Ovariole microstructure and vitellogenesis in Lygocoris pabulinus (L.) and other mirids (Hemiptera : Miridae)

J . A. W I G H T M A N Unicersity of Bristol Research Station, Long Ashton, Bristol"

S Y N O P S I S

The microstructure of the ovariole of Lygocoris pubulinus is described, and an account is given of the method of yolk production. The significance of variations in the structure of the germarium in Heteroptera is dis- cussed.

I N T R O D U C T I O N

Methods of yolk formation in insects have been reviewed by Telfer (1965). Most of the available evidence suggests that the ovary absorbs from the haemolymph specific proteins and carbohydrate, which pass into the oocytes either through or between the cells forming the follicular epithelium. King & Richards (1969) have followed the passage of C1*-labelled glycine from the mid-gut epithelium to the fat body and subsequently into the protein yolk spheres of the egg in Nusoniu vitripennis (Walker). This confirms the suggestion made by Telfer (1954) that protein yolk, or at least its precursor, is synthesised in the fat body before passing to the ovary. In most species yolk is in the form of membrane-delimited spheres of a protein-carbohydrate complex interspersed with lipid spheres.

As stated by Leston (1961), Miridae have been less popular as a source of experi- mental material than have other Heteropteran families. This study was carried out to compare the ovariole structure of Mirids with that of other Heteropteran species described by other workers, using histological and histochemical methods. Aspects of vitellogenesis were investigated with auto-radiographical and disc electrophoresis techniques.

M A T E R I A L S A N D M E T H O D S

.Uults and fifth instar larvae of Lygocoris pubulinus (L.) were bred in glass-house cages (Wightman, 1969) or were collected from the field in summer. Adults were dissected alive, in 0.7 per cent. saline containing 0.01 per cent. Ethylan C.P. (surfactant), by pulling the abdomen tip and thorax apart with watchmaker's forceps. The resulting split between the third and fourth abdominal segments exposed the ovaries, which were teased apart and processed. Complete abdomens of fifth instar nymphs were

Present address : Entomology Division, c/o Crop Research Division, D.S.I.R., Private Bag, Christchurch, New Zealand.

' 0 3

1 04 J.A. U’ightmun

examined. The histological and histochemical techniques of Pearse (1960), Pantin (1962) and Barka & Anderson (1963) were used except when another author is cited. Ovarioles were fixed and stored in aqueous Bouin’s fluid for general histology and histochemistry and Clark’s or Carnoy’s fluid for nucleic acid tests. The material was dehydrated and cleared in dioxan (1-4, Dioxane) and impregnated with ester or paraffin wax (MP 52’ C). Sections 6-10 p thick were cut in the normal way. Eggs and ovarioles to be tested for lipid were fixed in formol-saline for 24 hours and embedded in wot er- soluble wax from which water was expelled by heating to 175O C. for 30 seconds and storing at 60” C. for a minimum of four weeks before use (Firminger, 1950). The fixed material was washed in 0.7 per cent. saline for 1 5 minutes, infiltered with water wax for 24 hours and set in blocks which were chilled in a current of air; rapid cooling in a refrigerator did not prevent wax crystallising (cf. Wade, 1952). Blocks were stored in a desiccator over calcium chloride granules at room temperature. Sections 1 5 p thick were attached to grease-free slides by the method of Giovacchini (1958). Lipids were also stained in fresh tissue with lipophilic dyes. A 0.1 per cent. solution of Trypan blue was injected into the haemocoel of adult females, and the ovaries were fixed after 3 hours. Wax sections were counterstained with I per cent. alcoholic picric acid solution.

The following techniques were used for histological and histochemical investiga- tions.

Histology. Ehrlich’s haematoxylin with eosin, Mann’s methyl blue with eosin and Masson’s trichrome method proved satisfactory.

Proteins. The mercuric-bromophenol blue (Hg-BPB) and ninhydrin-Schiff (Itchi- kawa and Yatsuma) methods were used.

Carbohydrates. The periodic acid-Schiff (PAS) test, as modified by Demalsey & Callebaut (1967) with diastase and pepsin digestion, methanol-chloroform lipid extrac- tion and periodic acid omission controls were used as a general carbohydrate test. Glycogen was stained with Best’s carmine controlled by diastase digestion. Acid muco- polysaccharide was stained with alcian or astra blue.

