PARTICLES PRESENT IN THE HAEMOLYMPH AND DEFENSIVE ... · phoretic and ultracentrifugal propertie of...

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J. Cell Sci. 4, 369-379 (1969) 369 Printed in Great Britain PARTICLES PRESENT IN THE HAEMOLYMPH AND DEFENSIVE SECRETIONS OF INSECTS D. KAY Sir William Dunn Scliool of Patliology, Oxford MIRIAM ROTHSCHILD Ashton, Peterborough AND R. APLIN Dyson Perrins Laboratory, Oxford, England SUMMARY Defensive fluids obtained from the Garden Tiger, Scarlet Tiger and Burnet Moths and from the Monarch Butterfly and the Seven-Spot Ladybird Beetle, haemolymphs taken from the wasp, the Burying Beetle and the Rat Flea, and fluids from the eggs of the Garden Tiger, Gypsy Moth and Oak Eggar Moth were examined in the electron microscope after negative staining. Except for the ladybird all the defensive fluids, haemolymphs and egg fluids contained particles 100-150 A across which frequently had rectangular or square outlines. In some species the particles were occasionally arranged in groups of 2 and 4 and in the egg fluid of the Garden Tiger they were arranged in rows which were aligned side by side to form strands of considerable length. The particles are believed to be protein. They correspond in size to the 16 s component of the haemocyanin8 of Limulus and Homarus and the protein particles found in Calliphora by other workers. INTRODUCTION A not inconsiderable amount of information has been accumulated on the electro- phoretic and ultracentrifugal properties of the proteins of insect haemolymph, but there have been relatively few studies made, with the aid of the electron microscope, of the size and shape of the protein molecules found in insect body fluids. Certain invertebrates such as squids and snails (Mollusca) possess haemocyanins in their haemolymph, which are very large protein molecules, measuring up to 340 x 680 A (Fernandez-Moran, Van Bruggen & Ohtsuki, 1966), concerned with the transport of oxygen. In the Insecta no molecules of comparable size have been found in the haemolymphs or body fluids. In work described in the present paper the fluids from several species of insects were examined in the electron microscope and well defined particles, tentatively called protein molecules, have been observed. Generally the particles are roughly square in outline and are about 100 A across. Some- times they are seen in groups of 2 or 4, and in one species they aggregated in long chains which aligned themselves in exact register so that strands were formed with a 24 Cell Sci. 4

Transcript of PARTICLES PRESENT IN THE HAEMOLYMPH AND DEFENSIVE ... · phoretic and ultracentrifugal propertie of...

  • J. Cell Sci. 4, 369-379 (1969) 369Printed in Great Britain

    PARTICLES PRESENT IN THE HAEMOLYMPH

    AND DEFENSIVE SECRETIONS OF INSECTS

    D. KAYSir William Dunn Scliool of Patliology, Oxford

    MIRIAM ROTHSCHILDAshton, Peterborough

    AND R. APLINDyson Perrins Laboratory, Oxford, England

    SUMMARYDefensive fluids obtained from the Garden Tiger, Scarlet Tiger and Burnet Moths and

    from the Monarch Butterfly and the Seven-Spot Ladybird Beetle, haemolymphs taken fromthe wasp, the Burying Beetle and the Rat Flea, and fluids from the eggs of the Garden Tiger,Gypsy Moth and Oak Eggar Moth were examined in the electron microscope after negativestaining.

    Except for the ladybird all the defensive fluids, haemolymphs and egg fluids containedparticles 100-150 A across which frequently had rectangular or square outlines. In somespecies the particles were occasionally arranged in groups of 2 and 4 and in the egg fluid of theGarden Tiger they were arranged in rows which were aligned side by side to form strands ofconsiderable length.

    The particles are believed to be protein. They correspond in size to the 16 s componentof the haemocyanin8 of Limulus and Homarus and the protein particles found in Calliphoraby other workers.

