Three new species of mealybug (Hemiptera, Coccomorpha, Pseudococcidae) on persimmon fruit trees (Diospyros kaki) in southern Brazil

Abstract Brazil has the greatest insect diversity in the world; however, little is known about its scale insect species (Hemiptera: Coccomorpha). Mealybugs (Pseudococcidae) have been found in at least 50% of persimmon orchards Diospyros kaki L. in the southern part of the country. In this study three new mealybug species on persimmon trees located in the Serra Gaúcha Region, RS, Brazil, namely, Anisococcus granarae Pacheco da Silva & Kaydan, sp. n., Ferrisia kaki Kaydan & Pacheco da Silva, sp. n. and Pseudococcus rosangelae Pacheco da Silva & Kaydan, sp. n. are described. In addition, an identification key for the genera occurring on fruit orchards and vineyards in Brazil is provided, together with illustrations and molecular data for the new species.


Introduction
Southern Brazil is the third largest fruit-producing region in the country. It produces large amounts of temperate fruits, such as grape, apple, stone fruits and persimmon (Fachinello et al. 2011). Persimmon trees (Diospyros kaki L.) (Ebenaceae) were first cultivated in Brazil in the late 19 th century, but this crop expanded only after Japanese immigration, around 1920 (Neuwald et al. 2009). Persimmon is currently grown on about 9,000 ha and about 172,000 tons of fruits are produced annually, for domestic consumption and export (Fachinello et al. 2011). The São Paulo and Rio Grande do Sul states are the main producers of persimmon fruits in Brazil (Camargo-Filho et al. 2003). In Rio Grande do Sul, fruit production occurs mostly in the Serra Gaúcha Region, in which mealybugs (Hemiptera: Coccomorpha: Pseudococcidae) have been detected in at least 50% of production areas (Bavaresco et al. 2005), probably due to increases in insecticide application in recent years, leading to a decrease in the population of effective natural enemies.
Live mealybugs are small soft-bodied, sap-sucking insects with an oval, elongated to rounded body, often dorsoventrally compressed, pinkish to grayish in color, covered with a white powdery wax (the source of their common name) (Cox and Pearce 1983). They frequently have waxy filaments, those on the head being shorter than those close to the anus (Gimpel and Miller 1996). These filaments originate from the cerariigroups of trilocular pores, generally with two conical setae and, in some groups, also auxiliary setae (Williams and Granara de Willink 1992) predominantly found along the margin. The family to which mealybugs belong is the second largest family in infraorder Coccomorpha, in terms of the number of species it contains, almost 2020 species, distributed in 260 genera (García et al. 2016). In the Neotropical Region, only 223 species, from 44 genera, have been recorded (García et al. 2016).
In total, 153 species of Pseudococcus (Westwood) have been identified worldwide, 30 of which have been recorded in the Neotropical Region. It can be subdivided into two informal groups according to the presence or absence of simple pores associated with each eye (Gimpel and Miller 1996) -present in Pseudococcus maritimus complex.
It includes 33 species, 21 of which are present in the Neotropical Region (García et al. 2016), and is assumed to have originated from the New World, where some species, such as P. sociabilis Hambleton, P. viburni (Signoret) and P. maritimus (Ehrhorn), are considered to be major pests of fruit crops and vineyards (Casco Mila 2012;Correa et al. 2012;Daane et al. 2012;Pacheco da Silva et al. 2014).
The genus Ferrisia Fullaway, which is of New World origin, includes 18 species, most (12 species) of which occur in the Neotropics (Kaydan and Gullan 2012). The species from this genus are easily separated from the other genera in the Pseudococcidae by the presence of robust dorsal enlarged tubular ducts opening to the exterior via an irregularly circular sclerotized area bearing one or more setae and, often, one or more minute pores (Gullan et al. 2010). Furthermore, the living insects have long glassy filaments produced by the enlarged tubular ducts, and, depending on the species, may have typical dorsal patterns formed by dark areas of cuticle not covered by white wax (Kaydan and Gullan 2012).
The genus Anisococcus Ferris is also believed to have originated from New World and to be closely related to Ferrisia on the basis of both molecular phylogenetic studies (Downie and Gullan 2004;Hardy et al. 2008;Gullan et al. 2010) and morphological studies, as it has minute discoidal pores associated with enlarged tubular ducts and oral collar tubular ducts (Kaydan and Gullan 2012). This genus is found exclusively in the Americas, where 11 species have been described, nine in the Nearctic region and two from the Neotropical Region (McKenzie 1967;Williams and Granara de Willink 1992).
Brazil has the greatest biodiversity of any country worldwide and 13% of all species (including animals, plants, fungi and other organisms) are found only in Brazil (Lewinsohn and Prado 2005). Insect diversity is also greater in Brazil than in any other country, with almost 100 thousand species recorded (almost 10% of all insect species worldwide) (Rafael et al. 2009). It has been estimated that almost 11% of hemipteran insects are present in Brazil. However, only 3.8% of mealybug species have been recorded in this country, although this should probably be regarded as an underestimate of the percentage actually present.
Three new species of mealybugs sampled from persimmon orchards located in the Serra Gaúcha Region, Rio Grande do Sul, Brazil are described, and an identification key for the genera occurring in fruit orchards and vineyards in Brazil is provided, together with illustrations, molecular data and an identification key for the new species of Anisococcus, Ferrisia and Pseudococcus described here.