Nucleic acids. DNA was stained by the Feulgen technique as modified by Demalsey & Callebaut ( I 967) with deoxyribonuclease (DNase) digestion and hydrochloric acid hydrolysis controls. Methyl green-pyronin (MGP) was used for RNA and DNA and the toluidine blue-ribonuclease (RNase) test used for RNA.

Lipids. Sudan I11 and IV and oil red 0 were used to stain neutral lipid in water wax sections and whole mounts; luxol fast blue (Salthouse, 1962) was used to stain phospholipid.

Autoradiography. Two microlitres ( = 2 pc) of tritiated leucine and glucose were in- jected into the haemocoel of adult females, which were dissected after 3 hours. The ovaries were excised, fixed in Carnoy’s fluid and embedded in paraffin wax. Sections were mounted on “subbed” slides and fine grain autoradiographic stripping film (Kodak AR 10). The autoradiographs were processed after seven days incubation.

Electrophoresis. The protein content of male and female blood and of macerated eggs was compared by using the polyacrylamide-gel disc electrophoresis technique of Davis & Ornstein (1961). Blood and yolk were suspended in 10 per cent. sucrose solution.

Ovarioles of seven other mirid species from five of the six subfamilies found in Britain were also sectioned and stained with Ehrlich’s haematoxylin and eosin. These were Dicyphus epilobii Reuter (Dicyphinae), Deraeocoris ruber (I,.) (Deracocorinae), Orthotylus

Ot~ariole microstructure in Lygocovis pubulinus 105

pavospuvsus (Sahlberg), Heterotoma merioptera (Scopoli) (Orthotylinae), Plagiognuthus arbustorum (F.) (Phylinae), Liocoris tripustulutus (F.) and Calocoris norvegicus (Gmelin) (Mirinae).

0 €3 S E R VAT I 0 N S

L . pabulinus has two ovaries, each containing eight ovarioles. The pedicels fuse to form the lateral oviducts and join the genital chamber, which is the modified median oviduct. The morphology of the female internal genitalia of L. pubulinus is similar to that of Lygus lineoluris (Palisot de Beauvois) (Davis, 1955). The pedicels and lateral oviducts

I f l l 25 Micrcns I

TERMINAL FILAMENT

TROPHIC NUCLEUS

LINES OF FLOW

NTERFOLLICULAR

Fig. I. Diagram of anterior region of an ovariole.

100 y.i1. I V&ll ItIl(111

stretch to accommodate chorionated eggs prior to oviposition. In some young adults egg development \\.as proceeding in only one ovary at the time of dissection.

Germarium 1 he germarium is pyriform and dpproximately 0.15 mm. \\ ide and 0.18 mm. long (fig. I ) . In adult and fifth instar nymphs there are no zones of cell division. The amoeboid trophic nuclei are arranged in pockets around the basiphilic trophic core; no nucleoli were observed in these nuclei. In haemotoxylin-stained sections, lines of flow were detected running from the region of the trophic nuclei to the posterior of this region. Migratory nuclei were occasionally observed in the trophic core, appearing as if they had been cast from the trophic nuclei. The trophic core was highly basiphilic in adults, and even in fifth instar nymphs there was slight basiphilia. Histochemical tests (MGP and Toluidine blue-RNase) showed that this was due to large amounts of RNA, which was particularly dense in the peripheral regions. Globules of RNA were observed in the trophic nuclei close to the nuclear membrane, appearing as if they had been fixed just before passing through the membrane. The RNA in the trophic core was con- tiguous with that being passed to the oocytes in the trophic cord, which was also pyronin positive. The trophic nuclei were intensely positive to Feulgen and methyl green; no extra-nuclear DNA was detected. Occasionally spheres of unknown origin, 14-16 p in diameter and heavily stained by the Hg-BPB, ninhydrin-Schiff and PAS reactions, were seen in the posterior region of the germarium. The trophic core was heavily stained by luxol fast blue, indicating the presence of phospholipid. In whole ovarioles treated with oil red 0 and Sudan 111 and IV the trophic core region was lightly stained, indicating the presence of neutral lipid. Water wax section confirmed this observation and also indicated that there was a very small quantity of lipid in the trophic cord.

