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PLATE I

Fig. 1—Simple tertian parasite. Fresh preparation.
1 and 2, Young forms, pigmented, actively amoeboid.

3. Large form with ‘boiling’ pigment. Corpuscle
much enlarged.

Fig. 2.—Simple tertian parasite. Romanowsky stain.

1 and 2. Young and medium size forms, shewing
stippling of the red cell (Schiiffner’s dots).

3. Female gamete, with much stippling of red cell.
4. Segmenting stage.

Fig. 3.—Malignant tertian parasite. Fresh preparation.
Young parasites. Horse-shoe forms.
Mikrogametocyte (male crescent).
Makrogamete (female crescent).

Oval stage of crescent.

Spherical stage of crescent.

WEY Po

Fig. 4.—Malhgnant tertian parasite. Romanowsky stain.
tand 2. ‘Ring’ forms.
3. Large egg-shape form.
4. Large form shewing coarse stippling of red cell.
5. Segmenting form. Very rare in peripheral blood.

Fig. 5.—Quartan parasite. Fresh preparation.
Medium size.
Large size.

oN

Presegmenting form.
4. Segmenting, daisy, or marguerite form.

Fig. 6—Quartan parasite. Romanowsky stain.
1and2. Young forms.
3. Large form.
4. Segmenting form.
5. Mikrogametocyte (¢).

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BENIGN TERTIAN

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TERTIAN

TAN

THE PRACTICAL STUDY
OF MALARIA

AND OTHER BLOOD PARASITES

{

Kee WN BY

J: W: W. STEPHENS, M.D. Canvas, D.P.H.

WALTER MYERS LECTURER IN TROPICAL MEDICINE
UNIVERSITY OF LIVERPOOL

AND

hoa
S..RY CHRISTOPHERS, M.B. Vicr., IMS.

MEMBERS OF THE ROYAL SOCIETY'S COMMISSION ON MALARIA
IN AFRICA AND INDIA, 1898-1902

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PUBLISHED FOR

THE UNIVERSITY PRESS OF LIVERPOOL

BY
WILLIAMS & NORGATE
14 HENRIETTA STREET, COVENT GARDEN
LONDON, 1904

D

No. “Lle] e
At the University Press of Liverpool

No. 46. Nov., 1903. 500

Second Edition. December, 1904. 1,000

me
156
Sas
LG OR

KEAN 64 YO
7

PREFACE TO THE FIRST EDITION

In the authors’ experience many medical men
in the tropics are only deterred from undertaking
researches in tropical diseases by the impossibility
of obtaining the necessary knowledge of methods
apart from personal instruction in some laboratory.
Numerous works ‘on: technique exist, they are,
however, more adapted for work in a laboratory
than for the conditions under which the average
practitioner in the tropics must be prepared to
conduct his researches. As a result of an ex-
“perience of several years, during our work on the
Royal Society’s Commission on Malaria, of the
difficulties that Indian and. Colonial medical
officers experience in making the first start in what
must often be work of the greatest interest to
themselves and the utmost value to science,.we
have. deemed it wise to give instead of full and
elaborate technique, as usually given, only that
which we have found the best, the simplest, and
the most generally useful. In reality, the necessary
methods required to undertake research of the
highest value in Malaria are very simple, yet most
of these cannot be found in books, and they are
with considerable difficulty learnt except by the
personal direction of those who are familiar with
the small details which go to make success.

In the present handbook we propose to give
the essentially practical methods, by which those
not familiar with laboratory methods may, under

their own microscopes, follow all the most recent:
work on Malaria, and eventually be in a position
themselves to add new facts to our knowledge of
this important disease.
. For instance, with very little apparatus it is
possible to undertake many most important re-
searches, e.g., to work out the rationale of infection
in anystation or cantonment; the form of the para-
site present ; the percentage of adults and children
infected; the species of Anopheline; where each
species 1s found and where it breeds; the per-
centage of each species carrying sporozoits and
zygotes.

' In fact nearly the whole technique of Malaria
can be conducted with a microscopé, a few slides
and coverglasses, a needle, a stain, some: tubes,
pins, and cardboard: (Vide Appendix).

While our original intention was to write a
practical guide to Malarial Study solely, yet the
opportunities for research on other blood parasites
are so numerous in the tropics, that we have
thought it to be of practical value to add short
supplementary chapters on other Haematozoa and
on the Trypanosomidae, etc.

November, 1903

PREFACE TO SECOND EDITION

Our aim in writing this book was primarily to
assist the beginner who.had little or no experience
of the study of malaria or kindred diseases of man
and animals. While keeping this view steadily
in mind, we by no means intended that its
scope should be restricted solely to this, but
endeavoured to make the book a convenient and
complete reference book for those investigating
blood parasites. Progress in the study of blood
parasites is extremely rapid, and we have to add
to this new edition, besides many new Anophelines
and mosquito genera and details of mosquito life,
many new haemogregarines, trypanosomata, etc.,
and an account of ScHaupinn’s_ remarkable
investigations on ‘ Halteridium.’ Two new
additional chapters have also been added, one on
the LrrsHmMAN-Donovan bodies; a second on
spirillar fever. The chapter on biting flies and
fleas has been much expanded, and in response to
requests we have added four new coloured plates.

November, 1904

CONTENTS

CuapTerR | PAGE
Preparation of wet and dry blood films. Fixing, stain-
ing, Romanowsky stain - I
CuapTer II
Normal blood. Varieties of leucocytes. Platelets.
Abnormal constituents 14

CuHaprter III

Forms of the malaria parasite commonly seen in blood
~ films. Rings, large forms, crescent and spherical
bodies. Pigment (melanin). Flagellation. Dis-
tinction of species, simple tertian, malignant tertian,

and quartan. Gametes 23

Cuaprer IV

Subsidiary signs of malaria. Pigmented leucocytes.
An increase in the percentage of ie mononuclears.

Normal leucocytic values 39
CHAPTER V
Examination of tissues. Smears. Simple apparatus
for embedding in paraffin 43

CuapTer VI
Life history of malaria parasite. Explanation of terms 53

CHAPTER VII

Mosquitoes. Diptera. Life history of mosquitoes.
Culicina and Anophelina. Capture of mosquitoes 58

CuapTer VIII
Examination of eggs of Stegomyia, Culex, Taeniorhyn-
chus, Anopheles, etc. - - 68
CHAPTER IX

Larva and nymph. Respiratory syphons of larva and
nymph, generic differences 74

CHAPTER X

Feeding mosquitoes on man; on birds. Breeding out
mosquitoes 94

il

CuapTer XI
Dissection of mid-gut, viscera, etc. Detection of

zygotes. Salivary glands and sporozoits. To cut »

sections of mosquitoes

Cuapter XII
Internal anatomy. Histology

CuapTeR XIII
To collect and preserve mosquitoes -

CHAPTER XIV
External anatomy. Proboscis and palpi. Escape of
sporozoits and filarial embryo. Thorax. Wing
venation. Legs Z
CHAPTER XV
Classification and identification of the Culicidae.
Scale structure

CuarTeR XVI
Classification and identification of Anophelinae -

CuapTer XVII

Anophelinae, Habits, distribution, hibernation, dispersal.
‘Domestic’ and‘ wild’ species. Food. Fecundation

CuapTer XVIII
Anophelinae. Examination of eggs

CHapTeR XIX
Anophelinae. The larva. Clypeal and palmate hairs.
The nymph
CHAPTER XX
Anophelinae. Breeding-places. Selective action of species

CHAPTER XXI

Anophelinae. Identification of larvae. Larvae of Indian
Anophelinae

PAGE

104

129

157

173

186

209

221

229

241

244

ill

CHAPTER XXII
Anophelinae. Relation of species to malarial endemicity

CHAPTER XXIII

Endemic malaria. Endemic index. Mode of determin-
ation. Endemic areas of a country

CHAPTER XXIV

Clinical study of malaria. The leucocytes. The
urine. Determination of cycle of development of a
parasite. The absorption and elimination of
quinine. Post-mortemchanges

CHAPTER XXV
Blackwater fever. Examination of the blood and
urine. Post-mortem changes

CHAPTER XXVI

The haemamoebidae. Proteosoma, halteridium,
parasites of bats, monkeys, cattle, reptiles. The
haemogregarines of reptiles, fish, etc. The piro-
plasmata. Texas fever. African Coast fever.
Spotted fever, etc. Ticks, anatomy and classifica-
tion -

CuapTer XXVII

The trypanosomidae.

CuapTer XXVIII
The Leishman bodies

CuHaPTER XXIX
The spirochaetes. Spirillar fever .

CuapTER XXX
The filariae

APPENDICES

PAGE

254

310

35°

369

377

382

Tsetse and other biting flies and fleas. Stains. Weights and

measures. Apparatus.

The Practical Study of Malaria

Chapter I
TO PREPARE BLOOD FILMS

OR ordinary work we have no hesitation in
saying that it is better to use almost entirely
dry films. The advantages of using these

are many :—

1. They are much less trouble to make,
especially under the adverse conditions one has
to. contend with in the tropics.

2. They need not be looked at at once, but
can be put aside until one has the leisure to
examine them. .

3. Large numbers, fifty or one hundred, may
be taken at once, as will be found constantly
necessary, and examined weeks or months later,
a thing quite impossible with ‘ wet’ films.

4. hey give information as to the leucocytes
if this is needed, and may be examined over and
over again when some new point of view demands
a re-examination.

_ By the use-of exceedingly simple methods
the routine of blood examinations may be reduced
to the greatest simplicity.

For studying movement, delicacies of struc-
ture, for watching the process of exflagellation,

B

2

phagocytosis, fertilization, and other phenomena
of the living parasite, it is necessary, however, to
be able to make good wet films, and the points of
importance in the preparation of these will be
described.

THE PREPARATION OF Dry FILMS

The simplest and by far the best way of
making films is by the use of no other apparatus
than—

1. A straight surgical needle about two

inches in length.

2. Clean glass slides.

To CLEAN SLIDES

Slides should be dipped in water and rubbed
dry and clean with a solt cloth, e.g.,a clean hand-
kerchief. To ensure the best results it is well to
heat the slides in the flame of a spirit lamp, or
smokeless paraffin lamp, and allow them to cool on
a sheet of clean paper. For ordinary purposes this
is quite unnecessary. Butifa perfectly clean shde
is required, then heat it ‘red hot’ over a flame.
In this way greaseis completely removed.

