Rev. Biol. Trop., 47(4): 101 5-1020, 1999 www.ucr.ac.cr www.ots.ae.er www.ots.duke.edu

Pharmacological properties of the repellent secretion of (: Prygomorphidae)

A. B *Idowu and O. A Idowu

Department of Biological Scienees, University of Agriculture, Abeokuta, Nigeria. * Corresponding author

Received 10- IX-1998. Corrected 23-II1-1999 Accepted 16-IV-l999.

Abstrae!: The odours of the whole body and the secretion of Zonocerus variegatus were easily recognised and perceived by human volunteers. However, the secretion odour is not related to the odour of the food plant con­ sumed by the grasshopper. The repellency of Z. variegalus becomes more pronounced in the 6th. and adult instars whose gland lumens contain an appreciable volume of secretíon. The seeretion odour is so strong that even dilulion does no! affee! its repulsiveness to humans. The secretion had pharmacological properties: it indueed contraction in rat (Rattus rattus)stomach smooth musc1e preparations and guinea pig (Cavia porcellus) ileum, and induced oedema formation in the rat hind paw. The secretion was not lethal to the used in this study, effects were temporary and reeovery oecurs after a short time.

Key words: Z.variegatus, odour, seeretions, vertebrate, invertebrate, aetions.

INTRODUCTION ed in their death. Thesecretion of P. bufonis con­ taíns histanUne which causes the cOiitfaction of When molested, both sexes and an instarsof the smooth muscle of the guinea pig ileum (von the varíegated grasshopper, Zonocerus variega­ Euw et al 1967). The effects of the secretion of Z tus (L.) expel an odorous, milky secretions from variegatus on organisms under laboratory condi­ a gland opening on the dorsal intersegmental tions has not been reported, and there is no data membranebetween thefirst and second abdomi­ on the perception of the secretion's odour by nal tergites (Idowu 1995). Thesecretion contaíns human beings. AH reports on the repellent gland proteins, amino acids, glucose, alkaloids and studied merelystate that the odour is repulsi ve or ions (Idowu & Modder 1998). The penetrating unpleasant to human beings. and unpleasant odour of the secretion caused the grasshopper to be avoided by vertebrate and invertebrate predators (Idowu 1997). MATERIALS AND METHODS Injections of secretions of the grasshopper, Poecilocerus bufonis (Fishelson 1960) and P. Perception oC Odour by human beings hieroglyphics (Abushama 1970) into mice result- For this experiment, human volunteers were 1016 REVISTA DE BIOLOGÍA TROPICAL used. Their perceptions of the odour was by dropping the secretion on the body of ants, recorded as one of the follows: termites, mouth parts of the praying mantids i) Very Strong: The volunteer could perceive and the eyes and skin of mice and rats. The the odour immediately the tube containing response of the animals was recorded and the test sample was presented. compared with control animals. subjected to ii) Strong: when it takes about 5 - 15 seconds the same treatments with distilled water. for the odour to be recognised. iii) Weak: when it takes about 20 - 25 seconds Effects on tbe bind paw of rats (Rattus rat­ for the odour to be perceived. tus): Adult rats (sprague dawley) weighing iv) No effect: The volunteer did not perceive between 150g and 200g were selected divided the odour. into 4 groups based on the treatment below. They were fasted for 12-16 hours before the The intensity of the odour was alloted 3, 2, 1 treatment. The circumference of the hind paw and O scores respectively. The sets of experi­ was measured before and after the experiment. ments performed are: Each paw was measured three times and the Body Odour:- 10 of different mean size was obtained. developmental stages were placed separately in The hind paw of the rats in the differentgroups different stoppered conical flask covered with were infected separately with the following: foil papero The flasks were numbered 1-7 to correspond to the number of Zonocerusinstars. a) 0.lmm3 of al% (VN) suspension of car­ The flasks were thoroughly shaken before pre­ rageenin in 0.9% saline solution (a sus­ sentation for odour recognition by the human pension that is known to induce inflam­ volunteers. The order of presentation of flasks mation of the rat paw.) was change from one volunteer to the other. A b) O.lmm3 of the repellent