Pharmacological properties of the repellent secretion of Zonocerus variegatus ( Orthoptera : Prygomorphidae )

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 animal s used in this study, effects were temporary and reeovery oecurs after a short time.


INTRODUCTION
When molested, both sexes and an instars of the varíegated grasshopper, Zonocerus va riega tus (L.) expel an odorous, milky secretions from a gland opening on the dorsal intersegmental membrane between the first and second abdomi nal tergites (Idowu 1995).The secretion contaíns proteins, amino acids, glucose, alkaloids and ions (Idowu & Modder 1998).The penetrating and unpleasant odour of the secretion caused the grasshopper to be avoided by vertebrate and invertebrate predators (Idowu 1997).
Injections of secretions of the grasshopper, Poecilocerus bufonis (Fishelson 1960) and P. hieroglyphics (Abushama 1970) into mice result-ed in their death.The secretion of P. bufonis con taíns histanUne which causes the cOii tfaction of the smooth muscle of the guinea pig ileum (von Euw et al 1967).The effects of the secretion of Z variegatus on organisms under laboratory condi tions has not been reported, and there is no data on the perception of the secretion's odour by human beings.AH reports on the repellent gland studied merely state that the odour is repulsi ve or unpleasant to human beings.

Perception oC Odour by human beings
For this experiment, human volunteers were used.Their perceptions of the odour was recorded as one of the follows: i) Very Strong: The volunteer could perceive the odour immediately the tube containing the test sample was presented.ii) Strong: when it takes about 5 -15 seconds for the odour to be recognised.iii) Weak: when it takes about 20 -25 seconds for the odour to be perceived.iv) No effect: The volunteer did not perceive the odour.
The intensity of the odour was alloted 3, 2, 1 and O scores respectively.The sets of experi ments performed are: Insect Body Odour:-10 insects of different developmental stages were placed separately in different stoppered conical flask covered with foil papero The flasks were numbered 1-7 to correspond to the number of Zonocerus instars.The flasks were thoroughly shaken before pre sentation for odour recognition by the human volunteers.The order of presentation of flasks was change from one volunteer to the other.A time lag of 30 minutes was allowed between presentation of samples.Secretion Odour:-Another set of 68 volunteers chosen randomly was presented with sample bottle containing the pure repellent secretion for odour perception.Distilled water was used as the control.Effect of dilution on the recog nition of the secretion odour was also carried out.Undiluted and serial dilutions (0.8, 0.6, 0.5, 0.4, 0.3, 0.2 and 0. 1) of the secretion were presented to another set of volunteers.The vol unteers were also asked to compare the odour emanating from the botde containing the undi luted secretion with that of the body odour of living adult Z. variegatus.A time interval of 30 minutes was allowed between presentation of samples.The human volunteers used were between the age of 20-30 years.
Effects 01 the Secretion upon animals: This effect was tested on different animals by exter nal application and by injection.Different dosages were injected subcutaneously into mice and rats.External application was done by dropping the secretion on the body of ants, termites, mouth parts of the praying mantids and the eyes and skin of mice and rats.The response of the animals was recorded and compared with control animals.subjected to the same treatments with distilled water.
Effects on tbe bind paw of rats (Rattus rat tus): Adult rats (sprague dawley) weighing between 150g and 200g were selected divided into 4 groups based on the treatment below.
They were fasted for 12-16 hours before the treatment.The circumference of the hind paw was measured before and after the experiment.Each paw was measured three times and the mean size was obtained.
The hind paw of the rats in the different groups were infected separately with the following: a) 0.lmm3 of al % (VN) suspension of car rageenin in 0.9% saline solution (a sus pension that is known to induce inflam mation of the rat paw.) b) O.lmm3 of the repellent secretion.c) 0.lmm3 of the mixture of carr agaenin and the secretion.d) 0.lmm3 of distilled water.
The effect of the injected fluids on the hind paw of the rats was observed by measuring its circumference limb three hours, 6 , 12 and 24 hours after injections.
Stimulatory action on smooth muscle: Strips of smooth muscles of guinea pig ileum (Cavia porcellus), rat stomach and rat colon were used.The rats and guinea pig were killed by a blow on the head dissected for the parts were placed in dishes containing Tyrode's solution.The mesentric membranes were trimmed off along the intestine and stomach parts to free the muscles.Tissue threads were attached at each end by inserting a needle through the muscle wall.One end of the thread was tied to a fixed pin in the moist chamber and the other to a lever which has a fine writing point.The effect of the secretion on the smooth muscles strips was recorded using a Kymograph and a ." :' ;;?,{ ' ;;, ; .: � �; { .'::,< ./:.,,;;i�' �'< :, ',""<", j,; ... ) ;. )" ;, ', > •ttRl� ',' , ) smoked drum, with a¿etylcholine as stanC lard..�bservatiónduring the study also shoW�:mat'�ople in lkare, Owo and Oka .i,• all in QI1�9;isfáte. of Nigeria wherethe insect stu die�.i$i :)l�� as a•food iteQl (TqHle' 1).They,.
Effect on the hind paw of rat: The extent of the oedema produeed in the hind paw of rats infeeted with earraegenin and the repellent seeretion are given in Table 3.The oedema with a mixture of earraegenin and the repellent seeretion was more pronouneed than the oede ma with O.lmm3 portions of earraegenin and repellent seeretion administered separately.
The oedema resulting from earaegenin and the seeretion separately showed that the peak of oedema was 6 hours after injeetion.(Table 3) whíle the oedema produeed by the mixture of earr aegenin and seeretion peaked at the 3rd hour and persisted up to 6 hours.
On the contraction of smooth muscle in guinea"pig ileum, and rat stomach and colon: The muscle eontraetion indueed by dif ferent molarities of aeetyeholine (Ach) was eompared to that elicíted by different dílution's of the seeretion.The muscles of the guinea pig ileum and the rat stomaeh eontraeted in response to the secretion studied while that of the rat colon showed no response to the seere tion (Table 4).The response of guinea-pig ileum and rat stomaeh to Aeh at 8 x 10-8M was taken as maximum (100%), the amount of the other eontraetions are expressed as a percentage of it.The contraetions obtained from the guinea pig ileum following the addítion of the secretion at different were more pronounced eompared to the rat stomaeh.The response of the guinea pig ileum to 0.4 dilution level of the secretion showed a 50% inerease over that obtained at 0.2 dilution leve!.The same increased was observed for the contrae ion of rat stomaeh at 0. 1 and 0.2 dilution levels on one and, 0.4 and 0.8 dilution levels of the seeretion on another hand.

