ERRATA. Page 12, lines 16 and 17, for one hundred read three hundred and for one thousand read six hundred. Page 17, line 2, dele first letter in the line. Page 168, line 12, page 177, lines 13 and 14, and page 271, line 10, for Lemna tnsulca read Spirodela polyrMza. Page 209, line 2 of foot-note, after hut insert represents. Page 2;'56, line 7, and page 266, line 19: snoioi n. s. has been shown to be hieroghjijhka, (^. Page 257, insert as line 8 as follows: -ken to the office produced young in ten days. The Page 272, line 13, for P. higuUatus read Pompiliis higuttatus. Page 278, Plate Y., 16, after view insert as follows: a, mentum; 6, labial rudiment; c, maxillary palpi; d, maxilla; e, labrum; /, antenna; g, eye; h, mandible. Page 286, line 11, drop initial the one line. Page 386, line 1, for Comstocki read Comstock. Page 399, line 17, for specimens read specimen. Page 411, line 10, for Michaelson read Michaelsen. Page 441, line 3 from bottom, for 66 read 68. Page 445, line 10 from bottom, for 57 read 5S. Page 466, line 1 from bottom, for Cyima read Oypris. Article XJl.— On a Bacterial Disease of the Squash-hug {Anasa tristis DeG.). By B. M. Dik^gar. INTRODUCTORY. During July, 1895, while occupied as an Assistant in the Illinois State Laboratory of Natural History, in studying some fungous diseases of the chinch-bug I used many squash-bugs {Anasa tristis DeG.) for experimental purposes, their larger size making them convenient for use in certain investigations for which the chinch-bug is poorly adapted. These squash-bugs were kept in a lab- oratory breeding-cage, and daily supplied with fresh food and a suitable amount of moisture. They were soon observed to be dying in considerable numbers, although I could detect nothing unhealthful in their surroundings. A fresh lot of the insects was thereupon brought from the field July 23, put into a large breeding-cage, and kept as far as possible under normal conditions. A few of the bugs recently dead in the first outbreak were broken in pieces and scattered about the earth of this new habitation, or touched to the bodies of some of the health^' individuals, A much larger number of this fresh supply of insects was resei-ved for another small cage, which, with conditions otherwise similar, was left uncon- tarainated by the bodies of any of the dead or diseased insects. In three days one half of the insects in the in- fected cage were dead, while in the untreated cage, with so many more individuals, there were only two or three dead. The dead bugs in the infected cage presenting common characters, and a careful microscopic examina- tion showing a well-defined bacillus uniformly abundant, this preliminary test encouraged further experimentation with this disease from a strictly bacteriological stand- point, with a view to an elucidation of both its practi- cal and scientific features. Bacterial Disease of the Squash-hug. 341 PRIMARY INFECTION EXPERIMENTS. In order to refer all cultures and subsequent results to a definite and unmistakable source, a few preliminary infection experiments were begun, the results of which were recorded in detail. It seems well, therefore, to preface particulars concerning the culture and infec- tion work reported in this paper with the following fun- damental infection records: Experiment 1.—A box breeding-cage was stocked with about two hundred squash-bugs, both nymphs and adults, squash leaves being daily supphed as food, and kept fresh by inserting the stems through the cork of a small jar containing water. From the preliminary dis- ease-cage several dead bugs were taken August 1, the bodies torn apart, and the pieces scattered about the earth and food leaves in the new breeding-cage. In two days ten bugs were dead, and in five days a total of sixty dead was reached. At this date a few of the dead insects were again broken in pieces and scattered about the cage, while the remainder were piled in a corner. On the eighth day the mortality reached ninety, and the following day added ten more. The death rate was now much lessened, and from August 12 to August 15 only three deaths occurred. A few insects had been removed for various purposes, and on the last-named date the number of living bugs remaining was between sixt^' and seventy-five, while about one hundred and fifteen deaths had occurred. Experiment ^.—As a check on the preceding, about fifty squash-bugs of the same lot were put into a much smaller breeding-cage on the same date, August 1, and kept sub- ject to similar conditions, but without inoculation. The first death was noted August 7, but the body of the dead insect was normal. This cage was observed until August 17, with a record of but one additional death, and this not attributable to the disease. From the record of these two experiments it will be 342 Illinois State Laboratory of Natural History. seen that evidence of the contagious nature of the disease was clear, and after August 17 the cage used in Experi- ment 1 served as a general source of infection mate- rial. The observations show that the greatest mortality was probably from three to six days after the introduc- tion of the disease, and no dead insects were in any case found at the expiration of the first day. The proportion of deaths was undoubtedly greatest among the younger nymphs, but many adults also succumbed. Subsequent experiments likewise confirmed this conclusion, although the difference was not always very great. Cliaracters of the disease.—The effect on the insect and the post-mortem appearances were carefully noted in Ex- periment 1. A few hours before death the insect may be found in a sluggish condition, resting low on its ventral surface, and often apparentl}^ incapable of rais- ing itself erect, or of crawling without a marked drag. If placed on its back, it has no power to return to the normal position. As the disease progresses the insect loses nearly all muscular activity, and a slight waving of limbs and antennse may be the only indication of life. Squ£ish-bugs cannot attach themselves strongly to the leaves by their limbs; and as they usually fre- quent the under surface of the leaf, diseased bugs are commonly found on the earth of the cage. A few hours before death there is no marked discolor- ation of the body ; but the insect becomes slightly darker as death approaches, owing probably to changes in the body fluids. After death the changes are rapid. The nymphs soon assume a deep purplish black hue, the body does not shrink at all, but appears tense and slightly swollen, and in the course of twenty-four hours or more it becomes a mere sack of gruel-like fluids. In this con- dition the walls readily collapse, and the insect may not bear lifting without breaking. In the adults the body is observed to have a rather moist appearance at the time of death, especially in the cephalic region of the Bacterial Disease of the /Squash-lmg. 343 ventral surface of the abdomeu. Later, the wet appear- pearance is more evident throughout; but the hard chitinous crust does not shrink or collapse, and unless broken, the offensive fluids within are unnoticed. In all cases, the odor—more pronounced and putrefactive than the normal squash-bug odor—is especially characteristic. A short time after death the appendages are ver^'^ readily separable at the articulations, and it is almost impossi- ble to lift an insect by means of them. A long series of infection experiments will be described later, and in this connection it is only necessary to note further the post-mortem appearances of insects free from this disease. It has been established that bacteria are normalh' found in the ccecal appendages of