The germaria from insects injected with trypan blue had hea<y deposits in the trophic nuclei but none in the trophic cord. Similarly, small amounts of both tritiated tracers were detected in the periphery of the trophic nuclei. These observations indicate that germaria are capable of taking up material from the haemolymph.

r 7

Vitellariunz The oogonia are situated immediately posterior to the trophic core and are theoretically part of the germarium. However, as they are embedded in prefollicular tissue it is more convenient to consider them as part of the vitellarium in this species. Oocyte growth can be divided into six arbitrary stages, distinguished by the structure of the follicle cells (fig. 2).

Stuge I. Cell division takes place in the anterior region of the vitellarium. The oogonia and follicular mother cells can be distinguished, the former having larger nuclei than prefollicle cells of the same size. The cytoplasm and nucleoli of the pre- follicle cells are basiphilic, due to a high RNA content. Their cytoplasm was heavily stained by Hg-BPB and ninhydrin-Schiff, indicating the presence of protein. These characteristics are retained throughout development.

Stage 2. The follicle cells are columnar and are radially orientated around the developing oocyte, to which the trophic core is attached. They become binucleate, possibly by a form of endomitosis, as no mitosis was observed at this stage or in inter- mediate stages. KNX is concentrated in the bases of the follicle cells, but there is no

STAGE 2

STAGE 6

Not t o Scak

Fig. 2. Stages in oocyte development. (I) Oocytes and prefollicular tissue barely differentiated, except for larger nuclei in oocytes. (2) Follicle cells columnar and bi- nucleate. (3) Follicle cells columnar to cuboid, vitellogenesis commenced. (1) Follicle cells mostly binucleate, with villi extending to oocytes. ( 5 ) Chorion being deposited ; all follicle cells uninucleate. (6) Follicle cells disintegrate when chorion formed.

evidence to suggest that it was transmitted to the oocyte. The ooplasm becomes increas- ingly basiphilic/RNA positive during this stage, particularly at the point of entry of the trophic cord. In wax sections the egg had a reticulate appearance, possibly due to the removal during processing of lipid droplets which were the precursors of small ( I p diameter) lipid spheres appearing in the cytoplasm late in this stage. Small quantities of tritiated leucine and trypan blue were observed in the peripheral regions of the oocyte but no tritiated glucose was taken up.

Stage 3. As the oocyte grows, the follicle cells flatten until they are cuboid. The nuclei are large, with prominent nucleoli. Albuminous yolk spheres appear around the bases of the follicle cells, which are heavily stained by Hg-BPB, ninhydrin-Schiff and PAS ; all these heavily stained the follicle cell/ooplasm interface, suggesting the presence of a brush border. Yolk spheres are eosinophilic (acidophilic). The germinal vesicle (oocyte nucleus) is in a lateral position at this stage.

Stage 4. Some of the follicle cells become uninucleate and, characteristically, forni villi extending into the ooplasm, presumably increasing the surface area available for the transmission of yolk precursors. As the oocyte grows by yolk incorporation the follicle

cells become more squamous, except at the anterior pole, where cap formation will take place. Yolk spheres (3-5 p diameter) are present throughout the ooplasm by this stage. The histochemical tests (PAS and controls) suggested that the albuminous spheres are mostly mucoprotein. Glycogen, which was first detected at this stage, was in the form of granules in the ooplasm between the yolk spheres; it is, however, possible that the granular appearance was a fixation artefact. Follicle cell cytoplasm and nucleoli were also stained by Best’s carmine, the nuclei less so. The yolk spheres contained no glycogen. Tritiated glucose, the precursor of glycogen, was concentrated in the basal half of the follicle cells and between the yolk spheres in the oocyte. This suggests that glycogen is synthesised in the follicle cells before passing into the oocyte. In insects injected with trypan blue the dye penetrated between the follicle cells and into the peripheral ooplasm, although none was seen in the yolk spheres. l’ritiated leucine was concentrated in the follicle cells along the follicle cell/oocyte interface in the ooplasm and in smaller peripheral yolk spheres. Follicle cell nuclei did not take up the tracer. Acid-mucopolysaccharide had a similar distribution to that of glycogen. As well as staining all membranes, luxol fast blue visualised phospholipid in the peri- pheral ooplasm, indicating high concentration in that region. Neutral lipid was distri- buted throughout the yolk in 5-10 p diameter spheres.

