Wax produced by dermal pores in three species of mealybug (Homoptera : Pseudococcidae)

,X,2,, 7322/X3 $03 IW) + (Xl IYR3 Pcrgamon Prev I Id

WAX PRODUCED MEALYBUG

BY DERMAL PORES IN THREE SPECIES (HOMOPTERA : PSEUDOCOCCIDAE) M. Cox

JENNIFER British

Museum

(Natural

History),

OF

Cromwell

Road,

London

SW7 5BD, U.K.

and M. J. PEARCE Overseas

Development

Administration,

Tropical Development and Research Lane, London WR 5SJ, U.K.

(Accepted

4 April

Institute,

College House,

Wrights

1983)

dermal pores and the characteristic forms of wax that they produce are in three species of mealybug, Ferrisiu virgara, Phenacoccus manihori and PIanococcus cirri (Homoptera : Pseudococcidae), and illustrated with scanning electron photomicrographs. The trilocular and quinquelocular pores covering the bodies of most instars produce curled filaments of wax which are also found on the surfaces of droplets of honeydew and ostiole exudate. Their primary function is probably to protect the mealybugs from contamination by their own honeydew and defensive exudates. The paired marginal cerarian setae are secretory and produce long wax rods which support tangled masses of trilocular pore wax. These lateral wax projections may protect the mealybugs from predators and aid in the spacing of individuals within the colony. Tubular ducts and multilocular disc pores respectively produce long hollow and shorter curled filaments which make up the ovisac and the male cocoon. Solid rods of wax produced by an unusual type of tubular duct in F. virga/a have associated setae and a campaniform sensillum and probably have a sensory function. Adult males also have pores which produce filamentous wax, but these pores are of a different form from those of the other stages.

Abstract-The

examined

Index descriptors (in addition to those in title): Ovisac, morphology, scanning electron microscopy.

cocoons,

honeydew,

functional

INTRODUCTION SCALE insects and mealybugs are particularly noted for their production of integumentary secretions. These secretions take many forms such as the hard covering shields of the armoured scale insects (Diaspididae) or the felty coverings of the Eriococcidae. Voluminous watery wax covers the genus Ceroplustes (Coccidae), while fine powdery wax, often produced into long lateral filaments, gives the Pseudococcidae their common name of mealybugs (Figs. 4- 6). In addition, almost all mealybugs and scale insects produce wax in association with parturition and many produce extensive ovisacs into which the eggs are laid. The wax on the surface of the body is discarded along with the cast skin during ecdysis. Some of this wax is secreted as a relatively featureless film by subepidermal glands as in the smooth covering of the soft scales (Coccidae) (Tamaki et al., 1969; Foldi, 1978), but in most scale insects and mealybugs wax is secreted through distinct sclerotized structures that act as moulds to produce structurally different forms of wax at different positions on the body. These wax-producing structures have been termed pores and ducts by taxonomists who have found that the types and distributions of these structures as seen in 235

236

JENNIFER M. Cox and M. J. PEAKC~

stained macerated specimens under the light microscope, provide an invaluable aid for the identification and classification of these insects. Although the presence of filamentous wax on the surfaces of many species of scale insects and mealybugs is well documentated, there has been only limited speculation on its function and the reasons for the diverse variety of pores and ducts which are to be found within a single species. Suggestions concerning the functions of this wax have included disposal of excess material obtained during feeding (Pollister, 1937), protection from desiccation (Gullan, 1979; Hamon et al., 1975), preventing eggs from sticking together (Hamon et al., 1975) and ventilation of the stigmatic furrows (Tamaki et al., 1969). In our present study we have examined 3 species from 3 genera of Pseudococcidae and used the SEM to study both the external morphology of the pores and ducts which act as moulds for the wax produced, and the structure of the wax produced by each type of pore or duct. We have also attempted to locate and identify the various forms of wax after their departure from the mealybug in order to speculate as to their functions. Female mealybugs are neotenic. They lack wings, and although equipped with short legs, are relatively immobile throughout their 4 instars. Male mealybugs have 5 instars, the first 2 of which are similar to those of the female and are the stages in which feeding occurs. The 2nd instar spins a cocoon in which the prepupal and pupal stages are passed and from which emerges the relatively tiny, winged male. Mealybugs, like scale insects and aphids, are usually found in dense colonies. Most live in secluded positions, such as under bark, in leaf axils and under fruit calyxes. Some form and occupy galls or live underground on the roots of plants. Protection from natural enemies is provided by the production, when the mealybug is disturbed, of droplets of fluid from the 2 pairs of dorsal structures known as ostioles (Fig. 1). These droplets harden rapidly on exposure to the air, thus fouling the mouthparts of an attacking predator. Ostioles are generally considered to be homologous to the cornicles of aphids where the secretion involved has been shown to consist of triglycerides (Strong, 1967). MATERIALS