many Hemip- tera,* among- which are the squash-bugs. It conse- quently seemed of interest to ascertain if these bacteria might become truly pathogenic, or, at least, cause the peculiar post-mortem appearances under abnormal condi- tions—as of lessened food supply. Such investigation was quite superfluous in view of the check experiment above recorded, but, nevertheless, a few healthy indi- viduals were allowed to die from gradual starvation. Under these conditions the body cavity gradually dried out, and when death finally resulted the shell was greatly contracted in the abdominal region, and slightly drawn together ventrally. This was especially true of the nymphs, and later observations were to the same effect. Lessened vitality may encourage the disease, but it has nothing to do with a "spontaneous" occurrence. Moreover, the microscopic characters and the cultiva- tion experiments enumerated later show conclusively that the disease form is entirel.y distinct from the normal form. Other nymphs were killed b^' immersion in the toxic bacterial infusion from a pure culture of the disease or- ganism,—which toxic infusion will be subsequently de- * "Bacteria Normal to Digestive Organs of Hemiptera." By S. A. Forbes. (Bull, III. State Lab. Nat. Hist., Vol. IV., pp. 1-7.) 344 Illinois State Laboratory of Natural History- scribed,—but the usual shrinking of natural death oc- curred. It is probable that dead bugs placed on a wet surface might absorb moisture and show an appearance somewhat similar to the disease effect, but this is only a surmise. ISOL.\TION CULTURE AND GROWTH ON AGAR-AGAR. A number of dilution cultures were made during the progress of these preliminary experiments, but the re- sults were so uniform that only one such culture will be reported in detail. For some of the earliest dilution cultures dead insects were necessarily employed, and the body fluids were thus greatly mixed. Even with these, however, cultures were obtained which gave one form of colon}" in great preponderance, while a few other forms were confined to scattered colonies in the original dish. Here, also, the abundant form was identified as a bacillus. Preliminary experiments demonstrated that this form was an effective pathogenic agent for squash- bugs; and as soon as sick insects were available, every precaution was taken to secure a culture with the least practicable admixture of foreign forms. All the necessary apparatus having been properly sterilized, a sick nymph was thoroughly washed with corrosive sublimate solution, and a fold in the soft body wall of the abdomen was caught in the forceps, and slit with the scissors in such a manner as not to disturb the alimentary organs. A small amount of the exuded body fluid was then transferred with a platinum needle, in the usual manner, to a tube containing peptonized nutrient agar-agar at the prescribed temperature. After shaking well, several transfers were made from this first tube to a second, and finally to a third; and the con- tents of each were poured into a Petri dish. To the nutrient agar used in these tubes was added a small quantity of squash-leaf decoction, and as this was found to be of advantage, it was continued in all subsequent ao'ar cultures. Bacterial Disease of the 8quash-bug. 345 In four days the di.sh from the original tube gave abundant colony formation, and in the next dish about ten colonies appeared. The surface colonies were circu- lar, slightly yellowish white or dirty white, but with a distinct opalescence. The submerged colonies were ellip- tical or slightly pyriform in vertical projection, growing, toward the surface, more and more laterally compressfMJ. This peculiar growth of submerged facultative anaerobic bacteria has been well explaiued by Professor H. Mar- shall Ward,* who has studied in detail under high pow- ers the formation of micro-colonies in certain cell cul- tures. The horizontal long diameter of such elliptical colonies is in the plane of fission of the rodlets. The form of growth is due to the fact that as the elongating rods are broken up into daughter cells, these cells slip by each other (one over the other) under the pressure of the contracting gelatine or agar; and if in agar, evi- dently this form will be retained in the macro-colony. To return to the circular colonies, those that had room for the fullest development showed in a few days marked lobulations, and sometimes beautiful fan-like radiations. Furthermore, those submerged colonies that had reached the surface took on the circular form and very soon de- veloped some of the characteristic radiations, although the growth is thinner than when they develop superficially. All stages of these developments are evident in Plate XXVIII., Fig. 1. Submerged colonies appear deeper colored than the surface ones, but this is only an ap- parent coloration. It will also be seen that there is a film-like growth on the lower surface of the agar be- neath deeply submerged colonies. This, also, is the same form which has spread out between the contracted agar surface and the glass. The lobulated growth appearance is not always pres- ent, even in cultures direct from the insect, and like all such radiating bacterial growths, is probably greatly * "The Formation of Bacterial Colonies" (Annals of Botany, Vol. IX., No, XXXVI., Dec, 1895.) 346 Illinois State Laboratory of Natural History. affected by the amount of moisture, by the temperature, and by all conditions affecting the vitality of the organ- ism. These radiations are apparently more abundant where the amount of moisture is considerable and the temperature about the optimum, but with too much moisture the bacillus spreads over the surface as a con- tinuous sheet-like growth. Where the colonies are very abundant they remain small and circular, or become variously united in a complete network. At low tem- peratures the lobulations seldom occur. I have also made Petri-dish cultures from tubes kept for some time in the laboratory, and on agar containing varied quan- tities of water, bub these marked growth characters were then entirely absent. This may be due to a lessened motility of the bacillus, or to other causes not wholly understood. After being grown in the laboratory in several suc- cessive tube cultures, this organism seems to lose the power of producing the slight yellowish-white color, and the growth becomes a purer opal w^hite. It should be noted that in subsequent isolation cul- tures, it was found that the pathogenic form could often be secured pure, even in the first dish, by using sick insects, observing proper sterilization precautions in clipping off a leg in the region of the femur, and transferring to the tube with a platinum needle a little of the exuded fluid. The fluids of the diseased bug are almost pure cultures of the pathogenic organism, and unless the alimentary tract is badly broken down, I have seldom found many foreign germs. From the above notes it will be seen that with this bacillus there is apparently no tendency towards spon- taneous variation in the colonies growing side by side in a culture direct from the insect. I have also care- fully observed the growth in cultures from various sources, and the more marked opalescence after contin- ued growth on agar is the only variation noted. Bacterial Disease of the Squash-bug. 3-47 GROWTH CHAKACTEKS WITH VARIOUS NUTRIENT MEDIA. Solid media.