anicetus 715 715 Ceroplastes rubens Hunan, Chenzhou 10.xi.2006 ZYZ Anicetus 7981 7981 Ceroplastes rubens Jiangxi, Xinyu 13.v.2008 HDY Anicetus 7982 7982 Ceroplastes rubens Jiangxi, Xinyu 15.v.2008 HDY Anicetus 7983 7983 Ceroplastes rubens Jiangxi, Xinyu 15.v.2008 HDY Anicetus beneficus 824 Ceroplastes rubens Jiangxi, Xinyu 20.xi.2009 HDY Anicetus beneficus 825 Ceroplastes rubens Jiangxi, Xinyu 20.xi.2009 HDY Anicetus beneficus 8291 Ceroplastes rubens Zhejiang, Hangzhou 24.xi.2008 LHL Anicetus beneficus 8292 Ceroplastes rubens Zhejiang, Hangzhou 24.ix.2008 LHL Anicetus beneficus 8293 Ceroplastes rubens Zhejiang, Ningbo 28.v.2009 ZTX Anicetus beneficus 8294 Ceroplastes rubens Zhejiang, Ningbo 28.v.2009 ZTX Anicetus beneficus 8295 Ceroplastes rubens Zhejiang, Hangzhou 25.ix.2008 LHL Anicetus 8301 8301 Ceroplastes rubens Zhejiang, Hangzhou 25.ix.2008 LHL Anicetus 8302 8302 Ceroplastes rubens Zhejiang, Hangzhou 25.ix.2008 LHL Anicetus 8303 8303 Ceroplastes rubens Zhejiang, Hangzhou 25.ix.2008 LHL Anicetus 8321 8321 Ceroplastes rubens Sichuan, Chengdu 16.v.2009 LHL Anicetus 8322 8322 Ceroplastes rubens Sichuan, Chengdu 16.v.2009 LHL Anicetus 8323 8323 Ceroplastes rubens Sichuan, Chengdu 16.v.2009 LHL Anicetus 8324 8324 Ceroplastes rubens Sichuan, Chengdu 16.v.2009 LHL Anicetus 8324 8324 Ceroplastes rubens Sichuan, Chengdu 16.v.2009 LHL Anicetus 835 835 Ceroplastes rubens Jiangsu, Nanjing 13.vi.2009 LHL Anicetus 836 836 Ceroplastes rubens Jiangsu, Nanjing 15.vi.2009 LHL Anicetus 7961 7961 Ceroplastes rubens Shanghai (Tiyu park) 18.iv.2009 LHL Anicetus 7962 7962 Ceroplastes rubens Shanghai (Tiyu park) 18.iv.2009 LHL Anicetus 7963 7963 Ceroplastes rubens Shanghai (Tiyu park) 18.iv.2009 LHL Anicetus 7991 7991 Ceroplastes rubens Jiangxi, Yichun 13.xi.2008 HDY Anicetus 7992 7992 Ceroplastes rubens Jiangxi, Yichun 13.xi.2008 HDY Anicetus 7993 7993 Ceroplastes rubens Jiangxi, Yichun 13.xi.2008 HDY Anicetus 7994 7994 Ceroplastes rubens Jiangxi, Yichun 13.xi.2008 HDY Anicetus 7995 7995 Ceroplastes rubens Jiangxi, Yichun 13.xi.2008 HDY Anicetus 8231 8231 Ceroplastes rubens Jiangxi, Yichun 15.xi.2008 HDY Anicetus 8232 8232 Ceroplastes rubens Jiangxi, Yichun 15.xi.2008 HDY Anicetus 8281 8281 Ceroplastes rubens Shanghai (Binjiang park) 15.vi.2008 LHL Anicetus 8282 8282 Ceroplastes rubens Shanghai (Binjiang park) 15.vi.2008 LHL Anicetus 853a 853a Ceroplastes rubens Hunan, Changsha 11.xi.2006 ZYZ Anicetus 853b 853b Ceroplastes rubens Hunan, Changsha 11.xi.2006 ZYZ Anicetus 853c 853c Ceroplastes rubens Hunan, Changsha 11.xi.2006 ZYZ Anicetus ceroplastis 8031 Ceroplastes ceriferus Beijing (Haidian) 5.xi.2007 ZYZ Anicetus ceroplastis 8032 Ceroplastes ceriferus Beijing (Haidian) 5.xi.2007 ZYZ Anicetus ceroplastis 8034 Ceroplastes ceriferus Beijing (Haidian) 5.xi.2007 ZYZ Anicetus ceroplastis 8037 Ceroplastes ceriferus Beijing (Haidian) 13.iv.2008 ZYZ Anicetus ceroplastis 8038 Ceroplastes ceriferus Beijing (Haidian) 13.iv.2008 ZYZ Anicetus ceroplastis 8041 Ceroplastes ceriferus Shandong, Taian 10.v.2008 ZYZ Anicetus ceroplastis 8042 Ceroplastes ceriferus Shandong, Taian 10.v.2008 ZYZ Anicetus ceroplastis 8043 Ceroplastes ceriferus Shandong, Taian 10.v.2008 ZYZ Anicetus 8221 8221 Ceroplastes japonicus Shaanxi, Xian 23.v.2009 LHL Anicetus 8222 8222 Ceroplastes japonicus Shaanxi, Xian 23.v.2009 LHL Anicetus 071a 071a Ceroplastes japonicus ShanXi, Taiyuan 3.vi.2007 LJ Anicetus 071b 071b Ceroplastes japonicus ShanXi, Taiyuan 3.vi.2007 LJ Anicetus 071c 071c Ceroplastes japonicus ShanXi, Taiyuan 3.vi.2007 LJ Anicetus 071d 071d Ceroplastes japonicus ShanXi, Taiyuan 5.vi.2007 LJ Anicetus 071e 071e Ceroplastes japonicus ShanXi, Taiyuan 5.vi.2007 LJ

