ON THE NATURAL EQUILIBRIUM OF PYRAUSTA NUBILALIS HB.

By A. PAILLOT Southeastern Entomological Station, France

When, in 1927, 1 commenced my researches on the parasites and predators of Pyrausta nubilalis Hb., I planned to make a complete study of the natural equilibrium of that species, and to determine the relative importance of -the various factors of all kinds which limit its spread in France. It soon became evident, however, that the solution of a problem of such wide scope would require years of research. I have, therefore, limited my investigation to the central eastern region, and especially to the valley of the Saône, the Jura, the Dombes, the valley of the Rhône above Lyon, and the valley of the Grésivaudan above Grenoble, regions in which the growing of corn is rather important.

 The problem of the natural equilibrium of animals, and in particular of noxious insects, has been the topic for numerous researches in all countries, but the majority of the publications on that subject are incomplete and only consider some of the facts of the problem. How many authors, for instance, have determined the influence of such factors as bacteria or ultramicroscopic viruses in the natural destruction of a species ? Many limit themselves to a study of insect parasites and predators. Studying the problem of the natural equilibrium of Pieris brassicae1[1], I have previously called attention to the extreme complexity of the problem. I have demonstrated the importance of bacteria and especially of the filterable viruses which, at least in some years, play a leading part in the destruction of the cabbage butterflies. It follows from my own investigations, and from those of my former collaborator J. C. FAURE, that the facts of the problem differ from year to year, and that the relative importance of the factors, determining the equilibrium, is far from constant. A study of the kind I have undertaken, ought to cover a series of years and be extended to all corn growing regions.

 GENERAL RESEARCH TECHNIQUE

 My previous researches on the contagious diseases of insects have proved to me, that the study of the parasites can not give satisfactory results, if it is not undertaken at the place where the epidemic appears. The examination of the diseased insects in the locality, where they are collected, offers the opportunity for determining with certainty, whether the bacteria found in their body cavity are actually pathogenic or whether they are merely saprophytes. In case the study of the collections is undertaken after they have been brought to the permanent laboratory, the principal infection is often disguised by secondary infections, and errors are easily made in the determination of the significance of the isolated bacteria. In order to avoid these errors, while studying the diseases of the silk worm, I designed, in 1924, an automobile laboratory, the construction of which is illustrated in Figs. 1 and 2. The automobile carries the equipment for bacteriological investigations into the fields and contains the necessary apparatus for collection and fixation of samples for later histological investigations. The equipment includes a binocular microscope and a travelling microscope with ordinary and dark field illumination. The automobile laboratory, which served me so well during my studies' on the pathology of the silk worm, has been used extensively during the investigations on the equilibrium af Pyrausta nubilalis, and has enabled me to collect many data on the Corn Borer parasites.

 Corn Borer larvae were examined in the field where they were -collected. The first examination took place by simple dissection under binocular microscope. If the preliminary observations revealed the presence of interesting parasites, a more detailed and complete examination followed . The larvae were pinned to a plate of cork, one pin piercing the head, another the tail end. They were opened centrally with a pair of fine scissors, and the skin stretched out to both sides and fastened to the cork with pins. The examination with binocular microscope revealed the anomalies in the various tissues, and a description was made of the lesions. The larvae, showing signs of microbe infection, were placed in a fixing fluid and preserved for later

 Fig. 1 et 2

histological and cytological study. Preparations of the blood and the infected organs were fixed and stained with Giemsa (protozoa) or Gram (bacteria). In this way everything necessary for a complete study of any epidemic, is collected and it is possible to obtain quickly the exact data on the mortality due to parasites and on their distribution and importance. The use of the automobile laboratory makes it possible to examine, within a short time, a great number of corn fields and to make numerous observations, a point of considerable importance in investigations during developmental stages of short duration, such as the development of the eggs. The advantages of the automobile laboratory may be illustrated by the day's work on July 25, 1928. On this day I visited two large corn fields at Bletterans, 140 km. from the permanent laboratory, and 5 other fields, 25-30 km. further North. Important observations were made in these fields especially on the action of predacious insects, of which a new one was found on the Corn Borer eggs. In addition, material was collected to determine the degree of parasitism by microsporidia.

