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LONDON :

GEORGE PHILIP & SON 3Z FLEET STREET

LIVERPOOL :

PHILIP SON & NEPHEW 45 TO 51 SOUTH CASTLE STREET

MALARIA & MOSQUITOES

ABSTRACT OF A DISCOURSE DELIVERED BEFORE THE

ROYAL INSTITUTION OF GREAT BRITAIN

ON MARCH 2x0 1900

MAJOR) RONALD ROSS, D.P.H., M.R.C.S.

LECTURER TO THE LIVERPOOL SCHOOL OF TROPICAL MEDICINE

REPRINTED BY PERMISSION OF THE ROYAL INSTITUTION

Kajral Institution 0f (foat Britain.

WEEKLY EVENING MEETING,
Friday, March 2, 1900.

His GRACE THE DUKE OP NORTHUMBERLAND, KG. F.S.A.,
President, in the Chair.

MAJOR RONALD Ross, D.P.H. M.R.C.S.
Malaria and Mosquitoes.

OUB knowledge of the disease called malarial fever first emerges
from chaos in the seventeenth century, when, owing to the recent
discovery of quinine, the great Italian physician, Torti, was able
to differentiate this malady from other fevers, and to describe its
symptoms with accuracy. Next century, Morton, Lancisi, Pringle
and others observed the connection of the disease with stagnant water
and low-lying ground, and first emitted the theory — which in one
form or another has found general acceptance up to the present date
— that the fever is due to a miasm which rises from the soil or water
of malarious localities. The next great advance was made in the
middle of the nineteenth century by Meckel, Virchow and Frerichs,
who ascertained that the distinguishing pathological product of the
disease is a black substance, which is distributed in collections of
minute coal-black or brown granules in the blood and organs of
patients, and which is called the malarial pigment or melanin. This
line of research culminated in the great discovery of Laveran in 1880
— to the effect that the melanin is produced within the bodies of vast
numbers of minute parasites which live in the red blood-corpuscles
of the patient.

Ray Lankester had already opened the science of the parasitology
of the blood-corpuscle by his discovery of Drepanidium ranarum in
frogs ; and it was at once apparent that the parasites found by these two
observers are somewhat nearly allied — that is, that Laveran's parasite
is a Protozoal organism, and not a vegetable one like the pathogenetic
organisms recently discovered by Pasteur, Lister, Koch and many
others. And our knowledge of the subject was quickly increased by
the discovery of similar haematozoa in certain species of reptiles, birds,
monkeys and bats, and in cattle, by Danilewsky, Kruse, Labbe, Koch,
Dionisi, Smith and Kil borne. In 1885 a further advance was made
by Golgi, who ascertained that the human parasites propagate within
the body of the host by means of ordinary asexual spore-formation ;
that the exacerbations of fever in a patient are coincident with the
disruption of the clusters of spores produced by the organisms ; and
that there are at least three varieties of the parasites in man in Italy

2 Major Ronald Ross [March 2,

These observations were confirmed and extended by a large number
of persons working in various parts of the world — most prominent
among whom are Marchiafava, Celli, Vandyke Carter, Grassi, Osier,
Bignami, Antolisei, Councilman, Mannaberg, Rcmanowsky, Labbe,
Koch, Manson, Thayer and MacCallum. In short, the work of all
these observers, and of many others scarcely less meritorious, has not
only absolutely established the fact that the parasites are the cause
of malarial fever, but has given us a very thorough knowledge both
of the parasites themselves and of their pathological effects, direct and
indirect ; until the science of malaria — for it may almost be de-
scribed as a science in itself — has become a brilliant exemplar of the
modern methods of research as regards the science of .disease in
general.

But I am not here concerned with questions of pathology in
malarial fever. At tho conclusion of the labours to which I have
just referred, we had, it is true, grasped the nature of the disease
itself; but a question of the greatest moment still required an
answer. We had studied side by side the morbid process and the
parasites which cause it ; but we had still to find out how infection
is caused, how these parasites effect an entry. We had ascertained the
life-history of the parasites within man, and of the kindred parasites
within other animals ; but, even after all these investigations, the
life-history of the parasites outside man and outside other vertebrate
hosts remained to be discovered. Until this was done our knowledge
was not complete. It is now my privilege to describe the interesting
theories and investigations which led to the solution of this great and
difficult problem.

The importance of the problem need not be enlarged upon. In
the British army in India during the year 1897, out of a total strength
of 178,197 men, no less than 75,821 were admitted into hospital for
malarial fever ! Fortunately the death-rate of the disease is low in
most places ; but on the other hand the cases are so numerous that
in the aggregate the mortality from malarial fever is very large
indeed. For instance, in India alone, among the civil population
(who do not take adequate treatment), the mortality from " fevers "
during the single year 1897 amounted to the enormous total of
5,026,725 — over five million deaths — being nearly ten times that due
to any other disease. Although undoubtedly thousands of deaths are
wrongly attributed to fever in these statistics, such figures can point
only to a very great mortality due to malaria. Yet India on the
whole is not nearly so malarious as many localities — such, for in-
stance, as places on the coasts of Africa. In short, next perhaps to
tuberculosis, malarial fever is admittedly the most important of
human diseases.

But if the problem to which I refer was an important one, its
solution presented difficulties which I, for one, formerly thought to
be insuperable. It has been mentioned that Lancisi and Pringle
connected tho disease with stagnant water; and their views have

