WAGENINGEN AGRICULTURAL UNIVERSITY PAPERS 97-1(1997)
A histological description ofth e
alimentary tract and related organs
ofPhylloxerida e (Homoptera, Aphidoidea)
by
M.B. Ponsen
Date of publication: 31Marc h 1997
0000 0750 0339
Wageningen EM Agricultural University
•8 Key words: Phylloxeridae, life cycle, histology, alimentary tract, salivary pump,mycetome , oenocytes, phylogeny.
M.B. Ponsen Department of Virology Agricultural University Wageningen The Netherlands
A histological description of the alimentary tract and related organs of Phylloxeridae (Homoptera, Aphidoidea) / M.B.Ponse n ISBN 90-73348-64-1 NUGI82 3 ISSN0169-345 X
Distribution: Backhuys Publishers, P.O.Box 321, 2300A H Leiden, The Netherlands. Telephone:+31-71-517020 8 Fax: +31-71-5171856 E-mail: [email protected]
All rights reserved Printed in The Netherlands
BBL!OTH:;I:K LANDBOUWUN;VIR.G ! n..rr WAGEN1NGKN Contents
Introduction 5 Life cycle 5 Materials and methods 19 Biological observations 19 Alimentary tract 26 Sexuales 43 Salivary pump 50 Mycetome and oenocytes 54 Discussion 58 Phylogeny 66 Summary 68 Acknowledgements 69 References 70 Abbreviations used in figures 77 Introduction
The generic name Phylloxera (Greek:phyllos =leaf ; xeros = dry) was given by Boyer de Fonscolombe (1834) to an aphid, which he found all over the underside of oak leaves in Province (France) producing "rust spots" which caused the drying out and shedding of the leaves. The family name "Phylloxériens" was introduced by Planchon (1868), but was later changed to Phylloxeridae at the International Congress of Zoology in Paris (Dreyfus, 1889b). This family consists of about 14 genera (Grassi et al., 1912) the species of which are so small that they are classified by Borner (1952) as "Zwergläuse". These minute aphids initiate galls on the aerial parts of plants, necrotic lesions on leaves and fruits, necrotic areas on the bark of stems, or swellings on roots and rootlets. The most noxious species, the grape louse Daktulosphaira vitifoliae (Fitch) (Table 1), produces both leaf galls and root swellings. In the second half of the last century this aphid devastated the vineyards in many countries, especially Europe. Within the family Phylloxeridae the various internal organs of a number of species has been investigated. The alimentary tract of Phylloxera florentina (Targioni Tozzetti, 1877), Phylloxera punctata (Lemoine, 1893; Dreyfus, 1894), Daktulosphaira vitifoliae (Krassilstschik, 1893a, b; Grassi et al., 1912; Breider, 1952; Gersch, 1953; Federov, 1959; Rilling, 1960), and Phylloxera coccinea (Dreyfus, 1894; Kunkel, 1966) consists of a pharynx, a very short foregut, a somewhat dilated transparent stomach, a very short tubular intestine, a transparent "hindgut" which shows vigorous peristaltic movements, but lacks Malpighian tubules. The purpose of this study is to investigate the anatomy of the alimentary tract of the Phylloxeridae. This information is used to determine the phylogenetic relationships of the phylloxerids within the superfamily Aphidoidea.
Life cycle
Excellent biological studies, morphological descriptions and illustrations of species of the family Phylloxeridae have been produced by many investigators whose results are summarized in Table 2 and 7. Their life cycles are depicted in Figure 1-3. They characteristically produce eggs parthenogenetically and do not defecate (Borner, 1929).
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The feeding by Daktulosphaira vitifoliae stimulates the leaves to produce a gall and the second instar is enclosed within by the developing gall. Inside the gall it grows, matures, and deposits its eggs which give rise to the fundatrigeniae (Figure 3 C). As observed by Shimer (1866a), the presence of the aphid is necessary for the development of the gall, and as soon as the aphid leaves, the gall ceases to grow. The fundatrigeniae are dimorphic, viz. gallicolae or leaf gall formers and radicicolae, which initiate root swellings. The former appear in great numbers when the vine is actively growing. The gallicolae remain on the leaf, either feeding in the maternal gall or founding new galls on the same or other leaves. The radicicolae migrate as larvae down to the roots before taking food and producing several parthenogenetic generations which remain in succession on the roots. Their feeding stimulates the roots to produce swellings (gall tissue) and stunts root growth. In all phylloxerid species (Table 2) the fundatrices as well as the fundatrigeniae are wingless parthenogenetic oviparous females (Figure 1-3). The latter give rise to three or more generations, dependent on the weather during the summer and food quality. The number of generations of both gallicolae and radicicolae of Daktulosphaira vitifoliae that develop during hot summers might be as many as eleven (Grassi et ah, 1912). In the life cycle of Acanihocherm.es quercus, Phylloxera deplanata, Phylloxera caryaecaulis, Aphanostigma ulmifoliae, Phylloxera russellae, Phylloxera perniciosa, and Phylloxera devastatrix (Table 2) the fundatrigeniae are lacking; in the first three species the fundatrix is the sexupara (Figure 1A and C), but in the last four species the first generation of the fundatrigeniae (Figure 1 B and D). The fundatrix (= sexupara) of Acanthochermes quercus matures inside the gall and crawls out of the gall and dies after rapidly depositing about 15 eggs on an oakleaf (Kollar, 1848). Fundatrices of Phylloxera caryaecaulis (Figure 1 C) deposit white eggs of two sizes, both with a smooth chorion: small eggs may give rise to the alate form that produces only males, and large eggs may be future female producers, or the difference in size may reflect normal size variation (Caldwell & Schuder, 1979). Inside the galls on Ulmus and Carya caused by Aphanostigma ulmifoliae and Phylloxera russellae, respectively, three morphs occur, viz. fundatrices, wingless 8 Wageningen University Agricultural Papers 97-1 (1997) m u u Q Q ta W < m w www NO ON o vo > 55 ON o •~ Z. <* o •-* ^ r^- W as O T3 as "S O as J3 ON T3 CS c/3. 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S a a a a S S sa S a s a se fc £ se I 1 g se y y s y -> a S g ^ §H.y .S « c H-SH «--Swas;g«„S^ r-, *» O »Q U *Ç -£r (i *î 2* tA *-» *- ^ »Q "— ^ g u I~ o H PL, o m a o 3 P •c -s: -c -s? a, a, a, a, Wageningen Agricultural University Papers 97-1 (1997) 11 m u -o o BH O „ r» „ t~ •£3 "0^> 00 a> SI N N N O c o H 'E H c e c o JoS •?o„ 1 1 'S £5 5 o <>l E< s; B. S ^3 O o H JS o co E '5ö i—' 13 •S ca; C5r. 3 S -e Phylloxerina the sexuparae are wingless, but in Daktulosphaira vitifoliae, Moritziella corticalis, Phylloxera caryaecaulis, Phylloxera castanea, Phylloxera devastatrix, Phylloxera perniciosa, Phylloxera releyi, and Phylloxera texana they are winged. Both sexuparous morphs occur in Phylloxera coccinea, Phylloxera glabra, Phylloxera spinulosa, Phylloxera florentina, and Phylloxera quercus (Figure 1-3). The winged sexuparae of the last two species and those of Phylloxera castanea and Phylloxera texana return Wageningen Agricultural University Papers 97-1 (1997) 13 \ ï 1 1 t 9 9 9 9 9 9 A 1 A 1 t <=> C=Z> C=T> o CZZ> CZD C=D c=> 1 t 1 11 1 1 cf9 9 t f f 9 9 1 /\ 1 \ A 1 ^m o CZZ3 o < > <= cri CTD CZD 1 1 1 1 1 1 1 c? 9 cf9 CT 9 9 9 1 1 1 1 14 Wageningen University Agricultural Papers 97-1 (1997) 1 1 \ i 9 9 9 9 1 \ /\ 1 CZD <=ZD C=> CZD CZD t ! 1 1 1 9 9 9 f f 1 1 A 1 l CZD czz> c= CZZ> « > CZD \ t 1 1 1 1 9 9 cf 9 9 9 1 1 1 1 1 CZ3 <—> ^m •—i CZD 1 \ 1 1 9 9 9 9 /\ /\ 1 1 => <—> CZD CZD CZ3 CZD \ \ 1 1 1 1 9 f 9 f 9 f \ A A A A A ZZ3 CZ5 CZD o < > o ' > <=> CZD <=> CZT 1i 1 1 \ 11 1 1 1 1 9 cf 9 cf 9 cf 9 cf 9 cf 9 l 1 1 \ \ 1 =D ^m ^m ^m «•» ï 9 Figure 2. Schematic representation of the life cycle of Moritziella corticalis and Phylloxera rileyi (A), Phylloxeracoccinea an d Phylloxera glabra(B) ,Phylloxera notabilis(C) , Phylloxera texana and Phylloxeracastanea (D) . The winter eggs are laid in October and the fundatrices emerge in the following May.Th e letters A-D correspond with those inTabl e 2. See legend toFigur e 1. Wageningen Agricultural University Papers 97-1 (1997) 15 1 1 1 9 9 9 /\ / \ 1 <—> <—> c=> '—> «—» i * 1 1 i 9 t 9 t 9 \ 1 1 1 / \ C=D CZZ5 CZ3 CZZ> C=3 C=3 t 1 1 1 1 1 9 9 9 9 9 9 1 1 1 1 1 1 CZZ3 CZZ3 CTD \ 1 1 9 9 9 Figure 3. Schematic representation of the life cycle of Phylloxera quercus (A), Phylloxera florentina and Phylloxeraspinulosa (B), and Daktulosphaira vitifoliae(C) . The winter eggs are laid in October and the fundatrices emerge in the following May.Th e letters A-C correspond with those inTabl e 2. See legend toFigur e 1. 16 Wageningen University Agricultural Papers 97-1 (1997) from the summer host to the winter host (Table 2). The remaining winged sexuparae fly to branches of the same oak or to other oaks of the same species. The sexuparae of Daktulosphaira vitifoliae complete their larval stages on the roots of vine, emerge from the soil, and fly to the aerial parts of vines to lay their eggs. In the genera Aphanostigma and Phylloxerina winged morphs do not occur in the fundatrigeniae and in the sexuparae (Table 2). The eggs of the sexuparae are of two kinds, viz. the smaller give rise to males and the larger to females. Daktulosphaira vitifoliae produce more small eggs than large ones, but the latter hatch more rapidly than the former (Borner, 1908). On the other hand, the sexuparae of Phylloxerina produce three times more female eggs than male eggs (Iglisch, 1965). Among the sexuparae there are three morphs, viz. those that produce only small eggs (androparae), or only large eggs (gynoparae), or both small and large eggs. The latter morph occurs in all phylloxerid species, except Phylloxera caryaecaulis and Phylloxera devastatrix which only have androparae and gynoparae (Table 2; Figure 1C) , whereas in Daktulosphaira vitifoliae (Figure 3 C) all three morphs are present (Grassi, 1909b). All sexuales, summarized in Table 7, lack stylets, but in Southern Russia about 9% of the Daktulosphaira vitifoliae sexuales have well-developed mouthparts and are able to feed on the roots of vine (Nikolaev & Ass, 1973). According to Leclant (1963) and Geoffrion (1971) the larval sexuales of Aphanostigma piri moult four times within the egg, after which the larva is surrounded by four exuviae pressed together against the inner wall of the egg. The larvae are immobile, lack mouthparts, and the legs and antennae lie flat against the body. Within the egg of the three species of Phylloxerina (Iglisch, 1965; Table 2) the four exuviae are firmly telescoped into each other at the posterior end of the immobile, styletless larva. On the other hand, in the pecan phylloxerids, Phylloxera caryaecaulis, Phylloxera caryaevenae, Phylloxera deplanata, Phylloxera devastatrix, Phylloxera notabilis, and Phylloxera russellae the eggs hatch into sedentary, pupiform larvae which lack mouthparts, often stand vertically on the substrate, and make undulating movements as they moult. Each pupiform larva moults four times, and the exuviae pile up in circular layers at the posterior end of the abdomen (Stoetzel, 1981, 1985c). The larvae do not grow during their larval instars and after the last moult the adult sexuales are produced and mating occurs soon after. They are wingless, dwarfish individuals, which lack mouthparts and consequently do not feed. The male is smaller than the female and