 The results obtained by the use of the automobile laboratory prove, that this equipment must be considered indispensable for investigations on biological methods to be applied in the fight against injurious insects. The equipment was completed this year by the acquisition of a microcentrifuge, using the current from the storage batteries of the car (12 volts). It rotates 12,000 times a minute causing a rapid sedimentation in organic fluids. This is particularly important for insect blood, infected by bacteria. By using this microcentrifuge, expressly constructed by P. COUPRIE of Lyon, I was able, this year, to determine conclusively the epidemiological nature of a very important silk worm disease, the gattine.

 CORN GROWING IN THE INVESTIGATED TERRITORY

 In eastern France (Fig. 3), or more specifically in the central part of the Rhône basin, comprising the departments of Rhône, lsère, Ain, Saône, Loire and Jura, corn is not grown as extensively as in the Danube or Mississippi valleys. Farming is much diversified and the corn -fields are in most cases less than one hectar and separated by fields with other crops. In certain localities corn growing is more intensive, as in the regions of Bletterans and Pierre en Bresse, where thevery fertile, newer Alluvian, plains are particularly adapted to the growing of cereals. In the region of Bletterans, at the little village of Villevieu, for instance, the farm land is divided into three parts, alternatively planted with corn, small grains, and potatoes or beets. In the region of Pierre en Bresse, the crop rotation is often biennial so that corn is planted in the same field every second year. The Corn Borer causes the greatest losses in these two regions.

 In the very fertile valley of the Isère (Grésivaudan), the corn fields are numerous but separated by other crops such as grapes, potatoes, tobacco, beets, and vegetables. Corn only covers a small part of the land under cultivation. In this region, as well as. in Savoy and in the valley of the Rhône, it is customary to cut the top of the corn plants after fertilization and to use the tops for cattle feed. This practise must certainly serve to destroy a very large part of the Corn Borer larvae. In the other regions of the central East, this practise is unknown.

 After harvest, the corn stalks are cut by hand. In many localities they are used as cattle feed during the winter. For that purpose they are cut into small pieces. Certain farmers use the stalks to cover the beet pits, others burn them. The corn straw is seldom Just left near the fields or by the farm house. In regions where corn growing is relatively important, the destruction of the stalks in winter is never as complete as in other regions. That is one of the main reasons why the Corn Borer does more damage in these sections.

 THE NATURAL AGENCIES DESTROYING THE CORN BORER

 I - INSECT PARASITES

 J. C. FAURE [2]', working at the Southeastern Entomological Station oil the parasites of Piefis brassicae, has shown that "from the standpoint of their relationship to their host, the parasites may be divided into two large groups: normal parasites and incidental parasites. The normal parasites play the principal rôle in limiting the propagation of the host; occasionally the other group may exert an important influence too. The incidental parasites are observed to be present in increasing numbers as the host multiplies excessively. Their significance under such conditions is, however, small because the normal parasites develop parallel with the host in sufficient numbers to quench its excessive multiplication".

 The incidental parasites of Pyrausta nubilalis do not seem to play a rôle comparable to that played in certain years by those of Pieris brassicae. A study of them is, therefore, at the present time, only of secondary importance. We shall consider here only the effect of the normal parasites. Contrary to what might be expected, this is far from preponderant, and other natural agencies destroy many more Corn Borers than do the insect parasites.

 Four species of insect parasites have hitherto been found in the investigated region. One is an ichneumon fly, by THOMPSON & PARKER erroneously determined as Eillimneria crassifemur Thoinson. According to ELLINGFR & SACHTLEBEN it is a new species named Limnerium alkae. The second parasite, Microbracon brevicornis, belongs to the Braconidae. The third parasite, Lydella senilis Meigen, is a tachinid fly. Recently some specimens of Exerisles roborator have been collected. The other parasites, recorded by THOMPSON & PARKER, have not been found in the central East.