1900.] on Malaria and Mosquitoes 3

been generally endorsed by innumerable observations made since their
time — by tbe general experience of mankind, by statistics, and by
the fact that malaria can often be actually banished by means of
drainage of the soil. But Laveran had uo\v shown the disease to be
due to a parasite of the blood. How reconcile these facts ? There
appeared to be but one way of doing so — namely, by supposing that
the organism lives a free life in the water or soil of malarious places,
from which it enters man by the respiratory or digestive tracts. To
prove this it was necessary to discover it in the water or soil of
malarious places. But how make this discovery? The organism
is not a bacterium, but an animal parasite. It cannot be taken from
the living blood and sown on the surface of a gelatine film. Experi-
ments have proved that it can be inoculated from man to man by the
intravenous injection of fresh infected blood ; but this is a very
different thing to cultivating it in an artificial medium. At all
events, experiments in this line have always failed and are not in
the least likely to succeed. Tbe parasites simply perish when taken
from their natural habitation, the blood. It was therefore extremely
unlikely that we should ever be able to follow up their life-history
by this means — which has proved so successful as regards the bacteria.
It remained only to find them in the soil or water by direct search.
But how identify them among the host of Protozoa which live in
these elements? Certainly not by their form or appearance. As
known to us at that time, they were simply minute amoebae ensconced
in the red corpuscles and accurately adapted for such a life. Now
red corpuscles do not exist in soil and water ; if the parasites live in
the latter, they must possess some other form to that which they
possess in the blood, and the clue afforded by identity of appearance
fails us. The only remaining method open to us would have been
to attempt to produce infection by each one in turn of the numerous
species of Protozoa found in the water and soil of malarious places —
a task of great magnitude, and one which we now know would have
failed. Indeed, it was actually attempted by several observers, and
actually did fail.

Such was the state of things up to the end of the year 1894.
Speaking for myself, I can well remember the hopeless feelings with
which 1 then regarded the problem. Fortune, however, was to be
kinder to us than I had dared believe. At this very moment the key
to the solution of the problem had already been indicated by
Dr. Patrick Hanson.

I have said that since the original discovery of Ray Lankestcr
numerous haematozoa — or rather hrcmocytozoa — have been found in
man and various animals. All these are generally classed by zoolo-
gists in Leuckart's order of the Sporozoa, and are usually divided
into three groups — groups which are not very closely related, except
for the fact that all the organisms concerned are parasites of the red
corpuscles of the blood. One group — found in reptiles — consists of
parasites closely allied to the G-regarinidae, another is found iu oxen,

4 Major Eonald ROM [March 2,

and is the cause of Texas cattle-fever ; the third, for which I adopt
the name of Htemamoabidae Wassielewski — is found in man, monkeys,
bats and birds. It is to this third group — the Haemamcebidae — to
which we must now direct onr attention, because it includes the
parasites of malarial fever. There are, at least, two known species
found in birds, two in bats, one in monkeys, and three in man. The
human parasites are those which respectively cause the three varieties
of malarial fever — quartan, tertian, and remittent or pernicious fever.
For these three species I adopt the names Hsemam&ba malarise
(quartan), Haemamosba vivax (tertian), and Haemomenas prsecox
(remittent fever).* According to Metchnikoff the group belongs, or
is allied, to the Coccidiidae. All the species have a close resemblance
to each other, and all contain the typical melanin of malarial fever.
The youngest parasites are found as minute amoebulse living within
the red corpuscle and generally containing granules of this melanin
(which, indeed, is derived by the parasite from the haemoglobin of the
corpuscle within which it makes its abode). The amoebulaa grow
rapidly in size, until, after one or more days (according to the species)
they reach maturity. At this point many of them become sporocytes
— that is, give rise to ordinary spores by vegetative reproduction.
These spores presently attach themselves to fresh corpuscles, become
fresh amoebulae, and so continue the life of the parasites indefinitely
within the vertebrate host. Others of the amoebulaB, however, instead
of becoming gporocytes like the rest, become gametocytes.

Now it is to these gametocytes that an extreme interest attaches,
because it is to them, and to Manson's study of them, that we owe the
solution of the malarial problem. Numerous observers had examined
them before Manson's time, but all had failed in arriving at a correct
idea as to their function. It had been often observed that they circu-
late in the blood of the vertebrate hosts without, apparently, perform-
ing any function at all. As soon, however, as they are drawn from
the circulation — as when the blood containing them is made into a
fresh specimen for microscopic examination — they undergo the most
remarkable changes. They swell up and liberate themselves from the
enclosing corpuscle ; and then some of them are suddenly seen to
emit a number of long motile filaments. These filaments can easily
be watched struggling violently, and may sometimes be seen to break
from the parent cell and to dart away among the corpuscles, leaving
the residue of the gametocyte, with its melanin, an inert and apparently
dead mass.

Now it is not to be supposed that such an extraordinary pheno-
menon as this — which was observed by Laveran during his first
investigations — could be witnessed without exciting the liveliest
curiosity. As a matter of fact a hot controversy rose regarding it.
Laveran, Danilewsky and Mannaberg maintained that the phenomenon
is a vital one — that the motile filaments are living organisms, and

* Nature, August 3, 1899.

1900.] on Malaria and Mosquitoes. 5

constitute a stage in the history of the parasite. Antolisei, Grassi,
Bignamiand others of the Italian school fell back upon the old theory
— which we always like to employ when we cannot explain a pheno-
menon— that it is a regressive phenomenon, a disintegration of the
parasite due to its death in vitro. Here, however, the controversy
practically stayed. While the Italians, in conformity with their
views, attached no signification to the motile filaments, Laveran,
Dauilewsky and Mannaberg, who held an opposite opinion, did nut
expressly or exactly state what their signification is. Maunaberg,
indeed, held that they are meant to lead a saprophytic existence, but
did not explain how they could escape from the body in order to
do so.

It was reserved for Manson to detect the ultimate (though not the
immediate) function of these bodies. He asked why the escape of the
motile filaments occurs only after the blood is abstracted from
the host (a fact agreed upon by many observers). From his study of
these filaments, of their form and their characteristic movements, he
rejected the Italian view that they are regressive forms; he was con-
vinced that they are living elements. Hence he felt that the fact of
their appearance only after abstraction of the blood (about fiiteeii
minutes afterwards) must have sonic definite purpose in the life-
scheme of the parasites. What is that purpose ? It is evident that
these parasites like all others must pass from host to host ; all known
parasites are capable not only of entering the host. but. either in
themselves or their progeny, of leaving him. Manson himself had
already pushed such methods of inductive reasoning to a brilliantly
successful issue in discovering by their means the development of
Fiiaria nocturna in the gnat. He now applied the same methods to
the study of the parasites of malaria. Why should the motile fila-
ments appear only after abstraction of the blood '? There could be
only one explanation. The phenomenon, though it is usually observed
in a preparation for the microscope, is really meant to occur within the
stomach cavity of some suctorial insect, and constitutes the first step in the
lift-history of the parasite outside the vertebrate host.