MALE

I I

L FEMALE

1

2

3 4 5

6

7

EGG

4 5

Fig. 3. Diagram of disc-electrophoresis gels. Stippling and shading indicate the intensity of staining of the various bands.

Or3ariole microstructure in Eygocoris pabulinus 109

Stage 5. The chorion is formed during this stage. The follicle cells are all uninucleate and squamous, except in the cap region, where they are columnar. There were two layers in the chorion, but further subdivisions were not detected. The inner layer was positive to PAS, Hg-BPB, ninhydrin-Schiff, alcian blue, astra blue and Sudan I11 and IV, but the outer layer remained unstained in all tests.

Stage 6. The follicular epithelium disintegrates after the chorion is formed and ruptures, allowing the egg to pass down the lateral oviduct, leaving the remains of the epithelial tissue in the pedicel. Whole mounts stained with Sudan I11 and IV and oil red 0 showed that the pedicular plug contained relatively large deposits of lipid in the form of globules 5-20 p in diameter.

Seven proteins were detected in the female blood (fig. 3). Five of these were present in the male and two, with a very faint trace of a third (not figured), were found in the egg. The egg proteins (bands 4 and 5) were heavily stained in both egg extracts and the female blood, but were absent in the male blood. I t is assumed that these are the vitello- genic proteins.

Ovarioles rarely contained more than three follicles. In a typical mature female there might be three to five Stage I oocytes, with one Stage 3 and one Stage j oocyte, in an ovariole, and one or more eggs stored in the pedicel.

The ovarioles of the seven other mirid species that were examined histologically had the same basic structure as that of L. pabulirius, in that the germaria had no apical zones of cell division and the trophic nuclei were in pockets around the periphery of a basiphilic trophic core. The only apparent difference was morphological, L. pabulinus having eight overioles and the others seven, which is the normal number found in Mirids. Leptoterna dolobrata (L.), L. ferrwguta (FallCn), Calocoris hispanicus Gnielin and Deraeo- cork ruber (L.) also have eight ovarioles, and some other species of Leptoterna have five (Woodward, 1950; Carayon, 1950).

D I S C U S S I O N

Germarium The Heteroptera are usually divided into Hydrocorisae (water bugs), Amphibicorisae (water-surface bugs) and Geocorisae (land bugs) (Southwood & Leston, 19 59). The Geocorisae can be further divided into Pentatomomorpha and Cimicomorpha (Leston, Pendergrast & Southwood, I 954). The former includes such superfamilies as Pentato- moidea, Coreoidea and Lygaeoidea and the latter Reduvioidea and Cimicoidea. This system of classification is not totally accepted (see below) but is a useful guide.

The heteropteran ovariole has not been widely studied although, fortuitously, representatives of some major taxa have been investigated, i.e. the hydrocorisan Gerris remigis Say (Gerridae) (Eschenberg & Dunlap, 1966), the pentatomomorphan Onco- peltus fasciatus (Dallas) (Lygaeidae) (Bonhag & Wick, 1953 ; Bonhag, 195 j), Acantho- cephala spp. (Coriedae) (Schrader & Leuchtenberger, 1952), Pyrrhocoris apterus (L.) (Pyrrhocoridae) (Bielanska-Osuchowska, I 960) and the cimicomorphans Cimex lectu- Iarius I,. (Cimicidae) (Davis, 1956) and Rhodnius prolixus Stil (Reduviidae) (Vanderberg, 1963). The Miridae are included in the Cimicomorpha.

The germaria of the Pentatomomorphan species have several characteristics in common. They can be divided into a number of zones of development with dividing and newly formed cells in the apex with trophic nuclei, devoid of cytoplasm, growing

I I 6 J.A. IViglztman

either by fusion or endoniitosis as they progress posteriorly. DNA is released into the trophic core by two methods. In 0. fasciatus trophic nuclei migrate into the trophic core and breakdown, releasing their contents, whereas in P. apterzis droplets of DNA are extruded with no nuclear break down. Both processes occur in Acanthocephala. DNA has not been detected in the trophic cord, although all authors have stressed that RXA4 is present in relatively high concentrations. In C. lectulurius there is a gradation

COREIDAE -

LYGAEIDAE -

iil -I

2 5 0 a I

8 I-

-J

I-

z > v)

P

s

z 0

0 %

8 z z

2 O k w 2

?