    INTRODUCTION

    A not inconsiderable amount of information has been accumulated on the electro-phoretic and ultracentrifugal properties of the proteins of insect haemolymph, butthere have been relatively few studies made, with the aid of the electron microscope,of the size and shape of the protein molecules found in insect body fluids.

    Certain invertebrates such as squids and snails (Mollusca) possess haemocyanins intheir haemolymph, which are very large protein molecules, measuring up to340 x 680 A (Fernandez-Moran, Van Bruggen & Ohtsuki, 1966), concerned with thetransport of oxygen. In the Insecta no molecules of comparable size have been foundin the haemolymphs or body fluids. In work described in the present paper the fluidsfrom several species of insects were examined in the electron microscope andwell defined particles, tentatively called protein molecules, have been observed.Generally the particles are roughly square in outline and are about 100 A across. Some-times they are seen in groups of 2 or 4, and in one species they aggregated in longchains which aligned themselves in exact register so that strands were formed with a

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  • 370 D. Kay, M. RotJischild and R. Aplin

    cross-banded appearance. The square outline particles are similar in size to the 16 sprotein molecules found by Fernandez-Moran et al. (1966) in the haemolymph ofLimulus.

    Certain details could be observed in the fine structure of the square particles, andin some the arrangement of the subunits was just visible.

    MATERIAL

    It is well known that blood cells are frequently found in the defensive secretions ofvarious insects. Thus, for example, the Garden Tiger Moth (Arctia caja L.) initiallyexudes a colourless, pungent fluid from its cervical glands, but yellow haemolymphis subsequently mixed with it and blood cells are then found clotting in the secretion(Rothschild & Haskell, 1966). The nymphal stages of the grasshopper Poekilocerusbufonius Klug forcibly eject the contents of their poison glands in a well-directed jetand this fluid contains blood cells (Von Euw et al. 1967). The adult insect, however,only froths out the contents of the glands and this secretion is free of cells. Manyinsects which do not apparently have specialized defensive glands nevertheless possesspoisonous substances in their body fluids, and in such cases there are often specializedareas in the integument which bleed easily and freely. Such bleeding pointsare present, for example, in the cervical region and tarsal segments of certain moths,at the tip of the antennae and marginal wing-veins of various butterflies, and the legjoints of beetles and grasshoppers, etc. In some instances such bleeding points aresituated in the proximity of defensive gland apertures, so that when a predator seizesthe insect, the haemolymph and gland secretion mix on the surface of the body. Inother cases bleeding points occur within the lumen of a defensive gland.

    We obtained fluid exuded spontaneously from the defensive glands of the GardenTiger Moth (Arctia caja L.) and Scarlet Tiger Moth (Panaxia dominula L.), and theSeven-Spot Ladybird Beetle (Coccinella septempunctata L.) and fluid obtained fromspecific bleeding points, after pressure on the thorax, from the Six-Spot Burnet Moth(Zygaena filipendulae'L.) and the Monarch Butterfly (Danausplexippus L.). Haemo-lymph was extracted from the thorax of the Wasp (Vespula germanica L.), the BuryingBeetle (Necrophorus investigator Zett.) and the Rat Flea (Nosopsyllus fasciatus Bosc).Fluid was also obtained from freshly laid fertile eggs of the Garden Tiger, the GipsyMoth (Lymantria dispar L.) and the Oak Eggar Moth (Lasiocampa quercus L.). Thecontents of the eggs of the Garden Tiger were examined in the electron microscopeafter fixation and thin sectioning.

    METHODS

    The defensive fluid, and fluid from bleeding points, was collected in a fine glasscapillary and diluted in a drop of water or saline (0-85 % NaCl). Haemolymph wasobtained by inserting a fine capillary into the thorax of the insect, and egg fluidcollected by piercing the egg with a fine capillary.

    Specimens of fluid were prepared for electron microscopy by suitable dilution inaline and applied to carbon-coated grids. After a few moments the fluid was washed

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    off with three successive drops of uranyl acetate (1% in water) and the grids weredried by holding against filter paper. In this way the protein molecules and otherparticles were negatively stained which caused them to appear light against a darkbackground in the microscope. In true negative staining there is no chemical combina-tion between the stain and the particle concerned, which is merely surrounded by theelectron-dense stain and perhaps penetrated by the stain if its structure is sufficientlyopen.