DISCUSSION
The secretion of Z. variegatus was lethal or does it produced any pennanent effect on la boratory animals (mice, rats) and other insects (ants, tennites and praying mantids).These re sults differ from the effect produced by the se cretions of P. bufonis (Orthoptera: Prygomorp hidae) (Fishelson 1960) and P. hieroglyphics (Abushama 1972) which led to the death of the animals into which they were injected.
The secretion of Z. variegatus induced oedema in the hind paw of the rat similar to that induced by the kJown infIammatory agent I carraegenin (Table 3) ¡ while the additive effects of the carraegenin an� the secretion produced oedema that reached fts peak earlier, in the 3rd hour, earlier than the �wo separately.The secre tion also has similar I pharmacological proper ties to that of P. bufo�is (von Euw et al 1967) in its ability to indud, e contraction of smooth muscIes.Although, t�e factor responsible for this pharmacologicalproperty of Z. variegatus secretion to induce co' ptraction of the muscIes needs to be identified, !the susbtantial infIux of Ca2+ from extracellul� medium is known to cause the contractiori of smooth muscIe (Bolton 1979) and since secretion of Z. varie gatus is rich in Ca2+ (ldowu & Modder 1998), it is possible to assume that it is probably one of the factor responsibl� for the contraction of smooth muscIes.
The study showed that the odours of the whole body and the r l1P ellent secretion of Z. variegatus were cIearly recognised by human beings.No significant difference exist in the perception of t�e odours.The volun teers described the " odour as offensive, unpleasant and un�ttractive .. These same description were used for the odour of related grasshoppers (Whitman 1990).The study has also shown that the degree of perception of the odour was highest in the 6th and �dult instars.It is in these instars were recoi rded appreciable volume of secretion (Idowu 1996).Studies have also shown that the grasshopper is avoided by invertebrate and vertebrate predators because of its body odour (Idowu 1997).

TABLE2
Effect of infected repelle/JI secretion of adalt Z. variegatus on rat, Spraque dawley and albÍlIO mice, Mus muscalus for resumption of limb movements.

TABLE3
Oedema in the hind paw of wister rals, Spraque dawley induced by carragaenin and Ihe repellent secretion of Z. variegatus