14
Insect Venoms and Toxins TOM PIEK University of Amsterdam, Amsterdam, The Netherlands
1 Introduction
595
2 Venoms of Hemiptera, Coleoptera and Lepidoptera 2.1 Hemiptera 2.2 Coleoptera 2.3 Lepidoptera
596 596 596 596
3 Venoms of Hymenoptera Terebrantia 3.1 Ichneumonidae 3.2 Braconidae 3.3 Cynipoidea 3.4 Chalcidoidea
596 596 598 600 600
4 Venoms of Hymenoptera Aculeata 4.1 Bethyloidea 4.2 Scolioidea 4.3 Pompilidae 4.4 Sphecidae 4.5 Masaridae 4.6 Eumenidae 4.7 Vespidae 4.8 Apidae 4.8.1 Enzymes 4.8.2 Peptides 4.8.3 Melittin 4.8.4 Apamin 4.9 Formicidae
601 601 601 606 612 617 618 619 621 622 623 623 623 624
References
626
as crustaceans. The chapter will concentrate more on the physiological and (neuro-)pharmacological side rather than on the biochemical side; the latter will be treated by E. Zlotkin (volume 10). The class of Insecta can be subdivided into about 30 orders (Oldroyd, H., 1966), four of them being interesting because of the poisonous products they contain. These four orders are: Hemiptera, Coleoptera, Lepidoptera and Hymenoptera. Thefirstthree orders produce many poisonous products, some of which could be considered to be true toxins for vertebrates. Their insect pharmacology and
1 INTRODUCTION
The knowledge of natural toxins active in insects and related invertebrates has important physiological, pharmacological and biochemical implications. Furthermore such knowledge may be of great importance for the development of new principles for regulating insect populations, enabling the use of more specifically acting insecticides. This chapter deals with those venoms and their composing toxins produced by insects with their activity predominantly in insects and related animals such CIP VOL11-MM
595
596
Tom Piek
toxicology is only fragmentarily known; they will be briefly described. The Hymenoptera, however, produce true venoms, and these will be described in more detail.
2
2.1
VENOMS OF HEMIPTERA, COLEOPTERA AND LEPIDOPTERA
Coleoptera
Within this order twelve families have been studied with regard to the chemistry of their defensive secretions (Weatherston, J. and Percy, J., 1978b). Here too it is doubtful whether the terms venom and toxin used by these authors are always correct. The defensive secretions contain (aliphatic and aromatic) acids, aldehydes, and esters, as well as hydrocarbons, quinonoid compounds, steroids, terpenoid compounds and alkaloids. Nothing seems to be known about the insect pharmacology of the secretions. The biologically defensive effects on insects, such as the repellent effects and the vertebrate pharmacology, have been reviewed elsewhere (Weatherston, J. and Percy, J., 1978b).
Lepidoptera
Some butterflies as well as caterpillars contain toxic substances used by these insects in both active and passive defence against predatory vertebrates. The anthropotoxic Lepidoptera are reviewed by Delgado Quiroz, A. (1978). Nothing seems to be known about toxic effect on insects.
Hemiptera
The majority of insect species which belong to this order are terrestrial, many of these insects being phytophagous and others entomophagous. The minority is aquatic, all being predatory on other insects, small amphibians and fishes. Venomous substances are present in salivary secretions. The subject has recently been reviewed by Weatherston, J. and Percy, J. (1978a). The salivary secretions contain alkanes, alkanals, alkenals, alkanones, alkenones, alkanols, alkanolic esters, alkenonic esters, dicarboxylates, steroids and aromatic compounds. The secretions are considered to have defensive functions, but a lack of sufficient studies precludes generalization. It is doubtful whether all the compounds summarized above may be called toxins, as was proposed by Weatherston, J. and Percy, J. (1978a). Nothing seems to be known about the pharmacology of hemipteran secretions. 2.2
2.3
3
VENOMS OF HYMENOPTERA TEREBRANTIA
Hymenoptera are generally known as venom producers, but this general knowledge is often restricted to the pain- and inflammation-producing social wasps and bees. Yet out of the roughly 250,000 described species of Hymenoptera (Malyshev, S., 1966) the vast majority are solitary wasps (Evans, H. and Eberhard, M., 1970). According to Akre, R. and Davis, H. (1978) there are about 15,000 species of aculeate wasps (see below), 95% of which are solitary. The Hymenoptera can be subdivided into two groups: the Symphyta, or sawflies, which are phytophagous, and the Apocrita, most of which are entomophagous (Imms, A., 1960). The sawflies have no "wasp waist", such as the Apocrita. The latter group again is divided into two sections: the Terebrantia which have an ovipositor (terebra or drill), which is also used as ductus venatus, and the Aculeata, having an aculeus or sting, which is considered to be a fully modified ovipositor, no longer used for oviposition. The section of Terebrantia includes three important superfamilies: the Ichneumonoidea, with the families Ichneumonidae (section 3.1) and Braconidae (section 3.2), the Cynipoidea (Section 3.3), and the Chalcidoidea (section 3.4). 3.1
Ichneumonidae
All species of this family are internal or external parasites of larvae of Holometabola (i.e. insects that hatch from the egg into a form, for example a caterpillar, which is quite different in appearance from the adult, in this example a butterfly). The female ichneumon wasps have a strong preference for lepidopteran hosts. The first observations on the stinging behaviour
597
Insect Venoms and Toxins
FIG. 1. Paniscus ocellaris stings a larva of the moth Agrostis segetum. (After Shevyrev, 1912, (cf. Malyshev, S., 1966).)
of ichneumon wasps originate from Shevyrev (1912; cf. Malyshev, S., 1966), who described the stinging act of Paniscus ocellaris paralysing a larva of the moth Agrostis segetum (Fig. 1). The wasp thrusts the tip of her abdomen with the sting extended towards the caterpillar, and pierces one of the last body segments. The caterpillar's movements immediately become slower. After withdrawal of the ovipositor the caterpillar was no longer paralysed. According to Malyshev, S. (1966) the site of stinging shows that the wasp does not inject the venom especially into the victim's central nervous system. Table 1: References to observations ofparalysis in insects and spiders by a sting of wasps belonging to the Family Ichneumonidae Wasp genus
Host/prey taxum
Kind of References paralysis
Aenoplex Calliephialtes
Lep. Lep.
+1 +C
Ephialtes Nemeritis
Lep. Lep.
+c
Lep.
+ TI
Paniscus
Lep. Lep.
Pimpla Polysphincta Rhyssa Sericopimpla Tersilochus Zaglyptus
Col. Ara. Hym. Col. Col. Ara.
+T +P +T + +P +T +P
McClure, H., 1933 Nickels, C , et al., 1950 Juillet, J., 1959 Richards, O. and Thomson, W. 1932 Piek, T. and Simon Thomas, R. 1969 Shevyrev, 1912 (cf. Malyshev, S., 1966) Vance, A., 1927; Shevyrev, 1912 (cf. Malyshev, S., 1966) Speyer, W., 1925 Clausen, C , 1940 Spradbery, J., 1968 Iwata, K., 1976 Sweetman, H., 1958 Iwata, K., 1976
+ = paralysis, — = no paralysis, P = permanent, T = transient, C = complete, I = incomplete. Key to host or prey taxa: Ara. = Arachnida, Col. = Coleoptera, Hym. = Hymenoptera, Lep. = Lepidoptera.
Only a few species completely paralyse their hosts, the majority paralyses them only incompletely (permanently or temporarily) (Table 1). The prey of a number of ichneumonid wasps do not seem to become paralysed at oviposition. Richards, O. and Thomson, W. (1932), for example, reported that larvae of the mealmoth, Ephestia kuehniella, were not paralysed when they were stung by the ichneumonid Nemeritis canescens. However, Piek, T. and Simon Thomas, R. (1969) studied the effect of the sting of these wasps of different ages, and found that the larvae of E. kuehniella, as well as of the wax moth Galleria mellonella, were paralysed for about | h when stung by wasps aged 2-3 days, and not by younger or older wasps. Table 2: References to observations of paralysis in insects by a sting of wasps belonging to the Family Braconidae. W a s p genus Apanteles
Aphidius Bassus Cedria Coeloides Diachasma Heterospilus Iprobracon Leiphron Macrocentus Microbracon Microgaster Phδnomens Rhaconotus Rhogas Rhoptocentrus Stenobracon
Host/prey taxum
Kind of paralysis
References
+T
Nickels, C , et al, 1950 Piek, T. and Simon Thomas, R. 1969 Allen, W., 1958 Lep. Sweetman, H., 1958 Hem. Nickels, C. et al., 1950 Lep. +T Lep. + CD Beeson, C. and Chatterjee, S., 1935 Col. + CD Ryan, R. and Rudensky, J., 1962 Col. + ID De Leon, D., 1935 Pemberton, C. and Dip. +TI Willard, H., 1918 Sweetman, H., 1958 Hym. + Jaynes, H., 1933 Lep. +PI Waloff, N., 1967 Hem. +T Newman, L., 1965 Lep. + see Table 3 Vance, A., 1935 Lep. — Clausen, C , 1956 Hym. +PI Lep. + PD Cherian, M. and Israel, P., 1938a Chaterjee, P., 1943 Lep. Amad, M., 1943 Lep. +T Lep. +T Col. +c Kühne, H. and Becker, G., 1974 Lep. MashoodAlam,S.,1952; Narayanan, E. and Chaudhuri, R., 1955; Cherian, M. and Israel, P. 1938b Lep.
+ = paralysis, - = no paralysis, P = permanent, T = transient, C = complete, I = incomplete. Key to host or prey taxa: Col. = Coleoptera, Dip. = Diptera, Hem. = Hemiptera, Hym. = Hymenoptera, Lep. = Lepidoptera.
598 3.2
Tom Piek Braconidae
The first observation on the stinging behaviour of a braconid wasp was that of Hase, A. (1924) who found that Microbracon hebetor does not sting its prey at any specific location. Beard, R. (1952) confirmed this observation. The wasp does not assume a characteristic position on the host larva when it stings. Hase, A. (1924) measured the wasp's ovipositor and found that its length was about onethird of the diameter of the larva of the wax moth Galleria mellonella. In relation to the length of the ovipositor it is impossible for the wasp to sting into the host's ganglia when it attacks the larva from the dorsal side. The Braconidae are parasites of larvae of
Holometabola (see section 3.1) with a marked preference for lepidopteran hosts. In general the endophagous species parasitize free-living hosts and these are not, or only very temporarily, paralysed. In contrast the exophagous species parasitize hosts which have a cryptic lifestyle. The host is usually stung before oviposition and this often results in a complete and permanent or a longterm paralysis, especially in hosts of wasps of the genus Microbracon (Tables 2 and 3). It is interesting that the onset of paralysis may take some time in hosts of some of the exophagous species. For example Cedria paradoxa attacks caterpillars of Hapalia machaeralis and induces a paralysis which is only complete after 24 h (Beeson, C. and Chatterjee, S., 1935). The beetle larva Dendroctonus pseudotsugae
Table ?>\ References providing evidencefor a paralysing activity ofvenoms produced by Microbracon spp. (fromPiek, T. etal., 1982a)
Wasp species
Host species
References
Microbracon brevicornis ( = Habrobracon, = Bracon) Microbracon cephi Gahan ( = Bracon) Microbracon chinensis (Szepl.) Microbracon gelechiae (Ashm.) ( = Habrobracon johansenni Vier.) Microbracon greenii (Ashm.) Microbracon hebetor (Say) ( = Habrobracon, = Bracon)
complete and permanent paralysis of lepidopteran larvae permanent or no paralysis of hymenopteran larvae complete and permanent paralysis of lepidopteran larvae complete and permanent paralysis of lepidopteran larvae
Genieys, P., 1922, 1925; Taylor, J., 1932
Microbracon hylobii (Ratz.) Microbracon kirkpatricki (Wilkins) ( = Bracon) Microbracon lineatellae (Fischer) ( = Habrobracon) Microbracon lutus (Prov.) Microbracon mellitor (Say) ( = Bracon) Microbracon pectoralis (Wesm.) Microbracon piger (Wesm.) Microbracon pini Meus. Microbracon pygmaeus (Prov.) Microbracon terebella (Wesm.) Microbracon thurberiphagae Mues. ( = Bracon) Microbracon variabilis (Prov.)