—On iiutrieut gelatine this organism makes a rapid growth. Several stab cultures were made on Sept. 30, and the tubes were kept at a cool, living-room temperature. In two days the central needle-path showed a gi'owth throughout its whole extent, and on the third day liquefaction had begun. The appearance is at first that of a short cylindrical or rotund air cavity, from the lower surface of which projects the tapering liquefied portion for about one third of the inoculation line, show- ing a considerable precipitate in the lower part. On the third day the liquefied portion was considerably extended, occupying an area shaped like an inverted lamp chimney, alono* the middle line of which extended the undulating precipitate-like bacterial formation. In the course of one week, three fourths of the gelatine was liquefied, and the bacterial growth was scattered through the lower portion in a flocculent manner. The remainder of the gelatine was soon liquefied, the flocculent material set- tled to the bottom, and with the exception of a slight tui-bidity the color of the liquefied portion remained un- changed. After standing for about one month, and before any great amount of evaporation had taken place, the light amber-color was changed to a deep reddish amber, and the color became more pronounced as evaporation advanced. On slices of sterile potato kept in a moist chamber, this organism makes a profuse growth in two days. About this growth there is nothing especially typical; but the dirty-white color is well marked, and the thick film of growth shows a strong tendency to become lobulated on the margins. Liqvid media.—Fluid cultures were made to determine the nature of the growth, and also to ascertain how the organism might be propagated in considerable quantity for infection ex]ieriments, if the latter should prove de- sirable. In bouillon a considerable turbidity is produced 348 Illinois State Laboratory of Natural History. in the course of two da3's; and with further growth a slight film is developed on the surface, and a precipitate begins to accumulate at the bottom. This precipitation increases with the maturity of the culture, the superficial film disappears, and the liquid is left slightly clouded and very little darker than the uninoculated fluid. After the cessation of growth, there is no further change of color in the bouillon. Other cultures were made on de- coctions of green corn, of squash leaves, and also on a mixture of bouillon with each of these. The growth differed little from that on bouillon, except in a thicker, scum-like surface film. Sterile skimmed milk inoculated from a pure culture of the squash-bug bacillus shows considerable curdling, in twenty-four hours, and subsequently a rapid productioif of whey. In one week the curd is about half dissolved, and it soon assumes a somewhat rusty color, as also does the turbid whey. A few days after inoculation the odor from these milk growths is extremely disagreeable* and it becomes vile and penetrating. It resembles the odor of sulphureted hydrogen, and, while not so strong, it is more nauseating. An infusion of the bacillus from a growth on agar was sterilized at a low temperature, and then added to a small quantity of sterile skimmed milk. No change was produced in the milk, and a sub- sequent inoculation with the active bacillus gave a growth characterized as above. From the experiments recorded under the head of "Toxic Properties" it is noted that an infusion* of this organism from an agar culture contains some substance fatal to insects, and various media were employed in order to get this substance in a form suitable for chem- ical analysis. Buckraasterf has mentioned some nutrient media used by Uschinsky for the cultivation of certain * It is well to note here that in this paper I have used the word i; fusion only to denote the fluid in which bacteria have been diffused for inoculation or other such purposes, and no idea of heat is thereby implied. i Ursprw'ig unci Beschaffenheit gewii>ser Bakterlengifle. (Biol. Centr. Bd. XV., Nr. i, Feb.. 1895. JSacter'ial Disease of the Squash-bug. 349 pathogenic organisms, by means of which a toxic excre- tion of bacteria may be secured in a way convenient for chemical study, The mixture which I have tried is form- ulated as follows: Water 1,000 Glycerine 40-50 Sodium Chloride 5-7 Ammonium Lactate 10 Calcium Chloride 0.1 Magnesium Sulphate 0,2 Potassium Biphosphate l To the above formula Uschinsky added a little sugar for some organisms, and urea or uric acid for others. With the bacillus of this squash-bug disease an addition of 5% sugar gave an abundant growth, but the develop- ment was very slow. Three weeks after the inoculation a thick yellowish white film had formed on the upper eui-face, and the bottom of the flask was likewise thickly covered with a sedimentary deposit. During the first two weeks' of growth the liquid had a slight pinkish coloration. An addition of 10% sugar gave much less growth than the above, but the pink coloration was more marked. Urea to the amount of one half per cent, with the formula mentioned, also urea one half per cent, and sugar 5% in each of two other flasks, differed very little from the culture first described, although the pink color was more lasting, and possibly the growth of less extent. The value of these media for securing the toxic principle for analytical purposes cannot yet be determined, as the chemical work has not been completed. A culture consisting of fermentation broth in bent tubes gave no gas production. The ingredients of the broth used were as follows: Water 1,000 c.o. Glucose 20 grms. Peptone 10 grms. Sodium Chloride 5 grms. In a nitrate solution the organism grows well, but there is no reduction of the nitrates, as shown by nega- tive results (absence of red color) from the usual test — 350 Illinois State Laboratory of Natural Histm^y. the addition of a few drops of naphthylamiue chloride and a small quantity of sodium sulphanilate. The formula for the nitrate solution used is as follows: Water 1,000 c.c. Peptone 1 grm. Potassium Nitrate 0.2 grm. INFECTION EXPERIMENTS WITH THE SQUASH-BUG. Laboratory Experiments. In addition to the introductory experiments it is necessary to describe in some detail the numerous in- fections made in the laboratory under various condi- tions. It was my practice to accompany every experi- ment, or series of experiments, with a check subjected to similiar conditions, but without inoculation. Moreover, with all the cages and contents of cages, sterilization by means of heat and corrosive sublimate was employed as far as was compatible with the size and nature of the materials employed. Whenever possible, the bugs used for experimental purposes were first kept in the laboratory for two or three days, in order to watch for any "spontaneous" outbreak of the disease. Experiment 3.—In a breeding-cage which had served as a check on some previous work, there were twenty- eight adult squash-bugs and twelve large nymphs. These insects had remained perfectly healthy throughout, and on August 12 each of the individuals was inoculated by touching to its body the mixed fl.uids from a diseased bug out of Experiment 1. In four days eighteen bugs were dead, ten adults and eight nymphs; on the fifth day twenty-four were dead, fourteen being adults and ten nymphs; and during the next four days there were only six deaths, one nymph and nine adults remain- ing. A second inoculation was then made in the same manner as before, and five days thereafter one half of the remaining ten were dead. Four adults sur- vived both inoculations. In this experiment it was no- ticeable that the nymphs died rapidly during the first few davs. Bacterial Disease of tfoe Squash-bug. 351 Experiment If..