secretion. time lag of 30 minutes was allowed between c) 0.lmm3 of the mixture of carragaenin and presentation of samples. the secretion. Secretion Odour:- Another set of 68 volunteers d) 0.lmm3 of distilled water. chosen randomly was presented with sample bottle containing the pure repellent secretion The effect of the injected fluids on the hind for odour perception. Distilled water was used paw of the rats was observed by measuring its as the control. Effect of dilution on the recog­ circumference limb three hours, 6 , 12 and 24 nition of the secretion odour was also carried hours after injections. out. Undiluted and serial dilutions (0.8, 0.6, 0.5, 0.4, 0.3, 0.2 and 0. 1) of the secretion were Stimulatory action on smooth muscle: Strips presented to another set of volunteers. The vol­ of smooth muscles of guinea pig ileum (Cavia unteers were also asked to compare the odour porcellus), rat stomach and rat colon were emanating from the botde containing the undi­ used. The rats and guinea pig were killed by a luted secretion with that of the body odour of blow on the head dissected for the parts were living adult Z. variegatus. A time interval of 30 placed in dishes containing Tyrode's solution. minutes was allowed between presentation of The mesentric membranes were trimmed off samples. The human volunteers used were along the intestine and stomach parts to free between the age of 20-30 years. the muscles. Tissue threads were attached at each end by inserting a needle through the Effects 01 the Secretion upon animals: This muscle wall. One end of the thread was tied to effectwas tested on different animals by exter­ a fixed pin in the moist chamber and the other nal application and by injection. Different to a lever which has a fine writing point. The dosages were injected subcutaneously into effect of the secretion on the smooth muscles mice and rats. External application was done strips was recorded using a Kymograph and a .'::,< . " :' ;;?,{ ';;,;.:��;{ . /:.,,;;i�'�'< :, ',""<", j,; ... ) ;. )" ;, ',> ·ttRl� ',' , ) smoked drum, with a¿etylcholine as stanClard. .... l\lt��orw: ih t � lt 1 . yvlij�hJghes�t,a�flowe�t � The eX ent �. , . . P7rim w� �perfonned'�t 3'?°Ci� " levFt'ofdilJitiplh . . � . .',) i./it�Tyrode's·solUtion thto�gh which(�ii-, �'aSi . ,1li�¡�S�Iisp'\)f91e)�(li�te�rSs�9�9�that ' �.i " dose�, �n.· . ':'¡i P�SS�':: e�� ��C���.�lve th7;.li��º4e }[,qr!egat¡lsP!�t,lC�Sln9dof w.ruG�\�J�w .., ...• :s�' solution. w washed. ' .. off :and the muscle fue 1st, 2nd, 3td �d'4thlnstars. lntel'nlediate.In " aU9wed to relaxfo� 4�� min�tes. .. ' .' theSth �dhlgh.in·the6tÍl amIth�i� dult in�hu-s. �� - -� - . ���ReSu1t.obtained,:sho;w�(Ltha��dPlioa�c-7-_� . .. the,b o�Yocl0� óf the 6tll. i��•• iJltb.iMwt by .' , human voJl,lÓte��was no! sÍgruficantIy di1'fcrent' (t"'test, .P>b.OO$�;.�bservatión during the study . PerceRtion ofili�odourof the boc,íy-and also shoW�:mat'�ople in lkare,Owo andOka .i,· the. secretion of Z. vqriegatZf,s by Quman yol;. all in QI1�9;isfáte.of Nigeria wherethe insect unteers. ,:;iiíf.¡' studie�. i$i:)l�� as a·food iteQl (TqHle' 1). They,. . There w� instatlt.recognitjon of thefu\p�t' rec9�;Wli fIi�·fasrthat \ �ultz,i.Vo/:fe$dtU$;,l1as .. of the rep�llehrseH��op '¡�y the humáU v�iúp.� J an od,,-urw�h:according to their:>d��tripti�n, is teers uponp¡;es�n�op li.S strong and rep�l� bad,: pungen�'and repulsive. Hqwevér, fooked , . ,;sive. The volunteers wer�.aP1e to perceive the insect donotproduces such OO'our (Tap1e}). Th�,r ...... '.' 'bdour ofthe,secretion evén when diluted. The volunteer recorded l;h�perc�ptiQ� of the' OAOUf.,;, .�í of of fue whole . gtas ho f>erls 'rep�ll�n1::; ,: pel'cepti�n . tbe: uri4j� , � � anp Q.e " . '.' theQ4Jqrsflo��dd�a� .. "�;1,;�� ,'j" i.( ' J};�;;[)f�¡f 1018 REVISTA DE BIOLOGÍA TROPICAL

The subeutaneous injeetion of the seere­ and 0.5mm3 of the seeretion had no effeet on tion into the body of miee (M.musculus)and the rats, however, the injeetion of the same rats (Rattus rattus) resulted only in paralysis. amount of seeretion into miee eaused paralysis Paralysis was manifested as the inability of the (Table 2).The reeovery time was direetly pro­ animals to move their límbs after they were portional to the volume of seeretion injeeted injeeted. The subeutaneous injeetion of 0.1 into the miee and rats (Table 2).