Before proceeding to take specimens of blood,
the prepared slides may be placed in a small
pocket slide box or wrapped in a sheet of clean
dry white paper. A packet of half-a-dozen pre-
pared slides wrapped in a sheet of note-paper,
afterwards transfixed with the needle is a most
convenient form of carrying the necessaries for
taking specimens of blood. The needle should be
an ordinary triangular-pointed, straight surgical
needle. (It is best to nip off the eye with pincers).

3

To CLEAN THE Patignt’s FINGER

If the finger of one’s subject is obviously dirty,
and especially if damp with sweat, the finger
should be roughly wiped with a cloth. If con-
sidered necessary, precautions may be taken to
avoid all skin contaminations by the routine of
water, alcohol, and ether, but in ordinary exam-
inations for malarial parasites this is quite un-
necessary.

To Prick THE FINGER

The last phalanx of the finger (the third
finger of the right will be found most convenient
and the skin usually soft) is taken between the
finger and thumb of the left hand of the operator
and: gently pressed to force the blood towards the
pulp. A slight prick with the triangular pointed
needle will in most cases cause a fair-sized drop
of blood to exude.

To Make THE FILM

When the drop of blood reaches the size of
the head of a small pin, a slide is taken in the
right hand and lowered on to the drop (taking
care not to ‘dab’ it on the skin). Jf the drop is
too large, wipe it away and squeeze a small fresh
one. ‘The drop should be transferred to the slide
about one-third inch from the far end. The slide
is then changed to the left hand, the finger and
thumb grasping the end nearest to the drop. The
right hand again takes the needle, and holding
it by the pointed end lays the cylindrical shaft
transversely to the slide and across the drop of

4

blood. After waiting about a second, that ts
until the drop spreads to the extent of about
one-third inch between the slide and the needle,
the needle is evenly and not too quickly carried
to the right and so along the whole length of
the slide. ‘The right amount of pressure 1s very

Fig. 1. Authors’ method of making blood films

quickly learnt, and the making of a useful and
good film is far easier in this way than in any -
other known to us (Fig 1). Immediately the film
is made it should be waved to and fro until it is
seen to be quite dry. The quicker the ‘film. dries
the more perfectly preserved will be the red cells
of the blood.

THE CHARACTERS OF A Goop FILM

1. As a film is needed to detect even the
most minute forms of the parasite within the red
cell, it is necessary that the red cells be well spread
out and not distorted or lying over one another.
To ensure this the film must be uniform and as
thin as possible.

2. Films should be made so that, if desired,
the leucocytes may be differentially counted. A

little practice will enable oné to make films with
the upper and lower edges more or less parallel’
with the edges of the:slide, and terminating in a
pointed manner about half an inch from the right
hand end‘of the slide. Practically the whole of
the drop.of blood is then upon the slide, and the
edges to which the leucocytes tend to find their
Way are.in a suitable position for examination
(see differential counting of leucocytes, p. 41).
'..3. In the case of very anaemic bloods, e.g.,
those of ‘malarial cachexia,’ difficulty will arise
from the film being too thin. The needle in this
case must be carried very loosely and rapidly along
the slide and a thicker film thus made. When
blood with difficulty adheres to the slide, good
evidence of extreme anaemia is obtained.

THe PREPARATION OF WeT FILMS

A wet film is not so easy to make as a dry
film, and requires cleanliness and rapidity of
manipulation. Wet films are therefore difficult to
make in dusty countries, where a single particle of
grit will mar the process.

Before proceeding to make films, several glass
slides and coverglasses should be carefully cleaned
and polished with a dry pocket handkerchief, and
wrapped in clean smooth paper, to ensure the
absence of dust or grit. In making a wet film the
result may be marred by—

t. Too small or too large a drop of blood.

2. Tooslow manipulation allowing the drop
of blood to partially clot.

. An uneven coverglass or a coverglass with
a minute bubble or speck in its substance.
4. Dust of any kind.

6

To Maxe a Wet FI_m

The same procedure is gone through as in the
case of a dry film. It is well to polish the slide
and coverglass immediately before use with a clean
handkerchief. When the exuding drop of blood
reaches the size of a small pin’s head, a coverglass
is picked up rapidly with forceps, or the edges are
grasped between finger and thumb, and allowed
to touch the dsop without ‘ dabbing’ the skin, and
again rapidly placed drop downwards upon a
slide. A gentle tap or two with a needle or
forceps may aid in the film formation, but the
pressure must not be great or the corpuscles will
be found laked and invisible.

The vequivements of a suitable wet film are :—
There should be a central transparent area shew-
ing no sign of a granular appearance, and even
looking quite free from blood. If this appearance
is present, the film is probably a good one. If the
centre of the film appears reddish or granular, it
is useless to examine it (for young parasites), as
the corpuscles will be massed together and the
parasite not seen. Under the microscope a good
film should shew clear, even, circular discs, lying
side by side and not overlapping each other. It
may be necessary to make several before a suffi-
ciently good one is obtained.

To Lapet Fits

Films should always be labelled as soon as
possible, otherwise uncertainty and annoyance are
sure to arise. The use of paper labels is not at all
necessary in routine work,

7

1. The most convenient method is that of
writing on the end or back of the slide with
ordinary ink. This should be quite dry before
placing in alcohol. There is then no fear of its
coming off.

_ 2. Anexcellent and extremely simple method
of labelling has been described by Dr. Powe t,
(Bombay), viz:—After making a dry film, as
described above, the name, date, and other
necessary information, are scratched on the film
with the head or point of the needle. The films
used being extensive, the writing in no way injures
them. The first half inch or so of the film is
frequently rather thick, and much information as
to name, date, temperature, etc., may safely be
written on it.

To Store FitMs

Slide boxes may be used, holding the slide
vertically. These should be well cleaned out if
made of wood, otherwise fine sawdust accumulates
on the slide. A size which will go in the pocket,
and holds about twenty-five slides, will be found
a great convenience for daily work. Larger boxes
to hold one hundred or so are best for use at home;
half-a-dozen of these may be enclosed in a stronger
outside case. Ina square foot of space something
like 1,500 slides can be stored in this way.

If no box is at hand, films may be wrapped
in clean white paper; a fold of paper being placed
between each slide. For transmitting half-a-dozen
films or so this is quite the most convenient way,
the whole of course being packed in a box or tin
with wool.

8

Both fixed and unfixed films rapidly develop
moulds in damp and hot climates. Moulds appear .
as branching threads under the microscope.
certain amount of mould does not much interfere
with the utility of films if they are intended only
for detecting the presence of parasites, counting
leucocytes, etc.

Unstained films kept for six months or more
stain as a rule badly, and are not of much use.
Stained films (as we shall see, without putting on
Canada balsam and a coverglass) will shew ex-
cellent results after many years. Films that have
been unfixed at the time of making, and that may
have subsequently become damp, will have their
red cells destroyed, but the parasites will still
stain. ;

The method of wrapping one’s slides up in
paper (after duly labelling) is in practice the most
convenient one, especially if travelling, when not
unfrequently boxes of slides suffer much damage.

To Fix Fits

Until a film has been ‘fixed’ it is soluble in
water, and will be immediately washed off if
placed in water or any watery solution of a stain.
When fixed it is insoluble, and may be treated in
almost any way without destruction. Except
when using LeisHMan’s combined fixing and stain-
ing solution, the film must always be ‘ fixed.’

On returning home or to the laboratory, place
the films in a glass-stoppered cylindrical jar,
about four inches high and one-and-a-half inch
diameter, containing absolute alcohol. . When a
number of slides are taken, they are placed in

9

known order, and a blank one added at the last,
to avoid the possible mistake of reversing the series.
A few minutes is all that is necessary if one isin a
hurry. Otherwise they may be left in from one-
half to twenty-four hours as may be convenient.
They are then taken out, allowed to dry, and
placed in series in a slide-box, and labelled as
desired.

There is the possibility that absolute alcohol
may not at times be obtainable. Other fixing
methods are :—

1. Methylated spirits. (This is in fact quite
adequate for the fixing of blood films in the
absence of absolute alcohol).

2. By the use of Leisuman’s stain. (The
stain itself contains the fixative).

3. By heat. Heating to 100°—110° for five
minutes on a copper plate gives very beautiful
leucocyte preparations with Euruicn’s stain. This
method requires care, and a copper plate for
heating the slides. If greatly pushed, one may
often satisfactorily fix by passing the slides several
times through the flame of a spirit lamp.

4. For other methods, vide Appendix.

To Stain Fi_Ms

A large choice of methods is usually given for
the demonstration of the malarial parasite in
blood. There is one stain, however, so much
more strikingly effective and generally satisfactory
than other stains that, for routine use, no alter-
native method need be considered. This stain is
Romanowsky’s chromatin stain ; the modifications
of Le1sHMan will also be found simple and certain.

Io

In some circumstances the one will be found
more convenient; in others, the other. Both
methods are therefore given. The making up of
the stain, although apparently rather complicated,
is not in reality so, and the staining of blood films
is one of the simplest, most rapid, and certain of
methods. It is useless to attempt to prepare the
stain unless suitable methylene blue and eosin are
obtained. These stains are very inexpensive, and
the only care necessary is to order the exact stain
from a good firm (C. Baker, 244 High Holborn,
London).

By the use of Method 2, the staining of blood
films is so simplified that a bottle of stain anda
supply of water is all that is necessary for the pro-
cess. Process No. 1, however, is in some ways
very convenient and rather more certain, and was
largely used by us.

Method 1. The following materials are
necessary for the making of the stain, viz :—

‘Medicinal’ methylene blue.
Eosin extra (B.A. or A.G.) or simply pure
eosin for blood staining.
Sodium carbonate, pure.
Two stock solutions are made :—

Solution A. Methylene blue . 1’o part.
Sodium carbonate. 05 parts.
Water . : . 100 parts.

The solution is then placed in a hot incubator
or by the kitchen fire, or in the tropical sun for
two or three days. By this time a deep-purple
colour will be noticed at the edges of the liquid.
The colour depends upon the formation of a new
red body which, combined with eosin, forms the
active staining principle of Romanowsxy. Until

II

the purple colour is developed the solution is quite

useless.

Solution B. Eosin . 2 I part.
Water. . 1,000 parts.

For staining, these stock solutions are diluted
one in twenty, respectively, with water, 1.e., five
parts of the stock are made up to one hundred
parts with water.

To Stain.—Mix equal parts (say one c.c.) of
each solution and pour on the slides.