28S sequences were recovered from all specimens analyzed. 1986). However, the natural enemies, particularly those belonging There was 0.7–3.0% divergence in 28S between these haplogroups. to the superfamily Chalcidoidea, are difficult to study taxonomi- Intra-haplogroup polymorphism in the 28S D2 region was only cally (Noyes, 1982). Our results demonstrate that molecular tools found in the A. beneficus haplotype group, with 0.2% divergence. provide fundamental new approaches for helping to identify spe- The NJ analysis of the alignment of 28S recovered all haplogroups cies. DNA barcoding provides rapid, accurate and potentially auto- obtained with COI (Plate 5). mated species identifications by using short, standardized gene regions as internal species tags (Hebert and Gregory, 2005), and has been widely utilized to discriminate between cryptic species 4. Discussion and in phylogenetic analysis of morphologically similar taxa (Sperling and Hickey, 1994; Danforth et al., 1998; Lin and Wood, The correct identification of a natural enemy of pests is as 2002; Monti et al. 2005; Heraty et al. 2007; Triapitsyn et al. essential to the success of biocontrol programs as the correct iden- 2007). High genetic divergence was found among the Anicetus spe- tification of the pest species itself (Rosen and DeBach, 1973; Rosen, cies analyzed, with a mean value of 11.98%, in accordance to the 186 Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191

Plate 1. Female antennae and fore wings of specimens reared from Ceroplastes rubens. 1–3, A797a; 4–6, A835; 7–9, A824; 10–12, A8321. Scale bar = 0.5 mm. mean value known for the Hymenoptera (11.5%) (Hebert et al., DNA barcoding may also assist to distinguish morphologically 2003). The high interspecific sequence variation of the COI gene similar species, revealing cryptic species, or to provide an alterna- found in the genus Anicetus, coupled with a low intraspecific vari- tive set of characters to assist in inferring species boundaries. For ation, indicate its potential to diagnose species of Anicetus, as it has instance, based on morphological characters of frontovertex and been shown in other insects. Likewise, although the 28S-D2 region forewing, we can’t identify samples like A_8301 as A. beneficus. is more conserved than the ITS or Cytochrome Oxidase regions However DNA sequence analyses show they should be regarded (Heraty, 2004), it appears to be a very useful marker for identifica- as a member of beneficus. If the COI and 28S information indeed tion of species in Chalcidoidea (Babcock and Heraty, 2000; De Bar- is indication of the species boundaries, this would require a reap- ro et al. 2000; Babcock et al. 2001; Manzari et al. 2002; Pedata and praisal of the morphological characters usually used to define a Polaszek, 2003). Our results also support the use of 28S D2 rRNA species. The relative width of frontovertex, setae arrangement for distinguishing the Anicetus species. It is very interesting that proximal of linea calva, and setae on the scutellum are frequently in species of Anicetus, the COI gene groups have intra-haplogroup used in identification keys to separate Anicetus species of the cero- variation but intra-haplogroup polymorphism in the 28S D2 region plastis group (Tachikawa, 1958; Tachikawa, 1963; Annecke, 1967; was only found in the A. beneficus haplotype group. Moreover, NJ Hayat et al., 1975; Hayat, 2006). Our results show that these char- analysis of the alignment of 28S recovered all haplogroups ob- acters are not always reliable. For example, A. rubensi was proposed tained with COI, showing a perfect match of COI ‘‘haplogroups’’ by Xu and He (1997) based on material reared from C. rubens, who and 28S ‘‘genotypes’’. The congruence of unlinked gene markers stated it is similar to A. beneficus. It is our view that A. rubensi is confirms the existence of independent gene pools (Monaghan very similar to A. angustus (Hayat et al., 1975), which also exhibits et al., 2005). a single line of setae in the base cell of the forewings and the 4–6th Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 187