both morphs are active (Table 7), except the female sexuales of Wageningen Agricultural University Papers 97-1 (1997) 17 Phylloxerina, which areplum p and immobile (Pergande, 1904;Iglisch , 1965). The sexuales of Daktulosphaira vitifoliae are able to mate directly (Lichtenstein, 1878), or about five days (Moritz, 1908) after shedding of their eggshell. Males copulate with a maximum of three females and soon after this he dies. After mating the female seeks out a crevice in the bark and lays one egg, which is almost as large as the female. The female sexuales of Daktulosphaira vitifoliae (Kirk, 1897) and Aphanostigma piri (Leclant, 1963) deposit their egg with violent contractions of the mother's body, and die alongside the egg. The winter egg of Daktulosphaira vitifoliae expands slightly after having been laid (Kirk, 1897). In Phylloxerina (Iglisch, 1965) and the pecan phylloxerids (Stoetzel, 1985c) the egg retains within the body which then shrivels around the egg (see page 22). Overwintering eggs are oval, yellow when first laid and then darken to black. The unfertilized winter eggs shrivel within a few days (Borner, 1908). The winter eggs of Daktulosphaira vitifoliae (Lichtenstein, 1878), Phylloxera coccinea, Phylloxera foae, Phylloxera glabra (Borner, 1909), Phylloxerina (Iglisch, 1965), and Aphanostigma piri (Geoffrion, 1971) are attached to the bark by means of apedice l (seepag e22) . In general the overwintering eggs are laid in autumn and do not hatch until the following spring. The sexual females of Acanthochermes quercus, Phylloxera deplanata, Aphanostigma ulmifoliae, Phylloxera russellae, Phylloxera caryaecaulis, Phylloxera perniciosa, and Phylloxera devastatrix deposit their winter eggs at the beginning of summer (Figure 1 A-D). The number of eggs produced by the parthenogenetic females of Phylloxera quercus and Daktulosphaira vitifoliae diminishes with each generation. This is associated with an increase in the number of atrophied ovarioles. The fundatrix of Daktulosphaira vitifoliae produces about 450 eggs, the fundatrigeniae from 250 to 50 in successive generations, to 2-4 eggs for the sexuparae. The sexual female laysjus t one egg produced in a single ovariole, the counterpart of which having atrophied (Balbiani, 1884).Accordin g to Riley (1874) the Phylloxera species, with which Balbiani worked in Paris, is not quercus because "the tubercles which are characteristic of the species in Southern France are entirely wanting around Paris". The fundatrigeniae of Phylloxera releyi (Riley, 1875), Aphanostigma piri, Moritziella corticalis (Borner, 1908, 1909; Cheng and Yeh, 1992), Phylloxera florentina, Phylloxera spinulosa (Grassi and Foà, 1911), and the gallicolae and radicicolae of Daktulosphaira vitifoliae may hibernate as first and/or second instar larvae in crevices in the bark of the trunk, branches, or roots of their hostplants, or in the 18 Wageningen University Agricultural Papers 97-1 (1997) galls on the tendrils and limbs of vine (Shimer, 1866a; Planchon & Lichtenstein, 1869b; Carrière, 1888; Federov, 1959). The over wintering larvae mature the following spring, and give rise on the leaves and roots to a succession of parthenogenetic generations of fundatrigeniae, gallicolae, and radicicolae, during summer and autumn (Table 2; Figure 1-3). Materials and methods Specimens of the species listed in Table 3 were collected from the hostplants and put in Duboscq-Brasil's fluid. After fixation the aphids were dehydrated in a graded series of ethanol and in methyl benzoate, stored in methyl benzoate celluidin (2%) for three days or longer, and then in toluene and finally embedded in paraplast. Serial sections, 5-8 urn thick, were stained in 0.5% methylgreen aqueous solution, rinsed in tap-water, dehydrated in methanol and in methyl benzoate, cleared in xylene, and finally mounted in xylene-dammar. The sections were examined under a Wild phase microscope; the drawings were made with the aid of aWil d drawing tube. The morphology of the internal organs was reconstructed from the drawings of serial sections of a whole aphid viewed at a magnification of 600 times. The number of nuclei with their conspicuous big nucleoli, which correspond to the number of cells, were counted at a magnification of 1500 times. The length of the aphid and that of the digestive system was calculated by multiplying the number of serial sections by the thickness of each section. In order to dissect the internal organs, aphids were placed on double sided self-adhesive tape attached to ablac k plastic plate. Under a Carl Zeiss dissecting microscope (magnification 80x) each aphid was covered with a drop of Levy solution and dissected using watchmakers forceps. Biological observations In spring and during the summer of 1986 all Quercus robur oak trees in Wageningen were heavily infested with Phylloxera coccinea (Table 3). After hatching from the overwintering eggs the larvae settles at the margins on the underside of young growing leaves and starts to penetrate the parenchymous tissue close to a vein. Their feeding initiates a yellow spot in the parenchymous tissue and hyper- Wageningen Agricultural University Papers 97-1 (1997) 19 m u u u BO c 60 E X < et) «2 m SON E cSO\ < ex C3 VI <3\ k : 5 Uoo ofi 2 ö §" c •j^ CO i—l >> Sa o (j . •ad " 3 X a «Of Î2 C3 O 60 ao DO O « 8 8 s sa 5 'S ia •o !• o. 1t3u T3 o. o. e X 3 S C &3 3 eu 3 C/} (D X ,3 ,3 X CA X 3 c-t—1 > tM (U 00 tu tt-i tu e XI CU O CP cd «3 •s bß CO 4-T •v cd O tu O CD O t 3 m U oi PQ C3 c o c •o > £f •c o'> k. CU C X 5 S3 -0 eu tu jo & 5 S st O s<*1 ">. •S = • C s g J3 S S > S o ta •S •s.g O S •ïî •ï: et) s BC £ Es ^ S^U 3 X tu -C cod 5 O o X < oc E Ü eu tu tu cd Ç3 •S .t3 & CU «S ,t-3 O. -s •S?o p. c a. •s < H I I 20 Wageningen UniversityAgricultural Papers 97-1 (1997) < M (Tl r-H -0 en .0 Os OS (D h ta OS > Os © VO ON Ov OO Os ö _aS 'fi 'H S o. ta D. 3 -Co X S U <£ CA en CA CA Q. CA M O U O « ^ ü 60 >* 'M G c ü "5b C >- S c c £ '£ £ £ PQ AS ^ ^A o 1C 2 T3C « C « o •S ,3 t<-i ^ 3 S S "-o3 E3 & tu •S3 s "3" S 3 O) 3 Ol com c 0-. .JH o a se •J o>> 'C!»> ^—'ta « 3 g a §un CJ î3 tj t. c tu >U . s "S3 o. 2 <2u ^8 O1 ^H X .g _§ -2 Ü ^B -;*-c , -^C •S O a, Q- a. a. a. Pu Wageningen Agricultural University Papers 97-1 (1997) 21 trophy of the vein. Then the leaf margin folds back towards the underside forming a small fold gall with a diameter of about 4 mm. After the fourth moult the gall is completely folded and harbours one adult pyriform fundatrix. She lays many smooth, oval-shaped eggs in an irregular scattered pattern or in two to four half-concentric circles close together around the feeding puncture. After death of the adult the green fold gall becomes brown to black and shrivels finally. If the larva stops feeding or dies during gall development, the gall stops folding and shrivels. The fundatrices of Phylloxera glabra also initiate a fold gall on Quercus robur (Kunkel, 1967). Directly after hatching the larvae leave the gall and scatter over the underside of the leaves. They moult four times and after the first moult they change from oval or suboval to pyriform in shape. The adult fundatrigeniae are smaller than the fundatrices (Figure 9). They lay their smooth, oval-shaped eggs oneb y one in acircl eb y turning on their stylets which are inserted into the plant. If the circle of eggs is closed, she deposits a second circle of eggs around the first or she leaves the place, inserts her stylet at another site and deposits a new circle of eggs. Their feeding stimulates the leaves to produce yellow spherical spots, so that the dark green leaves become speckled in appearance. If the fundatrigeniae stop feeding or leave, the yellow spots gradually change into brown necrotic areas. Winged individuals are never observed in spring, indicating that Phylloxera coccinea does not have a secondary hostplant. Wax coating honeydew droplets, as inside the galls of the Adelgidae and Pemphigidae, are never observed in the fold galls. The oak leaves do not show any trace of honeydew, and the aphids are not attended by ants. As reported earlier the wingless fundatrigeniae of Phylloxera coccinea, Phylloxera glabra (Heyden, 1837; Kaltenbach, 1867; Kunkel, 1967; Barson & Carter, 1972), Phylloxera rileyi (Riley, 1874), Phylloxera quercus (Balbiani, 1874), Phylloxera punctata (Lichtenstein, 1877), and Phylloxera kunugi Shinjii (Miyazaki & Teramoto, 1991) deposit their eggs singly around the feeding site on the underside of oak leaves and their feeding initiates the formation of necrotic spots. Phylloxera florentina deposits its eggs on the winter- host in the same way as described above, but on the summerhost along the midrib on the underside of the leaves (Targioni Tozzetti, 1875). Phylloxera texana produces only one generation of galls per year on the leaflet surface of Carya illinoinensis, whereas feeding may cause discolouration and depressions on the leaves of several species of Quercus (Stoetzel, 1985b) (Table 2). Late in July of 1986 the leaves of a large number of trees were 22 Wageningen University Agricultural Papers 97-1 (1997) completely brown and dried out due to the multitude of necrotic lesions. Only very young, heavily infested trees were killed. Phylloxera coccinea was only found on Quercus robur, never on Q. cerris, Q. frainetto, Q. macon, Q.palustris, Q.petraea, and Q. rubra. All these species grew in the immediate neighbourhood of the Q. robur infested with Phylloxera coccinea. Observations during the autumn of 1993 in a heavily infested oak show that unlike the fundatrigeniae, the wingless sexuparous adults of Phylloxera coccinea leave the leaves and wander as white dots on the brown branches. The winged sexuparae migrate from the leaves to the branches of the same oak or to other trees of the same species. Presumably the sexuparae do not feed in the adult stage because in the majority of the sectioned adults their stomach lumen is less filled with fine-grained material or empty compared with the fundatrices and fundatrigeniae in which the stomach lumen is completely filled with fine-grained material (see page 32). According to Blankenhorn and Moritz (1875) and Kirk (1897) the sexuparae of Daktulosphaira vitifoliae stop feeding after the third moult, leave the roots and come up to the surface of the soil, crawl up the stem and after the final moult they fly to the aerial parts of the vine. At the end of the summer the sexuparae lay their eggs scattered in clusters of one to four eggs on the underside of the leaves and on the inner surface of the scales of the apical bud of last year. The smooth, white, and oval-shaped eggs are of two sizes: small eggs and large eggs with a ratio of 2 to 3, of which the former produce the males and the latter the female sexuales. The eggs are firmly attached to the leaves and scales with their posterior end. When the eggs are about three days old, the egg-shell, embryonic cuticle, and first instar cuticle are shed partly posteriad up to the sixth abdominal segment. Within about five days three more moults follow. The first instar larva positions its three pairs of legs and one pair of antennae immobile against the body; one pair of red-pigmented triommatidia is visible, but the larva lacks the moufhparts. During the fourth moult the sexuales raise and stretch their body several times perpendicular on their cauda (see Figure 18-3 