Leave the stain on the slides any time from
ten minutes to half-an-hour or longer. Wash off
the excess of stain with water, and allow them to
drain or dry them with blotting paper, but do
not dry them by heating over a flame. The red
corpuscles may have a bluish tinge. This can be
got rid of if desired by washing in water or very
rapidly in equal quantities of spirit and water.

Placed under the microscope, while still wet,
the blood platelets should appear as ruby red
granular masses, if they are bluish the film should
be replaced in the staining solution.

Slides may be decolourized to any required
extent by soaking in water, in fact, if left long
enough the stain is entirely washed out. Such a
specimen can, however, easily be stained a second
time.

The exact position and relations of pigment
are best seen in specimens lightly stained, as deep
Romanowsky staining may completely obscure
pigment.

Method 2. LEIsHMaN’s stain :—

LEISHMAN’S stain consists of the product of
inter-action of the eosin and the methylene blue
of the first method.

\

I2

Make the following solution, viz. :—
Leisumay’s stain . . O15 grammes.
Methyl alcohol . .. . TOO‘O C.C.

Place sufficient solution on the slide to cover
the film, and allow it to stand: for about half-a-
minute. Add about twice as much water. Mix
by moving the slide to and fro, or stir gently with
a glass-rod. Allow it to stain five minutes or
longer. ;

The stain is also sold in the form of ‘soloids’
(by BurRouGHS, WELLCOME & Co.), each ‘soloid’ =
oo15 grammes. If it is impossible to. procure
methyl alcohol (pure), dissolve the ‘soloid’ in
methylated spirit (ten c.c.) and proceed as above.
The results got with methylated spirit are per-
fectly satisfactory for diagnostic purposes. There
is no preliminary fixing.

TuLLocu recommends making a saturated solution of the
stain in twenty-five cc. of methylated spirit, to which two
drops of a ten per cent. solution of potassium bicarbonate have
been added,

After some minutes the slide will be stained.
The same red scum and precipitate are seen as
in Method 1, and are of the same significance.
The slide should, when stained, be washed in water
and allowed to remain in this for a minute or so.
This intensifies the Romanowsky staining and
removes the remains of the deposit. The red cells
also by this process are changed from greenish to
faint pink.

To obtain the most brilliant results with
these stains is perfectly easy, and no one who has
used them will, except for special reasons, use
any others at present in use.

oe

The advantages of the Romanowsky stain
(either method) as stated by LeIsHMAN are :—

1. The great beauty and brilliancy of the
staining.

2. The greater certainty of the detection of
young forms of the parasites.

3. The ease of application and certainty of
result.

4. The staining of the red cell in simple
tertian (SCHUFFNER’s dots).

In malignant tertian also a peculiar staining
of the red cell. is sometimes seen, especially
in overstained specimens not washed too much.
It consists of coarse blotches or clefts, and the cell
also as a whole stains a deeper tint than the
surrounding cells (Vide Fig. 4).

By fixing the films in chloroform instead of
alcohol the ‘malignant stippling’ is well brought
out.

14

Chapter II

NORMAL BLOOD *

It is difficult, without considerable experience,
to know exactly the interpretation to put upon
many appearances seen in blood films under the
microscope. The less carefully the film is prepared,
the more numerous are artifects and various
contaminations all broadly included under the
designation ‘ dirt,’ and, until one is used to recog-
nize these, mistakes from this cause are extremely
likely to happen. There is no way to get over
this difficulty except by experience. After a time,
however, artifects and ‘ dirt’ can never be mistaken
for a parasite.

We may point out some of the artificial
appearances that may be encountered :—

1. Ifin any portion of a film the red cells
shew as double outlined circles, or have a central
spot, or indeed give any other appearance than

* In examining unstained specimens use :—
1. Concave mirror.
2. The Iris diaphragm partly closed.
3. The condenser may be racked down or dispensed with.
For stained specimens use :—
1. The condenser racked up.
2. The flat mirror.
3. The Iris diaphragm almost wide open.

Always keep an eye-piece in the tube of the microscope, if a lens is at the other
end, to prevent dust getting in, and always wipe the oil off the oil-immersion lens
with a little xylol before putting away. An oil-immersion lens generally keeps
quite well in the tropics, especially if in use.

a3

that of perfectly uniform disks, this portion of the
film should be passed over. If the whole of the
film is of this character, it should be discarded.

2. It is well to have had the parasite
demonstrated to one both fresh and stained. After
this, little or no difficulty will be experienced in
recognizing the parasite when it is really present.
Should there be any doubt, the object seen is
probably not a parasite. A parasite stained pro-
perly by the Romanowsky stain has always a blue
body with a bright red dot or dots, and a more or
less clear, unstained, whitish (vacuolic) area. Its
definite outline, whether circular or elongated,
should be quite clearly made out—then no pos-
sibility of mistake should arise.

3. Artificial bodies in the red cells are gener-
ally to be detected by their occurrence in nearly
every corpuscle in one portion of the field, and
not in another. When a really well-spread portion
of the film is reached, they are no longer seen. A
common artifect of this nature is a small granular
mass stained reddish, apparently in the red cell.
It is caused by the staining of vacuoles in the cell.
The body has a granular appearance, but the blue
body, red spot, unstained area, and clear-cut out-
line of the parasite are quite wanting.

4. In fresh specimens, crenations or vacuoles
may simulate young ring parasites. It is, how-
ever, impossible to get a clearly defined edge to
these by focussing. Crenations appear as black
dots in one focus, and as bright dots in another.
Vacuoles have not the peculiar solid look of young
parasites, and cannot be clearly focussed.

| Masses of bright yellowish-brown pigment
derived from the skin are common in films made

I6

without cleansing the finger. They have no rela-
tion, however, to any surrounding parasite.
Micrococci, yeast, etc., are commonly found in
blood films, especially in the tropics. Further, we
have found from much experience that the com-
monest bodies to mistake for parasites are platelets
and, strange to say, leucocytes. Platelets (stained)

Poly morphonucleay Sma tt Large

Mmunonuclear

Eotimophyl Inlay medcalé
@)
9,
x 8
Fon wee 8B
e

Fig. 2. Novmal constituents of Blood :—
M = Mast Leucocyte

may show a great variety of shape. They may
be sausage-shaped, oval, or elongated, or they may
vary in size from about one-fifth of a red cell to
masses three or four times, or even more, as great

17

as the red cell. Frequently, too, platelets are
surrounded by what looks like a definite clear
outline, but a closer examination will show that
the resemblance to a parasite is only superficial.
The mass is gvanulay throughout, a parasite is
not. The staining is uniformly reddish or blotchy,
blue and red, it is not divided as in the parasite
between a definite blue area and a definite red
dot or dots.

Leucocytes, we have said, are also not
uncommonly taken for the larger forms of parasites
(e.g., gametes), but only a beginner could possibly
make such a mistake, as the leucocytes have a
large densely staining mass of red (the nucleus)
forming a considerable proportion of the whole
cell mass, whereas in the gametes there is only a
patch or so of red amidst the blue.

Dust on the eye piece is at once detected by
rotating the eye piece when the body shifts its
position.

THe NorMAL CONSTITUENTS OF THE BLOOD

Normal blood should be carefully studied in
fresh and stained specimens.

1. The Red Cells — With Romanowsky’s stain
these are only faintly stained reddish (Method 2),
greenish or bluish in colour (Method 1). Their
apparent size, 7.e., the area they occupy when flat-
tened out, depends upon the thickness of the film;
in well-made thin films they are large, and stain
with beautiful uniformity. In fresh specimens
(wet films) they ought to appear as perfect,
uniformly straw-coloured discs ; 1f crenated, it is
impossible for the beginner to detect parasites.

Cc

18 -

2. Leucocytes—The following types should
be clearly made out in stained films :— __

The Polymorphonucleay Leucocytes (Fig. 2).—
These are very characteristic, and a reference to
the diagram will make their recognition easy. It
will be noted that they have a very irregular
nucleus and fine granulations (stained red by
RoMANOWSKY).

The Small Mononuclear Leucocytes (Fig 2).—
These, the lymphocytes, are readily seen and can
scarcely be mistaken for other forms. Their ap-
pearance varies somewhat with the thickness of
the film. The thinner the film the larger they
appear and the greater the area of protoplasm
surrounding the nucleus. In typical forms the
nucleus is dark staining and nearly spherical.

In wet films, a proportion of .these cells shew
a dark refractile spot (Manson’s spot), which
might be mistaken for a pigment granule, but, as
we shall see later, they cannot possibly be confused
with a typical pigmented leucocyte.

The large Mononuclear Leucocytes (Fig 2).—
It is well to get thoroughly familiar with the
appearance of this type of leucocyte, as the per-
centage of these is of great diagnostic importance
in malaria.

Typical large mononuclear leucocytes are
readily and clearly distinguishable. They are the
leucocytes which may contain malarial pigment,
and the recognition of a pigmented leucocyte gives
a clear idea of their characters.

(i) They are in thin films of considerable size,
half as much again to twice the size of the small.

(ii) The nucleus is large, oval, eccentric, not
nearly so dense as in the case of the small—as is

19

shown by its less intense staining. They also
present indentations giving a partly bi-lobed
appearance.

(ii) The area of protoplasm surrounding the
nucleus is considerable. It is clear, and contains
at most a few scattered granules (ROMANOWSKY
stain). The only difficulty will be found to arise
in the case of a comparatively small number of
‘intermediate’ and ‘ transitional leucocytes.’

Intermediate Leucocytes (Fig. 2).—These are
forms intermediate between the large and small
mononuclear forms. They are usually classed
along with the large forms, the characteristics of
which they generally more nearly approach.

Transitional Leucocytes (Fig 2).—These are
very characteristic, and when seen will be at
once recognized. In shape, the nucleus approaches
that of the polymorphonuclear forms, being trident-
shaped or S-shaped. In consistence, however, it
is obviously related to the nuclei of the large
mononuclear cells. As a rule these cells are small
in number, and from their close resemblance to
the large mononuclears may be included with
these.

Eosinophil Leucocytes (Fig 2).— The large
granules with which these are packed suffice to
distinguish them. The granules are stained pink
or blue (peripherally) by Romanowsxy. The
nucleus in the eosinophil cells is frequently
characteristic, consisting of two spherical portions
united by a thin strand of nuclear material ; it is
really of the polymorphonuclear type. |

Mast Leucocytes (Fig. 2, M).—These, in a film
stained by Romanowsky, are cells crowded with
granules stained deep blue or nearly black.