 LIMNERIUM ALKAE Ellinger & Sachtleben.

 I have shown in my study [3] of the biology of Lininerium crassifemur Thomson, a parasite of Neurotonza (Lyda) nemoralis, that this parasite differs materially from the Corn Borer parasite, recorded by THOMSON & PARKER under the same name. I have particularly called attention to the fact that the ovipositor of the Corn Borer parasite is considerably longer than that of the Neurotoma parasite. Explaining the morphological differences by the different habits of the two parasites, I concluded that the Neurotoma parasite was a variety of Thomson's species. ELLINGER & SACHTLEBEN [4], have recently shown that the two parasites actually belong to two different species. The parasite of Neurotonza nemoralis is identical with the type studied and described by THOMSON under the name Linmeritun crassifemur, while the Corn Borer parasite constitutes a new species to which the two authors have given the name Lininerium alkae.

 The biology of Lininerium alkae differs materially from that of Limnerium crassifemur. The adults of the latter species emerge from the ground shortly before the emergence of Neurotoma and commence to oviposit in the hatching larvae of the saw fly. The life of the adult Limnerium crassifemur is short and does not ordinarily exceed one month; it is even shorter if the emergence of the host and the parasite coincide.

 In eastern France, the adults of Lininerium alkae generally emerge towards the end of April. Their emergence, therefore, preceeds the hatching of the Corn Borer larvae by almost three months. It is not likely. that the female parasites wait, so long before depositing their eggs. Furthermore, no adult parasites have been found in the corn fields before the Corn Borer moths emerge, and they do not become numerous there before the end of July or the beginning of August, when the first Corn Borer larvae appear. The first hatching larvae most frequently escape parasitism by Limnerium alkae, while the opposite condition exists in the case of Neurotoma nemoralis parasitized by Limnerium crassifemur. The existence of intermediary hosts, maintaining Limnerium alkae during the first months after emergence, is the most probable explanation. Unfortunately, it has not yet been possible to discover these hosts which seem to play a very important part in the maintenance of the species.

 The female Limnerium alkae introduces into the posterior part of the young Corn Borer larva an egg similar to that deposited by Limnerium crassifemur in the eleventh segment of the Neurotonia larva. While the female Limnerium crassifemur is in direct contact with the host larva during oviposition, that is not the case with Limnerium alkae. The position ot the Corn Borer larva in the corn plant is determined by the female parasite. The ovipositor is then pushed through the stem and into. the body of the host. I have observed how the female parasite has pierced the ovipositor through the corn stem several times before depositing the egg. Similar acts may be observed on a number of different ichneumon Ries. It is hardly possible to give an explanation for these movements of the ovipositor before the egg is placed in the host.

 It is very easy to find the grayish black egg of Limnerium alkae in the body cavity of the Corn Borer larva (Fig. 4). It is even possible to separate the infected larvae from the healthy ones by transparent light. Very frequently, several Limnerium alkae eggs are found in the same larva,

  fig 4, 5 et 6

but they are never deposited by the same parasite. The female parasites of both Limnerium species are unable to distinguish parasitized host larvae from healthy ones. THOMPSON & PARKER have counted up to 10 and even 15 parasite eggs in the body cavity of a single Corn Borer larva. Those are exceptional figures, as ordinarily but 3 or 4 eggs are found in each host, but it is evident that the number varies with the number of parasites.

 The form, structure, and color of the egg is identical with those of the Limnerium crassifemur egg. The hatching larva emerges through that end of the egg at which the head is located. The shell breaks transversally at hatching. The empty shell is not absorbed although a great many blood corpuscles are retained at its surface. Even in full grown host larvae the empty shells of the parasite eggs are found imbedded in a mass of closely united blood cells.