1 PYRRHOCORIDAE

i \\' CI

!

8 MlRlDAE

' CI M IClDAE

REDUVllDAE

\ \

\ ' ,:;\I \\

Fig. 4. A possible evolutionary sequence of several heteropteran families, based upon germarium structure and function.

Orariole nzirrostructzire in 1,ygororis pubzilinus 111

in nuclear size in the germarium, but no cell division v as observed in the adult; as in L. pabzilinus, RNA is extruded by the trophic nuclei with no nuclear break down. The Redu! iid, R. prolixus, is intermediate between the two types because the trophic nuclei synthesise RXA, but cell division occurs in the apex of the germarium. The germaria of G. remigis differ from those of the Cimicomorpha and Pentatamomorpha in that the trophocytes retain their individuality until they reach the posterior end of the germarium, and both RNA and DNA are apparently secreted into the trophic core, DNA being released by both nuclear break down and extrusion. Distinctions can therefore be made between the germarial structures of the major heteropteran taxa.

Most of the evidence available suggests the main function of the trophic tissue in telotrophic ovarioles is to synthesise the ooplasmic nucleic acid, which is probably only RNA in the Insecta. As no DNA has been observed in the trophic flow, it is assumed that RNA is formed in the trophic core in the pentatomomorphan species. In the Cimi- coidea and Redukioidea it is secreted by the trophic nuclei, a method which can be considered more efficient, as no nuclear break-down occurs; hence the lack of actively dividing cells in the apex of the germaria in C. lectularius and L. pabulinus. The mirid germarium, in which the trophic nuclei are arranged around the central core, is probably the most efficient.

Assuming that the species so far described are truly representative of their families or superfamilies, it is possible to construct an evolutionary series based mainly on the method of RNA synthesis in the germarium (fig. 4). The Cimicoidea are the most efficient, the Miridae being placed above the Cimicidae. Among the Pentatomomorpha, the Lygaeidae are allotted lowest place because a continuous replacement of trophic nuclei is required, the Pyrrhocoridae are the most efficient because no nuclear break down occurs, and the Coreidae are intermediate. The Reduviidae lie between the Cimicoidea and Pentatomomorpha. The Hydrocorisae (Gerridae) are the most primitive, as the trophic nuclei retain their cytoplasm.

Previous attempts to elucidate the phylogeny within the Heteroptera have been based on cytology, egg structure and the anatomy of genitalia (e.g. Leston, 1958; Manna, 1958, 1962; Bannerjee, 1958; IbIcDonald, 1966; Cobben, 1968) and are partially re- viewed by Cobben (1968). Ranerjee considered the Lygaeidae to be relatively primitive, but placed the Coreidae above the Pyrrhocoridae. Manna disagrees with Banerjee, in that he considers that the Miridae, Cimicidae and Reduviidae to have arisen from a Nabid stock, although the primitive nature of the Lygaeidae is upheld. Cobben states that the Amphibicorisae are primitive and may have given rise to the Geocorisae; he also separates the Reduvioidea from the Cimicomorpha.

Vitellogenesis

,\lthough the protein moiety of insect egg yolk at least sometimes includes material from an ovarian source such as the follicle cells (Bell, 1970; Anderson & Telfer, 1969), the bulk of this yolk protein has an extra-ovarian origin. This has been discovered by using labelled tracers in Panorpa comniunis I,. (Ramamurty, 1964) and Saturniid moths (Telfer, 1961), for example, and by the observation of the entry of yolk at the oocyte periphery by pinocytosis in electronrriicrographs in several species including IJygaezis kabnii Stil (Kessel & Beams, 1963), Bombus terrestris (L.) (Hopkins & King, 1964), Periplaneta americann (I,.) (Anderson, 1964) and .Vasoniu 7,itripenni.v (King & Richards,

I I 2 J.A. Wightman

I 969). The result of the present disc electrophoresis experiment confirms that there are “female” or vitellogenic proteins in L. pabulinus, and the autoradiography shows that they enter the egg cia the follicle cells. The presence of interfollicular spaces was shown by the penetration of trypan blue.