—One of the most successful experiments with the organism of this disease was the result of test- ting the bacillus found so abundantly and almost pure in one of the early isolation cultures. A young colony direct from a Petri dish was diffused in a small quantity of water, and each of five squash-bugs was thoroughly wet with the infusion. Three of the insects were dead on the morning of the second day, and at the end of two and one half days all were dead. A check lot of bugs used in conjunction with this experinient remained healthy. Experiment 5.—About forty squash-bugs were inoculated from diseased insects, the cage being without moisture except that furnished by the food leaves. The results indicate that the disease took Tapid effect, as fifteen bugs were dead at the end of five days. From this time the death rate diminished, but the bugs dropped off gradually until only two or three apparently resistant ones remained. In order to compare the effect of pure cultures of vari- ous ages with infusions direct from diseased insects as sources of infection, a series of experiments were made in each of which six bugs were used. When pure cultures were employed, some of the bacterial growth was diffused in distilled water, and in this the insects were momenta- rily immersed. With the diseased or dead bugs an infusion was likewise prepared by tearing the bodies apart in water. Healthy bugs from two different fields furnished the subjects, and a separate check was used for each lot. All dead insects were removed as soon as observed, in order that the results might follow only from the first infection. 352 Illinois IState Laboratory of Natural Jlidory. Table I. Ex- peri- ment No. Ko. Insects SouECE OP Intection. Number or Deaths. 2 days. 3 dajs. daj-s. 10 days. Total. Number of bugs remain- ing. 6 Bacterial Disease of the Squash-bug. 353 to yield the disease or^^anism ; and, moreover, one of the bug's immersed for a verv short time in the infusion from this grasshopper was killed. This grasshopper had been dead for about two daj's, however, and this may have injuriously affected the disease organism. Field Experiments. Experiment 15.—A careful examination of a squash patch on the Agricultural Experiment Station grounds on Sept. 17 resulted in a find of two dead squash-bugs; but microscopic examination showed that the bacteria in the bodies of these insects were different from the disease bacillus. It was doubtful if the disease existed in the field at all at this time, and a field infection test was greatly to be desired. For this purpose several dead bugs from Experiment 1 were teased out in about 40 cc. of water, and this infusion was sprayed upon a squash-vine containing about two hundred bugs. This vine was somewhat isolated from the remainder of the plat. No attempt was made to get the infusion on all of the bugs ; but the spray was simply directed to those leaves containing the largest number of insects. The result of the first infection alone was desired, so the dead bugs were removed as soon as found. Three dead insects and three distinctly sick, all n\'mphs, were re- moved on the second day, the true disease bacillus being found in all of their bodies. On the third da^' eight dead insects were removed, and the number of bugs on the vine was much reduced, owing to migration for fresher food supply. One week after infection the total number of diseased insects amounted to eighteen ; but before this time nearly all of the bugs had migrated, and the experiment was discontinued. Experiment 16.—Although the season was far advanced, and the weather probably too cool for the best results, I was encouraged to repeat Experiment 15, with pre- cautions against migration. An infusion of ten dead 23— 354 Illinois State Laboratory of Natural History. bugs out of previous experimental lots was employed in the same manner as above, in this case one gill of water being used. This was sprayed upon two hundred or more half-grown nymphs and adults infesting a squash-vine, and the vine then securely covered with mosquito netting. The weather continued quite cool, and on the fourth day after infection fourteen dead bugs were found, all but two of which showed the disease characteristics. On the seventh day there was a total of thirty-two dead bugs. A frost the previous day killed the food leaves, but the stems were still in good condition. This cold so chilled the bugs that they moved very little during the early part of the day; yet an examination of the field, as a check, gave no dead insects which might not have died from injuries by persons passing about. On the tenth day thirteen recent deaths had resulted ; and in spite of the precautions taken, many of the bugs had escaped, the wind having frequently displaced the netting. After the twelfth day, October 9, the netting was re- moved and only one additional count made, the small number of bugs remaining not justifying further observa- tions. A summary of the results is as follows: September 27, about two hundred squash-bugs were sprayed with infusion of diseased bugs. October 1, four- teen dead insects were counted, and later, additional ones, as follows : October 4, eighteen ; October 7, thir- teen ; October 9, seven ; and October 15, three—a total of fifty-five. Experiment 17.—Parallel with the above and on the same date (September 27), an experiment was made to test in the field the efliciency of old pure cultures of this organism. The growth from two tubes one month old was diffused in a gill of water, and sprayed upon the insects as before. There were, however, more than two hundred bugs present under the netting in this experi- ment. Four were dead October 1, and subsequent counts of additional dead were as follows: Oct. 4, two; Oct. 7, Bacterial Disease of the Squash-bug. 355 thirteen; Oct. 9, four; and Oct. 15, one—making a total of twenty-four. As the weather was unfavorable for the spread of the disease when once established, these experiments must be judged wholly in the light of results accruing from the original infection alone. Evidently the old cultures were not as effective as the infusions from dead bugs, and the difference is even greater than is apparent from the summaries, for there were more insects in Experiment 17 than in No. 16, and fewer bugs escaped towards the later periods of observation. There is every reason to believe that this disease may be spread among squash- bugs in the field. INFECTION EXPERIMENTS WITH THE CHINCH-BUG (jBUssus leuGopterus Say). The first infection experiments with chinch-bugs, made in the early part of August, were wholly unsatisfactory, owing to the spent condition of the bugs of the first brood, and the results are not included in this paper. When the second brood began to make its appearance, late in August, all previous experiments were repeated and new ones were begun. The majority of bugs involved in the series of experi- ments tabulated below were in the first stage—a few in the second. In each case a large tumbler about one third filled with moist sand and covered with a muslin cloth was the cage extemporized as best adapted for this purpose. The vessels and the sand were previously steril- ized, and stalks of Indian corn cut into suitable pieces were regularly supplied as fresh food. A small pill-box of the bugs, approximately five hundred, were then momentarily immersed in the infusions used, or immedi- ately put into the cage with the infection material. The number of dead bugs in each cage was ascertained by actual count, and is given below. 