 A considerable proportion of the Lininerium alkae eggs, found in the body cavity of the Corn Borer larvae prove to be dead. This phenomenon, which has not been mentioned by THOMPSON & PARKER, is very common and may each year be observed in many Corn Borers. Also many Limnerium crassifemur eggs die in their host larvae. The destruction of the parasite eggs seems due to their envelopment by blood cells. We are dealing here with a regular immunity reaction of the same kind, as is observed in the body of all other animals when foreign substances are introduced. Such reactions have been studied by numerous authors, but especially by METCHNIKOFF, whose experiments on inflammation reactions have become classical. The gathering of blood cells on the surface of the Lininerium eggs results in the formation of a rather substantial pseudo-cyst (Fig. 5) which remains connected with the posterior part of the digestive channel of the host. The cells which form the pseudo-cyst are all the same type with relatively large nuclei; they are morphologically identical with macronucleocytes. Two distinct layers are clearly visible in the cellular sheath around the dead eggs under moderate enlargement (Fig. 6). The inner layer is composed of round cells with indistinct borders and usually very distinct nuclei. The outer layer is composed of spindle-shaped, flattened cells, often somewhat resembling fibres. When suspended in fresh blood, these fusiform cells regain their form as macronucleocytes.

 The causes of the phagocyte reaction at the chorion of the eggs of Limnerium alkae can not be determined with certainty. This reaction may depend upon the special nature of the chorion, especially upon its surface tension; it may also depend upon certain physical and chemical properties of the blood of the larva in which the phenomenon is observed. It seems that the influence of the host is preponderant. It may be observed that the phagocyte reaction differs in the various host larvae, but that it is alike on eggs of different parentage deposited in the body cavity of the same host. If the influence of the parasite had been preponderant, a difference in the intensity of the phagocyte reaction on the eggs would have been noticeable. Furthermore, it is not unusual to find groups of eggs (Fig. 5) uniformly covered with a dead cellular layer.

 The phagocyte reaction undoubtedly causes the death of the eggs. The embryo first develops normally but succumbs when the cellular laver has reached its maximum thickness.

Phenomena of the same kind have been described in 1925 and 1926 by N. F. MEYER, who observed them on larvae of Pieris rapae parasitized by Apanteles glomeratus L and Angitia rapae Meyer. MEYER has pictured egg groups of Apanteles and single eggs of Angitia surrounded by cells, the small nuclei of which make them resemble micronucleocvtes more than macronucleocytes. No exact 'indication is given of the origin of the phagocytes. According to these observations, the eggs of Apanteles are digested in the midst of the cellular masses. The Limnerium eggs, on the other hand, remain intact within the pseudo-cyst.

 The relative immunity of the Corn Borer larvae against Limnerium alkae may indicate that this parasite is still poorly adapted to its host. The Corn Borer may really be but a potential host, while the principal host is still unknown. Limnerium crassifemur has been recorded among the parasites of Conchylis ambiguella and Polychrosis botrana. It ought, however, first to be checked up, whether the parasite of these two microlepidoptera is Limnerium crassifemur or Limnerium alkae. I believe that I am in a position to state that the two grape moths are not the principal hosts of Limnerium alkae, at least in the central eastern region. I have never, from these microlepidoptera, reared an ichneumon fly that might be identified as Uinnerium alkae. The principal host of this parasite must probably be found among the microlepidopterous larvae living on wild plants. The parasite fauna of the majority of these microlepidoptera is unknown, even when the hosts are abundant, especially in spring. The microlepidoptera probably play an important part in the propagation of parasites, useful to agriculture. It is important to fill this gap in our knowledge in order to obtain all the facts necessary for the intelligent application of parasites in the fight against noxious insects. How is it possible to think of multiplying parasites artificially without knowledge of the various factors which are indispensable for their propagation ?  

INFECTION OF PYRAUSTA NUBILALIS BY LIMNERIUM ALKAE (table 1)

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