BULLETIN

NATURAL HISTORY.

VOLUME II

Article IV.—Studies on the Contagious Diseases of

Insects.

By S. a. Forbes.
)

1886.

J. W. FRANKS A SONS, PRINTERS AND BINDERS
PEORIA, ILLINOIS.



EKEATA.^^

Page 5, line 3 of table, second column, for 39 read 38 ; line 6, second
column for 121 read 120.

Page U, line 17. for conjunction read conjuyation.

Page 21, line 13, for Ricciaci<v read Ricciacea'.

Page 67, line 17 from bottom, for fraligifolia read fraqilifolia.

Page 123, line 4 from bottom, and page 12(5, line 1, for Tricholeoi read
TrichoGolea\

Page 126, line 2, for Tricholea read Trichocolea.

Page 177, line 16, for Lecythia read Lecythea.

Page 333, line 1, after Tachidius add Lilljeb.

Page 33S, under Daphnella brachyura, line 16, insert ffa6.—Massa-
chusetts (Birge), Minnesota (Herrick).

Page 340, line 5, for Hcapaoleberis read Seapholeberis.

Page 389, line 7 from bottom, for carjiogonium read sporocarp ; lines

9, 12, 15, for ojgonium read carpogonmm.
Page 391, line 1, for Cessatii read Cesatii.

Page 400, line 4, for Myceliumin conspicuous read Mycelium incon-

spicuous ; line 14, for coleosporium read Coleosporitun.

Page 401, line 9, for connatas read connnta; line 12, for Taraxicum
read Taraxacum.

Page 408, line 15, for macrocarpa read macrospora; line 18, for Hy-
pohyllous read Hypophyllous.

Pages 470 and 471, head of column 11, for cyprinella read cypri-

nellus.

Page 503, lines 8, 14, and 17, for cyprinella read cyprinellus.

For additional errata see page 247.



Fig. I

Fig. 2.

Culture of Micrococus from diseased Cabbage Worm {Pieris rapes L.), in test tubes of sterilized beef

broth, commenced October 20, 1883 (see p, 275). Preserved over winter in plugged test tube, and

slides mounted April 10, 1884, in carbolized v?ater, after staining with brown aniline.

Photographed with lamplight by Dr. H. J. Detmers, with Spencer tV homogeneous immersion, X 1000.

Both figures are from different parts of the same slide, differing only in focal adjustment,- figure 1 being

a " positive," and figure 2 a "negative."



DIVISION OF
^^

Article IV.— Studies on the Contagious t)$$eaip?\Q/iMsScts:-^i. .'^/

By S. A. FoBBES. <^!,^j^^^y^^
Since August, 1883, the writer has used such opportunities

as came in his way for observation of the diseases of insects,

and for more or less careful and systematic work upon thera,

directed especially to the point of artificially propagating them
for the destruction of injurious insect species. While these

researches are not by any means completed, lacking especially

critical study of the bacterial forms dealt with, as botanical

species, and imperfect also on the side of field experiments on a

large scale. I have thought them worthy of present report as a

contribution to progress on a difficult but interesting subject,

especially as opportunity for further continuance of some of

these studies may not soon return.

My main object has been experimental and economical, and

I have adopted such methods of study as seemed to me to offer

the simplest means of surely ascertaining whether some of the

common diseases of our insects were of bacterial origin, whether

their germs were readily and conveniently cultivable, and

whether such cultures could be used to convey the original

affections to healthy insects.

This will serve to explain what may seem to some an ex-

cessive reliance on fluid cultures,—much more convenient for

my purpose in these preliminary studies than "solid cultures"

with gelatine films or tubes, and quite conclusive as to the iden-

tity of the forms dealt with, if the cultures are often enough

repeated and the results are closely scanned.

Without attempting at this time to summarize the literature

of the subject,—scattered and chiefly fragmentary, except as

relates to the silkworm and the honey-bee,—I give here only a

brief account of my own earlier notes and observations.

The appearance of what seemed to be an epidemic of con-

tagious disease among the chinch bugs of Central Illinois in the

latter part of the summer of the above year, gave rise to an

article on this subject, published in 1883 in my first report as



258 Illinois State Laboratory of Natural History.

State Entomologist of Illinois (pp. 45-57). This article con-

tained an account of a considerable series of microscopic obser-

vations on the fluids of chinch bugs apparently affected with

disease, and described some successful attempts at the culture of

the Micrococcus found invariably characterizing this insect

affection. Time failed for further experiments, and the chinch

bug has since been so scarce in my vicinity that no further op-

portunity has offered to complete the study of the subject. The

observations made amounted to a practical demonstration of the

occurrence of a " germ disease " in this insect species, iden-

tified the germ as a Micrococcus, since described as Micrococcus

insectorum, Burrill, and proved that this was easily and freely

cultivable in beef broth. The Micrococcus was shown to have

its seat in the alimentary canal of the insects, occurring most

abundantly in the posterior part of the same, to infest pupae and

adults more seriously than the younger stages, and to have the

apparent effect to retard the development of the brood as well

as to destroy a large percentage of them before they reached

maturity. This disease was apparently the representative of

flacherie or schlqfsucht in caterpillars, as described by previous

authors and in the following pages.

Next there appeared early in August, 1883, in our breeding

cages of Datana ministra (the yellow-necked apple caterpillar),

an outbreak of disease characterized by the occurrence, at first

in the alimentary canal and later in the blood, of immense num-
bers of micrococci of a form very different from the above,

and evidently quite readily conveyed from one insect to another.

Elaborate studies of this disease were made during the remain-

der of the season and the following spring, the bacteria associ-

ated with it were repeatedly cultivated with success in animal

infusions, and several experiments were made to convey the dis-

ease by their means to still healthy larvae. Tubes of the cul-

ture fluids were sealed up for preservation over winter, their

contents were cultivated again in June, 1884, and the resulting

cultures were used to infect the food of larvae of Mamestra
picta, with the hope of thus reproducing the original disease of

the Datana larvae of the preceding year.

Parallel with these experiments was a similar series made on

a frightfully contagious and destructive disease of the European



Contagious Diseases of Insects. 259

cabbage worm {Pieris rapce), first observed by us at Normal.

September 11, 18S3. The bacterial character of this disease

was ascertained, many attempts at cultures were made, some of

them successful, and the possibility of conveying the disease to

a distance by means of affected cabbage worms was tested by

us in Western Illinois and Iowa. Many of the observations

and experiments relating to fiacherie in this insect were re-

peated by me in 1884. and in the early summer of 1885 admi-

rable photographs of several of the slides were made for me by

Dr. H. J. Detmers, of Champaign.
" Jaundice " of the silkworm appearing in an experimental

nursery of this species, under the charge of Professor Burrill

at the State Industrial University, at Champaign, in June, 1884,

an opportunity was afforded me to study this affection. Many
successful cultures were made of the bacteria involved, and

several experiments were undertaken for the infection of healthy

cabbage worms with the contagion from these artificial cultures.

Succeeding in the laboratory, these experiments were can-ied

into the field, and attempted on the large scale of actual

practice.

An epidemic of muscardine appearing in certain breeding

cages of the forest tent-caterpillar (CUsiocamjm sylvatica) in

June, 1884, this disease was studied by us as there illustrated,

and connected more or less certainly with a destructive epidemic

of the preceding year, which had swept away vast numbers of

that species under my observation in Southern Illinois.

With the exception of the fiacherie of the chinch bug,

these observations have not hitherto been anywhere fully re-

ported, although brief notices and general accounts of a more

or less popular character have been printed in the scientific

journals and in some economic publications.

The chinch bug observations were published, as already

mentioned, in the Twelfth Report of the State Entomologist

of Illinois, the species of Micrococcus concerned having been

previously described by Prof. Burrill in the Report of the

Trustees of the Illinois Industrial University for 1882, and in

the " American Naturalist " for March, 1883.

A brief preliminary paper on fiacherie of Datana was read

to the entomolosical club of the American Association for the



260 . Illinois State Laboratory of Natural History.

Advancement of Science at its Minneapolis meeting in August,

1883, and of this a synopsis appeared in the " Canadian Ento-

mologist " for September, 1883. In the " Prairie Farmer

"

(Chicago) for October 6. 1883, and in "Science," also, for

October 5, 1883, brief notes occur with reference to this dis-

ease in the cabbage worm.

In the Transactions of the Illinois State Horticultural So-

ciety for 1883 (printed February, 1884) is a somewhat elab-

orate paper on the Contagious Diseases of Caterpillars, read

before this Society December 18, 1883, giving a general and

rather popular account of the character of the work done by me
on this subject, up to that time ; and a still more elabo-

rate paper (never published) was read before the State Nat-

ural History Society of Illinois, at its meeting in Peoria, July

8, 1884, in which a classification of insect diseases was pre-

sented, and a full resume of methods and results, up to that

date, was given. At a meeting of the State Horticultural So-

ciety, held at Champaign, December, 1884, I added some fur-

ther items relating to cultures and experiments, especially those

affecting the cabbage worm, and these notes were published in

April, 1885, in the Transactions for the year preceding.

It is my purpose, in this paper, to present the principal

results of the above studies,— both the successful and the un-

sucessful issues,— the latter so far as they have any significance

or value.

Disregarding the chronological order of my observations, I

shall first discuss fiacherie of the cabbage worm, and jaundice

of the silkworm, with experiments upon the former insect with

the artificial cultures derived from the latter. I will then take

up the longer and more complicated record of flacJierie in our

Datana larvse and the experiments drawn from it, and will con-

clude with a brief account of the muscardine of the forest tent-

caterpillar.

EUROPEAN CABBAGE WORM (Pieris rapce, L.)

In studying experimentally an insect disease, it is necessary,

in the present state of our knowledge, (1) to determine pre-

cisely the symptoms and character of the disease itself, in order

that it may be subsequently recognized with certainty; (2) to



Contagious Diseases of Insects. 261

learn whether it is characterized by bacteria ; and (3) whether

it is practically contagious. Determining these questions affirm-

atively, (4) cultures of the baqteria must be made artificially,

and (5) these cultures must be used to produce, in healthy

insects of the same or other species, a disease characterized by

the symptoms and results of the original affection. It is fur-

ther desirable that (6) second cultures should be prepared

from these cases of disease artificially produced, in order that

a strict comparison may be made of the bacteria concerned, as

they occur both in the bodies of the insects and in artificial cul-

ture fluids.

I propose to take up these points seriatim, (first with

respect to ftacherie in the cabbage worm), presenting separately

the facts bearing upon each, only premising that the proof of

one proposition is sometimes partly contained in the data relat-

ing chiefly to another, so that some repetition will be neces-

sitated by this mode of discussion; but this disadvantage will

doubtless be found insignificant, compared with the gain in

clearness and* cogency.

Description of Flacherie in the Cabbage Worm.

In this insect flacherie is distinguishable with great ease

and certainty by conspicuous external symptoms, the color

alone of affected larvae being, in fact, entirely characteristic and

unmistakable. The natural color of a healthy cabbage worm is

a light lively green, sometimes slightly tinged with yellowish,

but without any approach to an ashy or milky hue. As the

first symptom of ftacherie, however, the larva commences to

turn pale, this paleness increasing more or less rapidly until the

color is almost milky white, only slightly tinged with greenish.

This discoloration is uniform and simple, no other tint usually

appearing until after death. Then, however, the color deepens

to a sooty gray, commonly uniform, but sometimes appearing

first about the center of the length of the larva. Occasionally

this deeper color appears a little before death, but it is not then

of equal depth over the whole surface.

In the actions of the insect there is little to indicate any

change of state^ except a gradually increasing sluggishness,



263 Illinois State Laboratory of Natural History.

slowness of movement, and loss of appetite. These are later to

appear than the pale discoloration above mentioned, and even

shortly before death a larva may show considerable impatience

if rouo^hly handled. When the disease is well developed, the

caterpillar is very feeble, and will remain motionless for a

long time ; or if it attempt to crawl where some strength is

needed, as horizontally on a vertical surface, it may lose its

hold with its jointed limbs and cling only by its central prolegs,

the fore and hinder parts hanging limp and helpless at right

angles to the remainder of the body.

Most commonly an escape of fluid from the vent is among

the earlier symptoms of the affection, at first greenish or whit-

ish, and later a dirty gray, or even a chocolate brown. Rarely

this fluid exudes also from the mouth. The amount of it is

usually sufficient to stain considerably the surfaces over which

the larva crawls: but sometimes this symptom is wholly absent.

Occasionally the stomach is found empty after death, but almost

invariably it is well filled with food, much of which has not yet

lost its native color, digestion being, in fact, evidently sus-

pended during the course of the disease. I have found in only

a single instance an appearance of bubbles of gas in the alimen-

tary canal, such as Pasteur describes in the Jlacherie of the silk-

worm. Usually the mass of the alimentary contents seems to

lie inert in the stomach, undergoing neither digestion nor

decay.

The color of the fluids of the healthy larva is a very pale

transparent green, the blood containing only lymphoid cor-

puscles in greater or lesser number; but if a proleg of a dis-

eased specimen be snipped off, and a cover glass be pressed

against the cut surface, the droplet exuding will be of almost

milky whiteness, or, in the latest stages of the disease, a dirty

gray. Rarely, where there has been much escape of fluid from

the vent, the juices of the larva will be thick and scanty, so that

it requires some pressure to force out a very small quantity. If

a minute droplet of the milky fluid obtained by snipping off a

proleg be examined under a high power of the microscope, it

will found to contain innumerable myriads of very minute

spherules, varying in diameter, according to the individual, from

.b fitolfi' Usually their average size does not surpass . 7 /*. It is



Contagious Diseases of Insects. 263

the infinite multitude of these which gives to the fluids of the

diseased caterpilhir their milky look, and likewise, unquestiona-

bly it is they which cause the ashy appearance of the surface, the

skin being thin and delicate, so that the color of the fluid con-

tents shows through. The diseased blood is so thick with

these minute corpuscles that little else can be ordinarily seen in

it. Sometimes, however^ degenerated lymphoid corpuscles of

the blood will be noticed, recognizable by their size and spherical

contour, but differing from the normal corpuscles in their darker

tint and coarsely and irregularly granular structure. These

darker, granular corpuscles are always dead, no longer exhibit-

ing amceboid movement, and have usually a spherical form. Not

infrequently debris of the fatty bodies is apparent in the form

of large irregular cells, floating freely in the fluid, but these

cells themselves will be found to contain immense numbers of

the minute spheres already 'mentioned. In fact, if a little

portion of the soft remnant of the fatty bodies be removed,

spread upon a cover, and examined with a power of a thousand

diameters, it will be seen that the cells of these organs are the

seat of an extreme degeneration, the entire contents of many of

them being wholly replaced by the spherical granules mentioned

above. Occasionally a cell containing a nucleus will be found,

but more commonly all distinction of contents has disap-

peared.*

* As an example of the condition of the fatty bodies, I will

describe those of a larva examined October 9, whitish in color and
nearly dead, making little effort to escape. A droplet of the blood

exuding from a small cut made in the back was alive with the

minute spherules already mentioned, and contained also noticeable

numbers of dead blood corpuscles in a dark, spherical, granular

condition, together with a few unaltered examples still capable of

amoeboid movement.
A fragment of the fatty bodies examined, consisted chiefly of

pale spherical cells, 1.5// to 7.5/^ in diameter, resembling oil globules,

except that they had not the high refractive index of fat. A few
of these globular cells were very pale and indistinct, the contents

very indefinitely granular and often with a large spherical nucleus

likewise very pale ; but most of them were more or less completely

filled with dancing spherules, slightly different in size in different

cells, these differences having, however, no relation to the proper

size of the cells. Sometimes there were not more than twenty-five

or thirty such granules in an optical section of a large cell, the con-



264 Illinois State Laboratory of Natural History.

If the body of a diseased larva be cut across and a cover

glass be pressed against the cut end of the intestine, or, still

better, if the larva be opened lengthwise, the stomach removed

and laid open separately, so that a droplet of the pure contents

of the alimentary canal may be obtained, the fluid portion of

these contents will be seen to swarm with infinitesimal gran-

ules identical in appearance with those found in the blood, ex-

cept that they are, on an average, often appreciably larger and

are occasionally more or less oval in outline. These same forms

m ay also be found in the fluid excreta escaping from the vent of

the still living larva. If the specimen has been dead some time,

so that the sooty discoloration of the surface has occurred, the

fluids both of the alimentary canal and of the body at large

will often be found to contain, besides myriads of the above

spherules, various other forms clearly recognizable as septic

bacteria,—among these, members of the genus Bacterium, easily

distinguishable by their oval form and by the manner in which

they actively propel themselves across the field of the micro-

scope. Rod-like bacilli may also appear in the fiuids at this

time, equally active, and evidently moving by means of flagella,

especially in the vicinity of the bubbles of air which may be in-

cluded in the fluid under the cover glass. Occasionally these lat-

ter bacterial forms may be found in smaller numbers even before

death, very rarely in the perivisceral fluids, but not very uncom-

monly in the contents of the alimentary canal. Still they are

infinitely less abundant than the Micrococcus-like spheres

already mentioned, even long after the death of the larva.