35(5 Illinois State Lahoi^atory of Natural History. Table II. Ex- peri- Bacterial Disease of the Squash-bug. 357 thpy do not dry out as rapidlj as those dying naturally. Moreover, the color is apparently somewhat dulled. The success of the experiments in Table II. led to the institution of a series of tests dealing- with the effective- ness of chinch-bugs dead from the disease as a source of infection. The number of bugs, style of cage, and other conditions were similar to those in the preceding series. Man}"^ of the bugs had now passed the second molt. The following table summarizes the results. Table III. Ex- peri- 358 Illinois State Lahoratory of JSatural History. disease were broken in pieces and thrown about in the box, and numbers of young chinch-bugs were introduced. In a few days some dead insects were found-, but after a time the disease seemed to die out. Most of the insects were reaching the pupa state, and experiments were discontinued until the bugs reached the adult con- dition. When the adult bugs became abundant, Mr. W. G. Johnson, an Entomological Assistant in the Laboratory, conducted several infection-box experiments with various diseases, and in one of these the bacillus of the squash- bug disease was used. Every effort was made to get the bugs infected, and they were thoroughly wet with the spray of infusions rich with the bacillus direct from fresh pure cultures. Very few bugs died in this box; indeed, no more than died in other boxes with other diseases, and apparently no more than in the check box. The failure of these experiments with adult chinch-bugs, and on a scale so much larger than the tumbler experiments which I had previously concluded, somewhat chilled the prospect of pushing field experiments. It was now necessary to test the organism on adult chinch-bugs under the conditions prevailing in the ex- periments given in Tables II. and III., in order to compare by actual count the death rate of the old bugs w'ith that of the voung. In this instance two tumblers were employed for each experiment. The sand in both jars was very slightly moist at the beginning; but in one it was allowed to dry out gradually, while in the other it was kept moist. As before, about five hundred bugs were used in each tumbler. The bugs were brought from the field in quantity, and as some appeared to be stifled, all dead bugs were removed from each of the cages after twenty-four hours, in order to avoid any error from outside sources. When infusions are men- tioned, temporary immersion of the insects to secure infection must be understood. The following table gives all essential data. Bacterial Disease of the Squash-hug. Table IV. 359 Ex- peri- 3GO Jllitiois State Laboratory of Natural History. laboratory, the cold weather haviuo; caused them to fall to the ground. Their food supply was then very scant, as the leaves were drying rapidly; nevertheless, the bugs were used for experimental purposes, as it w^as desirable to test the squash-bug disease on as many Hemiptera as possible. Small box breeding-cages were used, but it soon became impossible to find suitable food. Post- mortem appearances and microscopic examination guided my opinions as to the presence of the disease, and the series was early abandoned on account of the condition of the bugs. The appended table is not, however, with- out interest. Table V. Ex- peri- ment Ko. Bacteria' Disease of the Squash-hug. 361 cage four grasshoppers were brushed externally with the body fluid from diseased squash-bugs, and in another cage the infection material was from a pure culture of the bacillus ten days old. In the course of ten days two hoppers were dead in each cage, as also in the check lot. With the hoppers from the inoculated cages, isolation cultures, growth on gelatine, and microscopic characters indicated that the bacillus was that of the squash-bug disease; but the only attempt made to inoculate squash- bugs from these dead grasshoppers gave very slight re- sult. INFECTION EXPERIMENTS WITH GRUBS AND CATERPILLARS. No true bacterial disease of Hemiptera has previously been reported, and, with few exceptions, these diseases have been confined, as far as known, to lepidopterous and coleopterous insects. In this connection it is to be remembered that Bacillus insectorum {Micrococcus insec- torum Burrill) of the chinch-bug was finally located as a "normal" form,—one of various normal forms common in the coecal appendages of the higher Hemiptera,—and it consequently cannot be termed pathogenic with our present knowledge of the subject. These facts make ex- periments with the squash-bug bacillus on gi'ubs of beetles and on caterpillars greatly to be desired. White grubs, probably larvae of Lachnosterna fusca, were inoculated externally both with fluids from diseased insects and with fresh pure cultures ; but in no case was there any successful infection. Four grubs were used in each experiment, and they were kept under observation for about thi-ee weeks. Fall web-worms were also exposed to this disease by touching to their bodies the fluids of diseased squash- bugs, smearing the material over the food leaves, and scattering bits of the diseased bugs about the cage; but the disease took no effect. A few 'other preliminary ex- periments were attempted, which are briefly summarized below. 362 lllinok State Lahoratorij of Natural History. A tomato-worm (Protoparce) nearly full grown was smeared along the line of spiracles with the diseased fluids. Suitable food could not be obtained, but the larva lived ten days, and gradually shrank in size and partly pupated. At this time a microscopic examination was made, but no bacteria could be found in the tissues or in the fluids. Following the above, a larva of the white-lined morn- ing sphinx, Deilephila lineata Fab., was inoculated by clipping off its horn and injecting into the body a small quantity of an infusion from a pure culture. The larva died in two days, filled with bacteria of several kinds, and four squash-bugs were then inoculated from this larva. At the end of two days one of these bugs was dead, but the others remained healthy. The evidence certainly indicates that this bacillus is not very effective on any insect yet experimented on out- side the order Hemiptera, and that the disease it causes is not likely to be confused with any disease previously described. The growth characters alone, indeed, would serve to distinguish the organism specifically. TOXIC PUOPERTIES. From one of the early isolation cultures I removed several colonies of the disease bacteria from the surface of the agar, and diffused these in a small quantity of distilled water to serve some inoculation purposes. On immersing young squash-bugs in this infusion, death fol- lowed almost immediately. With nymphs somewhat older the effect was not so rapid, but the bugs soon succumbed. Young chinch-bugs, flies, and other insects stiffened as if dead on being immersed from one to sev- eral minutes. Many of the hard-shelled insects, if re- moved immediately on becoming rigid, recover in a few minutes sufficiently to crawl away; but even these die if immersed in the infusion for some time. The rapid action of these infusions suggested that some poisonous principle was excreted by the bacteria, Bacterial Disease of the Squash-hug. 368 for it seemed impossible that an effect so marked could result from any circumstances concomitant to the mere presence of the bacteria in the water. Nevertheless, similar experiments were made with other active aerobic bacteria; but in these infusions no such marked or per- manent effects could be induced. In general, soft-skinned insects were much more readily affected; and the yellow-necked apple-tree caterpillar, Jjiitana mAnistra, proved to be an excellent subject for experimentation. I give in detail a record of the effect on one of these larvae, as made by Professor Forbes: The larva was dipped for