stings coleopteran larvae without paralysis
paralysis of lepidopteran, hemipteran, and coleopteran larvae complete and permanent or transient paralysis of lepidopteran larvae (except Ostrinia nubilalis)
Seamans, H., 1929; Nelson, W. and Farstad, C. 1953 Cherian, M. and Narayanaswami, P., 1942; Subba Rao, B., 1955 Trouvelot, B., 1921; Genieys, P., 1925; Piek, T. etal 1974 Glover, P., 1934; Angalet, G., 1964 Hase, A., 1922, 1924; Donohoe (see Clausen, C , 1940); Morrill, A., 1942; Ullyett, G., 1943; Beard, R., 1952; Apanna, M., 1952; Piek, T. et al., 1974 Munro, J., 1916 Azab, A. et al., 1968, Cross, W. et αί, 1969
complete and permanent paralysis of lepidopteran larvae complete and permanent paralysis of lepidopteran larvae complete and permanent paralysis of lepidopteran and coleopteran larvae complete and permanent paralysis of lepidopteran and coleopteran larvae paralysis of lepidopteran larvae
Willard, J., 1927; Folsom, J., 1936; Adams, C. et αί, 1969 Parker, H., 1951
paralysis of lepidopteran larvae
Parker, H., 1951; Clausen, C , 1956
transient paralysis of coleopteran larvae complete and permanent paralysis of lepidopteran larvae stings hymenopteran larvae without paralysis paralysis of lepidopteran larvae
Taylor, R., 1929 Doner, H., 1934
complete and permanent paralysis of lepidopteran larvae
Nickels, C. et al, 1950
Laing, D. and Caltagirone, L. J., 1969 Balduf, W., 1929
Salt, G., 1931 Bennet, F., 1959
599
Insect Venoms and Toxins
is completely paralysed after a period of several hours to 2 days after being stung by Coeloides dendroctoni (De Leon, D., 1935), or about 12 h after being stung by Coeloides brunneri (Ryan, R. and Rudensky, J., 1962). These observations are interesting in relation to what is now known about the paralysing effect of the venom of Microbracon hebetor. All Microbracon species are exophagous on hidden living hosts, and nearly all the species paralyse their prey (Table 3). The stings of Microbracon hebetor and Microbracon gelechiae cause complete and permanent paralysis of the prey within 5-15 min, but if a dilute venom solution is injected the paralysis is reversible (Beard, R., 1952; Piek, T. et al, 1974). M. hebetor and M. gelechiae venoms are slow-acting venoms. The paralysis persists over a long period of time without any apparent change in the ultrastructure of nerve endings being caused (Piek, T. et al, 1974; Rathmayer, W. and Walther, C, 1976). Paralysis caused by purified toxins isolated from the venom of M. hebetor (Spanjer, W. et al, 1977; Visser, B. et al, 1983) also shows a very slow time-course. The time-course of paralysis, as well as that of the recovery, shows S-shaped timepercentage effect curves, which become linear when plotted on a probability scale against log-time (Litchfield, J., 1949). Plotted in this way, the degree of paralysis against time of two toxins from M. hebetor venom (A-MTX and B-MTX, see below) is shown in Fig. 2. The values representing the degree of paralysis have been obtained using the score system according to Beard, R. (1952). Unaffected larvae were scored 0; larvae showing uncoordinated movements were scored 1; larvae unable to turn around but reacting to tactile stimulation were scored 2; completely paralysed larvae were scored 3. The activity of venom solutions was expressed in paralysing units, as defined by Drenth, D. (1974) as the amount of venom per 100 mg larva causing, after 2 h, an average score of 1.5, being 50% in Fig. 2. The moment of maximal effect has been estimated by extrapolation of the calculated regression lines. At all doses the peak paralysis lies at about 20 h. In Fig. 2 some of the regression lines are extrapolated. Other lines are not extrapolated, but it is obvious that all points of intersection have approximately the value of 20 h (Piek, T. et al, 1982b). This is similar to what has been described
/ * '.
I
I
I
I
I
2
6
12
24
48
I 96 time, h
I 220
FIG. 2. Time-percentage effect plots of the paralysis of waxmoth larvae (Galleria mellonella) injected with different doses of A-MTX or B-MTX. The degree of paralysis (see text) has been plotted on a probability scale against log-time. The doses are indicated as the number of units (see text) per 100 mg larva. Points of supposed maximal paralysis are indicated with arrows. (From Piek, T. et al, 1982b).
before, for paralysis caused by a sting of Cedria or Coeloides wasps, and might be a general phenomenon for venoms produced by braconid wasps. The time-courses of paralysis (onset) differ for the two toxins, the slopes are less steep for A-MTX than for B-MTX. Similar to what has been found with the crude toxins by Spanjer, W. et al (1977) the dose-response curve of A-MTX is also less steep than of B-MTX (Fig. 3, Piek, T. et al, 1982b). In the
600
Tom Piek
/B-MTX
0-44
067
voo
1-48
dose
units/100 mg
222
FIG. 3. Dose-effect curves of five A-MTX preparations and sixteen B-MTX preparations, plotted as the average score after 2 h (see text) against log-dose. The data are normalized to 1 U 100 m g - . The regression coefficients indicated are expressed as points per decade in dose. Vertical bars are SEM-values, n is 5 and 16 for A-MTX and B-MTX respectively. (From Piek, T. et al, 1982b.)
analytical gel chromatography elution pattern of AMTX the peak of paralysing activity was associated with a molecular weight of 42,000, in that of BMTX with 57,000. Both toxins are labile proteins (Visser, B. et al, 1983). The crude venom of M. hebetor causes a decrease in frequency of miniature excitatory postsynaptic potentials (MEPSPs) in lepidopteran muscle fibres (Piek, T. and Engels, E., 1969) and in locust muscle fibres (Rathmayer, W. and Walther, C , 1976) without affecting their amplitude. This has been confirmed for the venom of M. gelechiae (Piek, T. et al, 1974) and for the purified venom preparation of M. hebetor (Visser, B. et al, 1976) as well as for A-MTX and B-MTX (Spanjer, W. et al, 1977, Visser, B. et al, 1983). The effect of purified AMTX on MEPSPs recorded from a small accessory flight muscle of Pieris brassicae (see Spanjer, W. et al, 1977) is shown in Chapter 3, Fig. 39 (Piek, T. etal, 1982b). The venoms of Microbracon species cause flaccid paralysis in some insect groups (Lepidoptera, Hymenoptera), but are less active in other groups (Orthoptera) (Piek, T. et al, 1982b). The venom as well as both toxins of M. hebetor cause a presynaptic block of the glutamatergic excitatory transmission. The venom has no effect on the inhibitory
(GABAnergic) transmission (see chapter 3, section 3.4). This observation led Piek, T. and Mantel, P. (1970), and Walther, G. and Rathmayer, W. (1974) to suggest that this venom might specifically act on glutamatergic transmission processes. The fact that the venom also does not act on cholinergic transmission (frog muscle, guinea-pig ileum, squid stellar ganglion — see Piek, T. et al, 1982b) confirms this view, but the fact that the venom is not active in crustaceans (Rathmayer, W. and Walther, G., 1976), which are also thought to have glutamatergic transmission (cf. Chapter 3, section 3.3) is not in accordance with this notion, and remains to be explained. Microbracon venom can be used to selectively block excitatory neuromuscular transmission so that the inhibitory transmission can be studied separately. Examples have been described and reviewed recently by Piek, T. (1982a). Although the venom of Microbracon hebetor contains labile toxins of high relative molecular mass, production of crude venom is easy and purification is not necessary in order to achieve the block of excitatory neuromuscular transmission. 3.3
Cynipoidea
This superfamily includes about 1600 species of small insects (Imms, A., 1960). The subfamily of Figitinae normally consists of parasites of Diptera. Nevertheless Doutt, R. (1963) observed Figitis anthomyiarum to transiently paralyse larvae of the mealmoth Ephestia kuehniella. A transient paralysis of larvae of flies has been described by Sweetman, H. (1958). 3.4
Chalcidoidea
This superfamily is probably the largest in the order of Hymenoptera as regards number of species, and it also includes some of the smallest members of the Insecta (Imms, A., 1960). Within this superfamily larvae and sometimes adults of representatives of nearly all insect orders are accepted as hosts (Table 4). In most cases paralysis occurs, often incompletely and/or transiently. However, lack of paralysis is mostly not reported. Thus the number of wasps which do not paralyse their prey is probably much larger than suggested by Table 4. This superfamily
601
Insect Venoms and Toxins Table 4: References to observations of paralysis in insects by a sting of wasps belonging to the Superfamily Chalcidoidea
Wasp genus
Host/prey Kind of References taxum paralysis
Aphelinus
Hem.
+ PT
Brachyneria Cirrospilus
Dip. Lep.
+
Coccophagus Dahlbominus Dicladocerus
Hem. Hym. Lep.
+T +1 +
Elachertus
Lep.
+
Elasmus Eulophus
Lep. Col. Lep.
+1 +1 +
Euplectrus Eupteromalus Eurytoma Habrocytus
Lep. Lep. Col. Lep.
+P +? +P
Lariophagus
Col.
+ PC
Melittobia Microplectron Pleurotropis Rhopalicus Schizonotus Solenotus Tetrastichus
Lep. Hym. Col. Col. Col. Dip. Hym. Lep.
+D +1 + +P +1 +D — +
Hem. Col.
+T -
Trichomalus
Clausen, C , 1940; Wilbert, H., 1964 Roberts, R., 1933 Eveleens, K. and Evenhuis, H., 1968 Sweetman, H., 1958 Clausen, C , 1956 Dowden, P. and Carolin, V., 1950 Dowden, P. and Carolin, V. 1950 Mahdihassan, H., 1934 Taylor, T., 1937 Parker, H. and Smith, H., 1933 Noble, H., 1938 Clausen, C , 1956 Nuorteva, M., 1957 Noble, N., 1932; Fulton, B., 1933; Clausen, C , 1940 Hase, A., 1924; Piek, T., new record Buckell, E., 1929 cf. Clausen, C , 1940 Taylor, T., 1937 Taylor, R., 1929 Dowden, P., 1939 Doutt, R., 1957 Taylor, T., 1937 Dowden, P. and Carolin, V., 1950 Moran, V. et at., 1969 Sweetman, H., 1958
+ = paralysis, — = no paralysis, P = permanent, T = transient, C = complete, I = incomplete, D = delayed. Kev to host or prey taxa: Col. = Coleoptera, Dip. = Diptera, Hemiptera, Hym. = Hymenoptera, Lep. = Lepidoptera
also includes many families, which are entomophagous on eggs or pupae (Clausen, C , 1940). In a discussion on paralysing venoms, eggparasites could be excluded. However, it would be interesting to study the effect of "venom"secretions ofthese egg-parasites. One could imagine that parasites of insect pupae do not necessarily paralyse their hosts. However, Pleurotropis passei stings, and immediately paralyses beetle pupae (Taylor, T., 1937). Hase, A. (1924) observed that Lariophagus distinguendus stings its prey at random and not specifically in the direction of ganglia. Wilbert, H. (1964)
observed the behaviour of Aphelinus semiflavus as it stung into the leg of the aphid, Macrosiphon solani. After the onset of paralysis the sting was inserted further into the host before oviposition took place. The available data on the stinging behaviour of the Chalcidoidea support the conclusion that these wasps do not sting in, or adjacent to, the central nervous system. This might be generalized to the whole section of Terebrantia (see preceding section). It has been described before, in section 3.2, that as a rule ectoparasitic wasps paralyse their host prior to oviposition. An exception is the chalcidid wasp Trichomalus fasciatus, an externally living parasite of the beetle larva Ceutohorrhynchus assimilis, which is not paralysed by the sting of the wasp (Sweetman, H., 1958).