TABLE2

Effect of infected repelle/JI secretion of adalt Z. variegatus on rat, Spraque dawley and albÍlIO mice, Mus muscalus

Volume of secretion Rats (n=5) Effect Mean recovery time nfection (mm3) (mins+-S.E)

0.1 No effect 0.5 No effect 1 ParaJysis 7 +- 1.07 3 ParaJysis 19 +- 1.40 5 ParaJysis 36 +- 1.59 Mice (n-5) 0.1 ParaJysis 12 +- 1.26 0.5 ParaJysis 17 +- 1.14 l ParaJysis 36 +- 1.40 3 ParaJysis 50 +- 2.14 5 ParaJysis 99 +- 3.28

ParaJysis - inability to move Iimb Recovery time - Time required for resumption of limb movements.

The experimentalrats and mice used weighted between 1 00-120g and 35-50g respectively.

Effect on the hind paw of rat: The extent of eompared to that elicíted by different dílution's the oedema produeed in the hind paw of rats of the seeretion. The muscles of the guinea pig infeeted with earraegenin and the repellent ileum and the rat stomaeh eontraeted in seeretion are given in Table 3. The oedema response to the secretion studied while that of with a mixture of earraegenin and the repellent the rat colon showed no response to the seere­ seeretion was more pronouneed than the oede­ tion (Table 4). The response of guinea-pig ileum ma with O.lmm3 portions of earraegenin and and rat stomaeh to Aeh at 8 x 10-8M was taken repellent seeretion administered separately. as maximum (100%), the amount of the other The oedema resulting from earaegeninand the eontraetions are expressed as a percentage of it. seeretion separately showed that the peak of The contraetions obtained from the guinea pig oedema was 6 hours after injeetion. (Table 3) ileum following the addítion of the secretion at whíle the oedema produeed by the mixture of different were more pronounced eompared to earraegenin and seeretion peaked at the 3rd the rat stomaeh. The response of the guinea pig hour and persisted up to 6 hours. ileum to 0.4 dilution level of the secretion showed a 50% inerease over that obtained at 0.2 On the contraction of smooth muscle in dilution leve!. The same increased was guinea"pig ileum, and rat stomach and observed for the contraeion of rat stomaeh at 0.1 colon: The muscle eontraetion indueed by dif­ and 0.2 dilution levels on one and, 0.4 and 0.8 ferent molarities of aeetyeholine (Ach) was dilution levels of the seeretion on another hand. IDOWU & IDOWU: Phannacological properties of the repellent secretion of Zonocerus variega tus 1019

TABLE3

Oedema in the hind paw of wister rals, Spraquedawley induced by carragaenin and Ihe repellent secretion of Z. variegatus

Time (hr) Mean Diameter (mm) of Differences in mean % ¡nerease in diameter oedema +-S.E. diameter (mm) over initial diameter

S C C+S D S C C+S D S C C+S D

O 26.0+ 26.8+ 25.8+ 26.0+ -4.4 -0.95 -2.3 -2.08

3 34.4+ 35.5+ 39.1+ 27.0+ 7.5+ 8.7+ 13.3+ 1+ 28 32.5 52.0 3.90 -2.45 -3.94 -2.08 02.65 -1.94 -2.99 -0.22 -0.57

6 36.1+ 38.8 39+-1 26.7+ 9.2+ 11.5+ 13.3+ 0.7+ 34 43 52.0 2.70 -3.39 +-3.98 +-2.89 -2.89 -1.00 -3.03 -0.59 -0.8

12 31.6+ 34.5+ 36+ 26.0+ 4.7+ 7.2+ 10.2+ O 17 29 39.5 -1.51 -5.67 -3.51 -3.61 -2.88 -4.72 -1.21

24 28.4+ 28.6+ 34.3+ 26.0+ 1.5+ 1.8+ 8.5+ O 5.6 6.7 32.9 -2.28 -4.09 -2.08 -3.61 -2.12 -3.14 -0.22

(N=6)

S 0.1 mm3 of the repellent secretion of adult Z. variegatus C 0.1 mm3 of carragaenin suspension (1w/v in 0.9% saline solution) C+S 0.05 mm3 of carragaenin + 0.05mm3 of the repellent secretion D 0.1 mm of distilled water

TABLE4

Smoolh muscle contractions induced by ¡he repellent secretion ofZ.variegatus compared 10 Ihat by acetylcholine

Ach Size of Ach obtained Dilution factor Secretion (s) Contraction concentration Contractions (mm) of secretion obtained (mm) (M)

