First Description of the Adult Male of the Gall-Like Scale Insect Allokermes galliformis (Riley) (Hemiptera: Coccomorpha: Kermesidae)

The adult male of Allokermes galliformis (Riley, 1881) (Hemiptera: Coccomorpha: Kermesidae) is described for the first time in Colorado, United States of America. This scale insect species recently emerged as a significant pest of red oaks in Colorado through its causative role in drippy blight disease. A description and illustration of the adult male characterize its key external morphological characteristics.


Introduction
Kermes scales (Hemiptera: Kermesidae) are an understudied group of insects that feed on the sap of trees and shrubs in the family Fagaceae throughout the Nearctic, Oriental, and Palearctic regions [1,2]. In total, there are 92 species within Kermesidae [3], and in the Nearctic region there are 34 species representing five genera (Allokermes, Eriokermes, Kermes, Nanokermes, and Olliffiella) [4]. Kermes scales display marked sexual dimorphism [5]. Females have three nymphal instars and a pre-reproductive and post-reproductive adult stage, both of which have a gall-like form. Males typically exhibit four nymphal instars, with the "prepupal" and "pupal" instars occurring in a white cocoon-like test, and a winged adult stage [2,5].
Immature stages and the adult male kermes scales are minute in size and spatially separated throughout the landscape, which makes them difficult to collect. Furthermore, adult males are short-lived, lacking functional mouthparts. The sclerotized post-reproductive female is the most conspicuous and most readily collected life stage because they remain on branches long after oviposition. Kermes scale identification is largely based on morphological characteristics of pre-reproductive females, which can only be collected during a narrow phenological window, and their waxy covering may change throughout development [6]. Characterizing the morphology of the adult male life stage may help support the current phylogenetic placement of Kermesidae, which is based on morphological characteristics [7]. While host and locality information is available on each of the 12 species within the genus Allokermes, the specimens presented may also help disentangle the phylogenetic placement of species within Allokermes as well as aid in species identifications. A detailed description of the adult male is most needed for the type species of the genus Allokermes, the pin oak kermes Allokermes galliformis (Riley, 1881) [8]. Not only is A. galliformis one of the most widespread and well-studied species in the genus [1,4,[9][10][11], it was also recently reported in outbreak numbers in Colorado, United States of America, where it was documented damaging red oak trees (Quercus spp.) in conjunction with the bacterial pathogen involved in drippy blight disease (Lonsdalea quercina) [12,13]. In these drippy blight diseased systems, A. galliformis exhibited a univoltine life cycle similar to the previously reported life cycle of A. kingii Cockerell, 1898 [14,15]. Furthermore, adult males typically congregated on large branches and trunks of host trees and surrounding debris [11].
Allokermes galliformis was originally described from female syntypes from Iron Mountain, (St. Francois County) Missouri [4]. Morphological descriptions are available for all female life stages [9,10]. Although the second-instar male was described [9], the adult male has not been described. Information on the adult male stage for species of the genus Allokermes is generally lacking with only the male of A. kingii (Cockerell, 1898) described [15]. The abundance of adult male scales on drippy blight diseased trees in northeastern Boulder County, Colorado, on planted northern red oak (Quercus rubra L.) has allowed us to provide a detailed morphological description of the adult male of A. galliformis.

Materials
The identification of Allokemes galliformis females feeding on red oak trees in Boulder, Colorado, USA, was determined by the morphological characteristics of pre-reproductive adults [11]. Allokermes gillettei (Cockerell) can also be found in the region, but this species is host specific to gambel oak (Q. gambelii) [10]. In this study, adult males were field-collected in July 2015 in Boulder, Colorado, USA (Colorado, Boulder, 40 • 01 72.89" N, -105 • 25 99.04" W, 2.vii.2015, R. Sitz). Adult males were aspirated from the trunk of infested northern red oak trees or reared in the lab from tests. The phenology of the males in relation to A. galliformis females, coupled with the red oak host plant association, led to the male species determination of Allokermes galliformis.
Specimens were stored in 90% ethanol at 4 • C until slide mounting or scanning electron microscopy. All slide mounted specimens and additional voucher specimens preserved in 80% ethanol were deposited in the C.P. Gillette Museum of Arthropod Diversity, Colorado State University, Fort Collins, Colorado. Additional specimens are stored in the Department of Bioagricultural Sciences at Colorado State University in 95% ethanol at −20 • C, as they may be useful in future molecule-assisted species identifications.