4
VENOMS OF HYMENOPTERA ACULEATA
The Aculeata can be divided into two groups: the solitary aculeate wasps on the one hand, and the ants (Formicidae), bees (social and solitary Apidae) and social wasps (Vespidae) on the other hand. Members of the first group produce paralysing venoms, used to immobilize and not to kill a prey (insect or spider), which serves as a host for the parasitizing larva of the wasp. Members of the second group produce venoms, used by the insects for defence. The three latter groups are described in sections 4.7 (Vespidae), 4.8 (Apidae) and 4.9 (Formicidae). The paralysing effect of a sting by a aculeate wasp was already known by the ancient Chinese. The Erh-Ya (Kuo Po, A.D. 276-324) describes a green "worm" which was paralysed by a wasp. This knowledge has been overlooked by western science; the first subsequent reference to stinging behaviour was not until 1742 when Reaumur (Ferchauld de Reaumur, R., 1742) described an observation by Cossigni of a wasp stinging a cockroach, which resulted in a loss of the cockroach's forces. Bartram, J. (1744) described a mud-dauber wasp, which disabled spiders but did not kill them. However, Dufour, L. (1841) believed that weevils collected by the sphecid wasp Cerceris bupresticida were dead, and that the wasp's venom contained a preservative. Fabre, J. (1855) conclusively settled these conflict-
602
Tom Piek
Insect Venoms and Toxins
603
FIG. 4. Prey capture and stinging behaviour of aculeate solitary wasps (Sphecidae). Top left: Ammophila sabulosa stings a looper (inch-worm, Geometridae) at the ventral side. Top right: Cerceris rybyensis stings a solitary bee between the legs. Middle right: Mellinus arvensis stings a weevil in the thorax. Bottom right: Philanthus triangulum carrying a paralysed honey bee. Photographs J. M. C. P. Schoonen, Venlo, The Netherlands. Bottom left: Diagrammatic representation of the position of the CNS in the cricket, Gryllus domestica (in blue). The figure shows the characteristic stinging sites (squares) and directions at which the sting was introduced (blue arrows ending in the squares). Red points and lines show the variation of individual stinging sites and directions. S: suboesophageal ganglion; I—III: pro- meso- and metathoracic ganglion. (From Steiner, A., 1962).
ing views. He observed that after attack by C. bupresticida the weevils were able to perform contractions when stimulated by electrical currents. Fabre's conclusion was that the prey of solitary Aculeata were paralysed and not dead. In his Souvenirs Entomologiques, J. Fabre (1879-1910) presented the view that solitary Aculeata sting their victims in the central nervous system. He observed that the number of stings given by the wasp was correlated with the number of main nerve ganglia in the prey. From the work of many authors, which have confirmed Fabre's view (Table 5) attention is paid to the painstaking work of Steiner, A. (1962), who observed Liris nigra stinging the cricket (Gryllulus domesticus) in the four big ganglia (Fig. 4). 4.1
Bethyloidea
This superfamily consists of five families, the most important being the Dryinidae, Bethylidae, Chrysididae and Sclerogibbidae. The Dryinidae are parasitic upon nymphs of Hemiptera. Transient paralysis has been described (Swezey, 1919; cf. Sweetman, H., 1958; Clausen, C , 1940; Newman, L., 1965). Wasps belonging to the family Bethylidae attack lepidopterous or coleopterous larvae (Table 6). They sting their prey through the dorsal surface (Voukassovitch, M., 1924) or through the ventral surface of the thorax or neck, probably without great precision (Iwata, K., 1942). Clausen, C. (1940) has reported that Laelius anthrenivorus frequently directs its sting towards the central nervous system of the prey. The Chrysididae, or cuckoo wasps, are of a brilliant metallic coloration. These wasps are usually seen in the neighbourhood of the nests of various solitary bees and wasps where the cuckoo wasp's larva prey on those of the host (bee larva) or on the prey of the solitary wasp (Imms, A., 1960). Within the Chrysididae Chrysis shanghaiensis is exceptional in that it lays its eggs directly on caterpillars of Monemaflavescens, which it incompletely
paralyses (Du Buysson, R., 1898; Piel, R., 1933; Parker, D., 1936). The Sclerogibbidae are parasites of Embioptera. Ananthasubramanian, K. and Ananthakrishnan, T. (1959) reported transient paralysis of Oligotoma greeniana stung by Sclerogibba embiidarium. 4.2
Scolioidea
The most primitive members of Aculeata are found within this superfamily. The Scolioidea are subdivided into five families, two of which are of relative importance with regard to our knowledge of their venoms: these are the Scoliidae and Tiphiidae. Members of both families usually parasitize on larvae of Coleoptera (Table 7). An exception is Diamma bicolor, which paralyses molecrickets (Gryllotalpa coarctatd) incompletely and transiently (Hardy, A., 1911). Beetle larvae are paralysed by a sting before oviposition and the wasp's larva, on hatching from the egg, feeds on the living but immobilized prey. According to Passerini, C. (1840) Mr Piccioli found pupae of Megascoliaflavifrons in old tanning bark. In 1841 Passerini described that the host, larva of the beetle Oryctes nasicornis, was alive and paralysed. Fabre (1879-1910) in the third volume of his Souvenirs Entomologiques gives an excellent description of the behaviour of certain scoliid wasps (Scolia hortorum, Scolia haemorrhoidalis (=flavifrons), Scolia bifasciata, and Scolia interrupta ( = Campsomeris sexmaculata). Fabre described the way in which S. bifasciata stings the beetle larva Cetonia aurata once, resulting in an immediate paralysis of the larva. Piek, T. et al. (1983a) demonstrated that the wasp Megascolia flavifrons stings larvae of the beetle O. nasicornis on the ventral side of all segments except the last three, which do not contain nerve ganglia (Fig. 5). Using pharmacological analysis with different mammalian smooth muscle preparations they found histamine-like and bradykinin-like activity in the venom. No
604
Tom Piek
Table 5: References providing evidence for or against stinging by aculeate wasps into or in the direction of the central nervous system (CNS) of insects and spiders Bethylidae (stinging larvae of Lepidoptera or Coleoptera; stinging in CNS not certain) sting through the dorsal surface Voukassovitch, M. 1924 sting towards ganglia Clausen, C. 1940 sting through the ventral surface of thorax and neck Iwata, K. 1942 Scoliidae (stinging larvae of Coleoptera) one sting in the direction of the ganglion complex stings at the ventral side of ganglion containing segments
Fabre,J. 1879-1910 see section 4.2.
Pompilidae (stinging spiders) stings between legs and/or in the neck at the ventral side
Ferton, C. 1910; Petrunkevitch, A. 1926; Iwata, K. 1942; Williams, F. 1956
Sphecidae (stinging various insects or spiders) stings in the direction of the suboesophageal ganglion of bees stings between the legs of a bee stings between the legs of a beetle stings at the junctions of the coxae and thoracic segments of cockroaches stings at the ventral side of caterpillars (only in segments containing ganglia)
Peckham, G. and Peckham, E. 1898; Fulcrand, J. 1966; Gervet, J. and Fulcrand, J. 1970 Rau, P. and Rau, N. 1918; Claude-Joseph, F. 1928; Piel, R. 1933; Molitor, A., 1939b; Malyshev, 1941 (cf. Malyshev, S. 1966); Ristich, S. 1953; Steiner, A. 1962, 1976
stings toward thoracic and suboesophageal ganglia of locusts, grasshoppers, crickets and preying mantids
Eumenidae (stinging larvae of Lepidoptera; sting in the CNS not certain) stings at the ventral side stings at random locations
cholinergic or serotoninergic activity was found. As in bradykinin, the bradykinin-like activity on guinea-pig ileum was potentiated by the pentapeptide BPP5a (Fig. 6). The presence of histamine was confirmed by a radio-enzymatic method (Piek, T. et al.9 1983a). So far only one species of the superfamily Scolioidea, Diamma bicolor, seems to produce a venom which does not contain histamine (Owen, M., 1969), but this venom contains more than 1 //gml - 1 serotonin (5-HT). Except for/), bicolor all Scolioidea listed in Table 7 paralyse larvae of Coleoptera, and a number of references indicate that the paralysis is transient or incomplete. Piek, T. et al. (1983a) concluded from the stinging behaviour, as well as from experiments with beetle larvae injected with a venom extract, that M. flavifrons stings into the nerve ganglia where the venom seems to block nervous conduction or transmission. The insect CNS is affected by a large number of putative transmitters and other agonists, as well as antagonists, including polypeptides, such as enkephalin and /?-endorphine (Pitman, R., 1980).
Walckenaer, C. 1817; Marchal, P. 1887 Claude-Joseph, F. 1928; Rathmayer, W. 1962 Fabre,J. 1879-1910 Eberhard, W. 1974
Maneval, H. 1932 Raubaud, 1916
In the mammalian CNS, the presence of histamine H : -receptors is now firmly established both by direct binding studies with [3H]mepyramine and by the demonstration of biochemical responses to H ^ receptor activation (cf. Schwartz, J. et al., 1980). Although no transmitter or other role has been proposed for histamine, the widespread capacity to synthesize histamine in the CNS of Manduca sexta (Lepidoptera) supports its candidacy for a physiological role in the insect nervous system (Hildebrand, J. and Maxwell, G., 1980). For a hypothetical role of the agonists present in solitary wasp venoms see section 4.4. In Campsomeris sexmaculata venom which does not contain acetylcholine or serotonin, but a considerable amount of histamine (about lOOng per venom reservoir), an additional unknown smooth muscle-contracting factor has been discovered (Piek, T., new record). The venom caused contraction of the rat colon, which was not antagonized by mepyramine. The action of this unknown factor on vertebrate smooth muscle is comparable with that of angiotensin.
605
Insect Venoms and Toxins
m 3rV ©
„Jf *
**'■'"
'ί^í.|":*í·"'".
FIG. 5. Megascoliaflavifrons stings the larva of the beetle, Oryctes nasicornis in the direction of the metathoracic ganglion complex. (Photograph by M. S. J. Overzier, from Piek, T., 1978.) Table 6: References to observations of paralysis in insects by a sting of wasps belonging to the Family Bethylidae
Wasp genus Allepyris Cephalonomia Epyris Goniozus Holepyris Laelius Parascleroderma Perisierola Scleroderma
Host/prey taxum Col. Col. Col. Lep. Lep. Lep. Col. Col. Col. Lep. Lep. Col. Lep.
Kind of paralysis +c +
+1 +T + IP
+c
+ PC
+
+1 + PC + (T)C
+ +
References Iwata, K. 1942; Yamada, 1955 (cf. Iwata, K. 1976) Sweetman, H. 1958; Finlayson, L. 1950 Williams, F. 1919a Voukassovitch, M. 1924 Gordh, G. 1976 Bridwell, J. 1920b Vance and Parker, 1932 (cf. Clausen, C. 1940) Sweetman, H. 1958 Maneval, H. 1930 Nickels, C. et al. 1950 Sweetman, H. 1958; Iwata, K. 1976 Kühne, H. and Becker, G. 1974 Bridwell, J. 1920a
+ = paralysis, P = permanent, T = transient, C complete, I = incomplete. Key to host or prey taxa: Col. = Coleoptera, Lep. = Lepidoptera.
606
Tom Piek
MfV 0125
MfV •0250
FIG. 6. Potentiating effect of the pentapeptide BPPsa (lOOng ml - ) on contractions of the guinea-pig ileum induced by 1 ng ml - 1 bradykinin (BK) and the venom of Megascoliaflavifrons(MfV) at concentrations of 0.0025, 0.0125, and 0.0250 venom reservoirs per ml. Note that 100 ng ml ~ BPPsa causes comparable potentiation of contractions induced by 1 ng ml ~ bradykinin and 0.0125 v.r. ml - venom. W = wash. (From Piek, T. et al., 1983a.) Table 7: References to observations of paralysis of larvae of Coleoptera (Col.) and Orthoptera (Ort.) by a sting of wasps belonging to superfamily Scolioidea Wasps genus
Host/prey taxum
Kind of paralysis
Campsomeris
Col.
+ PC
Cosila Diamma Elaphroptera Elis Epartiothynnus Methoca
Col. Ort. Col. Col. Col. Col. Col. Col. Col. Col. Col. Col.
+
Col.
+ TC
Pterombrus Scolia Megascolia Tiphia
+ IT + PC(I) +1 +1
+
+ + + + + +
TI PC(I) PC PI PC T
References Clausen, C. et al. 1927; Illingworth, J. 1919; Jarvis, E. 1931; Sweetman, H. 1958 Janvier, H. 1933 Hardy, A. 1911 Janvier, H. 1933 Clausen, C. et al. 1927 Williams, F. 1919b Adlerz, 1903 (cf. Malyshev, S. 1966) Iwata, K. 1942; Palmer, M. 1976 Williams, F. 1928a; Palmer, M. 1976 Fabre, J. 1879-1910 Passerini, C. 1840, 1841 Piek, T. and Simon Thomas, R. 1969 Adlerz, 1916 (cf. Malyshev, S. 1966) Janvier, H. 1956 Clausen, C. et al. 1927
+ = paralysis, P = permanent, T = transient, C = complete, I = incomplete 4.3
Pompilidae
The Pompilidae and all the subsequently described families of solitary wasps differ from the Scolioidea in that they transport their prey to a nest. The Pom-
pilidae (= Psammocharidae) prey on spiders, most of which are temporarily paralysed (Table 8). If observations are not continued for long periods, permanent paralysis may be recorded erroneously since examples of long recovery periods are known.
Insect Venoms and Toxins
607
Table 8: References to observations of paralysis in spiders (Ar a.) by a sting of wasps belonging to the Family Pompilidae
Wasp genus Agenia Agenioideus Anoplius
Aporus Aporinellus Arachnophoctonus Auplopus Batozonellus Calicurgus Ceropalus Cryptocheilus Deuteragenia Dipogon Elaphrosyron Episyron Fabrogenia Hemipepsis Homonothus Notocyphus Pepsis Phanagenia Planiceps Poecilopompilus Pompiloides Pompilus
Priocnemis Priocnemioides Pseudagenia Sericopompilus Tachypompilus
Host/prey taxum
Kind of paralysis
References
Ara. Ara. Ara. Ara. Ara.
+ PI +1 + PC +P(i) + TC
Ara.
+T
Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara.
+ IT
Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara.