It is perhaps impossible for any one, except one who has spent
years in revolving this subject, to understand the full value and force
of this remarkable induction. To my mind the reasoning is complete
and exigent. It was from the first impossible to consider the subject
iu the light in which Maiisou placed it without feeling convinced that
the parasite requires a suctorial insect for its further development.
And subsequent events have proved Mausou to have been right.

The most evident reasoning — the connection between malarial
fever and low-lying water-logged areas in warm countries — suggested
at once that the suctorial insect must be the gnat (called mosquito
in the tropics) ; and this view was fortified by numerous analogies
which must occur at once to any one who considers the subject at all
and which it is not necessary to discuss in this place.

Needless to say, since Mauson's theory was proved to be right, it

6 Major Ronald Ross [March 2,

has been shown to be not entirely original. Nuttall, in his admirable
history of the mosquito theory, demonstrates its antiquity. Eleveu
years before Manson wrote, King had already accumulated much
evidence, based on epidemiological data, in favour of the theory. A
year later (1884), Laveran himself briefly enunciated the same views,
on. the analogy with Filaria nocturna. Koch, and later, Bignami and
Mendini, were also advocates of the theory — partly on epidemiological
grounds and partly because of a possible analogy with the protozoal
parasites of Texas cattle-fever which Smith and Kilborne had shown
to be carried by a tick. Hence many observers had independently
arrived at the same theory by different routes. But I feel it most
necessary to point out here that there is a difference between a
fortunate guess and a true scientific theory. Interesting and sug-
gestive as were many of the hypotheses to which 1 have just referred,
they were to my mind far from convincing. Filaria nocturna, and
even Apiosoma bigeminum, are not in close enough relationship with
the Haeuaamoabidse to admit of very forcible analogies in regard to
the respective life-histories. The epidemiological arguments of King
and Bignami (some of which were also used by Manson) were scarcely
solid enough to support by themselves a theory of any weight. All
these were hypotheses — little more: I can scarcely conceive a prac-
tical man sitting down to laborious researches on the strength of
arguments like these. On the other hand, Manson's theory was what
I have called it — an induction — a chain of reasoning from which it
was impossible to escape.

I have wished to defend this work of Manson's because it has been
much misunderstood and much misrepresented, and even (in a
somewhat amusing manner) completely ignored by some who, though
they once strongly opposed his theory, now, as soon as it has done
its work, wished to forget it. It is true that he endeavoured to
predict the history of the parasites a little too far, and that he was
in error (as will presently appear) regarding the immediate nature of
the motile filaments ; but the core of his theory was invaluable. I
have no hesitation in saying that it was Manson's theory, and no
other, which actually solved the problem ; and to be frank, I am
equally certain that but for Manson's theory, the problem would
have remained unsolved at the present day.

Dr. Laveran's theory was unfortunately enunciated with great
brevity ; but it appears to me to have been really founded on many
if not all the arguments independently advanced by King and Manson.
To him we owe not only the discovery which made all these re-
searches possible ; but also an early and correct prediction as to the
future life-history of the organisms with which his name will be
inseparably connected.

To leave these interesting theories and to return to actual observa-
tions— I should begin by remarking that Manson thought the motile
filaments to be of the nature of zoospores — that is, motile spores
which escape from the gametocytes in the stomach cavity of the gnat,

1900.] on Malaria and Mosquitoes. 1

and then occupy and infect the tissues of the insect. In this he was
proved, two years later, to have been wrong. The motile filaments
are not spores, but microgametes — that is, bodies of the nature of
spermatozoa. I have said that some of the amoebulae in the blood-
corpuscles of the host become sporocytes, which produce asexual
spores (nomospores) ; while other amrebulae become gametocytes,
which have no function within the vertebrate host. As soon, how-
ever, as these gametocytes are ingested by a suctorial insect they
commence their proper functions. As their name indicates, they are
sexual cells — male and female. About fifteen minutes after ingestion
(in some species), the male gametocyte emits a variable number of
microgametes — the motile filaments — which presently escape and
wander in search of the female gamet« >cy tes. These contain a single
macrogamete or ovum, which is now fertilised by one of the micro-
gametes, and becomes a zyrjote. \V'e owe this beautiful discovery to
the direct observation of MacCullum (1897), confirmed by Koch
and Marchoux, and indirectly by Bignami. Metchnikoff, Simond.
Schaudinn and Siedlecki have also demonstrated what are practically
sexual elements in some of the Coecidiidaa. Directly MacCallum's
discovery was announced Manson saw the important bearing of it on
the mosquito theory. Admitting that the motile filaments themselves
do not infect the gnat, he at once observed that it was probably the
function of the zygote to do so — and this time he was perfectly
right.

1 must now turn to my own researches. Dr. Manson told me of
his theory at the end of 1884, and I then undertook to investigate
the subject as far as possible. I began work in Secunderabad, India,
in April 1895 ; and should take the present opportunity for ac-
knowledging the continuous assistance which I received both from
Dr. Mausou and from Dr. Laveran, and later from the Govern-
ment of India. Even with the aid of the induction, the task so
lightly commenced was, as a matter of fact, one of BO arduous a
nature that we must attribute its accomplishment largely to good
fortune. The method adopted — the only method which could be
adopted — was to feed gnats of various species on persons whose blood
contained the gametocytes, and then to examine the insects carefully
for the parasites which by hypothesis the gametocytes were expected
to develop into. This required not only familiarity with the histology
of gnats, but a laborious search for a minute organism throughout
the whole tissues of each individual insect examined — a work of at
least two or three hours for each gnat. But the actual lalwur
involved was the smallest part of the difficulty. Both the form and
appearance of the object which I was in search of, and the species of
the gnat in which I might expect to find it, were absolutely unknown
quantities. We could make no attempt to predict the appearance
which the parasite would assume in the gnat ; while owing to the
general distribution of malarial fever in India, the species of insect
concerned in the propagation of the disease could scarcely be

8 Major Ronald Eoss [March 2,

determined by a comparison of the prevalence of different kinds of
gnat at different spots with the prevalence of fever at those spots. In
short, I was forced to rely simply on the careful examination of
hundreds of gnats, first of one species and then of another, all fed on
patients suffering from malarial fever — in the hope of one day finding
the clue I was in search of. Needless to say, nothing but the most
convincing theory, such as Hanson's theory was, would have sup-
ported or justified so difficult an enterprise.

