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Tijdschrift voor Entomologie 158 (2015) 1–19

Morphological and molecular studies of a new species of the root mealybug genus Ripersiella Tinsley (Hemiptera: Coccoidea: Rhizoecidae) from greenhouses in The Netherlands and a first incursion of the American root mealybug Rhizoecus keysensis Hambleton in Europe Maurice Jansen & Marcel Westenberg

Ripersiella emarai is described from Ficus cyathistipula Warburg, Ficus lyrata Warburg and Dieffenbachia sp. from Dutch greenhouses. The immature stages are described based on microscopic features and a key to the stages is provided for their separation. This species description is actuated by the necessity to distinguish the new species from the root mealybug Ripersiella hibisci (Kawai & Takagi), which is occasionally imported and shares host plants with the new species. An identification key to adults of the Dutch greenhouse species and species intercepted during import inspections based on microscopic morphological characters is given. A key to distinguish the nymphal stages is provided and keys are given to the identification of first, second and third instar nymphs of Ripersiella multiporifera Jansen, R. hibisci (Kawai & Takagi) and R. emarai Jansen. Rhizoecus advenoides Takagi & Kawai, 1971 is found to be a junior synonym of Rh. amorphophalli Betrem, 1940. Keywords: Pseudococcidae, Rhizoecidae, Ripersiella, new species, The Netherlands, greenhouses Maurice Jansen*, Ministry of Economic Affairs, Division Agriculture and Nature, Netherlands Food and Product Safety Authority, P.O. Box 9102, 6700 HC Wageningen, The Netherlands. [email protected]

Introduction an important milestone in the exploration of this In Dutch greenhouses root mealybugs (fam. species-rich subterranean fauna. Rhizoecidae are con- Rhizoecidae) are a common occurrence on the roots tinually transported all over the world on rooted of a large number of bonsai and pot plants. Several plants with soil. The Netherlands is a main port in factors influence their presence. Because of their hid- Europe for imports of plant material originating from den habits, it is to be expected that only a relatively different parts of the world. Most of these imported small percentage of the world fauna is described yet, consignments are intensively sprayed, killing the and as a result many new species are expected to be majority of specimens, but a small number of speci- discovered in the future. The overview of Kozár & mens or eggs might survive, giving rise to new popu- Konczné Benedicty (2007) of the world fauna is lation growth. Most of these revived populations

Tijdschrift voor Entomologie 158: 1–19, Figs 1–12. [ISSN 0040-7496]. brill.com/tve © Nederlandse Entomologische Vereniging. Published by Koninklijke Brill NV, Leiden. Published 20 October 2015. DOI 10.1163/22119434-15812049 *Corresponding author Downloaded from Brill.com09/29/2021 11:17:00PM via free access

2 Tijdschrift voor Entomologie, volume 158, 2015 are discovered much later by accident or might by Measurements and drawings were made using an overlooked by inspectors, which may enhance the ocular micrometer on an Olympus (JAPAN) BX50 risk of unseen spreading to other parts of Europe interference contrast microscope. or in the case of re-export even to other parts of DNA was isolated from single specimens of the world. The new species described here, Ripersiella R. emarai, R. hibisci, R. multiporifera and Rh. dianthi emarai, was only found on Ficus cyathistipula Warburg Green using the Mammalian Tissue protocol of and was successfully reared on F. lyrata Warburg as the High Pure PCR Template Preparation kit well, indicating that this new root mealybug species (Roche, Basel, Switzerland) and stored at –20°C might be able to propagate on many more host until use. Partial COI using primers PcoF1 plants. 5’-CCTTCAACTAATCATAAAAATATYAG-3’ The morphology of the pre-adult stages was com- (Park et al. 2010) and LepR1 5’-TAAACTTCT pared with the related Ripersiella multiporifera GGATGTCCAAAAAATCA-3’ (Park et al. 2011), Jansen (2008) and R. hibisci Kawai & Takagi (1971). an 18S region using primers 18S-2880 5’-CTGGT The last species has a quarantine status in Europe TGATCCTGCCAGTAG-3’ and 18S-B 5’CCG and is regulated by its presence on the Annex I/AII CGGCTGCTGGCACCAGA3’ (von Dohlen & list of species whose introduction into Europe is Moran, 1995) and a fragment of the D2 expansion prohibited. R. hibisci shares part of its hosts with region of 28S using primers 28S-D2F AGAGA that of the new species. As a result consignments GAGTTCAAGAGTACGTG and 28S-D2R TTGG with plants originating from East Asia are very often TCCGTGTTTCAAGACGGG (Belshaw & Quicke, heavily treated with pesticides. In most cases only a 1997) were amplified. The PCR reaction compo- very small number of the populations living on nents and final concentrations were 2.5 mM MgCl2, the roots survive this treatment resulting in the dis- 0.1 mM dNTPs, 0.2 μM each primer, 2.5 units Taq covery of a very small number of specimens during DNA polymerase with appropriate buffer (Qiagen, import interceptions. Therefore pre-adult stages Venlo, The Netherlands), and 2 μl DNA template in were studied to compare the morphology of related a final volume of 25 μl. The PCR cycling condition species with the description of those of R. hibisci for COI amplification was 3 min at 94°C; five cycles (Jansen 2001). Furthermore, molecular data of cyto- of 30 s at 94°C, 45 s at 45°C, 1 min at 72°C; forty chrome oxidase I (COI), 18S small subunit ribo- cycles of 30 s at 94°C, 45 s at 51°C, 1 min at 72°C; somal RNA (18S rRNA) and 28S large subunit 10 min at 72°C. For 18S and 28S the PCR cycling ribosomal RNA (28S rRNA) were gen;erated from conditions were 3 min at 94°C; forty cycles of 20 s at R. emarai to aid species identification at the molecu- 94°C, 1 min at 50°C, 1 min at 72°C; 10 min at 72°C. lar level. All PCR products were visualized in a 1% agarose gel stained with SYBR Safe (Lifetechnology, Carlsbad, CA, USA) and bidirectionally sequenced using Big Material and methods Dye Terminator v3.1 Cycle Sequencing Kit (Applied Populations of the new species originating from the Biosystems, Carlsbad, CA, USA) on an Applied finding sites in Berkel en Rodenrijs and Bleiswijk Biosystems 3500 Genetic Analyzer. Sequences from were reared on under quarantine conditions for fur- both strands were assembled, edited if necessary ther study on infested Ficus plants in a greenhouse in and aligned with MAFFT (Katoh et al. 2002) using Wageningen. Nymphs were collected several times to Geneious ver. 7.0.6 (Biomatters, Auckland, New obtain enough material. For microscopic slides, Zealand). All sequences were deposited in GenBank specimens were cleared in 10% KOH, ethanol 70%, under accession numbers KM453213–KM453227. and acetic acid, stained with a mixture of lignin pink and acid fuchsin, cleared in clove oil and mounted in Canada balsam. Specimens of the first and second Ripersiella emarai Jansen sp. n. stage tend to fold in antennae and legs when Figs 1–7 mounted in Canada balsam. Therefore part of the specimens of these stages was mounted in berlese Type material. Holotype, female: The Netherlands, solution. About 150 specimens in total were studied undamaged female on a slide containing two speci- and part was used for measurements. The descrip- mens. Left label: leg. M. Jansen/48/NVWA-kas, tion of each stage and the measurements are based on greenhouse, Wageningen, 29.iv.2013, reared, origi- multiple slide-mounted specimens only. Measure nal: leg. Sukul, 10.ii.2012, Berkel en Rodenrijs. ments are given as ranges. The characters of the holo- Right label: Ripersiella emarai Jansen, Holotype type fall within the range of the characters as given. [marked with red], 1738349. The slide is deposited The wrongly printed original figures ofRipersiella in the collection of the National Plant Protec multiporifera (Jansen 2008) are given here as well. tion Organization, Plant Protection Service (PPS),