AND

METHODS

The following species of mealybug were used: variegated mealybug, Ferririia virgata (Cockerell), from Trinidad: cassava mealybug, Phenacoccus manihori Matile-Ferrero, from Nigeria; and citrus mealybug, P/unococcus ci/ri(Risso), from England. All 3 species were maintained in culture at the British Museum (Natural History). F. virgara and P. manihori are parthenogenetic and P. cirri is bisexual. 1’7 virgara is viviparous: P. tnanihoti and P. cirri are oviparous. Both immature and adult females of all 3 species were examined together with 2nd instar and adult males of P. c-i/r;. Individuals were either washed with chloroform to completely remove wax from the pores, or lightly brushed with a fine paintbrush to partially remove the wax. In addition the ovisac, male cocoon and the surfaces of the eggs, honeydew droplets and ostiole exudates were examined.

OBSERVATIONS

Trilocular pores Trilocular pores are characteristic of the family Pseudococcidae and are generally abundant on both surfaces of the body in all the female instars and in the first 2 male instars (Figs. 1, 2). Under the SEM, they are seen to be in the form of a raised triangle situated in a more circular depression (Fig. 7). They measure about 3.0 urn along each edge and have 3 dumb-bell-shaped loculi, which act as moulds to produce 3 railway trackshaped wax strands about 1 urn across (Fig. 8). The loculi are orientated so that on extrusion, each wax strand curves over one edge of the triangular pore, thus avoiding contact with the others. Each wax strand twists in a clockwise direction, probably due to

Wax Produced

by Dermal

Pores in Three Species of Mealybug

237

DORSUM , , VENTER

spimcles

02 t A trilocular

03mm

pore

0 quinquelocular

01

4

k

0.5mm

multilocular

0

x

tubular

disc

pore

duct

FIGS. 1-3. Generalized schematic diagrams of 3 different instars of mealybugs, showing typical relative positions of cerarii and different types of wax producing pore. 1. Adult female. 2. 2nd instar male. 3. Adult male. Pores and cerarian setae have been shown comparatively larger in relation to body outline and in fewer numbers than in actual specimen. For clarity, all setae except cerarian and anal lobe setae have been omitted.

differential thickening, causing the strands to spiral when further wax is produced. As the body is densely covered with these pores (Figs. 1,2,9), a thick covering of wax is ensured (Figs. 4 - 6). Quinquelocular pores

These pores occur in certain genera of Pseudococcidae, including Phenacoccus, but not In P. manihoti they are scattered over the venter, particularly in

Ferrisia or Pianococcus.

238

JENNIFERM. Cos and M. J. PEARCE

FIG. 4. Adult P. manihoti showing ovisacs and a honeydew droplet. x 8. FIG. 5. Adult female P. cifri showing lateral wax filaments. x 20. FIG. 6. Immature F. virgara showing tangled wax rods. x 20.

the midregion of the thorax. They measure about 6.0 pm in diameter and have 5 loculi (Fig. lo), each of which produces a strand of wax measuring about 0.5 pm across. A bifurcate projection at the mouth of each loculus gives the wax a C-shaped cross section, causing it to curl into a spiral as it is produced (Fig. 11).

Wax Produced

by Dermal

Pores in Three Species of Mealybug

239

FIG. 7. Trilocular pore of an immature female P. citri. x 7000. FIG. 8. Trilocular pore with extruded wax filaments of an immature female P. cirri. x 7000. FIG. 9. General view of trilocular pores with extruded wax filaments in an adult P. manihoii. x 500.

Multilocular disc pores Multilocular disc pores are characteristic of the adult female mealybug where tlw occur predominantly around the vulva, and are generally more numerous in oviparc 3us species than in viviparous species; and of the 2nd instar male where they are scattered o ver the dorsum as in P. citri.