Slide Mounting
Males were cleared in a 5% KOH solution for 7-8 h [6], and then mounted on slides in Hoyer's mounting medium. The slides were left in an oven at 46 • C for 7-10 days, then the edges of the coverslips were sealed using clear nail polish. Measurements were conducted with an Olympus BHA Phase System compound microscope following previous descriptions [2,[15][16][17][18]. Measurements are in micrometers (µm), and 6 specimens (n = 6) were used for each measurement. Ranges are reported, followed by the mean in parentheses. Table 1 presents the abbreviations used for structures, and abbreviations follow previous literature [1,15,16]. Accurate measurements of the radius (rad) and media (med) of the wing could not be taken due to folding of the wings in slide-mounted specimens.

Scanning Electron Microscopy
For scanning electron microscopy, specimens were initially prepared by fixing in 2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4, at 4 • C for 24-48 h. Post-fixation was carried out in 1% buffered osmium tetroxide overnight at 4 • C, then samples were dehydrated through a graded ethanol series, followed by critical point drying using a BioRad E3000 critical point dryer (Quorum Technologies, East Sussex, England), or by dehydration with acetone and hexamethyldisilazane. All samples prepared for SEM were sputter coated with 10 nm gold using a Hummer VII (Anatech USA, Union City, California, USA). Micrographs were taken using a JEOL JSM-6500F Field Emission Scanning Electron Microscope at the Central Instrument Facility, Imaging Laboratory, located at Colorado State University. All images were captured digitally as tiff files, and graphically edited to input black image backgrounds (Adobe Photoshop, CS6 13.0.1). All measurements were taken at the widest or longest point of a given structure (Table 1), and are given in µm. Ranges are presented, followed by the means in parentheses.

Results
In life, adult males are orange to brown with clear to iridescent wings. Excluding the head, the body shape is robust, subtriangular with long, distinct legs, and an elongated genital segment ( Figure 1A,B, Figure 2). Many specimens observed were missing sections of antennae, legs, or wings. Fusion of consecutive antennal segments was observed in some specimens, but all antennal segment measurements were taken from antennae without fusion. Folding of the wings in mounted specimens did not permit accurate wing and wing vein measurements. Total body length from the apex of the head to the apical tip of the penile sheath is 976-1091 µm (mean 1022 µm); the width at the mesothorax is 277-395 µm (mean 325 µm) at the scutum. Insects 2019, 10, x 4 of 14