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Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 3

Wageningen. Paratypes, females: The Netherlands: Description of adult female Bleiswijk, 29 June 2006, Ficus cyathistipula, leg. A. Figs 1, 2 Emara, 1 slide: 2 adults, PPS3373646; Bleiswijk, 7 July 2006, Dieffenbachia sp., leg. A. Emara, 3 slides: Slide-mounted characters. Measurements based on 12 6 adults, PPS3373742; Bleiswijk, 7 July 2006, Ficus specimens. The holotype is a teneral adult female. It lyrata, leg. A. Emara, 1 slide: 2 adult females, is elongate-oval with almost parallel sides, length PPS3373726; Bleiswijk, 7 July 2006, Ficus cyathis- 0.72 mm (antennae and anal setae excluded), width tipula, leg. A. Emara, 21 slides: 17 adult females, 2 0.3 mm. Paratypes: adults are 640–1650 μm long adult males, 13 nymphs, PPS3373726; Bleiswijk, 21 and 275–960 μm wide. June 2007, Ficus sp., leg. A. Emara, 1 slide: 1 adult, Dorsum. Posterior end of body with well developed, PPS3379415; Wageningen, 20 September 2009, slightly projecting anal lobes on a slightly sclerotized Ficus lyrata, leg. M. Jansen, 5 slides: 4 adults, 3 plate; each lobe with 7 apical setae of varying length: a nymphs, 1 male nymph, PPS3373726; Berkel en group of 4 short setae 12–23 μm long near the edge Rodenrijs, 10 February 2012, Ficus cyathistipula, leg. of the lobe and three setae 65–85 μm long accompa- B. Sukul, 1 slide: 2 adults, PPS5395187; Berkel en nied by a group of 20–25 trilocular pores, mostly Rodenrijs, 10 February 2012, Ficus cyathistipula, leg. only half of them visible. Anal ring 48–53 μm in B. Sukul, 3 slides: 6 adults, 1 nymph, PPS5395187; diameter with 6 setae each 55–68 μm long, with an Wageningen, 29 April 2013, Ficus cyathistipula, outer row of 18 cells; up to 14 well-visible spiculae leg. M. Jansen, 12 slides: 17 adults, 6 nymphs, in outer row of cells. Flagellate setae in bands across PPS5395187; Wageningen, 9 October 2013, Ficus the segments but absent from intersegmental areas; cyathistipula, leg. M. Jansen, 25 slides: 2 adults, 29 length marginal setae around the entire body: those nymphs, 1 male nymph, PPS5395187. around the head up to 28 μm, of the thorax up to Etymology. The species epithet is named in honour 25–30 μm and from the abdomen up to 25 μm. On of Atef Emara, the collector of the first specimens of each abdominal segment usually the longest setae up the new species, in acknowledgment of his activities to 15–20 μm long, the shortest setae 8–10 μm long as an experienced phytosanitary field inspector. (including basal socket). Bitubular pores of one type, more or less oval-shaped, up to 6 μm wide and Field characters The body of the species is typical for the Rhizoecini: elongate to broadly oval depending of the age of the specimen, flattened ventrally and convex dorsally. Teneral females are more or less elongate and the hind legs reach the ostioles whereas egg-laying females possess a swollen abdomen and an oval- shaped body, the legs reaching the third or fourth abdominal segment. Normally they are very active, walking around. The body is lightly covered with a white powdery, mealy, wax secretion; legs and antennae are pale brown. Biology. Like all members of the family Rhizoecidae, the new species was found living subterraneously on the root hairs. Most species of Rhizoecidae tend to live oligophagously or polyphagously on their hosts. The description of the new species is based on specimens found on representatives of two families and two genera (Moraceae: Ficus cyathistipula; F. lyrata; Araceae: Dieffenbachia sp.). During all observations, most developmental stages were present and mixed, suggesting that there is more than one generation per year, which is in accordance with observations on the life cycle of Ripersiella hibisci (Jansen 2001). Major factors that influence the duration of the life cycle are the temperature and the host plant. The new species is bisexual with alate males being present in much smaller numbers Fig. 1. Ripersiella emarai Jansen, adult female, micro- than females. scopic view. Photo by Maurice Jansen.

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Fig. 2. Ripersiella emarai Jansen, adult female. Left side is dorsal, detail drawings from top to bottom: trilocular pore, tubular duct, circulus and anal ring. Right side is ventral, detail drawings from top to bottom: bitubular pore, trilocular pore, tubular duct and internal female genitalia.