Plate 2. Female antennae and fore wings of specimens reared from Ceroplastes spp. 13–14, A8301 reared from Ceroplastes rubens; 15–16, Anicetus ohgushii, 7961 reared from Ceroplastes rubens; 17–18, Anicetus ohgushii, 8231 reared from Ceroplastes rubens; 19–20, Anicetus ceroplastis, 8031 reared from Ceroplastes ceriferus; 21–22, 071b reared from Ceroplastes japonicus. Scale bar = 0.5 mm.

complexes of cryptic taxa, each of them restricted to only a few hosts (Herre, 2006; Wood et al., 2007; Smith et al., 2008; Li et al., 2010). A. beneficus has been recorded from different hosts in the lit- eratures (see Table 2). However, most of the publications regarding host–parasitoid records are of limited value because of the incon- clusive taxonomy on which these records are based (Askew and Shaw, 1986; Noyes, 1994). Our results indicate that Anicetus species may be adapted to much narrower niches or to be species- specific for particular hosts. Specifically, A. beneficus and A. ohgushii use the same host (C. rubens), and they have not been reared from other hosts so far by us in samples from across China. Our results suggest that A. beneficus, A. ceroplastis, and A. ohgushii are each confined to a limited set of host species, or even are monospecific in their host choice. This is consistent with the notion that endoparasitoids must overcome specific host defenses (Salt, 1968), where- Plate 3. 23, dorsal view of A8301 reared from Ceroplastes rubens. Scale by many physical and chemical factors may become increasingly bar = 0.5 mm. refined in the interactions with the hosts (Vinson, 1976). For example, Takabayashi and Takahashi (1985) found that A. beneficus funicular segments distinctly larger than the third funicular seg- showed the highest response to intact C. rubens among the three ment. Thus more efforts are needed in order to verify the true sta- host species tested. The host of A. dodonia Ferriere was verified tus of the species in this group by investigating all known species as Ceroplastes pseudoceriferus (Subba Rao, 1965), but it did not at- in the group or an even wider collection of material of Anicetus. tack the Florida wax scale in laboratory conditions when tested A recent result of DNA barcoding studies is that traditional spe- for biological control of C. floridensis introduced to Israel (Wysoki, cies of parasitoid wasps that use many different hosts are in fact 1979). 188 Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191

Plate 4. Neighbour-joining tree of Chinese Anicetus COI sequences, using Kimura-2-parameter distance. Bootstrap values 100 for each haplogroup were calculated in MEGA4.0 with 1000 replicates and hosts of each haplogroup are shown in the right column.

Efforts to estimate parasitoid host speciﬁcity that do not include single species (Bickford et al., 2007). Kankare et al. (2005) found DNA-based discrimination of the parasitoid species are likely to genetic differentiation within a group of Cotesia parasitoids is underestimate host speciﬁcity due to the incorrect labeling of mor- associated with host species rather than subdivided according to phologically similar but genetically isolated lineages as being a spatial isolation. Considering the large number of hosts recorded Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 189

Plate 5. Neighbour-joining tree of Chinese Anicetus 28S sequences, using Kimura-2-parameter distance. Bootstrap values 100 for each haplogroup were calculated in MEGA4.0 with 1000 replicates and hosts of each haplogroup are shown in the right column.

