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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 <UN> 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), Downloaded from Brill.com09/29/2021 11:17:00PM via free access <UN> 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.

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