Telfer (1965) queries the fate of the membranes enclosing pinocytotic vesicles in which yolk precursors pass into the oocyte and points out that an excess of membrane material will accumulate if yolk spheres are formed by the fusion of the pinocytotic vesicles. As biological membranes contain phospholipid, it is probable that peripheral staining in oocyte sections treated with luxol fast blue is due to this excess membrane being liberated in the ooplasm as a result of the pinocytotic uptake of yolk precursors.

The origin of lipid yolk in L.pabulinus is not known. Since Martin (19694 b) has shown that the fat body contributes lipid to the yolk in Pyrrhocoris apterus, the possibility of lipid synthesis in situ in L.pabulinus seems unlikely. Traces of lipid were detected in the germarium and trophic core, but it is not known what proportion of the deutero- plasmic lipid entered the egg by this route. The origin of the lipid in the pedicular plug, which is composed of degenerated follicle cells, is not known. The follicle cells may be involved in passing or metabolising lipid prior to its incorporation into the deuteroplasm, and the lipid in the pedicular plugs may be an excess of that material extracted from the haemolymph. Another possibility is that as the follicular epithelium secretes the chorion, in which lipid material is incorporated, the lipid could be a by-product of that process.

The last yolk component to enter the oocyte is glycogen. This tardiness has been observed in several species including Panorpa communis (Ramamurty, I 968), Apis mel1;Jica (L.) (Engles & Drescher, 1964) and Phyllobius urticae DeGeer (Bielanska- Osuchowska, 1960) ; in P.urticae glycogen did not appear until after chorion formation. I t is believed that there may be antagonism between the processes of protein and of carbohydrate formation (Engles & Drescher, 1964). In Musca domestica L. the enzyme responsible for glycogen formation (glycogen synthetase) is present in presumptive eggs from the early stages of oogenesis but is not activated until the protein yolk is formed (Engles & Bier, 1967). In Bombyx mori L., in which vitellogenesis occurs at the pupal stage, protein and glycogen are formed at the same time (Yamashita & Hase- gawa, 1969). The method of ooplasmic glycogen synthesis in L.pabulinus is different from that found in Panorpa communis by Ramamurty (1968), who found no evidence for the incorporation of precursors in the follicle cells. In the Mirid the results of the autoradiography and histochemistry imply that the inner region of the follicle cells is involved in glycogen synthesis. No labelled glucose was found in the yolk spheres in L.pabulinus, whereas in Panorpa communis labelled glucose was incorporated in the yolk spheres as well as in the ooplasm. Another variation was found in Bombus terrestris by Hopkins & King (1964) who observed two types of yolk; one contained glycogen and was thought to be the precursor of the other. I t appears that although most of the proteinaceous yolk enters the egg by a similar process throughout the Insecta, there is variation in both the nature and the source of the carbohydrate components.

S U M M A R Y

The ovariole structure of Lygocoris pabulinus (L.) and other Mirids differs from that of other Heteroptera. The germarium is pyriform with acellular trophic nuclei arranged

Ocariole microstructure in Lygoronk pabailinus 1'3

in pockets around the periphery. There are no zones of cell division and growth. The trophic nuclei secrete RNL4, which is passed to the developing eggs by the trophic cord. hlucoprotein and neutral lipid are the major yolk components, with glycogen interspersed in the ooplasm. T h e protein has an extraovarian source. Glycogen is probably synthesised in the follicle cells.

An e\ olutionary series based on the structure and function of the Heteropteran gzrmarium can be constructed. In the primitive (gerrid) form, trophocytes retain their cytoplasm, and the trophic nuclei release both D N A and RNL4. Among the Geocorisae, the more primitive forms such as Lygaeidae release D N A into the trophic core by nuclear break down. I n the higher Pentatomoidea, DIVA is secreted without nuclear break down. In the hliridae and Cimicidae, highest in the series, the trophic nuclei secrete RNAA, and there are no apical dividing cells in this type of germarium. T h e Keduviidae fall bctneen the pentatomoid and cimicoid lines.