The most characteristic post mortem phenomenon is the

rapid softening, decay, and deliquescence of the body, the whole

of which may be converted, in an hour or two after death, into

a dirty fluid mass which the rotten skin is barely sufficient to

hold together. This breaks at a touch, allowing the fluid con-

tents to escape.

tents being otherwise fluid. Many of the cells were not full, areas

occuring which the dancing particles did not invade. Occasionally

an unaltered nucleus would be seen in the midst of the corpuscular

contents of the cell. The fat globules intermingled were easily dis-

tinguished from the above cells by their very different refracting

power, and were always free from the spherical granules. They

were less than half as numerous as the pale cells. The average size

of the granules was not far from .66 u.



Contagious Diseases of Insects. 265

The Characteristic Bacteria.

As implied in the foregoing, I have no doubt that a large

percentage, at least, of the minute spherical granules abundant

in the fluids of the body and alimentary canal of the diseased

larvge are genuine bacteria, belonging to the genus Micro-

coccus I cannot hope to convey verbally the same conclusive

conviction of this fact which I have myself derived from long

study of these little forms under a great variety of conditions,

and the preparation and examination of a multitude of slides,

both recent, and permanently mounted. These latter were some-

times unstained, and again stained with a considerable variety

of aniline dyes,—brown, blue, violet, and magenta. Several

successful cultures have also assisted to confirm this view, the

products of the cultures being unmistakably the form originally

taken from the caterpillars under experiment.

In form the micrococci of the cabbage worm are usually

strictly spherical, although in the alimentary canal a patch will

occasionally occur in which they are of a slightly oval outline.

The micrococci of the fluids of the diseased larvae seen in the

field of the microscope are mostly separate spheres, but a con-

siderable percentage of them are attached in pairs, as if in pro-

cess of division. Rarely a short chain of four, six, or eight

may be seen. In the stomach they occur not infrequently in

compact patches or zoogloea-like masses. In size the individuals

vary from .5 ^ to 1.25 fi in diameter, the small forms being

those in the blood and the larger those in the stomach. Indi-

vidual larvaj differ, in fact, with respect to the size of their

micrococci, in some the average of those found in the blood

being not far from .75 ^ tol fi^ while in others they barely reach

.5 fi. Commonly, those of the stomach average 1 /t.

In addition to the direct evidence above adduced, the close

resemblance of these corpuscles to those occuring in other

larvge affected similarly to the cabbage worm, in which the

bacterial character was even less obscure, gave indirect and cu-

mulative evidence with respect to the nature of those forms

in the cabbage worm. Their reaction to the usual staining

fluids was such as the hypothesis of their bacterial character



266 Illinois State Laboratory of Natural History.

would require. Although staining with some difficulty, many
slides were prepared in which the individual bacteria were beau-

tifully stained and distinctly differentiated from an uncolored

film, by brown aniline, methyl violet, and magenta.

Although several of the attempts at artificial culture were

abortive, and although the cultures resulting were sometimes im-

pure and occasionally doubtful, enough cases of unquestionable

success occurred to give full effect to this mode of proof. The

details supporting this statement will be given under another

head. It is worthy of special remark that in no case did the

beef broth in which these cultures were made, although it be-

came densely milky with bacteria, give off the slightest smell of

decomposition. Only a faint, indescribable odor was percepti-

ble, but little different from that of the fresh liquid.

To the above we may add the association of these spherules

with diseased conditions and with post mortem phenomena

which could scarcely be accounted for at all, except on the sup-

position of the bacterial character of these excessively abundant

forms.

The proof of the contagious character of the disease in

question, next to be adduced, must also be taken as indirect,

or, at least, prima facie evidence, in the present state of our

knowledge, of the living organic character of these multi-

tudinous particles,—the only forms present which could in any

way be connected with the disease as agents of the contagion.

Contagious Character of the Disease.

Most of the considerations brought forward in the preced-

ing section apply with some force to the subject of this, for if

the fluids of the diseased and dead larvae swarm with micrococci

so minute (these appearing in the blood long before death),

and if these are shown to escape from the body by way of

the excrement and the fluids exuding from the vent, the pre-

sumption is strong that the disease which they characterize

would be conveyed to healthy individuals by their instrumen-

tality. But we must look for proof of contagion chiefly to the

conditions of the occurrence of the disease, to the phenomena



Contagions Diseases of Insects. 267

of its spread, and especially to the results of experiments for

conveying it artificially to localities or regions where it had not

before appeared.

That this affection, or one very similar to it, attacks the

cabbage worms of the old world, is made likely by a chance

remark in Curtis's ''Farm Insects" (p. 96), where he says of

several larvae of an allied species, Pieris brassicce: "On the

20th they appeared healthy, but inclining rather to a yellow

color: it rained during the night, and on looking at them in the

afternoon of the following day, I saw they had removed to a

leaf, to which they stuck by four of their hinder legs, and, to

my surprise, they were of a dirty color, and rotten, the skins

being lax, and lying just as the wind blew them about. I found

they only contained some cream-colored lluid, a portion of

which was scattered upon the leaves."

In this country the disease seems to have been first noticed

in the vicinity of Washington, in 1879, although little atten-

tion was paid to it, and its bacterial character was not then

ascertained. In Bulletin 3, of the United States Entomological

Commission, (pp. 69, 70), Dr. Riley remarks, while discussing

some experiments made with yeast on the cabbage worm:

"An incident connected with these experiments which I

made is, however, well worthy of being mentioned, because it

shows how very easily single experiments may lead to false

hopes and conclusions. A certain proportion of the last-named

larvte— the proportions differing in the different lots treated—
perished before or while transforming to the chrysalis state.

They became flaccid and discolored, and after death were little

more more than a bag of black putrescent liquid. I should

have at once concluded that the yeast remedy was a success,

had I not experienced the very same kind of mortality in pre-

vious rearing of this larvae, and had I not, upon returning to

the field from which the larvte in question were obtained, found

a large proportion similarly dying there."

Xo other notices of it have occurred in my reading, pre-

vious to those of its appearance in Illinois, already mentioned

(October 5, 1883). That it did not occur at Normal in 1882

is made certain by the fact that the cabbage fields there were

frequently visited in autumn by myself and my assistants dur-



268 Illinois State Laboratory of Natural History.

ing the progress of a series of experiments with insecticides

upon the cabbage worm, and that nothing of the sort was seen

by us.

When first noticed there, its distribution was peculiarly

irregular. In' certain small fields, for example, not one half

mile distant from those in which the disease was raging

violently, affecting one fourth to one half of the worms in

sight, not a single dead larva could be found by very careful

search. A few weeks later (October 4), larvae in these fields

were suffering as severely as the others, 20 per cent, of the

worms, 'on an average, showing signs of illness.

September 27, at Rosehill, near Chicago, I visited fields in

which, although the worms were fairly abundant, I could not

find a single diseased larva during a careful examination

of more than a hundred individuals ; while across a road

and a half mile away, the disease was fully at work in four

adjacent fields, and fully one fourth of the worms had been

attacked. These were in all stages of the disease, many of

them being dead and rotten. The identity of the affection

with that observed at Normal was established by careful micro-

scopic examination.

From correspondents to whom I had described the cabbage

worm mortality at Normal, I received various reports. Prof.

A. J. Cook, of the State Agricultural College of Michigan, wrote

me, October 2, that about 10 per cent, of the cabbage worms

near Lansing were affected by it. On the other hand. Prof.

Lintner, State Entomologist of New York, informed me, No-

vember 3, that it had not been noticed with him. Dr. E. R.

Boardman, in Stark county, sixty miles northwest of Normal,

reported, September 29, that the cabbage worm was there very

destructive, but that no appearance of the disease in question

was discoverable. October 5 he repeated this observation, but

on the 13th of that month he finally found a very few affected

larvae.

D. S. Harris, of Cuba, in Fulton county, nearly south of

Dr. Boardman, first wrote me on the 13th of October that no

disease had appeared among the cabbage worms about his place,

nor at adjacent towns, as he had learned by careful search and

inquiry, but on the 25th of the month he wrote: " That disease



Contagious Diseases of Insects. 269

attacking the cabbage worm has made its appearance in Cuba

at last. On the 21st I found one full-grown worm sick (head

downward), and in about five hours it was dead and decora-

posed, and several others were affected. To-day it is a difficult

matter to find a sound worm on the plants, while the remains

of dead worms are numerous."

From Prof. G. H.French, at Carbondale, and Mr. Frank

Earle, at Cobden, I learned that the disease had not appeared in

Southern Illinois as late as October 29, nor did it occur there

during the season. From Champaign, east of me, Prof. Burrill

wrote me, October 25, that he had not yet seen any of it in his

small garden patch of cabbages, although watching carefully

for it: but that an intelligent student had described it as oc-

curring in fields near the town.

In Iowa, to the westward, it seems not to have occurred

spontaneously that year, the only appearance of it noted by

Prof. Osborn, of the Agricultural College of that State, being

the result of an experiment, the material for which I furnished

him from Xormal. Wherever it once occurred it continued to

prevail throughout the season, as far as our observations Avent.

The facts clearly and positively negatived the supposition

that there was anything in the weather or local conditions to

explain either the presence or the absence of the disease, and

all bore out the hypothesis of a gradual progress from the east

Avestward. The same phenomena of irregular local distribution

were manifest the next year (1884). In certain large fields

almost daily observed, it was impossible to find a single diseased

larva at a time when, half a mile away, the cabbage worms of

small patches had been almost wholly destroyed, their black-

ened bodies, or the shriveled remnants of the same, being scat-

tered everywhere on the leaves.

I may say, incidentally, that the effect of the epidemic in lim-

iting the ravages of the worms, was very evident last year. For

the first time in several seasons large fields of late cabbage were

brought to full maturity without the loss or serious damage of

a head.

From the foregoing the conclusion is unavoidable that all

the circumstances of the natural occurrence and spread of the

disease are consistent with the hypothesis of its contagious

character, and wholly inconsistent with any other.



270 Illinois State Lahoratory of Natural History.

Two attempts were made to convey the contagion by

means of diseased larvae to localities not reached by it,— one

lot being sent October 3, to Dr. Boardmam, at Elmira, and

one to Prof. Osborn, at Ames, Iowa. The experiment of Dr.

Boardman was not wholly satisfactory, for the reason that

through an unfortunate delay of the package the worms which

I sent him did not arrive until October 22, at which time the

disease had appeared spontaneously, in a small way, in his vicin-

ity. Nevertheless he selected, October 23, two lots of twenty-

five worms each, all perfectly healthy to appearance, fed them

regularly, but exposed all of them to the contagion by enclos-

ing them in two boxes with the dead and sick caterpillars which

I had sent him. At the same time he secured ten healthy larvae

in a box by themselves and kept them free from infection. The

latter lot all pupated without accident, but were not followed

further. The first two lots commenced to show symptoms of

disease on the fifth day, and by the eighth day all of both lots

were dead, except three, only one of which finally reached pu-

pation. Even this pupa, in fact, afterwards died and decayed.

By this time, however, the disease was so violently raging in

the open fields that no great value can be attached to this ex-

periment, especially as the fluids were not microscopically

examined.

The material sent Professor Osborn, of Iowa, including

dead and dying worms and a mounted slide of the micrococci,

arrived October 5, and two cabbage heads were at once infected.

On the 7th one of the worms " had evidently succumbed to the

disease." The gathering of the cabbages under observation

during the temporary absence of Prof. Osborn necessarily inter-

fered with the further progress of the experiment, but he col-

lected such worms as he could from the stumps and fed them

in confinement. A number of these larvae died, and December

28 he wrote me that he had " found micrococci in a number of

sick and dead cabbage worms, which must certainly have taken

the disease from the ones sent."

Although these experiments, taken alone, could scarcely

be regarded as conclusive as to the contagious character of

flacherie, taken in connection with the other facts mentioned,

we must at least allow them some weight as cumulative evidence.



Contagious Diseases of Insects. ' 271

Artificial Cultuees of Bacteria.

Methods of Culture.—The modes of culture used in all the

experiments reported in this paper were based, unless otherwise

specified, on those of Klein, as described in his paper " On the

Relation of Pathogenic to Septic Bacteria," in the Journal of

the Royal Microscopical Society for January, 1883, differing

only by modifications which will appear in the description.

The cultures were usually made in beef broth (rarely in in-

fusion of cabbage) in test tubes plugged with sterilized cotton,

or in ordinary flasks similarly closed. The broth was prepared

by boiling lean beef from a half hour to an hour in a

porcelain-lined vessel, and then filtering and carefully neu-

tralizing wdth caustic soda. The tubes, flasks, and cotton

were sterilized by heating in a tin oven, over a gasoline stove,

for several hours, at a temperature of not less than 275 degrees

or more than 300, as determined by a thermometer inserted in

the oven. The heat was sufficient to considerably scorch the

cotton without actually charring it. While still within the hot

oven the tubes and flasks were securely plugged by means of

steel forceps freshly heated in the flame of an alcohol lamp, the

cotton plugs, from two to three inches in depth, being pushed

firmly in. Most frequently the mouths of these plugged

vessels were covered wath a cap of sterilized cotton, held in

place by an inverted beaker also carefully sterilized by dry heat.

In charging the vessels with the fluids the plugs were rapidly

withdrawn and as promptly returned, the infusions being intro-

duced boiling hot and afterwards boiled for several minutes to

destroy any germs which might have entered during the instant

before the plug was replaced. It was not found necessary to

test the sterilization of the tubes by protracted incubation, as

all the check tubes and the stock flasks in which the store of

prepared infusion was preserved, remained unchanged through-

out the entire season. Neither was any incubator required, the

ordinary temperature of the air during the weeks when these

experiments were in progress being never below 60^ by day, and

ranging commonly above 70" both day and night.

When a culture tube or flask was infected with the fluids

of a larva, the following process was invariably used. From



272 Illinois State Laboratory of Natural History.

small glass tubes, about a quarter of an inch in diameter,

pipettes were made in the flame of an alcohol lamp by drawing

out each end to a capillary filament, the tips being closed by

melting at the time of making. To charge these pipettes, the

end of a proleg of a caterpillar was usually cut off with steril-

ized scissors, the point of the capillary tube broken off with for-

ceps just flamed in an alcohol lamp, one of these points pushed

into the cavity of the proleg, and the pipette partially filled by

exhaustion of the air from the other end. To introduce the

droplet of fluid so obtained into the test tube we invariably, re-

moving first the beaker and the cap of cotton contained within

it, carefully forced down through the cotton plug the capillary

tube of the pipette containing the infection material, without

loosening at all the plug itself. Sometimes the tip of the tube

containing the fluid was broken off inside the test tube before

the withdrawal of the pipette, at other times the contents were

carefully forced out with the breath, pains being taken not

wholly to expel the fluid contents of the pipette. After with-

drawal of the tube, the cotton plug was grasped with sterilized

forceps and slightly twisted within the mouth of the test-tube

to close effectually any small opening through the plug which

might have been made by the introduction of the pipette.

