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Cytochemistry of the venom-secreting cells of the scorpion, Palamnaeus bengalensis
7oxlcon. 1976,Vol. 14, pla.449-450.PergamonPreu. Printed in GreatBritain.
CYTOCHEMISTRY OF THE VENOM-SECRETING CELLS OF THE SCORPION, P A L A M N A E U S B E N G A L E N S I S USHA KANWAR and NARINDER K. BRAR Department of Zoology, Panjab University, Chandigarh, India (Accepted for publication 1 June 1976)
U. KANWARand N. K. BRAn. Cytochemistry of the venom-secreting cells of the scorpion, Palamnaeus bengalensts. Toxicon 14, 449-450, 1976.--Venom glands of the scorpion, Palamnaeus bengalensis were studied by various cytochemical techniques. The venom cell secretion comprises mainly proteins and phospholipids. The protein moiety is rich in --SH and ---SS groups, and also exhibits traces of tryptophan and protein bound --NHI groups. The secretion of the mucus cells consists of proteins and polysaccharides. The lumen of the gland reveals a mixture of these secretions. INTRODUCTION THE VENOM of scorpions is produced by a pair of glands which are lodged in the telson. There has been no detailed cytochemical study on the venom glands of these arachnids. The work done so far is either of morphological or biochemical nature. Hence comprehensive cytochemical studies on the venom and venom-secreting cells of scorpions were considered essential. MATERIAL AND METHODS Adult specimens of the black scorpion, Palamnaeus bengalensts collected from areas surrounding Agra (Utter Pradesh) were used. The venom glands were dissected and fixed in Zenker, Bouin, weak Bouin, formaldehyde calcium and Lewitsky saline. The techniques employed for the demonstration of lipids include (1) Sudan black B (BAKER,1949), (ii) acid haematein along with pyridine extraction control (BAKJm, 1946), and (iii) Nile blue sulphate (CAIN, 1947). The tests for proteins include mercuric bromphgnol blue for localizing proteins in general, ferric-ferricyanide (Clt~vniMoN-rand Fa£OtmlC, 1943) for --SH groups, performic acid alcian blue for ---SS groups, DMAB-nitrite for tryptophan (PEAnSE, 1968) and ninhydrinSchiff (Pv.AI~, 1968) for protein-bound --NH2 groups. Periodic acid Sc,hiff (Horcttttsa, 1948) technique was employed for the identification of polysaccharides while KnA~R and Wn,OaRUM'Stoluidine blue method (1955) was employed for the determination of metachromasia. RESULTS AND DISCUSSION As has been described (KANWAR and BRAR, in press), the secretory epithelium of the v e n o m gland of the scorpion comprises two cell types: (i) venom-secreting cells and (ii) mucus-secreting ceils (Figs. 1 and 2). The cell cytoplasm of both cell types is full of secretory material which is usually in the form o f granules or globules of varying sizes or it may be in the form of a homogeneous material. The secretion granules of venom-secreting cells are stained deep blue after mercuricbromphenol blue staining (MAz1^ et al., 1953), thereby, confirming not only the presence but possibly the predominance of proteins in them (Fig. 3). Ferric-ferricyanide (CHI~VI~_.MONT and FRI~DE"RIC, 1943) and performic acid-Schiff/alcian blue (ADAMS and SLOPER, 1955, 1956) techniques further reveal that venom granules are rich in - - S H and - - S S groups (Fig. 4), respectively. Weak staining of the v e n o m granules after D M A B nitrite and ninhydrin-Schiff techniques (cf. PEARS)=, 1968) suggests that these are not rich in tryptophan and protein bound - - N H 2 groups. 449
450
USHA KANWAR and NARINDER K. BRAR
After Sudan black B (BAKER, 1949), which is a physical colorant for lipids in general,
secretion in the venom cells appears sudanophilic while the mucus in mucus cells remains unstained (Fig. 5). The presence of phospholipids in the sudanophilic venom is confirmed by acid haematein along with its usual pyridine extraction control (BAKES, 1946) and Nile blue sulphate (CAIN, 1947) techniques, both of which appreciably stain the venom granules and globules blue. Periodic acid-Schiff (I"IOTCHKIS$, 1948) stains only the mucus while the v e n o m cell granules are completely devoid o f any PAS positivity, suggesting the absence o f polysaccharides. The mucus globules turn violet after toluldine blue (KRAMES and WINDRUM, 1955), suggesting the presence o f 13-metachromasia in the mucus. These tests indicate that the secretion products o f b o t h v e n o m cells and mucus cells are complex mixtures rich in proteins. The protein moiety is rich in - - S S and - - $ H groups with traces o f tryptophan and protein-bound - - N H 2 groups. Secretion o f this same chemical nature is also observed in the lumen o f the gland. MCIh"rOSH and WATT (1966) during their biochemical studies on the v e n o m o f the scorpion, Centruroides sculpturatus, have shown that the toxicity o f the v e n o m is dependent on the presence ofsulphydryl groups. The mucus, as usual, is rich in proteins and carbohydrates. Biochemical studies have also revealed that scorpion venom is composed pri-
marily of proteins, mucin and nucleoproteins (BALOZET, 1971), the proteins of which have been suggested as being responsible for the venom toxicity (BALOZET, 1971). Acknowledgement--We are grateful to Dr. G. P. SI~ARMA,Professor and Head of Zoology Department, Panjab University, Chandigarh, for providing the necessary research facilities.