+ PT + TC
Hartman, C. 1905 Maneval, H. 1939 Evans, H. and Yoshimoto, C. 1962 Eberhard, W. 1970 Evans, H. and Yoshimoto, C. 1962; Krombein, K. 1952; McQueen, D. 1979; Soyer, B. 1953; Thijsse, J. 1907 Evans, H. and Yoshimoto, C. 1962; Krombein, K. 1953; Powell, J. 1958; Soyer, B. 1953; Wasbauer, M. 1957 Molitor, A. 1939a, b; Soyer, B. 1953 Maneval, H. 1939 Peckham, G. and Peckham, E. 1898 Evans, H. and Yoshimoto, C. 1962 Rau, P. and Rau, N. 1918 Evans, H. and Yoshimoto, C. 1962 Olberg, G. 1959; Tsuneki, K. 1968b Fabre, J. 1879-1910; Evans, H. and Yoshimoto, C. 1962; Evans, H. and Matthews, R. 1973a Ferton, C. 1897 Minkiewicz, 1934 (cf. Richards, O. and Hamm, A. 1939) Hingston, R. 1928; Evans, H. and Yoshimoto, C. 1981 Nielsen, E. 1932a Evans, H. and Yoshimoto, C. 1962 Evans, H. and Matthews, R. 1973a Evans, H. and Yoshimoto, C. 1962 Evans, H. and Yoshimoto, C. 1962 Veenendaal, R. personal communication Evans, H. and Matthews, R. 1973a MacNulty, B. 1961 Williams, F. 1956 Iwata, K. 1932 Nielsen, E. 1936 Williams, 1928 (cf. Sweetman, H. 1958) Rau P. and Rau, N. 1918 Petrunkevitch, A. 1926; Williams, F. 1956 Evans, H. et al. 1962 Williams, F. 1928a Evans, H. and Yoshimoto, C. 1962 Rau, P. and Rau, N. 1918 Ferton, C. 1897, 1902, 1905, 1910, 1923; Bristowe, W. 1948; Evans, H. and Matthews, R. 1973a Evans, H. and Yoshimoto, C. 1962 Maneval, H. 1939 Ferton, C. 1897, 1908, 1910, 1923 Ferton, C. 1905 Ferton, C. 1897, 1908, 1910 Ferton, C. 1891; Peckham, G. and Peckham, E. 1898; Rau, P. and Rau, N. 1918; Evans, H. and Yoshimoto, C. 1962 Peckham, G. and Peckham, E. 1898 Ferton, C. 1901; Bristowe, W. 1948 Ferton, C. 1897; Evans, H. and Yoshimoto, C. 1962 Ferton, C. 1897 Evans, H. and Yoshimoto, C. 1962 Grandi, G. 1926; Rau, P. 1928; Evans, H. and Yoshimoto, C. 1962 Maneval, H. 1939 Evans, H. and Yoshimoto, C. 1962 Evans, H. and Yoshimoto, C. 1962
+c
+TI +T
+c
+P
+c +P
+c
·. +
+P
+
+P + TC +TI
+ +
+T +T
+
+T +1 +T +P
+
+P +1
+
Ara. Ara. Ara. Ara. Ara. Ara.
+P
Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara. Ara.
+ CI
+c
+T +1 + IT + CT
+
+P + CT +T +1
+c +c +
+ = paralysis, P = permanent, T = transient, C = complete, I = incomplete.
608
Tom Piek
Peckham, G. and Peckham, E. (1898) described the slow recovery of spiders paralysed by Anoplius ( = Pompilus) biguttatus, which took 2 months. Figure 7 summarizes observations of spiders (Trochosa terricola), paralysed by Anoplius viaticus, which show an average recovery period of more than a month. A number of paralysed spiders died during the observation period. If death of the host (which was for the first time observed 10 days after the onset of paralysis) occurs in nature, the wasp's larva would have started to consume the host's tissues several days before this time. A similar slow recovery has been described for spiders paralysed by A. relativus (McQueen, D. 1979), Veenendaal, R. (personal communication) observed the time-course of paralysis of a spider (Araneus sp.) (Fig. 8) stung by Episyron rufipes. Directly after the sting he isolated the spider in a Petri dish with a humid cotton ball. The spider was paralysed but the antennae moved now and then. After 3 days all movement had ceased, but after
9 days the spider began to react on tactile stimulation with faint movement of its legs. After 4 weeks the spider had partly recovered, and tried to stand on its legs, although without success. One week later the spider died. In other cases spiders may revive soon after being paralysed, as for example the spider Cheiracanthium rufulum stung by Homonotus iwatai (Iwata, K. 1932). The pompilid wasps sting spiders in the cephalothorax by inserting their sting between the legs or in the vicinity of the mouth (Iwata, K. 1942). An example is shown in Fig. 8, where Episyron rufipes is stinging a spider. Another example has been described by Petrunkevitch, A. (1926), who observed the tarantula hawk (Pepsis marginata) to sting its prey (Cyrtopholis portoricae) in the articular membrane between the maxilla, first coxae and sternum. The tarantula was paralysed within a few seconds of being stung. When one of the legs of the spider still moved, the wasp returned, climbed onto the tarantula and inserted the sting between
Taken from wasps collected in the field • Taken from wasps 10 sec after one sting
20
40 t (min)
60
30
t (days)
FIG. 7. The time-course of the paralysis and recovery of spiders {Trochosa terricold) stung by the wasp Anoplius viaticus. The degree of paralysis is scored as described by Beard, R. 1952: completely paralysed spiders are scored '3', spiders unable to turn around when lying on their back, but showing movements, either spontaneously or in response to tactile stimulation are scored 2; spiders showing only uncoordinated movements are scored 1; and unaffected spiders are scored 0. Twelve spiders were taken from wasps collected in the field. These spiders were completely paralysed and are assumed to have been stung 1 h before the first observation. Six spiders were stung in the laboratory, and were isolated within 10 s after the wasp had inflicted a single sting. The degree of paralysis was determined 1,10,20 and 60 min after the sting, and subsequently every day. This leads to the conclusion that, if the spiders are stung once, complete paralysis appears in between 1 and 60 min. It appears that spiders are capable of recovering in up to 40 days. Many spiders died or decayed before that time (indicated by the end of lines on the plot). (From Piek, T. 1978.)
Insect Venoms and Toxins
sternum and coxa of the leg involved. A number of other observations of the stinging behaviour of pompilid wasps also suggest that these wasps sting into or in the direction of the spider's central nervous system (Ferton, C. 1910; Rau, P. and Rau, N. 1918; Williams, F. 1956). Our present knowledge on the active substances present in pompilid wasp venoms is extremely poor. In our laboratory we found evidence for small amounts ofhistamine-like activity in the venoms of Anoplius samariensis (40 ng per venom reservoir) and Batozonellus lacerticida (15 ng per reservoir). Both venoms induced contraction of rat fundus strip, guinea-pig ileum and rectum, without any effect on the tension of the rat colon. The venoms caused relaxation of the rat duodenum. Contrac-
609
tions of the ileum could be antagonised with mepyramine (2 x 10" 6 M), but not with atropine. The venoms also caused a phasic contraction which preceded the histamine-like contraction. This initial contraction was antagonized by pentolonium. It is not known if this effect, which might be initiated in the nervous network, is an effect of histamine or if it is 5-HT-like. In contrast to the above-described venoms of Anoplius species, which contain histamine-like activity and no cholinergic substances, the venom of Episyron rufipes contain no histamine (radioenzymatic assay, Piek, T. et al. 1983b) and no 5-HT (pharmacological analysis using the rat fundus preparation; Piek. T., new record), but a considerable amount of acetylcholine-like activity. This
FIG. 8. Episyron rufipes stings a spider (Araneus sp.). (Photograph by R. L. Veenendaal.)
610
Tom Piek
Table 9: References to observations of paralysis in insects and spiders by a sting of wasps belonging to the Family Sphecidae
Wasp genus Ammophila
Ampule x Aphilanthops Astata Bembecinus ( = Stizus) Bembix
Bembidula Bothynostethus Brachymeris Cerceris
Chlorion
Clypeadon Coelocrabro Crabro
Crossocerus Diploplectron Dolichurus Diodontus Eucerceris Fertonius Gorytes
Host/prey taxum
Kind of paralysis
Lep.
+1
Hym. Die. Die. Hym. Hym. Hem. Hem. Hem. Hem. Hem. Dip. Dip. Dip.
+ + + + +
(I) I(T) T C,I D
+
+1 + C(I)
+c +1
+c
+1 + PC
Dip. Hem. Hym. Neu. Hem. Col. Hym. Col.
+ CI +P
Col. Col.
+P +1
Col. Col. Col.
+T +D + CI
Col. Hym. Hym. Hym. Hym. Ort. Ort.
+ PC +1 +T + CI + TI
+P(D + +1 +1 +1
+
+
+1
Ort. Ort. Hym. Ort. Hern. Col.
+ PI + TI
Dip.
+1
Lep. Dip. Hem. Die. Die. Hern. Col. Hym. Hern.
+
+c
+1 +1 + C(I)
+1 +1 +1 + CT + PC(I) + D +1 +1
References Fabre, J. 1879-1910; Peckham, G. and Peckham, E. 1898; Hartman, C. 1905; Ferton, C. 1908, 1920; Claude-Joseph, F. 1928; Maneval, H. 1932; Molitor, A. 1939a, b; Evans, H. 1959; Malyshev, S. 1966; Tsuneki, K. 1963, 1968a; Krombein, K. 1972 Grandi, G. 1926; Evans, H. 1965 Hingston, R. 1925, 1928 Williams, F. 1929 Peckham, G. and Peckham, E. 1898 Wheeler, W. 1913 Ferton, C. 1901; Krombein, K. 1972 Evans, H. 1957; Tsuneki, K. 1969b Peckham, G. and Peckham, E. 1898 Evans, H. 1955 Ferton, C. 1902; Lüps, P. 1973 Ferton, C. 1899 Krombein, K. 1936 Fabre, J. 1879-1910; Wesenberg Lund, C.1891; Marchai, P. 1893; Ferton, C. 1899 Claude-Joseph, F. 1928 Hartman, C 1905 Evans, H. and Matthews, R. 1973b Evans, H. 1978 Claude-Joseph, F. 1928 Kurczewski, F. and Evans, H. 1972 Ferton, C. 1890; Emery, C. 1893 Bosc, cf, Walckenaer, C. 1817; Fabre, J. 1879-1910; Claude-Joseph, F. 1928; Cartwright, O. 1931; Krombein, K.1963 Strandtmann, R. 1945 Grandi, G. 1926; Hamm, A. and Richards, O. 1930; Krombein, K. 1936; Strandtmann, R. 1945; Alcock, J. 1974 Rau, P. 1928 Linsley, E. and MacSwain, J. 1956 Grandi, G. 1926; Claude-Joseph, F. 1928; Hamm, A. and Richards, O. 1930 Peckham, G. and Peckham, E. 1898 Molitor, A. 1939b; Bristowe, W. 1948; Tsuneki, K. 1965a Marchai, P. 1887; Hamm, A. and Richards, O. 1930 Ferton, C. 1910 Marchai, P. 1887 Strandtmann, R. 1945 Peckham, G. and Peckham, E. 1898; Rau, P. and Rau, N. 1918; La Rivers, I. 1945; Ristich, S. 1953 Woodbury, A. 1930; Frisch, J. 1937 Rau, P. 1928 Evans, H. and Eberhard, M. 1970; Alcock, J. and Gamboa, G. 1975 Bristowe, W. 1948 Claude-Joseph, F. 1928; Davidson, R. and Landis, B. 1938 Benoist, R. 1915; Grandi, G. 1927; Maneval, H. 1928; Claude-Joseph, F. 1928 Claude-Joseph, F. 1928; Simon Thomas, R. and Veenendaal, R. 1974 Peckham, G. and Peckham, E. 1898 Bristowe, W. 1948 Kurczewski, F. 1972a Ferton, C. 1923; Maneval, H. 1928, 1932 Benoist, R. 1927 Peckham, G. and Peckham, E. 1898 Linsley, E. and MacSwain, J. 1956 Ferton, C. 1896a Maneval, H. 1939
611
Insect Venoms and Toxins
Table 9: — Continued
Wasp genus Gorytes (continued) Harpactopus Harpactus Hoplocrabro Larra Lindenius Liris Lyroda Mellinus
Memesa Miscophus Nitelopterus Oxybelus
Palarus Paranysson Pemphredon Philanthus Pison Podalonia Podium Priononix
Psammophila Psen Rubrica Salius Sceliphron Solenius Sphecius Sphex
CIP V0L11-NN
Host/prey taxum Hem.
Ort. Hem. Dip. Ort. Ort. Dip. Ort. Ort. Ort. Dip.
Dip. Ära. Ära. Dip. Col. Hym. Hem. Ara. Ara. Dip. Dip. Dip. Dip. Dip. Hym. Hem. Hem. Hym. Hym. Hym. Ara. Lep.