E). During this process the cuticle breaks open in front of the head and the adult leaves its old cuticle with undulating movements of its flexible abdomen. Thereby, first the antennae are liberated and start to move, and subsequently the three pairs of legs. A part of the egg-shell (like an "egg-cup") remains behind firmly attached to the leaf or bud scale. The shrivelled exuviae are left attached to the inner wall of the posterior part of the egg-shell. When the egg is not firmly attached to the substrate, the sexuales are not able Wageningen Agricultural University Papers 97-1 (1997) 23 to emerge from the final cuticle and die. One bud scale was observed to have about 21 empty "egg-cups" on its inner surface. The empty "egg-cups", from which the sexuales of Daktulosphaira vitifoliae and Phylloxera quercus emerge, illustrated and named "les Pupes" by Lichtenstein (1878), suggest that these sexuales moult in the same way as those of Phylloxera coccinea. According to Moritz (1908) the sexuales of an oak Phylloxera become active a few days after shedding their egg-shell. The immobile larvae do not grow during their development. Soon after emerging from the final cuticle the adult sexuales mate and wander as minute wingless dots on the brown branches among the much bigger sexuparae. The males are light brown and somewhat smaller than the white transparent female sexuales. The latter have only one opaque white egg which during the adult life increases in size until it occupies nearly the whole body (Figure 15 N). Then the body of the female shrivels around the egg and disappears, but between the egg and the bud scale the shrivelled remnants of the legs and a part of the head with the antennae and triommatidia are still present. The cylindrical white winter egg is nearly twice as large as the egg from which the female sexuales emerge. The egg-shell has a very fine crenated structure. The anterior pole of the egg has a small, knob- shaped organ, presumably the germarium, which is firmly attached to the egg, but not to the bud scale. The eggs of the remaining morphs of Phylloxera coccinea lack this organ. The winter egg gradually darkens toblac k and does not hatch until the following spring. The knob-shaped organ is shriveled or has disappeared. The pale orange colour of the wingless morphs of Phylloxera coccinea is caused by the presence of dark orange spots inside their transparent bodies. These spots are the oenocytes, which are lacking in the transparent sexuales (see page 54). Both Phylloxera coccinea and Moritziella corticalis may feed on the same oak tree, viz. the former on the leaves and the latter on the trunk and branches in minute bark cavities. The eggs are laid singly under flakes of bark, in crevices, and under and between lichens growing upon the trunk. Winged sexuparae of Moritziella corticalis are only observed feeding on the thin bark of young oaks, never on old ones. Probably this species hibernates on young oaks as a winter egg, and on old oaks as first-instar larvae of the fundatrigeniae (Figure 2 A). In one instance Lady-bird beetles on the bark of a young oak indicated the presence of a colony of Moritziella corticalis. Colonies of this species are found in cracks in the bark of 8 to 50 years old Quercus robur oaks, as well as 12 to 18 years old oaks (Kaltenbach, 24 Wageningen University Agricultural Papers 97-1 (1997) 1867). As a result of a heavy infestation of Moritziella corticalis the bark of three oaks {Q. robur) of about 8 years old in the arboriculture at Lienden (Table 3) developed black cankerous spots and swellings that were filled with a clear liquid. A multitude of these swellings causes a spongy bark by which some areas of the bark release (Barson & Carter, 1972) resulting in the death of the tree. Similar black swellings are also caused by the feeding of Daktulosphaira vitifoliae on the roots of Vitis vinifera (Westwood, 1869; Riley, 1870; Blankenhorn & Moritz, 1875; Cornu, 1878; Millardet, 1898), Aphanostigma iakusuiense (Tanabe & Mishima, 1929) and Aphanostigma piri on the stem, branches, and fruit of Pyrus communis (Leclant, 1963; Geoffrion, 1971;Chen g & Yeh, 1992),Moritziella castaneivora on the burs of Castanea crenata (Miyazaki, 1968), and Phylloxerina Salicis on the stem of Salix alba. Phylloxerina Salicis has been found on Salix alba at Halle and Lienden (Table 3). At Halle the cracks in the bark of the bole were so heavily infested by this species, with its fine white waxy threads, that the bole had the appearance of a zebra. On some areas of the bole the bark was transformed into a black spongy mass, which came off without damaging of the tree. The dense colonies in the bark fissures consist of fundatrigeniae, sexuparae, and sexuales. The sexuparae have fewer wax gland plates and consequently produce less wax than the fundatrigeniae. The sexuales are very small and not coated with wax threads (Iglisch, 1965), like the sexuales of Pemphigus venafuscus (= Prociphilus americanus (Walker)) (Patch, 1909), Eriosoma pyricola (Baker & Davidson, 1916), Eriosoma lanuginosum (Fluiter, 1933), and Prociphilus (Stagona)xylostei (Heie, 1980). An interesting phenomenon was the absence of wax-coated honeydew droplets of various sizes among the wax threads of Phylloxerina Salicis. In contrast, honeydew droplets were observed among the wax threads of the wingless viviparous females of Phloeomyzus redelei Hille Ris Lambers feeding in dense colonies in the fissures of the bark of Populus nigra (Doom & Hille Ris Lambers, 1962) in the Millingerwaard (V.1993) at Kekerdom (Gelderland). The leafgall-inhabiting form of Daktulosphaira vitifoliae has been found in France on a cultivar of Vitis vinifera (Table 3). These galls contained one or two adults, young larvae, and eggs as previously observed by Fitch (1854), Shimer (1866a), and Planchon and Lichtenstein (1869b), as they do there on Caryacause d by Pemphigus caryaecaulis (= Phylloxera caryaecaulis), Phylloxera caryae-scissa, Phylloxera caryae-avellana (Fitch, 1854;Riley , 1880), and Phylloxera Wageningen Agricultural University Papers 97-1 (1997) 25 devastatrix (Stoetzel, 1985b). Alimentary tract In all phylloxerid morphs (Table 3), except the sexuales (see page 41), the ectodermal part of the anterior region of the alimentary tract consists of the stylets, pharynx, foregut, and the oesophageal valve. The alimentary tract starts with the food canal, which is formed by the interlocked maxillary stylets (Figure4) . The piercing and sucking organ of the phylloxerids consists of four stylets. Each of them originates from a retort-shaped organ situated in the posterior part of the head. The interior of these glands is made up of a matrix of cells that secrete the chitinous substance, which is formed into a new stylet at each larval moult. The two pairs of retort-shaped organs are previously reported for the radicicolae of Daktulosphaira vitifoliae (Krassilstschik, 1892, 1893; Grassi et al., 1912) and for Phylloxerapunctata (Dreyfus, 1894). From these organs the stylets run ventral to the pharynx (Figure 5 G and 6 C) and converge in the chitinous cavity of the epipharynx on the ventral face of the clypeo-labrum and labium to form the stylet bundle. The stylets fit snugly into the cavity, and are held firmly in position yet have freedom of movement for protraction or retraction. After leaving the head the stylet bundle crosses over into the longitudinal labial groove and runs along it to the extremity of the rostrum. In Daktulosphaira vitifoliae the stylet bundle shows in its way a torsion of 180° (Rilling, 1960). The stylet bundle consists of one pair of mandibular and one pair of maxillary stylets. The latter are firmly interlocked enclosing two canals formed by opposing grooves.Th e mandibular stylets are closely applied to the maxillary pair, as illustrated by Geise (1888) for the radicicolae of Daktulosphaira vitifoliae. The dorsal or food canal communicates with the pharyngeal duct, and the ventral one joins the efferent salivary duct. Moreover, within each mandibular stylet is a minute canal running from the base to the tip (see Figure 3 in Rilling, 1960). There is also a minute canal within each maxillary stylet, but this canal ends before entering into the cavity (Figure 5 G and 6C) . According to Foà and Grandori (1908) and Janicki (1908) the stylets of the radicicolae are longer than those of the gallicolae, varying between 122 and 163 urn for the gallicolae, between 190 and 215 urn for the spring radicicolae, and between 150an d 170\xm for the overwintering radicicolae. Moleas et al. (1992) found that the length of the stylets in seven generations of gallicolae varied from 90 to 160 26 Wageningen UniversityAgricultural Papers 97-1 (1997) ego phd Figure 4. Sagittal section of the head of anadul t winged sexuparous Moritziella corticalis showing the 14pair s of muscles of the pharynx (M4) and the three pairs of retractor muscles of the salivary pump (M17).Not e the situation of the sensillary pores in the epipharynx, pharyngeal duct, and at the foot of the pumpstem. Barrepresent s 10p.m . For list of abbreviations seepag e77 . Wageningen Agricultural University Papers 97-1 (1997) 27 um. The stylets of the fundatrigeniae of Phylloxerina capreae and Phylloxerina Salicis have an average length of 294 urn and 355 urn, respectively (Borner, 1942). There are two muscles attached to the basal end of each stylet: a retractor muscle on the inner side originating from the tentorial bar (Ml5 and M16), and a protractor muscle (Ml3 and M14) inserted on the outer side of the stylet and originating from the ventral wall of the maxillary sclerite. More proximally on the outer side of the stylet another muscle is inserted originating from the lateral (M8 and M9, Figure 6 C) wall of the maxillary sclerite. On the inner side there are muscles attached to the pharynx (M23 and M24, Figure 5 G) floor (Figure 8 and 13 in Ponsen, 1972). The skeleton and musculature of both the gallicolae and radicicolae of Daktulosphaira vitifoliae were studied by Grassi etal. (1912) and in detail by Rilling (1960). According to Millardet (1898) the radicicolae of Daktulosphaira vitifoliae take their nourishment from the cortex of the roots and not from the vascular tissue cells. The stylets penetrate intracellularly through the cortex and parenchyma of vine roots (Petri, 1909) and leaves (Koch, 1952). Moreover, the radicicolae can feed directly on callus tissue cultures indicating that they do not need the sieve tubes for their nourishment (Rilling & Radier, 1960). Phylloxera coccinea and Phylloxera glabra ingest their food mainly from the non-vascular tissue cells (Kunkel, 1967) and Phylloxera devastatrix larvae feed in the phloem cells by puncturing between cuticular cell walls (Hedin et al, 1985). The food canal leads into the pharyngeal duct, which is formed by the epipharynx and the lip of the hypopharynx (Figure 4). This portion does not exert any sucking action, simply forming a duct, which conveys the plant-sap to the pharyngeal pump. The pharyngeal duct is separated from the pharyngeal pump by a valve, of which both the dorsal and ventral walls are marked by two cuticular dome-shaped prominences (Figure 5 and 6). These structures are present in Daktulosphaira vitifoliae and named "Protuberanzen" by Krassilstschik (1892), "Naroïden" by Dreyfus (1894), "Bottom" by