As a matter of fact, for nearly two and a half years, my results
were almost entirely negative. I could not obtain the correct
scientific names of the various species of gnats employed by me in
these researches, and consequently used names of my own. Unats of
the genus Culex (which abound almost everywhere in India) I called
" gre7 " an<l " brindled " mosquitoes ; and it was these insects which
I studied during the period I refer to. At last, the persistently
nugatory results which had been obtained with gnats of this genus
determined me to try other methods. I went to a very malarious
locality, called the Sigur Ghat, near Ootacamund, aud examined the
mosquitoes there in the hope of finding within them parasites like
those of malaria in man. The results were practically worthless
(except that I observed a new kind of mosquito with spotted wings) ;
and I saw that I must return to the exact method laid down by
Manson. The experiments with the two commonest kinds of Culex
were once more repeated — only to prove once more negative. The
insects, fed mostly on cases containing the crescentic gametocytes of
Hsemomenas prsecox, were examined cell by cell — not even their
excrement being neglected. Although they were known to have
swallowed living Haemamoebidse, no living parasites like these could
be detected in their tissues — the ingested Hseuiamoebidffl had in fact
perished in the stomach cavity of the insects. I began to ask
whether after all there was not some flaw in Hanson's induction ;
but no — I still felt his conclusion to be an inevitable one. And it
was at this very moment that good fortune gave me what I was in
search of.

In a collecting bottle full of larvze brought by a native from an
unknown source, I found a number of newly-hatched mosquitoes like
those first observed by me in the Sigur Ghat — namely, mosquitoes
with spotted wings and boat-shaped eggs. Eight of these were fed on a
patient whose blood contained crescentic gametocytes. Unfortunately
I dissected six of them either prematurely or otherwise unsatisfac-
torily. The seventh was examined, on August 20, 1897, cell by cell ;
the tissues of the stomach (which was now empty owing to the meal
of malarial blood taken by the insect four days previously being
digested) were reserved to the last. On turning to this organ I was
struck by observing, scattered on its outer surface, certain oval or
round cells of about two to three times the diameter of a red blood-
corpuscle — cells which I had never before seen in any of the hundreds
of mosquitoes examined by me. My surprise was complete when I

1900.] on Malaria and Mosquitoes. 9

next detected within each of these cells a few granules of the character-
istic coal-black melanin of malarial fever — a substance quite unlike
anything usually found in mosquitoes. Next day the last of the
remaining spotted-winged mosquitoes was dissected. It contained
precisely similar cells, each of which possessed the same melanin;
only the cells in the second mosquito were somewhat larger than those
in the first.

These fortunate observations practically solved the malaria
problem. As a matter of fact, the cells were the zygotes of the
parasite of remittent fever growing in the tissues of the gnat ; and the
gnat with spotted wings and boat-shaped eggs in which I had found
them belonged (as I subsequently ascertained) to the genus Anopheles.
Of course it was impossible absolutely to prove at the time, on the
strength of these two observations alone, that the cells found by me
in the gnats were indeed derived from Haomamoebidse sucked up by
the insects in the blood of the patients on whom they had been fed —
this proof was obtained by subsequent investigations of mine ; but,
guided by the presence of the typical and almost unique melanin in
the cells, and by numerous other circumstances, I myself had no
doubt of the fact. The clue was obtained ; it was necessary only to
follow it up — an easy matter.

The preparations of the stomachs of the two Anopheles were
sealed, and were afterwards examined by Drs. Smyth, Mauson, Thin
and Bland-Sutton ; and an account of the work, and of the observa-
tions of these gentlemen, was published a little later. Unfortunately,
my labours now met with a serious interruption ; but not before I
had succeeded again in finding the zygotes in two other mosquitoes
— one, another species of Anopheles, also bred from the larva, and also
fed on a case containing crescentic gametocytes ; the other, a " grey
mosquito " (Culex pipiens type), which had been caught feeding on a
case of tertian fever, and which I now think had become previously
infected from a bird with Hsemamasba relicta.

Early in 1898, mainly though the influence of Dr. Hanson, Sir
H. W. Bliss and the United Planters' Association of Southern India,
I was placed by the Government of India on special duty in Calcutta
to continue my investigations. Unable to work with human malaria
— chiefly on account of the plague scare in Calcutta — I turned my
attention to the Hsemamoebidae of birds. Birds have at least two
species of Heeniauicebidae. I subjected a number of birds containing
one or the other of these parasites to the bites of various species of
mosquitoes. The result was a repetition of that previously obtained
with the human parasites. Pigmented cells precisely similar to those
seen in the Anoplieles were found to appear in gnats of the species
called Culex fatigans, Wiedemann, when these had been fed on
sparrows and larks containing Hsemamceba relicta. On the other
hand, these cells were never found in insects of the same species when
fed on healthy birds or on birds containing the other parasite, called
Hsemamceba danilewskii.

B 3

10 Major Ronald Boss [March 2,

It will be evident that this fact was the crucial test both as regards
the parasitic nature of these cells and as regards their development
from the haemocytozoa of the birds ; and it was not accepted by me
without very close and laborious experiment. The actual results
obtained were as follows : —

Out of 245 Culex fatigans fed on birds containing H. relicta, 178,
or 72 per cent., contained " pigmented cells." But, out of 41 Culex
fatigans fed on a man containing crescentic gametocytes, 5 on a man
containing immature tertian parasites, 154 on birds containing H.
danilewskii, 25 on healthy sparrows, and 24 on birds with immature
H. relicta — or a total of 249 insects, all carefully examined — not one
contained, a single " pigmented cell."