REFERENCES ADAMS,C. W. M. and SLopzg, J. C. (1955) Techniques for demonstrating neurosecretory material in human hypothalamus. Lancet 1, 651. ADAMS,C. W. M. and SLOPER,J. C. (1956) The hypothalamic elaboration of posterior pituitary prinoiples in man, rat and dog. Histochemical evidence derived from performio acid-alcian blue reaction, for cystine. J. Endocr. 13, 221. BArring,J. R. (1946) The histochemical recognition of lipine. Q. Jl microsc. Sci. 85, 1. BAKL~,J. R. (1949) Further remarks on Golgi elements. Q. JI mtcrosc. Sei. 90, 293. BALOZET, L. (1971) Soorpionism in the old world. In: Venomous Animals and their Venoms, Vol. Ill, (BuCn~RL, W. and Bucrd.a~, E. E., Eds.). New York: Academic Press. C~N, A. J. (1947) The use of Nile blue in the examination of lipids. Q. Jl. micrusc. Sci. 88, 383. Cngx,g~otcr, M. and FRIgI3tgxc,J. (1943) Une nouvelle methode histoohemique de mise en evidence des substances a fonotion sulphydrille. Application/I l'epidermis, an poll et b la levure. Archs Biol..54, 580. HoTcmcJss, R. D. (1948) A microohemical reaotion resulting in the staining of polysaechafide stn~ures fixed in the tissue preparations. Archs Biochem. 16, 31. KA~rVW,g, U. and BRAg,N. K. (in press) Cytoarchitecture of the venom glands of the scorpion, Pulamnaeus bengalensls. Res. Bull. Pan]. Univ. ~ , H. and Wtm3RUM,G. M. (1955) The metachromatic staining rea~ion. J. Histochem. Cytochem. 3, 227. MA,7~IA,D., B~w'~t, P. A. and AJ.Z~gT, M. (1953) The cytochemical staining and measurement of protein with merourio-bromphenol blue. Biol. Bull. 104, 57. Mclwro~, M. E. and WATr, D. D. (1966) Biochemical-immunochemical aspects of the venom from scorpion Centruroides sculpturatus. In: Animal Toxins, (RUSSELL,F. E. and SAUCERS, P. R., Eds.). Oxford: Pergamon Press. I ~ A ~ , A. G. E. (1968) Hlstochemistry, Theoretical and Applied. Boston: Little, Brown & Co.
-vc
F
-MC
MS-
.m
CTL
MS-
FIGS. 1
AND
2.
SEMIDIAGRAMMATIC
REPRESENTATION
OF DIFFERENT
CELL
TVPES
IN THE
VENOM
Palamnaeus bengalends. The secretory epithelium which forms villi shows secretion in the form of small and large granules, globules, flakes or frothy droplets. CI’L-connective tissue layer; FVWfrothy venom droplet; LVG-large venom granule; MC-mucus-secreting cell; MF-mucus flake; MCIuscular sheath; SE -secretory epithelium; SVG-small venom granule; V-villi and W-venom-secreting cell. 0uND
OF
f.p. ,450
. 3--5. PHOTOMICROGRAPHSOFIHEVENOMGLANDCELLSOF
Palamnaeusbengalen
FIG. 3. E~WINS-MERCURICBROMPHENOLBLUE. V ‘enom granules are darkly stained as compared to mucus droplets. >: 100. FIG. 4. E~WINS-PERFORMIC ACID SCHIFF. dtlcus isobserved in the form of flakes venom granules darkly stained. x 900. FIG.~. FORMALDEHYDECALCIUM POSTCHROMED~UDANBLACK B. Venom granules darkly stained. Mucus unstained. v 100.
