A survey of scale insects in soil samples from Europe (Hemiptera, Coccomorpha)

Abstract In the last decades, several expeditions were organized in Europe by the researchers of the Hungarian Natural History Museum to collect snails, aquatic insects and soil animals (mites, springtails, nematodes, and earthworms). In this study, scale insect (Hemiptera: Coccomorpha) specimens extracted from Hungarian Natural History Museum soil samples (2970 samples in total), all of which were collected using soil and litter sampling devices, and extracted by Berlese funnel, were examined. From these samples, 43 scale insect species (Acanthococcidae 4, Coccidae 2, Micrococcidae 1, Ortheziidae 7, Pseudococcidae 21, Putoidae 1 and Rhizoecidae 7) were found in 16 European countries. In addition, a new species belonging to the family Pseudococcidae, Brevennia larvalis Kaydan, sp. n. and a new species of Ortheziidae, Ortheziola editae Szita & Konczné Benedicty, sp. n. are described and illustrated based on the adult female stage. Revised keys to the adult females of Brevennia and Ortheziola are presented.


Introduction
Several expeditions were organized since the 1950's mainly within mainland Europe by the Hungarian Natural History Museum to collect snails, aquatic insects and soil animals (mites, springtails, nematodes, and earthworms). More recently, these studies were focused on the Balkan Peninsula and the Carpathian Region (Csuzdi et al. 2011;Dányi 2010;Kontschán 2010;Mahunka and Mahunka-Papp 2010;etc.). For these studies, a total of 2970 soil samples were collected from different habitats such as forest litter, moss, agricultural areas etc. in 16 European countries. Although visual sampling is a widely used method and often very effective for collecting scale insect species (Hemiptera: Coccomorpha), other collecting methods such as Berlese funnel and D-Vac are also useful as both provide plenty of scale insect species living in leaf litter, soil and under moss (Kozár 2004;Kozár and Konczné Benedicty 2007).
Scale insects are small, sap-sucking true bugs, sister to Aphidoidea, Aleyrodoidea and Psylloidea (Gullan and Martin 2009). Scale insect taxonomy is generally based on the microscopic cuticular features of the adult female which are paedomorphic, maturing in a juvenile form (Kosztarab and Kozár 1988). García et al. (2015) indicated that more than 8000 species have been described up to now. Among these are many agricultural pests (Miller and Davidson 1990) and invasive species (Miller et al. 2005, Ouvrard et al. 2013. It has been argued (Koteja 1985) that the evolution of the scale insects occurred in two stages. In the first stage, the scale insects split from the homopteran stock (in the Carboniferous or Permian) prior to the appearance of flowering plants (Jurassic), living in the forest litter on a "mixed" diet and feeding on the sap of various plants at the surface and from living and decaying plant tissues. The legs became modified as a digging organ (one claw, one segmented tarsus, functional tibiotarsus), the females lost their wings and became paedomorphic and the males became dipterous. They also diverged into numerous groups at that time. The second evolutionary trend commenced with the appearance of the flowering plants in the Jurassic and continues to the present. As a result of these latter trends, the coccoids became true plant parasites and most scale insect groups started to live on the aerial parts of the plants and acquired their own endosymbionts (Koteja 1996). As a result, the level of specialization in the recent scale insects is great although some species still continue the primary, hypogeal mode of life, e.g. in the families Ortheziidae (Nipponortheziinae, Newsteadinae, Ortheziolinae) and Rhizoecidae (Koteja 1986;Vea and Gimaldi 2012).
Although the pest scale insect species found on the aerial parts of agricultural and horticultural plants are well studied in Europe, there is a great gap in the knowledge on the hypogeal scale insect fauna in Europe. The aim of this study was to investigate the hypogeal scale insect fauna of Europe by studying the scale insect specimens found in the soil and litter samples of the Acarology Collection of HNHM, because hypogeal species are indicators of the ecological richness and biodiversity of the soils and provide useful information about the comparative ecologies of the regions, and about the evolution of soil animals.