Another experiment was as follows : — Three sparrows, one con-
taining no parasites, another containing a moderate number of JJ.
relicta, and the third containing numerous H. relicta, were placed in
separate cages within three separate mosquito curtains. A number
of culex fatigans, all bred simultaneously from larvae in the same
breeding bottle, were now liberated on the same evening partly
within the first mosquito netting, partly within the second, and partly
within the third. Next morning many of these gnats were found to
have fed themselves on the birds during the night. Ten of each lot
of gnats were dissected after a few days, with the following result : —

The ten gnats fed on the healthy sparrow contained no " pigmented
cells." The ten gnats fed on the sparrow with a moderate number of
parasites were found to contain altogether 292 " pigmented cells " ;
or an average of twenty-nine in each gnat. The ten gnats fed on the
sparrow with numerous parasites, contained 1009 " pigmented cells " ;
or an average of 100 cells in each gnat. These thirty specimens
were sent to Manson in England, who made a similar count of the
cells.

I may mention one more out of several experiments of the same
kind. A stock of Culex fatigans, all bred from the larva, were fed on
the same night partly on two sparrows containing H. relicta, and
partly on a crow containing H. danilewskii (placed, of course, under
separate mosquito-nettings). Out of twenty-three of the former lot,
twenty-two were found to have pigmented cells ; while out of sixteen
of the latter, none had them.

Hence no doubt remained that the "pigmented cells," really
constitute a developmental stage in the mosquito of these parasites ;
and this view was accepted both by Laveran and Manson, to whom
specimens had been sent. In June 1898, Manson published an illus-
trated paper concerning my researches, and showed that the pigmented
cells must in fact be the ysygotes resulting from the process of
fertilisation discovered by MacCallura.

It remained to follow out the life-history of the zygotes. For
this purpose it was immaterial whether I worked with the avian or
the human parasites, since these are so extremely like each other. I
elected to work with the avian species, chiefly because the plague-

1900.] on Malaria and Mosquitoes. 11

scare in Bengal still rendered observations with the human species
almost impossible. By feeding Culcx fatigans on birds with II. relicla
and then examining the insects one, two, three and more days
afterwards, it was easy to trace the gradual growth of the zygotes.
Their development briefly is as follows : After the fertilisation of the
macrogamete has taken place in the stomach-cavity of the gnat, the
fertilised parasite or zygote has the power of working its way through
the mass of blood contained in the stomach, of penetrating the wall
of the organ, and of affixing itself on, or just under, its outer coat.
Here it first appears about thirty-six hours after the insect was fed,
and is found as a " pigmented cell " — that is, a little oval body, about
the size of a large red corpuscle or larger, and containing the granules
of melanin possessed by the parent gametocyte from which the macro-
gamete originally proceeded. In this position it shows no sign of
movement, but begins to grow rapidly, to acquire a thickened capsule,
and to project from the outer wall of the stomach, to which it is
attached, into the body cavity of the insect host. At the end of six
days, if the temperature of the air be sufficiently high (about 80° F.),
the diameter of the zygote has increased to about eight times what it
was at first ; that is, to about 60 /z. If the stomach of an infected
insect be extracted at this stage, it can be seen, by a lower power of
the microscope, to be studded with a number of attached spheres,
which have something of the appearance of warts on a finger. These
are the large zygotes, which have now reached maturity and which
project prominently into the mosquito's body-cavity.

All this could be ascertained with facility by the method I have
mentioned ; and it should be understood that gnats can be kept alive
for weeks or even months by feeding them every few days on blood —
or, as Bancroft does, on bananas. But a most important point still
required study. What happens after the zygotes reach maturity?
I found that each zygote as it increases in size divides into meres,
each of which next becomes a blastophore, carrying a number of blasts
attached to its surface. Finally the blastophore vanishes, leaving the
thick capsule of the zygote packed with thousands of the blasts. The
capsule now ruptures, and allows the blasts to escape into the body
fluids of the insect.

These blasts, when mature, are seen to be minute filamentous
bodies, about 1^-16 /j. in length, of extreme delicacy, and somewhat
spindle-shaped — that is, tapering at each extremity. Just as the
zygotes recall the shape of the Coccidiidas, so do these blasts recall
the "falciform bodies." Prof. Herdman and I have adopted this
word " blast " for these bodies after careful consideration — but others
prefer other names. They are, of course, spores, but spores which
have been produced by a previous sexual process — and are, in fact the
result of a kind of polyembryony. Just as a fertilised ovum gives rise
to blasts which produce the cluster of cells constituting a multi-
cellular animal, so, in this case the fertilised ovum or zygote gives
rise to blasts, each of which, however, becomes a separate animal.

12 Major Ronald Ross [March 2,

Prof. Eay Lankester suggests for the blasts of the Haemamcebidse the
simple term " filiform young."

At this point the investigations took a turn of extreme interest
and importance, scarcely second even to what attached to the first
study of the zygotes. Since the blasts are evidently the progeny of
the zygotes, they must carry on the life-history of the parasites to a
further stage. How do they do so ? What is their function ? Do
they escape from the mosquito, and in some manner, direct or in-
direct, set up infection in healthy men and birds ? Or, if not, what
other purpose do they subserve ? It was evident that our knowledge
of the mode of infection in malarial fever — and perhaps even the
prevention of the disease — depended on a reply to these questions.