GPI RS GPI RS

10-8 23.3+-1.73 23+-2.0 0.1 10+-2.1 (14%) 6+-0.45 (10.2%) 2 x 10-8 35.7+-0.89 23.3+-3.6 0.2 15+-1.96 (21) 12+-1.8 (20.4%) 4 x 10-8 50.3+-3.2 33.7+-1.50 0.4 30+-2.3 (42%) 15+-0.31 (25.4%) 8 8 X 10- 71.7+-1.65 (100%) 59+-2.4 (100%) 0.8 40+-1.0 (56t%) 30+-0.69 (50.8%)

(n = 5)

Percentages in brackets are comparing with maximal contraction obtained(Ach concentratíon of 8 x 10-8)

S Repellent secretion of adult Z. variegatus GPI = Guinea pig ileum RS = Rat stomach 1020 REV ISTA DE BIOLOGÍA TROPICAL

DISCUSSION appreciable volume of secretion (Idowu 1996). Studies have also shown that the The secretion of Z. variegatus was lethal grasshopper is avoided by invertebrate and or does it produced any pennanent effect on la­ vertebrate predators because of its body odour boratory animals (mice, rats) and other insects (Idowu 1997). (ants, tennites and praying mantids). These re­ sults differ from the effect produced by the se­ cretions of P. bufonis (Orthoptera: Prygomorp­ REFERENCES hidae) (Fishelson 1960) and P. hieroglyphics (Abushama 1972) which led to the death of the Abushama, F.T.E. 1972. The repugnatorial gland of the animals into which they were injected. grasshopper Poecilocerus hieroglyphicus (kIug). J. Entomol Ser. (A) 7: 95-100. The secretion of Z. variegatus induced

oedema in the hind paw of the rat similar to Euw, J.V.L. FisheIson, L, Parsons, J.A. Reiehstein & M. Rothsehild 1967. Cardenolides (Heart poisons) in a that induced by the kJI own infIammatory agent carraegenin (Table 3) while the additive effects grasshopper feeding on mulkweeds Nature, (Lon­ ¡ 214, 35- 39. of the carraegenin an� the secretion produced don) oedema that reached ts peak earlier, in the 3rd f Fishelson, L. 1960. The biology and the behayiour of hour, earlierthan the �wo separately. The secre­ Poekilocerus bufonis (klug), with speeial referenee tion also has similar Ipharmacological proper­ to the repellent gland (Arth. Aerididae) Eos. Madr. ties to that of P. bufo�is (von Euw et al 1967) 36: 41-62. in its ability to indud,e contraction of smooth Idowu, A.B. 1995. The strueture of the repellent gland is muscIes. Although, t�e factor responsible for Zonocerus variegatus (Orthoptera: Pyrgomorphi­ this pharmacologicalproperty of Z. variegatus dae) 1. Afr.Zool. 109: 247-252. secretion to induce co'ptraction of the muscIes needs to be identified, !the susbtantial infIux of Idowu, A.B. 1996 The growth pattem of the repellent Ca2+ from extracellul medium is known to gland in Zonocerus variegatus (L.) (Orthopetera: � ) Bio. Seienee Comm. 8:1-6 cause the contractiori of smooth muscIe Z. (Bolton 1979) and since secretion of varie­ Idowu, A.B. 1997 The defensiye meehanism of Zonocerus gatus is rich in Ca2+ (ldowu & Modder 1998), variegatus (L) 1. Afr. Zool. II l: 199-203 it is possible to assume that it is probably one 1998 of the factor responsibl� for the contraction of Idowu A.B. & Modder, w'W.D. Preliminary analyses of the ehemical constituents of the repellent secre­ smooth muscIes. tion of Zonocerus variegatus (Orthoptera: Prygo­ The study showed that the odours of the morphidae). Inseet Sei. Appl. 18:- whole body and the rl1Pellent secretion of Z. variegatus were cIearly recognised by Pasteels, 1.M. Gregoire, lC. & Rowell - Rahier, M 1983 human beings. No significant difference exist The ehemical eeology of defenee in . Ann. Rey. Entomol. 28:263-289 in the perception of t�e odours. The volun­ teers described the " odour as offensive, Qureshi, & Ahmad, 1. 1970. Studies on the funetional ana­ unpleasant and un�ttractive .. These same tomy and histology of the repellent gland of Poeki­ description were used for the odour of related locerus pictus (F.) (Oithoptera: Pyrgomorphidae). 45: 149-155. grasshoppers (Whitman 1990). The study has Proe. R. Ent.Soc.lond. (A) also shown that the degree of perception of Whitman, D.W. 1990 Grasshopper chemical communica­ the odour was highest in the 6th and �dult tion, p. 357-391 in: R.F.Chapman &A.loefli (eds.). instars. It is in these instars were recoirded Biology of Grasshopper.