257

Spittle-production and tube-building by cercopoid nymphs(Homoptera). i. The cytology of the Malpighian tubules

of spittle-bug nymphs

By A. T. MARSHALL

(From the Department of Zoology, University of Hong Kong)

With 2 plates (figs. 2 and 3)

SummaryUse of the basic dyes toluidine blue and alcian blue reveals stacked circular granules,1 to 3 /A in diameter, and a highly vacuolated material within the cells of the proximalsegments of the nymphal Malpighian tubules.

It is suggested that cyclical secretory activity occurs, involving coalescence of thegranules and vacuolation, to produce a secretion which is liberated into the tubulelumen and then passes into the hindgut.

IntroductionT H E Malpighian tubules of the Homoptera are of particular interest in thatthey often show morphologically differentiated segments, presumably for theelaboration of special secretions (Licent, 1912; Day and Briggs, 1958; Smithand Littau, i960). The superfamily Cercopoidea is divisible into 2 families,Cercopidae and Machaerotidae (Maa, 1963). Within the Cercopidae the sub-families Cercopinae, Aphrophorinae, and Clastopterinae contain the truespittle-bugs, having nymphs living more or less permanently in a covering offroth or spittle. Production by the nymphs of a surface tension depressantto stabilize the froth or spittle has been previously accredited to a clearlydifferentiated proximal segment of the Malpighian tubules (Licent, 1912;Kershaw, 1914; Pesson, 1956), which produces a mucocomplex secretion.The histochemistry of these segments will be described elsewhere (Marshall,unpublished).

Almost nothing has been previously reported of the cytology of thisproximal secretory region, which is described here. The cytology of theremainder of the Malpighian tubule has been described in some detail byLicent (1912) and Kato (1958).

Materials and methodsThe nymphs examined belong to the subfamilies Cercopinae and Aphro-

phorinae.

Subfamily Cercopinae

1. Aeneolamia varia saccharina Distant (Trinidad)2. Locris sp. (Africa)

[Quart. J. micr. Sci., Vol. 105, pt. 2, pp. 257-62, 1964.]

258 Marshall—Spittle-production by cercopoid nymphs

Subfamily Aphrophorinae

1. Philaenus leucophthalmus spumarius Linnaeus (England)(Philaenus spumarius)

2. Neophilaenus sp. (England)3. Poophilus sp. (Africa)4. Ptyelus sp. (Africa)5. Clovia lineatocollis de Motschulsky (Hong Kong)6. Bathylus albicinctus Erichson (Australia)

Dissections of nymphs of P. leucophthalmus, Neophilaenus sp., C. lineato-collis, and B. albicinctus were made in insect saline for observations on andsquashes of proximal segments. Nymphs of A. varia, P. leucophthalmus, andC. lineatocollis were fixed in formaldehyde-calcium, dehydrated in tetra-hydrofuran (Salthouse, 1958), embedded in 550 C paraffin wax, and sectionedat jfi. Specimens of Ptyelus sp. and Locris sp., fixed in Bouin, were alsoexamined. Various other fixatives were tried but fixation of the proximalsegments did not appear to be as good as that yielded by formaldehyde-calcium. Tetrahydrofuran was used as a dehydrating and clearing agent, inpreference to graded alcohols and xylene, because it resulted in improvedstaining of the cell contents of the proximal segments.

Sections were stained with toluidine blue (Kramer and Windrum, 1955),alcian blue (McManus and Mowry, i960), Heidenhain's Azan, and the Massontrichrome techniques.

Supravital staining of proximal segments was achieved by applying o-i %aqueous neutral red to freshly dissected segments and also by injecting livingnymphs with 1 microlitre of 1 % indigo carmine in insect saline and dissect-ing after 10 min.

ObservationsThere are 4 Malpighian tubules, each consisting of 3 distinct regions: a

lobulated distal segment, a smooth proximal segment, somewhat larger indiameter, and a slender Malpighian duct (fig. 1, A, B).

Each Malpighian tubule terminates in an ampoule closely adhering to therectum. The Malpighian ducts enter the posterior end of the filter chamberpouch, run anteriorly along it but not to the anterior end, return upon them-selves and enter the midgut (fig. 1, A).