'l'his research was carried out during the tenure of a Boots Scholarship and forms part of a dissertation submitted to the University of Bristol for the Doctor of Philosophy degree. I am grateful to Professor H.G.H.Kearns and his successor Professor J.P.Hudson for the provision of laboratory facilities, to M.Wright for carrying out the disc electro- phoresis, and to D r P.E.King, University College, Swansea, and M r M.E.Solomon Long Ashton Research Station, for their comments on the manuscript.

REFERENCES

ANDERSON E. I 964. Oocyte differentiation and vitellogenesis in the roach, Periplaneta anzericana.

ANDERSON L.M. & TELFER W.H. 1969. A follicle cell contribution to the yolk spheres of moth

BANERJEE h1.K. 1958. A study of the chromosomes during meiosis in twenty-eight species of

BARKA T. & ANDERSON P. J. 1963. Histochemistry theory, practice and bibliography. New York. BELL J.W. 1970. Demonstration and characterization of two vitellogenic blood proteins in Peri-

planeta americana: an immunochemical analysis. J . insect Physiol. 16 : 291-9. BIELANSKA-OSUCHOWSKA Z. I 960. Histochemical studies on insect telotrophic ovaries. Zool.

Poloniae 10 : 131-63. BOXHAG P.F. 1955. Histochemical studies of the ovarian nurse tissues and oocytes of the milk-

weed bug, Oncopeltus fasciatus (Dallas). J . Morph. 96 : 381-439. BONHAG P.F. & WICK J.R. 1953. The functional anatomy of the male and female reproductive

systems of the milk weed bug, Oncopeltus fasciatus (Dallas) (Heteroptera : Lygaeidae).

CARAYON J. 1950. Nombre et disposition des ovarioles dans les ovaires des Hemiptirres-HCtirrop- tttres. Bull. Mus . natn. Hist . nat. Paris 22 : 470-75.

CORBEN R.H. 1968. Ecolutionary trends in Heteroptera, I . Eggs, architecture of the shell, gross embryology and eclosion. Agricultural Research Report 707, Agricultural University, Wageningen.

J . Cell. Biol. 20 : 131-55.

oocytes. Tissue Cell. I : 633-44.

Hemiptera (Heteroptera, Homoptera). Proc. zool. Sac. Calcutta 11 : 9-37.

J . Morph. 93 : 177-283.

DAVIS B.J. & ORNSTEIN L. 1961. Disc electrophoresis. Rochester. D.AYIS N.T. 1955. hlorphology of the female organs of reproduction in the Rliridae (Hemiptera).

DAVIS N.T. 1956. The morphology and functional anatomy of the male and female reproductixre

DEMALSEY P. & CALLEBALJT M. 1967. Plain water as a rinsing agent preferable to sulfurous acid

Ann. ent. Soc. Ain. 48 : 132-50.

systems of Ciinex lectzrlarius L. (Heteroptera, Cimicidae). Ibid. 49 : 466-93.

aftrr the Feulgen nucleal reaction. Sta in Technol. 42 : 133-6.

8

j. A. Wightman

ENGLES W. & BIER I<. 1967. Zur Glykogen-Speicherung wahrend der Oogenese und ihrer vor- zeitigen Auslxung durch Blockierung der RNS-Versorgung (Cntersuchungen an Musca domesticus L,.). Arch. Entzcick. Mech. Org. 158 : 64-88.

ENGLES W. & DRESCHER W. 1964. Einbau von H3-D-Glucose wahrend der Oogenese hei Apis mellifica L. Experientia 20 : 445-6.

ESCHENBERG K.M. & DUNLAP H.L. 1966. The histology and histochemistry o f oogenesis in the water strider, Gerris remigis Say. J . Morph. 118 : 297-316.

FIRMINGER H.I. 1950. Carbowax embedding for obtaining thin tissue sections and the studying of intracellular lipids. Stain Technol. 25 : 121-4.

GIOVACCHINI R.P. 1958. Affixing carbowax sections to slides for routine staining. Stain Technol.

HOPKINS C.R. & KING P.E. 1964. Occurrence of microvilli and micropinocytosis in trophocytes

KESSEL R.G. & BEAMS H.W. 1963. Micropinocytosis and yolk formation in oocytes of the small

KING P.E. & RICHARDS J.G. 1969. Oogenesis in Nasonia zitripennis (Walker) (Hymenoptera :

LESTON D. I 958. Chromosome number and the systematics of Pentatomomorpha (Hemiptera).