Material and methods
The specimens described and recorded in this study were all obtained from the soil samples in the Hungarian Natural History Museum (HNHM) collection (2970 samples in total). The samples were extracted by Berlese funnel. This is an apparatus widely used to extract living organisms, particularly arthropods. It works by creating a temperature gradient over the sample such that mobile organisms will move away from the higher temperatures and fall into a collecting vessel, where they are preserved for examination (Southwood and Henderson 2000). The Berlese funnel is a suitable device with which to collect and sort hypogeal and ground-dwelling animals, and also those which live in the lower herb layer of different habitats. It will also occasionally collect species living on higher aerial parts of plants that have fallen to the ground on plant material, such as leaves, twigs, etc.
All measurements and counts were taken from all the available material, and the values are given as a range for each character.
Holotypes of the new species are deposited in the Hungarian Natural History Museum (HNHM). Paratypes are deposited in the HNHM and in the Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences (PPI).
Detailed locality and collection data have been provided for the new and some rare species only. For a host plant list of each species see García et al. (2015). Distribution data for each species have been provided, with new country records in bold. However must take into consideration, that these new country records are all relative to García et al. (2015) and latest available checklists (Fetykó et al. 2010;Masten Milek and Simala 2008a;Pellizzari et al. 2015;Trencheva et al. 2012), as to create new country checklists is out of the scope of this work.
Comments. N. floccosa is the most common species in the collection. Although there is some variability in the number of antennal segments and in the size of the individuals examined in this study, all specimens above are considered to be part of the morphological variation of N. floccosa.
Diagnosis of genus. Adult female in life with a series of marginal, mediolateral and medial waxy protrusions, corresponding to wax plates on slide-mounted specimens. The distribution of these protrusions and wax plates ( Fig. 1) differs between species in the genus (Kozár 2004).
Slide-mounted adult female with three-segmented antennae; third antennal segment with a slender apical seta, flagellate sensory seta and small subapical seta; second segment with one sensory pore. Eye stalk protruding, thumb-like, fused with sclerotized area at base of each antenna (sometimes called the pseudobasal antennal segment). Legs well developed; leg setae robust, spine-like; trochanter and femur fused, tibia and tarsus fused; tibia with one sensory pore and at least one fleshy sensory seta; tarsus without digitules; claw digitules hair-like, claw without a denticle. Labium onesegmented, with many setae; labium with three long setae near apex, very close together, all situated in a single setal socket . Anal ring situated in a dermal fold on dorsal surface, ring bearing six setae. Sclerotized plate present on dorsum anterior to anal ring, wider than long. Modified pores, each with two, three or four loculi, scattered over surface, appearing like microtubular ducts. Thumb-like pores forming a cluster on each side of anal ring. Abdominal spiracles ventral on anterior segments, with at least one present on each side of segments I, II or III; when present, posterior abdominal spiracles located on dorsum near anal ring, surrounded by a cluster of multilocular pores (Kozár 2004).
Distribution. The 13 species of Ortheziola are found in the Palaearctic and northeastern part of the Oriental Regions. For detailed distribution data of the twelve previously known species, see ScaleNet (García et al. 2015). New locality records for several Ortheziola species were discovered during the study of the HNHM collection, which is listed below. The distribution patterns of the species may imply the existence of several other species in these regions, which would be worth further study.
Comments. The genus Ortheziola resembles the genera Ortheziolacoccus and Ortheziolamameti in having three-segmented antennae, with the basal part of the antenna fused to the eye. However, Ortheziola differs from Ortheziolacoccus and Ortheziolamameti in having only a single spine band inside the ovisac band, and these genera have different geographic distribution: Ortheziola species are distributed in the Palaearctic and north east part of Oriental Regions, Ortheziolacoccus species occur only in Ethiopian Region, while Ortheziolamameti species in the Oriental and Ethiopian Regions. Venter. Labium 120-148 µm long. Stylet loop about as long as labium. Leg segment lengths: front coxa 107-127 µm, middle 117-133 µm, hind 122-127 µm; front trochanter-femur 291-332 µm, middle 321-357 µm, hind 316-362 µm; front tibia-tarsus 357-372 µm, middle 357-388 µm, hind 438-454 µm; front claw 46-54 µm, middle 43-51 µm, hind 51-54 µm long; claw digitules spine-like, 7-12 µm long; legs with rows of robust setae; longest seta on trochanter-femur, each 12-14 µm long; with one flagellate sensory seta on each of femur and tibia, 10-12 µm long; each trochanter with four sensory sensilla on each surface. Wax plate 11 and 12 present at marginal areas of head; marginal wax band surrounding each thoracic spiracle (plates 15 and 16); wax plates in front of coxae absent (plates 13, 14, 17 and 18 absent), plate 19 absent; with scattered clusters of spines between hind legs and ovisac band. Anterior margin of ovisac band with three waves; with one band of spines within ovisac band, with quadrilocular pores predominant near anterior edge of spine bands and scattered within the spinebands, each pore 3.5-4 µm in diameter. Thoracic spiracles each with scattered quinquelocular pores loosely associated with spiracle opening, each group contains 10-13 pores, each pore 5-6 µm in diameter (several of these pores present on dorsum); diameter of opening of anterior thoracic spiracle 13-20 µm. Setae few, scattered in medial areas of thorax, with several setae present near anterior margin of ovisac band (some capitate), several associated with anterior and posterior multilocular pore rows, several more associated with posterior multilocular pores surrounding vulva. Multilocular pores each 8-9 µm in diameter, with 7-9 (mainly 7) loculi around perimeter and one loculus in central hub; partial band of multilocular pores near anterolateral edge of spine band, also scattered around vulva and near ovisac band, almost forming a row on the apical abdominal segment. Abdominal spiracles present, two pairs on each side of body anterior to ovisac band and one pair situated inside ovisac band, near anterolateral angle; each abdominal spiracle with sclerotized vestibule. Dorsum. Wax plates covering two-thirds of marginal area; mediolateral thoracic plates (3, 5 and 6) present; waxplate 3 small, containing only a few spines and pores; medial area of thorax and abdomen with a few scattered spines and pores. Spines at margin of wax plate 4 each 15-16 µm long, those in middle of wax plate each 16-18 µm long; spines truncate and expanded at apex. Flagellate setae present in very small numbers on each wax plates and in medial bare area, each seta 17 µm long. Quadrilocular pores, each 3.0-3.5 µm in diameter, with four loculi, present at the margins of all waxplates and scattered within the waxplates. Quinquelocular pores, each 5.5-6.0 µm in diameter, present in marginal areas of abdomen, between the waxplates; also present in a cluster near anal ring. Sclerotized plate on abdomen 63-77 µm long, 230-251 µm wide; with a few setae with pointed apices situated at posterior edge of plate. Anal ring with incomplete triple rows of circular pores, each pore 1.5-3.0 µm in diameter; longest anal ring seta 72-74 µm long; anal ring 60-67 µm long, 50-55 µm wide. Thumb-like pores, each 6 µm long. Abdominal spiracle present in center of multilocular pore cluster situated laterad to anal ring.