Methods
Mealybugs were collected from persimmon orchards during the harvest period in the years 2013-2015. Specimens were collected on fruits and leaves of the trees. Insects at all stages of development were collected (nymphs and adult females) and taken to the laboratory for examination. Nymphs were reared until adulthood on persimmon fruits. Labeled specimens were stored in 95% ethyl alcohol.

Molecular characterization
DNA characterization was performed using the nondestructive method described in Malausa et al. (2011). The DNA region studied was a ~ 760 bp fragment within the mitochondrial region of Cytochrome Oxidase Subunit I previously used in molecular studies on mealybugs (Malausa et al. 2011;Pacheco da Silva et al. 2014). DNA was extracted using the Qiagen DNEasy Tissue kit, following the manufacturer's recommendations. For amplification were used the primers 5' CCTTCAACTAATCAT-AAAAATATYAG 3' (Forward) and 5' TAAACTTCTGGATGTCCAAAAAATCA 3' (Reverse) PCR was performed using the Qiagen Multiplex PCR kit (QIAGEN, Valencia, CA), with a 23 mL reaction mixture and 2 ml of diluted DNA (1-20 ng of DNA matrix). PCR conditions were as follows: initial denaturation at 95°C for 15 mn; 35 cycles of denaturation at 95°C for 30 s, hybridization at 48°C for 90 s, elongation for 60 s; and final extension at 72°C for 10 mn. PCR-amplified fragments were analyzed with the QIAxcel Advanced System with QIAxcel DNA Fast Analysis cartridges (QIAGEN). PCR products were sent to Beckman Genomics (Takeley, United Kingdom) for bidirectional sequencing on ABI automatic sequencers (Applied Biosystems, Foster City, CA, USA). Consensus sequences and alignments were generated and checked with Bioedit version 7.01. We carried out BLAST searches (MEGABLAST method) on the NCBI GenBank database (http://www.ncbi.nlm.gov/BLAST).
The DNA results are shown for each species after the morphological descriptions. Additionally, all sequence data are available as Suppl. material 1 (FASTA format).

Morphological identification
The DNA voucher specimens plus other preserved adult females were slide-mounted and identified by light microscopy in the Plant Protection Department of Çukurova University, Adana, Turkey and ANSES, Laboratoire de la Santé des Végétaux, Montferrier-sur-Lez, France, according to a slightly modified version of the method of Kosztarab and Kozár (1988). The mealybugs were examined under a LEICA DM 2500 phasecontrast compound microscope and identified with the keys of Williams and Granara de Willink (1992), Gimpel and Miller (1996) and Kaydan and Gullan (2012).

Morphometric analysis
Mealybugs were measured and the main taxonomic characters evaluated and quantified under the Leica microscope. Measurements were taken from all the available material. The morphological terms used here are those used by Williams (2004) and Williams and Granara de Willink (1992). All the measurements given are the maximum dimensions (e.g. body width was recorded at the widest part) and are expressed as ranges. Tarsal length excludes the claw. Setal length includes the setal base. Cerarii are numbered as described by Williams and Granara de Willink (1992), with cerarius 1 on the head, anterior to the antenna, and cerarius 17 being on segment VIII.
Illustrations are provided for each species. Each figure represents a generalized individual based on several of the specimens used for description. Each illustration is split longitudinally, with the left half representing the dorsum and the right half the venter. Structural details are shown as enlargements around the central drawing, and are not drawn to the same scale. The translucent pores on the hind legs are mostly found on the dorsal surface, but they are illustrated ventrally on the main figure for convenience. The illustrations and description were prepared by MBK and VCPS.