Grassi etal. (1912), and "Mundknöpfe" by Rilling (1960). The dorsal wall of the pharyngeal valve is controlled by two pairs of divaricator muscles, each pair being attached to a short tendon. The muscles diverge from each side of the tendon and are attached bilaterally of the clypeo-labrum. On the lateral side of the valve a muscle (lateral pharyngeal valve muscle, M10) is inserted, which originates from the lateral wall of the clypeus. In a closed position the pharynx protuberances and the valve fit closely together (Figure 5 E-G 28 Wageningen UniversityAgricultural Papers 97-1 (1997) Figure 5. Oblique section of the pharyngeal system (A) of a winged sexuparous Phylloxera caryaecaulisan d the pharyngeal duct with two hypopharyngeal sensillary pores (B) of a wingless fundatrigenious Aphanostigma piri. Note the 8 sensillum pores in the epipharynx, the 6 pores of the pharyngeal valve, and 2 hypopharyngeal sensillary pores. Transverse sections of the pharyngeal duct (C) and the pharyngeal duct anterior to the valve (D) of a brachypterous Phylloxera punctata, the pharyngeal valve in an open (E) and closed position (F), and the pharyngeal pump posterior to the valve (G) of a wingless fundatrigenious Moritziella castaneivora,th e anterior region of the pharyngeal pump in an open (H) and closed position (I), the posterior region of the pharyngeal pump (J) of a radicicolous Daktulosphaira vitifoliae,an d the pharyngeal valve (K) of a wingless viviparous Fordamarginata Koch (Family Pemphigidae). Barrepresent s 10urn .Fo r list of abbreviations see page77 . Wageningen Agricultural University Papers 97-1 (1997) 29 and 6C) . The pharyngeal pump extends from the valve across the middle region of the head through the oesophageal connectives and joins the foregut in front of the tentorial bar. The movement of the flexible dorsal wall of the pump is controlled by 12 pairs of muscles (Figure 4). In the anterior region they are attached to 10 short tendons arising from the midline of the dorsal pump wall. The muscles diverge from each side of the tendon and have their origin on each side of the mediodorsal line of the wall of the clypeus (Figure 5 H-I). Both in the middle and posterior regions of the pump one pair of fan-shaped muscles is attached to the edges of the flexible dorsal wall and originates from the junction of the clypeus and epicranium, and the middle region of the epicranium, respectively (Figure 5J) . Only the 10pair s of muscles that are inserted on the dorsal wall of the pharyngeal pump via a cuticular tendon are responsible for the pumping action. Contraction of these muscles pulls the invaginated dorsal wall (Figure 5 H) upwards. During this process, the lumen increases in size and the reduced pressure draws plant-sap into the pharynx lumen by opening of the pharyngeal valve. When the muscles relax, the dorsal wall springs back to expel the sap into the foregut and the pharyngeal valve is then closed (Figure 5I) . Close to the tentorial bar, muscles are attached to the floor of the pharyngeal pump: one pair originates from the tentorium (ventral pharyngeal pump muscles), and one pair from the salivary pump (retractor muscle of salivary pump, M17) (Figure 4 and 5J) . The epipharyngeal gustatory organ has 14 sensillary pores. Eight pores are arranged in a row in the epipharynx of the pharyngeal duct (Figure 4, 5 A, and 6 A) as previously reported for Phyllloxera punctata (Dreyfus, 1894) and in both gallicolae and radicicolae of Daktulosphaira vitifoliae (Grassi et ah, 1912; Rilling, 1960). Six sensillary pores are located in the dorsal wall of the pharyngeal valve, three on each side of the two insertions of the short cuticular tendons to which the divaricator muscles of the pharyngeal valve are attached (Figure 5 A and 6 A). These six pores are illustrated by Grassi et al. (1912) in a drawing of the pharyngeal valve of Daktulosphaira vitifoliae. The hypopharyngeal gustatory organ has four sensillary pores: two in the hypopharynx of the pharyngeal duct just anterior to the valve (Figure 5 B and D) and two at the foot of the salivary pumpstem (Figure 4 and 17).Th e location of the 18 sensillary pores is identical in all aphid species (Ponsen, 1991). The pharyngeal pump passes into the digestive system which consists of the foregut, midgut, and rectum, and terminates at the anal opening. The midgut is composed of the stomach, crenated intestine, 30 Wageningen University Agricultural Papers 97-1 (1997) r- CS CO q •* CN ^ q ^ TT, es Ov ö es' ^H ^H •* ^H av O VO Ov VO O (N Ov OO r- o es «asS CO in CM VO CS i3c CO TT TT Se VO ta « S es „su es. ' +i u • ' ' ' ' ' ' ' ' ' ' ' es 'Cx S> ° in JfSjä Ov M o 00 OV VO ^r h G M H u u « O 2 2 B 'S '8 tù .s e^: 60 cd 60 60 O o o (U eu cd to Wageningen Agricultural University Papers 97-1 (1997) 31 and caecal intestine. The total length of the digestive system is about one and a quarter that of the aphid's body length (Table 4; Figure 18- 2). In dissections of living specimens of Moritziella corticalis, Phylloxera coccinea, and Phylloxerina Salicis (the remaining species in Table 3 were not dissected), both the stomach and the caecal intestine are relatively big transparent structures, which show vigorous peristaltic movements. The very short tubular crenated intestine is opaque and nearly completely covered with white dots that are the triplets. The foregut, stomach, and the crenated intestine have only circular muscles; movement of the caecal intestine is generated by circular and four longitudinal muscles, the latter are external to the circular muscles. The foregut (oesophagus) runs posteriad from the tentorium and terminates in the oesophageal valve (Figure 4 and 7 A). Of all aphid species investigated, the phylloxerids have the shortest foregut. Its length is about one tenth of that of the foregut of Myzus persicae (Sulzer) (Table 4). This thin tube is made up of squamous epithelium of which the nuclei protrude into the narrow lumen. The oesophageal valve is a short invagination of the foregut into the stomach. It consists of two layers of simple epithelium, which are continuous with each other. The epithelium of the inner layer is a continuation of that of the foregut, whereas the outer layer consists of cuboidal cells each of which has a relatively large spherical nucleus. The cells of both the foregut and the oesophageal valve secrete a chitinous intima. The two cell layers of the valve are separated by an intravalvular space which lacks muscles (Figure 7G) . Just before the stomach the foregut is supplied by a short nerve originating from the medial dorsal nerve, which runs alongside the dorsal vessel (Figure 4). Working with Daktulosphaira vitifoliae, Anders (1961) concluded that amino-acids in the stomach fluid are responsible for the formation of the leafgalls. Presumably this nerve regulates the oesophageal valve so that fluid from the stomach can be regurgitated into the non-vascular tissue cells of the host. An oesophageal valve has been reported in gallicolae of Daktulosphaira vitifoliae (Gersch, 1953) and Phylloxera coccinea (Kunkel, 1966), but is absent in radicicolae of Daktulosphaira vitifoliae (Krassilstschik, 1893),Phylloxera punctata (Lemoine, 1893), and Phylloxera coccinea (Dreyfus, 1894). The midgut is the endodermal part of the alimentary tract and consists of stomach, crenated intestine, and caecal intestine. The stomach is the biggest organ of the alimentary tract and in length is about half that of the aphid's length, and as long as the stomach of Myzus persicae (Table 4). It runs posteriad from the tentorium, 32 Wageningen University Agricultural Papers 97-1 (1997) Figure 6. Oblique section of the pharyngeal system of an adult fundatrix of Phylloxera coccinea (A) and a wingless oviparous Israelaphiscarmini Essi g (Family Greenideidae) (B). Note the 14 pairs of pharyngeal pump muscles of P. coccinea and the 30 pairs of I. carmini. Transverse section of the pharyngeal valve of a gallicolous Daktulosphaira vitifoliae(C) . Bar represents 10 urn.Fo r list of abbreviations seepag e77 . Wageningen Agricultural University Papers 97-1 (1997) 33 between the accessory salivary glands in the prothorax, between the principal salivary glands in the meso- and metathorax, and dorsal to the suboesophageal- and thoracic ganglia. At the end of the thoracic ganglion in the anterior part of the metathorax, the stomach gradually bends ventrally and subsequently joins the crenated intestine in the first, second, third, or fourth abdominal segment (Table 5;Figur e 8A) . The transition from the stomach to the crenated intestine is marked by a sharp loop, which is situated either to the right or to the left of the stomach (Figure 8-12). At the end of the stomach the two lateral oviducts unite into the common oviduct, which terminates at the gonopore. In the Adelgidae (Figure 17 in Ponsen, 1991) and Phylloxeridae (Figure 8-12) the stomach is situated ventral to the caecal intestine, but in the other families of the Aphidoidea the stomach is dorsal to the caecal intestine. The stomach is tubular or more or less dilated in structure and consists of two types of cells. The first type of cells is situated in the anterior region of the stomach and varies from 13 to 15 cells. These small columnar cells do not show any cytological evidence of secretory activity (Figure 7 and 13E-F) .Th e remainder of the stomach is made up of relatively large triangular cells, which vary from about 40 inMoritziella corticalis to 87 inPhylloxera caryaecaulis (Table 6). The triangular cells have small vacuoles and granules some of which are situated inside vacuoles. The spherical to oval nuclei contain one or more nucleoli, which have small spherical cavities (Figure 7 and 13 E-F). The basal cell membrane is strongly invaginated and the apical cell membrane presents a labyrinthine system of closely packed irregular invaginations of different dimensions (Figure 5 B and 19 C in Ponsen, 1991). The triangular cells produce a merocrine secretion, which involves breaking off of parts of the labyrinthine system. These cells continue secreting throughout larval and adult life without any degeneration of cells. The presence of only one type of gland cells in the stomach of the phylloxerids, viz. merocrine cells, indicates that these species belong to the monophagous aphids (see page 44i n Ponsen, 1990). The stomach lumen is in open connection with the very narrow lumen of the crenated intestine. It is completely filled with a uniform mass of fine-grained material, as illustrated by Grassi et al. (1912) for Acanihocherm.esquercus and Daktulosphaira vitifoliae. This material is only found in the lumen of the stomach and does not occur in the lumen of the crenated and caecal intestine, which has a clear appearance. In the majority of adult sexuparae the stomach lumen is more or less filled with fine-grained material or empty, indicating that they take no nourishment in the adult stage, as reported for the 34 Wageningen University Agricultural Papers 97-1 (1997) Figure 7. Longitudinal section of the stomach and oesophageal valve of a gallicolous Daktulosphaira vitifoliae (A).Transvers e sections of the stomach of a gallicolous D. vitifoliae (B), a winged sexuparous Phylloxera caryaecaulis (C) and Phylloxera devastatrix (D), a wingless fundatrigenious Moritziella castaneivora (E), a winged fundatrigenious Phylloxera caryaeseptum (F), the anterior region with corpus allatum and dorsal vessel (G) and middle region (H) of the stomach of a second larval stage fundatrigenious Phylloxera coccinea. Note the first type of cells in the anterior region and the second type of cells in the remaining part of the stomach. Bar represents 10um .Fo r list of abbreviations see page77 . Wageningen Agricultural University Papers 97-1 (1997) 35 T3 tu <ë" "S o J3 'S cd "tu B Ö tu t« J3 O tfl en * e e O.I CD tu C •5'3 CN — HCNtNcNtnrOCN^fcN ~* *o« S Cu <« 3 ri CL) C CO 5 *C ^C r*~i ^t Tt- ^f m m rn m ^f Tf m m m -t— *i •r 3 vi CO °s^e Mg ta U > H M n ö ä ä ^ S SI S S S S S S S C S co-r" s > w60 cod 'gt«Cä ^o o •«Se cS.S 2. ses -o r-» e- C °^ co ao o-o C !0 ca? Is T3.S > c/> 'S -Û ö ccï 3 «^ > J3 cd <"£ <3 •a'S S) S u G. Ta3 ft cd o.S? c 3 c 3 -a C eu X1 3 c co tri C3 co ,3 CA EB« O vi »•S J3 •— eu Js « >in " Kfi fTt S? c .y Ul 00 00 Où tu-S-~ i "3 ^ s a -3 c goo [x. D-, S D S £ * £ £ £ £ C BO tu'-T't^ 'C * t5û-i| O 1-, fi 3cd6 £ H u Wageningen University Agricultural Papers 97-1 (1997) E-J3PQ + + + + + + + + + + «o fi + + + + + + + + + + + + + + + + + + + + + + + + r-~ t— oo oo r-- r- c-~ h h h oo « _, ~ CO CO ^ CN CO CO -*t CO CO CO CO ^J- CN CN ö ö Ö ö ö ^ ii ö i ö ö ö s* a CO CO - 00 (5JÛ C3 c3 1 3 CS? 