for Anicetus, the group is likely to include cryptic species confined Acknowledgments to certain hosts. Thus more molecular taxonomic work should be carried out to verify these cryptic species. A survey of successful The project is supported by the National Natural Science Foun- projects in biological control shows that most effective natural dation of China (NSFC Grant No. 30870321, 31071950), by Public enemies either are virtually monophagus or narrowly oligophagus Welfare Project from Ministry of Agriculture of the People’s Repub- (Rosen and Debach, 1973). Our results also support that in future lic of China (Grant No. 200803002) and partially by Ningbo Science biocontrol programs endoparasitoids from the same insect pest and Technology Bureau (2009C10004). The authors are grateful to species are the best choice (Phillips et al., 2008). COI sequences Dr. John S. Noyes (Natural History Museum, London) provided can detect substantial genetic diversity of endoparasitoids wasps invaluable assistance in studying the encyrtids during ZYZ’s visit and DNA barcoding approaches can therefore help to assess the to the Natural History Museum, London. Thanks to Prof. San-An intraspecific diversity of problematic taxa to reveal host specific Wu, Beijing Forestry University, for identifying scale insects, par- lineages not separable by morphological characters. ticularly Ceroplastes spp. 190 Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191

References Herting, B., 1972. Homoptera. A catalogue of parasites and predators of terrestrial arthropods. Section A. Host or Prey/Enemy.2.. Commonwealth Agricultural Bureaux, Slough, England, vol. i, pp. 210. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., Hunter, S.J., Goodall, T.I., Walsh, K.A., Owen, R., Day, J.C., 2008. Nondestructive DNA 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens search programs. Nucleic Acids Research 25, 3389–3402. for DNA barcoding projects. Molecular Ecology Resources 8, 56–61. Annecke, D.P., 1967. The genera Anicetus Howard, 1896, Paracerapterocerus Girault, Ishii, T., Yasumatsu, K., 1954. Description of a new parasitic wasp of Ceroplastes 1920, and allies, with descriptions of new genera and species (Hymenoptera: rubens Maskell. Mushi 27, 69–74. Encyrtidae). Transactions of the Royal Entomological Society of London 119, Jiang, H., Gu, H.G., 1988. The bionomics of Ceroplastes japonicus Green and its 99–169. parasitoids. Insect Knowledge 25, 154. Askew, R.R., Shaw, M.R., 1986. Parasitoid communities: their size, structure and Kajita, H., 1964. On the Parasites of Ceroplastes pseudoceriferus Green. Science development. In: Waage, J., Greathead, D. (Eds.), Insect Parasitoids. Academic Bulletin of the Faculty of Agriculture, Kyushu University, vol. 21, pp. 7–11. Press, London, pp. 225–264. Kankare, M., van Nouhuys, S., Hanski, I., 2005. Genetic divergence among host Babcock, C.S., Heraty, J.M., 2000. Molecular markers distinguishing Encarsia formosa specific cryptic species in Cotesia melitaerum aggregate (Hymenoptera: and Encarsia luteola (Hymenoptera: Aphelinidae). Annals of the Entomological Braconidae), parasitoids of checkerspot butterflies. Annals of the Society of America 93, 738–744. Entomological Society of America 98, 382–394. Babcock, C.S., Heraty, J.M., De Barro, P.J., Driver, F., Schmidt, S., 2001. Preliminary Kim, H.S., Moon, D.Y., Park, J.S., Lee, S.C., Lippold, P.C., Chang, Y.D., 1979. Integrated phylogeny of Encarsia Forster (Hymenoptera: Aphelinidae) based on control of citrus pests: 2. Control of ruby scales (Ceroplastes rubens) on citrus by morphology and 28S rDNA. Molecular Phylogenetics and Evolution 18, 306– introduction of a parasitic natural enemy, Anicetus beneficus (Hymenoptera, 323. Encyrtidae). Korean Journal of Plant Protection 18, 107–110. Barrett, R.D.H., Hebert, P.D.N., 2005. Identifying spiders through DNA barcodes. Kim, H.S., Moon, D.Y., Kwon, H.M., 1994. Studies on integrated control of citrus Canadian Journal of Zoology 83, 481–491. pests: 3. Suppressive effects of the red wax scale (Ceroplastes rubens) density by Ben-Dov, Y., 1993. A Systematic Catalogue of the Soft Scale Insects (Homoptera: a parasitic natural enemy, Anicetus beneficus (Hymenoptera, Encyrtidae). RDA Coccoidea: Coccidae) of the World with Data on Geographical Distribution, Host Journal of Agricultural Sciences, Crop Protection. 