Body Setae and Pores
Long, conspicuous setae are present on most of the head, body, and appendages. Much of the exoskeleton has a granular surface, including the simple eyes ( Figure 6). Polygonal reticulation is present on the head (Figures 3-5). Waxy excretions are especially prominent on the genital segment, tarsus (tar) near the claw (cl) (Figure 7), and on and around the glandular pouch (glp) and glandular pouch setae (glps) (Figure 8). The genital segment also bears setae (gts). Glandular pouch setae (glps)are made of wax filaments (Figures 8 and 9).
Laterally, the mesepisternum (eps2) is proximal to the subepisternal ridge (ser). Postalare (pa) is situated dorsal and posterior to the mesepisternum. The basisternum (stn2) is a large, subrectangular, shield-like plate with margins strongly sclerotized, 105-143 μm (mean 128 μm) in length and 202-231 μm (mean 214 μm) in width at the widest point. The anterior edge of the basisternum is bordered by the marginal ridge; the posterior edge is bordered by the precoxal ridge (pcr2), and partially covers the furca (f) and furcal pit (fp).
Laterally, the mesepisternum (eps 2 ) is proximal to the subepisternal ridge (ser). Postalare (pa) is situated dorsal and posterior to the mesepisternum. The basisternum (stn 2 ) is a large, subrectangular, shield-like plate with margins strongly sclerotized, 105-143 µm (mean 128 µm) in length and 202-231 µm (mean 214 µm) in width at the widest point. The anterior edge of the basisternum is bordered by the marginal ridge; the posterior edge is bordered by the precoxal ridge (pcr 2 ), and partially covers the furca (f) and furcal pit (fp).
Wings: Each wing is 820-943 μm (mean 868 μm) in length, extending past the apex of the abdomen and with a distinct alar lobe (al) ( Figure 1A). Small, numerous microtrichia cover all wing surfaces and surround the wing margins. Media arise along the radius at approximately 1/4 the total length of the radius, distal to the costal complex of the wing veins. The radius extends approximately 4/5 the entire length of wing, with a slight proximal curve in the first 1/4, before conjunction with the media. Hamulohalteres are finger-like, measuring 78-86 μm (mean 82 μm) in length and 17-23 μm (mean 21 μm) in width, with an apically hooked hamulus seta arising at the distal apex, approximately equal in length to the hamulohaltere. The mean ratio of total body length to wing length is 1:1.18 μm.
Legs: Pro-and mesothoracic legs are almost subequal in length, with the metathoracic legs being shortest. The mean ratio of prothoracic leg to total body length is 1:0.58 μm. The mean claw length is about 22 μm, similar to the width of the tarsal base. The tarsus has two tarsal digitules (tdgt), each 25-32 μm (mean 29 μm) in length, arising from the dorsal to the claw. The claw has two ungual digitules (ugdt), each approximately the same in length as the claw, arising ventrally at the point of attachment of the claw to the tarsus (Figure 7). Both tarsal and ungual digitules are knobbed apically ( Figure 1C; Figure 7). All leg segments, except for the claw, are covered with numerous setae ( Figure  1A; Figure 2). Table 3 provides the mean length and width of each leg segment. Metathorax: Indistinct and weakly sclerotized. The metasternal plate is not visible. There are 11-19 (mean 16) ventral setae. Dorsal setae are absent.
Wings: Each wing is 820-943 µm (mean 868 µm) in length, extending past the apex of the abdomen and with a distinct alar lobe (al) ( Figure 1A). Small, numerous microtrichia cover all wing surfaces and surround the wing margins. Media arise along the radius at approximately 1/4 the total length of the radius, distal to the costal complex of the wing veins. The radius extends approximately 4/5 the entire length of wing, with a slight proximal curve in the first 1/4, before conjunction with the media. Hamulohalteres are finger-like, measuring 78-86 µm (mean 82 µm) in length and 17-23 µm (mean 21 µm) in width, with an apically hooked hamulus seta arising at the distal apex, approximately equal in length to the hamulohaltere. The mean ratio of total body length to wing length is 1:1.18 µm.
Legs: Pro-and mesothoracic legs are almost subequal in length, with the metathoracic legs being shortest. The mean ratio of prothoracic leg to total body length is 1:0.58 µm. The mean claw length is about 22 µm, similar to the width of the tarsal base. The tarsus has two tarsal digitules (tdgt), each 25-32 µm (mean 29 µm) in length, arising from the dorsal to the claw. The claw has two ungual digitules (ugdt), each approximately the same in length as the claw, arising ventrally at the point of attachment of the claw to the tarsus (Figure 7). Both tarsal and ungual digitules are knobbed apically (Figures 1C and 7). All leg segments, except for the claw, are covered with numerous setae (Figures 1A  and 2). Table 3 provides the mean length and width of each leg segment.