'S'a G. o. T) •n es 3 h 3 C c X 3 2 cd X 2'S 1 3 eu X •s s O O CO CO CN a\ O ^ »n o in ^H c s -<3 a. Wageningen Agricultural University Papers 97-1 (1997) 37 sexuparae of Daktulosphaira vitifoliae (Blankenhorn & Moritz, 1875; see page 21). According to Krassilstschik (1889) and Petri (1909) there are no microorganisms in the lumen of the digestive system of Daktulosphaira vitifoliae. In dissection the stomach of aphids (Ponsen, 1991)usuall y appears opaque, but in Moritziella corticalis, Phylloxera coccinea, and Phylloxerina Salicis (the remaining species in Table 3 were not dissected) it is transparent. Although the average body length of phylloxerids is about half that of Myzus persicae, the stomach of the latter is as big as that of the former (Table 4). The stomach of Myzus persicae consists of about 347 epithelial cells whereas the average number of stomach cells in phylloxerids is 80 (Table 6). Consequently the stomach cells of phylloxerids are much bigger than those of Myzus persicae, which accounts for the transparent stomach in phylloxerids. The most characteristic feature of the digestive system of the phylloxerids (Table 3) is the very short crenated intestine (Figure 8- 12), which runs between the mesothorax and fourth abdominal segment without any coil or loop (Table 5). It consists of about 8 triangular cells and about 9 triplets. The crenated intestine of Myzus persicae is about 22 times longer than that of phylloxerids (Table 4) and has 32 times more triangular cells, but the same number of triplets (Table 6). The triangular cells are strongly vacuolated and their apical cell membranes consist of a labyrinthine system of closely packed irregular invaginations of different dimensions (Figure 6C and 20 F in Ponsen, 1991). Each triplet consists of three conical-shaped cells; the middle cell is very large with a large nucleus and the other two are very small each with a relatively small nucleus. The cytoplasm contains numerous minute vacuoles; the apical cell membrane has a small labyrinthine system and the basal cell membrane is strongly invaginated (Figure 11 C-Handl3G). The caecal intestine starts between the mesothorax and the fourth abdominal segment (Table 5) and runs directly caudad dorsal to the stomach to open into the rectum (Figure 8-12). Its length is about one third of that of the body length (Table 4). It is a subdivision of the midgut since the luminal surface of the epithelium lacks a cuticular lining (Ponsen, 1991). The caecal intestine is in open connection with the crenated intestine and consists of only squamous cells; on the side adjacent to the stomach the filterchamber cells are lacking. The squamous cells contain granules and irregular membraneless "vesicles", and nuclei that protrude into the lumen (Figure 11I-K) . The apical cell membrane consists of irregular minute projections (Figure 20 I in Ponsen, 1991). 38 Wageningen University Agricultural Papers 97-1 (1997) Figure 8. Sagittal view (A) and dorsal view (B) of the digestive system of a wingless fundatrigenious Phylloxeraquercus, an d dorsal view of the digestive system of a brachypterous Phylloxerapunctata (C), reconstructed from serial sections showing the topographical position of oenocytes. Bar represents 30 um.Transvers e sections of the rectum of a wingless fundatrigenious Phylloxera coccinea (D), PhylloxerinaSalicis (E), and Moritziellacastaneivora (F) . Transverse sections of the anterior (G) andposterio r (H)regio n of the anal opening of awinge d sexuparous P. coccinea, and the anal opening of abrachypterou s P.punctata (I ) and wingless fundatrigenious P. quercus(J) .Ba rrepresent s 10urn .Fo r list of abbreviations seepag e77 . Wageningen Agricultural University Papers 97-1 (1997) 39 D Figure 9. Dorsal view of the digestive system of six adult morphs of Phylloxera coccinea, reconstructed from serial sections: fundatrix (A), wingless fundatrigenia (B), wingless sexupara (C), winged sexupara (D), female sexuales (E), and male (F).Th e adult fundatrigeniae are smaller than the fundatrices; the sexuales are dwarfish, wingless, and stylefless individuals. Bar represents 30|jm .Fo r listo f abbreviations seepag e77 . 40 Wageningen University Agricultural Papers 97-1 (1997) Figure 10. Dorsal view of the digestive system and topographical position of the oenocytes of a wingless fundatrigenious larva ofMoritziella corticalis (A),a wingles s third stage fundatrigenious larva of Phylloxera coccinea (B),an d a wingless fundatrigenious adult ofPhylloxerina Salicis (C), reconstructed from serial sections. 1-2, meso- and metathoracic spiracles; 3-8, abdominal spiracles.Ba r represents 30urn .Fo r listo f abbreviations seepag e77 . WageningenAgricultural UniversityPapers 97-1 (1997) 41 The cells of the caecal intestine increase in size as the larva grows. The total number varies from 10 cells for Phylloxera caryaecaulis to 25 for Phylloxerina salicis (Table 6). Halfway, on its ventral side, the caecal intestine is innervated by abranc h of themai n abdominal nerve. In both the fundatrices and sexuparae of Aphanostigma ulmifoliae the stomach passes caudad gradually into the crenated intestine, which ends blindly in the fourth or fifth abdominal segment. In the fundatrigeniae of Aphanostigma piri the caecal intestine ends blindly in the sixth abdominal segment and the transition from the stomach to the crenated intestine is marked by a sharp loop (Table 5; Figure 11L and 13). Dorsal to the termination of the caecal intestine, the dorsal vessel starts blindly and extends forwards above the caecal intestine, stomach, and foregut, and discharges into the head (Figure 4). The ventral wall of the funnel-shaped mouth terminates half-way above the corpus allatum (Figure 7 G and 15 A), and is connected with its ventrolateral sides to this body. The dorsal wall of the mouth extends further and is attached to the corpora cardiaca. The medial dorsal nerve, originating from the corpora cardiaca, runs alongside the dorsal vessel giving off branches to the wall of this organ and the pericardial cells. In the anterior region of the dorsal vessel of Myzus persicae there occur three pairs of ostia (Ponsen, 1972), but in Daktulosphaira vitifoliae there is only onepai r (Breider, 1952). All organs including the alimentary tract are held in position by strands of connective tissue cells, which are anchored to the epidermis (Figure 7 C and 11A) . The very short ectodermal part of the posterior region of the alimentary tract consists of a rectum, epidermal invagination, and the anal opening (Figure 8-12). The rectum is situated in the seventh abdominal segment and passes into the epidermal invagination (Figure 12 J and 14). It consists of small cuboidal cells with spherical nuclei; the apical cell membrane is lined with an intima (Figure 8I and 20 J-K in Ponsen, 1991). The muscular coat of the rectum is connected to the abdominal body wall by two thin dorsal visceral muscles and one pair of lateral visceral muscles (Figure 8E- F and 12G) . The epidermal invagination is a very thin tube and its structure varies from an S-shaped flexure (Figure 14 B) to a straight canal (Figure 12 J). The cellular structure of the invagination is similar to that of the foregut. In longitudinal sections it is clear that the cuticular layer lining the rectum, the epidermal invagination and that of the body wall form one continuous layer as illustrated in Figure 12J after moulting. The caecal intestinal lumen is in open connection with the exterior via the anal opening. The latter is situated in a somewhat 42 Wageningen University Agricultural Papers 97-1 (1997) terminal position ventral to the cauda. The anal opening is controlled by two pairs of lateral anal muscles. Each pair of muscles originates at the junction of the seventh and eighth sternites just lateral to the gonopore, and are inserted on the dorsolateral and ventrolateral walls of the anus, respectively (Figure 12H and 14C) . The cauda is very short and broadly rounded forming two or three lobes posterior to the anal opening (Figure 8G-J , 121, and 16F) . Oak leaves and bark, infested with Phylloxera coccinea and Moritziella corticalis, respectively, do not show any trace of honeydew. Observations on these two species carried out under a dissecting microscope reveals that they will excrete a minute droplet of honeydew from the anus if the aphid's body is pinched with a pair of forceps. The droplets are very small, clear, and evaporate rapidly, and can only be observed at a magnification of 80x. Touching the surface of the body with a hair reveals that it only sticks to the distal end of the cauda, presumably due to the presence of "sugar" or "protein" left by the evaporated "honeydew". The pyriform body of Phylloxera coccinea has a very flexible abdomen which can be pointed in any direction (Figure 18-3). When placed upside down on a smooth surface (A), this species can flex its abdomen (B and C) so that the distal region of the broadly rounded cauda sticks to the surface (C), then it raises its body slowly (D) until it stands perpendicular on its cauda (E), and finally lowers onto its legs (F-G). After this the three pairs of legs come into action (H), and the cauda is disengaged from the substrate with difficulty and the aphid walks away (I). It can repeat this many times. Moreover, this behaviour has also been observed on an oak leaf. The aphids Moritziella corticalis and Phylloxera coccinea can walk vertically up and on the underside of a smooth plastic or glass plate. Similarly Moritziella corticalis can attach its cauda to bark to enable it to crawl into a cavity. This and the acrobatic behaviour of Phylloxera coccinea is possibly facilitated by adhesive fluid in the cuticle of the distal region of the cauda similar to that described by Dixon etal. (1990) for the adhesive feet ofAphis fabae Scopoli. Sexuales The sexuales of the Phylloxeridae (Table 7) as well as those of the Pemphigidae (Heie, 1980) and the males of Stomaphis (Tóth, 1933; Sorin, 1995) are characterized by the absence of functional mouth parts. In