ten seconds in a strong in- fusion of the bacteria, and then removed to a piece of filter paper for observation. On removal it was quite rigid, but in two minutes there were slight signs of life, and in three minutes it was wriggling and tossing, con- tinuing these incoherent movements until after the fifth minute, when it lay quietly upon its back. Six minutes after removal from the infusion, the larva ceased en- tirely to respond to touch, and was apparently dead. It was kept for twenty-four hours more, but gave no evidence of recovery. Such insects as squash-bugs, flies, etc., often make characteristic movements when about to succumb to this poison; and in the stiffness that finally ensues, the legs are often closely drawn together. As mentioned before, many insects will recover from the effect if removed as soon as they become stiff, I shall have further occasion to refer to this stiffness produced in insects previous to death, and as it is doubtless analagous to certain effects of heat, cold, etc., we may conveniently employ for it the term toxic rigor. In order to avoid any possibility of confounding with these toxic phenomena those incident to drowning, a number of water-beetles {Dineutes discolor) were secured for experimentation. It was first ascertained that a water-beetle easily lives in pure bouillon many hours — thirty in the case recorded; and after such a length of 364 ILinois iState Laboratory of JSataral History. time decomposition would have advanced considerably. In dilute squash-leaf decoction this beetle also survives an immersion of many hours; and it is therefore evident that the ingredients which might be dibsolved from agar cultures in making the infusions would play no part in the result. In all subsequent cases the infusions were obtained from agar cultures in slanting tubes, or from the Petri dishes, by adding a small quantity of distilled water, and then with the needle diffusing in this some of the bacterial growth. This infusion was then trans- ferred to a deep Petri dish. It is probably well to give in detail the results of a few experiments with this water-beetle. {a) Three minutes after immersion in an infusion from an isolation culture the beetle became somewhat sluggish, although it made a few rapid dives when touched, and in ten minutes no movement could be induced. After re- maining thus immersed five minutes longer, the beetle was transferred to filter paper. Ten minutes later there was slight sign of revival; but this was only temporary. The insect in this case was immersed in an infusion from pure cultures. ih) In seven minutes rapid diving about the vessel had ceased ; in nine minutes there was only a slight movement of the limbs ; and in fifteen minutes the beetle was apparently dead. It was kept in the infusion for fifteen minutes after the toxic rigor was produced, and was then removed to filter paper; but there was no re- covery. (c) A tube culture was sterilized by exposing it to a temperature of 125° F. for one hour on two successive days. An inoculation from this tube showed that it was perfectly sterile, and an infusion was then prepared, in which a beetle was placed. Sluggishness was manifest in seven minutes, and in fifteen minutes there was no sign of life. Fifteen minutes thereafter the insect was re- moved to filter paper; but the only sign of recovery was a temporary twitching of the limbs. BacteHal Disease of the tSquash-buy. 365 The above results and a few others are briefly indi- cated in the table below. Table VI. No. of ex- peri- ment. 3GG Illirtois State Laloratory of Natural History. tire than pure cultures made from it. To determine if the organism was thus attenuated by continuous growth in the laboratory, ten successive cultures were made, the first proceeding immediately from an isolation culture, and the growth in each tube being permitted to mature before the succeeding one was inoculated from it. The first series was kept for one month, and the second series was prepared from this just two days before the experi- ment was to be made. At this time water-beetles were not procurable, and adult squash-bugs were necessarily used. The latter are rather difficult to operate upon, and recover more readily than water-beetles; but the relative activity of these solutions could be tested by a comparison of the time required to produce the toxic rigor. The results are indicated by the following table. Table YII. Bacterial Disease of the Sqiias/t-hug. 367 This record .seems to iudicate that there is no deterio- ration in the properties of this organism when grown successively on agar cultures. The individual variations in the above table are probably due entirely to a differ- ence in the resistance of the bugs themselves. Chemical analyses of the pathogenic principle produced by this bacillus have been kindly undertaken by Profess- ors A. W. Palmer and H. S. Grindley, of the University of Illinois. MICROSCOPIC CHARACTERS. Slides of this organism direct from the insect, if prop- erly stained, show a short bacillus, single or in pairs, usually 1.2-1.8/s, Nature, and Prevention." (Bull. Neb, Agr. Exper. Station, Vol. 2, Pt. I., p. 104, etc.) tFor this stain and for various technical susrsiestions, I am indebted to Professor T. J. Burrill. •-5G8 Illinois kState Laboratory of Natural History. Some of the squash-bug uymphs sick with this disease^ or recently dead, were fixed in hot water, hardened, de- hydrated, and imbedded in paraffin hj the usual ])roces8. Sections were then made, generally Q% pt in thickness, with the idea of ascertaining as well as possi- ble the general distribution of the bacteria within the tissues. It is difficult to find stains that will differentiate an organism under such circumstances. On finding that anilines, haematoxylins, and combinations of these worked to little advantage, Loffler's alkaline meth^'lene blue was tried with fairly good results. This was used alone, or after previous staining with eosin. A few hours after the death of an insect, the tissues are so badly broken down that little of interest is to be gained from a study of such specimens. In sections of an individual fixed just at the time of death, the bacteria will be found in great abundance in all parts of the perivisceral cavity and well differentiated from the blood coagulum. (See Plate XXVII., Fig. 2.) The blood, indeed, appears to have been like a pure cul- ture of the disease organism. At this stage, moreover, the adipose tissue and the hypodermis are considerably broken down, and thoroughly penetrated by the para- site. The cells of the cardiac tissue also show the pres- ence of the bacteria; but the structure of these cells and the form of their nuclei have suffered very slight disturbance at this stage. There are very few structures unattacked besides the muscles and the stout walls of the alimentary organs; and surrounding both of these the bacteria are often found in great abundance. Sections of a very sick nymph, killed probably not more than an hour before death would have occurred from the disease, show little that is different from the preceding, except that the tissues are somewhat more nearly normal. (See PlateXXVIl., Fig. 3.) Another nymph of the lot thus prepared was fixed while in a very early stage of the disease, the only sign of indisposition Bacteria' Disease of the Squash-hug. i5G9 on the part of the insect being a slight sluggishness, a touch causing it to respond actively. An examination of sections made frrtm this individual showed the bacteris. in the blood to some extent (see Plate XXYII, Fig 4) but they were apparently quite as abundant in the hypo, dermis. In the