CYTOCHEMISTRY OF THE VENOM-SECRETING CELLS OF THE SCORPION, P A L A M N A E U S B E N G A L E N S I S USHA KANWAR and NARINDER K. BRAR Department of Zoology, Panjab University, Chandigarh, India (Accepted for publication 1 June 1976)
U. KANWARand N. K. BRAn. Cytochemistry of the venom-secreting cells of the scorpion, Palamnaeus bengalensts. Toxicon 14, 449-450, 1976.--Venom glands of the scorpion, Palamnaeus bengalensis were studied by various cytochemical techniques. The venom cell secretion comprises mainly proteins and phospholipids. The protein moiety is rich in --SH and ---SS groups, and also exhibits traces of tryptophan and protein bound --NHI groups. The secretion of the mucus cells consists of proteins and polysaccharides. The lumen of the gland reveals a mixture of these secretions. INTRODUCTION THE VENOM of scorpions is produced by a pair of glands which are lodged in the telson. There has been no detailed cytochemical study on the venom glands of these arachnids. The work done so far is either of morphological or biochemical nature. Hence comprehensive cytochemical studies on the venom and venom-secreting cells of scorpions were considered essential. MATERIAL AND METHODS Adult specimens of the black scorpion, Palamnaeus bengalensts collected from areas surrounding Agra (Utter Pradesh) were used. The venom glands were dissected and fixed in Zenker, Bouin, weak Bouin, formaldehyde calcium and Lewitsky saline. The techniques employed for the demonstration of lipids include (1) Sudan black B (BAKER,1949), (ii) acid haematein along with pyridine extraction control (BAKJm, 1946), and (iii) Nile blue sulphate (CAIN, 1947). The tests for proteins include mercuric bromphgnol blue for localizing proteins in general, ferric-ferricyanide (Clt~vniMoN-rand Fa£OtmlC, 1943) for --SH groups, performic acid alcian blue for ---SS groups, DMAB-nitrite for tryptophan (PEAnSE, 1968) and ninhydrinSchiff (Pv.AI~, 1968) for protein-bound --NH2 groups. Periodic acid Sc,hiff (Horcttttsa, 1948) technique was employed for the identification of polysaccharides while KnA~R and Wn,OaRUM'Stoluidine blue method (1955) was employed for the determination of metachromasia. RESULTS AND DISCUSSION As has been described (KANWAR and BRAR, in press), the secretory epithelium of the v e n o m gland of the scorpion comprises two cell types: (i) venom-secreting cells and (ii) mucus-secreting ceils (Figs. 1 and 2). The cell cytoplasm of both cell types is full of secretory material which is usually in the form o f granules or globules of varying sizes or it may be in the form of a homogeneous material. The secretion granules of venom-secreting cells are stained deep blue after mercuricbromphenol blue staining (MAz1^ et al., 1953), thereby, confirming not only the presence but possibly the predominance of proteins in them (Fig. 3). Ferric-ferricyanide (CHI~VI~_.MONT and FRI~DE"RIC, 1943) and performic acid-Schiff/alcian blue (ADAMS and SLOPER, 1955, 1956) techniques further reveal that venom granules are rich in - - S H and - - S S groups (Fig. 4), respectively. Weak staining of the v e n o m granules after D M A B nitrite and ninhydrin-Schiff techniques (cf. PEARS)=, 1968) suggests that these are not rich in tryptophan and protein bound - - N H 2 groups. 449
450
USHA KANWAR and NARINDER K. BRAR
After Sudan black B (BAKER, 1949), which is a physical colorant for lipids in general,
secretion in the venom cells appears sudanophilic while the mucus in mucus cells remains unstained (Fig. 5). The presence of phospholipids in the sudanophilic venom is confirmed by acid haematein along with its usual pyridine extraction control (BAKES, 1946) and Nile blue sulphate (CAIN, 1947) techniques, both of which appreciably stain the venom granules and globules blue. Periodic acid-Schiff (I"IOTCHKIS$, 1948) stains only the mucus while the v e n o m cell granules are completely devoid o f any PAS positivity, suggesting the absence o f polysaccharides. The mucus globules turn violet after toluldine blue (KRAMES and WINDRUM, 1955), suggesting the presence o f 13-metachromasia in the mucus. These tests indicate that the secretion products o f b o t h v e n o m cells and mucus cells are complex mixtures rich in proteins. The protein moiety is rich in - - S S and - - $ H groups with traces o f tryptophan and protein-bound - - N H 2 groups. Secretion o f this same chemical nature is also observed in the lumen o f the gland. MCIh"rOSH and WATT (1966) during their biochemical studies on the v e n o m o f the scorpion, Centruroides sculpturatus, have shown that the toxicity o f the v e n o m is dependent on the presence ofsulphydryl groups. The mucus, as usual, is rich in proteins and carbohydrates. Biochemical studies have also revealed that scorpion venom is composed pri-
marily of proteins, mucin and nucleoproteins (BALOZET, 1971), the proteins of which have been suggested as being responsible for the venom toxicity (BALOZET, 1971). Acknowledgement--We are grateful to Dr. G. P. SI~ARMA,Professor and Head of Zoology Department, Panjab University, Chandigarh, for providing the necessary research facilities.