    In an attempt to study the arrangement of the protein molecules in situ in the eggsof the Garden Tiger, whole eggs were fixed in Caulfield's osmium tetroxide fixative,dehydrated in alcohol and embedded in Araldite. The attempt failed because therewas no penetration of the resin into the eggs. To overcome this, eggs were cut openand immediately placed in ice-cold Caulfield's fixative for 1 h, after which they weredehydrated and embedded in Araldite by the standard procedure. Sections werestained with lead citrate.

    RESULTS

    The Six-Spot Burnet Moth

    Bright yellow fluid obtained from specific bleeding points on the thorax revealed alarge number of negatively stained particles (Fig. 1). They frequently showed arectangular, almost square outline, and were 100-120 A across (a). Other particlesabout the same size, but of rounded or irregular outline, were also present (b). Occa-sionally particles of circular outline with open centres were seen (c). In some particlesthe arrangement of the subunits was just visible (arrows).

    The Garden Tiger Moth

    Particles of various sizes from 150 to 300 A were seen in the spontaneous emissionsof odoriferous, pale yellow, defensive fluid from this moth (Fig. 2), but the majoritywere about 150-180 A across and some showed a rectangular outline. A tendency forthe particles to form pairs and groups with their edges aligned was noticed.

    The Monarch Butterfly

    The particles seen in the body fluid obtained from specific bleeding points of thisbutterfly were of a fairly uniform size, about 150 A across (Fig. 3). They were some-times rectangular in outline, but more often they were rounded. Frequently theyaggregated in pairs and in groups of four in such a way as to give a close-packedstructure of equal-sized, square-outlined particles (arrow).

    The Scarlet Tiger Moth

    Rectangular particles were abundant in the spontaneous emissions from theprothoracic glands of this moth (Fig. 4). They were generally about 100-120 Aacross, but occasionally particles up to 200 A were seen.

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    The Ladybird Beetle

    Spontaneous emission of pungent, bright yellow defensive fluid was obtained bytapping the insect with the finger. It was collected either by capillary pipette or byabsorption on to filter paper, from which it was later extracted with saline. None ofthe characteristic rectangular particles was seen and it is presumed that they areabsent. No blood cells were found in the spontaneous emission when the defensivefluid was examined by the light microscope.

    On one occasion, when the insect was tapped more vigorously than usual, bleedingprobably occurred as one particle of rectangular outline was seen (Fig. 5) and a few ofroughly circular outline were found. One particle with an open centre 145 A acrosswas noted (arrow). In addition there was much amorphous material lying in largeclumps.

    The Burying Beetle

    The haemolymph was found to contain a variety of particles of ill-defined size andshape, but about 130 A across (Fig. 6).

    The Wasp

    The haemolymph contained particles of irregular outline (Fig. 7) often clumpedtogether in small groups in which individual particles showed no alignment like thatfound in the defensive fluid of the Monarch Butterfly.

    The Rat Flea

    The haemolymph of Nosopsyllus fasciatus Bosc. contained well defined particlesof rectangular outline (Fig. 8) which varied from 85 x 105 A to 100 x 130 A in size.This variation is small and suggests that one species of particle predominates.

    Eggs of the Garden Tiger

    The eggs of this moth are known to be extremely toxic (Frazer & Rothschild, i960).A milky, white fluid, obtained from fertile eggs not more than 24 h old was dilutedand negatively stained in the standard way. A tangle of strands of various widths andindeterminate lengths was seen. A regular arrangement of dark and light cross-bandswas clearly visible along all the strands (Fig. 9).