After this the cap of cotton and the beaker glass were restored,

and the tube was set aside with a companion precisely like it in

all respects, except that it had not been infected. During the

latter part of our investigations these check tubes were them-

selves operated upon with capillary pipettes, distilled water only

being introduced, at the time that the experimental tube was

infected.

In withdrawing portions of the products of the culture for

inspection a similar process was used, the freshly made pipettes

being introduced as for infection and partially filled by exhaust-

ing the air from the upper end. After withdrawal, the cot-

ton plug was again twisted as already described and the cap

returned. The cover glasses used in the examination and prep-

aration of the material, whether this was derived directly from

the larvae or from artificial cultures, were flamed with an alco-

hol lamp immediately before using, after being thoroughly

cleaned by rubbing with a linen cloth. Slides were similarly



Contagious Diseases of Insects. 273

treated for study with the microscope. A droplet of the fluid

was allowed to flow from the tip of the pipette upon the

cover glass, spread in a thin film by means of the capillary

glass tube, and either placed at once upon the slide for imme-

diate examination, or laid aside under a glass shade to dry.

After drying, if it was desired to stain and permanently mount
the specimen, the cover with the film attached was passed

repeatedly through the flame of an alcohol lamp, covered for

some minutes with a drop of the staining fluid (the glycerine

aniline colors recommended by Prof. T. J. Burrill*), and then

thoroughly washed with distilled water. The covers thus pre-

pared were often mounted in balsam, but most frequently, at

first, in carbolized water in very shallow cells made with white

zinc cement, this cement being also used to fasten the covers

to the slides. For microscopic study of the material, my
principal reliance was a superb xV-inch homogeneous immersion

objective, made to order for the purpose by Herbert R. Spencer

& Company, of Geneva, New York. This objective was used

with Bulloch oculars, giving powers ranging from 500 to 1,450

diameters. Some of the more interesting or difiicult slides were

also studied under a js-inch homogenous immersion of Zeiss.

The measurements here reported were originally made by

means of an eye-piece micrometer so graduated that with the

highest powers used, each space equalled 2 fi^ the micrococci

being commonly measured in doubles and chaplets. Many of

these measurements were verified by repetition with a more

finely divided micrometer, the spaces of which, with a power of

1,000 diameters, had a value of .3 ;tt; but practice with the more

coarsely spaced scale enabled me to measure as accurately with

this as with the other, and with much greater convenience.

The products and results of the fluid cultures were com-

monly so satisfactory that I rarely resorted to solid cultures

upon gelatine films. A few of these were made, however, but

not with the micrococci of the cabbage worm; and they will be

described under the head of the Datana larvae. As I was pri-

marily interested only in the disease and secondarily in the

bacteria, cultures on films were less essential to my purpose
1

* Proceedings Amer. Society of Microscopists, 1883, p. 79.



274 Illinois State Laboratory of Natural History.

than if I had wished to discriminate and describe the various

forms appearing. I depended upon frequent repetition of the

experiments and uniformity of results, rather than upon the

more critically exact cultures and continuous observation of

current methods with gelatine films and masses.

Culture Experiments.—Concerning our first cultures, the

fact should be remembered that one could rarely expect to find

a perfectly pure culture in the body of a diseased insect,

exposed as it is by way of the food ingested to invasion by

bacteria in great variety. I consequently did not find it

remarkable that several of our unquestionably sucessful infec-

tions were not really pure cultures, other bacteria developing

than those most abundant in the original fluids. For example,

in the very first culture made,— one in beef broth begun Sep-

tember 16, when the infection process was very carefully man-

aged without the slightest accident, and when the check tube

remained clear indefinitely,— the culture became turbid the fol-

lowing day, and by October 3, was nearly as yellow as cream,

with a thick yellowish felt on top and an abundant precipitate.

The greater part of the product of this culture consisted of

micrococci like the larger of those of the cabbage worm, the

spherules, in singles and doubles, averaging 1 ^w in diameter; but

the surface film consisted largely of a Saccharomyces embed-

ded in the Microccocus. Individuals of Bacterium also oc-

curred in the slides. The check tube, as already mentioned,

was quite clear to the end.

The second culture was still less conclusive and satisfac-

tory. A cabbage worm which, on the afternoon of the 17th

September, was noticeably paler than its companions, was iso-

lated and watched. At 9 p. m. it seemed a little stupid, but

otherwise unchanged. At 9 a. m. of the 18th, however, it was

dead, blackened, and very soft, the contents evidently little

better than fluid. These fluids contained two micrococci,—one

a larger spherical or slightly quadrate form, 1 ;«. in diameter, and

the other a minute spherule .5 fi to .75 n.

A small flask of rather weak beef infusion was infected

from these fluids in the usual manner, and the next day, the

19th, it was already (^ecidedly milky. Examined, it was found

to contain Bacterium and the larger Micrococcus above men-



Contagious Diseases of Insects. 275

tioned, a slender Bacillus, and another Bacillus-like form, of

which there will be further question hereafter,—a short, broad

form with rounded ends and a paler center,—but nothing re-

sembling the smaller Micrococcus. These organisms were quite

possibly all septic bacteria, derived from the deca,3'ing body of

the caterpillar.

The first unmistakable culture of the Micrococcus of the

cabbage worm was made October 20, in a test tube of beef

broth infected from the blood of a larva about half grown, de-

cidedly pale, but far from dead. The slide representing the

blood of this larva is not stained, but is in good condition.

There were two bacterial forms visible in it,—a spherical Mi-

crococcus .7 fi in diameter, and, very rarely, a slender Bacillus.

The flask in which the culture was made was poured from a

stock flask into a sterilized tube from which the ping of cotton

had just been removed, plugged again, boiled thoroughly about

three minutes and left to cool completely. The blood was

obtained by snip])ing the skin of the back with sterilized

scissors, and drawing up with a fresh pipette a little of the

thick fluid exuding. The tube was infected in the usual man-
ner, and not examined until two days thereafter, when it was

found decidedly turbid, although not extremely so. The Mi-

crococci were strictly spherical, 1 ^ in diameter, very uniform

and abundant, usually in doubles, but often single. The slides

made were excellent and well stained, some violet and some

brown. The bacteria differed from their originals only in being

somewhat larger. Still they were not larger than the Micrococ-

cus of the cabbage worm is often found, especially in the intes-

tines. The check flask remained wholly clear.

Four other successful cultures of this Micrococcus were

made, so similar in all respects to the preceding that it is not

worth while to repeat details. It must be admitted, however,

that the minute blood form did not certainly reappear in its

original size in any of my cultures, if we except one case where

its numbers were relatively so few (about 100 to the field with

a power of 1,000) that it is barely possible that all were intro-

duced in the original infection. This fact is capable of either

one of three interpretations : (1) We may suppose that the

proof is incomplete that these smallest spherules from the blood



276 Illinois State Laboratory of Natural History.

were micrococci at all, notwithstanding their uniform shape,

size, and character, and the fact that they were repeatedly dis-

tinctly stained ; or (2), taking for granted their bacterial

nature, we may suppose them insusceptible of culture under

the conditions supplied; or, finally (3), we may assume that

the conditions of tube culture in beef broth were so different

from those occurring within the blood of the insect as to in-

crease the size or even modify the form of the Micrococcus in

question. In favor of the latter hypothesis we have the fact

of the generally larger size and often slightly oval form of the

micrococci found in the intestinal fluids, as compared with

those in the blood of the same specimen.

These considerations apply, however, only to the minute

blood form, and not at all to the intestinal Micrococcus. This I

have cultivated repeatedly with indubitable success in this

insect, and, still more frequently, forms indistinguishable from

it occurring in other species. I venture to add that the

frequency with which certain bacteria, different from the

infection material, appeared in the test tubes when these were

infected from the cabbage worm, suggested repeatedly the

hypothesis of an alternation of certain forms which were in this

way frequently connected,—a point on which I shall have more

to say when describing the Datana bacteria. Especially was

this true of the larger Micrococcus and of the short, broad

Bacillus (?) with pale center and rounded ends (here called, for

convenience. Bacillus intrapallens) . The latter, I shall presently

show, behaved precisely like a pathogenic form,—giving no

odor of putrefaction in fluids swarming with it, killing insect

larvae whose food was treated with it, and certainly multiplying

for some days within their living bodies.

The late period at which successful cultures of the cabbage

worm Micrococcus were made precluded attempts at artificial

infection by their means, and with respect to this particular

insect this part of the proof is consequently wholly wanting.

When the evidence is given respecting the reproduction of

what wa,s clearly the same disease in other insects, I think that

no reasonable doubt will remain that Jiacherie of the cabbage

worm may be conveyed through artificial cultures of its Micro-

coccus.



Contagious Diseases of Insects. 377

THE SILKWORM (Bomhyx man, L.)

Late in July, 1884, I heard from Professor Burrill, of the

State Industrial University, that a lot of silkworms which

were being reared under his direction for experimental purposes

were dying rapidly from an apparently contagious disease

resembling the flacherie of the old world, and wishing to im-

prove the opportunity thus afforded to determine the possibility

of conveying this affection to our native Lepidoptera, I had,

July 30, some of the dead and dying larvae sent me by mail

from Champaign. From our correspondence at the time and

from an account of the experiment by Prof. Burrill, published

in the Twelfth Report of the Board of Trustees of the Illinois

Industrial University, 1884, we learn that the lot of worms

(about 80,000 in number) in which this disease broke forth

were Raised from eggs derived from a perfectly healthy brood

of the preceding year ; that they commenced to hatch June 21
;

that they were kept in a clean and thoroughly ventilated build-

ing set aside for their use on the University grounds ; that they

began to spin July 25 ; that between this date and the 29th

183 cocoons were produced, but that in consequence of the out-

break of this disease among them only a single additional

cocoon was made during the season. The entire remainder of

the 80,000 worms perished,—commencing to die July 23, and

continuing until the latter part of August.

Desceiption of the Disease.

In a note of July 23, Prof. Burrill says of the affected

larvae that they " become yellow, shorten up; the skins become

very tender so that they can hardly be picked up without burst-

ing; body flaccid; the blood loses its clearness and becomes

thick with a dirty yellowish color.'' Again, July 26, he writes:

" They first refuse food and uneasily creep around, then become

yellowish and flabby."

In the article above cited. Prof. Burrill distinguishes two

forms of disease among the larvge, as follows:

" In one case the affected larvae became restless, ceased

eating, the skin assumed a decidedly yellowish tint and ulti-



278 Illinois State Laboratory of Natural History.

mately became very tender and easily ruptured, while the blood,

unusually copious, was thin and yellow instead of its normal

limpid or grayish color. Other larvae became slug.gish, con-

tinued to eat, but consumed only a small quantity of food, the

body gradually became flaccid, the skin wrinkled and tough,

and the color a grayish or leaden tint, and finally nearly black.

These, hours or even one or two days before their death,

adhered by their prolegs, or some of them, to a support, and

remained quiet, at length only showing signs of vitality when

touched, and at last dying while still firmly anchored to the

limb or other object upon which they rested. After, and for

some time before, death, the flaccid body hung directly downward

from the point of attachment. If this latter happened to be

near the middle of the body, the two ends hung down, the parts

nearly parallel with each other. From these dead and black-

ened worms a decided and characteristic odor of putrescence

was perceptible, tainting, when numerous, the air of the well-

ventilated room."

The first of these diseases was also characterized in the

Statistical Record of the State Board of Agriculture for Au-

gust, 1885, by Mr. Woodworth. who conducted the experi-

ment for Prof. Burrill. " This disease," he says, '' does not

make its appearance until the worms are about ready to

spin, that is, near the end of the last age. The body of the

affected worms assumes a somewhat granular, yellow color,

instead of the natural, bright semi-transparent hue. This

change of color also differs from the normal change, in that

the yellow is first on the middle of the segment instead of at

the ends. The skin becomes soft and tender, breaking at the

least fall, and allowing the yellow body fluid to escape more

readily than wounds of equal size would in healthy worms.

The affected worms become very restless, crawling about and

shrinking in size from loss of blood until they finally die. A
few spins cocoons, which are generally soft, often bright

orange, and sometimes so thin that the pupa or dead worm may
be seen within. Some of the worms even pupate without spin-

ning, and from these pupae moths may emerge, which will

sometimes deposit their eggs. When a brood of worms is

attacked by this disease generally very few survive."



Contagious Diseases of Insects. 279

Several lots of the larvae were sent me in July and August,

representing both the above-described affections, the difference

between which was easily discernible. The former disease was

apparently that known to the French as Jaunes (sometimes

calbd jaundice by the English writers and by some consid-

ered the same as grasserie), and the latter was unquestionably

fiacherie or mortS'Jiats of the Fi'ench — the schlaffsucht of the

Germans.

The yellow color of the "jaundiced" worms was evidently

due to the tint of the blood, and this, again, was as clearly

derived from the great numbers of peculiar cellular bodies with

which the blood was always loaded, these originating chiefly,

if not wholly, in the fatty bodies, as a result of that form

of degeneration of those organs in the larva which attends

pupation. These bodies, when entire, consisted usually of

masses of spheres, each 4 ;U or 5 ^ in diameter, the aggregate

attaining a diameter of 30 /a — 40 fx.. The individual spheres

often presented a slightly angular outline, as if modified by

mutual pressure, and they took no aniline color with which I

tried to stain them. These bodies are evidently the mulberry

cells and granules of Viallanes, as described in his admirable

memoir on the histolysis of insects.* That they originated

chiefly in the fatty bodies, I demonstrated by finding masses of

them in portions of the fatty bodies themselves and by deter-

mining the substantially unaltered condition of all the other

tissues of the affected silkworms.

In the blood of these larvae no bacteria were found, as a

general rule, although Professor Burrill occasionally recognized

a Bacillus in it ; but in the alimentary canal I never failed to

discover great numbers of micrococci and often also numerous

examples of Bacterium and Bacillus.f These bacterial forms

* Ann. Sci. Nat, Zool., xiv. 1,—Art. 1. August, 1882.

t A transverse section of a jaundiced larva mounted in balsam

without staining, shows great numbers of spherical micrococci, some-

what unevenly distributed throughout the entire thickness of the wall

of the intestine, and fully as abundant in the outer portion of this wall

as within. The same micrococci occur in the perivisceral spaces^

being accumulated especially upon the free surface of the organs con-

tained therein. A very few are apparent also in the sections of the

fatty bodies, and occasionally in the muscles, but none occur in the skin



280 Illinois State Laboratory of Natural History.

were not different from those observ^ed in eases of undoubted

flacherie, but they were usually far less auinerous,—afact which

has suggested to me the following theoretical explanation of

the supposed jaundice of the silkworms at the University.

Assuming that the mortality was originally caused by the

intestinal bacteria, we may suppose that this infection was not

sufficiently overwhelming to destroy life by direct action, as

seems to be the case in flacherie, but that it nevertheless had

the effect to so disturb the balance of physiological functions

as to retard the development and preparation for pupation of

some of the organs, while the fatty bodies, being special stores of

material accumulated for use in pupation, and so less promptly

and easily affected by causes attacking the general health of

the larva, went on to pupation and experienced the histolysis

characteristic of that phenomenon. In other words, we may

suppose, quite consistently with all the facts, that a relatively

slight bacterial attack took uneven effect on the various parts

of the animal and not immediately destructive effect on any
;

that it retarded the preparations for pupation of the great

vital organs, but that the fatty bodies, as if unaware of this

fact, continued their course of maturation and histolysis, reach-

ing a condition of pupal disorganization before pupation had

actually occurred.