REFERENCES ADAMS,C. W. M. and SLopzg, J. C. (1955) Techniques for demonstrating neurosecretory material in human hypothalamus. Lancet 1, 651. ADAMS,C. W. M. and SLOPER,J. C. (1956) The hypothalamic elaboration of posterior pituitary prinoiples in man, rat and dog. Histochemical evidence derived from performio acid-alcian blue reaction, for cystine. J. Endocr. 13, 221. BArring,J. R. (1946) The histochemical recognition of lipine. Q. Jl microsc. Sci. 85, 1. BAKL~,J. R. (1949) Further remarks on Golgi elements. Q. JI mtcrosc. Sei. 90, 293. BALOZET, L. (1971) Soorpionism in the old world. In: Venomous Animals and their Venoms, Vol. Ill, (BuCn~RL, W. and Bucrd.a~, E. E., Eds.). New York: Academic Press. C~N, A. J. (1947) The use of Nile blue in the examination of lipids. Q. Jl. micrusc. Sci. 88, 383. Cngx,g~otcr, M. and FRIgI3tgxc,J. (1943) Une nouvelle methode histoohemique de mise en evidence des substances a fonotion sulphydrille. Application/I l'epidermis, an poll et b la levure. Archs Biol..54, 580. HoTcmcJss, R. D. (1948) A microohemical reaotion resulting in the staining of polysaechafide stn~ures fixed in the tissue preparations. Archs Biochem. 16, 31. KA~rVW,g, U. and BRAg,N. K. (in press) Cytoarchitecture of the venom glands of the scorpion, Pulamnaeus bengalensls. Res. Bull. Pan]. Univ. ~ , H. and Wtm3RUM,G. M. (1955) The metachromatic staining rea~ion. J. Histochem. Cytochem. 3, 227. MA,7~IA,D., B~w'~t, P. A. and AJ.Z~gT, M. (1953) The cytochemical staining and measurement of protein with merourio-bromphenol blue. Biol. Bull. 104, 57. Mclwro~, M. E. and WATr, D. D. (1966) Biochemical-immunochemical aspects of the venom from scorpion Centruroides sculpturatus. In: Animal Toxins, (RUSSELL,F. E. and SAUCERS, P. R., Eds.). Oxford: Pergamon Press. I ~ A ~ , A. G. E. (1968) Hlstochemistry, Theoretical and Applied. Boston: Little, Brown & Co.
-vc
F
-MC
MS-
.m
CTL
MS-
FIGS. 1
AND
2.
SEMIDIAGRAMMATIC
REPRESENTATION
OF DIFFERENT
CELL
TVPES
IN THE
VENOM
Palamnaeus bengalends. The secretory epithelium which forms villi shows secretion in the form of small and large granules, globules, flakes or frothy droplets. CI’L-connective tissue layer; FVWfrothy venom droplet; LVG-large venom granule; MC-mucus-secreting cell; MF-mucus flake; MCIuscular sheath; SE -secretory epithelium; SVG-small venom granule; V-villi and W-venom-secreting cell. 0uND
OF
f.p. ,450
. 3--5. PHOTOMICROGRAPHSOFIHEVENOMGLANDCELLSOF
Palamnaeusbengalen
FIG. 3. E~WINS-MERCURICBROMPHENOLBLUE. V ‘enom granules are darkly stained as compared to mucus droplets. >: 100. FIG. 4. E~WINS-PERFORMIC ACID SCHIFF. dtlcus isobserved in the form of flakes venom granules darkly stained. x 900. FIG.~. FORMALDEHYDECALCIUM POSTCHROMED~UDANBLACK B. Venom granules darkly stained. Mucus unstained. v 100.