    Closer examination showed that the cross-banding was due to the arrangement ofthe small subunits from which the strands appeared to be built (Fig. 10). These couldbe seen arranged in lines closely touching each other and these lines of subunits layside by side in such a way that the subunits in adjacent lines were in exact register.It was the arrangement of the subunits across the strands which gave rise to thelight-coloured cross-bands, while the negative stain infiltrating between them pro-duced the dark lines. The centre-to-centre distance of the subunits was about 145 Aand it is likely that the overall size of the subunits is about this value. Occasionallysingle particles were seen but most of them were involved in strand formation. Somelarger particles about 350 A across were noted.

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    In order to determine whether the strands were an artifact caused by the negativestaining procedure the contents of Garden Tiger eggs were examined in the electronmicroscope after thin sectioning. Again the strands could be seen with the subunitsin register across numerous strands. The egg contents appeared to consist of largeparacrystalline arrays of protein molecules. It therefore appears that the strands seenin the negatively stained preparations are not artifacts but are fragments from muchlarger paracrystalline masses.

    Gipsy Moth eggs

    The egg fluid contained particles 100 A across of ill-defined shape but in additionthere were a number of very fine filaments 60 A wide but of great length (Fig. 11).On low-magnification prints some filaments could be traced for more than 20 fi.

    Ladybird eggs

    Only one egg of Coccinella septempunctata L. was examined and this yielded a yellowfluid. It contained particles of a fairly homogeneous size about n 5-130 A across andoften showing a rectangular outline (Fig. 12).

    Oak Eggar eggs

    A purple fluid was obtained from the eggs of this moth. Small particles of irregularshape about 130 A were found in it.

    DISCUSSION

    The elegant and technically excellent studies of Fernandez-Moran et al. (1966) onthe haemocyanins of certain arthropods and molluscs have shown that these verylarge macromolecules consist of small protein subunits arranged in a very precisemanner. These produce the cylindrical molecules, 340 A diameter and up to 680 Along, in, for example, Busycon, and smaller rectangular-outlined molecules 240 Aacross in Limulus. Further studies (Van Bruggen & Fernandez-Moran, 1966) havedemonstrated that the protein subunits of these large molecules can be dissociated bytreatment with alkali, but that after neutralization the subunits reaggregate to producelarge structures indistinguishable from the original ones.

    In the haemolymph, defensive fluids and egg fluids of the insects no large moleculeswere found, but, with the exception of the ladybird defensive fluid, protein moleculesof comparatively small size were always seen. The majority of these particles were100-150 A across but, considering the likely errors in measurement, it is probablethat most of the particles were members of one size group.

    Frequently the particles showed a rectangular or even square outline (Figs. 1,4, 8,12) and occasionally hexagonal and circular outlines were noted. Particles exhibitingthese shapes were described by Fernandez-Moran et al. (1966) in the 16 s ultra-centrifugal component of L. polyphemus and H. americanus haemocyanin where, forexample, small squares or rectangles 80-100 A across, a near-rectangle 105 A across,and hexagons 120-150 A in diameter were seen.

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    Van Bruggen (personal communication) has also found small square particles about120 A across in the body fluid of Tipula (Diptera), and Munn, Feinstein & Greville(1967) have found similar particles in extracts of the pupae of Calliphora erythrocephala(Diptera). These latter particles had an s value of 18-3, a molecular weight of 5-1 x io6

    and were about 100 A across. Greville, Munn & Feinstein (1967) believe that thisprotein may be a storage material as it is found only in larvae, prepupae, pupae andnewly emerged adults. These authors have found similar proteins in Sarcophaga(Diptera), Tenebrio (Coleoptera) and Pieris (Lepidoptera).

    In contrast to the findings of Munn et al. (1967) and Greville et al. (1967) we haveobserved similar particles in specimens of the Monarch which were by no meansfreshly emerged. In this case the protein cannot be assigned a storage role as it appearsto be a normal constituent of the adult insect fluid.