36, 373–377. Plants, Biology and Economic Importance. Sandhill Crane Press, Inc., Gainesville, Kimura, M., 1980. A simple method for estimating evolutionary rates of base p. 536. substitutions through comparative studies of nucleotide sequences. Journal of Bickford, D., Lohman, D.J., Sodhi, N.S., Ng, P.K., Meier, R., Winker, K., Ingram, K.K., Molecular Evolution 16, 111–120. Das, I., 2007. Cryptic species as a window on diversity and conservation. Trends Krull, S.M.E., Basedow, T., 2005. Evaluation of the biological control of the pink wax in Ecology & Evolution 22, 148–155. scale Ceroplastes rubens Maskell (Hom., Coccidae) with the introduced Campbell, B.C., Steffen-Campbell, J.D., Werren, J.H., 1993. Phylogeny of the Nasonia parasitoid Anicetus beneficus Ishii & Yasumatsu (Hym., Encyrtidae) in the species complex (Hymenoptera: Pteromalidae) inferred from an internal Central province of Papua New Guinea. Journal of Applied Entomology 129, transcribed spacer (ITS2) and 28S rDNA sequences. Insect Molecular Biology 323–329. 2, 225–237. Li, Y.W., Zhou, X., Feng, G., Hu, H.Y., Niu, L.M., Hebert, P., Huang, D.W., 2010. COI and Clare, E.L., Lim, B.K., Engstrom, M.D., Eger, J.L., Hebert, P.D.N., 2006. DNA barcoding ITS2 sequences delimit species, reveal cryptic taxa and host specificity of fig- of Neotropical bats: species identification and discovery within Guyana. associated Sycophila (Hymenoptera, Eurytomidae). Molecular Ecology Molecular Ecology Notes 7, 184–190. Resources 10, 31–40. Danforth, B.N., Levin Mitchell, P., Packer, L., 1998. Mitochondrial DNA Liao, D.X., Li, X.L., Pang, X.F., Chen, T.L., 1987. Hymenoptera: Chalcidoidea (1). differentiation between two cryptic Halictus (Hymenoptera: Halictidae) Economic Insect Fauna of China 34, 1–241. species. Annals of the Entomological Society of America 91, 387–391. Lin, C.P., Wood, T.K., 2002. Molecular phylogeny of the North American Enchenopa Dasmahapatra, K.K., Elias, M., Hill, R.I., Hoffman, J., Mallet, J., 2010. Mitochondrial binotata (Homoptera: Membracidae) species complex. Annals of the DNA barcoding detects some species that are real, and some that are not. Entomological Society of America 95, 162–171. Molecular Ecology Resources 10, 264–273. Liu, Z., Liu, W., Xu, A., 1983. Life history and behaviour of Anicetus ohgushii De Barro, P.J., Driver, F., Naumann, I.D., Schmidt, S., Clarke, G.M., Curran, J., 2000. Tachikawa (Hymen.: Encyrtidae). Natural Enemies of Insects 5, 135–137. Descriptions of three species of Eretmocerus Haldeman (Hymenoptera: Loch, A.D., 1997. Natural Enemies of Pink Wax Scale, Ceroplastes rubens Maskell Aphelinidae) parasitizing Bemisia tabaci (Gennadius) (Hemiptera: (Hemiptera: Coccidae), on Umbrella Trees in Southeastern Queensland. Aleyrodidae) and Trialeurodes vaporariorum (Westwood) (Hemiptera: Australian Journal of Entomology 36, 303–306. Aleyrodidae) in Australia based on morphological and molecular data. Manzari, S., Polaszek, A., Belshaw, R., Quicke, D.L.J., 2002. Morphometric and Australian Journal of Entomology 39, 259–269. molecular analysis of the Encarsia inaron species-group (Hymenoptera: Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for Aphelinidae), parasitoids of whiteflies (Hemiptera: Aleyrodidae). Bulletin of amplification of mitochondrial cytochrome c oxidase subunit