20

These cells occur.as isolated specimens in normal
blood. They form about 0’5 per cent. of all white
cells.
3. Platelets (Fig. 2)—Bodies of various
sizes up to one-third diameter of the red cell,.
nearly always lying in clumps of from six to fifty,:
and stained bright crimson. They often show a.
considerable amount of differential staining, but
differ entirely in appearance from parasites, more
especially in having no blue stained mass. An
isolated platelet lying upon a red cell may
simulate a parasite. I*or the difference between
it and a parasite, vide. above, platelets often:
occur in large numbers in cases of malaria and,
perhaps, especially in blackwater fever.

4. Blood Dust or Granules.—Small granules,
smaller than micrococci. In fresh films they exhibit
active motion (? Brownian). :

Rk
Aormoblast” mega loblast”
Fig. 2a

Among abnormal constituents of blood we
may mention :—

1. Nucleated Red Cells.—In conditions of loss or
destruction of blood cells, e.g.,.malaria, it is com-
mon to see nucleated forms of the red cell in the
blood. They are characterized by a small globular
nucleus with sometimes one or more little buds,
staining almost black with Romanowsxy. If the

21:

film be counterstained with eosin, the fact that
the surrounding pale area is red cell will become
evident.

Two forms may be seen :—

(a) Normoblasts, 7.e., nucleated red cells the
size of a red cell (Fig. 2a).

(b) . Megaloblasts, oe nucleated red cells
much larger than a red cell (Fig. 2a).

Myelocytes

Fig. 28

Normoblasts are the form usually seen.
Megaloblasts in excess are found in ‘pernicious
anaemia.’

2. Deformed and Small Red Cells may be seen.
—These are known as poikilocytes and microcytes.
They are common in severe anaemias, especially
pernicious anaemia. It is quite exceptional to
find deformed cells in blackwater fever. The red
cells are generally quite normal in shape, though
anaemic in varying degree.

3. Abnormal Leucocytes.—Under certain con-
ditions, e.g., malaria, but especially myelogenous
leukaemia abnormal leucocyte forms are seen
which normally are only found in the marrow, 2.e.,
myelocytes. These belong to the large mono-
nuclear class, and may be of two kinds, either with
large eosinophil granules as in the eosinophil
cell, or fine neutrophil granules as in the poly-
morphonuclear leucocytes (Fig. 2B). If large

22

mononuclear forms are seen crowded with |
granules, films should be stained with EsR.icn’s
triacid stain, in order to accurately determine the
forms of leucocyte present.

4. Frequently in malaria films (stained)
large open meshworks of nuclear matter are seen
with little or no surrounding protoplasm. These
are degenerated or dropsical, or, according to
others, mechanically damaged leucocytes, and
often occur in great numbers.

5. RedCells with LongWavy Processes.—These
are seen especially in anaemic bloods after the
fresh film has been under examination for some
time. They occasionally break off and float about.
Shorter and more granular processes emitted by
the red cell are even commoner.

6. Further, we must point out an extra-
ordinary appearance of the red cells in stained
films, so far as we are aware not hitherto described.
In anaemic (malarial) bloods, we find red cells, ten,
thirty, or forty times the diameter of a normal
cell, and these huge swollen structures shew at
one side a crescentic area which is granular, and
is the only remaining part of the red cell that can
be recognized ; the remainder is practically un-
stained. These gigantic structures may or may
not be occupied by parasites.

a

Chapter III

THE DETECTION OF THE MALARIA
PARASITE

EXAMINING THE FILM

After staining and drying, the film is ready
for examination. No Canada-balsam or coverglass
need be applied. A drop of cedar-wood oil is
placed upon the film and the oil immersion lowered
into it.

After the examination is completed, if it be
desired to keep the film, the cedar oil is dissolved
off by dropping a little xylol over the film and
allowing this to drain off and then to dry. After
drying, the film can be put away and kept in-
definitely. If not needed the slide is: placed on
one side with others and eventually cleaned.

To CLEAN Dirty SLIDES

1. Rub with turpentine (benzine or xylol) to
remove any adherent oil.

2. Wash with soap and water.

3. Rinse in water.

4. Dry and rub well with a clean cloth.

24
Tue DETECTION OF THE MALARIA PARASITE

We propose to describe first the actual
appearances which are likely to meet the eye, and
later to give a systematic description and mode
of distinguishing the various forms of parasite.
A stained specimen (RomaNnowsky) should always
be used for the purpose of making a diagnosis.

6) ey) ©
O &

Suacl forms Gmuchoud forms harge forms

Creseenl Oval hody Round body

Fig. 3. Forms of the malaria parasite commonly met with in the
blood :——The dark dots in the first line represent chromatin,
the fine dots, pigment.

We may first note that it is not necessary, as 1s
often thought, to examine the blood at any par-
ticular time, but it is very necessary that the
patient should not have taken quinine previously.
Even five grains of quinine may so diminish the
number of parasites as to make detection a
laborious task, and anegative result under these
conditions is not conclusive.

In examining the slide it is a very convenient
method to begin at the edge of the film and to
work systematically towards the ‘ tail’ end.

a)

The following forms of parasites may be
seen :—

(i) Small forms looking more or less like rings,
or stained streaks lying across or apparently stuck
to the side of the red cell.

N.B.—Parasites free in the plasma are practically never
seen.

(ii) Larger stained bodies of various shapes
and sizes more or less filling the cell.

(iii) Crescents or large round or oval bodies
with a cluster of coarse pigment placed more or
less centrally.

1. Ring Forms (Fig. 3)—These may be
quite small, one-sixth of a red cell in diameter,
or much larger, one-third in diameter.

Rings are parasites of very distinct outline
and structure. The part of the parasite that will
first be noticed in a RomMANowsky specimen will
be the red nucleus (chromatin), a clearly stained
bright red dot (or dots). This is generally situated
on the margin of the blue ring, which is equally
distinct in outline, though often only a faint blue.
The blue ring encloses an unstained vacuolic area.
These rings stand out so sharply that they appear to
project from the corpuscles. The red dot gener-
ally forms the signet of the ring (signet forms),
but also may occur in the centre of the vacuole.
The red nucleus or dot is often also rod-shaped
or angular. The rings may shew a very faint
blue outline or a thicker portion on the side
opposite to the nucleus.

Though generally called ‘rings,’ these para-
sites are really discs, or saucer-shaped bodies,
adhering to the sides of the red cells.

Besides these young rings, we have irregular
forms of considerable variety, e.g., a mere faint

26

bluish line stretching across the corpuscle, yet
always shewing somewhere a red nucleus, or a
mere streak along the margin of the red cell, with,
however, a red nucleus in the blue protoplasm
(accolé forms).

Finally,no smallstructure should be diagnosed
as a parasite unless it is clearly made out that it
has three distinct parts.

(i) A red nucleus.

(11) Blue protoplasm or body. ;

(iii) A vacuolic area within the ring (in the
irregular forms this cannot be distinguished).

No confusion can then possibly arise with a
platelet or stained vacuole or dirt.

A nucleated red cell has not these characters.
Nor, again, has a red cell shewing polychrome or
basophil staining, 7.e.,a purplish or bluish mottling
all over. In fact, no other body has the definite,
quite easily distinguished characteristics of a
parasite.

2. Large Intra-corpusculay Forms (Fig. 3).—
They appear as more or less extensive areas of
blue protoplasm, with one or more distinct, red
areas. Pigment may be seen scattered over the
parasite. These large forms are generally simple
tertian or quartan parasites.

3. Crescent and Crescent-derived Bodies—T hese
are most definite bodies, and readily recognized
by the coarse pigment granules centrally situated.
The presence of this pigment should absolutely
preclude the possibility of mistaking distorted
red cells crescentic in shape, or a crescentic mass
of platelets, for parasites. In neither of these is
there a definite central pigment mass, nor should
a foreign body be mistaken for a crescent.

a7

Moreover, crescents again have quite definite out-
lines, and shew a red-stained central portion and
blue extremities.

Fig. 3a. Pigmented Large Mononuclear Leucocytes

The same criteria apply to the spherical form
of the crescent.

4. Pigmented Leucocytes (Fig. 3a).—Large
leucocytes with a large nucleus. Pigment (melanin)
may occur scattered about the periphery of the
cell, or in little clumps, or even in very fine powdery
grains. The pigment is brownish-black in colour.
Skin pigment may. be seen in epithelium scales or
free in the plasma, but the definite position of
the pigment in the protoplasm of the leucocyte
characterizes melanin.

APPEARANCES IN A FRESH SPECIMEN

1. Rings.—The very small forms of these
are characteristic of malignant tertian infection.
They measure about one-seventh the diameter of
a red cell. A ‘ring’ is characterized by its rather
opaque white look, its very definite contour, and
by the fact that the central portion is of the same
colour as the red cell, which isin fact seen through
its substance. The ring has often a thickening
at one point giving the ‘signet ring’ appear-
ance. On watching sucha ring from time to time

28

it is seen to alter its shape. The diagnosis of a
ring from a vacuole is not difficult if the latter 1s
carefully focussed,when it will be found impossible .
to get a sharp outline as in the parasite, the outline
on the contrary ‘opens out.’ Crenated corpuscles
are very frequently taken to be parasites. The
crenations focus alternately as dark and bright
points, and they have no clear ring outline.

‘e. @@ %
OO ofa Bp

ae ae O®

eo, 1 @
5 & @

Fig. 33. 1. Thin portion of film. 2. Thicker portion:
(a) a distorted corpuscle with no pigment; (b) a crescent with
pigment. 3. Cvrenated corpuscles. 4. Commencing crenation.
5. (c) Red cells with processes; (d) a male gamete with flagella
and pigment. 6. (e) vacuoles; (f) acrack; (g) ayoung parasite
with very fine pigment; (h) a segmenting parasite with central
pigment mass.

Isolated platelets in the plasma or lying on a
corpuscle appear as little granular masses, but
again they have not the opaque white look of
parasites nor the definite ring form, nor are they
amoeboid. Here, as in the stained specimen, the

definiteness of the body should prevent mistakes
being made.

29

_ In young tropical or malignant tertian rings
pigment is very rarely seen. In larger rings pig-
ment may be seen, e.g., in simple tertian rings.

2. Large Forms.—They may occupy the
whole of the cell and may shew active amoeboid
movement and pigment in active motion may also
be seen. The pigment may be reddish-brown, very
fine, or rather coarser black pigment. The
oe of these forms will be considered
aver.

3. Crescents and Spherical Bodies (Fig. 3).
The former are at once as in the stained
specimen characterized by their shape. They are
distinct, fat, plump-looking bodies, unmistakable
when once seen. They always have, besides, a
central ‘clump of distinct pigment. Stretching
across between each end of the crescent is seen the
curved edge of the red cell. ‘The spherical bodies
also: possess this definite, easily seen pigment mass.

In fresh specimens, further, the extremely
beautiful and striking process of flagellation can
be readily seen, provided a suitable case is used.

FLAGELLATION

Select a case of (simple tertian or) malignant
tertian infection, in which parasites have been
found. On examining the latter about a week later,
crescents will be found in the blood.. In about
twenty minutes, or in hot weather in England in
five minutes or less, many of these will be seen to
become spherical and to get free of the corpuscle
in which they were situated. Two varieties may
be distinguished—the male in which the-pigment
is distributed over the whole of the parasite, and

30

the female in which the pigment is concentrated
into aring or figure of 8. Observe that attached to:
these spherical bodies small ring-like bodies occur,
one to two in number, about as big as a pin’s
head. These are the so-called ‘polar’ bodies.
They occur in the male and female, and, as seen
in stained specimens, consist of little circular
masses of chromatin. These changes orcur in
the tropics very rapidly, so that the examination
must be commenced as rapidly as possible.

On watching these spheres, the pigment in
some will be seen to be in active motion—this
probably indicates the internal changes prepara-
tory to extrusion of flagella. Suddenly one of
these spheres is perceived to be oscillating violently,
and in amoment three or four or more pale, long
processes are emitted. The red cells all around
are put in motion by their violence, and it may
be only after a time, when the activity has grown
less, that the flagella are actually seen. Nodosities
will be observed in the flagella, and occasionally
a speck of pigment at their extreme end. The
flagella, after a time, break off, but they have
only once, by MacCattum, been seen penetrating
the female gamete.

Under certain unknown conditions the cres-
cents do not become spherical and eventually
flagellate, but remain as crescents.

Breathing on the slide, adding a trace of
water, etc., have been recommended to produce
the change more certainly, but it is probable that
the real cause lies in the state of development of
the crescent for certain observations, e.g., those of
Major Bucuanan, I.M.S.,shew that there is a certain
time after the fever when a maximum number of
gametes flagellate.

a1
To Stain FLAGELLATED BopIiEs.

1. When flagellation is observed the cover-
glass is forcibly ‘smeared’ off, and slide and
coverglass are then fixed and stained with Roman-

~ OWSKY.

2. A number of rather thick drops of blood
are placed on a series of slides. These are inverted
over rectangular holes cut in blotting paper,
moistened with water, and spread on a sheet of
glass. A series of moist chambers is thus made.
A dozen or more films are made, and each one is
removed at intervals of five minutes, dried
(spreading out somewhat if necessary), fixed, and
stained with Romanowsky; or dry the thick
film, decolourize with water, stain with Roman-
owsky (without fixing), as in Professor Ross’
method of making thick films.

To DETERMINE THE SPECIES OF PARASITE
PRESENT

Three forms are recognized—simple tertian,
malignant tertian, and quartan. The malignant
tertian can, as we shall see, produce a quotidian
temperature with only a single generation of
parasites. Whether or no there is a true quo-
tidian parasite, one or more, is extremely doubtful.
tr. Minute rings, one-sixth to one-seventh
the diameter of a red cell, showing the signet
ring shape, are characteristic of malignant tertian
(Tig. Su
Large rings.—If, when the temperature
of te patient is still high, the rings are of con-
siderable size, one-fourth to one-third of the red

32

cell, they are, probably, simple tertian or quartan.
If, on the contrary, the temperature is low and
the febrile attack finished, these forms correspond
to fully developed malignant tertian parasites.

Terlian
2oarlan
Creseenl™
Terlian,

Fig. 4. The three species of Malarial Parasites

The large malignant tertian rings have a
characteristic appearance. They are oval, with a
thicker layer of protoplasm (blue) opposite the
nucleus (red).

3. Large forms, with considerable blue
protoplasm and with pigment granules, are, prob-
ably, simple tertian or quartan.

oo

The tertian parasite is an irregular and
flimsy-looking body, and the medium sizes may
show several pseudopodia (Fig. 4). Pigment
is scattered throughout and is actively motile,
while the quartan parasite is oval or globular, of
compact appearance, with darker, coarser pig-
ment, shewing but slow motion (Fig. 4).

The enlargement of the cell in which the
simple tertian lies is-also very characteristic.

In a well-stained specimen we have the
further characteristic differences.

1. Simple Tervtian—The cell is dotted all
over with fine red granules (ScHtrFNeErR’s dots),
these cells strike the eye during the microscopic
examination and are diagnostic (Fig. 4).

2. Malignant Tertian.—In specimens deeply
stained with Romanowsky, the malignant tertian
parasite also produces changes in the red cell
(Fig. .4). These consist of coarse dots or
clefts, especially around the parasite. They are
few. in number and are equally characteristic of
this parasite. Their appearance is quite different
from SCHUFFNER’S dots.

Maurer recommends the following method
of developing them :—

10 drops of methylene blue (stock solution)

+ 25 c.c. of tap water.
15 drops of eosin (stock solution) + 25 c.c.
of water. ,
Mix and stain for five minutes; shake actively |
the whole time. ee

3. Quartan.—The red cell shows no altered
staining characters, but it may appear even
smaller than normal. The parasite is not irregu-
lar in shape, but compact, oval, or globular. (In

D

ae

the fresh specimen the parasite is very refractile
and has a peculiar opaque look) (Fig. 4).

The finding of crescents, is, of course, diag-
nostic of malignant tertian, but the possibility
of a double infection, e.g., simple and malignant
tertian, must be borne in mind.

1 Quarvtan Parasites: segmenting forms _
2. Simple Tertian
3. Malignant Tertian 6
4 Quotidian (after Z1EMANN) ,,

”

We have so far described the forms generally
encountered during a febrile attack and the
means of making a diagnosis, but it is necessary
to consider other forms, e.g., the sporulating forms,
and more especially the gametes.

Sporulating Forms.—Besides the sporulating
forms or segmenting forms, we can recognize also
the presegmenting forms, in which the pigment
begins to collect into a single mass and the.
chromatin gets split into a number of fragments.
These are even commoner than the final or ,
sporulating forms, in which the segments or |
spores are arranged around acentral mass, though
frequently here also the appearances do not ‘cor-
respond with the diagrammatic exactitude of the
text-books. The segmenting and presegmenting
forms are best seen in a case of regular quartan.

35

_ _Quartan Sporulating Forms—The pigment
is placed centrally or often laterally, and grouped
around.it can be seen several, six to eight, chro-
matin masses. In the presegmenting forms the
pigment has not yet condensed into a single bloc k,
and the distribution of the’ chromatin masses is
still irregular.. In fresh preparations the typical
‘daisy’ forms can be clearly'seen (Fig. 5).

Simple Tertian Sporulating Forms.—Here the
whole parasite mass is larger, and fifteen or more
chromatin segments can be distinguished
(Fig. 5).

Malignant Tertian Sporulating Forms.—
Rarely seen inthe circulation. There are eight
to ten chromatin masses (Fig. 5).

GAMETES

Simple Tertian—The young forms which,
under certain unknown conditions, also appear in
the circulation are characterized by the fact that
the chromatin appears in the centre of the vacuolic
area (RuGE), while in the asexual forms (schizonts)
it is applied laterally.

The full-grown gametes are much more easily
distinguished. The female gamete (¢?) is charac-
terized by the possession of much protoplasmic
matter, staining deep blue with Romanowsky and
little chromatin; in the ? the chromatin is laterally
placed, and is generally surrounded by a thin
vacuolic area, the pigment is black in colour, and
is irregularly scattered over the whole protoplasm
(Fig. 6).

The male gamete(s). The chromatin is more
voluminous than in the female, it is of a looser

30

texture, that of the @ being compact; the chro-
matin is centrally placed or extends in a broad
band across the cell. The male gamete stains
a characteristic greyish-green or greyish-red
colour with Romanowskxy. It has little blue,
so that the pigment is clearly seen, yellowish-
brown in colour, while the female stains a deep
blue, more deeply than the schizonts (z.e., asexual
forms) (Fig. 6).

Inale form

fem ale form

Simple Tether

Fig. 6. Male and Female Gametes (after Scuaupinn)

Further, there is the difference that asexual forms
of a corresponding size would show several
portions of chromatin corresponding to pre-
segmenting forms.

Malignant Tertian.—The young gametes also
occurring in the circulation are characterized
according to MauRER :—

(i) By their accurately spherical form.

(11) The ring is of the same thickness all
round.

(iii) The nucleus forms a portion of the ring
and does not project in various forms as

in the schizonts.

(iv) The absence of ‘coarse’ stippling in the

red cells.

Aromalin
artes

A

Fig. 7.

Malignant Tertian Gametes (partly after MauRER).

Right-hand corner: Spherical body (fresh)

(v) A circumferential red-staining line soon
develops as in the mature gametes.
The adult gametes.—(Vig. 7).

MixrocamerocyTe (g) Mare
Crescent

1. The chromatin of the
nucleus occurs in an extensive
loose network, occupying tle
greater portion of the parasite.

2. Comparatively little
blue staining protoplasm.

3. ‘The pigment is scat-
tered.

The shape is some-
what kidney-shaped, shorter
and broader than in the 9.

Maxrocamete (9) Femae
CRESCENT

1. ‘Vhe chromatin occurs
in a compact mass more or less
centrally p'aced.

2. Muchmore bluestain-
ing protoplasm.

3. ‘The pigment is con-
centrated into a ring around
the nucleus or heaped up into
mass.

4. Crescentic in share,
longer and narrower than the
male.

38

STAINING REACTIONS OF Gametes (MALIGNANT
TERTIAN)

‘Arcutinsky,’ who has. lately ‘studied this
question of stippling by means of a modification
of the Romanowsky stain comes to the following
- conclusions. By his-miethod he is unable to obtain
stippling of the young asexual forms, which we
have ourselves often obtained, and which. MaurErR
also confirms, but he obtains stippling ofthe red
cell’ infected by the adult gametes (crescents).
These forms were also seen by us in West’ Africa
while using a very active haematein stain ;* but
they are certainly not shown by the usual Roman-
‘owsxy methods in use. ARGUTINSKY’s results
depend upon his fixative acetic-osmic vapour ;
while then he differs in not having obtained stip-
pling of cells containing ordinary ring forms, yet
his criteria for distinguishing the gametes agree
with those generally received. With regard to
the stippling in the mikrogametocyte (male) there
isa single peripheral row of finer and less intensely
stained dots than in the red cell surrounding the
makrogamete. The rim of red cell surrounding
the parasite is also narrower in the case of the
male.

Quartan Gametes.—Presumably similar differ-
ences exist as in other forms of the parasite, but
they have not yet been described, and, in fact,
gametes are often very rare in quartan Cases.

_ Note.—Gametes (crescents) may be but rarely found in
the blood of Europeans in the tropics (West Africa).

1 C. fiir Backteriologie, Bd. xxxiv, S. 144.
2 Reports to the Malarial Committee. Harrison & Sons. Series iii, p. 8.

39

‘Chapter IV
THE SUBSIDIARY SIGNS OF MALARIA

When patients have taken quinine it is not
uncommonly impossible to find parasites in the
peripheral blood. Apart from the actual presence
of the parasites, one may still derive evidence of
malarial infection from

t. The presence of pigmented leucocytes.

2. An alteration in’ the proportion of the

leucocytes.

Pigmented Leucocytes——Pigmented leucocytes,
even in severe malaria, are often very few, and
often require for their detection prolonged search
in large films. In other cases, however, they are
abundant. The presence of very few is quite
compatible with a severe malarial infection.
For instance, in two cases seen by us only very
few were found in the peripheral blood, but in the
spleen, post-mortem, enormous numbers occurred.
To detect them, it is necessary to make large and
good films by the method already described. By
following the margins and termination of the
film, the majority of leucocytes in the film will
have passed beneath the eye, and pigment, if
present, is readily seen.

In the vast majority of cases, the pigment
will be found in the large mononuclear forms, and

40

only very rarely indeed in the polymorphonuclear
forms. As a rule, a pigmented large mononuclear
(Fig. 3) is crowded with granules of pigment,
the presence- of only a few grains, or a single
granular clump, is exceptional. The appearance
of the clearly defined yellowish-brown or black
pigment granules in the clear protoplasm is so
characteristic, that no doubt ought to exist. It
should be remembered, however, that in dirty
films, specks of dirt may be over a leucocyte, and
so resemble pigment. In this case, similar specks
will be found lying free. The occurrence of
malarial pigment free in the blood has never been
seen by us.

Leucocytic Variation.—Often in cases where
pigmented leucocytes are difficult to find, there is
a very obvious increase in the percentage of the
large mononuclear leucocytes. ‘This change, which
is usually very pronounced in the apyretic periods
of an attack of malaria, is, however, most fre-
quently absent during pyretic periods. If, during
a period of low temperature, this change is not
found, there is a strong presumption that the case
is not malarial. If the blood be taken at the
height of the fever, a negative result does not ex-
clude malaria, and a further examination should
be undertaken, if possible, during an apyretic
period. In some cases, the change can be detected
even during the pyretic periods, but in these it is
always more marked in the apyretic. In some
cases, during the course of the fever, no such
change occurs, but appears immediately the tem-
perature subsides, and diminishes as convalescence
proceeds. Perhaps the cases where this test is of
the greatest value, are those where the patient has

41

already been treated with quinine, and one can
scarcely hope, even if the disease be malaria, to
find parasites in the blood.

To Make a DirreRENTIAL CouNT OF THE
LEUCOCYTES

Large films are necessary, especially .in
malaria where, during the apyretic period, there
is a distinct diminution in the total number of the
white cells. It is important, in making films for
leucocyte counting, that the margins and terminal
points of the film’ be regular, and so in a
convenient position for examination (Fig. 1).
The margin of the film is focussed and passed
beneath the objective. By passing along one-
half or the whole of the margin of the film, the
great majority of the leucocytes in the film are
seen. In order to obtain accurate results, one
thousand leucocytes should be counted, but a
count of three or four hundred is generally suffi-
cient for diagnostic purposes. Counts of a smaller
number of leucocytes are valueless, as too great
variations will occur.

As a leucocyte is seen, it is marked under the
heading, large mononuclear, intermediate, transi-
tional, small mononuclear, polynuclear, eosinophil,
as the case may be. As many as ten to twenty
or more are mentally noted before making each
record in its column.

From the results obtained by blood counts of
a considerable number of Europeans living in the
tropics, we found that an increase beyond fifteen
per cent. of the large mononuclear forms is proof
of an actual or recent malarial infection, whereas

42

with a value of twenty per cent. it is almost
always possible, by long search, to find an
occasional parasite or pigmented ‘leucocyte. A
value of over twenty per cent. probably implies
actual infection at the time of observation.
The Normal leucocyte values are :-—

Polymorphonuclear leucocytes, 65-70 per cent.
Large mononuclear 2

Intermediate sala #

Small mononuclear 20-25 2
or lymphocytes

Eosinophil 2-4,

Other forms of leucocytes, e.g., ‘mast’ ‘cells,
are always in extremely small numbers in health
(o'5 per cent.)

w™

43

Chapter V
THE PARASITE IN THE TISSUES

Tissues may be readily examined for the
presence of parasites or pigment in the following
way :—Place a minute portion of the tissue on a
slide, and with the end of another slide spread it

out as evenly and thinly as possible. Dry, fix,
and stain in the same way as a blood film. Para-
sites, if present, are in this way much more easily
and clearly seen than in sections. Spleen pulp,
bone marrow, kidney, liver, etc., give beautiful
results, and in the same way any secretion or fluid
can be examined. For certain tissues, e.g., bone
' marrow, it is advisable to fix in—

Absolute alcohol, 1 part,

Ether . : . 2 parts,
in order to dissolve out the fat present.

To PREPARE TISSUES

In preparing tissues for examination :--

1. Use as small pieces as possible, 1.2. at
most five mm. thick.

2. Use plenty of the fixing and hardening
fluid because the tissues contain much water, and
if the fixing fluid becomes dilute it acts as a
macerating agent.

3. See that the separate pieces do not cohere
to one another. Place some cotton wool on the
bottom of the vessel.

44

Corked collecting tubes will be found most
convenient and will hold ample material. The
large masses of tissues sometimes sent home are
of far less value to the pathologist than much
smaller pieces well fixed and hardened. Always
put a label zn the fluid, with the data written on
it in pencil, as well as the outside label.

FIXING

t. Alcohol is on the whole the most useful
fixing fluid. Small pieces of tissue should be put
directly into absolute alcohol. Larger pieces
should be placed in ninety-five per cent. alcohol
for two or three days, and then for twenty-four
hours in absolute alcohol. Intestine should be
spread on filter paper, as also nerves, or other
tissue, which it is desired to keep flat. When
removing the tissue from the paper, care should
be taken that no fibres of the paper adhere, as
they may prevent the proper cutting of sections.

For other modes of fixing vide appendix.

To Srore Tissuzs

Keep tissues in diluted alcohol (seventy-five
per cent. about). If kept long in absolute alcohol,
many tissues become very hard.

To EmsBep Tissues ror SECTION CuTTING

Except for very special reasons, embedding
in paraffin should always be the method employed.
Very general misconceptions exist as to the time

45

and trouble necessary to prepare tissues in this
way. It may, be pointed out :—

1. That the times usually given for immersion
in paraffin and other reagents are unnecessarily
long.

2. That the use of two paraffins for
embedding, a soft anda hard, is an unnecessary
and even harmful procedute.

3. That an elaborate apparatus for the
paraffin bath is unnecessary (vide later).

4. By using flat and very thin pieces of
material, sections of considerable area may be
obtained in a minimum of time. It is necessary
to cut thin slices of the raw material (1 mm. or
less in thickness), and place these upon a small
piece of paper or coverglass before placing in the
alcohol to harden. The paper keeps the slab from
becoming distorted, and enables one to cut sections
of the full area of the slab, say two-fifths inch
square.

5. By placing minute pieces of tissue (in
slabs on paper, if-a section of some size is needed)
directly into absolute alcohol, fixing, hardening,and
dehydration can be accomplished within an hour.

NEcESSARY APPARATUS FOR PARAFFIN SECTIONS

1. Cambridge Rocking Microtome.—The or-
dinary form is all that is necessary, costing about
five pounds. It is convenient to have a ball and
socket adjustable holder, which enables one to
change the angle of the block without remelting
the paraffin.

2, Razovs-—These may be hollow-ground
on one side, or on both, to a varying depth. For

46

general use a moderately hollow-ground razor is
used. Examine the edge under a low power
to see if any notches exist, if so they must bé
ground out ona hone. A ‘water of Ayr’ stone,
as long as possible, should be used and kept |
absolutely free from grit during use. The stone |
should be soft, capable of being scratched with a ~
pin, and as a lubricant water or filtered kérosene
oil may be used. After honing, the razor should
be stropped. On one side of the strop a minimum
amount of razor paste should be rubbed in
and the leather side should be kept scrupulously
clean and dry. If the razor is hollow-ground on’
one side only, it should only be honed on this
side.

Examined under the microscope the edge
should now present a clear, sharp line. It may be
tested on a thin hair, which it should easily cut.

lin Vesse( av Klateh Ylasses

a
Care ttn
° a y
—3 => ‘ /

A Capper plate

Fig. 8. Simple Embedding Apparatus

3. Embedding Apparatus.—A slab of metal
(copper), 12 x 3 x dinches. Heat this at-one
end, and place the vessel containing the paraffin
at a point on the slab where the paraffin is just

47

kept melted. This is the temperature for embed-
ding. This simple device serves all the purposes
of an elaborate paraffin oven (Fig. 8).

4. Alcohol.—Absolute alcohol in the tropics
has absorbed a good deal of water, and it is
necessary to dehydrate it.

Heat crystals of CuSO, till a white mass is
formed. Allow to cool, and place in a tall bottle
of alcohol. Allow to settle and decant off alcohol
as required. Add fresh anhydrous CuSO, if a
marked blue tint develops, or tie up the anhydrous
copper sulphate in a muslin bag, and place in the
alcohol pot.

Gelatine may be used to dehydrate alcohol ;
it must previously be washed free from salts by
soaking in water.

Xylol.—Xylol is the most generally satis-
factory agent for displacing the alcohol and allow-
ing the paraffin to permeate the tissue. Chloro-
form, wood naphtha, turpentine, oil of cloves, and
other substances may be used.