The condition of the fatty bodies of the larva affected by

the supposed jaundice is well illustrated by slide 4732 of our

collections, containing portions of the fatty bodies of larvae

received from Prof. Burrill on the 30th July. The cells of

these organs, when examined under a power of 500 diameters,

were found, nearly all of them, to have undergone a remarkable

change. The contents of a few still remained minutely granu-

lar, a large nucleus being also occasionally visible, but the Con-

or in the silk tubes. These micrococci are very distinctly visible, shin-

ing with a recldisli light when slightly out of focus, not being rendered

transparent by the mounting medium as are the tissues of the larva.

They are arranged in patches and strings, the former of irregular shape,

the latter sometimes containing as many as eight or ten spherules. The
fatty bodies of this larva are almost solid masses of mulberry granules.

The Malpighian tubules of another specimen show also, besides their

normal crystalline contents, great numbers of these mulberry granules,

formed within the cells or derived from outside sou^rces.



Contagions Diseases of Insects. 381

tents of the greater number had been converted into very

distinct pale granules, varying in size in the different cells from

2 n in diameter to 4 u or 5 /t. About 20 or 25 of the larger size

were usually contained in a single cell, and a multitude—too

numerous to count—of the smaller ones. Here and there in

the area of the object were large irregular lacunae evidently

filled with liquid fat, as shown by the slightly crystalline

character of their contents.

Whatever we may assume with respect to the bacteria

infesting these worms as a cause of the premature pupal degen-

eration, I do not know that we have any reason to suppose that

they are the only possible cause of such a catastrophe to the

insect. Other influences tending to disturb seriously the

balance of functions at the critical period when larval life is

about to terminate in pupation might not impossibly have

the same effect.

Additional details respecting this peculiar catastrophe to

maturing larvaa will be given further on, under the head of

Mamestra picta.

The Characteristic Bacteria.

As an illustration of some of the conditions characteristic

of this disease, I give descriptions of well-mounted slides pre-

pared from the fluids of one of the larvae received from Prof.

Burrill on the 30th July. The larva was dead when exam-

ined, but perfectly fresh. In the blood I found only the mul-

berry granules, some free and others still enclosed in their

mother cells, as already described, together with blood cor-

puscles in various stages of degeneration. My notes at the

time and a recent examination of carefully prepared slides

show that no bacteria occurred in the blood.

In slide 4603, material for which was obtained by touching

a cover glass to the cut end of a divided worm, I find great

nuuibers of the mulberry granules, varying in size from 2.5 /*

or 3 /* to 6 iit, the more usual diameter being, however, 4.5 (i to

5 fji. With these occurred, everywhere, myriads of micrococci,

probably one fifth of the area of the field of the microscope

being occupied by them where the film is of moderate thick-

ness. These micrococci vary in form from exact spheres,



282 Illinois State Laboratory of Natural History.

usually in doubles, to broad ovals, with the transverse diame-

ter about three fourths the longitudinal, these likewise usually in

doubles. Occasionally pairs of doubles are joined end to end

in four's, but longer chains than these were not observed. The

micrococci frequently occurred upon the slide in patches of

fifty to one hundred, in which most of the individuals were

seemingly single. The ovals above mentioned have the same

transverse diameter as the spheres, differing only in length.

This diameter varies but little from .75 /t, although slightly

smaller singles are not infrequently found. Many of these

small, as well as larger, singles are scattered separately through

the field. Besides the ovals above described, occasional ovals

larger than these are seen, closely resembling, in fact, Bacterium

termo, and probably to be considered as belonging to that genus.

These are about 1.5 ft. in length (doubles 3 ;tt) by 1 |tt in trans-

verse diameter.

In the thicker part of the film very considerable numbers

of excessively minute spherules were discernible, deeply stained,

.5 w in diameter, apparently identical with those described under

Pieris rapes, on a preceding page*, and clearly the same as those

appearing in the culture described on page 286.

The slide from which the above description is taken was

deeply stained with methyl violet July 30, and mounted in

dammar.

Another slide, 4612, derived from the same lot of worms

and similarly treated, differs only in the fact that the micro-

cocci average somewhat smaller; that nearly every one is almost

strictly spherical; and that an occasional small Bacillus occurs,

2 /* to 3 |U in length by about .66 fi in width. The ends are

broadly rounded, the sides parallel, except in the shorter speci-

mens where they are slightly convex. These bacilli are some-

times single, more commonly attached eudwise in pairs. The

smaller oval forms, possibly distinct, frequently show a pale cen-

ter with ends heavily stained.f In this slide are a considerable

*Is it perhaps possible that the silkworm affection had its excit-

ing cause in the disease of the cabbage worm, which made its first ap-

pearance in this region the year before ?

t To this form a peculiar interest attaches in some of my other

studies, reported on a later page.



Contagions Diseases of Insects. 883

number of large, regularly elliptical bodies, about 5 /* in length

by 3 ;U in transverse diameter. As they do not stain, they are

probably crystalline, especially as it is well known that larvae

about to moult or pupate often have the blood loaded with crys-

tals of uric acid of which the form is often not different from

that here noted.

As characteristic of the second form of disease, flacherie^

that distinguishable in the living larvae by the pale color of the

surface as compared with the lemon-yellow of jaunes, I have

selected slide 4727. derived from the fluids of a freshly dead

larva. In the blood of this specimen no bacteria were discern-

ible, but in this slide, prepared from the mingled blood and

alimentary fluids, they occur in innumerable myriads. The
slides are, however, instantly distinguishable from those derived

from the yellow-skinned larvae, by the complete absence of the

mulberry granules. The bacteria from the selected slide are

not by any means so uniform as those in the one previously

described, but vary from perfectly spherical micrococci to ovals,

double ovals, and elongate bacillar rods. The spherical and

oval forms of micrococci are, however, the predominant bac-

teria. The spheres in this slide are commonly wider than the

ovals, measuring about .75 fi, while the smaller ovals are not

more than .5 ft in their short",er diameter. The spheres vary in

arrangement from singles to chains of considerable length, but

the latter aggregates may be due to an accidental running

together in the drying film. The bacilli are not distinguishably

different from those described for the other form of disease.

Besides the above, occasional larger broad ovals appear, similar

to those doubtfully determined above as Bacillus intrapallens.

Judging, in short, from this representative slide, one would

say that the bacteria of Jiacherie of the silkworm consist of a

varied mixture of round and oval micrococci of different sizes,

of species of bacteria, and of small bacilli. Some of them, how-

ever, may have been of pout mortem origin. The slide in ques-

tion is beautifully stained with methyl violet, and mounted in

dammar.



284 Illinois State Laboratory of Natural History.

Contagious Character of the Diseases.

I had no opportunity to observe the progress of these

diseases in the silkworm, but Professor Burrill was entirely

confident of their contagious character as exhibited under

his observation. On this point he says *
:
" That the worms

came from good eggs, and were, for a considerable time, per-

fectly healthy and wholly free from the malady which finally

overtook them, we have the best of evidence. The disease

which carried them off was not hereditary. It was not lurking

unobserved during the more favorable weather in the living or

dying worms. Its introduction occurred about, and probably

at, the time of the first heavy rains spoken of, but we con-

fidently know that it could have been artificially introduced

without the rains or the wet weather at all. Moreover, the

worms continued to die after the weather cleared up, and after

every precaution had been taken to put them under the best

possible conditions. We constructed new racks in a room not

previously used, picked out the healthiest worms and moved
them to the new and clean quarters, where, afterward, the tem-

perature and other conditions were as favorable as could be

desired ; but the ravages of the disease continued with no

perceptible abatement. To further test the matter, other

apparently healthy worms, voracious feeders, growing rapidly,

were put out upon the open hedge, where they were watched

from daylight until dark to keep ofE the birds, and where, for

a time, they seemed to thrive under the favorable skies and

wide isolation ; but here, too, they gradually fell victims to the

destroyer. In each of these places about five hundred worms
were placed, from which, as was before said, one cocoon only

was secured, and this from the out-of-door lot. The latter did

live longer than any of the others, but at length as surely suc-

cumbed. Another experiment proved equally futile ; viz. that

of spraying the food with an aqueous solution of carbolic acid.

No apparent improvement followed this treatment.

It maybe said that our disaster followed in consequence of

retarding too long the hatching of the eggs by keeping them in

* Twelfth Rep. ol the Board of Trustees of the Illinois Industrial

University, pp. 90, 91.



Contagious Diseases of Insects. 285

an ice-house, thus pushing the feeding season out of the

natural time and subjecting the worms to unfavorable summer
heat, or providing them with leaves too far advanced towards

maturity. This might, indeed, seem plausible had not several

other lots, fed in the vicinity, but not so retarded, died in the

same way. It is interesting to note that in some of these small

and isolated experiments in silkworm feeding, certain lots from

the same kind of eggs as our own, produced from the same lot

of moths, fed on the same kind of food, remained perfectly

healthy and produced good cocoons, while others totally failed.

It seemed that in every case where what appeared to be the

disease called in this paper fincherie became once introduced,

few or none of the worms lived to spin passably good cocoons.

Most of them died after the third or fourth moults, and after,

therefore, no little care had been bestowed upon them."

My own observations on this phase of the subject were of

an experimental character, and will be found in detail under the

head of Experiments for Artificial Infection. Here I need

only say that they demonstrated the possibility of affecting with

disease healthy larvae of the common cabbage butterfly ( Pieris

rapip) by means of artificial cultures of the bacteria occurring

in the sick silkworms,—these cultures being made in beef broth

and applied to. the cabbage worms in confinement by sprinkling

or spraying their food.

Artificial Cultures.

Our first cultures of the bacteria of the silkworm were

made July 30, in test tubes of beef broth, by the methods

described above, in my account of the cabbage worm disease,

the material for infection having been obtained from a yellow-

skinned larva (affected by jaundice^ received on the same date

from Professor Burrill, of Champaign. The larva used was

recently dead, but still perfectly fresh. Two cultures were

made, one from the blood and one from the alimentary fluids.

Xo bacteria were discernible in the blood, either in fresh

preparations or in mounted films, the latter presenting only

numerous and excellent examples of the mulberry cells and

granules characteristic of the disease. The slides prepared from

the fluids of the alimentary canal, however, exhibit numerous



286 Illinois State Laboratory of Natural History.

specimens of a strictly spherical Micrococcus, occurring usually

in doubles, measuring 1 /j- in diameter, with an occasional oval

example apparently elongating for division, and then about

1.5 fi in length. These micrococci stained readily with methyl

violet.

In the test tube infected from the blood, curiously enough

this Micrococcus reappeared in a perfectly pure culture. The

fluid, infected July 30, was seen to be milky on the 1st of

August, and many micrococci were visible in doubles and

chains, the latter being unusually abundant. On the prepared

slides, less heavily stained than the originals from the silkworm,

these micrococci measured a little less than those of the

alimentary canal, the diameter usually falling between .75 ft

and 1 fi, rarely attaining the latter dimension. Chains of six

or eight were not uncommon.

The culture derived from the alimentary canal of this larva

was unexpectedly impure and not altogether comprehensible.

The fluid was observed to be milky August 1, and many

micrococci appeared in fresh slides, both in doubles and chains.

A perfect film, distinctly stained, but rather pale, shows, how-

ever, a variety of forms. Most conspicuous, but not the most

abundant, are doubles and short chains of three to six of a

strictly spherical Micrococcus, deeply stained, entirely similar

to those above described, but averaging smaller, their mean

diameter being a scant .75 jw. Besides these are short, broad

ovals, a little less deeply colored than the above, of the same

transverse diameter, but a fair 1 ^ in length, some, indeed, fall-

ing scarcely short of 1.25 ju,. In addition to these and of the

same transverse diameter, we see, rarely, rod-like forms, appa-

rently bacilli, measuring from 3 ^^ to 4 /« in length ; and,

finally, thickly scattered, everywhere more abundant than any

oval form, are very minute spherules, always in singles (except

in now and then an instance seemingly accidental), measuring a

scant .5 i^ in diameter. These are well stained and conspicuous,

and unquestionably do not belong to the film. They are

extremely like the smaller form of cabbage-worm micrococci

which I have already described. Their appearance under the

circumstances suggests the possibility of their being bacillar

spores, but the bacilli in the film are far too few to permit this



Contagions Diseases of Insects. 287

explanation ; nor did any of those noticed seem to be spore-

bearing. The impurity of this culture makes the supposition

plausible that some of the bacteria of the orignal infection

were introduced by accident and not derived from the silk-

worm. Tlie check tube, however, remained unaltered, as usual;

and it seems to me more likely that the originals of all these

forms were really derived from the alimentary canal. It is not

to be supposed that the alimentary contents of a larva long

diseased, and, indeed, actually dead, should remain wholly free

from invasion by bacteria other than those strictly characteris-

tic of its disease.

The cultivation of bacteria from the blood, although none

were microscopically demonstrable in the latter itself, seems to

me not a remarkable phenomenon (especially as the fluid was

derived from a dead larva), since it could scarcely be credible

that the circulatory fluids shcmld, under such circumstances,

be entirely free from the peculiar germs of the disease to which

the larva had succumbed. It must be remembered that a single

individual Micrococcus would be sufficient to start the culture

in the tube, and that the quantity introduced into the beef

broth was much greater than that represented by the films

microscopically examined. Furthermore, an occasional Micro-

coccus in a stained film may readily be overlooked or passed as

doubtful, since the difficulty of distinguishing single individuals

from accidental granulations of the film itself forbids positive

identification of the micrococci unless they occur in numbers
sufficient to make their character unmistakable.

Another culture, commenced July 30, from the silkworm

4603, the bacteria from which were described under this number
on page 281, was examined August 1, at which time the fluid

was observed to be milky and found swarming with micrococci

and a few examples of Bacterium (?). (The latter, it will

be remembered, were also observed in the original material.)

The resultant culture was possibly impure, the two forms appear-

ing on the slides being distinguished, however, only by the posi-

tive strong stain of one and the very delicate stain of the other,

shapes and sizes not being appreciably differeni .* That distinctly

*Those lightly stained were probably the empty walls of dead
examples.



288 Illinois State Laboratory of Natural History.

stained unquestionably agreed in every particular with the com-

mon spherical Micrococcus of the original silkworm material,

except that it measured a trifle smaller, scarcely averaging 1 jU,

although many individuals and doubles were fully that size.

This culture was preserved for experiment and used as an infec-

tion fluid on the 9th August. The results of this attempted

infection will appear under another head.

Still another culture, commenced and examined upon the

same dates, yielded an abundance of the spherical Micrococcus

most frequently mentioned above, together with occasional

examples of a Bacillus 3 jw or 4 /* in length and about 1 /* in

transverse diameter. These last were, however, too rare to have

any special significance, except as a slight adulteration of the

culture.

The next culture attempted, commenced July 31 and

examined August 4, is of especial interest, as it resulted in the

complete displacement of the normal Micrococcus of the silk-

worm by another organism present in its fluids (the question-

able Bacillus intrapallens already mentioned*), but in small

num bers.

This culture was made from a silkworm of the original lot

received from Professor Burrill, July 30, the beef infusion being

infected from a dead worm. The fluids of this larva contained

vast numbers of the ordinary silkworm Micrococcus, somewhat

under the usual size, averaging, indeed, only about .75 /*. An
occasional large Bacillus, 4.5 ^ long and T /* wide, also occurs

on the slides made from this individual. Besides the above

is the organism already mentioned, varying in form from a

broad oval to a Bacillus - like rod, characterized by a pale

center staining little or none, and heavily stained extremities.