Kind of paralysis +C +TI +1
+
+ IT + TC +1 +D +1 +1 +P + PI
f
+ + + +
QI) C(I) C(I) C(I)
+
+P
+
t
+
+c
+1 + TC
+c
+1 + PI +P +T + TI
+c +
Lep. Die. Ort. Ort.
+P + IT TI +1
Ort. Lep. Hem. Dip. Odo. Lep. Ara. Ara. Ara. Ara. Dip. Hem. Ort.
+ D
+
Ort. Ort. Ort. Ort. Lep.
+1 + TC +T + ID +1
+
+P
-(t?) -(t?) -(t?)
+P
+
+ TC +1
+
+ PC
References Claude-Joseph, F. 1928 Peckham, G. and Peckham, E. 1898 Maneval, H. 1928 Maneval, H. 1928 Williams, F. 1928a; Smith, C. 1935 Malyshev, 1941 (cf. Malyshev, S. 1966) Bristowe, W. 1948 Steiner, A. 1958, 1962 Williams, F. 1928a Peckham, G. and Peckham, E. 1898 Rabaud, E. 1917; Spooner, G. 1928; Molitor, A. 1939a; Bristowe, W. 1948 Adlerz, G. 1903 Goodman, W. 1970 Claude-Joseph, F. 1928
Janvier, H. 1955 Ferton, C. 1896a Krombein, K. and Kurczewski, F. 1963 Wesenberg Lund, C. 1891 Bohart, R. and Marsh, P. 1960 Ferton, C. 1902, 1923 Krombein, K. and Kurczewski, F. 1963 Ferton, C. 1902; Kurczewski, F. 1972b; Steiner, A. 1979 Cherian, 1937 (cf. Clausen, C. 1940) Bequaert, J. 1933 Janvier, H. 1961 Evans, H. 1959 Fabre, J. 1879-1910; Roth, P. 1917; Rathmayer, W. 1962 Claude-Joseph, F. 1928 Claude-Joseph, F. 1928 Williams, F. 1928b; Hicks, C. 1932b; Krombein, K. 1936; Steiner, A. 1974 Gervet, J. and Fulcrand, J. 1970 Williams, F. 1928a Peckham, G. and Peckham, E. 1898 Hartman, C. 1905; Rau, P. and Rau, N. 1918; Evans, H. 1958 Rau, P. and Rau, N. 1918 Hingston. R. 1928 Janvier, H. 1955 Evans, W. et al 1974 Ferton, C. 1910 Rau, P. and Rau, N. 1918; Rau, P. 1928; Aptel, E. 1929 Peckham, G. and Peckham, E. 1898 Claude-Joseph, F. 1928 Krombein, K. 1936 Riley, C. 1892; Hartzell, A. 1935 Fabre, J. 1879-1910; Picard, F. 1903; Ferton, C. 1909; Claude-Joseph, F. 1928; Berland, L. 1938, 1959; Newman, L. 1965 Berland, L. 1958; Claude-Joseph, F. 1928; Piel, R. 1935 Peckham, G. and Peckham, E. 1898 Hingston, R. 1928 Fabre, J. 1879-1910; Molitor, A. 1939a Strandtmann, R. 1945
612
Tom Piek Table 9: — Continued
Wasp genus
Stizus Tachysphex
Trigonopsis Trypoxlon
Host/prey taxum
Kind of paralysis
Lep. Die. Hem. Ort. Ort. Ort.
+T +1 + +1 + +1
Hem. Die. Die. Hym. Die. Ara. Ara. Ara. Ara. Ara.
+c
+ + PC
+
+1
+
+P +1 + PI + IT
References Hicks, C. 1932a Claude-Joseph, F. 1928 Ferton, C. 1908, 1910 Ferton, C. 1908, 1910; Tsuneki, K. 1965b Ferton, C. 1923; Claude-Joseph, F. 1928; Krombein, K. 1972 Ferton, C. 1908; Rau, P. and Rau, N. 1918; Strandtmann, R. 1945; Evans, H. and Kurczewski, F. 1966; Kurczewski, F. 1966; Tsuneki, K. 1969a Claude-Joseph, F. 1928 Ferton, C. 1901, 1911 Cros, A. 1936 Evans, H. 1964 Williams, F. 1928a; Richards, O. 1937; Eberhard, W. 1974 Newman, L. 1965 Peckham, G. and Peckham, E. 1898 Hamm, A. and Richards, O. 1930 Malyshev, 1911 (cf. Malyshev, S. 1966) Hartman, C. 1905; Kurczewski, F. 1963; De Groot, W. 1971
+ = paralysis, - = no paralysis, P = permanent, T = transient, C = complete, I = incomplete, D = delayed, f = dead. Key to host or prey taxa: Ara. = Arachnida, Col. = Coleoptera, Die. = Dictyoptera, Dip. = Diptera, Hem. = Hemiptera, Hym. = Hymenoptera, Lep. = Lepidoptera, Neu. = Neuroptera, Odo = Odonata, Ort. = Orthoptera
activity, which is probably due to acetylcholine (ACh), was equivalent to 400 ng ACh per reservoir. A comparable situation is described in the next section (Sphecidae) where one species produces a venom containing ACh and no histamine, and several others contain histamine and no ACh. It is by no means probable that only these active substances are responsible for the paralysing effects, as will be shown in the next section. 4.4
Sphecidae
The majority of the sphecid wasps are fossorial, but some genera such as Sceliphron construct free mudcells. The family in its totality preys on nearly all insect orders as well as spiders, but Table 9 shows that some genera are specialized to prey on only one insect order. Prey specialization is sometimes more specific, such as in Philanthus triangulum which preys exclusively on workers of the honeybee, Apis mellifera (Fig. 4). Walckenaer, C. (1817) observed that Cerceris ornata stung Halictus bees in the direction of the suboesophageal ganglion. Fig. 4 shows Cerceris rybyensis stinging a solitary bee. Peckham, G. and Peckham, E. (1898) described the behaviour of Ammophila urnaria while stinging caterpillars (Fig. 9). In three captures they observed that the prey was
stung in a number of segments in succession through the ventral integument although the stinging sequence showed marked variations. Fig. 4 shows Ammophila sabulosa stinging a looper. Fabre, J. (1879-1910) observed that Cerceris tuberculata stings its prey, a buprestid beetle, between the legs. Cerceris arenaria stings the weevil Br achy der is incanus through the ventral side of the soft membrane between head and thorax. The discolourations of the weevil's integument are indicative of the sting locations, which may be found close to the suboesophageal ganglion (Fig. 10). Figure 4 shows Cerceris quadricentata stinging a weevil. Table 5 summarizes a number of references which provide evidence of the stinging of sphecid wasps into, or in the direction of, the central nervous system of insects or spiders. The question now arises as to whether the venom of these wasps, when brought into the ganglia, can interfere with synaptic transmission or nerve conduction. Rathmayer, W. (1962) found that honeybee workers, 2 h after being stung in the first thoracic ganglion by Philanthus triangulum, show morphological changes in this ganglion close to the site of the sting. Morphological changes in the second thoracic ganglion were not seen until 24 h after the sting. The central action of the venom of P. triangulum has been studied using the cereal nerve-giant axon preparation of the sixth
Insect Venoms and Toxins
j»&
o~^V <<£--< ry^r~J-'€r
Λ\Í ***' ^
- ^ s / ///
'ÖË, *
FIG. 9. Ammophila urnaria stinging a caterpillar. (From Peckham, G. and Peckham, E. 1898.)
abdominal ganglion of Periplaneta americana, and using the mannitol-gap technique described by Callec, J. and Sattelle, D. (1973). At concentrations lower than the wasp may inject into the ganglion mass of honeybee workers, the venom causes a considerable depolarization of the giant neuron, and a concurrent depolarization of presynaptic terminals (Piek, T. et al.9 1980a, 1982a). The venom contains ACh varying from about
613
600 ng per venom reservoir in P. triangulum from Egypt to about 200 ng in the same species collected in Europe (Piek, T. et al., 1983b). The venom of five other sphecid wasps did not contain ACh but various amounts of histamine-like activity, which was not present in P. triangulum venom. The histamine-like activity per venom reservoir was equivalent to 22 ng histamine in Cerceris arenaria, 173 ng in Mellinus arvensis, 540 ng in Bembix rostrata, 732 ng in Sceliphron spirifex, and 1800 ng in Palmodes occitanicus. The histamine-like effect on vertebrate smooth muscle was antagonised with mepyramine. The presence of histamine in these venoms was confirmed by a radioenzymatic method (Piek, T. et αί, 1983b) It can be concluded that venoms of solitary aculeate wasps, such as members of the family of Sphecidae, produce centrally acting neurotoxic venoms. Although our knowledge of these toxins is fragmentary, some pharmacological active substances are present in venoms at concentrations which are high enough to cause a transient but dramatic change of nervous function in the ganglia. However, these venoms may also contain toxins of a quite different nature. One, that of
F I G . 10. Discolorations in the integument of the ventral surface of the neck of the beetle Brachyderis incanus paralysed by Cerceris arenaria. The discolorations indicate the sting sites. (Photograph by M. S. J. Overzier.)
614
Tom Piek
Philanthus triangulum is now known to contain not only ACh, but also at least three paralysing toxins. One of these philanthotoxins, ä-ÑÔ× blocks ion channels in the insect muscle fibre membrane. The ion channels are only blocked in the open conformation (see chapter 3, section 3.2.2). Similar action of ä-ÑÔ× has been demonstrated recently in the cockroach CNS (Piek, T. et al., 1984). It can be speculated that if ä-ÑÔ× also causes an activationinduced block in the insect central nervous system, the role of ACh might be to initiate massive opening of cholinergic channels in the postsynaptic membranes. In general, sphecid venoms are less potent than those of the Terebrantia. This was demonstrated by Ferton, C. (1902) in his observations of Sphex subfuscatus italicus stinging crickets. The first cricket to be stung was completely paralysed, the second was only partly paralysed. Ferton, C. (1910) also showed that small Halictus bees, attacked by Cerceris emarginata, were completely paralysed while bigger bees were incompletely paralysed. This observation has been confirmed by Claude-Joseph, F. (1928) who showed that when Cerceris gayi attacks beetles, only the smaller species attacked were completely paralysed (the venom of some terebrant wasps such as Microbracon hebetor (section 3.2) is much more potent. Beard, R. (1952) calculated that venom of a single M. hebetor wasp could theoretically paralyse 5000 wax moth larvae). Transient paralysis lasting 5-10 min has been described for Larva analis and Larra anathema (Smith, C , 1935; Malyshev, 1941: cf. Malyshev, S., 1966; Nambu, 1970: cf. Iwata, K., 1976). Reversible paralysis lasting a number of months has been observed for caterpillars collected by Sphex aberti
(Hicks, C , 1932a). Permanent paralysis has been described for a large number of prey (Table 9). The prey commonly die after a relatively short time, but this is not always the case. Riley, C. (1892) described how the cicada Tibicen pruinosa, after being stung by Sphecius speciosus, remained in a state of suspended animation which, under favourable conditions, lasted for a year and potentially longer. A delay between the time of stinging and the onset of paralysis has been observed in the hosts of a number of Sphecidae (Table 9). A delay of several hours has been reported for the onset of paralysis in crickets stung by Liris nigra (Steiner, A., 1958). Wheeler, W. (1928) described how queen ants, stung by Aphilanthopsfrigidus, continued to move their palpi, legs and antennae, either spontaneously or when touched. These movements continued for several hours, or sometimes even for a few days, after the ants had been captured. All movement then ceased, although the insects retained a fresh appearance and limb flexibility with no indications of the tissues drying up. From the fragmentary knowledge of paralysis caused by the sting of sphecid, and also other solitary aculeate wasps, it is obvious that this paralysis is the result of a complex pharmacological phenomenon. It is also probable that the complex actions found for the venom of one species cannot be generalized for other species, even closely related ones. The best-known sphecid wasp venom is that of the bee wolf, Philanthus triangulum. The effects of one of its composing toxins, (S-philantho toxin or 5-PTX is described in chapter 3 (section 3.3.2); a general description is presented here. Although the venom of P. triangulum contains a centrally active component and a considerable
FIG. 11. Effect of 3 bee units per ml (BU ml ) venom of Philanthus triangulum from Europe on miniature excitatory postsynaptic potentials (horizontal arrows) and miniature inhibitory postsynaptic potentials (vertical arrows) recorded from a coxal muscle of the metathoracic leg of the locust, Schistocerca gregaria. The bee unit (BU) is defined as the activity of a P. triangulum venompreparation, injected in equal amounts into ten honeybee workers, causing five of these bees to remain paralysed for at least 1 h (Piek, T. et al., 1971). (1) Control, (2) 10min in venom, (3) after 60min wash. (Calibration 10//V and 100ms.)