adipose tissue there was occasiona,lly found a small colony of the bacteria; but here there was at the time no general and uniform distribution. From these results it was quite impossible to decide whether the blood became infected by the entrance of germs through the spiracles, or whether there was direct penetration of the hypodermis. The former would seem to be most natural, and 1 am inclined to suppose that the fluid of the perivisceral cavity is the seat of first action. On this ground, however, the more marked effect on nymphs, both of squash-bugs and of chinch- bugs would necessarily be explained on a truly physio- logical basis, rather than on the ground of the more penetrable character of the chitinous outer coat. TEMPERATURE EXPERIMENTS. For a further knowledge concerning the conditions of growth of this bacillus and for a guide to any field ap- plications of the organism, a series of temperature tests was planned, by means of which it was hoped to ascer- tain (1) the period of exposure to various temperatures necessary to kill this organism when mature, and (2) the range of optimum, temperature relative to its growth and development. In order to secure a mature growth, fresh slantino- agrar tubes were inoculated, and bv means of the water of evaporation the bacteria were distributed as much as possible. The tubes were kept at about 80° F. for two days, when the growth was abundant over the whole surface, and then the exposures were made as indicated in the table. On last removal from the incuba- tor, other slanting tubes of fresh agar were in a similar way inoculated from these, and the new tubes placed under observation for growth developments. 24— 370 Illinois State Laburatory of Natural History. Table VIII. Bacterial Disease of the Squash-hug. 871 Summarizing, then, it appears that this organism will not survive at 125° F. a continuous exposure of two hours, nor an exposure of one hour on more than one day. At a temperature of 115° F. it will withstand an exposure of one hour for several successive days, but no continuous exposure of three hours or more. It survives in great part an exposure of six hours at 105° F. on four successive days, but with a continuous exposure of four days at this temperature the organism is usually killed. I have not yet had an opportunity to experiment with this disease while keeping the bugs at different tempera- tures; but such a line of work would be interesting to supplement the above temperature tests, and to advance our knowledge as to the relationship of conditions re- quired by host and parasite. Neither have I yet deter- mined precisely the range of ojJtimum temperature for this organism ; but such results as are recorded seem to indicate that it is between 83° and 90° F. SPECIFIC CHARACTERS. I have carefullv compared the biological characters of this squash-bug organism with those enumerated for the various entomogenous bacteria already' described; but there is no form with which it agrees in detail. Its ac- tion on the insect and its characteristic pathogenic properties seem to be quite distinct; hence I have given to it the name Bacillus entomotoxicon, n. sp. This disease bacillus has also been carefully compared with the organism "normal" to the cce^^al appendages of the squash-bug. There is considerable structural dif- ference, and the normal form is cultivated on nutrient media with difficulty. I have secured cultures of the latter on media strongly alkaline; but further details of such work are not included in this paper, as the coecal form is hardly to be confused with the disease organism. 872 Illinois State Laboratory of Natural History. Bacillus entomotoxicon Duggar. Occurrence.—In the blood and tissues of diseased squash- bugs. Morpkology .—'t^\\o\% bacilli 1.2-1.8 ix X0.()-0.8 }x, single or in pairs, motile, not producing spores. Preparations stain well in most of the anilines, the bacilli often staining much more deeply at the poles, consequently showing a banded or belted appearance. Growth and pathogenic charactevK.—An aerobic and fac- ultative anaerobic organism, producing on nutrient agar- agar a dirty white colony often characterized by promi- nent fan-like radiations. Stab cultures on nutrient gela^ tine give liquefaction on the second or third day, soon assuming the shape of an inverted lamp chimnej'', and after standing one month the gelatine is colored wine- red. Milk is rapidly coagulated and the coagulum in great part dissolved, the odor attending this growth on milk being exceedingly vile. Nitrates are not reduced. It grows well at living-room temperature, but is easily killed by exposures to high temperatures. The infected insect becomes sluggish a few hours before death, and at death it is slightly darker and softer. After death the insect is shghtly swollen, darkens rapidly, and soon contains only a mass of gruel-like fluids. Sterile or active infusions from the growth on agar contain a principle toxic for many species of insects, as shown by temporary immersion. SUMMARY. Under the specific description are summarized the leading facts relative to the structure of this organism and to its growth on the usual culture media. It is necessary to add a few brief statements embodying some of the results of general oecological interest. Bacillus entomotoxicon is the cause of a characteristic disease of the squash-bug, first observed as an epidemic anions: bugs in a laboratory breeding-cage. Bacterial Disease of the Squash-hiig. 373 Both laboratory and tield experiments show that the disease is readily communicated to healthy squash-bugs by contact with the fluids of infected insects, nymphs being- more easily affected than adults. Fresh agar cultures of the bacillus are effective as sources of infection. The disease may be communicated to young chinch- bugs either from diseased insects or from cultures; but adult chinch-bugs are strongly resistant. With the grubs and other larvae hitherto experimented upon, external applications of infection material have given no successful results. Infusions from the growth on agar contain an active principle which kills many insects after a very short period of immersion. Sections of diseased squash-bugs show that the bacillus is present in the blood at all stages of the disease. The hypodermis, adipose tissue, and cardiac tissue are also early affected. When death ensues the body fluids are like pure cultures of the disease organism; and accom- panying saprophytic germs are seldom found. ACKNOWLEDGMENTS. To the Director of the State Laboratory of Natural History, Professor S. A. Forbes, I owe many thanks for the opportunity of woi'king upon the disease herein dis- cussed, as it is to him that the Department of Insect Disease Work owes its origin. I would also express my thanks to Mr. W. G. Johnson for valuable assistance with various entomological details. BIBLIOGRAPHICAL The list appended deals only with bacterial or so-called bacterial diseases of insects, and in it are included all works and articles to which I have had access, together with the majority of references definitely given in these 374 Illinois State Laboratory of Natural History. articles. Some papers are listed which really embody no original work, nor even a critical analysis of previous work; but such papers are often valuable to students who have not access to the original sources of publica- tion. Altum.—Ueber den Erfolg der Kiinstlichen Verbreitung der Flacheriebacillen gegen die Nonne in den Herzoglich Ratibor'scheu Bestanden. Zeitschrift fiir Forst-und Jagdwesen, Januar, 1893, p. 21. Balbiani.