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6–7 μm long (including internal part) and 3–3.75 μm each side one seta of about 20 μm. Legs well devel- high. Position and number of bitubular pores are oped: hind trochanter + femur 125–140 μm long; as follows: head 2 on submargin near eyes and one hind tibia + tarsus 165–185 μm long, hind claw slen- in posterior median position which is sometimes der, 25–29 μm long with a few short claw digitules of lacking; prothorax 2 marginal, 2 submedian and 1 about 18 μm long, in most cases about half this length median; mesothorax 2 marginal, 1 median; metatho- as a result of damage. Anterior width coxa 55–65 μm; rax 2 marginal, 2 submarginal and 1 median; abdomi- trochanter (measured outside) 40–47 μm long; tibia nal segments: I: 2 submedian, II: 3 or 4 divided over 83–95 μm long, tarsus 75–88 μm long. Ratio of the segment, III: 2 submedian or 3–4 in total, IV: 2 lengths of hind tibia + tarsus to hind trochanter + marginal and 1 median; V: 2 submedian, VI: 2 mar- femur 1.25–1.32. Lip-like triangular structure poste- ginal, 1 median; VII: 2 marginal, 1 median; VIII rior to vulva absent. Labium (three segments com- without ducts. Ostioles well developed with edges bined) 67–83 μm long, width of clypeolabral shield weakly sclerotized; anterior pair with the border of 58–78 μm. Circulus with cell-like reticulations at its both lips beset with up to 4 triloculars and 4 setae; truncate distal plate, shape of distal half variable from those of posterior pair with up to 6 triloculars and 5 conical to almost parallel, distal and proximal half of setae. Trilocular pores 2.5–3.5 μm wide, evenly scat- same length, width of circulus 30–37 μm at base, tered on surface, on abdominal segment I–VII: height 15–16 μm. Eyes present. Tubular ducts 4–5 more than 70, on VIII: 18–29, several between anal μm long, present in small numbers in most abdomi- lobe setae, the median part anterior of anal ring may nal segments, a single one on one or more thoracic possess an anterior row of three triloculars and a segments. Internal genital organ chitinized, length posterior row of several triloculars following the edge about 38 μm, width 40 μm. of the anal ring; sometimes the triloculars are scattered Diagnosis. Ripersiella emarai can be distinguished on the whole median surface of the segment, however. from female adults of the other members of the genus Tubular ducts 4–5 μm long, present in small numbers by the following combination of characters: bitubular in most abdominal segments, a single one on one or pores present, ventral and dorsal claw digitules setose, more thoracic segments. Multilocular pores absent. shorter than claw, multiloculars absent, oral collar Venter. Antennae relatively slender, 6-segmented, tubular ducts present, dorsal multiloculars absent, 148–168 μm long. Length of segments: I (lateral eyes present, antennae 6-segmented. Adult females inner side) 28–40 μm, II–IV (medially) 58–63 μm, V can be identified using the key of Kozár & Konczné (medially) 18–23 μm, VI 43–49 μm. The apical seg- Benedicty (2007) up to couplet 22 and key out ment has three long and one short sensory falcate against R. loksae Konczné Benedicty and Kozár. setae, segment five has one falcate sensory seta and Ripersiella emarai is distinguished from R. loksae by there is one sensory pore on the second segment. the shorter bitubular ducts which are 9 μm in length Segments I, II, IV and V have one row of setae, seg- in R. loksae and up to 6 μm in length in R. emarai, the ment III has two rows of setae. Trilocular pores shape of the internal genital organ, the cephalic plate numerous, about 3 μm wide. Bitubular pores of one which is not visible in R. loksae but present in R. ema- type present, more or less oval-shaped, each about 6–7 rai, width of circulus which in R. loksae is 14 μm wide μm wide and 3 μm high. There is one in the frontal and in R. emarai 30–37 μm wide at base, anal ring of margin of the head between the antennae. Thoracic R. loksae with 26 cells in outer row whereas there are segments: prothorax: 0; mesothorax: 2 submarginal 18 cells in the outer row of R. emarai and the anten- and metathorax: 2 submarginal. Absent on abdominal nae of R. loksae which are about 130–140 μm long segment I, II and IV. Present on abdominal segment whereas those of R. emarai are 148–168 μm long. III: 2 on the margin, V: 2 on the margin, VI: 0–2 on Adult females of R. emarai can be distinguished the margin, VII: 2 on the margin, VIII: 2 on the mar- from the preceding nymphal stage by the presence of gin. Flagellate setae in bands across the segments but a vulva, tubular ducts, ventral bitubulars and a larger absent from intersegmental areas and in small clear number of bitubulars on the dorsum. Additional areas in the ventral thoracic region; length longest lat- characters are the smaller number of trioculars and eral setae of head 27 μm, of thorax 30 μm, of abdomi- setae on dorsal abdominal segments and the absence nal segment I–V 20 μm and from last segments 25 of bitubulars on dorsal abdominal segment VI. μm. Length of setae of the head variable, up to 30 μm, those next to cephalic plate about 25 μm, several setae in interantennal space up to 33 μm; thoracic setae less Description of first instar variable, up to 15 μm long, abdominal setae up to 23 Figs 3–5 μm long. Multilocular disc pores absent. Width of mesothoracic sternal apophysis 15 μm. Spiracles Microscopic characters; 240–445 μm long (antennae 30–33 μm long, peritreme 13–15 μm wide. Cephalic and anal setae excluded) and 143–242.5 μm wide. plate variably developed, variable in shape; with on Measurements based on 16 specimens. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

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inner side) 18–25 μm, II–IV: (measured medially) 27–35 μm, V: (measured medially) 8–10 μm, VI: 35–45. Segment I–V have one row of setae. The apical segment has three sensory falcate setae at the distal half of the segment with lengths: 29 μm, 23 μm and 20 μm. Segment five has one falcate sensory seta about 10 μm long and there is one sensory pore (campaniform sensilla) on the second segment. Trilocular pores about 3 μm wide. Bitubular pores absent. Flagellate setae in a single row across the abdominal segments but absent from intersegmental areas and in small clear areas in ventral thoracic regions. Cephalic plate present but very weakly developed and hardly visible. Length of setae on the head, thoracic and abdominal segments up to 12 μm. Multilocular disc pores absent. Width of mesothoracic sternal apophysis 12–17 μm. Posterior spiracles 15–19 long, peritreme 3.8–5 μm wide. Legs well developed: hind trochanter + femur 52.5–67.5 μm long; hind tibia + tarsus 77.5–90 μm long, hind claw slender, 15–18 μm long with thin claw digitules as long as claw. Length trochanter (measured outside) 22–26; anterior width coxa 33–37.5. Ratio of lengths of hind tibia + tarsus to hind trochanter + femur 1.29–1.47. Hind tibia 31–35 μm long, hind Fig. 3. Ripersiella emarai Jansen, first-stage nymph, tarsus 42–45 μm long. Labium (three segments com- microscopic view. Photo by Maurice Jansen. bined) 40–55 μm long, width of clypeolabral shield 35–45 μm. Circulus at base 13–15 μm wide and 7–10 μm long; height 3–6 μm; 4 cell-like reticula- Dorsum. Posterior end of body with well-developed tions present at its truncate distal end, shape of distal membranous, slightly projecting anal lobes; each half variable from conical to almost parallel, distal lobe with 3 apical setae of varying length: 40–44 μm, and proximal half of same length. No variation in the 35–38 μm and 15–18 μm. One pair of tritubular number of circuli was observed. Eyes present. pores on the margin of abdominal segment I, 5–6 Tubular ducts and internal genital organ absent. Disc μm wide; bitubular pores absent. Anal ring 23–36 pores (Fig. 4) flat, circular with a more or less three- μm in diameter with 6 setae each 31–35 μm long, sided structure in the centre, 4–5 μm in diameter, outer row of cells with spiculae. Each abdominal seg- present in five pairs: on the head at the antennal base, ment with one row of flagellate setae, which are on the posterior margin of the prothorax behind the absent from intersegmental areas. Length of abdomi- labium, on mesothorax and metathorax anteriorly of nal setae on segment I–VI up to 10 μm, on segment the coxa of the leg, between two setae, on the second VII–VIII 10–13 μm, anal lobe setae up to 31–38 abdominal segment anteriorly of the circulus. μm. Trilocular pores 3–3.5 μm wide, numbering on Diagnosis. The first-instar nymph possesses five pairs head about 10, the numbers on the abdominal seg- of disk pores of a type that seems distinctive of this ments are as follows: I: 2 submarginal; II: 4, 2 sub- species and this character has not yet been found in marginal and 2 marginal; III: 2–4, 2 submarginal any other mealybug (Fig. 4). Because of their flatness and 0–2 marginal; IV: 2–4 at the edge of submargin the pores can only observed from above and may be and submedian; V: 4 evenly distributed; VI: 4 evenly easily missed when observed laterally. The first stage distributed; VII: 2 submarginal; VIII: 2 submedian. can easily be distinguished from subsequent stages by Ostioles membranous, anterior pair often barely per- the presence of a marginal tritubular on the dorsal ceptible, without related triloculars and setae; ante- second abdominal segment and the absence of bitu- rior lip of posterior pair related with 1 seta and 1 bulars. The number of triloculars on dorsal abdomi- trilocular pore. Tubular ducts and multilocular pores nal segments is mostly less than 10 whereas the absent. number of triloculars in the second stage always is at Venter. Antennae 6-segmented, 75–88 μm long, bor- least 12. The first-instar nymphs of both sexes are der between segment II and III not well indicated. indistinguishable; no morphological groups could be Length of the segments: I: (measured laterally at the distinguished.

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Fig. 4. Ripersiella emarai Jansen, first-stage nymph. Left side is dorsal. Right side is ventral.

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30–43 μm. Segments I–V have one row of setae. The apical segment has three sensory falcate setae, the longest one almost half way or just below the middle of the segment, length 9–11 μm, two others at the distal half of the segment 8–9 μm long. Segment five has one falcate sensory seta about 4–5 μm long and there is one sensory pore (campaniform sensilla) on the second segment. Trilocular pores about 3–3.5 μm wide. Bitubular pores absent. Flagellate setae in a single row across the abdominal segments but absent from intersegmental areas and in clear areas in ventral head and thoracic regions. Cephalic plate present, length 17–20 μm, width 32–45 μm with two setae at its border. Maximum length of setae on the head 12–22 μm, those of thoracic and abdominal Fig. 5. Ripersiella emarai Jansen, first-stage nymph, segments up to 15 μm. Multilocular disc pores disc pores. Photo by Maurice Jansen. absent. Posterior spiracles 20–25 long, peritreme 7–8 μm wide. Legs well developed: hind trochanter + femur 76–85 μm long; hind tibia + tarsus 93–103 μm Description of second-instar female long, hind claw slender, 16–20 μm long with thin Fig. 6 claw digitules as long as claw. Length trochanter (measured outside) 25–36 μm; anterior width coxa Length 450–710 μm (antennae and anal setae 33–40 μm. Hind tibia 35–48 μm long, hind tarsus excluded) and width 190–348 μm. Measurements 52–63 μm long. Ratio of lengths of hind tibia + tarsus based on 11 specimens. to hind trochanter + femur 1.20–1.38. Labium (three Dorsum. In most abdominal segments several rows of segments combined) 50–60 μm long, width of cly- spinules are present. Posterior end of body with well peolabral shield 46–55 μm. One circulus between developed slightly sclerotized and projecting anal third and fourth abdominal segment, width at base lobes; each lobe with 3 apical setae of about same 15–23 μm and 13–18 μm long; height 12–13 μm; 9 length, 45–58 μm. Between anal lobe setae are 3 tri- cell-like reticulations present at its circular truncate loculars. Bitubular pores present on the head: 2 sub- distal end which is about 5 μm in diameter, shape of marginal and 1 frontal in median position between distal half variable from conical to almost parallel, dis- antennae; prothorax: 1 in median position; mesotho- tal and proximal half of same length. Eyes present. rax: 2 on the margins of each side of the body; meta- Tubular ducts and internal genital organ absent. thorax: 2 marginal, rarely an additional median one; Diagnosis. Two forms were found in second instars: on abdominal segments confined to segment IV: 2 in one specimen was found with a small number of marginal and 1 in median position and on segment tubular ducts but no other specimens of this instar VII: 2 median. Anal ring 30–40 μm in diameter with possessed these ducts. The gender of the second-instar 6 setae each 38–58 μm long, outer row of cells with nymph is based on the assumption that specimens spiculae, cell structure as adult. Most abdominal seg- without common ducts are females (see e.g. Gullan ments with one to two irregular rows of flagellate 2000, Kondo et al. 2004). One specimen with a low setae, which are absent from intersegmental areas. number of ducts on the whole body was observed. A Length of abdominal setae on segment I–VI up to third- and fourth-instar male nymph were observed as 10–18 μm, on segment VII–VIII 10–16 μm; anal well suggesting that males are fewer in number than lobe setae up to 52–60 μm. Trilocular pores 3–3.5 females. No observations of the pharate third–instar μm wide, numbering on head about 15, evenly dis- female were made. The male instars are not described. tributed on abdominal segments, 8–12 on abdominal segment I–VII and on VIII, 2–3 at the anal lobes and 1 in median position. Ostioles membranous, Description of third-instar female without related triloculars and setae. Tubular ducts, Fig. 7 disk pores and multilocular pores absent. Venter. Antennae 6–segmented, 87–112 μm long, Length 570–780 μm (antennae and anal setae border between segment II and III not well indicated. excluded) and width 250–365 μm. Measurements Length of segments. I: (measured laterally at the based on 8 specimens. inner side) 17–25 μm, II–IV: (measured medially) Dorsum. In most abdominal segments several rows 40–54 μm, V: (measured medially) 10–15 μm, VI: of spinules are present. Posterior end of body with

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Fig. 6. Ripersiella emarai Jansen, second-instar female. Left side is dorsal. Right side is ventral.