240

JENNIFER M. Cox and M. J. PEARCE

measure approximately diameter and have 6 - 10 10 being the most common number encountered, situated around the circumference bifurate projection, similar observed in the quinquelocular loculus gives the wax a C-shaped section, it to curl as it is produced. As the strand curls small sections produced measures surface (Fig. 13). Tubular ducts

Like multilocular disc pores, tubular ducts are primarily found in the adult female, particularly on the abdominal venter, and are generally more numerous in oviparous species than viviparous species; and in the 2nd instar male. Externally, as revealed by the SEM, they appear merely as circular apertures with a raised rim about 4.0 urn in diameter (Fig. 14), but in macerated specimens viewed under the light microscope they have a characteristic tubular appearance. They produce long hollow tubes of wax about 1.5 urn in diameter, each of which in the species studied here bears 8 longitudinal ridges on its external surface (Fig. 15). These ridges probably increase the strength of the wax tube without adding substantially to its bulk. The relative sizes of the 4 pore types described above are shown in Fig. 16. Modified tubular duct in F. virgata

This species possesses a curious pore type, which in macerated specimens viewed under the light microscope appears as a very long, heavily sclerotized tubular duct. These ducts produce long solid rods of glassy appearance that protrude from the dorsum, frequently breaking off and forming a dense tangle that can be seen with the naked eye (Fig. 6). The rod itself is about 3.0 urn in diameter and has 10 longitudinal ridges visible on its surface (Fig. 18). Unlike the wax produced by the more usual type of mealybug tubular duct, these rods have a solid centre which may have been produced by the amalgamation of the exudates of separate pores at the base of the duct. The large rim surrounding the aperture of the duct has a campaniform sensillum and 2-4 setae in close proximity (Figs 17, 18). Anal ring

A tube of densely matted, fine wax filaments supported protrudes from the anal ring (Fig. 19).

by the six anal ring setae

Cerarii

The lateral protrusions of wax shown in Figs. 4 and 5 correspond in position to the structures on the body termed “cerarii” by taxonomists. Cerarii typically consist of pairs of short conical setae closely surrounded by groups of trilocular pores (Fig. l), positioned around the perimeter of the body. SEM examination of the lateral protrusions of wax (Figs. 4, 5) shows that they consist largely of entangled spirals of trilocular pore wax supported by 2 long rods (Figs. 20, 21). However, these supporting rods are not the short cerarian setae illustrated in Fig. 1, although they correspond with them in position. It appears, therefore, that the setal bases are secretory and are responsible for the production of these wax rods. The close concentration of trilocular pores around the setal bases produces a dense network of broken spiral lengths entangled together, which extends along the wax rods, so forming the substantial lateral wax protrusions characteristic of most mealybug species.

Wax Produced

by Dermal

Pores in Three Species of Mealybug

FIG. 10. Quinquelocular pore of an adult P. manihofi. x 7000. Quinquelocular pore with extruded wax filaments of an adult P. manihoti. x 5000. FIG. 12. Multilocular disc pore of an adult P. manihori x 6000. Fro. 13. Multilocular disc pore with extruded wax filaments of an adult P. manihori. x6000 Foci. 14. Apertures of tubular ducts of an adult P. manihori. x2500. FE. 15. Wax filaments produced by tubular ducts of an adult female P. citri. x 7000. FIG.

II.

241

242

JENNIFER M. Cox and M. J. PEARCE

FIG.

Trilocular pores extruded wax wax filaments middle), multilocular (upper middle right, right) of

(lower right), pores (left) apertures of adult P. manihoti. x 4000.

pores with ducts

Honeydew droplets and ostiole exudates Small balls consisting of either hardened droplets of honeydew or the exudate from the ostioles are readily apparent amongst colonies of mealybugs (Fig. 4). Under the SEM the surface of these balls can be seen to be covered with trilocular pore wax (Figs. 22, 23). Ovisacs Most oviparous species of mealybug produce large ovisacs into which the eggs are laid (Fig. 4). These ovisacs are made of wax filaments (Fig. 24), which under high magnification are seen to consist of long strands of the flexible tubular duct wax and short curls from the multilocular disc pores (Fig. 25). Male cocoons These are produced by the 2nd instar males and are very similar to the female ovisac in that they consist of a framework of tangled tubular duct wax intermingled with the wax

Wax Produced

by Dermal

Pores in Three

Speciesof

Mealybug

243

FIG. 17. Aperture

of a modified tubular duct with 3 setae and a campaniform sensillum of an adult F. virgara. x 6000. FIG. 18. Wax rod extending from aperture of a modified tubular duct of an adult F. virgafa. x 4000. FIG. 19. Filamentous wax tube surrounding anal ring of an adult female P. citri. x 1000.

produced by the multilocular disc pores. These multilocular disc pores are more flexible than those produced in the adult female and remain as long spirals rather than breaking up into short curls.