Key to species of Ortheziola, based on adult females
Host plant. Unknown. Distribution. Bulgaria, Croatia. Etymology. The new species is dedicated to Edit Horváth, who has worked as an assistant in the Acarology Collection of the Hungarian Natural History Museum, Budapest for many years and helped our work in extracting the specimens and finding locality data.
Comments. Ortheziola editae is characterized by the presence of (i) dorsal wax plate 3 being only slightly developed, (ii) ventral plates 11 and 12 present at the base of antennae, and (iii) plate 19 absent from near the body margin. This species is very close to O. marottai but differs in having (O. marottai values in brackets): (i) only one or two multilocular pores anterior to vulva (plenty of multilocular pores); (ii) multilocular pores near each thoracic spiracle, each pore with five loculi (four loculi) and (iii) anterior margin of ovisac band with six waves (at least eight waves).

Ortheziola marottai Kaydan & Szita
Material examined. Greece: 1 ♀ -Ioannina regional unit, Kalpaki, Vellas Monasteri. Comments. The type locality of O. vejdovskyi is in Czech Republic, originally: Bohemia, Bechlin; Králové Dvur n. L. east Bohemi (Šulc 1895), and this is the only report from the area of former Czechoslovakia (García et al. 2015), thus the current data from Slovakia can be considered as a new country record. O. vejdovskyi was reported from Yugoslavia by Kosztarab and Kozár (1988) (García et al. 2015), without detailed locality data, therefore we have no exact information which current successor state(s) could have been the actual locality(ies) in that report. Accordingly we list the current localities by states, without considering these as new country records, thus it was not unequivocally proven. Adult female. Labium three-segmented, longer than wide. Posterior pair of spiracles always larger than anterior spiracles. Circulus present or absent. Legs well developed, claw with or without denticle; tarsal digitules hair-like, not capitate; claw digitules knobbed, claw digitules broader than tarsal digitules. Only posterior ostioles developed; anterior ostioles absent. Anal lobes poorly developed. Anal ring oval, with one inner row of pores and one or two outer rows of pores plus with six setae. Minute discodial pores present of various sizes, scattered throughout.

Pseudococcidae
Dorsum. Antennae 6-8 segmented. Eyes oval, each on a small basal cone. Cerarii present numbering 1-4, only on posterior abdominal segments. Dorsal body setae spinelike. Multilocular disc pores present or absent. Quinquelocular pores present, scattered all surface. Oral collar tubular present in transverse rows on body segments. Trilocular pores absent. Minute discodial pores present, from a few to scattered on the surface, variable in sizes.
Venter. Most ventral setae slender and hair-like, of various sizes. Oral collar tubular ducts of one or two sizes, each varying in length and width. Multilocular disc pores present on posterior abdominal segments, especially around vulva or absent. Quinquelocular pores present, scattered throughout. Trilocular pores, each 2.5-5.0 µm in diameter, only around atrium of both pairs of spiracles. Minute discodial pores present, of variable sizes, scattered through.
Etymology. This species is named because of absence of multilocular pores on venter and dorsum, which is a character of larval (nymphal) stages.
Comments. Occurring on needles and in bark crevices of conifers. Biology in Italy studied by Sampo and Olmi (1979). Life history discussed by Kosztarab and Kozár (1988).
Distribution. Albania, Hungary, Kazakhstan (García et al. 2015).  Distribution. United States of America (García et al. 2015), Spain. Comment. This species is characterized by the lack of multilocular pores on both the dorsum and venter and in having very few oral collar tubular ducts on the dorsum. This species is similar to R. cacticans and R. leucosomus, but differs from both in having more anal ring pores. In addition, this species is also similar to R. nakaharai but differs in having a longer labium. However, these are poor characteristics upon which to base species differences and so a drawing of this species is presented here. Detailed descriptions of the above mentioned species are available in Kozár and Konczné Benedicty (2007).

Discussion
In this study, 43 scale insect species were found in 16 different European countries. Despite scale insects being found in only 10% of the 2970 samples collected, the Berlese funnel collection method has revealed new species and widened distribution records for known species. It is believed that the use of diverse collecting methods can provide researchers with additional sources of information about species distribution and diversity.