the sexuales of both Daktulosphaira vitifoliae and Phylloxera Wageningen Agricultural University Papers 97-1 (1997) 43 quercus the digestive system is completely lacking (Balbiani, 1874; Planchon, 1877). In Phylloxera punctata (Lemoine, 1893), Acanthochermes quercus, and Acanthaphis spinulosa (Grassi et al., 1912) the digestive system is arudimentar y structure without any trace of a loop or coil. A rudimentary digestive system occurs in the sexuales of Prociphilus nidificus-poschingeri (= Prociphilus fraxini (Fabricius) and Prociphilus bumeliae (Schrank)) (Nüsslin, 1910), Pemphigus spirotheca Passerini (Totti, 1938, 1939) and Eriosoma lanigera (= Eriosoma lanigerum (Hausmann)); the latter is illustrated by Baker (Plate VI A and B, 1915) as a shriveled and shrunken structure. Derbès (1872) reports the presence of a digestive system in the female sexuales of Aphis terebinthi (= Baizongia pistaciae (Linnaeus)) feeding on Pistacia terebintus. However, in the sexuales of Aphanostigma ulmifoliae, Moritziella corticalis, Phylloxera coccinea, Phylloxera devastatrix, and Phylloxerina Salicis (Table 3) the stylets and - associated - the two pairs of retort-shaped organs with their muscles (see page 24) are absent. Moreover, the pharyngeal duct, pharyngeal valve with its two pairs of divaricator muscles and one pair of lateral pharyngeal valve muscles, and the gustatory organs with their 18 sensillum pores are also absent (Figure 15 A). And in the sexuales of Phylloxerina Salicis the wax glands are completely lacking (see page 23). The "pharyngeal pump" starts as a closed tube, although the cuticular lining of the "pharyngeal pump" and that of the body wall forms one continuous layer. It traverses the middle region of the head, through the oesophageal connectives tojoi n the foregut in front of the tentorial bar. The 10 tendons and their divaricator muscles which are responsible for the pumping mechanism of the "pharyngeal pump" are absent. Only the two pairs of muscles which are directly attached to the edges of the "pharyngeal pump" (Figure 4 and 5) are present and keep the "pharyngeal pump" in position (Figure 15A) . Associated with the absence of stylets, is the lack of salivary glands and ducts and of a salivary pump with its muscles originating from the tentorial bar and from the ventral wall of the "pharyngeal pump". These sexuales also lack the labium, clypeo-labrum, labium, and hypopharynx (Figure 15 - see Figure 4). The absence of salivary glands has been reported for the sexuales of Daktulosphaira vitifoliae (Balbiani, 1874; Grassi et al., 1912), Phylloxera quercus (Balbiani, 1874), Phylloxera punctata (Lemoine, 1893), Acanthochermes quercus, anaAcanthaphis spinulosa (Grassi etal., 1912), as well as for the sexuales of Pemphigus spirotheca (Tóth, 1938, 1939) and the styletless males of Stomaphis quercus. The foregut is a continuation of the "pharyngeal pump" and passes 44 Wageningen University Agricultural Papers 97-1 (1997) eu en 0 .O vo m -3- r- co 00 en ^ t-- VO O CD VO •* vo l> co CN m 8 8.3 +1 +1 +1 +1 •H +1 +1 w in co CS r^ c'a en O >o m U.Ï3 o ~ CN 00 CS c Xi eu Sj« •c , , 1 , , , +1 u E tin o o to X O .2 « vo VO t- 0\ co en tN tN tN -H CL, O —, ,-, ,-, •ftS +1 +1 +1 +1 +1 +1 +1 S c» Tf 00 O o\ ON 0\ O vo VO r~ CT\ co co ja« TD eu CD C co CM tN tN eo ^H O C3 +1 +1 ^+1 +1 +1 +1 +1 •«'S 0 10 •* p- •* 00 m CL.CU o >, tu—. e" C SJ2 1 1 1 1 +1 P «3 S — ' ' ON ^^CO ,O , vo eo rr t-~ CO tN -—1 CL >> vo co o\ in CO •* >n a +1 ro +1 +1 +1 +1 +1 +1 +1 in O r- VO 0 VO tN sä VO "sT 00 VO r- VO m tu u °U c«o •~ CA T.2<3 SNB VO co Tf t-~ CO (N — C«T-J CO CS -H tN —1 (N Ov T3 vU +1 +1 +1 +1 +1 +1 +1 in Tl- CO Tf VO a « •* •* 00 >-S cU £ t/i <^ CU vvj •S-s C eu o .5* a CCI CÖ •a -o OCU. 5Ü g<0 c a C4-. ^> S <2 °s.g ^H (U Ä »— •S9 «•°2§ 3T3X1 O £ £ &: ^ C eu o GBO cd Figure 11. Transverse sections of the crenated intestine of a wingless viviparous larva of Phloeomyzus passerinii (Signoret) (A), and Thelaxes dryophila (Schrank) (B), wingless fundatrigenious Aphanostigma piri (C), wingless second larval stage fundatrigenious Phylloxera coccinea(D) , wingless fundatrigenious Phylloxerina Salicis with only triplet cells (E) and without triplet cells (F), winged sexuparous Phylloxera caryaecaulis with only triplet cells (G)an d without triplet cells (H). Transverse sections of the caecal intestine of a wingless fundatrigenious Ph. Salicis (I), wingless second larval stagefundatrigeniou s P. coccinea (J),an d winged sexuparous P. caryaecaulis (K). Bar represents 10 um. Dorsal view of the digestive system and topographical position of oenocytes of a wingless fundatrigenious Aphanostigma piri (L), gallicolous Daktulosphaira vitifoliae (M), and wingless fundatrigenious Moritziella castaneivora (N), reconstructed from serial sections. 1-2, meso- and metathoracic spiracles; 3-7, abdominal spiracles.Ba rrepresent s 30um .Fo r list of abbreviations seepag e77 . 46 Wageningen University Agricultural Papers 97-1 (1997) Wageningen Agricultural University Papers 97-1 (1997) 47 :0 m O CQ si C/J 0\ tu 00 „, Ui •J ~* C3 m 3 et) -tt o -a o e O 3 CN r»" 2 Si 30 to ON Tf ^f CO o CJ •if ^J- -a .. r P -a c tu S- .c 00 3 a S s Ol £ S a Ol a oi a a •S« u. c_l o « J3 "o i: "O £ ,£2 -C < <<-l es o O C/l CU ta >> a. a. a, J3 00 2 CU 00 >>cU s £ •c "2 >, 2 to .egu S to > S O 'J2 M-H o M o ^3 .ti u O eu g •a < E a pu. t« to to tu ON o O o\ w*" CU 'o e N :0 a> 03 o (N o -a c c CD cel 'S •o 00 C/) C C-i C a CD CD 00 CL, SCJ co CD •S .o li "a eu .ca 3,co .c 53 a 'G 5, to g. "S o C3 ^C a, co C3 o ka. !«eu. ^eu eu K K H .O _o „O -^\C -c ->C . a. a. a. G o Salivary pump The salivary gland system of the phylloxerids (Table 3) consists of one pair of accessory glands situated in the prothorax, and one pair of Figure 12. Dorsal view of the digestive system of an adult fundatrix (A) and a winged fundatrigenious larva (B) of Phylloxera caryaecaulis, a winged fundatrigenious larva of Phylloxera caryaeseptum (C), a winged sexuparous larva (D), a female sexuales (E), and a male (F) of Phylloxera devastatrix, reconstructed from serial sections. 1-2, meso- and metathoracic spiracles; 3,firs t abdominal spiracle. Barrepresent s 30 um.Transvers e sections of the rectum (G), anal musculature (H), and anal opening (I) of P. caryaecaulis. Longitudinal section of the posterior ectodermal part of the digestive system of a winged fundatrigenious larva of P. caryaeseptum (J) showing the old exocuticle of therectum ,epiderma l invagination, and epidermis. Barrepresent s 10urn .Fo r list of abbreviations seepag e77 . 50 Wageningen Agricultural Papers 97-1 (1997) Wageningen Agricultural University Papers 97-1 (1997) 51 principal glands in the meso- and metathorax. The ducts from both lateral glands join and run ventrally around the junction of the suboesophageal and thoracic ganglia and together with the contralateral duct form a common salivary duct. The common duct runs forwards medio-rostrally beneath the suboesophageal ganglion, and turns downwards to the hypopharynx where it passes into the afferent salivary duct following the median line beneath the salivary pump (Figure 4 and 17G-H) . The salivary pump is a relatively large structure of which the three pairs of well-developed muscles are inserted on the dorsal wall of the piston (Figure 17). Before entering the pumpcylinder the afferent salivary duct turns upwards and then downwards, forming an S-shaped flexure. At the place of entry the opening is controlled by a muscle originating from the middle of the tentorial bar. After leaving the pumpchamber the duct turns upwards and then downwards via a short duct into the pumpstem (Figure 17H) .A t the foot of the pumpstem the duct is provided with two sensillary pores corresponding with the hypopharyngeal gustatory organ. The duct passes into the efferent salivary duct and runs in the median line on the ventral side of the hypopharynx lip and terminates in the salivary canal enclosed by the maxillary stylets (Figure 4). Contraction of the three pairs of muscles of the salivary pump (Ml7) pulls the piston upwards. During this process the lumen increases in size, reducing the pressure, which draws saliva into the pumpchamber when the opening in the S-shaped flexure is opened by contraction of the muscles of the afferent salivary duct (M20). Relaxing of the three pairs of muscles forces the saliva into the efferent salivary duct as the S-shaped flexure is then closed. The S- shaped flexure has the same function as the pharyngeal valve, but the structure of the latter is more complicated (Figure 5 E-F). Morphologically and histologically the salivary gland system of all phylloxerid species (Table 3) is identical to that of the gallicolae and radicicólae of Daktulosphaira vitifoliae, described and illustrated by Krassilstschik (1892, 1893), Grassi et al. (1912), Breider (1952), and in detail by Rilling (1960). The salivary glands of both these morphs produce two types of saliva, viz. a viscous substance which hardens to Figure 13. Dorsal view of the digestive system of an adult fundatrix (A) and that of a wingless sexuparous larva (B), male (C), and female sexuales (D) of Aphanostigma ulmifoliae, reconstructed from serial sections. 1-2, meso- and metathoracic spiracles; 3-7, abdominal spiracles. Bar represents 30 um. Longitudinal section of the stomach and crenated intestine (E), and transverse sections of the stomach (F), crenated intestine (G), oenocyte, basophilic mesodermal cell, and fat cells (H) of a wingless sexuparous larva, and the stomach of amal e of A. ulmifoliae (I).Ba r represents 10um .Fo r list of abbreviations see page77 . 52 Wageningen Agricultural University Papers 97-1 (1997) Wageningen Agricultural University Papers 97-1 (1997) 53 form the sheath and a watery substance (Rilling, 1967).O n thebasi s of the histological studies on Myzus persicae (Ponsen, 1972), it is likely that the accessory glands are responsible for the production of the salivary sheath, which protects the stylets. The principal gland cells secrete granules (= enzymes) into the lumen of the salivary canal where they dissolve in haemolymph pumped up by the myoepithelioid cell situated at the distal end of each principal gland; this watery substance is injected into the sievetubes todiges t thephloe m sap. Mycetome and oenocytes Except the Hormaphididae and the Phylloxeridae, all families within the superfamily Aphidoidea, viz. Adelgidae, Anoeciidae, Aphididae, Chaitophoridae, Drepanosiphidae, Greenideidae, Lachnidae, Pemphigidae, Phloeomycidae, and Thelaxidae possess a mycetome. It is a complex organ consisting of a number of cells (mycetocytes) of which the scanty cytoplasm is completely filled with symbionts (cell organelles). The Hormaphididae have no mycetome, but the symbionts occur free in the haemolymph (Profft, 1937; Büchner, 1953; Ponsen, 1991). Each mycetocyte has an irregularly shaped nucleus due to the pressure of the multiplying symbionts, by binary fission (as in Hyalopterus) and a budding process (as in Subsaltusaphis ornata). However, all phylloxerids (Table 3) have no symbionts housing in a mycetome or loosely scattered in the haemolymph. Witlaczil (1886) was the first who reported the absence of a pseudovitellus (= mycetome) in the eggs of Phylloxera quercus. Later on, several authors confirmed the absence of a mycetome and symbionts in both gallicolae and radicicolae of Daktulosphaira vitifoliae (Dreyfus, 1894; Profft, 1937; Koch, 1952; Breider, 1952), Phylloxera punctata (Dreyfus, 1894), Acanthochermes quercus, and Phylloxera coccinea (Profft, 1937). On the other hand, Krassilstschik (1892, 1893) found "Secundäre Dotter" cells (= pseudovitellus cells = mycetocytes) with fine- granulated cytoplasm in groups of 2-4 cells or individually among the fat cells of radicicolae of Daktulosphaira vitifoliae, and Peklo (1953) Figure 14. Median longitudinal sections of the rectum and epidermal invagination of an adult wingless sexuparous (A) and wingless fourth larval stage fundatrigenious (B) Phylloxera coccinea. Note the open connection of the digestive system with the exterior via the anal opening. Transverse sections of the anal musculature of a wingless second larval stage fundatrigenious P. coccinea (C) and a wingless viviparous Mexicallis analiliae Remaudière (Family Drepanosiphidae) (D).Ba rrepresent s 10urn .Fo r list of abbreviations seepag e77 . 