I from diverse Entomological Research 92, 173–175. metazoan invertebrates. Molecular Marine Biology and Biotechnology 3, 294– Meier, R., Shiyang, K., Vaidya, G., Ng, P., 2006. DNA barcoding and taxonomy in 299. Diptera: a tale of high intraspecific variability and low identification success. Gimpel, W.F., Miller, D.R., Davidson, J.A., 1974. A systematic revision of the wax Systematic Biology 55, 715–728. scales, genus Ceroplastes, in the United States (Homoptera: Coccoidea: Monaghan, M.T., Balke, M., Gregory, T.R., Vogler, A.P., 2005. DNA-based species Coccidae). Agricultural Experiment Station of the University of Maryland delineation in tropical beetles using mitochondrial and nuclear markers. Miscellaneous Publication, vol. 841, pp. 1–85. Philosophical Transactions of the Royal Society of London. Series B, Biological Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and sciences 360, 1925–1933. analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, Monti, M.M., Nappo, A.G., Giorgini, M., 2005. Molecular characterization of closely 95–98. related species in the parasitic genus Encarsia (Hymenoptera: Aphelinidae) Hayat, M., 1986. Family Encyrtidae. In: Subba Rao, B.R., Hayat, M. (Eds), The based on the mitochondrial cytochrome oxidase subunit I gene. Bulletin of Chalcidoidea (Insecta: Hymenoptera) of India and the Adjacent Countries. Part Entomological Research 95, 401–408. II. Oriental Insects, vol. 20, pp. 67–137. Moura, C.J., Harris, D.J., Cunha, M.R., Rogers, A.D., 2008. DNA barcoding reveals Hayat, M., 1999. Taxonomic notes on Indian Encyrtidae (Hymenoptera: cryptic diversity in marine hydroids (Cnidaria, Hydrozoa) from coastal and Chalcidoidea) V. Oriental Insects 33, 349–407. deep-sea environments. Zoologica Scripta 37, 93–108. Hayat, M., 2006. Indian Encyrtidae (Hymenoptera: Chalcidoidea). Published by M. Noyes, J.S., 1982. Collecting and preserving chalcid wasps (Hymenoptera: Hayat, Department of Zoology, Aligarh Muslim University, p. 496. Chalcidoidea). Journal of Natural History 16, 315–334. Hayat, M., Alam, M., Agarwal, M.M., 1975. Taxonomic Survey of Encyrtid Parasites Noyes, J.S., 1994. The reliability of published host–parasitoid records: a (Hymenoptera: Encyrtidae) in India. Aligarh Muslim University Publication, taxonomist’s view. Norwegian Journal of Agricultural Sciences 16, 59–69. Zoological Series on Indian Insect Types, vol. 9, p. 112. Noyes, J.S., 2010. Universal Chalcidoidea Database. http://www.nhm.ac.uk/jdsml/ Hebert, P.D.N., Gregory, R., 2005. The Promise of DNA Barcoding for Taxonomy. research-curation/res-earch/projects/chalcidoids/, accessed October, 2010. Systematic Biology 54, 852–859. Noyes, J.S., Hayat, M., 1994. Oriental mealybug parasitoids of the Anagyrini Hebert, P.D.N., Ratnasingam, S., deWaard, J.R., 2003. Barcoding animal life: (Hymenoptera: Encyrtidae). CAB International, Oxon, UK, vol. viii, pp. 554. cytochrome c oxidase subunit 1 divergences among closely related species. Ohgushi, R., 1958. Studies on the host selection by three species of Anicetus wasps Proceedings of the Royal Society of London Series B 270 (Suppl 1), 96–99. (Encyrtidae) parasitic on Ceroplastes scales (Coccidae). Memoirs of the College Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004a. Ten of Agriculture, Kyoto University (B) 25 (1), 31–38. species in one: DNA barcoding reveals cryptic species in the neotropical skipper Pedata, P.A., Polaszek, A., 2003. A revision of the Encarsia longifasciata species group butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences (Hymenoptera: Aphelinidae). Systematic Entomology 28, 361–374. of the United States of America 101, 14812–14817. Phillips, C.B., Vink, C.J., Blanchet, A., Hoelmer, K.A., 2008. Hosts are more important Hebert, P.D.N., Stoeckle, M.Y., Zemlak, T.S., Francis, C.M., 2004b. Identification of than