244

JENNIFER M. Cox and M. J. PEARCE

FIG. 20. Cerarius of an adult female P. citri with one cerarian seta partially exposed, other supporting a wax rod. x 3500. FIG. 21. Pair of wax rods extending from cerarian setae of an adult female P. citri, supporting entangled threads of trilocular pore wax. x 1000. FIG. 22. Surface of a droplet of honeydew from a culture of P. cirri showing a covering of wax filaments. x 500. FIG. 23. Surface of a droplet of honeydew from a culture of P. citri showing wax to be of the type produced by trilocular pores. x 1500. FIG. 24. Egg and part of ovisac of P. cifri showing presence of both long and short wax filaments. x 150. FIG. 25. Surface of an egg of P. citri showing wax to be of the types produced by tubular ducts and multilocular disc pores. x 3000.

Wax Produced by Dermal Pores in Three Species of Mealybug

245

Wax produced by adult male P. Citri Although the immature males possess the same kinds of pores as the females, the adult males of P. citri have only 2 kinds of pore, neither of which is found in the females. Along the pleura are groups of pores, each pore about 4.0 pm in diameter and with 4 - 5 loculi (Figs. 3, 26). These loculi each produce a spiral strand of wax measuring about 1 pm across and with 3 longitudinal ridges (Fig. 27). Around the single pair of caudal cerarii are clusters of pores, each pore about 2 pm in diameter and with 4 - 5 loculi (Figs. 3, 28). Each loculus produces a spiral wax strand measuring about 5.0 pm across and with 2 longitudinal ridges (Fig. 29). As observed in the cerarii of the females, these wax strands extend along the wax rods produced by the secretory cerarian setae. DISCUSSION

Comparison of the wax produced by different families of mealybugs and scale insects Although only a limited number of species have so far been examined, some comparisons can be drawn amongst the different families. Two basic forms of wax filaments are found throughout this insect group: long hollow filaments and shorter curved filaments. The former type has been found in the ovisacs and male cocoons of Margarodidae (Hashimoto and Kitaoka, 1971; Foldi, 1981; Ben-Dov, 1981) and Pseudococcidae, and in the ovisacs of Coccidae (personal observation). The felty sac covering at least one species of Eriococcidae is formed of this kind of wax filament (Waku and Manabe, 1981). These long hollow filaments are known to be produced by tubular structures in the Pseudococcidae, Coccidae (personal observation) and Eriococcidae (Waku and Manabe, 1981). In some members of the more primitive family Margarodidae, however, these filaments are produced by disc-type pores (Foldi, 1981) or by bilocular pores (Ben-Dov, 1981). The shorter curved type of wax filament has been found on the egg surfaces of most families of mealybugs and scale insects (Hashimoto and Kitaoka, 1971; Hamon et al., 1975; Gerson, 1980) and appears to be universally produced by some form of multilocular or quinquelocular pore., In some Pseudococcidae at least, multilocular pores are also present in the 2nd instar male where the wax produced forms part of the cocoon. Some Eriococcidae and most Pseudococcidae are covered with loose dermal wax which in at least one genus of Eriococcidae, is produced by multilocular pores (Gullan, 1979), in the more primitive mealybug genera at least partially by quinquelocular pores, but in the majority of mealybugs by trilocular pores. Even in families which do not usually have a loose wax covering, pores producing short wax filaments may be found in restricted areas such as the quinquelocular pores along the stigmatic furrows of the Coccidae (Tamaki et al., 1969; Millar and Gimpel, 1974) and the pores around the vulva in the Coccidae (personal observation) and Diaspididae (Davidson, 1973). Possible functions of filamentous wax in mealybugs Insect cuticle contains a thin layer of wax (Wigglesworth, 1953; Locke, 1974) which is thought to prevent desiccation. However, the loose filamentous nature of the dermal wax of the Pseudococcidae, as well as many other Homoptera, discussed here suggests that this wax may have other specific functions. It is unlikely that the copious wax covering produced by mealybugs is merely an excretory product as suggested by Pollister (1937), since other plant-sucking Homoptera