Results and discussion
Key to identification of Pseudococcinae genera occurring on fruit trees and in vineyards in Brazil, adapted from Williams (2004) and Williams and Granada de Willink (1992).  Kenzie 1967). Body narrowly to broadly oval, 2.0-3.8 mm long, 1.1-2.8 mm wide. Labium with three segments, about as long as the clypeolabral shield. Antennae, 8-segmented. Circulus present or absent. Legs well-developed, without translucent pores; apparently with a small denticle on the claw. Both ostioles well developed. Anal lobes well developed. Anal ring rounded, usually large and cellular with six long setae, but sometimes reduced, non-cellular, more or less removed from the posterior apex of the abdomen (Anisococcus ephedrae (Coquillett)).
Dorsum. Dorsal tubular ducts with or without a rim, each orifice associated with one or more minute discoidal pores. Cerarii 13-17 pairs. Anal lobe cerarii, each with 7-20 conical setae on a sclerotized area, often with 3-7 auxiliary setae, remaining cerarii smaller, each with two or more conical setae plus an associated cluster of trilocular pores. Preocular cerarius always absent. Dorsal setae, slender and flagellate. Trilocular pores evenly distributed. Discoidal pores scattered and associated with tubular ducts, each smaller than trilocular pores. Multilocular disc pores absent.
Venter. Body setae flagellate. Trilocular pores evenly distributed. Discoidal pores scattered or associated with tubular ducts. Multilocular disc pores absent. Oral collar tubular ducts of one or more sizes, of various lengths and widths, with largest ducts, when present, on body margin, often associated with minute discoidal pores.
Key to adult females of Anisococcus found in the Neotropical Region (adapted from Williams and Granada de Willink (1992) (190,191,192  Diagnosis. Anisococcus granarae Pacheco da Silva & Kaydan, sp. n. is characterized by the following combination of features: (i) dorsal oral collar tubular ducts of 2 sizes, the large type with an indistinct rim, the small type without a rim (but with a sclerotized area next to the ducts opening); (ii) ventral oral collar tubular ducts of two sizes, smaller ducts present in rows across medial areas of abdominal segments, and larger ducts in body margin.
Description. Adult female. Appearance in life. Body oval, up to 4 mm long at maturity, covered in a layer of white wax; with two longitudinal lines of dorsal patches without wax on the intersegmental areas of the abdomen, exposing areas of dark gray-to-black subcutaneous pigment (Fig. 1). The margins have 14 small thin lateral filaments plus a long filament produced by anal lobe cerarii.
Description. Dorsum. With long enlarged ducts, each with the orifice surrounded by a circular sclerotized rim, either containing short setae or with setae just outside border. In living insects, these ducts secrete long glassy filaments typical of the genus. Cerarii confined to anal lobes; each anal lobe usually with 2 enlarged conical setae (more on some specimens of F. dasylirii Cockerell and F. virgata (Cockerell)) plus an associated cluster of trilocular pores and a few auxiliary setae. Body setae slender and flagellate, bluntly tipped to slightly capitate, and of various sizes. Trilocular pores each 3-5 μm in diameter, often slightly larger (4-5 μm diameter) than ventral trilocular pores (typically 3-5 μm), scattered over the dorsum. Minute discoidal pores on the dorsal submargin of the head at base of antennal segment I, usually in a small tight cluster of 3-8 pores (often difficult to see), and also associated with enlarged tubular ducts (generally present within sclerotized area surrounding duct rim). Enlarged tubular ducts present mostly on body margin and submargin in segmental clusters, but often also present medially and submedially; duct opening of each tubular duct with a sclerotized rim surrounded by a circular sclerotized area bearing 0-3 (generally 1 or 2) minute discoidal pores (appearing as clear areas in the cuticle) and with 1-7 (generally 3-5) blunt-tipped to slightly capitated setae. Oral-collar tubular ducts and multilocular pores absent.
Venter. Body setae slender, blunt-tipped to slightly capitate, and of various sizes. Trilocular pores each 2.5-5.0 μm in diameter, scattered over surface. Minute discoidal pores scattered throughout the venter, almost always associated with ventral oral-collar tubular ducts. Enlarged tubular ducts absent. Oral-collar tubular ducts of one or more sizes, of various lengths and widths, shortest ducts often present in marginal clusters, at least on posterior abdominal segments; ducts on anterior abdomen and margins or submargins of posterior abdomen often associated with a minute discoidal pore (rarely 2 pores), usually appearing as a clear circular to oval area in cuticle. Multilocular disc pores generally present (absent in F. meridionalis Williams) on posterior abdominal segments, especially around the vulva.