6. Paraffin —F or use in the tropics, paraffin
melting at sixty degrees will scarcely be found too
hard. At high altitudes a softer will be required,
and the right degree of softness must be deter-
mined and produced by mixtures of paraffin melt-
ing at 60° C. and paraffin of lower melting point,
say 50° C., such as is suitable for use in temperate
climates. .

To obtain paraffin suitable for use in a given
temperature place a block of paraffin in holder
and cut thin sections.

(a) If the sections curl very much the paraffin
is too hard.

48

(b) If the sections formed are forced together
(telescoped) the paraffin is too soft. ‘:

(c) Acertain amount of crinkling is usual
with thin sections, and can subsequently be got
rid of before mounting.

To Emsep TISSUES:

1. Alcohol.—Two to three hours, using dehy-
drated alcohol (vide antea) in excess, and changing
two to three times. If soft tissues, e.g., liver,
spleen, time is unimportant so long as dehydration
is complete. If fibrous organs, the least possible
time that will ensure dehydration (using ’ thin
slabs). Fibrous tissue becomes excessively hard if
left too long in alcohol, xylol, or paraffin; thus
skin and connective tissue require great care in
preparation.

2. Xylol (or oil of cloves).—Ten to twenty
minutes. When the tissues become transparent
they are ready, and should be transferred without.
delay to melted paraffin.

3. Paraffin.—Ten to thirty minutes. If a
tin trough be used, the tissues should not be
allowed to rest upon the bottom of the trough,
but be supported upon a strip of paper kept in
place by folding the ends over the edge of the
trough. A watch glass generally suffices.

Prepare a block for cutting by one of the
following methods :—

(i) Ifthe piece of tissue be small, smear a
watch glass with glycerine, fill with melted para-
ffin and add the piece of tissue picked out of the
bath with forceps, warmed by passing through
the fame.

49

(ii) Fold a piece of paper, so that by folding
a trough of required size is made. If an extra
length of paper be left at each end of the trough
it can be folded down and holds the rest in posi-
tion. Fill with freshly melted paraffin and
add the piece of tissue picked up with warmed
forceps.

_ (iii) Use metal pieces, supplied with most
microtomes, upon a slab of glass.

The following points should be borne in
mind :— .

(i) Fresh paraffin should be melted for the
block, as paraffin frequently melted, or kept melted
for long periods, does not form so uniform a mass
when cooled as freshly melted paraffin.

(ii) The more rapidly the paraffin is cooled,
the more uniform is the resulting mass. It is well
therefore, as soon as a well-marked surface crust
appears, to plurige the watch glass or trough into
cold water.

When cold, cut out a square block with the
tissue arranged in the position required for the
sections.

; . Cut sections.
Note (i) The angle the knife is placed at is
important, and must be found by experience.

(ii) It is well to use pads of paper to protect
the edge of the razor, where it presses against the
iron of the microtome.

(iii) To cut in ribbons, the top and bottom
edges of the block must be parallel and horizontal.
It is well to dip the block in soft paraffin, or
merely to smear the top and bottom surfaces of
the block with soft melted paraffin.

E

50

(iv) Cut as thin sections as will remain
intact.

To Strain aND Mount TiISsuEs

1. Ifthe sections are crumpled, float them
upon water just hot enough not to melt the
paraffin. They will become quite flat. Float the
flattened sections on to a clean slide. Remove
excess of water, and firmly press a piece of filter
or blotting paper over the section. Thoroughly
dry by holding a few minutes over the flame (care
being taken not to melt the paraffin), or by placing
for twenty-hours in a dessicator or warm oven.
No further fixative is generally needed. If neces-
sary, the shde may previously be smeared with
the merest trace of egg-albumen fixative. ; It must,
in this case, be dipped rapidly into the water and
quickly withdrawn. (Appendix.)

2. If the sections are flat they may be placed
directly upon a slide slightly smeared with fixative.
In this case, celloidin in oil of cloves is the best
fixative. (Appendix.)

3. Hold the slide or coverglass. with the
section over the flame till the paraffin melts.
Dissolve off the paraffin with xylol, and then
drop alcohol over the section. Place the slide
or cover-glass in water.

4. Stain.—The best stains for general use
are -—
(i) Haematein purissimus, saturated
solution in 70 per cent. alcohol 10 cc.
Alum solution (alum 50 grammes,
water 1,000 cc.) . : : 50 cc.

51

Stain for five to twenty minutes, according to the
depth of colour of the sections.

(ii) Methylene blue, or gentian violet.

(ai) _Counterstain, if desired, with watery
eosin. For the detection of pigment it is well to
stain a section faintly with eosin alone.

5. Pass through alcohol, oil of cloves, to
Canada balsam. In hot moist climates, the cold
produced by the evaporation of the alcohol
causes dew to be deposited upon the slide. When
the xylol or oil of cloves is added, this produces a
troublesome milkiness and may spoil the section.
To avoid this, all excess should be rapidly wiped
up after the use of alcohol, and the oil of cloves
added as quickly as possible.

To stain sections with Romanowsky

A—1. Fix and dehydrate small pieces of tissue, one to
two millimetres thick, in absolute alcohol (one to
four hours) Xylol, fifteen minutes, Hard para-
fin, twenty minutes

Cut sections as thin as possible,

3, Stain with the undiluted eosin solution ten to
fifteen minutes. Blot off excess,

4. Stain with the diluted methylene blue solution
fifteen to twenty minutes Pour off excess,

5. Pass rapidly through 70 per cent alcohol Place
at once in water,

6. Treat with one in four hundred acetic acid
momentarily, and place at once in distilled
water. Examine with a low power, and when
the nuclei appear bright red, and the section con-
tains but little blue, blot off excess of water.

7, Allow to dry on the slide
8, Examine by placing oil on the section without a
coverglass,

lo

52

B.—Lezisuman, before staining, places some fresh blood
serum on the section for five minutes, Stain with
LEISHMAN’S Stain, using two or three lots of fresh
stain for one or two hours, Differentiate alter-
nately with two solutions, I in 1,500 acetic acid
to remove excess of blue, and I in 7,coo caustic
soda to remove excess of eosin. Watch effect
with a low power. Dehydrate rapidly with
alcohol. Xylol. Balsam. ,

53

Chapter VI

THE MALARIAL PARASITE

Lire History

Among the groups into which the protozoa
are divided we find such well-known classes as
the Sarkodina, e.g., Amoeba Coli, the Mastigo-
phora, possessing flagella, €.£., Trypanosomes, and
the Sporozoa. It is these last that chiefly concern
us. The Sporozoa include such orders as the
Gregarines (e.g., monocystis in the testes of the
earth-worm) and the Haemosporidia (which in-
clude the malaria parasites of man, and blood
parasites of birds, etc.) There is a close relation-
ship between the coccidia and the haemosporidia
(malaria parasite), the delevopmental cycles of the
two being almost identical. The developmental
cycle in the blood (the febrile cycle) of the malaria
parasites was first demonstrated by Gora, the
further cycle in the mosquito by Ross. The cycle
of Goria! is the asexual cycle, producing auto-
infection of the patient ; the cycle of Ross is the
sexual cycle, producing a new infection in a
healthy subject.

The sexual cycle, it has been thought, com-
mences in the blood when the conditions are un-
favourable for the continuance of the asexual cycle,
and, in fact, has been taken as a sign that the

54

patient has already developed an immunity against
the fever-producing young parasites (spores).
Thus it is well known that in malignant tertian
the sexual forms, gametes or crescents, first appear
about a week to ten days after the first febrile
attack. If this view be true, then it follows that
the gametes develop from forms already present in.
the system, viz., the asexual forms, and possibly the
divergence into sexual forms takes place from the
youngest form of the parasite, z.e., the spore. But
it is possible that the divergence takes place at a
stage previous to the youngest form of parasite,
7.e.,at a stage immediately succeeding the entry
of sporozoits into the blood, so that we have from
the first asexual and sexual forms present. Sexual
development has been supposed to proceed. mainly
in the internal organs, e.g., bone marrow; but it
is being gradually recognized that young forms of
gametes are also found in the circulation; the
characters of these have already been noted (p. 35).
Let us suppose, however, that we are now dealing
with fully developed gametes in the blood. We
shall proceed to describe the further changes
undergone in the mosquito. The male cell is, as
we have seen, called the mikrogametocyte; the
female cell, the makrogamete. These we can
distinguish in the blood. Further flagellation can
be observed, 7.e., the protrusion of so-called
‘flagella,’z.e., mikrogametes or spermatazoa. These
‘flagella’ break off and fertilize the female cell,
the makrogamete, a process which has been seen
in halteridium of birds, but only once in man.
This fertilized female cell or egg is known asa
Zygote, though it is more convenient to reserve this
term for a little later stage, and to call this stage

a9

the Vermiculus or Ookinet (Figs. 6 and g). Both
these terms are suitable ones, for the first describes
the fact that the fertilized female becomes worm-
like in shape, and the second that the fertilized

Dr Inan.

—_—_—_——_—_—.
.

@
; Q sexvac <
ee (2)

°

ou & ae . Creseenl
Qt

a 2 ek 9 Ss ty
Yt Spore zoils
\\ Sex val * © ©

Cyele

Jngees Au_Inosquile

EF
a 5 >

a Ms

Fi Life Cycle of the Malaria Parasite in Man
oe ! and the Mosquito.

56

egg moves. The vermiculus stage can be seen on
the slide in the case of halteridium, but in the case
of malaria parasites, only by taking the blood
from the stomach of the mosquito after a suitable
lapse of time. The vermiculus now finds its. way _
through the epithelium of the stomach, and then |
lies in the external muscular layers as a spherical |
or ovoid body, the zygote. A kind of capsule is |
formed around it by these tissues, and so at this °
stage it is also called the Oocyst. Growth proceeds, :
and signs of division into several masses appear
in the protoplasm. These masses are termed
sporoblasts. Then we reach the stage of large
zygote (with sporoblasts), and by this time the
masses of the sporoblast have undergone division ~
into a number of fine curved thread-like bodies, ©
the sporozoits, so that eventually the large cyst is
almost entirely filled with sporozoits. The capsule
of the cyst eventually ruptures, and the sporozoits
pass from the tissues of the stomach to the thorax,
being found at first amidst the muscles, but
eventually all collected in the salivary glands.
From:here they are injected into the blood by the
mosquito, and they then attach themselves to and
penetrate the red cells (as has been actually
observed under the microscope by ScHauDINN),
producing a new infection.

We may briefly summarize these various
steps :— ;

1. Mikrogametocyte, and makrogamete in
blood.

_ 2. Development of mikrogametes = flagella-
tion, on the slide and in nature in stomach of an
Anopheline.