54 Wageningen Agricultural University Papers 97-1 (1997) Wageningen Agricultural University Papers 97-1 (1997) 55 found 5-9 mycetocytes filled with spherical microorganisms in the body cavity of the same species. Pseudovitellus cells are also present in the body cavity of both gallicolae and sexuparae of Daktulosphaira vitifoliae, Acanthochermes quercus, and Paraphylloxera glabra (Grassi et al., 1912). From their descriptions and illustrations it can be concluded that their pseudovitellus cells or mycetocytes are in fact oenocytes. In all phylloxerids (Table 3) the oenocytes are situated mainly laterad on either side of the body cavity between the mesodermal tissue and the internal organs of the thorax and the first seven abdominal segments (Figure 8 B-C, 10, 11 L-N, 12 B, and 13 B). In the sexuales the oenocytes are lacking (Figure 12E-F , 13C-D , and 15 L-O) as well as in the styletless males of Stomaphis quercus (Toth, 1933). The majority of the oenocytes occur as single cells, but also sometimes in groups of 2-3 cells in intimate contact with each other. The cytoplasm is filled with vacuoles and granules; each oenocyte has a somewhat irregularly-shaped nucleus which decreases in size due to the production of granules which are released into the cytoplasm (Figure 6 A and 13 H). The total number of oenocytes varies from 11 for wingless sexuparous Aphanostigma ulmifoliae to 44 for winged fundatrigenious Phylloxera caryaecaulis. These polygonal cells are anchored to tracheae or other internal organs by strands of connective tissue cells. Oenocytes has been described in both gallicolae and radicicolae of Daktulosphaira vitifoliae (Profft, 1937; Breider, 1952; Rilling, 1967), Acanthochermes quercus, and Phylloxera coccinea (Profft, 1937). The basophilic mesodermal cells are individually dispersed among the fat cells (Figure 4, 6 A, 13 H, and 14A) . In the appendages of the aphid these polygonal cells are lacking. They have a structureles homogeneous basophilic cytoplasm and a distinct spherical nucleus. Similar cells have been illustrated as x-cells in Acanthochermes quercus and in the sexuparae of Daktulosphaira vitifoliae (Grassi et al, 1912). Figure 15. Sagittal section of the head and anterior region of the thorax of a female sexuales of Moritziellacorticalis (A) . The epidermal cell layer is omitted. Transverse sections of the anterior (B), middle (C), and posterior region (D) of the pharyngeal "pump", the stomach of a female sexuales of Phylloxeracoccinea (E) ,M. corticalis(F) , and a male of Phylloxera devastatrix(G) , the crenated intestine of a male of M. corticalis(H) , the caecal intestine of a female sexuales of Phylloxerina Salicis (I) and P. devastatrix(J) , and the rectum of a male of P. coccinea(K) . Bar represents 10 um. Dorsal view of the digestive system of a female sexuales (L) and male (M) of Ph. Salicis,a female sexuales of P. coccinea(N) , and a male of M. corticalis(O) , reconstructed from serial sections. 1-2, meso- and metathoracic spiracles; 3-8, abdominal spiracles. Bar represents 30 urn.Fo r list of abbreviations see page77 . 56 Wageningen Agricultural University Papers 97-1 (1997) D C B « { I Wageningen Agricultural University Papers 97-1 (1997) 57 During larval life the oenocytes and basophilic mesodermal cells gradually increase in size. These cells have not been observed to divide and consequently the total number of these cells remains constant. Discussion In all aphid species (Ponsen, 1991) the pharynx consists of the pharyngeal canal, valve, and pump. The pharyngeal valve is opened by two pairs of dorsal muscles, and closed by the contraction of the lateral pharyngeal valve muscles (Figure 6 C). The ventral wall of the pharyngeal pump is attached by two pairs of muscles to the tentorium and the salivary pump (Figure 4). However, in phylloxerids (Table 3), other than the sexuales, the flexible dorsal wall of the pharyngeal pump is controlled by 10 pairs of muscles (Figure 4, 5 A, and 6 A), whereas in both Myzus persicae (Ponsen, 1972), Aphis species, and Israelaphis carmini Essig (Figure 6 B), there are more than twice as many pairs of muscles, viz.27 . In aphids the stylets penetrate intercellularly through plant tissues until they reach the phloem cells on which they feed (Tjallingii & Hogen Esch, 1993). However, the phylloxerids take their nourishment from non-vascular tissue cells by intracellular penetration (Millardet, 1898; Petri, 1909; Koch, 1952; Kunkel, 1967), although the musculature of the stylets is identical to that of Myzus persicae (Ponsen, 1972; see page 26). Presumably fewer muscles are needed to pump food from the damaged parenchyma cells than from the vascular tissues. Moreover, the non-vascular cells contain less liquid food than the vascular tissues, with their continuous flow of sap, but the former have a higher content of proteins than phloem sap which mainly contains carbohydrates. The ingestion of small quantities of cell sap would account for the possible excretion of very few minute "honeydew" droplets, and the high protein content of the food for the very short crenated intestine (see page 36). In general, insects feeding on a largely protein diet tend to have a shorter gut than those feeding largely on carbohydrates. The length of the digestive system of the phylloxerids is about one and a quarter that of their body length, but in other aphid species it varies from twice for the Thelaxidae to three times for Myzuspersicae (Table 4), and four times for Phloeomyzuspasserini (Signoret) (Figure 18-2). The salivary gland cells of the phylloxerids (Rilling, 1960) are histologically quite different from those of Myzus persicae (Ponsen, 1972). The structure of the salivary pump in phylloxerids (Table 3) is 58 Wageningen Agricultural University Papers 97-1 (1997) >T3 ca T3J3 tu cu= g. o ca 0 °o t~a ufio HFH o « y +1 vo u-s g £5«^ I ^UT3 •ê-S^g-SgoE o u S,tS £ o c CN CN CN CN CN CN CN o O O o O o O O O o o o O O o +1 CN vo 00 CN CN en vo vo cri CO o CN 't VO OO m 0\ in CN en oo CN oo o eO O ci J3 c/i H.23 OCH C •-H Ö u UJ: O-S T3^.S2.S Mäiä 13 o o u-S £ o J'ïSccUcïï g St«e e - ~-~ „,. ;= y^ S ra K^ -yj 0) ~ D 0) t — o o CN -et -et en -et -et en o o f t o r- -g Vf) CN en 00 o m o O l~~ o CN oo -et g HJ 3 ° u S " 60 i> t~ o\ o\ oo Ov en oo vo oo vo C^l -et „ÜS ca-° o >, ^ •g e»j3 u o £ c« u -a SQ,c§ z> SU *S'Cc ^s«a - > o •STJ3"" ," B -.CS p. J3 o u o L- C Cß'bi) n•i 2— u'S 'a S %•&£ 3a •S " s Sä oTa'CXixit; e« 3 a >- S .S- 2 Q S •Rtse « g* oc .STri) C tu Gq^H -0 Ü O -et S b a a S Sj a giO b w o U ,) •*- . r-i ni *J Q •â^S:« S S'a u S eu >\3 S t« J3 en a S£ ca CD S J3 5 K a C3 B Î3 ce! '-' c .as s u £ I "S 13 H u ca B3 -ÎSS CV Ü 'C .Ü 00 ^3 •~ S g -a -2 -a S CL, oo cis eg >aT3 "^ c^ "« -^ ~2 "a tU'-Ö 53 'C tu .2 (U S ^c tn t^ « QV - g X) CU-Cs (U y BO CL -o -o rca g a -g 60 Wageningen Agricultural University Papers 97-1 (1997) VO l~ ON CU _c c -o CA c CU CA csX> 13 CU o CA O s H-C CA eu O JU 2 CA "t3 CA 0) e CS CA w CA 2 CD CU a>T* * tJO IM CU c •s IZ-S DO CA o s CU o 'CA Ol Ol a, 0, 3 Xj O CT3 JS S euX S (J e g CA eou CU o eu >-.>O> S O 1 CS i/i u 62 Wageningen Agricultural University Papers 97-1 (1997) Figure 16. Median longitudinal sections of a male of Moritziella corticalis (A) and a female sexuales of Phylloxerina Salicis (B). Anal opening in ventral position. Transverse sections of the rectum of amal e of Ph. Salicis (C),th e anal musculature of afemal e sexuales ofM. corticalis (D), the anal musculature (E) and anal opening (F) of afemal e sexuales of Phylloxera coccinea. In the sexuales the fat cells and haemocoel contain protein globules with one or more cavities (pre), but the waxy droplets are lacking.Ba rrepresent s 10|im .Fo rlis t of abbreviations seepag e77 . Wageningen Agricultural University Papers 97-1 (1997) 63 deplanata Pergande the anus is bluntly rounded and furnished with four hairs around its edge (Pergande, 1904). According to Federov (1959) the anal aperture is absent in all five morphs of Daktulosphaira vitifoliae. Working with radicicolae of Daktulosphaira vitifoliae, Anders (1957a) supposed that the waste products are discharged via the salivary glands, or as in Phylloxerina via the wax glands (Iglisch, 1965). However, in most phylloxerid species (Table 3) the lumen of the digestive system is in open connection with the exterior via the anal opening which is situated in a terminal position ventral to the cauda (Figure 12J and 14A-B) . The exceptions areAphanostigma ulmifoliae and Aphanostigma piri: in the former the crenated intestine ends blindly and in the latter it is the caecal intestine (Figure 13A- B and 11 L). The anal opening in all aphid species investigated is opened by contraction of six dorsal muscles and closed by two pairs of lateral muscles (Figure 14 D; Ponsen, 1990, 1991). In phylloxerids (Table 3) the dorsal anal muscles are lacking as in the styletless males of Stomaphis quercus, which does not excrete honeydew. Presumably contraction of the two pairs of lateral anal muscles opens the anus slightly; pinching the body of both Moritziella corticalis and Phylloxera coccinea with fine forceps resulted in them extruding a minute clear droplet through the anal opening (see page 41). Possibly during ecdysis a small amount of honeydew is excreted along with the cuticular lining of the epidermal invagination and that of the rectum (Figure 12J and 14A-B) . The absence of the dorsal anal muscles is associated with the fact that: 1) the leafgalls on Vitisvinifera cv. Clinton and Vitis cardifoliae caused by Daktulosphaira vitifoliae, those on Carya alba caused by Dactylosphaera caryae-venae Fitch, and those on Carya glabra (= Carya amara (Russell, 1974)) caused by Dactylosphaera globosum (= Phylloxera caryaesemen Walsh (Pergande, 1904)) are "alike in being free from any of the sugary dust, so common among the gall- producing Aphididae" (Walsh, 1866; Shimer, 1867); 2) Phylloxera vitifoliae "does not secrete any sugary or flocculent substance as do most gall-inhabiting aphids" (Riley, 1870); 3) Dreyfus (1894) and Kunkel (1966, 1972) could not find any trace of honeydew on oak leaves infested by Phylloxera coccinea and Phylloxera punctata, and 4) there is no honeydew on Chestnut burs infested with Moritziella castaneivora (Miyazaki, personal communication 1994). In addition, Bazille et al. (1868) were surprised that the radicicolae of