As I have said, the zygotes become ripe and rupture about a week
after the insect was first infected — scattering the blasts into the body-
cavity of the host. What happens next ? It was next seen that by
some process, apparently owing to the circulation of the insect's
body-fluids (for the blasts themselves appear to be almost without
movement), these little bodies find their way into every part of the
mosquito — into the juices of its head, thorax, and even legs. Beyond
this it was difficult to go. All theory— at least all theory which I
felt I could depend upon — had been long left behind, and I could
rely only on direct observation. Gnat after gnat was sacrificed in
the attempt to follow these bodies. At last, while examining the
head and thorax of one insect, I found a large gland consisting of a
central duct surrounded by large grape-like cells. My astonishment
was great when I found that many of these cells were closely packed
with the blasts — (which I may add are not in the least like any
normal structures in the mosquito). Now I did not know at that
time what this gland is. It was speedily found, however, to be a
large racemose gland consisting of six lobes, three lying in each side
of the insect's neck. The ducts of the lobes finally unite in a common
channel which runs along the under surface of the head and enters the
middle stylet, or lancet, of the insect's proboscis,

It was impossible to avoid the obvious conclusion. Observation
after observation always showed that the blasts collect within the
cells of this gland. It is the salivary or poison gland of the insect,
similar to the salivary gland found in many insects, the function
of which, in the gnat, had already been discovered — although I
was not aware of the fact. That function is to secrete the fluid which
is injected by the insect when it punctures the sJcin — the fluid which
causes the well-known irritation of the puncture, and which is pro-
bably meant to prevent either the contraction of the torn capillaries
or the coagulation of the ingested blood. The position of the blasts
in the cells of this gland could have only one interpretation —
wonderful as that interpretation is. The blasts must evidently pass
down the ducts of the salivary gland into the wound made by the
proboscis of the insect, and thus causes infection in afresh vertebrate
host.

1900.] on Malaria and Mosquitoes. 13

That this actually happens could, fortunately, be proved without
any difficulty. As I had now been studying the parasites of birds
for some months, I possessed a number of birds of different species,
the blood of which I had examined from time to time (by pricking
the toes with a fine needle). Some of them were infected, and some,
of course, were not. Out of 111 wild sparrows examined by me in
Calcutta, I found JET. relicta — the parasite which I had just culti-
vated in Culex fatigans — in 16, or 13 • 5 per cent. As a rule, non-
infected birds were released ; but I generally kept a few to use for
the control experiments mentioned above, and the blood of these
birds had consequently been examined on several occasions, and had
always been found free from parasites. At the end of June I pos-
sessed five of these healthy control birds — four sparrows and one
weaver-bird. All of them were now carefully examined again and
found to be healthy. They were placed in their cages within
mosquito-nets, and at the same time a large stock of old infected
mosquitoes were released within the same nets. By " old infected
mosquitoes " I mean mosquitoes which had been previously fed re-
peatedly on infected birds, and many of which on dissection had been
shown to have very large numbers of blasts in their salivary glands.
Next morning, numbers of these infected gnats were found gorged
with blood, proving that they had indeed bitten the healthy birds
during the night. The operation was repeated on several succeeding
nights, until each bird had probably been bitten by at least a dozen
of the mosquitoes. On July 9, the blood of the birds was examined
again. I scarcely expected any result so complete and decisive.
Every one of the five birds was now found to contain parasites — and
not merely to contain them, but to possess such immense numbers of
them as I had never before seen in any bird (with H. relicta) in
India. While wild sparrows in Calcutta seldom contain more than
one parasite in every field of the microscope, those which I had just
succeeded in infecting contained, ten, fifteen, twenty and even more
in each field — a fact due probably to the infecting gnats having been
previously fed over and over again on infected birds, a thing which
can rarely happen in nature.

The experiment was repeated many times — generally on two or
three healthy birds put together. But I now . improved on the
original experiment by also employing controls in the following
manner. A stock of wild sparrows would be examined, and the
infected birds eliminated. The remainder would then be kept apart,
and at night would be carefully secluded from the bites of gnats by
being placed within mosquito nets. These constituted my stock of
healthy birds. From time to time two or three of these would be
separated, examined again to ensure their being absolutely free from
parasites, and then subjected to the bites of " old infected mosquitoes,"
and, of course, kept apart afterwards for daily study. Thus my stock
of healthy birds was also my stock of control birds. Until they were
bitten by gnats, I found that they never became infected (except in a

14 Major Ronald Ross [March 2,

single case in which I think I had overlooked the parasites on the
first occasion), although large numbers of healthy birds were kept in
this manner. The result in the case of the sparrows which were
subjected to the bites of the infected gnats was different indeed. Out
of 28 of these, dealt with from time to time, no less than 22, or
79 per cent., became infected in from five to eight days. And, as in
the first experiment, all the infected birds finally contained very
numerous parasites.

It was most interesting to watch the gradual development of the
parasitic invasion in these birds; and this development presented
such constant characters that, apart from other reasons, it was quite
impossible to doubt that the infection was really caused by the
mosquitoes. The course of events was always as follows. The blood
would remain entirely free from parasites for four, five, six or even
seven days. Next day one or perhaps two parasites would be found
in a whole specimen. The following day it was invariably observed
that the number of the organisms had largely increased ; and this
increase continued until in a few days immense numbers were present
— so that, finally, I often observed as many as seven distinct parasites
contained within a single corpuscle ! Later on, many of the birds
died ; and their organs were then found to be loaded with the
characteristic melanin of malarial fever.

I also succeeded in infecting on a second trial one of the six
sparrows which had escaped the first experiment ; and also a crow
and four weaver-birds ; and lastly, gave a new and more copious
infection to four sparrows which had previously contained only a few
parasites.

These experiments completed the original and fundamental
observations on the life-history of the Hsemamcebidse in mosquitoes.
The parasites had been carried from the vertebrate host into the
gnat ; had been followed in their development in the gnat ; and had
finally been carried back from the gnat to the vertebrate host. The
theories of King, Laveran, Koch and Bignami, and the great
induction of Manson, were justified by the event ; and I have given
a detailed historical and critical account of these theories, and of my
own difficulties and experiences, in the hope of bringing convict'ou
to those who might, perhaps, otherwise think the story to be too
wonderful for credence.

But work of great importance remained to be done. I had
intended, immediately after making this study of one of the parasites
of birds, to extend the investigation more fully to those of man — a
work which now presented no difficulty, since both the kind of
mosquito hospitable to them (Anopheles) and the form of the parasites
in the mosquito were well known to me. Unfortunately I was
obliged to attend to other and less important duties, which kept me
fully occupied for several months — an interruption which practically
put an end to my own study of the mosquito-theory at a very
interesting point. No time, however, was really lost In December

1000.] on Malaria and Mosquiiocs. 15

1898, Dr. Daniels, of the Malaria Commission of the Royal Society
and the Colonial Office, arrived in Calcutta to examine and report
upon my results. After carefully repeating the various experiments
he fully confirmed the statements made by me.* At the same
moment, the work was taken up with great brilliance and success by
Dr. Koch and by Prof. Grassi and Drs. Bignami and Bastianelli, in
Italy. I must now describe the investigations of these observers —
though I have scarcely space to do so at the length they deserve.