The culture examined August 4 contained vast numbers of this

organism and apparently nothing else. Most of those appear-

ing in the films from this culture were much smaller than the

original, all the stages, in fact, appearing, from a simple sphere

scarcely, if at all, distinguishable from a Micrococcus, to the

* This organism displaced similar cultures made from the larva of

Batana angusi presently to be reported on, was preserved through the

winter, cultivated the following season, and then applied effectively to

the destruction of larvse of other species.



Contagions Diseases of Insects. 289

rod-like form or double elongate oval, the paler centers com-

mencing to appear in the oval and becoming more conspicuous

as this elongates.

A single somewhat later culture, commenced August 4, did

not differ materially in results from those preceding. No
bacteria were discoverable in the blood of the larva, used by

prolonged and careful search, but the alimentary fluid contained

the usual Micrococcus. Five days later the infected infusion

was decidedly turbid, but without either film or sediment.

Besides an occasional short Bacillus in active movement, it con-

tained only the spherical Micrococcus of the usual size.

The slides of these various cultures clearly demonstrate the

presence of a spherical Micrococcus, varying in diameter from

.75 jtt to 1 |tt, as the characteristic Bacterium of the disease from

which these silkworms were perishing, and likewise the prac-

ticability of artificially cultivating this Micrococcus in neutral-

ized beef broth by infections from the alimentary canal and

from the blood. Although the Micrococcus itself was not

demonstrable in the blood by the microscope, it was obtained

therefrom by cultures in which it appeared without admixture

of other forms. Intestinal cultures were, however, liable to

contamination by other bacteria but doubtfully connected with

the disease, among which was the form last described.

Infection Experiments.

I found it by no means easy to provide means for testing

satisfactorily the possibility of conveying the disease of the silk-

worm above described to other demonstrably healthy insects.

The late period of the occurrence of the disease under my obser-

vation made it impossible to use other lots of the silkworm

itself in the experiment, and no other lepidopterous larva

was sufficiently abundant at the time, except the cabbage

worm. This, however, had alread been found, the previous

year, to suffer extensively from an extremely destructive dis-

ease of its own, and although at the time the experimental

stage of my studies of the sick silkworms had been reached, no

evidence of disease among the cabbage worms had yet appeared

in the fields^ I had every reason to anticipate its outbreak among

them,—a fact which made me very doubtful of really bringing



290 Illinois State Laboratory of Natural History.

the matter to a decisive test on that species. The occurrence

of a spontaneous outbreak of the common cabbage worm
flacherie among the lot under experiment, would of course

arrest the progress of the experiment, and might even so mask

the result as to mislead.

This accident, in fact, occurred to my first two experiments,

begun August 9 and 10. Not only did the cabbage worm
affection appear in both the experimental breeding cages and

the checks, but the latter lot as well as the former gave evi-

dence of infection from our silkworm material. The latter fact

convinced me that my arrangements were inadequate for the

protection of my check lots against accidental infection with

the experimental material. These lots were placed at a distance

from those purposely exposed to disease, but in another part of

the same large hall, and were attended by the same assistant.

In previous experiments, not yet detailed, with other larvse, I

had already had evidence of slight unintentional infection of

the check lots by this too close association with those under

treatment, and now arranged another experiment on a wholly

different plan.

Careful examination was made of all the cabbage fields near

Normal, and one was selected which showed no trace of the

proper disease of the cabbage worm. From this field two lots

of caterpillars were selected, twenty-five in each, those for

experiment by the assistant whose duty it was to make the infec-

tions, and the check lot, by an intelligent student of the Normal

school, who did not visit my zoological laboratory at all. The

first lot was brought to the ofiice and placed in a clean and dis-

infected cage in the usual place, but the second or check lot was

taken by the student mentioned directly to his own home and

confined in a new breeding cage. Care was taken that both lots

should be fed and treated alike, except for the infection, but no

opportunity was given for any communication between them.

The results in this case were more satisfactory, and confirmed

my suspicion that our check lots had not before been sufficiently

isolated.

History of the Infected Lot.—The food of the twenty-five

cabbage worms selected especially for experiment, was sprayed on

the 6th September with beef broth infected nearly a month pre-



Contaf/ioti!^ Diseases of Insects. 291

viously from the fluids of a silkworm recently dead from jaun-

dice. Unfortunately, from some oversight, neither slides nor

detailed notes were made of this culture until the experiment

upon the cabbage worms was instituted. The beef broth,

nearly a gallon in quantity, contained in a large receiver, the

tube of which was closed with a sterilized cotton plug nearly

six inches in length, had promptly become turbid, as usual, and

was soon opaque with bacteria. By the 6th September the

development of the bacteria had apparently nearly ceased, a

thick deposit covering the bottom of the jar. The fluids at

this time contained vast numbers of spherical micrococci .7 (* to

.8 fi in diameter, mostly in doubles, apparently identical with

those occurring in the silkworm. The culture, however, which

had been several times opened for examination, was not at this

time wholly pure, but contained likewise bacteria and large and

small bacilli. These occurred, however, in relatively insignifi-

cant numbers, and the fluids when poured out presented no

odor of putrefaction, but had, on the contrary, only the faint

indescribable smell characteristic of the cultures of all our

insect bacteria.

After infection on September 6. the cabbage worms were

fed with fresh food collected for them daily. Their cage was

kept in a large room, before an open south window, was

thoroughly cleaned each day, the paper covering the floor of

the cage being removed and burned, all the litter and dehris

destroyed, and the larva3 carefully transferred to fresh food

upon clean paper.

A single individual died September 8, evidently from

accidental injury. Three of the larvee pupated on the 10th.

On the 11th two died, apparently of disease. The fluids of

these were carefully examined and found to swarm with

micrococci. Of these covers were prepared in the usual form.

The first slide, made from the blood, contains large spherical

micrococci, nearly all in doubles, 1 ^ in diameter, excellently

stained with violet. The bacteria of the second slide, repre-

senting the contents of the alimentary canal, were more various

in form. In addition to tlie al)ove large Micrococcus, 1 jU in

diameter, many slightly double ovals of about the same trans-

verse diameter occurred, together with several .7 ^ wide, most



292 Tllinois State Lnhoratorj/ of Nattirat Ttistorj/.

commonly arranged in small groups; occasionally, also, an

unsegmented rod, possibly Bacillus. Nothing representing the

minute spherical micrococci characteristic of the native disease

of the cabbage worm occurred in this specimen. The next day,

September 12, one larva pupated and four perished. The first of

these examined was already blackened and deliquescent. It con-

tained nothing but large and small micrococci strictly spherical

in form, the large one 1 ju. in diameter, the other about .6 or .7 /i.

Both occurred usually in doubles, but not unfrequently in

singles or short chains. Both stained well in methyl violet,

and good slides were prepared. The smaller form of the above

micrococci was found only in the blood, and the larger only in

the intestine, as indicated by the stained slides from these two

sources,

The second larva studied was soft and grayish green, but

the skin was tougher than usual, and showed little tendency to

the characteristic deliquescence of the cabbage worm disease.

The fluids were yellowish white, and contained great numbers

of large and small spherical micrococci, the larger 1 /tt in

diameter, the smaller .6 or .7 (*.

The third specimen, smaller than the preceding one, was a

little darker in color, the fluids yellowish green and containing

identical micrococci. Both forms were spherical and of the

same dimensions as those just described. A single Bacillus was

also noted, 2.5 fi in length, and an occasional double oval occurs

upon the slides (probably Bacterium) each oval element about

.8 fi long.

The fourth specimen was flaccid, but bright green, its

fluids thick and milky white. It contained a moderate number

of large spherical micrococci, identical in appearance with those

described above, varying in character from .8 ;u. to 1 ft. Besides

these, the blood was literally loaded with large spheres, evi-

dently mulberry granules, occurring singly and in masses, the

diameter varying from 2 to 4 /it. A close correspondence in

the condition of this larva to that of the silkworm affected with

jaundice will at once be noted.

Four other larvae, two of which died September 13 and two

on the day following, were briefly examined, but not carefully

studied. Their fluids presented no considerable differences from



Contagious Diseases of Insects. 293

those already treated. On the 15th another larva pupated, and

a seeoncl died during the night which had been reported slug-

gish the previous day. The body was shrunken, not very soft,

a little brown, but the general color was still the usual green.

The fluids of the specimen were very white and thick, aud con-

tained vast numbers of mulberry granules, both singly and in

clusters, together with great quantities of oval micrococci (some

in chaplets of four) and occasional individuals of Bacterium,

some of the latter in actual luotion. The mulberry granules

were strictly spherical, and varied in size from 1.5 ju. to 3 ^u in

diameter.

Another larva which died was originally paler than

natural, but not white. Before examination it bad blackened

and turned very soft, but was not deliquescent. Slides pre-

pared from it contained dehris of tissues, muscular and other,

and vast numbers of minute spherical micrococci from .5 f*. to

.7 A* in diameter. No flagellar motion was detected in the fresh

slides, and no other forms are apparent in the stained mounts.

Another example, small and shrunken, a little discolored,

dried up in a few hours, and became hard and brittle. It was

not especially studied. On the 17tli of the month the last

remaining larva died. It was not discolored, and I could find

no bacteria in the blood or other fluids. The cause of its death,

in fact, was not apparent. At this date a blackened pupa from

the cage, evidently not long dead, was found full of a blackish

fluid, which contained vast numbers of a small spherical

Micrococcus (.6 ;i* in average diameter, commonly in doubles)

and nothing else, except occasional mulberry granules 2 ^a in

average diameter. Of the individuals which puj^ated, six

emerged successfully, three were deformed, and two failed to

complete their transformations.

History of the CJieck Lot.— This lot, placed in a new breed-

ing cage September 10 with fresh cabbage, was kept under

continued observation until the 28th. One of the specimens

died the first day from an accidental injury; one pupated on

the 12th; and two others were necessarily crushed in opening

the cage, having commenced to pupate on its sliding glass front.

On the 14th four examples pupated, and two more upon the

15th, at which time fifteen healthy larva? remained. The more



294 Illinois State Lahoratonj of Natural History.

rapid pupation of these specimens will be noticed, as compared

with those treated with the infection material,— a fact

consistent with what I have uniformly observed with regard to

the effect of these diseases.

On the 17th four worms were drowned in a dish of water

containing the food plant in the breeding cage. The fluids of

these worms were carefully examined with a microscope, and

careful studies were made of stained covers of their blood and

alimentary contents, but no possible bacteria of any sort were

detected in them. On the 21st three more larva pupated, and

on the 23d three died. Unfortunately; the latter fact was not

reported by the assistant in charge in time to permit an exam-

ination of these dead worms. All the remaining larvae pupated,

the images commencing to emerge on the 26th.

Although the results of the foregoing experiments were

somewhat less definite than might be desired, yet they clearly

indicate the transference of the disease affecting the silkworm

to healthy larvas of Pieris rapm. It would perhaps have

been difficult to establish by a study of the bacteria alone any

marked difference between the disease resulting from this

experiment and that native to the cabbage worm, but the

symptoms of the two diseases were so unlike as to make

it impossible to confound them. The general absence of the

peculiar discoloration of the common fiacherie of the cabbage

worm, and of that rapid post mortem deliquescence even more

characteristic of it, leave no doubt as to the actual difference

between this induced disease and the spontaneous affection.

That the artificial disease was identical with that of the silk-

worm, differing only in such a degree as was to be expected

when attacking such widely different larvee, is rendered

probable, not only by all the attending circumstances, but also

by the occurrence in the cabbage worm of the myriads of

mulberry granules characteristic of the affection in the silk-

worm. This fact is especially significant, since in all our

numerous examinations of the native Jiacherie of the cabbage

worm this condition of the fluids was not once observed.

I followed this experiment with a similar one in the field,

applying the same fluid to a number of cabbages infected by



Contagious Diseases of Insects. 295

the worms aud selecting others as a check on those treated, but

the appearance in this fiekl, at al^out this time, of the common
Jiacherie of the cabbage worm, and the death, from this cause, of

several of both lots of larvae interrupted the experiment. The
general outbreak, also, of the same spontaneous affection of the

Pieris larva? elsewhere in the vicinity, precluded all attempts

at a repetition of these field experiments.

THE YELLOW -NECKED APPLE CATERPILLAR.
(Datana ministra, Drury.)

On this species my first studies of the bacterial diseases of

caterpillars were made in the autumn of 1883. The affection

which attracted my attention broke out in our breeding room

shortly after the larvie were collected, but was not seen among
the species anywhere in the field. It probably was not differ-

ent from the disease well known to entomologists who rear

caterpillars to the imago, especially liable to appear in close and

sultry weather, and when the breeding cages are insufficiently

ventilated.

A lot of the larva?, two or three hundred in number,

obtained July 23, was reported to me, August 1, to have been

mysteriously dying for several days at the rate of two or three

a day. The small room in which they were kept was open to

the south by a large window, and breeding cages of ample size

were used, so placed as to be well ventilated. The larva3 were

fed and the cages cleaned daily.

Description of the Disease.

Esce])t that no change of color was usually perceptible, the

symptoms of this disease were not especially different from

those which have been already given for the silkworm and

cabbage worm. Sluggishness aud evident weakness and loss of

appetite were the first noticeable phenomena. A larva while

resting upon a vertical surface would often partly lose its hold,

and hang only by a few of the legs,— this occurring long before

the power of active locomotion was lost. As a very common
thing a discharge of a brownish fluid from the vent occurred

early in the disease, but occasionally this symptom was not



296 Illinois State Laboratory of Natural History.

observed. As a consequence of this purging, the body would

become soft and flaccid and somewhat shrunken,—an appear-

ance not presented by those in Avhich the purging did not

occur. Occasionally some portion of the body, usually the

central or posterior part, became darker before death, but much

more commonly the larva retained its natural hue. The

approach of death was gradual, the affected insect becoming

more and more sluggish and insensible to irritation. Post

mortem changes were neither so rapid nor so extreme as in the

cabbage worm, owing probably, in part, to the thicker and

tougher skin.

The fluids escaping from the vent were microscopically

examined, and found always swarming with bacteria,— many

of them not infrequently having the flagellate motion of

Bacterium proper, but the greater number of them being

clearly Micrococcus. If a droplet of the blood were obtained

before death, it rarely gave any evidence of bacterial affection,

the only cases in which this was seen being those in which an

ante-mortem blackening of the body was observed. After death,

however, the blood invariably swarmed with the same bacterial

forms which were found earlier in the intestine, the ordinary

septic species soon developing rapidly. The alimentary canal

usually contained, both before and after death, vast numbers of

Micrococcus, and also, not infrequently, true Bacterium, but

bacilli or other bacterial forms were rarely found. The micro-

cocci occurring were not by any means as uniform as in the

cabbage worm and silkworm, both spherical and oval species of

various sizes often appearing on the same slides. The intestine

was commonly filled with food little, if at all, digested. In only

one instance was the alimentary canal empty and partly filled

with gas.

The Charactekistic Bacteria.

The bacteria which, from their abundance and uniform

presence, must be regarded as characteristic of this affection,

occurring as they did in the still living larvae almost to the

exclusion of other forms, Avere oval and spherical micrococci,

—

sometimes one, sometimes the other, and sometimes both com-

mingled in variable proportions. The oval micrococci were



CoHfa(/ioi(s Diseases of Insects. 297

usually in singles and doubles, the spherical ones commonly in

doubles and short chains of four to six ; in the latter case,

often taking on a quadrate form. The ovals varied in length

from 1 fi to 1.4 fi, and in transverse diameter from .8 /» to 1 m-

The spherical and quadrate forms were nearly always under

1 fi in diameter, usually averaging about .8 i^. Both forms

stained readily with both methyl violet and brown, and occurred

frequently in patches or colonies in the intestinal canal.