LESTON D. 1961. Testis follicle number and the higher systematics of Miridae (Hemiptera-

LESTON D., PENDERGRAST J.G. & SOUTHWOOD T.R.E. 1954. Classification of the terrestrial

MCDONALD F.J.D. 1966. The genitalia of North American Pentatomoidea (Hemiptera : Heter-

MANNA G.K. 1958. Cytology and interrelationships between various groups of Heteroptera.

MANNA G.K. I 962. A further evaluation of the cytology and inter-relationships between various

MARTIN J.S. 1969a. Lipid composition of fat body and its contribution to the maturing oocytes

MARTIN J.S. 1969b. Studies on assimilation, mobilization, and transport of lipids by the fat body

PANTIN C.F.A. 1962. Notes on microscopical technique f o r zoologists, Cambridge, University Press. PEARSE A.G.E. 1960. Histochemistry, theoretical and applied, London. RAMAMURTY P.S. 1964. The contribution of the follicle epithelium to the deposition of yolk to

the oocyte of Panorpa communis. Expl. Cell. Res. 33 : 601-4. RAMAMURTY P.S. 1968. Origin and distribution of glycogen during vitellogenesis of the scorpion

fly, Panorpa communis. J . Insect Physiol. 14 : 1325-30. SALTHOUSE T.N. 1962. Luxol fast blue ARN: A new solvent azodye with improved staining

qualities for myelin and phospholipid. Stain Technol. 37 : 313-16. SCHRADER F. & LEUCHTENBERGER C. 1952. The origin of certain nutritive substances in the eggs

of Hemiptera. Expl. Cell Res. 3 : 136-46. SOUTHWOOD T.R.E. & LESTON D. 1959. Land and water bugs of the British Isles. London. TELFER W.H. 1954. Immunological studies of insect metamorphosis; 11. The role of a sex-limited

blood protein in egg formation by the cecropia silkworm. J . gen. Physiol. 37 : 539-58. TELFER W.H. 1961. The route of entry and localisation of blood proteins in the oocytes of

saturniid moths. J . biophys. biochem. Cytol. 9 : 747-59. TELFER W.H. 1965. The mechanism and control of yolk formation. A . Rev. Ent. 10 : 161-84. VANDERBERG J.P. 1963. Synthesis and transfer of DNA, RNA and protein during vitellogenesis

WADE H.W. 1952. Notes on the carbowax method of making tissue sections. Stain Technol.

WIGHTMAN J.A. 1969. Rearing and feeding Lygocoris pabulinus (Heteroptera : Sliridae). Rep.

33 : 247-8.

of Bombus. Nature, Land. 204 : 298-9.

milk weed bug (Lygaeus kalmii Stil.). Expt. Cell. Res. 30 : 440-3.

Pteromalidae). Proc. R. ent. Sac. Land. (A). 44 : 143-57.

Proc. 10th int. Congr. Ent. Montreal (1956) 2 : 911-8.

Heteroptera). Proc. zool. Sac. Land. 137 : 89-106.

Heteroptera (Geocorisae). Nature, Land. 174 : 91-2.

optera). Quaest. Ent. 2 : 7-150.

Proc. 10th int. Congr. Ent. Montreal (1956) 2 : 919-34.

groups of Heteroptera. Nucleus 5 : 7-28.

in Pyrrhocoris apterus. J . Insect Physiol. 15 : 1025-45.

and haemolymph of Pyrrhocoris apterus. J . Insect Physiol. 15 : 2319-44.

in Rhodnius prolixus (Hemiptera). Biol. Bdl. 125 : 556-75.

27 : 71-9.

Long Ashton Res. Stn. 1968 : 157-60.

Ovariole microstructure in Lygororis pabulinus I I j

WOODWARD T.E. I 950. Ovariole and testis follicle numbers in the Heteroptera. Entomologist’s

YAMASHITA 0. & HASEGAWX I<. 1969. Oocyte age and glycogen synthesis in the pupal ovaries of mon. .Wag. 86 : 82-4.

the silk worm, Bombyx mori L. (Lep., Bombycidae). Appl. entomol. Zool. 4 : 203-10.

Manuscript received 7th July, 1971