    A protein molecule in the shape of a cube with a side of 120 A would have amolecular weight of about one million and would almost certainly consist of severalsubunits. Some evidence of subunits about 45 A across is given by Fernandez-Moranet al. (1966) and even in these some finer substructure is evident. In the insect proteinssome substructure is shown in the square-shaped molecules (Fig. i,a) and in thecircular molecule (Fig. i,c), which appears to have eight or nine lobes and has adiameter of 125 A. Further information on the fine structure of the insect proteinsmight be obtained from micrographs at higher resolution but with the micrographsillustrated here no further comment on the fine structure is justified.

    The significance of the cross-banded strands in the Garden Tiger egg fluid is notknown but we can be certain that they are not formed during the treatment the fluidreceived after removal from the egg because they can be seen in thin sections of theegg contents. It should be noted that Fernandez-Moran et al. (1966) found moleculesarranged in single rows in the haemolymph of H. americantts.

    REFERENCES

    FERNANDEZ-MORAN, H., VAN BRUGGEN, E. F. J. & OHTSUKI, M. (1966). Macromolecularorganization of hemocyanins and apohemocyanins as revealed by electron microscopy.jf. molec. Biol. 16, 191-207.

    FRAZKR, J. F. D. & ROTHSCHILD, M. (i960). Defence mechanisms in warningly-colouredmoths and other insects. Proc. Xlth Int. Congr. Ent. Vienna, B, in, 249-256.

    GREVILLE, G. D., MUNN, E. A. & FEINSTEIN, A. (1967). A major soluble protein (calliphorin)of developing Calliphora erythrocephala (Diptera). Abstracts, Vllth Int. Congr. Biochem.A-41. Tokyo: Maruzen.

    MUNN, E. A., FEINSTEIN, A. & GREVILLE, G. D. (1967). A major protein constituent of pupaeof the blowfly Calliphora erythrocephala (Diptera). Biodiem.J. 102, 5 p.

    ROTHSCHILD, M. & HASKELL, P. T. (1966). Stridulation of the Garden Tiger Moth (Arctiacaja L.) audible to the human ear. Proc. R. ent. Soc. Lond. A 41, 167-170.

    VAN BRUGGEN, E. F. J. & FERNANDEZ-MORAN, H. (1966). Re-association of hemocyanins fromsubunit mixtures. .7. molec. Biol. 16, 208-211.

    VON EUW, J., FISHELSON, L., PARSONS, J. A., REICHSTEIN, T. & ROTHSCHILD, M. (1967).

    Cardenolides (heart poisons) in a grasshopper feeding on milkweeds. Nature, Lond. 214,35-39-

    {Received 20 May 1968—Revised 16 September 1968)

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    Fig. i. Six-spot Burnet Moth (Zygaena filipendulae L.). Fluid obtained from bleedingpoints after pressure on the thorax, (a, rectangular particle; b, rounded or irregularparticle; c, circular particle with open centre.) x 520000; inset x 400000.

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    Fig. 2. Garden Tiger Moth (Arctia caja L.). Defensive fluid, x 300000.Fig. 3. Monarch Butterfly (Danausplexippus L.). Fluid obtained from bleeding pointsafter pressure on the thorax. Arrow indicates equal-sized, square-outlined particles,x 220000.

    Fig. 4. Scarlet Tiger Moth (Panaxia dominula L.). Defensive fluid, x 400000.Fig. 5. Ladybird Beetle (Cocdnella septempunctata L.). Haemolymph. Arrow indicatesa particle with open centre, x 150000.Fig. 6. Burying Beetle (Necropliorus investigator Zett.). Haemolymph. x 280000.Fig. 7. Common Wasp (Vespula germanica L.). Haemolymph. x 520000.

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    Fig. 8. Rat Flea (Nosopsyllus fasciatus Bosc). Haemolymph. x 150000.Fig. 9. Garden Tiger Moth (Arctia caja L.) Egg fluid, x 40000.Fig. 10. Garden Tiger Moth (Arctia caja L.). Egg fluid, x 400000.Fig. 11. Gypsy Moth (Lymatitria dispar L.). Egg fluid, x 130000.Fig. 12. Ladybird Beetle (Coccinella septempunctata L.). Egg fluid, x 400000.

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