Daktulosphaira vitifoliae are not attended by ants, as many aphid species feeding on subterraneous parts of plants are attended by ants. 64 Wageningen Agricultural University Papers 97-1 (1997) Figure 17. Transverse sections of the salivary pump of a second larval stage fundatrigenious Phylloxera coccinea (A), a wingless sexuparous Aphanostigma ulmifoliae (B), a winged fundatrigenious Phylloxera caryaeseptum (C), awingles s fundatrigenious Moritziella castaneivora (D), a wingless viviparous Fordamarginata Koch (Family Pemphigidae) (E), and a winged male of Greenideaformosana (Maki) (Family Greenideidae) (F). Median longitudinal sections of the salivary pump of a wingless viviparous Thelaxesdryophila (Schrank) (Family Thelaxidae) (G) and a winged sexuparous Phylloxera caryaecaulis (H).Not e the three pairs of retractor muscleso f salivary pump piston in species of the Phylloxeridae and the two pairs of muscles in species of the remaining families of the superfamily Aphidoidea (M17). Bar represents 10 um. For list of abbreviations see page77 . Wageningen Agricultural University Papers 97-1 (1997) 65 Later on, this was confirmed by Mordwilko (1907). According to Koch (1952) and Baumann et al. (1995) the symbionts play a very important role in the supply of various substances which are absent or in short supply in phloem sap. All phylloxerids (Table 3) lack a mycetome (see page 52), presumably because they feed on a protein-rich diet ingested from non-vascular tissue cells. The styletless males of both the Pemphigidae and Stomaphis species lack a mycetome, whereas in the styletless female sexuales of the Pemphigidae the mycetome is reduced to some mycetocytes (Table 9). In Geoica setulosa (Passerini) the stomach and the crenated intestine are absent and consequently the mycetome, but the embryos in various stages of development in their mother's ovarioles all have a mycetome (Ponsen, 1990, 1991). Summarizing, it appears that the absence of a mycetome or symbionts is correlated with: 1) a protein-rich diet, 2) the absence of mouth parts, or 3) the absence of a stomach and crenated intestine. Phylogeny Anatomically and histologically the family Phylloxeridae belongs to the superfamily Aphidoidea. According toHill e Ris Lambers (1964) and Heie (1967) the Adelgidae and Phylloxeridae are phylogenetically the oldest family of the Aphidoidea. However, the presence of a caecal intestine in the Phylloxeridae indicates that this family may have evolved from the Drepanosiphidae. In addition, the average number of cells in the caecal intestine of both the Phylloxeridae and the Drepanosiphidae is similar (Table 8). Within the Drepanosiphidae there are species which have two types of smooth transparent intestines: the descending intestine and the caecal intestine, both showing vigorous peristaltic movements generated by circular and longitudinal muscles, but the cellular structure of both types is quite different. The descending intestine is a continuation of the crenated intestine forming one straight tube with the rectum, which terminates at the anal opening. The caecal intestine is a blindly ending evagination of the rectum and forms a concentric filtersystem with the stomach. The descending intestine forms a parallel filtersystem with the ascending intestine, but in both filtersystems the filterchamber cells are absent (Ponsen, 1979). During evolution the caecal intestine developed after the descending intestine, which was subsequently replaced by the caecal intestine, which forms the ultimate digestive system in the Phylloxeridae. Within the Aphidoidea there occur five families with a caecal 66 Wageningen Agricultural University Papers 97-1 (1997) A B C D E F y orm^am^orri^^f^^^sw ^mip DE F G Figure 18. 1.Diagram s of the digestive system of the family Phylloxeridae illustrating acomplet e digestive system (A), one without the rectum and epidermal invagination (B), one without the caecal intestine (C), and a hypothetical digestive system of which the stomach and crenated intestine are lacking (D). 2. Actual length of an aphid (A) and that of the digestive system of the family Phylloxeridae (B), Thelaxidae, Chaitophoridae, Greenideidae, and Anoeciidae (C), Aphis sp., Eulachnus sp., and Callaphididae without a filtersystem (D), Cryptomyzus ribis Linnaeus, Myzuspersicae (Sulzer), and Subsaltusaphis ornata (Theobald) (E), and Phloeomyzus passerinii (Signoret) (F). 3. Diagrams illustrating the righting behaviour of a wingless fundatrigenious Phylloxera coccinea (A-I, see page 41).Fo r list of abbreviations seepag e77 . Wageningen Agricultural University Papers 97-1 (1997) 67 Table 10.Majo r differences between thePhylloxerida e which presumably takethei r nourishment from thenon-vascula r tissuecell s andMyzus persicae (Sulzer),a nultimat e phloem-feeding aphid Phylloxeridae Myzuspersicae Page 10pair so fpharyngea l pump muscles 27pair s ofpharyngea l pump muscles 30,58 large salivary pump small salivary pump 50,60 3pair s ofsalivar y pump muscles 2pair s ofsalivar y pump muscles 50,60 short crenated intestine long crenated intestine (± 8triangula r cells) (± 253 triangular cells) 38,62 no filterchamber cells filterchamber cells 38,62 no mycetome mycetome 54,66 no analdorsa l muscles 6 anal dorsal muscles 43,64 nohoneyde w much honeydew 43,64 intestine: Phylloxeridae, Drepanosiphidae with a filtersystem, Phloeomyciidae, Lachnidae, and Aphididae (except the Aphis-group) of which only thelas t twofamilie s have filterchamber cells (Ponsen, 1979, 1981,1991) . The remaining families: Adelgidae, Mindaridae, Thelaxidae, Chaitophoridae, Greenideidae, Drepanosiphidae without a filtersystem, Anoeciidae, and the Aphis-group have a descending intestine inwhic h the filterchamber cells arelackin g (see Figure 16i n Ponsen, 1991). Probably aphids originally fedo nparenchymou s cells butevolve d to feed from vascular tissue cells, which contain a relatively low concentration ofproteins , ahig h concentration ofcarbohydrates , anda relatively high proportion of water. As a consequence of phloem feeding, the aphids evolved an association with bacterial symbionts (mycetome), a long crenated intestine, filterchamber cells (filter system) to drain off the excess water, additional pharyngeal pump muscles, andsi xana l dorsal muscles toexpe l the excess ingested food (carbohydrates) ashoneyde w (Table 10). Summary In allmorphs , except the sexuales, ofth e family Phylloxeridaeth e alimentary tract consists of a food canal formed by the interlocked 68 Wageningen Agricultural University Papers 97-1 (1997) maxillary stylets, pharyngeal duct, pharyngeal valve, pharyngeal pump, foregut, oesophageal valve, stomach, crenated intestine, caecal intestine, rectum, and an epidermal invagination which terminates at the anal opening. In Aphanostigma ulmifoliae the crenated intestine and in Aphanostigma piri the caecal intestine end blindly (Table 3 and 5). The most characteristic features of the Phylloxeridae are the 12 pairs of pharyngeal pump muscles {Myzus persicae 29), 3 pairs of salivary pump muscles {Myzus persicae 2), the very short crenated intestine, no filterchamber cells, an anal opening that lacks the six dorsal muscles, no expelling of honeydew droplets, and no syrribionts (mycetome), indicating that the phylloxerids must take their nourishment from non-vascular tissue cells (Table 10). In the sexuales (Table 3 and 5), the stylets, retort-shaped organs, pharyngeal duct, pharyngeal valve, gustatory organs, salivary gland system, symbionts (mycetome), and oenocytes, are lacking. The "pharyngeal pump" starts blindly, but lacks the 10 pairs of muscles, except the 2 pairs of "supporting" muscles. The digestive system is somewhat degenerated and terminates at the anal opening. In Aphanostigma ulmifoliae and Phylloxera devastatrix the stomach ends blindly. The presence of a caecal intestine in the Phylloxeridae indicates that this family may have evolved from the Drepanosiphidae. Acknowledgements I wish to express my thanks to Professor Dr. C.C. Yeh (National Chung Hsing University, Taichung, Taiwan, Republic of China),Dr . J. De Benedictis (University of California, Davis, California, U.S.A.), Dr. F.A. Ilharco (Estacao Agronomica Nacional, Oeiras, Portugal), Dr. M. Miyazaki (National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki, Japan), Dr. G. Remaudière (Museum National d'Histoire Naturelle, Paris, France), and Dr. M.B. Stoetzel (Systematic Entomology Laboratory, Beltsville, Maryland, U.S.A.) for collecting and sending me specimens. I am indebted to Professor Dr. A.F.G. Dixon (University of East Anglia, Norwich, England) for valuable suggestions and for correcting the English. Special thanks are due to Dr. P. Harrewijn for his enthusiastic cooperation and to Ing. P.G.M. Piron (Institute of Phytopathological Research, Wageningen) for his patience with the typing of the manuscript. Wageningen Agricultural University Papers 97-1 (1997) 69 References Anders, F., 1957a. Über die gallenerregenden Agenzien der Reblaus (Viteus (Phylloxera) vitifolii Shimer). Vitis 1: 121-124. Anders, F., 1957b. Neuere Auffassungen über die Reblaus-Resistenz. Vitis 1: 142-152. Anders, F., 1961. Untersuchungen über das cecidogene Prinzip der Reblaus (Viteus vitifolii Shimer). III. Biochemische Untersuchungen über das galleninduzierende Agens. 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Zeitschrift für wissenschaftliche Zoologie 43: 149-174. 76 Wageningen Agricultural University Papers 97-1 (1997) Abbreviations usedi nfigure s a antenna M19 retractor muscle of salivary pump adm anal dorsal muscles wall aim anal lateral muscles M20 retractor muscle of afferent ao anal opening salivary duct opening asd afferent salivary duct M21 ventral pharyngeal pump muscle bmc basophilic mesodermal cell M23 mandibular muscle to pharyngeal ca corpus allatum pump floor ci caecal intestine M24 maxillary muscle to pharyngeal cl clypeus pump floor clb clypeo-labrum N13 medial dorsal nerve cri crenated intestine oc ocellus csd common salivary duct oen oenocyte etc connective tissue cell ov oesophageal valve dv dorsal vessel p protocerebrum e epipharynx pb proboscis ego epipharyngeal gustatory organ pc pumpcylinder ei epidermal invagination pec pericardial cell encu endocuticle pch pumpchamber ep epidermis pg prothoracic ganglion epi epicranium phd pharyngeal duct esd efferent salivary duct php pharyngeal pump excu exocuticle phpr pharynx protuberance f foregut phv pharyngeal valve fac fat cell pre protein crystal fag female accessory gland ps pumpstem fc food canal pt piston go gonopore r rectum hp hypopharynx sep sensillary pore hpgo hypopharyngeal gustatory organ sog suboesophageal ganglion hpl hypopharynx lip st stomach is intravalvular space t tritocerebrum lb labrum tb tentorial bar lec larval compound eye te tendon loc larval ocellus tg thoracic ganglion md mandibular stylet to triommatidion mx maxillary stylet tpc triplet cells M2 circular muscle fibres ts testis M4 divaricator muscles of the pharynx tu tube M6 elevator muscle of tentorial bar vm visceral muscles M7 lateral muscle of clypeus vr ventral rod (chitinous ridge) M8 lateral muscle of mandibular stylet w waxy droplet MIO lateral pharyngeal valve muscle we winter egg Mil longitudinal muscle fibres wi wing-pad M17 retractor muscle of salivary pump piston Wageningen Agricultural University Papers 97-1 (1997) 77