Key to adult females of Ferrisia from the Neotropical Region (adapted from Kaydan and Gullan (2012)). The key includes only species displaying the following combination of features: (i) ventral oral-collar tubular ducts of at least 2 sizes, smaller ducts present singly or in segmental clusters on the body margin, at least on the last 2 or 3 abdominal segments, and (ii) minute discoidal pores in sclerotized area of enlarged tubular ducts, touching the sclerotized rim of the duct opening.  (2267).
Diagnosis. Ferrisia kaki Kaydan & Pacheco da Silva, sp. n. is characterized by the following combination of features: (i) ventral oral-collar tubular ducts of two sizes, smaller ducts present singly or in segmental clusters on the body margin, on the last two or three abdominal segments; (ii) minute discoidal pores on the sclerotized area of enlarged tubular ducts, almost always touching the sclerotized duct rim, and (iii) both anterior and posterior pairs of ostioles present and well-developed.
Comments. Ferrisia kaki Kaydan & Pacheco da Silva, sp. n. most closely resembles F. cristinae Kaydan & Gullan, in having few ventral oral-collar tubular ducts on the abdominal submargin (not those in posterior marginal clusters), and often with a circular discoidal pore in the sclerotized rim of the duct or on nearby derm. However, F. kaki differs from F. cristinae in having: (i) multilocular disc pores only on abdominal segments VII and VIII+IX (VI-VIII+IX in F. cristinae) and (ii) 87-99 enlarged tubular ducts on the dorsum (95-113 in F. cristinae). F. kaki is also similar to F. terani Williams in having a small number of multilocular disc pores and a slender body shape, but F. kaki can be readily distinguished from F. terani in having: (i) two sizes of oral collar tubular ducts on the venter (only one size in F. terani); (ii) enlarged tubular ducts with a minute discoidal pore touching the sclerotized rim of duct opening.
Etymology. This species was named after its host plant, to reflect the high levels of infestation in persimmon orchards.
Host plant. Diospyros kaki. Distribution. Brazil (Caxias do Sul, Farroupilha, Rio Grande do Sul). Molecular characterization. No intraspecific variation was observed at COI (7 replicates). A BLAST hit with sequence similarity of 98% was obtained with a sequence assigned to Ferrisia terani Williams & Granara de Willink from Pacheco da Silva et al. (2014). The alignment between F. kaki and F. terani is shown in Figure 4. Generic diagnosis (adapted from Gimpel and Miller 1996;Williams and Granara de Willink 1992). Adult female. Body normally broadly oval, 1.2-4.3 mm long, 0.6-2.6 mm wide. Antennae each normally 8-segmented, occasionally with 7 segments. Labium 3-segmented, always longer than wide. Legs well-developed, claw without a denticle; translucent pores generally present on hind legs, on coxae, and/or femora and/or tibiae, rarely on trochanter; tarsal and claw digitules both capitate, claw digitules thicker than tarsal digitules. Circulus usually present, well-developed and divided by an intersegmental line; rarely small and not divided, usually wider than long. Quinquelocular pores always absent.
Description. Dorsum. Dorsal setae flagellate. Anterior and posterior ostioles present, well-developed. Cerarii present, 12-17 pairs, preocular pair always absent, each cerarius normally with two conical setae, except for 1 or 2 on head and thorax, each often with 3 or 4 conical setae plus an associated cluster of trilocular pores; anal lobe cerarii welldeveloped, each often sclerotized, usually with two enlarged conical setae; usually all cerarii with auxiliary setae, but occasionally auxiliary setae absent anterior to the penultimate pair. Anal ring typically with six setae. Trilocular pores scattered over dorsum. Minute discoidal pores usually present, sometimes situated adjacent to rim of oral rim tubular ducts. Oral rim tubular ducts present on body margins and medially and submedially, or in rows across abdominal segments, sometimes associated with minute discoidal pores and setae. Oral-collar tubular ducts often present. Multilocular pores rarely present on dorsum.
Venter. Body setae flagellate. Trilocular pores scattered over entire surface. Minute discoidal pores scattered throughout the venter, often of two sizes, larger pores frequently present next to eyes and on venter of anal lobes, sometimes also situated adjacent to rim of oral rim tubular ducts. Oral rim tubular ducts occasionally on venter only. Oral-collar tubular ducts of one or more sizes, of various lengths and widths, shortest ducts often present medially on abdominal segments, and larger ducts often present on margins of abdomen. Multilocular disc pores present on posterior abdominal segments, especially around vulva.
Comments. Pseudococcus rosangelae Pacheco da Silva & Kaydan most closely resembles P. peregrinabundus, P. nakaharai and P. dasyliriae in having dorsal multilocular disc pores, but P. rosangelae can be distinguished from other species in having: (i) oral collar tubular ducts present over the entire dorsum (on other species not scattered all over the dorsum) and (ii) no translucent pores on the hind legs (present in the other species).
Etymology. This species is named after Rosangela Leme do Prado, mother of the author VCPS.