Ever since the discoveries of Laveran and Golgi, the Italian
observers of the Roman school have done much important work on
malaria, facilitated by the well-known prevalence of the disease near
Kome ; work, if not of much originality, yet full of careful detail.
More recently, however, this work had been practically arrested by
their theory — wholly gratuitous, but which they accepted as a dogma
— that the motile filaments are forms of disintegration in vitro. When
Manson propounded his theory, Bignami, for instance, rejected it on
this ground. But at the same time he evolved a gnat-theory of his
own — a theory that malarial fever is inoculated by gnats which carry
the parasite from marshy areas. The arguments he used were the
epidemiological ones already advanced by King, and which can
scarcely be said to amount to more than a plausible hypothesis : the
only solid basis for the theory — that of Manson — was opposed by
him. Later, however, the work of Simond, Schaudinn, Siedlecki,
MacCallum and myself, explained by Manson, rendered the Italian
position concerning the motile filaments quite untenable ; and
Bastianelli, Bignami and Grassi now undertook a study of the
mosquito-theory on sound principles. My own results, with descrip-
tions of the technique employed and with illustrations of the zygotes,
had been published from time to time ; a summary of them had been
given by Manson in June 1898, and another, including the infection
of healthy birds, before the British Medical Association, early iii
August ; and there could therefore be no difficulty in following up
the observations therein recorded. In September, Grassi published
a paper in which he described certain investigations made in Italy
with a view to ascertaining the species of gnats which are associated
with the prevalence of malaria in that country. Such investigations
are not, I think, trustworthy ; and as a matter of fact two out of the
three species of gnat then selected by Grassi as being malaria-bearing
ones, have now been rejected by him. The third species was an
Anopheles, namely A. claviger, Fabr.

At the same time Biguami resumed his study of the subject.
Some years previously, following his theory, he had endeavoured to
infect healthy persons by the bites of gnats brought from malarious
places. He had failed and abandoned his efforts — and I believe that
his method would of itself never had led to a solution of the problem.
In the autumn of 1898, however, he renewed his efforts ; but was

* Nature, August 3. 1899.

16 Major Ronald Ross [March 2,

again unsuccessful until he used a number of Anopheles claviger,
brought from a house containing infected persons. The result was
successful, the subject of the experiment becoming infected after some
time. This important experiment gave the first confirmation with
human malaria of my previous inoculation experiments with the
malaria of birds ; bat since other species of gnats as well as
A. claviger had been employed, it failed to fix suspicion entirely on
the latter. In order to obtain this result, these observers were finally
obliged to resort to the correct method of Manson and myself —
namely that of direct cultivation of the parasites in the gnat. Success
was now immediate. The zygotes and blasts of the parasites were
found, exactly as previously described by me, in the tissues of
A. claviger ; and lastly, healthy persons were infected by the bites of
these insects. Pushing forward with admirable rapidity, the Italian
observers next found that all three species of the human Haemamoebidffl
are cultivable in A. claviger ; and not only in this, but in other
Italian species of Anopheles, while, like me, they failed in cultivating
the parasites in Culex.

Almost simultaneously Koch repeated and confirmed with the
weight of his authority most of the results which had been obtained
as regards both the human and avian parasites. In August 1899, the
malarial expedition sent to Sierra Leone by the Liverpool School of
Tropical Medicine (of which expedition I was a member), found the
human parasites in two species of Anopheles in that colony, namely
A. costalis, Loew, and A. funeslus, Giles. I hear also that the same
result has been obtained with Anopheles in two other parts of the
world, so that it would appear that something like nine species of
Anopheles have now been inculpated — while as yet every species of
Culex which has been tried has failed to give positive results.

From this point it becomes impossible to follow in detail the
researches carried out in connection with the mosquito theory in
various parts of the world. The facts already collected would fill
a small volume ; and every month witnesses additional publications
on the subject. I shall therefore, in conclusion, content myself with
a brief reference to three points of leading importance.

I shall first try to indicate how completely the recent discoveries
explain the well-known laws regarding the diffusion of malaria. As
mentioned at the beginning of this lecture, malarial fever has long
been known to be connected with the presence of stagnant water.
That is to say, we generally, though not invariably, nud that the
disease is associated with low-lying flat areas, where water tends to
collect to a considerable extent. It was indeed the general apprecia-
tion of this law which led to the old miasma-theory of the disease —
the theory on which the word " malaria " was based. We assumed
that the poison is one which rises from marshy areas in the form of
a mist, and which thence infects all living within a given distance.
Later, when the pathogenetic parasite was discovered in the blood of
febricitants, many observers, still clinging to this conception, thought

1900.] on Malaria and Mosquitoes. 17

that the parasite is an organism which in its free state dwells in such
places, and diffuses itself in such mists. It is interesting to note
how near to the truth this almost instinctive conception took us. It
is right in idea, wrong in fact. It is not the parasite itself which
springs from the marshy ground, but the carrier of the parasite.