Proximal segment

The general histological staining techniques, e.g. Heidenhain's Azan,employ acid dyes for the staining of cytoplasm. With such techniques cyto-plasmic structures in the cells of the Malpighian tubules were unstainedexcept for a membranous network. However, the cells were revealed as beingrelatively exceedingly large and containing a large nucleus. The nucleusoften, but not always, had an irregular stellate appearance. In phase contrastobservations of freshly-dissected proximal segments the nuclei were seen to

Fie. z

A. T. MARSHALL

Marshall—Spittle-production by cercopoid nymphs 259

be oval and regular in outline (fig. 3, c), and this must be accepted as beingtheir normal condition. No brush borders were apparent.

Staining with alcian blue and toluidine blue, which are basic dyes, revealed

oesophagus

midgut

distal segment

FIG. I. A, diagrammatic representation of the nymphal gut complex. B, diagrammaticrepresentation of the nymphal Malpighian tubule.

a complex of cytoplasmic inclusions. In some sections granules in the formof circular platelets ranging in diameter from 1 to 3 /x were present. Their

FIG. 2 (plate), A, C, D, E, transverse sections of proximal segments of Aeneolamia variasaccharina nymphs, stained with alcian blue and photographed with a red filter.

A, stacked granules (s).C, radial rows of granules (i) and vacuoles (v). Patches of coalesced mucocomplex (?;i) are

evident near the lumen {I).D, areas of mucocomplex secretion (m) containing numerous large vacuoles (v).E, vacuolated granules (iav).B, squash of proximal segment of Bathylus albicinctus nymph, showing granules. Phase

contrast.

260 Marshall—Spittle-production by cercopoid nymphs

arrangement appeared to be in stacks, resembling the rouleaux of red cor-puscles seen in blood smears (fig. 2, A). Orientation of the stack axis appearedto be principally radial (fig. 2, c).

Numerous granules 1 to 3 /x in diameter were observed by phase contrastin squashes of freshly dissected proximal segments (fig. 2, B).

Sections of the proximal tubules of some specimens revealed the cytoplasmof the cells to be almost completely occupied by granule stacks. It is note-worthy that in specimens in which the proximal segments appeared in thiscondition, no material possessing similar histochemical properties could bedetected either within the lumen of the Malpighian tubules or in the gutposterior to the point of entry of the Malpighian tubules.

Specimens in which the granule stacks occupied progressively less of thecytoplasm were also obtained and others in which virtually no granule stackswere present. When granules were almost absent the cells either containedan intensely staining, highly vacuolated mucocomplex (fig. 2, D), or werehighly vacuolated and stained faintly. In the latter specimens material havingidentical histochemical reactions to the intensely staining mucocomplex andgranules was observed in the hindgut lumen.

The appearance of the cells when both granules and vacuolated muco-complex are present is shown in fig. 2, c. It is to be noted that the vacuolatedmucocomplex is near to the lumen and that granules lie remote from thelumen. Fig. 3, c, shows part of a living, freshly dissected proximal segment,clearly showing some of the structures apparent in the fixed and stainedsection (fig. 2, c).

Intracellular 'canals' were seen in proximal segments after staining freshdissections with neutral red or injecting nymphs with indigo-carmine. Thesemay perhaps be identical to the radial lines of vacuoles seen in fig. 2, c.

The condition of a proximal segment appeared to be the same throughoutits length and the same condition usually prevailed within the proximal seg-ments of each of the 4 Malpighian tubules.

The appearance of granules and vacuolated mucocomplex differed insections from different specimens as follows:

(a) granules separated and arranged in stacks (fig. 2, A);(b) granules of one stack connected by fine filaments or membranes to

granules of adjacent stacks and to superjacent granules of the samestack (fig. 3, A);

(c) granules stacked and vacuolated (fig. 2, E);

FIG. 3 (plate), A, B, transverse sections of proximal segments of Aeneolamia varia saccharinanymphs, stained with alcian blue and photographed with a red filter.

A, granules of adjacent stacks connected by filaments or membranes ( / ) . Superjacentgranules are also connected by filaments (p).

B, coalesced superjacent granules (c).c, freshly dissected proximal segment of Bathylus albincinctus nymph. Phase contrast.

n, oval nuclei; s, granule stacks; v, vacuoles.

FIG. 3

A. T. MARSHALL

Marshall—Spittle-production by cercopoid nymphs 261

(d) granules which appear to have recently coalesced and with vacuolesapparent in the coalesced material (fig. 3, B);

(e) vacuolated mucocomplex present but no granules (fig. 2, D).