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Fig. 7. Ripersiella emarai Jansen, third-instar female. Left side is dorsal. Right side is ventral. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 11 well-developed membranous, slightly projecting anal hind leg trochanter + femur 100 μm long, length tro- lobes; each lobe with 3 apical setae of about same chanter (measured at the outside) 35–40 μm, diago- length, 50–70 μm. Bitubular pores present on the nal length femur 50 μm; hind tibia + tarsus 122 μm head: 1 frontal in median position between anten- long, hind tibia 60–63 μm long, hind tarsus 45–50 nae and 2 marginal, occasionally an additional median μm; hind claw slender, 16–19 μm long with thin posterior one; prothorax: 2 on margin and occasion- claw digitules as long as claw. anterior width coxa ally 1 median; mesothorax: 2 on margin and occa- 33–35 μm. Ratio of lengths of hind tibia + tarsus to sionally 1 in median position; metathorax: 2 marginal, hind trochanter + femur 1.07–1.33. Labium (three rarely an additional median one; on abdominal seg- segments combined) 60–70 μm long, width of cly- ments the numbers are as follows, I: on each side 1 in peolabral shield 62–72.5 μm. One circulus between submarginal/submedian position; II: 0 or 1 marginal third and fourth abdominal segment, at base 20–30 and/or 1 median, III: 2 marginal, occasionally an μm wide and 15–20 μm long, height 12.5 μm, 8–10 additional marginal one, IV: 0 or 1 median, V: 2 mar- cell-like reticulations present at its circular truncate ginal and 1 median, VI: 0 or rarely 1 marginal, VII: 2 distal end which is 6–8 μm in diameter, shape of dis- marginal and 1 median, VIII: 0. Anal ring 37–43 μm tal half variable from conical to almost parallel, distal in diameter with 6 setae each 47–53 μm long, outer and proximal half of same length. Eyes present. row of cells with spiculae. All setae flagellate, the Tubular ducts and internal genital organ absent. abdominal ones evenly distributed but absent from Diagnosis. The third nymphal stage is distinguished intersegmental areas, those from segment I–VI up to from the second stage and the adult stage by the 13 μm, those from segment VI–VIII up to 20 μm number of triloculars on the dorsal abdominal seg- long. Thoracic setae up to 14 μm, those on the head ments. It has twice as many triloculars as the preced- up to 18 μm long. Trilocular pores 3–4 μm wide, num- ing stage and half the number of the adult. In bering more than 20 on head, evenly distributed on contrast to the second stage bitubulars are present on abdominal segments: I: 13–18; II: 14–20; III: 16–20; dorsal abdominal segments I, II, III and V. The adult IV: 15–19; V: 13–20; VI: 15–21; VII: 10–17; stage possesses bitubulars on dorsal abdominal seg- VIII: 8–10, 3 or 4 between each lobe and 2 in ante- ment VI and the numbers are regularly twice the rior position with the preceding segment. Ostioles number of the third-instar nymph. The third-instar membranous, without related triloculars and setae, nymphs of R. hibisci and R. multiporifera are easily 30–35 μm. Tubular ducts, disk pores and multilocular distinguished by the presence of eyes and six-segmented pores absent. antennae in R. emarai. Venter. Antennae 6-segmented, 122–140 μm long, border between antennal segment II and III not well indicated. Length of segments: I (measured laterally Molecular diagnosis at the inner side): 25–30 μm, II: 13–17.5, III: The COI sequence of Ripersiella emarai (Genbank 10–12.5, IV (measured medially): 12.5–16 μm, Acc. No. [GBN] KM453213) is 81% identical to V (measured medially): 15–18 μm, VI: 40–45 μm. R. hibisci (GBN KM453214 and KM453215), 82% Segments I–V possess one row of setae. The apical to R. multiporifera (GBN KM453216) and 85% to segment has three sensory falcate setae, the longest Rhizoecus dianthi (GBN KM453217). This indicates one almost halfway or just below the middle of that R. emarai can be distinguished from R. hibisci, 20–25 μm, two others at the distal half of the seg- R. multiporifera and Rh. dianthi on the basis of their ment 16–22 μm long. Interantennal width up to 50 COI sequences. Although COI is the DNA barcode μm. Segment V has one falcate sensory seta about 10 for species identification in the animal kingdom μm long. There is one sensory pore (campaniform (Hebert et al. 2003), no other COI data for the sensilla) on segment II. Trilocular pores about 3–4 genus Ripersiella is present in GenBank or any μm wide. Bitubular pores absent. A double row of other public database. Therefore additional sequence longer setae medially of abdominal segment II–VI of data of 18S rRNA and 28S rRNA for R. emarai 15–18 μm long and the rest of the segments setae up (GBN KM453218; KM453223), R. hibisci (GBN to 10–13 μm long. Flagellate setae across the abdom- KM453219, KM453220; KM453224, KM453225), inal segments but absent from intersegmental areas R. multiporifera (GBN KM453221; KM453226) and and in clear areas in ventral head and thoracic regions. Rh. dianthi (GBN KM453222; KM453227) were Cephalic plate present, length 25 μm, width 75 μm generated and compared to GenBank accessions of with two setae at its border with a length of about Rh. amorphophalli (GBN JQ0855361), Rh. cacticans 15–30 μm. Maximum length of setae on the head up (Hambleton) (GBN EU188591; EU188485), Rh. to 20–25 μm, from thoracic region around coxae up floridanus (GBN EU188592; EU188486), Rh. maya- to 11–13 μm. Multilocular disc pores absent. nus (Hambleton) (GBN EU188593; EU188487) Posterior spiracles 25–27.5 μm long, peritreme 6–9 and R. gracilis McKenzie (GBN AY426074; μm wide. Legs well developed, total length 243 μm AY427367). The interspecies variation for 18S rRNA Downloaded from Brill.com09/29/2021 11:17:00PM via free access