destinations: What genetic variation in Microctonus aethiopoides birds through DNA barcodes. PLoS Biology 2, e312. (Hymenoptera: Braconidae) means for foreign exploration for natural Heraty, J.M., Woolley, J.B., Hopper, K.R., Hawks, D.L., Kim, J.W., Buffington, M., 2007. enemies. Molecular Phylogenetics and Evolution 49, 467–476. Molecular phylogenetics and reproductive incompatibility in a complex of Rosen, D., 1986. The role of taxonomy in effective biological control programs cryptic species of aphid parasitoids. Molecular Phylogenetics and Evolution 45, Agriculture. Ecosystems & Environment 15, 121–129. 480–493. Rosen, D., DeBach, P., 1973. Systematics, morphology and biological control. Herre, E.A., 2006. Barcoding helps biodiversity fly. Proceedings of the National Entomophaga 18, 215–222. Academy of Sciences of the United States of America 103, 3949–3950. Y.-Z. Zhang et al. / Biological Control 58 (2011) 182–191 191

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for geographical populations of Anagyrus pseudococci (Hymenoptera: Encyrtidae), reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425. parasitoids of Planococcus spp. (Hemiptera: Pseudococcidae), with notes on Salt, G., 1968. The resistance of parasitoids to the defense reactions of their hosts. Anagyrus dactylopii. Biological Control 41, 14–24. Biological Reviews 43, 200–232. Trjapitzin, V.A., 2010. A Review of Encyrtid Wasps of the Genus Anicetus Howard, Santos, A.M.C., Besnard, G., Quicke, D.L., 2011. Applying DNA barcoding for the 1896 (Hymenoptera, Chalcidoidea, Encyrtidae) of the New World, Hawaiian study of geographical variation in host–parasitoid. Molecular Ecology Resources Islands, and Australia with Description of New Species from Mexico. 11, 46–59. Entomologicheskoe Obozrenie 89, 438–453. Shaw, M.R., 1994. Parasitoid host range. In: Hawkins, B.A., Sheenan, W. (Eds.), Vinson, S.B., 1976. Host selection by insect parasitoids. Annual Review of Parasitoid Community Ecology. Oxford University Press, New York, pp. 111–144. Entomology 21, 109–133. Smith, D., 1986. Biological control of Ceroplastes rubens Maskell, by the introduced Wang, T., 2001. Homoptera: Coccoidea: Pseudococcidae, Eriococcidae, Coccidae, parasitoid Anicetus beneficus Ishii and Yasumatsu. Queensland Journal of Asterolecaniidae, Lecanodiaspididae, Cerococcidae, Aclerdidae. Fauna Sinica Agricultural and Animal Sciences 43, 101–105. Insecta, vol. 22. Science Press, Beijing. Smith, M.A., Woodley, N.E., Janzen, D.H., Hallwachs, W., Hebert, P.D.N., 2006. DNA Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., Hebert, P.D.N., 2005. DNA barcoding barcodes reveal cryptic host-specificity within the presumed polyphagous Australia’s fish species. Philosophical Transactions of the Royal Society of members of a genus of parasitoid flies (Diptera: Tachinidae). Proceedings of the London. Series B, Biological sciences 360, 1847–1857. National Academy of Sciences of the United States of America 103, 3657– Whitworth, T.L., Dawson, R.D., Magalon, H., Baudry, E., 2007. DNA barcoding cannot 3662. reliably identify species of the blowfly genus Protocalliphora (Diptera: Smith, M.A., Wood, D.M., Janzen, D.H., Hallwachs, W., Hebert, P.D.N., 2007. DNA Calliphoridae). Proceedings of the Royal Society B: Biological Sciences 274, barcodes affirm that 16 species of apparently generalist tropical parasitoid flies 1731–1739. (Diptera, Tachinidae) are not all generalists. Proceedings of the National Wiemers, M., Fiedler, K., 2007. Does the DNA barcoding gap exist? – a case study in Academy of Sciences of the United States of America 104, 4771–4772. blue butterflies (Lepidoptera: Lycaenidae). Frontiers in Zoology 4, 8. Smith, M.A., Rodriguez, J.J., Whitfield, D., Janzen, D.H., Hallwachs, W., Hebert, P.D.N., Wood, D.M., Janzen, D.H., Hallwachs, W., Hebert, P.D.N., 