T mention here a point of especial interest in relation to

subsequent atteuipts at culture and infection. I studied on the

morning of the 5th August the fluids of a larva which had died

during the night. The blood obtained by snipping a proleg

was thick and gray with bacteria, as were also the intestinal

fluids, many in both blood and alimentary canal having the

form and flagellate movement of Bacterium. Occasionally a

string of four, attached end to end, would be seen in serpentine

movement across the field. Well-stained and permanently

mounted slides of their fluids show three bacterial forms: one

large oval, undoubtedly Bacterium termo; one a smaller oval

(the Micrococcus already described); and the third a somewhat

peculiar oval form which might be understood as a single oval

1.5 ju long, with a pale center, or as a short double oval whose

division was indicated, not by indentations of its margins, but

by a thinning of its central part. The study of slides subse-

quentl}^ made under other circumstances enables me to say that

this form last mentioned is really a developing Bacillus of a

peculiar character which, matured, is short, broad, and quadrate,

its central portion pale when stained, and the ends contrasting

by a positively darker tint. Unable to identify this form with

anything described, or to obtain through my botanical friends

any specific determination of it, I shall refer to it in this paper,

merely for convenience sake, under the provisional name of

Bacillus intrapallens.^

*I do not know that this is a distinct species, or intend so to

imply. Bacillus suhtilis sometimes presents the peculiar segregation

of its contents here described, under what peculiarities of circum-

stance I do not know, but never, as far as I have observed or can
learn, until the full size of the cell has been reached. In the above
Bacillus, on the other hand, it was usually evident as soon as the

young cell was large enough to show it.



298 Illinois State Lahonifori/ of Natural History.

Contagious Chaeactee of the Disease.

I made no effort to determine experimentally the question

of the contagious character of this disease in Datana ministra,

and can only report that it gradually invaded all the breeding

cages of this and an allied species, Datana angusi, which we

found during the season. Many of these were kept at a dis-

tance from those suffering from the disease, either as reserve

or check lots, with the hope of protecting them from its oper-

ations ; but as they were, at farthest, in adjacent rooms, and as

we passed freely from one to the other, none of them can be

said to have been isolated. The bacteria appearing in the

walnut Datana (D. angusi) were not different from those

infesting the other species, except that in our observations the

spherical form was usually the characteristic one for this

species. Still, both spherical and oval micrococci were noted

in a multitude of instances.

Artificial Cultures.

Our first culture illustrating this disease was commenced
September 6, 1883, with material obtained from an example of

Datana angusi seriously affected, but not yet dead. The slide

made from the fluids of this larva is not by any means pure. It

shows in nearly equal quantities the spherical and oval micro-

cocci described in the preceding section, the oval form mostly

in doubles, each pair varying from 2.5 ^w to 3 ^a in length, and

being .75 ^t, in transverse diameter. The spherules were mostly

in doubles (the pairs somewhat under 2 ;tt in length) and in

chains of four or more, the elements of which were sometimes

quadrate. Many of both ovals and spheres were aggregated

in large, dense patches. Very rarely, also, occurred a larger

form, not measured, apparently a Bacterium.

Sterilized and neutralized infusion of beef was infected

with fluids from this larva, by the methods and with all the

precautions already described. This infusion speedily became

milky, and slides made a few days after the culture was begun
show clearly a reproduction of the spherical Micrococcus of the

original fluid, but of no other form. In size, general appear-

ance, and reaction to staining fluids, this differed in no par-



Contaf/ioHs Diseases of Insects. 299

ticnlar from the original. Singles occurred occasionally, but

most of the specimens were in doubles, no chains being noticed.

Additional slides, mounted October 2, show likewise the

same spherical Micrococcus without admixture, or change in

size or mode of aggregation ; and still another series mounted

from the same tubes, April 9, 1884, represent a still pure cul-

ture of what was probably this same Micrococcus. The speci-

mens differ only by the somewhat smaller size, rarely surpassing

.8 ^,— a difference jn'obably to be accounted for by an exhaus-

tion of the nutritive fluids, certain to have occurred during the

seven months which had elapsed since the culture was begun.

It should be said, also, that the slides of this last stage are less

distinctly stained thau the ])receding, the micrococci very prob-

ably being dead.

After a careful re-examination of these materials I do not

doubt that this was a successful culture of the spherical Micro-

coccus, preserved through the winter, practically unaltered, in

a test tube plugged with cotton. It should be added that the

check tube remained throughout unchanged.

An interesting culture was begun September 8, the

material Ijeing obtained from a larva of Datana luinistra dead

several hours. The slides representing this larva are impure,

the fluids from the alimentary canal containing not only

spherical micrococci, but also a few ovals, and great numbers of

bacilli. The spherical micrococci range in diameter from 1 fi

to 1.25 jit, and are occasionally indistinctly quadrate, especially

when occurring in chaplets (as they frequently do). A few

doubles measure 3 |tt. The bacilli are all slender, varying

greatly in length (from 3 ^u, to 5 ft), but all .7 t^ in transverse

diameter.

The beef broth infected with this material on the 8th

September was observed on the 15th to have become slightly

milky, and, examined, was found to contain micrococci in

couples and chaplets, chiefly arranged in the latter form. The

slides made from this culture contain no bacilli, but only

spherical or subquadrate micrococci in doubles and strijigs.

These average a scant micro in diameter, some, however, reach-

ing 1.25 fi. October 2 these fluids were found to contain only

the same Micrococcus, not distinguishable in any way from



300 Illinois State Lahoyatorij of Natural History.

those on the slide already described ; and even on April 24 of the

following year, the test tube, which had been preserved over

winter, yielded only the same Micrococcus, as shown by well-

stained and mounted slides prepared at that time. Magnified

1400 diameters and carefully measured, the single spherules

vary from 1 /t to 1.25 ^ti in diameter.

From the foregoing I infer a verification of the experiment

just reported, by a second successful culture of the spherical

Micrococcus of the Datana larva and its preservation, uncon-

taminated, until the following year.

The only gelatine film cultures made with this material

were begun September 8. Six films of solid beef gelatine,

touched with a needle point dipped into the fluids of a larva

of Datana ministra and inverted over a deep cell containing a

droplet of distilled water to prevent drying out, exhibited Sep-

tember 10 a rapid growth of the infection,—each, originally a

mere point, being now about the diameter of a pin head, and

some having penetrated upwards the thickness of the film.

The growth of this mass was in the form of thick finger-like

processes, extending upwards through the gelatine film,—the

marginal increase however being uniform and continuous.

When warmed, these gelatine-film bacteria took on the flagel-

late motion of Bacillus, and the stained slides made from them

strongly indicate that they are young individuals of Bacillus

intrapallens.

Infection Experiments.

A few experiments with cultivated material were made
upon other Datana larvae obtained from time to time out of

doors, these being divided into experimental and check lots, and

the food of the former treated with infusions containing the

cultivated bacteria. These were among our first experiments,

and the control cages were evidently imperfectly isolated. As
a consequence, the experiments were brought to naught by the

appearance of flacherie in all the cages with which we had to

do. In each instance, however, the mortality was more imme-
diate, and at first much greater, among the lots treated with the

bacterial cultures than among those not purposely infected
;

but the results arrived at are not insisted on, and no detailed

account of these experiments is deemed advisable.



Contagious Piseases of insects. 301

THE WALNUT CATERPILLAR.
{Datana atic/usi, G. & R.)

I have to report under this species a series of observations,

cultures, and experiments, the longest which I attempted.

Although these failed, in part, of their original purpose, they

brought out incidental and unintended results of considerable

interest, and seem to me worthy of somewhat detailed description.

On the 14:th of August, 1883, a lot of the larva3 of Datana

angusi were collected from a black walnut tree (Juglans nigra)

in the university grounds at Normal, and brought to the office

for experiment. Seven of these were placed in a breeding cage

in the further end of the Laboratory, somewhat removed from

all the other experimental lots. On the 30th of August one of

these was found dead in the cage, having certainly perished since

the preceding day. The body of this individual was very limp and

flaccid and considerably shrunken, and no food occurred in the

alimentary canal. Mounted slides of the blood show vast

numbers of the short, broad Bacillus, with rounded or sub-

truncate ends and pale central area, which I have distinguished

as Bacillus vntrapallens. The blood was, in fact, a nearly or

quite pure culture of this organism, only some smaller and

apparently undeveloped forms being possibly micrococci, but

more probably the above Bacillus in its earlier stages. These

bacilli measured upon an average 1.25 i^ by 2.5 /«, and occurred

singly and in doubles, the doubles with truncate oppostd ends

and broadly rounded free extremities. Besides the above, the

intestinal contents presented spherical micrococci, usually

single, but occasionally in process of division, .8 ^ to 1 /it in

diameter. I strongly suspect that these apparent micrococci

also were the above Bacillus intrapallens, undeveloped.

The next morning a second larva of this lot was found

dead, having apparently succumbed several hours previously.

The intestinal fluids contained a great variety of bacteria,

including Bacterium, and multitudes of minute spherical

micrococci; but no slides or precise descriptions were prepared.

On September 2 another larva died which had been ailing

for two or three days. But very few bacteria were found in

the blood, while the intestinal fluids were full of double ovals,



302 Illinois State Lahoratory of Natural History.

not flagellate. Mounted slides show numerous spherical or

slightly quadrate micrococci, with many single and double ovals.

The spherical form is .75 a*' to 1 jtt in diameter, some of the

single ovals attaining a length of 1.5 i*. The usual length of

the latter is, however, about 1.25 ju..

Another larva of this lot died during the night of the

3d September, and was examined on the following morning.

Its intestinal contents were brown and nearly solid, requiring

to be moistened for examination. They were noted as "full of

single and double micrococci," but the slide prepared is so

excessively poor that nothing satisfactory can be determined

from it.

From this last larva a culture was made as follows : On
September 1 freshly prepared strong beef broth was filtered,

while hot, through sterilized filter paper into a four-ounce flask

which had just been heated for an hour in an oven at 275°-300°

Fahrenheit. This was stopped at once with a three-inch plug

of raw cotton, freshly sterilized by several hours' heating as

above, and was boiled with the plug inserted. This flask was left

undisturbed until the 4th September, when it remained perfectly

clear. It was then boiled five minutes without removing the

plug and left to cool. A particle of the alimentary contents of

the above larva, about as large as the head of a pin, was now
taken up on the point of a recently heated needle. The plug

of the flask was removed, the infection material introduced, and

the flask plugged again with fresh sterilized cotton still hot

from the oven. A check flask was set aside at the same time.

On the 5th September the fluid was evidently turbid

throughout, but especially so at the edges, and a slight film was

apparent upon the surface. The plug was loosened, and a

droplet of the fluid was obtained upon a freshly heated glass

rod. The mounted slide of this material was, unfortunately,

worthless, but, from notes made at the time, it appears that the

bacteria occurring were rather large " double ovals," nearly

all motionless, but with an occasional flagellate individual.

Compared with the original infection material, there was no
question of the identity of the two.

On the 6th Septeml)er these fluids were milky, and a film

had formed on the glass at the edges, where the fiuid had a



Contagious Diseases of inserts. 30S

somewhat ropy appearance when shaken. The check flask was

perfectly clear. On September 8 the infected infusion was

covered with a thick white surface-scum and the whole mass of

the fluid was strongly turbid. A droplet of the liquid contents

was now drawn out for examination, with a freshly-made capil-

lary tube pushed down through the plug. The thin film upon

the slide was milky with bacteria, which presented, under the

microscope, an appearance of double ovals with occasional

small clusters or patches of the same object, and occasional

strings of three. No other form was seen among myriads

passed under the eye, and no flagellate motion was detected ; this

was, consequently, an unmistakably pure culture of this single

organism. Admirable slides of this material, prepared at this

time, further illustrate the purity of the culture, and show that

many, perhaps all, of the so-called " double ovals " of my notes

were immature Bacillus intrapallens^ in most of which the pale

center was but just beginning to show. On the 13th of Sep-

tember a number of additional slides were made from this same

flask, the contents of which were now extremely turbid, the

lower half thick with a whitish sediment, and the surface and

the flask about the edges covered with a scum. These slides

contain only the above Bacillus, somewhat increased in size, and

showing the characteristic pale center more distinctly. Con-

sidering the frequency with which this form occurred in the

dead Datana larvae of this lot, I have no doubt that this was a

successful culture of this particular Bacillus.

On the 17th September these fluids were selected for an

experiment intended to test the possibility of preserving

throughout the winter the bacteria contained in them, and a

number of films were spread upon glass slides previously steril-

ized by heating, dried immediately with moderate warmth, and

laid away for preservation. At the same time small glass

tubing was taken, heated thoroughly in the flame of a lamp,

and divided by melting, while still almost red hot, into short

tubes closed at both ends. As soon as cooled, these partially

exhausted tubes were first filled with the bacterial culture by

breaking ofE beneath the fluid, with sterilized forceps, the tip

of the tube, which then filled by atmospheric pressure; and

were then immediately re-sealed by heat and laid away in



304 illinnis State Laboratory of N'aturai History.

cotton for the winter. Several of them were opened in the

spring and summer of 1884, at various dates, and found always

to contain only a pure culture of the original Bacillus, the

results of the first examination, made April 4, not differing in

this respect in any particular from the last, made July 30.

These bacteria stained much less freely than those in the fresh

culture, — a fact probably to be accounted for by their dormant

condition. Occasionally a spherical or subquadrate form, 1 fi

to 1.25 ^, is distinguishable in the field by a deeper stain,

—

possibly a spore of the preceding.

Next came a culture in beef broth made by the usual method

from the contents of these tubes on the 23d of June, 1884.

Two days later this was slightly turbid, decidedly so on the 26th,

and on the 27th, when slides were made and the material was

used for an infection experiment, they were almost milky. The

contained bacteria now consisted of two forms: that frequently

mentioned above as Bacillus intrapallens., and a spherical form

indistinguishable from rather large micrococci. The bacilli

occurred singly, doubly, and in strings, were 1 fi by 3 ;u

in typical specimens, but varied considerably, especially in

transverse diameter, reaching sometimes a width of 1.5 f^. The

spherules, on the other hand, averaged about 1 ;W in transverse

dimension. These occurred in various arrangement, but

especially in long chaplets. Many of them presented a slightly

quadrate outline and in a great number of instances strings of

these were continuous with shorter filaments of the bacilli.

Occasionally I satisfied myself that two or three of these

spherical forms were contained within the Bacillus cells; that

they were probably, indeed, to be considered as spores of the

cells or, as seems to me more consistent with the facts, as an

alternate form of the Bacillus. They seemed not to be devel-

oped by the transformation of the contents of an entire Bacillus

filament, but rather to be separated off from the end of such a

filament by a transformation of the protoplasm in the thickened

ends of the cells.

Numerous other cultures were made from this same

material. One commenced July 30 was found, August 1, to be

decidedly turbid, and on the 2d to have formed a thin trans-

parent pellicle over the whole surface. On the 3d this tube



Contagious Diseases of Insects. 305

was opened. The fluid was covered with a rather thick til in

made up wholly of the above BaciUus intrapaUens.^ as deter-

mined at the time and as shown by beautifully stained and well-

mounted slides which I have studied recently. Many of these

were in long filaments, but none showed any sign whatever of

flagellate motion. This culture, like the preceding, was subse-

quently used for an infection experiment.

Similar cultures from the same material were made April

21 and 24, three tubes being inoculated on the latter date.

From all these was obtained the same bacillar form, having

occasionally associated with it the sphericals already mentioned,

and in a single instance containing also a small Micrococcus

about .5 itt in diameter.

The general results of these cultures unquestionably estab-

lish the possibility of preserving through several months the

bacterial form here dealt with, and afterwards cultivating it

successfully in beef broth.

I have next to describe the infection experiments with this

Bacillus, showing the possibility of instituting disease in healthy

larvie by means of it, and of procuring its multiplication within

their bodies for some days subsequent to the infection.