615
Insect Venoms and Toxins
****WU^ Control
0-500 Hz
^^U^JJJIJ^^ Control
002-100 Hz
\i^J^^ 4B.U./ml,2min
ImV
^-Uwji^^ 5 min
^
■
^
^
^^
Wash , 20min
Ajji
n) 40 min
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FIG. 12. Effect of the venom of Philanthus triangulum from Egypt (4 BU m l - , see Fig. 11) on excitatory miniature potentials recorded from a short mesothoracic dorsal longitudinal flight muscle of the cabbage white (Spanjer, W. et al, 1977). The signals were recorded on tape with a bandwidth of 500 Hz (upper control) and fed into the computer with a bandwidth from 0.02 to 100 Hz (lower control). Note the reversible decrease in amplitude and the seeming decrease in frequency (see also Fig. 13 for computer analysis).
amount of acetylcholine (Piek, T., 1982b, Piek, T. et al, 1980a, 1983b), the most obvious effects are on the skeletal neuromuscular transmission in insects (Piek, T., 1966, Piek, T. et al, 1971). The venom blocks excitatory as well as inhibitory neuromuscular transmission (Fig. 11). It was found (Piek, T. et al, 1971; Piek, T. and Njio, K., 1975) that the venom causes a decrease in the frequency of spon-
taneous quantal release, but at relatively low concentrations also caused a decrease in amplitude of miniature postsynaptic potentials. The venom of P. triangulum collected in Egypt showed a more pronounced effect on amplitude (Figs 12 and 13) than the venom of wasps collected in Europe (Piek, T. et al, 1980a). This indicates that the venom may have a presynaptic as well as a postsynaptic effect,
616
Tom Piek
and that quantitative differences exist between races of one species. In the locust, Njio, K. and Piek, T. (1979) demonstrated that the nerve terminals of the extensor muscles, paralysed with Philanthus venom, showed a significantly lower density of presynaptic vesicles than those in the untreated muscle. The conclusion was that the venom inhibits the supply of transmitter substance to the nerve terminals. Recently Van Marie, J. et al. (1982) found a 50% inhibition of glutamate uptake in nerve terminals as well as in glial cells of the retractor unguis muscle of locusts, treated with P. triangulum venom at a concentration just enough to paralyse the muscle completely. It can be concluded that the presynaptic action of the venom is a reuptake inhibition of the transmitter substance, which probably is L-glutamate (see chapter 3, section 3.3). Philanthus venom also caused a decrease in amplitude and half-decay time of extracellularly recorded miniature potentials from the locust retractor unguis muscle (Fig. 14; Piek, T. et al., 3000
1000
V
300
100h c £30
% % • Control 5min,4B.U./ m | .
g.10 A
40 min Wash
-Ù 3
E 3
z
1 003
01
-L 0-3
10 mV 30
Amplitude FIG. 13. The effect of the venom of Philanthus triangulum on the inverse cumulative amplitude plots of miniature potentials shown in Fig. 12. Note the shift along the X-axis, indicating a change in amplitude without a marked effect on the frequency. (From Piek, T. et al, 1980a.)
FIG. 14. Effect of 0.4 BU ml ~ of venom of Philanthus triangulum on amplitude and half-decay time of excitatory miniature potentials externally recorded from a nerve-muscle junction of the locust (Schistocerca gregarid) retractor unguis muscle. A: Computer plot of the average of 50 miniature potentials before application of venom; B: average of 50 potentials after 10 min exposure of the muscle to the venom solution. Note the decrease in amplitude and the increase in decay rate. (From Piek, T. et al., 1980a.)
1980a,b). This has led to the suggestion that the venom potentially blocked thejunctional current by shortening the open time of the ion channel in the postsynaptic membrane. The first indication for a postsynaptic effect of the venom was found by May, T. and Piek, T. (1979) who showed that, in the locust retractor unguis muscle, the venom antagonizes bath-applied and iontophoretically evoked glutamate potentials. Bath-applied glutamate potentials were antagonized by the venom in a dosedependent manner. Although the effect was reversible the shift in the glutamate dose-response curves showed that the antagonism was not competitive (Fig. 15, left). These results indicated that the postsynaptic block was probably not caused by an antagonism of glutamate binding at the level of the glutamate receptor. The venom decreased the amplitude of iontophoretically evoked glutamate potentials to a plateau (Fig. 15, right). Comparable effects are described in chapter 3 for the toxin (δPTX) present in P. triangulum venom (Figs 32 and 33 of that chapter). The plateau value decreased with increasing venom or toxin concentrations. The view that Philanthus venom blocks open ion channels was born out of the results of May, T. and Piek, T. (1979) and Piek, T. et al. (1980b) and confirmed for (S-PTX, using the metathoracic extensor tibiae muscle of the locust and iontophoretically applied glutamate currents and patch clamp techniques (Clark, R. et al, 1981, 1982) (see chapter 3, Figs 32-36, for a detailed description).
617
Insect Venoms and Toxins
this plateau level is dose-independent (Dunbar, S. and Piek, T., 1982). The venom had no effect on bath-applied proctolin (see chapters 3 and 13 for proctolin), but responses to bath-applied Lglutamate were inhibited. It was concluded that the venom-resistant plateau contractions were the result of excitation by non-glutamatergic transmission, and were possibly the result of proctolin release.
10
4.5 of glutamate(iO- 2mol/l )
Fig. 15. Effects of the venom of Philanthus triangulum on Lglutamate potentials from fibres of the locust retractor unguis muscle. Left: effect of different doses of venom (0.0 to 2.0 BU m l - ) on the log-dose-response curve of bath-applied glutamate potentials. Right: effects of 1 BU m l - 1 of the venom on iontophoretically evoked glutamate potentials induced by 0.6 nC pulses (A) pen record; B, C: superimposed oscilloscope records of corresponding parts in A. (From May, T. and Piek, T., 1979.)
In the locust {Locusta migratorid) rectum the venom of P. triangulum also inhibits nerve-evoked contraction to a plateau (Figs 16 and 17), but at venom concentrations higher than 1.5BUml _1
Masaridae
The families Masaridae, Eumenidae and Vespidae are easily distinguished from other aculeate wasps by the longitudinal folding of their wings. These "true wasps" are classified by Imms, A. (1960) into a single family (Vespidae) and three subfamilies (Masarinae, Eumeniae, Vespinae). Separation of these (sub)families seems to be difficult. However, one useful behavioural feature is that all the Masaridae and Eumenidae are solitary wasps, whereas the Vespidae form social colonies. Our knowledge about the venoms of Masaridae is extremely poor. Only one species has been described, by Williams, F. (1927): Euparagia scutelaris Cresson, which incompletely paralyses the larvae of beetles (Curculionidae).
x1(T 6M L-Glu
FIG. 16. The effect of Philanthus venom (2BUml~ ) on L-glutamate- (5-7 x 1 0 - 6 M ) and neurally evoked responses. Neural stimulation was stopped for 2 min prior to, and 5 min after, each concentration of L-glutamate. Note the total inhibition of Lglutamate-elicited responses by the venom and the plateau of venom-independent contractions (asterisks). Downward arrows represent the removal of L-glutamate. (From Dunbar, S. and Piek, T., 1982.)
618
Insect Venoms and Toxins
Proctolin
FIG. 17. The effect of Philanthus venom ( 2 B U m l - 1 ) on proctolin- and neurally evoked contractions. Neural stimulation was stopped for 2 min prior to, and 5 mins after, the addition of each concentration of proctolin. Note the lack of action of the venom on proctolin-evoked contractions and the venom-insensitive plateau (asterisks). Downward pointing arrows represent the removal of proctolin. (From Dunbar, S. and Piek, T., 1982.) Table 10: References to observations of paralysis in insects by a sting of wasps belonging to the Family Eumenidae
Wasp genus Ancistrocerus Discoelius Eumenes Manobia Odynerus
Pterochilus Raphiglossa Rhynchium Synagris
Host/prey taxum
Kind of paralysis _?
Lep. Lep. Lep. Lep.
+P(i) + PI +1
Lep. Lep. Lep.
+1 + +1
Lep. Lep. Col. Col. Col. Col. Lep. Col. Lep. Lep. Lep.
+ PI +T + +1 +P + PI +1 +1
+c
+1 + C(I)
References Rau, P. and Rau, N. 1918 Taylor, L. 1922 Piek, T. and Veenendaal, R., new record Fabre, J. 1879-1910; Chretien, P. 1896; Ferton, C. 1902; Roubaud, E. 1916; Deleurance, E. 1945 Rau, P. and Rau, N. 1918 Adlerz (cf. Nielsen, E. 1932b); Spooner, G. 1934 Audouin, V. 1839; Ferton, C. 1896b, 1901; Hartman, C. 1905; Peckham, G. and Peckham, E. 1905; Roubaud, E. 1916; Claude-Joseph, F. 1928 Rau, P. and Rau, N. 1918 Mauvezin, M. 1886; Cooper, K. 1953; Medler, J. and Fye, R. 1956 Nielsen, E. 1932b Bristowe, W. 1948 Fabre, J. 1879-1910 Fabre, J. 1879-1910 Ferton, C. 1909 Ferton, C. 1923 Roubaud, E. 1916 Maindron, M. 1882 Roubaud, E. 1916
+ = paralysis, — = no paralysis, P = permanent, T = transient, C = complete, I = incomplete. Key to host or prey taxa: Lep. = Lepidoptera, Col. = Coleoptera. 4.6
Eumenidae
Most of the eumenid wasps are predacious upon small lepidopteran larvae; others prey on coleopteran larvae (Table 10). Odynerus gracilus stings its prey through the ventral side (Maneval, H., 1932). However, Roubaud, E. (1916) has described Synagris calida stinging its prey in random locations.
Audouin, V. (1839) described a eumenid wasp, Odynerus parietum, which attacked caterpillars. These caterpillars did not metamorphose, their bodies could contract, but they were not capable of locomotion. As is shown in Table 10, nearly all records of the paralysis of prey of euminid wasps testify to incomplete (or transient) paralysis. Nothing is known about the mode of action of these venoms.
619
Insect Venoms and Toxins 4.7
Vespidae
This family of well-known social wasps is subdivided into three subfamilies. The Stenogastrinae are little-known wasps of India, Indonesia and New Guinea. Nothing is known about their venoms. The Polistinae are found all over the world. This subfamily is tropical in distribution, except for the genus Polistes which also occurs in temperate regions. The Vespinae are wasps of the northern hemisphere, although also of Indonesia and New Guinea. The subfamily of Vespinae is usually subdivided into four genera: Provespula, Vespa, Vespula and Dolichovespula. There are no reports on the venom of Provespula. This section deals with venoms of hornets {Vespa) and yellow-jackets (Vespula and Dolichovespula). The pharmacologically active substances identified in the venoms of a number of genera belonging to the last two subfamilies are summarized in Table 11. According to Roubaud, E. (1916) wasps belong-
ing to the genus Belonogaster (Polistinae) do not use their sting for prey-capture, but only for defence. Also the sting of Vespa species is not used to kill prey unless the wasp loses the initiative and a struggle ensues (Rau, P. and Rau, N., 1918; Richards, O., 1953). The venoms of social wasps, like those of bees, are therefore considered to be used by the wasp predominantly against vertebrates and only occasionally against other insects. The extensive literature of vertebrate pharmacology of venoms of social wasps has been reviewed by Edery, H. etal. (1978). Schmidt, J. and Blum, M. (1979) claimed shortterm (10 min) paralysis in insects injected with venom of Dolichovespula maculata. However, this "paralysis" was lethal and internal autolysis followed shortly. They concluded that the venom possesses a short-term, but apparently non-lethal toxin for insects, but this conclusion has to be confirmed by separation of the toxin from lethal factors.
Table 11: References to identification of pharmacologically active substances in venoms of Vespidae. Subfamily, genus Vespinae Dolichovespula Vespula
Vespa
Polistinae Polistes
Polybia Ropalidia Synoeca
ACh
Hist
5-HT
Cat.
Kin.
MCD
+ + + + +( + ) + +++
+
++
+!
+ + + + + ( + ) + + + + +( + ) + +
+!
+ + ++( +)
++
+ ++ +++
+++
+!