246

JENNIFER M. Cox and M. J. PEARCE

FIG. 26. Group of pores on pleurum of an adult male P. ci~i. x 2000. FIG. 27. Pleural pore with extruded wax of an adult male P. citri. x 8000. FIG. 28. Cerarius of an adult male P. citri showing a cluster of wax producing pores around bases of cerarian setae. x 3000. FIG. 29. Cerarian pore with extruded wax filaments of an adult male P. cirri. x 8000.

such as aphids have both wax covered and naked species feeding on the same plant. Broadbent (1951) demonstrated that aphid species which live in enclosed situations, such as between closely packed leaves or in galls, are typically wax covered, while species living in more exposed situations are not noticeably covered in wax. He suggested that the function of the wax was to prevent contamination by honeydew by coating the sticky surface of the droplets with loose wax. The problems of honeydew contamination are obvious. Apart from the mechanical difficulties of living in a sea of sticky liquid, especially for the newly hatched 1st instars, honeydew is an ideal substrate for many fungi, some of which are entomophagous (Nixon, 1951). It is evident that all sedentary, have evolved some mechanism for avoiding honeydew excreting Homoptera contamination. Coccidae generally live on the undersides of leaves where they are protected from the falling spray of honeydew, and have a device for propelling their own excreta for a considerable distance (Williams and Williams, 1980); some cyst-forming Margarodidae which live under the bark of trees have long wax tubes extending from the anal ring which carry the honeydew to the exterior (personal observation); free-living aphids generally have long caudae which flick honeydew droplets away from their bodies,

Wax Produced

by Dermal

Pores in Three Species of Mealybug

247

while those living in more confined situations typically have short caudae and are attended by ants which remove the honeydew (Way, 1963) or are covered in loose wax. Mealybugs which are typically ant-attended may have very small numbers of trilocular pores on their bodies (Williams, 1978), and produce little wax. Most species of mealybugs are protected from their own defensive exudate by the wax produced by the trilocular pores on the ostiole lips. Loose tdocular pore wax forms lateral projections supported by the rods of wax

produced by the cerarian setae. These lateral projections may provide the mealybug with some protection against predators, as any attacking insect would initially bite into the wax and not the mealybug itself. It is also possible that these filaments have a sensory function as the rods of cerarian wax are rigid and may act as extensions of the setae and so aid in the spacing of individuals and in the detection of natural enemies. A sensory function of wax is more evident in F. virgata. The solid rods of wax, which can reach lengths of up to 6 mm, produced by the large dorsal tubular ducts differ markedly from those usually produced from mealybug pores or ducts. The rim around the aperture of each pore bears a campaniform sensillum with 2 - 4 setae in close proximity, which together with the long rigid wax rod would provide an effective device for detecting disturbances around the dorsum of the insect. In addition, parasites and predators may be deterred on encountering such a bristly surface. The wax rods that break away from the body of the mealybug and those that are discarded with the cast skins at ecdysis form dense tangles that partially cover the mealybug colony (Fig. 6). As this viviparous species does not produce an ovisac, the tangles of wax rods would provide some protection for the newly born 1st instars. The ovisac produced by the adult female and the cocoon produced by the 2nd instar male of P. cifri are both composed of a mixture of long flexible strands formed by the tubular ducts and shorter curled filaments formed by the multilocular disc pores. Both ovisacs and male cocoons probably have similar functions: to protect their contents from the environment including rain, desiccation, honeydew contamination and natural enemies, and to provide an attachment to the substrate. It is more difficult to determine the functions of the loose dermal wax found on the adult male. In the male of P. citri, wax production is largely confined to the powdery wax along the pleura and to the caudal filaments which trail behind the body. Possibly it is merely a vestige from the immature stages, although the pores which produce the wax are distinctively different in the adults from those in the immature male instars. The long trailing wax filaments seem too well developed to be completely nonfunctional and may be an aid to balancing in flight.

Acknowledgements-We

thank the staff of the British Museum (&atural History) Photographic and Electron Microscope Units for assistance in the preparation of the photomicrographs, especially Mr D. Claugher for permission to use the photomicrograph shown in Fig. 19 and Mr J. H. Martin, British Museum (Natural History) for information on aphids. The mealybugs from Trinidad and Nigeria were reared under licence number PHF 135/19 issued under the Import and Export (Plant Health) (Great Britain) Order 1980 and the Plant Pests (Great Britain) Order 1980. REFERENCES V,Y. 1981. Redescription of Mafsucoccus josephi Bodenheimer and Harpaz (Homoptera : Coccoidea Margarodidae). Israel J. Enromol. 15: 35 - 5 I. BROA~BENT, L. 1951. Aphid excretion. Proc. R. Enromol. Sot. Land. (A) 26: 97 ~ 103. DAVIDSON, J. A. 1973. Scale insects. Newsl. Electron Microsc. Cent. Facil. Univ. Maryland 1: 4.

BEN-D•

:

248

JENNIFERM. Cox and M. J. PEARCE

FOLDI, 1. 1978. Ultrastructure des glands tegumentaires dorsales, secretrices de la “Laque” chez la femelle de Coccus hesperidum L. (Homoptera : Coccidae). Inr. J. Insect Morphol. Embryol. 7: I55 - 63. FOLDI, I. 1981. Ultrastructure of the wax-gland system in subterranean scale insects (Homoptera, Coccoidea, Margarodidae). J. Morphol. 168: 159 - 70. GERSON,U. 1980. Wax filaments on coccid eggs. Israel J. Entomol. 14, 81 - 5. GULLAN,P. J. 1979. The wax-exuding, cuticular pores of Apiomorpha rubsaamen (Homoptera, Coccoidea): A light microscopy and scanning electron microscopy study. Aust. Enromol. Mug. 6: 3 - 9. HAMON,A. B., P. L. LAMBDINand M. KOSZTARAB.1975. Eggs and wax secretion of Kermes kingi. Ann. Entomol. Sot. Amer. 68: 1077 - 8.

HASHIMOTO,A. and S. KITAOKA.1971. Scanning electron microscopic observation of the waxy substances secreted by some scale insects. Jpn. J. Appl. Entomol. Zoo/. 15, 76 - 86. LOCKE, M. 1974. The structure and function of the integument in insects, pp. 123-213. In M. Rockstein (ed.) The Physiology of Insecta. Vol. 6. 2nd edition. Academic Press, New York, London. MILLAR,D. R. and W. F. GIMPEL. 1974. Scale insects. Newsl. Electron Microsc. Cent. Foci/. Univ. Maryland 2: 5.

NIXON, G. E. J. 1951. The Associafion of Ants with Aphids and Coccids. Commonwealth Institute of Entomology, London. POLLISTER,P. F. 1937. The structure and development of wax glands of Pseudococcus maritimus (Homoptera, Coccidae). Q. J. Microsc. Sci. 80: 127 - 52. STRONG, F. E. 1967. Observations on aphid cornicle secretions. Ann Entomol. Sot. Amer. 60: 668 - 73. TAMAKI, Y., T. YUSHIMAand S. KAWAI. 1969. Wax secretion in a scale insect, Ceroplasles pseudoceriferus Green (Homoptera : Coccidae). Appl. Enfomol. Zool. 4: 126 - 34. WAKU, Y. and Y. MANABE.1981. Fine structure of the wax gland in a scale insect, Eriococcus Iagersrruemiae Kuwana (Homoptera : Eriococcidae). Appl. Enfomol. Zool. 16: 94 - 102. WAY, M. J. 1963. Mutualism between ants and honeydew producing Homoptera. Annu. Rev. Enlomol. 8: 307 - 44. WIGGLESWORTH, V. B. 1953. The Principles of Insecr Physiology. 5th edition. Methuen, London. WILLIAMS, D. J. 1978. The anomalous ant-attended mealybugs (Homoptera : Pseudococcidae) of south-east Asia. Bull. Br. Mus. Nat. His!. (Entomol.) 37: I - 72. WILLIAMS, J. R. and D. J. WILLIAMS.1980. Excretory behaviour in soft scales (Hemiptera : Coccidaeh Bull. Entomol. Res. 10: 253 - 7.