THE ZEBRA CATERPILLAR.

{Mamestra picta, Harris.)

A small colony of zebra caterpillars found on cabbage near

Bloomington was brought to the Laboratory June 1, for infec-

tion experiments with one of the above cultures,— that begun

June 23 and found to contain the BaciUus intrapallens and

the spherical Micrococcus, as detailed above. A quantity

of this fluid was poured into a dish June 27, and a single

cabbage leaf was soaked in it for an hour and then fed to the

larvae. These ate freely of it, and were thereafter fed daily

with fresh cabbage and carefully attended, this first infection,

being the only one purposely made. A check lot of the same

brood was placed in a separate cage, but unfortunately removed

only a few yards from that infected.

On the next day a single larva of the first lot was found

aluiost dead, and, being isolated, died during the night.



306 Illinois State Lahoratorij of Natural History.

Examined June 29, at nine o'clock a. m., the fluid obtained by

snipping off a proleg was found swarming witb large bacilli,

motionless at first, but beginning to move actively in all direc-

tions when exposed to; the air under the cover. These bacilli

measured from 2.5 ^u to 5 ;it in length, one apparently undivided

reaching a length of 8 ;tt, with a transverse diameter of 1.5 fi.

These presented no appearance of spores; the ends were broadly

rounded, the sides parallel. Small numbers of micrococci

occurred in the same slides, about .7 /* in diameter, strictly

spherical, in singles and doubles. An examination of carefully

stained slides leaves]little room for question of the identity of

these bacilli with some of those introduced with the food, but

the interval Vasj tooj short ^ to make it certain that they had

multiplied since ingestion. Their occurrence, however, in such

vast numbers in the blood so soon after death, makes it very

unlikely that they merely represented an escape of the intes-

tinal fluids, especially as we shall soon see that the same bacilli

occurred abundantly in the blood of larvse not yet dead. The

intestinal contents were full of the above Bacillus and the

usual Micrococcus, 1 ,«. in diameter, in singles, doubles, and

patches. The food contents were partially digested.

Besides the above bacteria, the blood was yellow with

masses of cells with granular contents, many with a large

nucleus each. These cells were apparently derived from the

fatty bodies, which seemed to be in process of disorganization,

but differed from the usual mulberry bodies which result from

pupal histolysis, by the fact that there was no appearance of

the division of the cell contents into mulberry granules.

Another larva observed this day, June 29, evidently torpid

and apparently sick, seemed to have moulted imperfectly, frag-

ments of the skin still clinging to the shrunken posterior seg-

ments. The body was flaccid, but not discolored. A proleg

being snipped off, no flow of blood followed, but the fluid pressed

out contained a moderate number of the above bacilli, no

micrococci, but many well-defined mulberry cells and

granules. Each of the cells contained from ten to fifteen

or twenty of the latter. The alimentary contents contained

micrococci with an occasional Bacillus, but none of the mul-

berry granules, both forms of bacteria being in this larva much



Contagions Diseases of Insects. 307

less abundant than was usually the case with individuals so

seriously affected. The epithelial cells of the intestine con-

tained granular masses, seemingly of the micrococci, and the

fluid bathing them was thick with the same objects. Occasion-

ally patches or clusters of the micrococci occurred in contact

with the food. The stained and mounted slides of the blood

show chiefly mulberry granules, spherical or somewhat angular

in outline, 1.5 /t to 3 ^ in diameter. A small number of

spherical micrococci also occurred, many of them minute,

ranging from .6 fi to .8 fi. These appear in all the usual forms

of aggregation, including doubles, short chaplets, and patches

of considerable size. Bacilli also occasionally occur, with

parallel sides and rounded ends, from 1.25 fn to 1.5 ^ in trans-

verse diameter, and from 3 /* to 4 ^ in length. A single BariUus

intrapallens was noticed in process of development, measuring

1.75 fi hj 2 ft.

On the 30th June still another larva died, the grayish

fluids of which contained immense numbers of the spherical

micrococci, single and double, with vast quantities of the bacilli

above described,— motionless at first, but soon, near the edges

of the cover or in the vicinity of a bubble, commencing active

flagellate movements. The body of the next larva to die, (July

1,) was flaccid, and contained little fluid. Immense numbers

of spherical micrococci, 1 /i* in diameter, occurred in the blood,

mostly in doubles, together with many ovals about 1.5 a* long.

Neither Bacterium nor Bacillus were detected in this specimen.

On the 2d, a caterpillar, evidently diseased, shrunken, and

shortened, but with colors yet bright, was found lying upon

the floor of the cage, able to right itself when turned over, but

making no effort to escape. Blood from a foot of this larva

contained a great number of unsegmented cells, similar to blood

corspucles, but of variable size and shape, some with and some

without nuclei. A few hours later, when the blood was

examined again, besides these cells were found a considerable

number of segmented bodies and mulberry cells, the latter

evidently due to dissolution of the former. The next day

this segmentation of the cells in question had gone still farther

in this larva, and very many mulljerry cells were distinguish-

able, together with others but partly segmented.



308 Illinois State Lahoratoyy of Natiiml History.

Now killing the larva, I found the fluids full of mulberry

cells and granules, together, with a great number of spherical

micrococci,—so determined by staining coagulated films.

On the evening of July 1 a number of larvae in this cage

were curiously affected, the prolegs, except the anal pair,

being enlarged and swollen, with a slight reddish discoloration.

These larvae were evidently greatly annoyed by their condition,

and dragged themselves clumsily about as if half paralyzed.

One was seen to turn violently upon itself, and bite the

swollen prolegs, as if in pain, so that the blood flowed from

them freely. On the following morning one of these cater-

pillars was crawling about with the abdomen twisted and the

prolegs turned almost upwards. Carefully snipping one of

these swollen legs, I found in the blood an extraordinary

number of lymphoid corpuscles, and a very considerable number

of mulberry cells, but little, if any, larger than corpuscles of the

blood, varying from circular to oval in optical section.

Frequently a nucleus was visible in the midst of the mul-

berry granules, but no cell walls were distinguishable. The

unstable character of the segmentation of these cells was

unexpectedly demonstrated by the effect of a little carbolized

water run under the cover. As a consequence, the segmentation

entirely disappeared, the mulberry cells being all re-converted

into simple nucleated corpuscles with granular contents. In

fact, I happened to witness this retrogression of a mulberry

cell,— a mass of distinct granules with a nucleus dimly seen

among them, converted, with a curious internal commotion,

into a common lymphoid corpuscle, of rather large size, with

clearly distinguishable nucleus. In this condition the cells

were indistinguishable from dead blood corpuscles. No bacteria

were visible in these fluids.

On the 3d July one of these larvae died. The body con-

tained but little fluid, but this was loaded with cells, some

unsegraented nucleated sphericals of various sizes, without

trace of cell wall, staining deeply with aniline; and others

well-developed mulberry cells, but so similar to the forego-

ing as to have been apparently derived from them. On the

mounted slides of this material are also great numbers of sepa-

rate mulberry granules and the usual spherical micrococci, the



Confagiovs Diseases of Insects. 309

latter averaging 1 ;it in diameter, with an occasional Bacillus

like those already several times mentioned. Micrococci and

bacilli Avere, however, less abundant in these fluids than is com-

monly the case with larvas destroyed by bacterial disease.

In a peculiar larva which died July 2, a small specimen

that had scarcel}^ grown since it was first placed in the cage, a

few micrococci were found, and a considerable quantity of the

mulberry granules, although this individual caterpillar must

have been far from the pupal stage of development.

In another larva examined at the same time, likewise

dwarfed, although larger than the preceding, the blood was

gray with the usual Micrococcus, both free and in masses, and

contained likewise great numbers of mulberry cells and gran-

ules. On the 12th July a larva died in whose blood no bac-

teria were detected, save a few of the usual bacilli. Its fluids

contained, however, an immense number of mulberry cells and

granules.

From the 12th to the 14th July eight more larvas died in

this lot with symptoms and microscopic characters like those

already described,— the body usually somewhat shrunken and

flaccid and the colors unchanged. The blood was occasionally

gray with micrococci, but more commonly differed in appear-

ance from that of healthy larvte, only by the slightly yellowish

or whitish tinge. The original Bacillus found in the earlier

specimens occurred but once in these, and then in trifling quan-

tity. The ordinary Micrococcus was more commonly present,

sometimes, indeed, profusely abundant, but at other times in

relatively trivial numbers. The unvarying and characteristic

feature was the number of free cells in the blood, of variable

form and size, some of them being altered blood corpuscles and

others evidently derived from the fatt}^ bodies. These occurred

in all stages of segmentation, from a mere trace of commenc-
ing subdivision to a complete separation of the entire contents

of the cell into more or less equal granules. The absence of an

enclosing wall was unquestionably evident, granular masses

being occasionally found from which a single one of the mul-

berry granules had broken away, leaving the remainder undis-

turbed. When the segmentation of these cells was incom])lete

or indefinite, they readily reverted to nucleated cells with gran-



^10 Illinois State Lahoratory of Natural tfistorij.

ular protoplasm, if treated with alcohol or carbolized water.

In many of the mulberry cells the nucleus persisted, surrounded

and obscured by completely formed granules, but in others this

seemed likewise to have participated in the metamorphosis of

the body of the cell. The number of granules in a single

cluster varied from three or five to fifteen or twenty in an

optical section of the mass. The few remaining larvae of this

lot were now transferred to alcohol and glycerine for histo-

logical study.

In the meantime matters had taken a sornewhat unfortu-

nate course in the so-called check lot, these larvae commencing

to die mysteriously on the 30th of June. The first victims

were two dwarfed specimens which had evidently moulted very

imperfectly, being still covered with fragments of the old integu-

ment. An examination of the fluids of these specimens afforded

no explanation of their death, as they contained neither bacteria

in any appreciable number nor any cellular bodies. Another

affected larvae proved to have been parasitized.

Next two larvge were found dead upon the morning of

July 3, the fluids of which were grayish in hue. These con-

tained no recognizable bacteria whatever, but were loaded with

segmented mulberry cells.

On the 10th of July a larva died whose blood contained a

moderate number of micrococci in doubles and chains, concern-

ing which no further notes were made at the time and the slides

illustrating which were lost.

A larva evidently diseased on the evening of this day was

noticed the next morning with several spherical masses of

excrement clinging to the vent, connected with each other by

a delicate film. This film was dissected off, stained and mounted,

and found to consist of an exceedingly delicate, structure-

less, but rather firm, membrane (doubtless the cuticle of the

intestine) through which were dispersed great numbers of

micrococci,—unquestionably a pure culture. These were mostly

collected in patches, some compact and well defined, others

more or less diffused. The compact clusters varied in outline

from nearly circular to elongate oval. One of the latter was

35 ft long by 8 /i wide; others were respectively 18 i^ by 20 fi,

16 fi by 16 fi, and 12 jw by 20 /j.. The micrococci composing



Contagious Diseases of Insects. 311

them were 1 /^ in average diameter, slightly oval to the eye,

though not measurably so.

On the morning of the 12th two other larva? were dead.

The blood of one contained only immense numbers of mulberry

granules with a moderate number of possible spherical micro-

cocci,— not positively distinguishable in our slides, however,

from the smallest mulberry granules. The blood of the other

larva was in a similar condition, heavily loaded with mulberry

cells and the results of their disintegration, but contained, like-

wise, a small number of various bacteria,—rarely a short, broad

Bacillus, apparently identical with that first used in the experi-

ment ; more abundantly a small spherical Micrococcus, difEering

in appearance from the usual form ; also a double oval Micro-

coccus, and an occasional patch of the true spherical so abund-

ant in these experiments. These last were sometimes associated

on the slides with patches of unsegmented cells, which evidently

had their origin in the fatty bodies.

The third larva dead this day was soft, shrunken, and

nearly dry. The scanty fluids were full of micrococci and thick

with mulberry cells and granules. The effect of carbolized

w%ater upon the cells was, in this case, to cause separation into

their constituent particles.

The results of all the above observations and experiments

upon the zebra caterpillar may be summarized as follows : At

least one of the bacillar forms occurring in the culture used

in this infection was conveyed to the larvas under experiment

with fatal effect, and probably multiplied there successfully.

This Bacillus almost wholly disaapeared, however, in the later

stages of the experiment, and so is not certainly a true patho-

genic form. Associated with this in the fluids of the larvae

treated were the usual spherical micrococci of this disease,

clearly identical with those applied to the food, and certainly

multiplying freely in the bodies of the larvae. These presented,

consequently, the characteristics of a pathogenic microbe.

A curious change was observed in the phenomena of the disease

in the experimental lot. Death seemed at first occasioned by

the immediate action of the bacteria ingested or cultivated in

the blood and alimentary fluids ; but at a later period after the



312 illinois State Lahoratory of Natural History.

infection, these bacterial forms became less abundant, and the
^ blood was loaded with the products of histolysis, partly, in all

probability, of the blood cells and partly of the fatty bodies.

There seems to have been in general an inverse relation between

the abundance of the bacteria and the abundance of these his-

tolysis products, the former becoming less numerous with lapse

of time and the latter more so. These facts have an interest-

ing application to those observed in the silkworm, as detailed

on previous pages, the condition of the later examples of the

zebra caterpillar being, in fact, almost precisely similar, so far

as microscopic appearances go, to that of silkworms supposed

to be suffering from jaundice.

I have, consequently, to suggest a similar explanation of

these phenomena ; viz., that in the case of the latter larva the

bacterial affection largely lost its power, but still retained suf-

ficient energy to overthrow the physiological balance as the

larvae approached the age of pupation, death resulting from the

premature histolysis of certain of the larval structures,

—

notably the fatty bodies.

The history 'of the check lot gives no evidence of serious

bacterial infection, but rather of that modified form of it which

produces premature pupal histolysis. Reviewing the entire

series of slides and cultures, I have no doubt that these indicate

the successful preservation through the winter and transference

to the bodies of the zebra caterpillars of certain of the forms

characteristic of fiacherie in the walnut caterpillar, Dafana

angusi.

THE EUROPEAN CABBAGE WORM.

(Pieris rapw, L.)

A second infection experiment was begun with the same

fluids as the foregoing upon fifty cabbage worms, twenty-five

of which were selected for treatment, and an equal number
isolated as a check.

On the 6th August, four days after the infection, a larva

was found dead upon the bottom of the cage. On punctur-

ing the back a clear, greenish fluid exuded, which was swarm-

ing with a large and very active Bacillus, occurring usually in



Contagions Diseases of Lispcfs. 313

doubles. Stained slides of this exhibit the same characteristics

as those made directly from the culture used for the infection,,

but nothing else is evident.

On the same day another larva was found dead and black-

ened, clinging to the side of the cage, in quite different con-

dition, however, from cabbage worms affected by their own
peculiar disease. The body contained but little fluid, and that

was of a paste-like consistence, full of the above bacilli, which

the mounted slides show to be an absolutely pure culture.

Another larva, which died the following day, August 7,

was found to present precisely the same microscopic characters,

only large bacilli occurring in the slide. By the 10th ten of

the specimens under experiment had either pupated or were

evidentl}' making preparations for that change. But two were

apparently diseased. One of these last perished on the 12th,

its body soft, pale, blackened posteriorly, but not deliquescent.

The blood contained a multitude of minute spherical granules,

some Bacillus-like structures, more slender than those previously

occurring, and also floating cells of the fatty bodies containing

mulberry granules, irregular in size, and sometimes showing

also a central nucleus. With these were many large micro-

cocci, 1 ;tt in diameter, circular, or sometimes slightly oval,

commonly in singles or doubles, with rarely a chaplet of four.