12 Tijdschrift voor Entomologie, volume 158, 2015 is rather small as the difference between R. emarai Key to first instars and the other species is only 2 to 5%. The 28S rRNA 1. Antennae six-segmented. Five ventral pairs of gene is less conserved and therefore better suited for disc pores present on the ventral side at the species identification. For 28S rRNAR. emarai has antennal base, thoracic segments and first the highest similarity (91%) to Rh. floridanus. In con- abdominal segment. Length of marginal setae clusion R. emarai can at least be distinguished molec- up to 15 μm �� Ripersiella emarai sp. nov. ularly from eight members of Ripersiella and – Antennae five-segmented. Disc pores absent. potentially from all other members. Length of marginal setae up to 50 μm �� 2 2. Circulus absent. Dorsal setae long: those from Provisional key to the female nymphal thorax up to 40–58 μm long, of head 15–20 stages of Ripersiella emarai μm. Marginal setae on thorax up to (21) 40–58 μm long, on abdomen up to (22) 22–38 μm 1. Tritubular ducts present on the first segment long ��Ripersiella hibisci (Kawai & Takagi) of the dorsal abdomen, no bitubular ducts – Circulus present. Dorsal setae short: those of present. Body length 240–445 μm, on each thorax up to 5–13 μm, of head 13–15 μm long. dorsal abdominal segment never more than 4 Marginal setae on thorax up to 23 μm long, triloculars �� first instar those of abdomen up to 20–25 μm (Fig. 8) – Tritubular ducts absent, bitubular ducts pres- ��Ripersiella multiporifera Jansen ent; on most dorsal abdominal segments much more than 4 triloculars, generally at least 9 �� 2 Key to second instars 2. Bitubular ducts present on dorsal abdominal segments on the margin of segments IV and 1. Antennae six-segmented. Circulus and eyes VII and absent on the other abdominal seg- present �� Ripersiella emarai sp. nov. ments. Generally 9–12 triloculars on dorsal – Antennae five-segmented. Eyes absent, circulus abdominal segment I–VII and one trilocular absent or present �� 2 on the median of dorsal abdominal segment 2. Circulus absent. Dorsal abdominal segment I VIII. Body length 450–710 μm. Ventral bitu- with 12–16 triloculars �� bulars absent, one frontal bitubular on the ��Ripersiella hibisci (Kawai & Takagi) head ��second instar – Circulus present. Dorsal abdominal segment I – Bitubulars present at least on dorsal abdomi- with 19–24 triloculars (Fig. 9) �� nal segments I-III-V-VII. More than 12 tri- �� Ripersiella multiporifera Jansen loculars on dorsal abdominal segment I–VII, on VIII at least 2 submedian triloculars and an additional few between anal lobe setae. Key to third instars Ventral bitubulars present or absent �� 3 1. Antennae six-segmented. Eyes present. 3. Ventral bitubulars confined to a frontal Circulus present �� Ripersiella emarai sp. nov. one on the head and absent on the rest of – Antennae five-segmented. Eyes absent. the body. Dorsal bitubulars present on Circulus absent or present �� 2 abdominal segments I-III-V-VII, generally 2. Circulus absent. Antennal length 250–180 2 on the margin and in most cases one in μm. Hind trochanter + femur 115–155 μm median position. Triloculars on dorsal long ��Ripersiella hibisci (Kawai & Takagi) abdominal segment I–VII generally num- – Circulus present. Antennal length 130–145 bering 14–18, on VIII 2 submedian tri- μm. Hind trochanter + femur 105–112 μm loculars near anterior edge with the long (Fig. 10). Ripersiella multiporifera Jansen preceding segment, an additional 2–4 triloculars between anal lobe setae. Body length 570–780 μm. Vulva or internal gen- Key to the adult females of ital organ absent �� third instar subterranean mealybugs of The – Ventral bitubulars present on meso- and Netherlands, predominantly found at metathorax and on most abdominal segments; dorsal bitubulars numbering 3–4 in import interceptions and in greenhouses most dorsal abdominal segments but absent 1. Bitubular or tritubular ducts absent, common on dorsum of segment VIII. Body length tubular ducts present �� 2 640–1650 μm. Vulva or internal genital – Bitubular or tritubular ducts present, common organ present �� adult female tubular ducts present or absent �� 3

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Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 13

Fig. 8. Ripersiella multiporifera Jansen, first-instar nymph. Left side is dorsal. Right side is ventral. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

14 Tijdschrift voor Entomologie, volume 158, 2015

Fig. 9. Ripersiella multiporifera Jansen, second-instar female. Left side is dorsal. Right side is ventral. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 15

Fig. 10. Ripersiella multiporifera Jansen, third-instar female. Left side is dorsal. Right side is ventral. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

16 Tijdschrift voor Entomologie, volume 158, 2015

2. Posterior abdominal segments terminating in 13. Labium 60–70 μm long �� a pair of strongly produced sclerotized anal �� Rhizoecus albidus Goux (Note 3) lobes fused at basis. Large translucent pores on – Labium 75–90 μm long �� the hind tibiae absent �� Rhizoecus cacticans (Hambleton) ��Geococcus coffeae Green 14. Circulus absent, tritubular pores of one or – Posterior abdominal segments not terminated three sizes �� .15 in a pair of strongly produced sclerotized anal – One or two circuli present, tritubular pores of lobes. Large translucent pores on the hind two sizes: larger ones on dorsum, smaller ones tibiae with heavily sclerotized rims. At least on ventral side; tubular ducts with or without three pairs of cercarii of which a few have coni- a narrow oral rim �� cal setae. Large tubular ducts �� Rhizoecus amorphophalli Betrem �� Trochiscococcus speciosus (De Lotto) (= advenoides Takagi & Kawai, syn. nov.) (Note 4) 3. Bitubular pores present ��Ripersiella 4 15. Tritubular pores of two sizes, on dorsum larger – Tritubular pores present ��Rhizoecus 10 than on venter �� 16 4. Eyes absent. Antennae five-segmented �� 5 – Tritubular pores of three sizes �� – Eyes present. Antennae six-segmented �� 7 ��Rhizoecus americanus (Hambleton) 5. Multilocular pores present on venter and dor- 16. Tubular ducts present on venter of last abdom- sum; 0–2 circuli �� 6 inal segment. A group of 5–15 multilocular Multilocular pores confined to ventral pores present around vulva. Labium 88 μm abdominal segments VII–IX; 2–6 circuli (Fig. 12) �� Ripersiella campestris (Hambleton) ��Rhizoecus nemoralis (Hambleton) (Note 5) 6. Multilocular pores absent on head. Small – Tubular ducts absent. A few multilocular pores type bitubular pores present on venter and on posterior abdominal segments only. Labium dorsum ��Ripersiella hibisci (Kawai & Takagi) 98 μm ��Rhizoecus dianthi Green – Multilocular pores present on venter and dorsum of head. Small type bitubular pores con- Note 1. Ripersiella planetica Williams (2004) was fined to venter (Fig. 11) �� intercepted by M. van Merriënboer, phytosanitary �� Ripersiella multiporifera Jansen inspector in The Netherlands, on Livistona plants 7. Dorsal multiloculars present in small number. originating from Sri Lanka on 4 September 2003. Oral collar tubular ducts present �� 8 This was recorded by Kozár & Konczné Benedicty – Dorsal multiloculars absent. Oral collar tubu- (2007) but the host plant was not given and it was lar ducts present or absent �� 9 suggested to be Cactaceae. On 19 December 2013 a 8. Bitubular ducts on dorsal abdominal segments population was discovered in a greenhouse at Ede on 4–6 ��Ripersiella mexicana (Hambleton) Opuntia subulata, leg. W. den Hartog. Malumphy – Bitubular ducts on dorsal abdominal segments (2012) recorded the species from Malta, not known 2–3 �� to be established, but expected to survive for the ��Ripersiella planetica (Williams) (Note 1) immediate future. 9. Ventral multiloculars present on last abdomi- Note 2. Specimens of Rhizoecus keysensis were found nal segments. Oral collar tubular ducts absent living on the roots of two plants of Euphorbia neoar- ��Ripersiella maasbachi (Jansen) borescens Bruyns (Euphorbiaceae) and unidentified Ventral multiloculars absent. Oral collar tubu- Cactaceae in the greenhouse of a grower at lar ducts present (Fig. 1) �� Kudelstaart in The Netherlands on 15 November �� Ripersiella emarai sp. nov. 2007 and 19 February 2008, PPS402661, 10. Multilocular pores absent from venter �� 11 PPS4026582 and PPS4160263. This is the first dis- – Multilocular pores present on venter �� 14 covery of Rh. keysensis in The Netherlands. The 11. Median and submedian areas of dorsum Euphorbia plants originated from the Canary Islands with tritubular pores. Labium 60–85 μm but it is unknown if both plants were already infested long �� 12 during import. A possibility is that one of the plants – Median and submedian areas of dorsum with- was infested and the population on the second plant out tritubular pores and no more than four is the spread resulting from the infestation. The pop- tritubular pores on dorsal margins. Labium ulation of Rh. keysensis was mixed with that of 90–105 μm long ��Rhizoecus elongatus Green Ripersiella mexicana (Hambleton 1979). Rhizoecus 12. Length antennae 175–210 μm. Length anal keysensis is near to Rh. cacticans and measurements lobe setae 75–95 μm �� 13 and study of the observed specimens of Rh. keysensis – Length antennae 120–150 μm. Length anal reveals that the labium is 65–85 μm long, length of lobe setae 35–50 μm �� antennae 120–150 μm long, tibia + tarsus of hind �� Rhizoecus keysensis Hambleton (Note 2) legs 95–118 μm long, the apical antennal segment is Downloaded from Brill.com09/29/2021 11:17:00PM via free access

Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 17

Fig. 11. Ripersiella multiporifera Jansen, adult female. Left side is dorsal: detail drawings from top to bottom: trilocular pore, big type bitubular pore, multilocular pore and anal ring. Right side is ventral: detail drawings from top to bottom: trilocular pore, small type bitubular pore; circulus and internal female genitalia. about 1.5–1.7 times as long as wide, width of anal antennae is 170–210 μm, tibia + tarsus of hind legs ring (56) 60–65 μm, anal lobe setae are thin, hair-like are 140–195 μm long, the apical antennal segment and their maximum length is 33–55 μm, anal ring 2–2.5 times as long as wide, width of anal ring 62–75 with a double ring of cells, the outer one with (20) μm, anal lobe setae are flagellate and their maximum 25–28 cells and generally three cells between ante- length 75–95 μm, anal ring with a double ring of rior pair of setae. Rhizoecus cacticans on the other cells, the outer one with 25–32 cells and generally hand has a labium of 82–100 μm long, the length of 4–6 cells between anterior pair of setae. Downloaded from Brill.com09/29/2021 11:17:00PM via free access

18 Tijdschrift voor Entomologie, volume 158, 2015

ber of tritubular pores on the dorsal abdominal segments is between 2 and 7, the shape of the claw digitules is variable and the third antennal segment is not constant within populations of both species. The conclusion is that Rh. advenoides Kawai & Takagi is a junior synonym of Rh. amorphophalli, which represents a rather variable taxon. Note 5. Rhizoecus nemoralis (Hambleton) is recorded here for the first time for The Netherlands. Specimens were observed by B. Sukul, phytosanitary inspector, on plants of Agave filifera in a greenhouse at Honselersdijk on 19 June 2012. Danzig et al. (2008) presume that Rh. nemoralis and Rh. cyperalis are identical with Rh. dianthi. Only a part of the Rh. nemoralis specimens have tubular ducts, so it is pos- Fig. 12. Field view of Rhizoecus nemoralis (Hambleton) sible that Rh. nemoralis and Rh. dianthi represent the on Agave in a greenhouse. Photo by Maurice Jansen. same species.