All these stages were found in every species examined.

DiscussionLicent (1912), Kershaw (1914), Cecil (1930), and Kato (1958) give detailed

anatomical accounts of the nymphal cercopid gut complex. Of these authorsCecil and Kato did not describe a distinct proximal segment of the Mal-pighian tubules. Although Kato demonstrated photographically the transitionof the distal segment into the proximal segment, he apparently consideredthe proximal segment to be merely the beginning of the Malpighian ductproper.

Licent in describing the cytology of the proximal region notes merely thepresence of a cytoplasmic network of strongly staining fines trabecules and thepresence of a stellate nucleus. With the use of acid cytoplasmic dyes, as inthe usual histological techniques, a cytoplasmic network is the only structurerevealed. It is only after the use of basic dyes that the cytoplasmic structureis fully revealed. The nuclei in sections do appear to be stellate but in thefreshly dissected living tubule they are clearly seen to be smooth in outline.

The observations on the granules and vacuolated mucocomplex suggestsa secretory cycle. It is evident that the disappearance of granules coincideswith the appearance of a vacuolated mucocomplex. Since the appearance insome sections is suggestive of coalescing granules it is tentatively suggestedthat the vacuolated mucocomplex is formed by the coalescence and vacuola-tion of the granular mucocomplex and that this progresses outwards from theluminal cell border. It may be that the granular mucocomplex 'swells' orthat the granules serve as 'centres' for the formation of additional muco-complex, but whatever the chemophysical nature of the process is, the result-ing cytological picture might be the same.

The observations can be interpreted so as to yield 2 alternative ways inwhich granules could be transformed into vacuolated mucocomplex.

First, intragranular vacuolation (fig. 4, B) followed by coalescence (fig. 4, c)could produce vacuolated mucocomplex (fig. 4, D). Vacuolated granules,however, were rarely observed in sections and even more rarely in squashes.

Secondly, coalescence of superjacent granules (fig. 4, F, G), followed byintergranular vacuolation, occurring between granules of adjacent stacks(fig. 4, G, H), could give rise to vacuolated mucocomplex (fig. 4, H).

The possibility of both processes occurring simultaneously in differentgranules is not discounted.

The mucocomplex is then extruded into the Malpighian tubule lumen andpasses into the hindgut.

It is suggested that the events of the secretory cycle occur more or lesssynchronously in all 4 proximal segments of the nymph.

262 Marshall—Spittle production by cercopoid nymphs

Part of this work was carried out at the Department of Zoology and AppliedEntomology, Imperial College, London, during the tenure of a D.S.I.R.studentship. I am grateful to Professor 0 . W. Richards (Department ofZoology and Applied Entomology, Imperial College) for facilities in his.

INTRAGRANULAR VACUOLATION

A B C

INTERGRANULAR VACUOLATION

F

FIG. 4. Schematic representation of the coalescence of granules, illustrating the hypotheses ofintragranular and intergranular vacuolation.

Department, where this work was commenced. My gratitude is expressed toDr. D. F. Waterhouse (C.S.I.R.O., Division of Entomology, Australia) forconstructive criticism of the manuscript and I am indebted to Mr. T. C. Jim(Department of Zoology, University of Hong Kong) for photographic assis-tance. My thanks are also due to Dr. D. Fewkes (Central Agricultural Re-search Station, Trinidad) for specimens of A. varia.

ReferencesCecil, R., 1930. Ohio J. Sci., 30, 120.Day, M. F., and Briggs, M., 1958. J. Ult. Res., a, 239.Kato, K., 1958. Science Reports of Saitama University (Ser. B), 3, 33.Kershaw, J. C, 1914. Psyche, Camb., Mass., 21, 65.Kramer, H., and Windrum, G. M., 1955. J. Histochem. Cytochem., 3, 227.Licent, E., 1912. Cellule, 28, 7.Maa, T. C , 1963. Pacific Ins. Monogr., 5, 1.McManus, J. F. A., and Mowry, R. W., i960. Staining methods, histologic and histochemical.

New York (Hoeber).Pesson, P., 1956. Boll. Lab. Zool. Portici, Naples, 33, 341.Salthouse, T. N., 1958. Canad. Ent., 90, 555.Smith, D. S., and Littau, V. C, i960. J. biophys. biochem. Cytol., 8, 103.