This was one of the many interesting points made by King in his
mosquito-theory of seventeen years ago. But King fell into an error
which could have been used as a powerful argument against his
hypothesis. He seemed to have assumed that all mosquitoes rise
from marshes. Hence, he said, malaria exists in the presence of
marshes ; hence it is a disease of the country, rather than of towns,
and so on. As a matter of fact, mosquitoes as a rule do not rise from
marshes at all ; they do not all even rise from pools of water on the
ground ; the commonest species, at least of those which habitually
annoy human beings, spring from tubs and pots of water in the
vicinity of houses, and are indeed more common in cities than in
country places, at any rate in the tropics. Now it is not the least
interesting feature of recent researches that they have shown where
the error lay. As soon as I have succeeded in cultivating the human
parasites in my " dappled-winged mosquitoes," which were really
gnats of the genus Anopheles, I began to study the habit.s of these
insects, and soon ascertained the remarkable fact that while gnats of
genus Culex generally breed, in India, in vessels of water round
houses, gnats of genus Anopheles, which I had just connected with
malaria, breed in small pools of water on the ground. This point was
made the subject of a special investigation by the recent expedition
to Sierra Leone ; and we found that the law holds good there as in
India. While Culex larvaa wore to be seen in almost every vessel of
water, or empty gourd or flower-pot in which a little rain-water
collected, in only one case did we find Anopheles larvae in such. On
the other hand, Anopheles larvae occurred in about a hundred small
puddles scattered through the city of Freetown — puddles mostly of a
fairly permanent description, kept filled by the rain, and not liable
to scouring out during heavy showers. What was almost equally
significant, the larva) seemed to live chiefly on green water-weed.
Hence it follows that while Culex, the apparently innocuous genus
of gnats, are essentially, or at least often, domestic insects, Anopheles,
the malaria-bearing genus, are essentially gnats which spring from
stagnant water on the ground. And numerous other facts in the
history of malaria can be explained by the same discovery. It is
supposed, for instance, that malaria originates from freshly-turned
earth ; now we actually noted examples where railway embankments
and the like had produced Anopheles pools ; and it is easy to see that
disturbance of the soil may often produce depressions in the ground
capable of holding a little rain-water suitable for the larva? of these
insects. Again malarial fever often appears on board vessels which
have touched at malarious ports ; as an explanation of this we ascer-
tained that Anopheles visit ships from the shore. In short, on study-

18 Major Ronald Ross [March 2,

ing the matter from every point of view, I must confess to being
ignorant of any well-established fact about malarial fever which is
not explained by the mosquito-theory.

This brings me to the subject of objections to the mosquito- theory.
In view of the exact and copious microscopical and experimental
evidence which has now been collected in proof of the theory, it is no
longer permissible to doubt the main facts ; and the objections which
one still finds, both in the lay and the medical press, are generally
based on a complete ignorance of these facts, and need not be dis-
cussed here. But there is one objection — frequently made, in spite
of corrections as frequent, by persons who reside in malarious places
— which deserves comment. This is, that malaria exists where there
are no mosquitoes, and that so-and-so has had fever without being
bitten by gnats at all. Generally speaking, we must always remember
that malarial fever is a disease in which relapses occur perhaps for
years after the first infection, and that it is this first infection and
not the relapses which are due to the bite of Anopheles. It is thus
possible to suffer from any number of attacks of fever without being
bitten by Anopheles (except on one occasion), and without invali-
dating the theory — a fact of which those who argue in this manner
are generally ignorant. Again, it is well known that one may be
bitten without perceiving it ; that some persons are singularly callous
to the punctures of these insects ; and, lastly, that many others have
very limited powers of observation. I may say at once that, person-
ally, I cannot accept any statement to the effect that gnats are absent
in any locality in the tropics, until such a statement is made by a
competent observer after direct search ; because I have never been in
any place in the tropics — and I have been in a large number — where
there were no gnats. On the other hand, I have often found numer-
ous gnats in localities where I was previously told there were none.
I was once actually informed that there are no mosquitoes in Sierra
Leone ! The fact is that those who will trust the statements of the
general public on such matters must be very credulous.

I turn lastly to the all-important subject of prevention, but can do
no more than touch upon it here. Two methods suggested themselves
at once. I need not refer to that of guarding against the bites of
these insects by the use of mosquito-nets and so on — an obvious and,
I believe, an exceedingly useful measure, which may reduce the
chances of infection to a small fraction. Unfortunately such methods
will never be employed on a large scale in the majority of malarious
localities ; and we must resort to the destruction of malaria-bearing
species of gnats. Early in 1892 I reported to the Government of
India that it may be possible to exterminate Anopheles in some locali-
ties— especially some towns, cantonments and plantations — owing to
the habit the insects have (in some places) of breeding only in
selected pools. Since then, a considerable literature has already
grown up round the subject. Ee vie wing this literature, it seems
probable that we may be able to exterminate Anopheles or at least

1900.] on Malaria and Mosquitoes. 19

largely reduce their numbers, in towns where, owing to the confor-
mation of the ground, the low level of the subsoil water or the small
rainfall, surface pools suitable for the insects are comparatively few.
The methods which can be adopted against the larvae are numerous
— such as brushing out the pools with a broom, draining them away,
filling them up, or treating them with various culicides, such as
paraffin and numerous other substances (recently investigated by
Celli and Casagrandi). On the whole the most promising method
which suggests itself is the employment of some cheap solid material
or powder which dissolves slowly, which kills the larvae without
injuring higher animals, and which renders small pools uninhabitable
for the larvaa for some months. If, for instance, a cartload of such
a material would suffice to extirpate the larvae for a square mile of a
malarious town, the result would be a large gain to its healthiness.
Dr. Fielding-Ould has lately reported favourably on tar. Grillet
recently reports a case in France where a large district was rendered
free of malaria by the extensive use of lime for agricultural purposes.
Gas-lime, or even common salt, may be suggested. In short, though
the question of the possibility of attacking these insects with success
is still entirely in the experimental stage, we may reasonably hope
that the mosquito-theory of malaria may some day prove to be as
useful to humanity as it certainly has proved interesting to the
student of science.

In conclusion, however, I should add that this result is not likely
to be attained unless we, as a nation, determine to pay more attention
to scientific discoveries in the field of tropical medicine than hitherto
we have done. During the last fifty years discovery after discovery
in this field has been made without finding any adequate reflex in
medical and sanitary practice in our tropical possessions. The dis-
coveries, for instance, of Losch, Davaine, Dubini, Bilharz, Bancroft,
Koch, Laveran, Manson, Carter and Giles, though nearly concerned
with the lives of thousands of human beings, have been generally
treated either with scepticism or neglect — have been neither suffi-
ciently followed in the laboratory nor sufficiently acted upon in the
region of practical sanitation.

LONDON: PHINTED BT WM. CLOWES AND SOSS, LIJIITKD STAUFOiiO STllKEt
AND CHAKIXC CROSS.

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