NT
References Geller, R. et al 1976; Ishay, J. et al 1974; Owen, M. 1971; Welsh, J. and Batty, C. 1963 Geller, R. et al 1976; Hirai, Y. et al 1979a; Ishay, J. et al 1974; Jaques, R. and Schachter, M. 1954; Schachter, M. and Thain, E. 1954; Yoshida, H. et al 1976 Abe, T. et al 1980; Abrahams, G. 1955; Albl, F. 1956; Bhoola, K. et al 1961; Edery, H. et al 1972; Edery, H. and Ishay, J. 1965; Hirai, Y. et al 1979b; Ishay, J. et al 1974; Kawai, N. and Hori, S. 1976; Schachter, M. 1970; Yasuhara, T. etal \911 Erspamer, V. and Falconieri Erspamer, G. (1962); Geller, R. et al 1976; Hirai, Y. et al 1980; Owen, M. 1979; Pisano, J. 1968; Prado, J. et al 1966; Udenfriend, S. et al 1967; Watanabe, M. et al 1976; Welsh, J. and Batty, C. 1963 Welsh, J. and Batty, C. 1963 Owen,M. 1979 Welsh, J. and Batty, C. 1963
- = <10ng; + = 10-100ng; + + = 100-1000ng; + + + = 1-100//g per mg or per μ\ or per venom reservoir; + ! = no quantification. ACh = acetylcholine-like activity; Hist. = histamine-like activity; 5-HT = 5-hydroxytryptamin-like acivity, Cat. = catecholaminelike activity; Kin. = kinin-like activity; MCD = mast cell degranulating peptide; NT = other neurotoxins.
620
Tom Piek
The low molecular weight components demonstrated by various authors in venom preparations of vespid wasps are shown in Table 11. A number of authors have found a considerable amount of acetylcholine (ACh) or ACh-like activity in venoms of Vespa crabro and Vespa orientalis, but Yasuhara, T. et al. (1977) reported that the venom reservoir of Vespa xanthoptera contains only 3 ng ACh. In other vespid genera ACh-like activity has not been found, except in Polistes hebraeus where Joshi, G. and Hurkat, P. (1975) found an agonist for the frog rectus abdominis which was antagonized by d-tubocurarine. However, they did not give quantitative results. In all vespid wasps histamine or a histaminelike activity was demonstrated. Most records testify to more than 10//g histamine per mg (or per ìÀ, or per venom reservoir, which is approximately the same). Similar results have been obtained for 5-HT (serotonin). The presence of catecholamines seems to be restricted to the venoms of Vespa, Dolichovespula and Vespula; dopamine and noradrenaline have been found in all and adrenaline in most of these wasp venoms. Jaques, R. and Schachter, M. (1954) found that Vespula vulgaris (they used the name Vespa) venom contained large amount of histamine, 5-HT and a substance that resembled bradykinin, i.e. substances which may be involved in anaphylactic reactions in vertebrates. Bhoola, K. et al. (1961) found
in the venom of Vespa crabro not only 5-HT and histamine, but also ACh and a kinin which was not identical to the bradykinin-like peptide from V. vulgaris venom. A number of more or less identified peptide toxins from wasp venoms are summarized in Table 12. Most of them are bradykinin-like, in that they contain bradykinin (BK) or Thr 6 -BK. A hydroxyproline containing nonapeptide Gly(Hyp 3 BK)-Ile-Asp has been isolated from the venom of Vespa mandarinia. Except Thr 6 -BK all wasp kinins possess an amino acid or an amino acid chain attached to the N-terminal of BK. The venoms from Vespa mandarinia and V. xanthoptera have an additional amino acid chain at the C-terminal, which has also been found in several BK-like peptides isolated from frog skins (Table 12). The venoms of Asian hornets (Vespa mandarinia, Vespa xanthoptera and Vespa analis) cause, in the nerve muscle preparation of the stretcher muscle of the walking leg of the lobster, a complex effect consisting of an initial increase in amplitude of the EPSP and IPSP followed by a decrease in amplitude to zero, and a concurrent increase in membrane conductance of the muscle fibre to about 50% of control levels (Kawai, N. and Hori, S., 1976). These authors isolated several fractions and concluded that the increase in PSP-amplitude was a presynaptic 5-HT-like effect. A different component caused depression of the EPSP, while the IPSP was un-
Table 12: Bradykinin-like peptides Arg-Pro-Pro-Gly-Phe- ■Ser-Pro-Phe-Arg Bradykinin Thr*-Thr*-Arg-Arg-Arg-Gly-Arg-Pro-Fro-G\y-PhQ- •Ser-Pro-Phe-Arg Thr-Ala-Thr*-Thr*-Arg-Arg-Arg-Gly-Arg-Pro-Pro-G\y-PhQ- ■Ser-Pro-Phe-Arg A la-A rg- Arg-Pro-Pro-Gly-Phe- TVzr-Pro-Phe-Arg Arg-Pro-Pro-Gly-Phe- 77zr-Pro-Phe-Arg (Thr 6 -bradykinin) Pyr-Thr-Asn-Lys-Lys-Lys- Leu- Arg-Gly- Arg-Pro-Pro-Gly-Phe- ■Ser-Pro-Phe-Arg G/y-Arg-Pro-Pro-Gly-Phe- ■Ser-Pro-Phe-Arg G/y-Arg-Pro-//y/?-Gly-Phe- ■Ser-Pro-Phe-Arg-Z/e- Val Ë/a-Arg-Pro-Pro-Gly-Phe- ■Ser-Pro-Phe-Arg-//^- Val Ftf/-Pro-Pro-Gly-Phe- r/zr-Pro-Phe-Arg Arg-Pro-Pro-Gly-Phe ■Ser-Pro-Phe- Axg-Ile- Tyr(S03H) Arg-Pro-Pro-Gly-Phe- Ser-?ro-Phe-Arg-Val-Ala-Pro-Ala-Ser Arg-Pro-Pro-Gly-Phe- Thr-Vro-Phe-Arg-Ile-Ala-Pro-Glu-Ile-Val Arg-Pro-Pro-Gly-Phe- Ser-Pro-Phe-Arg-Gly-Lys-Phe-His
Vespula kinins Polistes kinins Vespa kinins
frog kinins
The amino acid residues which are not common to the bradykinin molecule are shown in italics *Carbohydrate-containing peptides 1,2: from Vespula maculifrons (Yoshida, H., et al 1976) 3,4: from Polistes rothneyi (Watanabe, M., et al. 1975, 1976) Thr 6 -bradykinin has also been found in frog (Rana rugosa) skin 5: from Polistes exclamans (Udenfriend, S., et al. 1967; Pisano, J., 1970) 6: from mixture of venom reservoirs of Polistes annularis, Polistes fuscatus and Polistes exclamans (Prado, J., et al. 1966) 7: from Vespa mandarinia (Kishimura, H., et al. 1976) 8: from Vespa xanthoptera (Yasuhara, T., et al. 1977) 9-13: from frogs (Rana, Phyllobates, Bombinus) see review by Bertaccini, G. (1976)
Insect Venoms and Toxins
altered and no appreciable change was found in the resting membrane potential and conductance. From the venom of Vespa insularis (Kawai, N. et al., 1979) and V. mandarinia (Kawai, N. et al, 1980a) toxins, called HTX-E and VTX-E respectively, were isolated. These toxins reversibly suppressed both EPSPs and IPSPs without affecting the presynaptic spike of lobster muscle fibres. The muscle fibre membranes were hyperpolarized with a concurrent increase in membrane conductance. This conductance increase was probably due to a chloride activation of the membrane. Kawai, N. et al. (1980b) concluded that the venom of V. mandarinia contains a GABAnergic agonist, which is not destroyed by GABA-transaminase and therefore not identical to GABA. Moreover, the venom of V. mandarinia contains a high molecular weight (21,000) basic polypeptide that blocks neuromuscular transmission in the lobster as well as in mealworm larvae, Tenebrio molitor, by a reduction of the sodium current in the nerve terminal (Abe, T. et al., 1980; Kawai, N. et al, 1980b, 1981). In similarity with bee-venoms (next section) some social wasp venoms contain mast cell-degranulating (histamine-releasing) peptides, called mastoparans. V. mandarinia contains in its venom sac approximately 20nmol mastoparan M, and the peptide degranulated the rat peritoneal mast cells at a concentration of 0.5nmolml _1 (Hirai, Y. et al, 1981). These peptides do not cause contraction of rat uterus or guinea-pig ileum, and do not affect arterial blood pressure when injected intravenously into anaesthetized rats. Okumura, K. et al. (1981) found that addition of 1 ìgmà 1 of Vespula lewisi mastoparan on both sides of an artificial lipid membrane caused a 100-fold increase in cation permeability. They suggested that such an enhancement of cation permeability may also occur at mast cell membranes. The mastoparans did not potentiate bradykinin (Hirai, Y. et al, 1979a, 1980). The structures of mastoparans are shown in Table 13. Mastoparans from Polistes jadwigae and from Vespa xanthoptera have been synthesized (Yajima, H.etal, 1980a,b). It is obvious that social wasp venoms contain interesting compounds, some of which are toxic for insects. The presence of bradykinin-like peptides may be the most interesting fact. These peptides produce pain in vertebrates and may be considered
621
Table 13: Mast cell degranulating peptides (mastoparans) from venoms ofVespidae (from Hirai, Y. et al. 1979a,b, 1980, 1981) Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu (mastoparan, Vespula lewisi) Ile-Asn-Trp-Lys-Gly-Ile-Ala-Ala-Met-Ala-Lys-Lys-Leu-Leu (mastoparan X, Vespa xanthoptera) Ile-Asn-Leu-Lys-Ala-Ile-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu (mastoparan M, Vespa mandarinia) Val-Asp-Trp-Lys-Lys-Ile-Gly-Gln-His-Ile-Leu-Ser-Val-Leu (Polistes mastoparan, Polistes jadwigae)
to be effective in the defence of the wasps against vertebrates. However, a bradykinin-like activity has also been demonstrated for the venom of a solitary wasp (see section 4.2), which does not use the venom for defence, but for paralysing its insect prey. For discussion of a possible role of bradykinin as a neurotoxin see section 4.2. 4.8
Apidae
All social and solitary bees are included in the superfamily of Apoidea, which is subdivided into six families. There is no information about the venoms produced by families other than Apidae, except the simple demonstration that here is a venom. For example bees belonging to the Megachilidae produce a venom which is less painful than that of honeybees (Fabre, J., 1879-1910). The family of Apidae includes both solitary and social bees. There are three important subfamilies: the Anthophorinae, the Xylocopinae, and the Apinae. Nothing seems to have been reported about venoms of the first group. The venom of Xylocopa violacea (Xylocopinae) was described for the first time by Bert, P. (1865) who observed that a sting from this bee could kill small birds within a few hours, but frogs seemed to be much less sensitive. The venom contained an acid (tested on "sunflower paper"). This acid was not identical to formic acid since it was not volatile (Bert, P., 1865). The sting of X. violacea seems to be extremely painful. It causes a local paralysis, and oedema (Hardouin, R., 1948). The subfamily of Apinae is subdivided into three classes: the Meliponini or stingless bees, the Bombini (bumble or humblebees) and the Apini. Our knowledge of bumblebee venoms is poor and restricted to the work of O'Connor, R. et al. (1964) and Mello, M. (1970), who found proteins in the venom of Bombus species. Of all the work done on
622
Tom Piek
venoms of Hymenoptera the majority has been devoted to the honeybee, Apis mellifera. In addition to what is known about A. mellifera venom, fragmentary information has been gathered about the venoms of the three other species of the Apini: the Indian honeybee Apis cerana ( = Apis indica), the giant honeybee or Bombara, Apis dorsata, and the little honeybee, Apisflorea. The venom-producing apparatus of a honeybee worker is shown in Fig. 18. The venom reservoir is thin-walled and contains, in a foraging worker, between 1 ìÀ and 3 μ\ of venom (Owen, M. and Bridges, A., 1974). The gland is a tubular structure which is about 1 cm long and bifurcates at its tip. Venom is produced by secretory cells distributed along the venom gland and part of the wall of the reservoir (Owen, M. and Bridges, A., 1974, Fig. 19). The venom of honeybee workers (A. mellifera) contains a complicated mixture of pharmacologically active substances: enzymes, peptides and small molecules. 4.8.1
ENZYMES
Several enzymes have been identified: hyaluronidase, phospholipase A and phosphatases. For a summary of references up to 1976 see O'Connor, R. and Peck, M. (1978). Bousquet, J. etal (1979) tested both honeybee venom collected by electrical stimulation and dried venom apparatus extracts on
FIG. 19. Diagrammatic reconstruction, from electron micrographs, of the arrangement of a cuticular duct which runs from the lumen of the venom gland, or reservoir, to a complex end-organ located within the secretory cell. (From Owen, M. and Bridges, A., 1974.)
FIG. 18. Diagrammatic representation of the venom gland and venom reservoir of a worker of the honeybee Apis mellifera. (From Owen, M. and Bridges, A., 1974, 1976.)
several combinations of substrates and buffers for the determination of 58 enzymes. They found in the venom collected by electrical stimulation a relatively high amount of acid phosphatase, less alkaline phosphatase, and some phosphoamidase and C4esterase. Moreover, this venom preparation contained jß-glucosaminidase and glycyl-proline arylamidase (Bousquet, J. et aL, 1979). The venoms of queens of A. mellifera contains much less phospholipase than the venom of workers (März, R. et aL, 1981). The earliest indication of hyaluronidase activity in bee venom came from the description of a "spreading-factor" present in body extracts of bees (Duran-Reynolds, F., 1936). This factor was identified as hyaluronidase (Chain, E. and Duthie, E., 1940). Hyaluronidase is present in the honeybee venom for at least 4 days prior to hatching (Owen, M., 1979).