This larva soon became deliquescent, as if affected by the

original ffacherie of the cabbage-worm ; its condition, in fact,

indicating a mingling of two diseases,—that conveyed by the

infection to the larva?, just described, and the one native to the

species. It will be noted that one of the effects of the

original infection seemed already to have waned, and that the

development of the mulberry cells and granules characteristic

of this condition had already occurred,— a phenomenon

especially significant, since in the native disease of these

cabbage caterpillars no similar condition of the fluids was

ever seen. Another larva, dead this day, presented appearances

so precisely similar to the preceding that no special descrip-

tion of it was made. The check lot, in the meantime, had

progressed without injury. August 14 this experiment was

interrupted, owing to a discovery of the fact that, through some

oversight of the attendant, the full number of the larva? placed



3l4 Illinois State Laboratory of N'attirat History.

in the breeding cages could not be accounted for, several

having, apparently, been allowed to escape as the food was

changed. This partial experiment can, consequently, only be

held to verify the conclusion drawn from the one just previously

described, to the effect that the Bacillus used for infection may

be at least temporarily propagated in healthy larvae with

destructive effect. It is proper to add that in the remnants

of both the infected and check lots, the common fiacherie of

the cabbage worm afterwards broke out, showing that these

insects had been exposed to this disease before they were brought

to the office for the experiment.

MuSCji-RDINE.

This disease, long well known in the silkworm, is not a

bacterial affection, but is due to an invasion of the body of the

insect by the filaments of a "thread fungus" (Hyphomycetes),

whose spores germinate on the surface. These send thread-

like processes through the skin which at first bud off from their

free ends, within the body, short cells (sometimes called

"conidia") with which the blood of the diseased insect speedily

becomes loaded. These multiply by division, and finally result

in a thread-mycelium which makes its appearance on the

surface of the insect, and bears vast numbers of spores, white

or green, with which the body becomes covered as with a fine

dust. An affected larva is commonly flaccid and shrunken at

death, but finally, as a consequence of the post mortem develop-

ment of the fungus, becomes filled with threads and spores, and

distended to its original size, drying without shrinkage into a

hard and brittle mummy.
These later stages of the development of the fungus are

greatly affected by the weather, a drouth preventing the con-

spicuous external appearance of the mycelium and the develop-

ment of spores, and thus limiting the spread of the disease.

Every experienced collector finds occasional examples of

this disease in the field in the form of stiff and mummified

insects, often covered with a dense white or greenish bloom;

but few observations of any wholesale destruction of a super-

abundant species by it have been recorded,— none for America



Contagions Diseases of tnseets. 315

as far as I am aware. The following observations on the history

of a tremendous outburst, in southern Illinois, of a species of

caterpillar, one of the most destructive insects known, and of

the means by which this irruption was apparently terminated,

will consequently be of considerable interest.

In April and May, 1883, the extreme southern part of the

state, from Cobden southward, was the scene of one of the

periodical uprisings of the forest tent caterpillar {Clisiocampa

si/lrafica), which have doubtless occurred at intervals in that

region from time immemorial. Vast numbers of forest trees

in the southern counties of Illinois and in the adjacent parts of

Missouri and Indiana were as completely defoliated as if raid-

winter had suddenly burst upon them in May, and whole

orchards of many acres of apple trees were left without a single

green leaf. Oak, hickory, the black and sweet gum, and dog-

wood were the trees especially selected for destruction in the

forest, and the apple on the fruit farms,— the foliage of the

peach being scarcely touched, even when the trees were covered

with the caterpillars. Strawberry fields were likewise vigor-

ously attacked,— young fields being occasionally nearly eaten

up.

By the 18th May, when my visit there was made, the larvae

had nearly all attained their growth and were travelling rest-

lessly about by myriads, in every direction, in search of suit-

able-places for pupation,—a few having, in fact, already trans-

formed along the tops of fences and under rubbish on the

ground. As I walked along the road sides my attention was

immediately caught by the great numbers of dead larva3 dried

against the boards of the fences, usually in a vertical position,

and the multitudes apparently in a diseased condition, traveling

more or less feebly, or resting motionless with the head down-

ward. These larvae were usually flaccid and shrunken pos-

teriorly, but not especially discolored.

It was, unfortunately, impossible for me to make any care-

ful examination of the disease at this time, and no other oppor-

tunity offered during the season.

Revisiting this region on the 11th July, an assistant found

that the moths had all emerged sometime previously, but that

from one half to three fourths of the cocoons had never yielded



Sl6 IlUnois State Laboratory of Natural History.

the imago. From a few of these, parasites had evidentlj^

escaped, but in most cases there was nothing in the external

appearance of the cocoon to explain the failure of its develop-

ment. Returning to this region June 3d of the following year,

we learned from A. J. Ayers, Esq., of Villa Ridge, that a suf-

ficient number of larvae hatched that spring to do considerable

damage, but that when they were a little over one half an inch

long they died and dried upon the leaves, sometimes whole
colonies being found dead together. Occasional examples of

larvae in this condition could even then be found on the apple-

trees. A few apparently healthy examples were collected at

this date and brought to the Laboratory at Normal. These

were carefully fed and attended, with the expectation of obtain-

ing the imago, but all died, without exception, with symptoms
precisely resembling those of the year before, as they then

came under my observation.

The first of these larvae was seen to be sick on the 27th

June, ejecting from the mouth and vent a 'fluid which contained

great numbers of oval corpuscles, not unlike those character-

izing pebrine^ bat varying appreciably in size and shape.

Examples were found in process of sub division, or even, in

occasional instances, short strings of three not wholly separated;

and other examples occurred where a spherical lobe was borne

upon the end of an oval cell, as if the latter were badding end-

wise. All these appearances were inconsistent with the hypoth-

esis of the presence of pebrine, the characteristic "corpuscles
"

of which develop by internal segmentation of spherical masses

(Sporozoa) and are never connected in doubles nor multiply by
fission. Dissections of these larvae afforded evidence that they

were attacked by muscardine. In specimens which had lain

some time it was not difficult to identify a scanty mycelium in

the body, although, owing probably to the dry and warm
weather at this season, there was no external development of

the fungas either in the form of threads or spores. These
larvae continued to die until July 5, at which time the last

perished.

The individual cells found in the blood varied from 2 ^ to

3.5 ^c, and in length from 3.5 /a. to 5 ;u. They differed also in

shape, some being a rather broad symmetrical oval, and others



Cottfrif/ioHS Dif^casrs of Insects. 317

narrower towards one extremity. Nuclei about one half as

long and wide as the cells containing them were visible in

most. Neither cells nor nuclei stained readily with aniline.

The blood of many of the larvae examined contained also

considerable numbers of mulberry cells of rather large size,

composed of granules averaging about 2 fx. in diameter.

As no insects affected by muscardine had been handled by

us at the time these caterpillars were received at the office, it is

certain that they brought the infection with them; and as all

perished, without exception, from this same disease, and this

without the development of spores by which the contagion

might have been conveyed from one to another, the presump-

tion is very strong that the affection illustrated by these indi-

viduals was that which had swept away the greater part of the

entire brood of the preceding year, and especially that which

had caused the death of the young larvae as reported by Mr.

Ayres.

SUMMARY AND CONCLUSION.

The circumstances under which the studies above described

have been made; the fact that they belong to a field of research

so difficult that new .comers are very properly viewed with a

certain suspicion until they have clearly demonstrated their

right to labor in it; and the further fact that my results have

not always emerged from the cloud of experiment with per-

fectly clear and definite outline, have seemed to me to require in

this paper a quantity of detail sometimes amounting, perhaps,

to wearisome prolixity; and the following summary of the

principal features and results of my research has been prepared

in the hope that it may serve to make this mode of treatment

less objectionable.

I have first attempted to characterize a common and highly

destructive disease of the European cabbage worm (Pieris

rap(e)., by whose ravages the injuries of these pests have received

a very important check,—a disease especially marked by the

whitish color of the living larvae, amounting before death to

{an ashy or almost milky hue, and by a rapid i^ost niorteui black-

'{ening and decay. The distinguishing microscopic appearances

i|are, first, a remarkable whiteness and opacity of the circulating

iffluids which are early loaded with immense numbers of very



318 Illinois State Lahoratory of Natural History.

minute spherical granules from .5 fi-io .1 iJi in diameter, staining

with aniline fluids, although sometimes with difficulty, and less

highly refractile than ordinary micrococci ; second, a great

degeneration of the mucous membrane of the chiliferous

stomach producing before death a marked diminution in the

thickness of the epithilial layer ; and third, the appearance in

the alimentary fluids, and usually also in the blood, of spheri-

cals and ovals (especially the former), presenting every char-

acteristic of unmistakable micrococci. Few if any of the blood

granules are affected by ether, and they dissolve in hot caustic

potash little, if at all, more readily than known micrococci,

bacilli, and bacillar spores,* but they are not all of them cer-

tainly to be understood as of bacterial character. The fatty

bodies are the next organs to suffer, after the alimentary canal,

and speedily undergo an immense degeneration.

That this disease is contagious is shown by its unequal dis-

tribution in the neighborhoods affected by it ; by its gradual

though rapid progression from one part of the field to an-

other ; by its evident independence of locality, climate, and

weather ; by its apparent progress across the country from east

to west ; by the probable success of experiments made to con-

vey it from infected regions to others at a distance, not previ-

ously invaded by it ; and, finally, by its evident bacterial char-

acter.

In 1883 and 1884, numerous cultures Vi^ere attempted in

beef broth by the strictest methods of fluid culture in tubes

and flasks, the accuracy of which was attested by the fact that

the check tubes in every instance remained unchanged through-

out. Not all the cultures were successful,—several careful

infections from the blood especially being without result ; in

other cases, however, such infections from the blood of still

living larvae yielded the spherical micrococcus figured in the

plate, identical in appearance with that observed in the fluids of

the diseased larvae, but larger in average size than the supposed

'"'Contrary to the statement frequently made respecting the eflfects

of alkahes upon bacteria, I have found that hot solutions of cau^tic

potash rapidly attack both the cells and spores of Bacillus suhtilis and

the common micrococci of fermentation. Two or three times heating

to a boiling point. in a strong solution is sufficient in most cases to com-
pletely destroy these microbes.



Contagious Diseases of Insects. 319

blood form. Cultures from the alimentary fluids were never

without result, although occasionally impure ; but the com-

monest forms there were micrococci like the above, and the

next commonest an oval micrococcus of nearly the same size

and yjeneral appearance. Specimens of Bacillus and Bacterium

were frequent in these alimentary cultures, but far less constant

than the micrococci. No opportunity offered for experimental

infection of healthy larva3 of this or other species with the cab-

bage worm microbes, either native or cultivated, and conse-

quently it must be confessed that, strictly speaking, the proof

is incomplete that this affection of the cabbage worm is a germ

disease, although it certainly amounts to very strong probable

evidence.

More complete and conclusive studies were made of a dis-

ease of the silkworm apparently identical with that known to

the French as jaunes, and called jaundice by English and Amer-

ican writers. This disease, distinguished especially to the eye

by the decided yellow color and restless activity of the larvte,

by the tender skin, easily broken, and by the free flow of thin

yellow blood, is microscopically characterized by an abundance,

in the blood, of the spherical or polygonal granules and clusters

of the same, resulting from the peculiar degeneration of the

larval tissues proper to pupation, — these being in this case

derived chiefly from the fatty bodies and in part also from the

blood corpuscles. This disease, therefore, seems to be essen-

tially a premature pupal histolysis of the fatty bodies,—or,

more properly, to be due to a retardation of the pupation of the

larva which takes unequal effect on the different tissues, the

fatty bodies breaking down before the muscles and membranes

are ready for pupal transformation.

Spherical micrococci .75 ;it to 1 m in diameter occur in the

walls of the alimentary canal as accompaniments of this dis-

ease, and are believed to be one, at least, of the exciting causes

of it, although it seems not impossible that other retarding

influences may produce a similar effect in overthrowing the

normal physiological balance as pupation approaches.

That this supposed jaundice was contagious, was shown

by the phenomena of its occurrence at Champaign, and that

the bacteria accompanying it were capable of exciting disease

in other larvte Avas proven by first cultivating them repeatedly



320 Illinois State Laboratory of Natural History.

in beef broth and then producing in cabbage worms (Pieris

rapw) a similar disease by moistening their food with the cul-

ture fluids containing the bacteria. While this disease, arti-

ficially induced, in some cases came so near that of the native

cabbage Avorm as to suggest that the bacterial treatment served

only to excite the natural disease of the larvae, in other cases it

was clearly different from the above and presented characters

so clearly like those of the silk worm jaundice that there could

be little doubt of an actual transference of the original disease,

especially when the blood of the sick cabbage worms was found

loaded with the mulberry cells and granules of pupal histolysis.

I have next reported at length on a breeding-cage disease

attacking the yellow-]^ecked apple catekpillar (Datana
ministra) and the walnut caterpillar {Datana angusi), so

similar to the well-know flacherie of the silkworm that I have

not hesitated to call it by that name. Its principal symptoms
are those indicating a gradual weakening of the larvae, usually

accompanied by brownish fluid discharges from the vent and a

consequent shrinking and softening of the body. The ali-

mentary canal contains always great numbers of microbes, com-
monly of considerable variety,—including bacilli, bacteria, and

micrococci, the most abundant and characteristic being oval and
spherical micrococci not distinguishable from those mentioned

above. The method of the appearance and spread of the dis-

ease in our breeding room indicated a contagious character
;

and this conclusion was verified by culture of some of the bac-

terial forms encountered and their successful use as an experi-

mental virus.

The cultures (in beef broth and on thin gelatine films)

related to both micrococci and bacilli, and both were preserved

over winter in plugged test tubes and in small sealed tubes,

cultivated the following season, and applied to the food of

another species of larva,—the zebra caterpillar {Mamestra
picta). The first result of this treatment was the destruction

of several of the larvae, in from two to six days, with a disease

marked by the appearance in their intestines of great numbers
of bacilli (in the specimens first to succumb) and micrococci

(later). The affection seemed then to change its character to

one resembling jaundice of the silkworm, the characteristic



Contagious Diseases of Insects. 321

histolysis granules commencing to appear in the blood of

slightly affected larvge as early as the fourth day after infection.

Caterpillars thus attacked did not commence to die until the

sixth day, and most lived until the 15th. As in the case of the

silkworm jaundice with which this is compared, the bacterial

affection was less evident than in more rapid and pronounced

cases of disease, but the usual intestinal micrococci were always

present in varying numbers.

The last infection experiment I had to report, began August

2, 1884, with the same fluid, applied to the food of the European

cabbage worm, was abandoned August 14 because the assistant

in charge was unable to account for all the larvge,—some having

evidently been allowed to escape when the food was changed.

As far as carried, it tended to confirm the indications of the

pi'eceding experiment, the blood of those dying up to the 7th

August being full of a large active Bacillus only, similar to

that used in the infection, and those perishing later containing

chiefly large micrococci together with mulberry cells and gran-

ules. Later the commonJtacherie of the cabbage worm appeared

in the remnants of both the infected and check lots.

Finally in a note on muscardine I have attributed largely

to this affection the disappearance of a vast host of the

forest tent caterpillar (Clisiocampa sylvatica) which devastated

the forests and orchards of a part of southern Illinois in 1883,

basing this conclusion upon the observed phenomena of the

disease appearing among them as compared with those accom-

panying the death of larvae of this species from the same local-

ities, perishing in our breeding cages the following year of

demonstrated muscardine.

There now remains to me only the pleasing duty of

acknowledging ray grateful obligations for aid in this work to

my first assistant, Mr. W. H. Garman, to whose faithful care

and unimpeachable accuracy of manipulation the larger part of

the bacterial cultures were due ; to Prof. T. J. Burrill, who has

had the kindness to examine many of my slides, giving me the

benefit of his extensive acquaintance with the bacteria ; and

to Dr. H. J. Detmers, now of the State University of Ohio, to

whom I owe, among many other favors of this character, the

excellent photographs of micrococci reproduced in the plate.