2007. DNA barcodes affirm 2008. Extreme diversity of tropical parasitoid wasps exposed by iterative that 16 species of apparently generalist tropical parasitoid flies (Diptera, integration of natural history, DNA barcoding, morphology, and collections. Tachinidae) are not all generalists. Proceedings of the National Academy of Proceedings of the National Academy of Sciences of the United States of Sciences of the United States of America 104, 4967–4972. America 105, 12359–12364. Wysoki, M., 1979. Introductions of beneficial insects into Israel by the Institute of Sperling, F.A., Hickey, D.A., 1994. Mitochondrial DNA sequence variation in the Plant Protection Quarantine Laboratory, ARO, during 1971–1978. spruce budworm species complex (Choristoneura: Lepidoptera). Molecular Phytoparasitica 7, 101–106. Biology and Evolution 11, 656–665. Xu, Z.H., 1985. The parasitic wasps of wax scale (Genus Ceroplastes Gray) Subba Rao, B.R., 1965. A key to species of Anicetus Howard, 1896 (Hymenoptera, from Zhejiang province with notes of five first recorded species from Encyrtidae) and descriptions of new species from India. Proceedings of the China (Hym. Chalcidoidea). Acta Agriculturae Universitatis Zhejiangensis Royal Entomological Society of London (B) 34, 71–75. 11, 411–420. Tachikawa, T., 1955. Some notes on the Japanese species of the genus Anicetus Xu, Z.H., He, J.H., 1997. Two new species of the genus Anicetus from China Howard (Hymenoptera: Encyrtidae). Japanese Journal of Applied Zoology 20, (Hymenoptera: Encyrtidae). Acta Zootaxonomica Sinica 22, 90–94. 173–176. Xu, Z.H., Huang, J., 2004. Chinese Fauna of Parasitic Wasps on Scale Insects. Tachikawa, T., 1958. On the Japanese species of the genus Anicetus parasitic on wax Shanghai Scientific & Technical publishers, Shanghai, pp. 524. scales of the genus Ceroplastes. Mushi 32, 77–82. Xu, Z., Li, X., 1991. A new species of the genus Anicetus (Hymenoptera: Encyrtidae) Tachikawa, T., 1963. Revisional studies of the Encyrtidae of Japan (Hymenoptera: from China. Entomotaxonomia, 13, 219–221. Text in Chinese. Chalcidoidea). Memoirs of Ehime University, vol. 9, 6, pp. 1–264. Xu, Z.H., Zhang, L.L., Wang, H.M., 2003. Revision on species of parasitoids on pink Takabayashi, J., Takahashi, S., 1985. Host selection behavior of Anicetus beneficus wax scale (Ceroplastes rubens) (Hymenoptera: Chalcidoidea). Forest Pest and Ishii et Yasumatsu (Hym.: Enyrtidae). III. Presence of ovipositional stimulants in Disease 22, 1–5. the scale wax of the genus Ceroplastes. Applied Entomology and Zoology 20 (2), Yang, Z.X., Ren, Y.S., 1999. Studies on the different kinds of parasitic wasp of 173–178. Ceroplastes rubens Mask. and C. japaonica [sic] Green and their killing effect on Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary the pests. South China Fruits 28, 16–18. Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Yasumatsu, K., 1958. An interesting case of biological control of Ceroplastes rubens Evolution 24(8), 1596–1599. Msakell in Japan. Proceedings of 10th International Congress of Entomology 4, Thompson, W.R., 1954. A Catalogue of the Parasites and Predators of Insect Pests. 771–775. Section 2. Host Parasite Catalogue. Part 3. Hosts of the Hymenoptera Yasumatsu, K., 1969. Biological control of citrus pests in Japan. Proceedings of the (Calliceratid to Evaniid). Commonwealth Agricultural Bureaux, First International Citrus Symposium 2, 773–780. Commonwealth Institute of Biological Control, Ottawa, pp. 191–332. Zhang, Y.Z., Yu, F., Zhu, C.D., 2008. Phylogeny of Copidosoma spp. (Hymenoptera, Triapitsyn, S.V., González, D., Vickerman, D.B., Noyes, J.S., White, E.B., 2007. Encyrtidae) associated with Noctuidae (Lepidoptera) based on 28S rDNA D2 Morphological, biological, and molecular comparisons among the divergent sequences. Acta Entomologia sinica 51, 992–996.