623
Insect Venoms and Toxins
Shkenderov, S. (1973) found a protease inhibitor in honeybee venom. Thin-layer chromatography on Sephadex G-50 showed that the molecular weight of the inhibitor was 9000 + 1000. He suggested that the role of the protease inhibitor may be the protection of enzymes (such as hyaluronidase and phospholipase) as well as of polypeptides (such as melittin and apamin) against protease activity of the animal which has been stung. 4.8.2
PEPTIDES
Peptides present in honeybee venom are in the molecular weight range of 2000-6000 (Peck, M. and O'Connor, R., 1974) and are strongly basic. These peptides account for 50-60% of the dry venom. An extensive summary of references dealing with studies of these polypeptides from A. mellifera venom has been given by O'Connor, R. and Peck, M. (1978). The polypeptides are classified as the melittins, the apamins including the mast celldegranulating (MCD) peptide, minimine and the above-described protease inhibitor. 4.8.3
MEUTTIN
Melittin was identified by Neumann, W. and Habermann, E. (1954). Kreil, G. and Kreil-Kiss, G. (1967) have found a second component the Naformyl-melittin. Bachmeyer, H. et al. (1972) found evidence for a precursor, promelittin, which was synthesized well before conversion to the pharmacologically active melittins. The structure of melittin from A. mellifera is compared in Table 14 with the structures of melittins from the three other species of Apis. Exposure of a lecithin bilayer to A. mellifera melittin resulted in the formation of channels that
are more permeable to anions than to cations (Tosteson, M. and Tosteson, D., 1981). Unilateral addition of melittin produced a voltage-dependent increase in membrane conductance when the side where the polypeptide was present was made positive, but not when it was negative. At a fixed voltage the conductance increased with the fourth power of the melittin concentration in the aqueous phase, indicating that four melittin monomers were needed to form a channel. Furthermore the conductance increased approximately e-fold per 6 mV increase in the electrical potential difference across the membrane, provided a fixed peptide concentration was maintained. Therefore, a minimum of four equivalent electronic charges were needed to be displaced by the electrical field, to explain the voltagedependence of the conductance (Tosteson, M. and Tosteson, D., 1981). According to these authors it is possible that the channel-forming properties of melittin may account for some of the physiological effects of the compound. In particular, the fact that melittin forms channels at aqueous concentrations well below those required to produce lysis, makes it attractive to speculate that this property of the molecule might also be important biologically, particularly in its capacity to stimulate neurons. Melittin is toxic to insects (Drosophila melanogaster) in amounts of more than 0.1 μg per animal (Mitchell, H. et al., 1971), and this action could explain the general toxicity of honeybee venom to insects (Fabre, J., 1879-1910). 4.8.4
APAMIN
This is the second polypeptide neurotoxin present in the venom of A. mellifera. Apamin was discovered by Neumann, W. and Habermann, E. (1954) and isolated by Habermann, E. and Reiz, K. (1965).
Table 14: Amino acid sequence of melittins in the venoms offour Apis species MELITTINS A me///feran)(Mi-GW)-
Äiä|- Leu-
lie-Ser-Trp- lie- (Ly:
Gin
Gln-CONH?
lle-Gly- Ala -
-Leu- (Lys)-Val-Leu-
-Thr
Gly- Leu-Pro-
A.cerana
(2)(iN-Sj)-lle-Gly-Ala-
-Leu- (Lys)-Val- Leu-
-Thr
Gly- Leu-Pro- Ala -Leu-lie-Ser-Trp-lie-(Lys) -
Gin
Gln-CONH?
A.dotsata
(»(HgN-Gly)-lle-Gly-Ala-
-Leu- (Lys)-Val-Leu-
-Thr
Gly -Leu-Pro- Ala - Leu-He - Ser-Trp - lie-(Lys)
Gin
A.florea
(2)(H^N-Gly)-lle-Gly-Ala-
-Leu- (Lys) -Val - Leu-
-Thr
Gly -Leu-Pro- Thr - Leu- lie - Ser -Trp - lie- (Lys)
(£)
- |Glu-CONH2| Gln-CONH,
(1) Habermann, E. and Jentsch, J. (1967); (2) Kreil, G. (1973); (3) Kreil, G. (1975). Non-homologous positions within the different melittin peptides are enclosed in boxes. Properties of individual amino acid residues are indicated as follows: a circle for each net basic functionality; a square for each net acidic functionality; an underline when the residue is neutral but polar. (From Schmidt, J. 1982)
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Tom Piek
Like melittin, apamin is not a single compound in the bee venom. Apamin chemically resembles the mast cell-degranulating (MCD) peptide, which was first described by Fredholm, B. (1966), and isolated by Breithaupt, H. and Habermann, E. (1968) (Fig. 20). Both toxins are minor compounds in the venom, each representing a small percentage of the dry weight of the venom (compare the melittins, representing about 50% of the dry weight). The MCD peptide may be responsible for the massive release of histamine in vertebrates. Its structure resembles somatostatin, the natural antagonist of somatotropin in vertebrates. Somatostatin causes release of histamine in mast-cells. Theoharides, T. and Douglas, W. (1981) compared the structure of somatostatin with the powerful mast cell stimulator, the polyamine 48/80, and concluded that cationic groups are essential. However, this is not true for social wasp MCDs (section 4.7). Apamin, however, is a neurotoxic peptide, and is a potent blocker of inhibition of mammalian smooth muscle, which is possibly purinergic in nature (Vladimirova, A. and Shuba, M., 1978; Maas, A. and Den Hertog, A., 1979). Evidence has been presented that apamin possesses potassium channel blocking properties (Banks, B. et aL, 1979; Maas, A et aL, 1980; Maas, A., 1981). Besides the above peptides with a marked pharmacological action honeybee venom contains other peptides in various amounts. Melittin F proved to be a fragment of melittin, secapin shows no obvious structural similarities with the other basic peptides in bee venom and seems to be non toxic (Gauldie, J. et aL, 1978). Another "minor" peptide has been prepared from bee venom, after the observation that larvae of Drosophila melanogaster treated with small amounts of bee
/ \
\ Arg
9 / Arg
c5s-Lys-Ar37s His
/ /
^- L y s ^ 5 / Ala As'n \
Ar
r
lle
CysH
º
Cys'
||e
, , le LY/
Ü01
Ala \
°
Cy
V
C S
/
y^s
Gl
^
S Apamin
FIG. 20. The amino acid sequence and disulphide bridge positions of the MCD-peptide (peptide 401) and Apamin. (From Banks, B. et al., 1978.)
venom stop growing, which after metamorphosis results in miniature flies, as small as one-quarter of the normal size. Since this basic peptide was not identical to any known basic peptide present in honeybee venom, the new peptide was called "minimine" (Mitchell, H. et aL, 1971; Lowy, P. et aL, 1971). About 3% of the original dry mass was recovered in the minimine-fraction. The basic polypeptide (6000 MW) contains all of the amino acids normally present in proteins. According to Habermann, E. (1972) minimine should be checked for phospholipase activity, since it appeared, during gel filtration, in the same region as phospholipase A. The larger peptides present in bee venom are accompanied by small fragments (Gauldie, J. et aL, 1978), but other small peptides have also been described. Studies of small peptides in bee venom (Nelson, D. and O'Connor, R., 1968) revealed the first natural source of histamine-terminal peptides in bee venom such as Ala-Gly-Pro-Ala-Glu-Histamine and Ala-Gly-Glu-Gly-Histamine ( = procamine) (Peck, M. and O'Connor, R., 1974). It seems doubtful that histamine is formed from these peptides. On the contrary it seems more likely that histamine is formed from histidine. Owen, M. and Braidwood, J. (1974) found that both histamine and histidine levels rose during development until the 21st day (moment at which honeybee worker leaves the hive), then the histidine level decreased while that for histamine still rose (Fig. 21). 4.9
Formicidae
This is the family of ants, which is subdivided into nine subfamilies, four of which have been more or less intensively studied in relation to their venoms: Myrmeciinae, Ponerinae, Myrmicinae and Formicinae. The venom of Myrmecia gulosa has been studied by Cavill, G. et al. (1964); that of Myrmecia forficata by De la Lande, I. et aL (1965), and Lewis, J. and De la Lande, I. (1967). These venoms contain histamine, hyaluronidase and a kinin-like activity. Wanstall, J. and De la Lande, I. (1974) found that the non-histamine-like smooth muscle-contracting substance was enzyme free, red cell lysing, and histamine releasing, and thus resembled melittin from bee venom. The subfamily of Ponerinae forms one of the most important groups of the Formicidae. Maschwitz, U. et aL (1979) observed that
625
Insect Venoms and Toxins
"Jf"
1600
X
•
1200
_
C
¸
« x
800
D
400
0
■—_ 1. 7
1
1
14
21
1 28
«· 35
1 42
1
Age in days
FIG. 21. Changes in histamine and histidine content of honeybee worker venom with age. (From Owen, M. and Braidwood, J., 1974.)
ponerine ants, Harpegnathus saltator, stung cockroaches, Blattella germanica, in the ventral thoracal region producing an immediate immobilization. The motionless cockroaches were then carried into the nest. Almost all cockroaches still reacted to mechanical stimuli by feebly moving their legs or antennae. After 3 days, more than half of the animals still showed reactions. One adult cockroach exhibited responses for 2 weeks; not a single cockroach recovered from the sting. Maschwitz, U. et al. (1979) concluded that workers of H. saltator paralyse their prey permanently as do many solitary wasps. In the Myrmicinae extensive studies have been done on the venoms of fire ants (Solenopsis sp.) and the harvester ant (Pogonomyrmex badius). The fire ant (S. saevissimd) has become an insect of considerable economic importance in the USA. It produces a highly toxic venom causing paralysis in insects, such as the boll weevil and the rice weevil (Blum, M. et al, 1958; Sonnet, P., 1967). The active substances are a's + /r
Tragus that flowers of Cichory (Chicory), and some other blue flowers, change their colour into a red one when brought into contact with disturbed ants. Wray, J. (1670) also referred to Fischer, who stirred a heap of ants with a stick and observed a liquor on the stick. Fischer compared the acid with that from vinegar and found a great similarity. Wray, J. (1670) found it strange "that nature should prepare and separate in the body of Pismires, without any sensible heat, and that in good quantity considering the bulk of the animal, a liquor the same for kind with those acid spirits which are by art extracted out of some minerals, not with great force of fire". This problem has been solved recently by Hefetz, A. (1977) and Hefetz, A. and Blum, M. (1978a,b). They found that formic acid synthesis in formicine ants is closely related to the C-l metabolism of the c · ^ Serine N-C-OO
Haemolymph
Venom gland cell
loFormyl H4F0late
ADP ATP^
Jif
Formic Acid
ADF Glandular Lumen
Formic Acid
FIG. 22. Diagrammatic representation of the regulating mechanism for the biosynthesis and transport of formic acid in the venom gland of formicine ants, as has been postulated by Hefetz, A. (1977). Serine is a C-l donor to form formic acid from several tetrahydrofolate intermediates (5,10 methylene H 4 folate; 5,10 methenyl H 4 folate, and 10 formyl H 4 folate). (From Hefetz, A., 1977.)
626
Tom Piek
glandular cells. Serine, glycine and histidine are potential C-1 donors to form formic acid by several tetrahydrofolate (H4F) intermediates (Fig. 22). This coenzyme form of pteroylglutamate (foliate) can serve as a shuttle to which the one-carbon group is covalently but transiently attached (Lehninger, A., 1975). A desalted gland extract of venom glands of Camponotus pensylvanicus lacking any endogenous H 4 F is totally dependent on exogenous H 4 F for the synthesis of formic acid. Incorporation studies utilizing differentially labelled serine revealed that it contributes both its a and /? carbons to formic acid, but not its carboxyl carbon. Further studies with H 4 F derivatives strongly suggest that the H 4 F derivatives tested indeed act as intermediates in the synthesis of formic acid (Hefetz, A. and Blum, M., 1978b). Moreover, the venom gland contains four enzymes catalysing the transfer of the ß carbon of serine to formic acid via H 4 F intermediates. Since the equilibrium between formic acid and 10-formyl H4-folate is 1:20, only small amounts of formic acid are produced in the glandular cells. For the acid to accumulate it is therefore necessary to transfer it to a second compartment (the venom gland reservoir), which is insulated by a cuticular intima (Hermann, H. and Blum, M., 1968). ACKNOWLEDGEMENTS
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