—Recherches sur les Corpuscules de la pebrine. Journ. d' Anat. de M. Robin, 1866, p. 599, et 1867, pp. 263 et 329. Etudes bacteriologiques sur les Arthropodes. Comptes Rendus de 1' Acad, de !Sci., CIII., 1886, p. 953. Benton, F.—The Honey Bee. Bull. No. 1, New Series, Div. Ent., U. S. Dept. Agr., pp. 112-113, 1895. Bollinger.—Ueber Pilzkrankheiten niederer und hor- erer Thiere, 1880. (Chapter II. of "Zur Aetiologie der Infectionskrankheiteu." Miinchen, 1881.) Chaerel.—Acetrophie ou Gatine des vers a sole, 1857. Cheshire, F. R., and Cheyne, W. W.—The Pathogenic History and History under Cultivation of a New Bacil- lus {B. alvei), the Cause of a Disease of the Hive Bee hitherto known as Foul Brood. Journ. Roy. Micr. Soc, Vol. v., Aug., 1885, pp. 581-601. CiEsiELSKi.—" Foul Brood" of Bees. 1884. (In Polish. See Botanisches Centralblatt, XXVII., 1886, p. 346.) CooK, A. J.—Foul Brood. Bull. No. 61, Mich. Agr. Exper. Station, April, 1890. CusHMAN, S.—Foul Brood. Bull. No. 9, R. I. Agr. Exper. Station, June, 1891. DoRRER.—Dasendeder Nonnencalamitat in Wiirttemberg. Forstwiss. Centralblatt, XV., 1893, pp. 73-89. Bacterial Disease of the Squash-hug. 375 Eckstein.—Das auftreten der Flacherie in der Ge^end von Kberswalde. Zeitsohr. f. Forst- und Jagdwesen, XXV., 1893; und Allg. Holzverkaufsanzeiger, XVIII., 1893, Nr. 22, p. 60. Untersuchuugen iiber die in Raupen vorkom- menden Bakterien. Zeitschr. f. Forst- und Jagdwesen, XXVI., 1894, pp. 3, 228, 285, 413. Fertjy, C. de.—Contribution a 1' etude de la flacherie, causes et traitement. Actes et Memoires du 4me Con- gres Sericicole, Oct., 1874. Kecherches experimentales sur les causes de la flacherie du ver a sole. Comptes Rendus Stenograph- iques, Congres International Sericicole, No. 23, p. 113, Paris, Sept., 1878. Forbes, S. A.—Studies on the Contagious Diseases of In- sects. Bull. 111. State Lab. Nat. Hist., Vol. II., Art. IV., 1886. • x\ Contagious Disease of the European Cabbage Worm, Pleris rapcB, and its Economic Application. Thirteenth Rep. Board of Trustees, Univ. 111., 1886, p. 294. Reports of State Entomologist of Illinois: 12th, 1882; 16th, 1887-88; 17th, 1889-90. Gagnot.—De la Maladie des vers a, soie dite Gatine. Le Commerce Sericicole, 1858. Garm.^n, H.—[Diseases of P/d7Y".s ra/>fle believed to be non- transferable to other species.] Insect Life, Vol. III., Nos. 7 and 8, Apr., 1891, p. 333. Gehren, von.—Bekampfung der Nonne durch Impfung mit dem Hofman 'schen Bacillus. Zeitschrift fur Forst- und Jagdwesen, XXIV., 1892, p. 501. Bekampfung der Nonnenraupen durch Infection mit Baeillen. Forstwiss. Centralblatt, XV., 1893, pp. 343-347. 376 Illinois State Laboratory of Natural History, Goldberg.—Zur Bekampfung der Nonneiiraupe. Leipzig Tageblatt und AUg. Holzverkaufsanzeiger, XVIII., Nr. 27, p. 300, Juli, 1893. Haberlandt, F.—Die seuchenartige Krankheit der Seid- enraupen. Wien, 1866: HoFMANN.—Insektentodtende Pilze mit Besonderer Be- rucksichtigung der "Nonne." Sondbd. aus dem Woch- enblatt fiir Forstwirtschaft "Aus dem Walde." Dez., 1890. Die Schlaffsucht {FlacTi&rie) der Nonne {Liparis monacha) nebst einem Anhang: Vortrag iiber Insek- tentodtende Pilze. Frankfurt, 1891. Jager.—Bakteriologische Untersuchungen von Nonnen- eiern. Aus dem Walde, 1893, Nr. 18, p. 69. Jeanjean.—La Maladie des vers a soio: Conseils aux educateurs. Montpellier, 1862. JoLY.—Sur les maladies des vers a sole, et sur la color- ation des cocons par 1' alimentation du chica. Mem. de r Acad, des Sci., 30 aout, 1858. Klamann.—Ueber die Faulbrut der Bienen. Bienenwirt- schaft. Centralblatt, Hannover, 1888, Nr. 18, u. 19. Der gelbe Faulbrutbacillus {Bacillus flavidus alvei). Bienenwirtschaft Centralblatt, Hannover, 1890, No. 2. Krasilshtshik, I.—La Graphitose et la Septicemie chez les insects, deux maladies des larves des Lamellicornes causees par des Bacteries. Mem. de la Soc. Zool. de France, VI., 1893, 3e pt. pp. 244-285. Lebert.—Ueber die gegenwartig herrschende Krankheit des Insekts der Seide. 1858. — : De la maladie de 1' insects de la soie. Berlin, 1858. Levert.—De la maladie des vers a soie dans F Ardecbe, 1858. Bacterial Disease of the Squash-hu^g . 377 LoRTET.—La Bacterie loquese; traitement de la loque par le naphthol. Kevue Internationale d'agriculture, fevrier, 1890. Macchiati, L.—Lo Streptococcus homhycis e la flaccidezza del baco da seta. Le Stazioui speriment. Agrarie itaU iane; Vol. XXIII, Asti, 1892. Maillot, E.—LeQons sur le vers a sole du Murier. Paris, 1885. Mally, F. W.—Bacteriological Experiments with Insect Diseases. Bull. No. 29, Div. Eat., U. IS. Dept. Agr., 1893. Meritau.—De la Gatine, son origin, les causes qui Tout produite, et son invasion epidemique. 1858. Metschnikov.—Les Maladies des larves du Hauneton du Ble (la muscardine vert et le Bacillus salutarius). Odessa, 1879. (En Russe.) Metz(ier, a., und MiJLLERi N. J. C—Die Nonnenraupe und ihre Bakterien. Miindener forstliche Hefte, I Bei- heft. Berlin, 1895. OsiMO.—Rechercbes sur la maladie actuelle des vers a sole. Padoue, 1859. Pasteur.—Etudes sur la Maladie des vers h. Sole. Tomes I. et II. Paris, 1870. Paulys.—Die Nonne in den Bayrischen Waldungen, 1890. Quatrefages, de.—Etudes sur les maladies actuelles du ver a sole. Mem. de 1' Acad, des Sci. de 1' Institut Im- perial de France, Tome XXX., 1860. NoUvelles Recherches faites en 1859 sur les mala- dies des vers a soie. Rev, et. Mag. Zool., 1860, T. XII., pp. 412-415. Reuter, O. M.—Patogena Bakterier i Landtbrukets Tjanst. Ofvertrvck ur Finska. Vet.-Soc. Ofversigt., H. XXXVI., 1894. Riley, C. V.—The Mulberry Silkworm. Bull. No. 9, Div. Ent., U. S. Dept. Agr., 1888, pp. 32-40. 378 Illinois State Laboratory of Natural History. Robin, Ch.—L' histoire natur. He des vegetaux para- sites. Paris, 1853. Schmidt, A.—Die Nonne {Liparis monacha). Darstellung der Lebensvveise mid BekampfuDg der Nonne nach den neuesten p]rfaliruDgen, u. s. f, Ratibor, 1893. Die Bekampfung der Nonne. Zeitschr. f. Forst- und Jagdwesen, XXV., 1893, pp. 218-222. Simon.—Reisebericht an die Regierung in Potsdam von Juli, 1892, p. 48. Bekampfung der Nonne durch Impfung mit dem Hofmann 'schen Bacillus. Zeitschrift fiir Forst- und Jagdwesen, 1892, p. 499. Snow, F. H.—Contagious Diseases of the Chinch-bug. First Ann. Rep. Director Kan. Agr. Exper. Station. Lawrence, 1892. Tangl, F.—Bakteriologischer Beitrag zur Nonnenrau- penfrage. Forstwiss. Ceutralblatt, XV., pp. 209-230, 1893. TuBEUF, C. VON.—Die Krankheiten der Nonne {Liparis ononacha). Beobachtungen und Untersuchungen beim Auftreten der Nonne in den oberbayerischen Waldun- gen, 1890-1891. Die Krankheiten der Nonne {Liparis monacha). Forstlich-Naturwiss. Zeitschr., Januar, 1892, Heft I., pp. 34-47. Weitere Beobachtungen fiber die Krankheiten der Nonne. Forstlich-Naturwiss. Zeitschr., 1892, Heft I., p. 277. Ueber die Erfolglosigkeit der Nonnenvernichtung durch kiiustliche Bakterien-Infektionen. Forstlich- Naturwiss. Zeitschr., 1893, Heft 3, pp. 113-126. Verson, E.—Etudes sur la flacherie des vers a sole. Journal Sericicole du Dr. Haberlandt, 15 aout, 1869. Wachtl und Kornauth.—Beitrage zur Kentniss der Morphologic, Biologic, und Pathologic der Nonne Bacterial Disease of the Squash-bug. 379 {PsUwa mo7iacha'L.) unci Yersnchsergebnisse ilber den Gebrauchswerth einig'er Mittel zui- Vertilgung der Raupe. Milth. aiis deiii forstlichen Versuchswesen Oes- terreichs. Der ganzen Folge, Heft XVI. Wien, 1893. Walker, Philip.—Grasserie of the Silkworm. Insect Life, Vol. 3, Nos. 11 and 12, Aug., 1891, p. 445. EXPLANATION' OF PLATES. Plate XXVII. Fig. 1. Bacillus entomotoxicon from the blood of a dis- eased squash- bug. Zeiss V12 horn, imm., oc. 4, tube length 205 mm. Fig. 2. Distribution of bacteria in the hypodermis and fatty bodies of a squash-bug at the time of death: