George Salt

THE RESISTANCE OF INSECT PARASITOIDS TO THE DEFENCE REACTIONS OF THEIR HOSTS

Some of the following propositions are to be read as suggestions or hypotheses, supported by circumstantial or direct evidence, but not yet rigorously demonstrated. An estimate of the significance to be attached to each should be gathered from the body of the paper rather than from the following brief statements.

The defence reactions of insects to metazoan parasites.

1. Defence reactions to metazoan parasites have been reported in fourteen orders of insects. The observations are brought together and reviewed in the first part of the paper. 2. Examination of the various accounts that have been given shows that blood cells are always involved in insect defence reactions. They act by forming a cellular capsule, from which a connective-tissue envelope is usually developed, and in which melanin is often deposited. 3. The reaction of the epidermal cells at perforations made by parasites is of the nature of wound-healing, and plays no part in defence against metazoan parasites after they have entered the body. 4. Although several other tissues have been implicated, there is insufficient evidence to show that any of them make defence reactions, their response being limited to processes of regeneration. 5. It is concluded that the blood cells of insects are their only known agents of defence to internal metazoan parasites. 6. The principal groups of metazoan parasites infesting insects are considered in the third part of the paper, in order to see how the defence reactions made to them are related to their mode of attack and to the nature and consequences of their parasitism. 7. Most parasites elicit a defence reaction when they are in unusual hosts. 8. Some parasites, at certain stages of their life-history, are able to avoid eliciting a defence reaction in their usual hosts. 9. Some parasites elicit a defence reaction in their usual hosts but are able either to endure it in a dormant state or to resist it. 10. General problems of host specificity in relation to defence reactions are discussed. It is concluded that analysis of the stimuli that produce defence reactions has not yet gone far enough to explain the phenomena. 11. The effects of insect defence reactions on metazoan parasites range from no perceptible effect to destruction of the parasite. 12. The defence reactions of insects are influenced by the species, genetic strain, stage, instar, size, health and physiological state of the host; and by the species, genetic strain, physical and physiological activity, and health of the parasite. Environmental temperature and the presence of other parasites of the same or different species also have effects on the reactions. 13. A brief survey of defence reactions made by invertebrates other than insects shows that encapsulation has been reported in Annelida, Mollusca, Crustacea, Acarina, and larval echinoderms. So far as it goes, the survey does not reveal in these other groups any reaction to metazoan parasites of a kind radically different from the reactions observed in insects. 14. The historical development and present state of our knowledge of insect defence reactions is traced. 15. The reactions made by insects to innocuous parasites are of theoretical interest but of little consequence to the species concerned. It is their effect on potentially dangerous parasites that determines the value of defence reactions. Consideration of the evidence suggests that the protection afforded to insects by their defence reactions is greater than has been generally supposed. 16. The review makes apparent many gaps in our knowledge of the phenomena. A few of the outstanding problems are mentioned. I am indebted to Mr R. T. Hughes of the Balfour Library for helping me to obtain journals not available in Cambridge; to Mr M. J. Ashby for the photography necessary in the preparation of the figures; and to Miss G. M. Edwards for her careful typing from my manuscript. The paper would not have been completed without the goodwill and assistance of two persons: Professor D. Keilin, F.R.S., encouraged me to continue and finish it when my effort flagged; my wife not only gave me positive help in many ways but also exercised great forbearance in allowing me to devote vacations and spare time to it.

Experimental Studies in Insect Parasitism. XIV. The Haemocytic Reaction of a Caterpillar to Larvae of Its Habitual Parasite

Although caterpillars of Ephestia kuehniella promptly encapsulate alien parasites and other foreign bodies in their haemocoele, they do not normally encapsulate larvae of their habitual parasite Nemeritis canescens, which develop unhindered and eventually destroy their host. The larva of Nemeritis does not achieve this immunity by repelling the blood cells, or by physically dislodging them. It is immune because it is able to live in the haemocoele of Ephestia without evoking a haemocytic reaction; presumably, that is, because it is not recognized as a foreign body. That ability is due to a property of its surface. So long as its surface remains unaltered, the larva, alive or dead, evokes no haemocytic reaction. When its surface is altered whether by perforation, abrasion, or chemical treatment, the living larva evokes a haemocytic reaction in Ephestia and becomes encapsulated. The protective property of its surface is acquired by the larva very late in its embryonic development, between 62 and 66 hours of age at 25 °C. This is about the same time as, or a little later than, the cuticle of the embryonic larva becomes impermeable to water. Four fat solvents were found to deprive the living larva of its immunity, but they may have affected the protective surface by disrupting the underlying wax layer of the epicuticle. Treatments and substances that did not affect the protective surface give some crude indications of its properties, but its ultimate characterization must be in terms of insect immunology. Observations incidental to the main theme of the paper show that the cuticle of the larva is impermeable to water; that ionic exchange takes place through the anus and wall of the rectum, where some food substances may also be absorbed from the blood of the host; and that the order of formation of the cuticulin and wax layers of the embryonic larva is the same as that in ecdysis from instar to instar in other insects. They also provide information on the longevity of bitten supernumerary larvae.

Experimental Studies in Insect Parasitism. IX. The Reactions of a Stick Insect to an Alien Parasite

Eggs and larvae of an ichneumon fly, Nenteritis canescens, injected into adults of a stick insect, Carausius morosus, elicited two defence reactions. Dark material, probably melanin, was deposited on their surface; and they were invested with haemocytes which, within 24 h, formed unorganized clumps of irregular shape about them. The haemocytic reaction was less vigorous in confined spaces than in the main blood stream, and several observations support the view that the haemocytes did not flock to the parasite from a distance but accumulated by chance encounter of, at most, a small chemotactic field. The size of the clumps reached a maximum about 24 h after injection of the parasite. The melanin reaction, on the other hand, was as strong in confined spaces as in the main blood stream, and was progressive, eventually encasing the parasite in a brittle black sheath. In nymphs of 1/2, and in young nymphs of 1/20, adult weight, the two reactions were essentially the same as in adults; the melanin reaction was indistinguishable from that in the adult; the haemocytic reaction in the large nymphs was a little, and in the small nymphs was much, less massive. The deposition of melanin about the parasite was inhibited by simultaneous injection of phenylthiourea. The haemocytic reaction could not be inhibited by injections of Chinese ink, because blood cells blackened by the carbon particles they had engulfed nevertheless invested the parasite. The two reactions were elicited both by living and by dead parasites; the haemocytic reaction appeared to be elicited by clean glass rods, but the melanin reaction was not. Most Nenteritis eggs failed to hatch in Carausius although the fully developed larva could be seen moving within them, and no Nenteritis larva was observed to live longer than 48 h in this host. But larvae that had hatched in Carausius and after 24 h were transferred to the normal host, Ephestia, lived and developed. Moreover, eggs hatched normally in the blood of Carausius in hanging-drop cultures, and the larvae lived as long as 75 h. The death of this parasite in Carausius does not appear to be due either to toxic properties of the blood as a fluid, or to its unsuitability as food, but to active processes, the haemocytic and melanin reactions, that take place in the living host.

Experimental Studies in Insect Parasitism. VIII. Host Reactions Following Artificial Parasitization

A method of artificial parasitization has been devised which allows eggs of insect parasitoids to be injected into any given species of host. By this means, eggs of an ichneumon fly, Nemeritis canescens, have been put into larvae of eight species of Microlepidoptera, none of which has ever been recorded as a host of that parasite. Three species encapsulated the parasite egg and prevented its hatching. In three species the egg hatched but the host reacted to the parasite larva: in two species by encapsulation, in one species by the blocking of its alimentary tract with melanin. Two species gave feeble reactions or none, and were partly or wholly susceptible to the parasite. The defence reactions of all these hosts to the same parasite took two principal forms: encapsulation and the deposition of melanin. Each species of host was capable of both forms, but each species reacted in a manner that was characteristic and peculiar to it. Nevertheless, hosts belonging to the same family reacted to the parasite in similar ways so that, within the limits of this preliminary survey, the defence reactions of a group of hosts to the same parasite reflect the relationships of the hosts.

Experimental Studies in Insect Parasitism. XI. The Haemocytic Reaction of a Caterpillar under Varied Conditions

Eggs or young larvae of an ichneumonid parasite, Nemeritis, injected into caterpillars of the tomato moth, Diataraxia, evoked a rapid reaction of the haemocytes, which led to the encapsulation of the parasite within 4 h. The further development of the capsule is described, and evidence is adduced that the encapsulated parasites were killed by asphyxiation. Encapsulation was blocked by injection of carbon particles, but only when the doses were so large that they affected the general health of the hosts; it was temporarily less intense in superparasitized hosts; and it appeared to be slightly depressed by cortisone. Attempts to induce tolerance in the host by various methods—by repeated parasitization, by inoculation with an homogenate of parasite larvae, by inoculation with the blood of a susceptible host and by embryonic inoculation of substances from an homogenate of parasite larvae—all failed to protect the parasite, which was encapsulated as thickly and rapidly as in untreated caterpillars. When the parasite was given prior experience of Diataraxia blood by two different methods it acquired no immunity. The haemocytic reaction was similarly evoked by other species of parasites, by dead parasites, by glass rods and pieces of nylon thread, by injured tissues of the caterpillar itself, and by organs transplanted from caterpillars of another species. Organs transplanted from other caterpillars of the same species attracted haemocytes only to injured surfaces. Haemocytic capsules formed in one caterpillar of Diataraxia and transplanted to another caterpillar of the same species were accepted as intraspecific implants and aroused no reaction; capsules formed in another species and transplanted to Diataraxia evoked a strong haemocytic reaction in the second host. It is concluded that surface properties of the parasite or implant determine whether or not it shall be encapsulated.

Experimental Studies in Insect Parasitism. XVI. The Mechanism of the Resistance of Nemeritis to Defence Reactions

The egg of the ichneumon wasp Nemeritis canescens is known to be resistant to the defence reactions of its usual host, Ephestia kuehniella, by virtue of a coating on its surface. The coating is here shown also to endow Nemeritis with resistance to the defence reactions of Achroia grisella and, by implication, several other species of hosts. Three ways in which the coating might act are: (1) passively, by mimicking the lining of the haemocoele; (2) indirectly, by preventing the modification of substances in the haemolymph; (3) directly, by inhibiting the adhesion of blood cells. Mimicry of the host’s internal lining is ruled out by experiments showing that the blood cells of Ephestia react to the internal lining of Achroia, and vice versa, although neither host reacts to Nemeritis. The idea that the coating might prevent modification of substances in the plasma, and so indirectly inhibit encapsulation, is not completely eliminated; but several experiments provide no evidence for it, and several observations favour a hypothesis of direct action by the particles on the blood cells. The nature of that direct action is inconclusively examined. Preliminary experiments suggest that the particles dissociate the cells of young capsules of Ephestia and may, therefore, act to inhibit the adhesion of cells to form capsules. Cells of capsules formed in Tenebrio molitor, which always encapsulates Nemeritis, were not dissociated; which indicates that this mode of action has the required element of specificity. The possibility that substances chemically similar to the particles of Nemeritis might inhibit cell adhesion and aggregation in vertebrates is briefly discussed.

Experimental Studies in Insect Parasitism. X. The Reactions of some Endopterygote Insects to an Alien Parasite

Eggs of an ichneumon fly, Nemeritis canescens, have been injected into endopterygote insects to which this parasite was alien. In larvae, pupae and adults of Tenebrio molitor (Coleoptera) almost all the eggs hatched; and within 24 h haemocytes enveloped the parasite larvae and melanin was deposited over their mouth and anus. The pattern of these reactions was the same in all cases and at all stages. When individuals of Tenebrio were given repeated injections at weekly intervals, they reacted to the last parasite exactly as to a first. Female individuals that had been repeatedly injected were allowed to develop and to lay eggs; when larvae reared from those eggs were injected they made the same characteristic reactions. No trace of increased tolerance or resistance on the part of Tenebrio was observed in either experiment. Larvae and pupae of Diataraxia oleracea (Lepidoptera) rapidly encapsulated parasites injected into them; adults made no visible reaction. Nemeritis larvae fed, grew and ecdysed in these adults, and only the premature death of the moth prevented the parasite’s development. In feeding larvae, resting larvae and adults of Calliphora erythrocephala (Diptera), the haemocytic and melanin reactions to injected parasites were both weak; in pupae the haemocytic reaction was weak but the melanin reaction strong. In the adult blowflies, parasite larvae lived as long as 7 days, but did not grow and seemed unable to use the food they had ingested. Observations recorded in this and the two preceding papers of the series are compared and discussed. The haemocytic reaction to Nemeritis, as observed in several orders of insects, is a true defence reaction and, when strong enough, leads to the death of the parasite. Deposition of melanin acts as a defence reaction only fortuitously, when the deposit is so sited as to prevent a vital activity, such as the hatching or the feeding, of the parasite. The reactions to Nemeritis of the different kinds of hosts so far investigated are distinguishable, but no constant difference has been observed that would serve to distinguish the reactions of exopterygote from those of endopterygote insects.

The egg-parasite of Sialis lutaria : a study of the influence of the host upon a dimorphic parasite.

1. Nearly a quarter of a million eggs of Sialis lutaria were collected at Cambridge in 1936. About 0·6 per cent of them were attacked by a parasite. 2. The egg-parasite of Sialis is distinct from Trichogramma evanescens , and is to be called T. semblidis (Aurivillius). 3. The male of Trichogramma semblidis occurs in two forms. Neither consists merely of imperfect or degenerate individuals of the other, for the two forms are equally large and differ constantly and fundamentally in several characters. The species, therefore, exhibits true dimorphism. 4. Rearing experiments involving isolated pure lines show that it is principally the host that determines which form of the parasite shall emerge. Males reared on Sialis are of the apterous form; those reared on three species of Lepidoptera are of the winged form. 5. The dimorphism of T. semblidis is discussed in relation to other examples of dimorphism in the Hymenoptera. It is shown to have several features of special interest.

Experimental studies in insect parasitism—XII. The reactions of six exopterygote insects to an alien parasite

Abstract When mature eggs of the ichneumon-wasp Nemeritis canescens were injected into crickets (Acheta domesticus), cockroaches (Blattella germanica), stick insects (Carausius morosus), and earwigs (Forficula auricularia), the parasite larvae were quickly encapsulated by blood cells and partly encased in melanin; and usually died within 48 hr. The hosts appeared to suffer no lasting disability. Eggs and larvae of Nemeritis were not encapsulated or melanized in larvae of Rhodnius prolixus, and only occasionally elicited any visible defence reaction; but the parasites did not feed or grow in this host, and died after 3 days. When the same parasite was injected into locust hoppers (Schistocerca gregaria) no visible reaction took place until about the fifth day, after which the parasite was incompletely encapsulated but never melanized. Parasite larvae lived in these hosts as long as observations were continued (25 days). They fed and grew, but at a subnormal rate; and they remained in the first instar. In adult locusts no reaction was observed within 5 days, but the parasite larvae did not grow. Locust hoppers quickly encapsulated other foreign bodies. First-instar Nemeritis that had lived up to 25 days in Schistocerca caused no reaction when transferred to their normal host, caterpillars of Ephestia; but ganglia of Schistocerca transferred to Ephestia were at once encapsulated. The time-lag in the reaction of Schistocerca to Nemeritis is due to the parasite, which at first elicits no reaction but becomes evocative at a particular state of development within the first instar. First-instar larvae of Nemeritis did not ecdyse to the second instar in Schistocerca. Their failure to moult might be due to hormones in the blood of the host; but when Nemeritis was injected into decapitated hoppers they did not ecdyse and failed to feed or grow, acting as they did in adult locusts. Moreover, larvae retrieved from decapitated hoppers had lost their acceptability to caterpillars of Ephestia, in which they excited a haemocytic reaction. This propensity persisted through transfer at 48 hr intervals to three successive caterpillars. It appears that both the feeding and growth of first-instar Nemeritis and the haemocytic reaction of their normal host are affected by substances released from the head of locust hoppers.