Note 3. Rhizoecus albidus is the only species occurring in the open in The Netherlands and was once Acknowledgements recorded on the roots of Ballota nigra (Jansen 2001). The late Dr Ference Kozár (Plant Protection Institute, The species was observed by the first author living on Hungarian Academy of Sciences, Budapest, Hungary) the roots of the grass Puccinellia maritima (Huds.) recognized specimens to belong to an undescribed Parl at the sea dike near Koudekerke-Burghsluis on species, gave valuable suggestion and information 16 April 2003. Later on high numbers were found about the new species and Rh. advenoides. We thank living on the roots of the grass Elythrigia maritima by Dr Gregory A. Evans (United States Department of A. Ivens (RUG Groningen) on 5 July 2010, 8 July Agriculture Systematic Entomology Laboratory, 2010, 21 July 2010 and 14 July 2011. This popula- Beltsville, MD, USA) for the loan of specimens of tion was living in association with the ant Lasius fla- Rhizoecus keysensis and Dr Sadao Takagi (Systematic vus (Fabricius) in the transition zone of a dune Entomology, Hokkaido University, Sapporo, Japan) towards a salt marsh on the Dutch Wadden Sea for the loan of Rhizoecus advenoides. Valuable sugges- island Schiermonnikoog. It has also frequently been tions were given by Prof. Giuseppina Pellizzari found in greenhouses (Jansen 2005). It was inter- (University of Padova, Italy), Dr Demian Kondo cepted on imported Eugenia plants originating from (Entomology Laboratory, CORPOICA, Palmira, China on 24 October 2008, leg. W. Zijlstra, Colombia) and an unknown reviewer. PPS4308854, PPS4354991. Only one dead specimen was observed which could not be properly pre- pared and therefore the plant on which it was found References was followed and reared. In June 2010 living speci- Belshaw, R. & D.L.J. Quicke, 1997. A molecular phylog- mens were observed on the roots of the plant. eny of the Aphidiinae (Hymenoptera: Braconidae). – Note 4. Type material of Rhizoecus advenoides Molecular Phylogenetics and Evolution 7: 281–293. Takagi & Kawai was studied. Takagi & Kawai Betrem, J.G., 1940. A new Rhizoecus species. – Treubia 17: (1971) summarised the differences between Rh. 267–270. advenoides and Rh. advenus Beardsley, which were Danzig, E.M., I.A. Gavrilov & I.V. Trapeznikova, synonymised by Hambleton (1979). Important 2008. A new pest from a greenhouse of St. Petersburg, Rhizoecus dianthi Green (Homoptera, Pseudococcidae), discriminating characters of Rh. advenoides and Rh. with karyotype data. – Entomological Review 88: amorphophalli Betrem (1940) are the labium, 676–678. which is as long as wide, and the tubular ducts, Gullan, P.J., 2000. Identification of the immature instars of which should miss a pale circular area around its mealybugs (Hemiptera: Pseudococcidae) found on orifice. Rhizoecus amorphophalli on the other hand Citrus in Australia. – Australian Journal of Entomology should have a labium which is longer than wide 39: 160–166. and the tubular ducts possess a pale circular area Hambleton, E.J., 1979. The status of Rhizoecus amorphophalli around the orifice. These characters actually repre- Betram, a little-known Oriental mealybug (Homoptera: sent variability within populations of both species. Pseudococcidae). – Journal of the Washington Academy Besides, the number of circuli in both species is of Science 69: 62–64. variable and may be 1 or 2 and the circulus has a Hebert, P.D., A. Cywinska, S.L. Ball & J.R. deWaard, reticulated distal plate (Williams 1985). The num- 2003. Biological identifications through DNA Downloaded from Brill.com09/29/2021 11:17:00PM via free access

Jansen and Westenberg: Morphological and molecular studies of a new Ripersiella species 19

barcodes. – Proceedings of the Royal Society London Kozár F. & Z. Konczné Benedicty, 2007. Rhizoecinae of Series B: Biological Sciences 270: 313–321. the World. – Plant Protection Institute, Hungarian Jansen, M.G.M., 2001. Instar identification and some Academy of Sciences, Budapest, Hungary, 617 pp. notes about the life cycle of Rhizoecus hibisci Kawai & Malumphy, C., 2012. First incursion of the Asian root Takagi (Coccoidea: Pseudococcidae). – Bollettino di mealybug Ripersiella planetica in Europe (Hemiptera, Zoologia Agraria e di Bachicoltura 33: 53–66. Coccoidea, Rhizoecidae). – Bulletin of the Entomological Jansen, M.G.M., 2005. An updated list of scale insects Society of Malta 5: 171–173. (Hemiptera, Coccoidea) from import interceptions Park, D.S., K.J. Hong, S.J. Suh, P.D.N. Hebert, H.W. Oh and greenhouses in The Netherlands. – Proceedings of & K.J. Hong, 2011. DNA barcodes for two scale insect the Xth International Symposium on Scale Insect families, mealybugs (Hemiptera: Pseudococcidae) and Studies held at Plant Protection Research Institute armoured scales Hemiptera: Diaspididae). – Bulletin of Adana, Turkey, 19th–23rd April 2004. Entomological Research 101: 429–434. Jansen, M.G.M., 2008. A new species of the genus Ripersiella Park, D.S., S.J. Suh, H.W. Oh & P.D.N. Hebert, 2010. Tinsley (Hemiptera: Coccoidea: Pseudococcidae) from Recovery of the mitochondrial COI barcode region in import interceptions in The Netherlands. – In: M. diverse Hexapoda through tRNA-based primers. – Branco, J.C. Franco & C. Hodgson (Eds), Proceedings BMC Genomics 11: 423. of the XI International Symposium on Scale Insect Takagi, S. & S. Kawai, 1971. Two new hypogeic mealybugs Studies (ISSIS XI), September 24–27, 2007, Oeiras, of Rhizoecus from Japan (Homoptera: Coccoidea). – Portugal, pp. 39–49. ISA Press, Lisbon, Portugal. Kontyû 39: 373–378. Katoh, K., K. Misawa, K. Kuma & T. Miyata, 2002. von Dohlen, C.D. & N.A. Moran, 1995. Molecular MAFFT: A novel method for rapid multiple sequence phylogeny of the Homoptera: A paraphyletic taxon. – alignment based on fast Fourier transform. – Nucleic Journal of Molecular Evolution 41: 211–223. Acids Research 30: 3059–3066. Williams, D.J., 1985. Hypogeic mealybugs of the genus Kawai, S. & K. Takagi, 1971. Descriptions of three eco- Rhizoecus (Homoptera: Coccoidea) in India. – Journal nomically important species of root–feeding mealybugs of Natural History 19: 233–241. in Japan (Homoptera: Pseudococcidae). – Applied Williams, D.J., 2004. Mealybugs of southern Asia. – The Entomology and Zoology Tokyo 6: 175–182. Natural History Museum, London: Southdene SDN, Kondo, T., P.J. Gullan & D.R. Miller, 2004. A new hypo- BHD, Kuala Lumpur, Malaysia, 896 pp. geal species of Oregmopyga Hoy (Hemiptera: Coccoidea: Eriococcidae) from southern California, U.S.A., and a Received: January 9, 2015 key to species of the genus. – Zootaxa 784: 1–12. Accepted: July 12, 2015

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