57

3. Fertilization of the makrogamete = ovum
or copula, on the slide and in nature in mosquito.

4. Vermiculus or ookinet. Only in mosquito
stomach.

5. Zygote or oocyst. In stomach wall.

6. Medium or large zygote with sporoblasts.

7. Sporozoits. In salivary glands.

The sexual cycle is known also as sporogony
or amphigony, while the asexual cycle is known
as schizogony or monogony. These two cycles
and their relation to one another are shewn in
the figure (Fig. 9).

Further, there is a certain amount of evidence
to shew that a gamete (2) in the blood can undergo
a kind of retrogressive development, and give rise
by parthenogenesis to young -parasites (iZ.e.,
schizonts). If this is so, it would explain the
supposed function of ald attributed to crescents
(gametes) of producing relapses.

Chapter VII

MOSQUITOES

Mosquitoes belong to the order of Diptera,
or true flies, which are characterized by :—

1. Asingle pair of membranous wings.
2. Suctorial mouth.
3. Complete metamorphosis.

In all mosquitoes, except the genera, Corethra
and Mochlonyx, there is a long piercing proboscis,
which is characteristic of the Culicidae. Mosquitoes
usually are about five mm. in length, but certain
species, e.g., Megarhinus, are much larger.

Flies which may be mistaken for mosquitoes
are :—

1. Chivonomidae (Midges), e.g. Chivonomus.—
Costal vein does not extend beyond the tip of the
wing. Antennae of male densely feathered. Legs

Fig. 10. Chivonomus.

long and slender (Vide Appendix). They do not
possess the characteristic proboscis of mosquitoes.
The veins of their wings are more complex, and

59

are quite devoid of scales. The absence of scales
upon the veins of the wings at once distinguishes
these from true mosquitoes (Fig. 10).

Enormous numbers of Chironomidae are found
near water, especially sedgy rivers and swamps.
They are attracted by light, and are constantly
seen around lamps and candles, a position in
which true mosquitoes are scarcely ever found.

Fig. 11. Trichocera.

2. Tipulidae (Daddy long-legs).—Some small
Tipulidae often possess a considerable superficial
resemblance to mosquitoes, as, for example, the
winter gnat (Trichocera). When at rest their
bodies lie parallel with the surface, and upon it.
They have no distinct proboscis (Fig. 11).

3. Cecidomyidae, or gall midges.—These have
a simple wing venation, and there are no forked
cells. In most species the wings and bodies are
hairy, not scaled.

. Rhyphidae—Wings have a discal cell
(below the anterior cross vein). They may have
spotted wings.

5. Simulidae, or sand-flies (sometimes also
called midges).—These are minute flies which
suck blood voraciously. They have a short and

60

stout proboscis. The salivary glands are very
large in proportion to the size of the fly, and the
bite is as severe as that of a mosquito. The
males are harmless (Fig. 12).

The larvae of the Simulidae are aquatic,
cylindrical in shape, and live in the stems of
water plants. The imago hatches beneath the
water.

6. Psychodidae (or owl midges), e.g. Phlebotu-
mus.—Small fluffy-looking flies which suck blood
readily. They are most readily detected after
feeding, when the abdomen is swollen with blood.
They have very hairy wings and body, and a short
powerful proboscis (Fig. 12). The larvae are
aquatic but can also exist in air.

Fig. 12. Sand Fly (left). Owl Midge (right).

LirE History oF THE Mosquito

In common with all other insects shewing
complete metamorphosis, the mosquito passes
through four stages :—

The egg.
The larva.
The nymph.
The imago.

61

_ The Imago.—The imago is the well-known
winged insect. The emergence of the imago may
be seen on the surface of almost any collection of
foul water. Shortly after hatching, the insect
may be seen resting quietly upon the surface of
the water, and does not fly away when disturbed,
or only very feebly. For some considerable time
after hatching (twenty-four hours) the insects
refuse to feed.

Fig. 13. Heads of Male (8) and Female (9) Culex.

In the imago there are marked differences
between the male and the female insect.

The Male—In the male the antennae are
markedly plumose. The palps also are long and
hairy. The effect is to make the ‘head’ of the
male mosquito very conspicuous (Fig. 13).

The male mosquito, with the exception of
certain species, does not feed upon blood, and the
proboscis is only used to suck in vegetable juices.
The male of Stegomyia mosquitoes, however, is said
to suck blood like the female.

The Female.—In the female the antennae are
inconspicuous and have only short lateral hairs.
The palps are also less conspicuous than in the

“male (Fig..13).

62

The female feeds upon blood, and is frequently
seen with the stomach distended with blood, more
or less digested.

The female is also seen with the abdomen ~
more or less swollen, with the greatly enlarged
ovaries, which give a whitish and opaque colour
to the mosquito, and often make the insect’ much
more conspicuous in its flight than it otherwise
would be.

The commonest species of mosquitoes belong
largely to the following genera or closely related
forms :-—

1. Anophelina (sub-family).

4. Culex.

3. Stegomyzta.

4. Taeniorhynchus and Mansonia.
Uvanotaenia.

The sub-family, Anophelina, is in many ways
the most distinct of these groups. Not only are
the adult insects highly characteristic in appear-
ance, but the ovum and larva are quite unlike
those of any other genus. One, indeed, can
recognize the Anophelinae at a glance merely by
their characteristic general appearance, once the
peculiarities of this genus are known.

The points which serve to distinguish the
Anophelinae from other groups of mosquitoes are:—

1. The character upon which the sub-family
is founded, viz., the relative length of the palps
and proboscis. In both the sub-families, Culicina
and Anophelina, the palps in the male are long”
plumose structures, as long or longer than the
proboscis. In the female of the Culicinae, however,
the palps are quite short and insignificant structures,
whereas in the female Anophelinae these are scaled

63

and as long as the proboscis. An examination of
the femaic proboscis will at once determine whether
an insect belongs to the sub-family, Anophelina, or
one of the other genera (Fig. 14).

2. The wings in nearly all species of Ano-
phelines are ‘spotted.’ The spots can be seen with
the naked eye. By the use of the low power of
the microscope or an ordinary lens, these spots are
seen to be due to the presence of areas of dark
scales upon the wing veins, elsewhere covered with
light scales.

Fig. 14. Shewing distinction between palpi of Female Anopheline
(vight) and a Culicine (left).

There are a few members of the Anopheline
group which, however, have not spotted wings
(e.g., A. bifurcatus, and the Indian A. 7mmaculatus).
Also there are other flies than Anophelines which
havespots(e.g., Rhy phus, C. mimeticus (costal spots),
the genera Theobaldia and Lutzia). Nevertheless,
as a general practical rule, mosquitoes with spotted
wings are Anophelines.

3. The angle which the proboscis makes
with the rest of the body is very different in
Anophelines from that of other mosquitoes. In Culex,
Taeniorhynchus, or Stegomyia, the proboscis forms
a distinct angle with the line of the body (Yae-
niovhynchus, forty-five degrees). In the case of
Anophelines, the proboscis continues on in the line
of the body (P. stephensi, fifteen degrees). The

64

result is to give to an Anopheline. mosquito a
peculiar and very characteristic awl-like appear-
ance (Fig. 15).

4. The attitude adopted by Anophelznes is, as
a rule, characteristic. When an Anopheline rests
upon.a wall, its body projects so as to form a
distinct angle withit. In some cases the angle
assumed is almost a right-angle. In the case of
almost all other mosquitoes, the body is held
either parallel with the wall, or what is more
frequent, the tail approaches the wall, giving the

Fig. 15. Shewing distinction between resting attitude of
an Anopheline (left) and Taeniorhynchus (vight).

insect a ‘hunchbacked.”’ appearance. This differ-
ence is readily seen by any careful observer, and
is a practical and useful distinction. A charac-
teristic of an Anopheline is that it rests by preference
on the first two pairs of legs only, and keeps the

65

last pair stretched out stiff and straight, or they
slowly oscillate to and fro. Many mosquitoes
wave the hind legs, notably Stegomyia, but they
are held with the tarsi curved backwards.

The exact attitude adopted depends upon
the species and the situation, whether a vertical
or horizontal surface on which the Anopheline is
resting. One very common species (M. culicifacies)
at least, when sitting on a wall, looks exactly like
asmall brown Culex, since it holds its body parallel
with the wall as a Culex does.

Culex.—Mosquitoes of the genus Culex are
many of them brown mosquitoes of sober hue,
e.g., the common house Culex, C. fatigans, which
is uniformly brown without markings. The genus,
however, contains a very large number of species.
In Culex mosquitoes the attitude when resting is
‘hunchback.’

Stegomyia.—The genus Stegomyia is of the
greatest interest and importance, since it is this
form which is concerned in the transmission of
yellow fever (Stegomyia fasciata).

These mosquitoes are generally black and
white, with banded legs and abdomen, and spots
on the thorax. They are found in houses, and
are most troublesome mosquitoes from their habit
of feeding. in the day, and their great alertness
and persistence. Stegomyia are also very common
in woods and forests.

CapTuRE OF MosQuiTOES AND [LIES

1. Place a lamp upon a sheet of white
paper, and note the insects which are attracted
by the ight. Note insects belonging to the

F

66

orders Lepidoptera (moths), Hemiptera (aphides,
green flies, etc.), Heteroptera (plant bugs), Neu-
roptera (caddis flies, stone flies, white ants). Pick
out any mosquito-like flies. They will probably
belong to the Chironomidae. Note the absence of
proboscis, the delicate transparent structure. Note
the plumed head of the male (as in the true
mosquito) and the less conspicuous antennae of
the female. Examine the wings under a strong
lens or a low power of the microscope, and note
that the wing veins are bare and do not carry any
scales. Note that true mosquitoes are not seen
around the lamp.

2. Examine, with a light, some wall which
has been only dimly illuminated by the lamp, z.e.,
some wall at the distance of several yards, and
note true mosquitoes resting upon this. Capture
several of these by placing a tumbler over them,
and kill them by puffing in a little tobacco smoke.
Observe that they have a distinct proboscis.
Observe the ‘pluined’ male and the female without
plumes. Examine the wings under a strong lens
or low power objective, and note the scales
attached to the wing-veins. '

The specimens caught will probably be speci-
mens of Culex. If near a swamp or jungly place
there may be Taentorhynchus, Mansonia, and
possibly Anophelines. Observe the hunchback
attitude in the case of most of the mosquitoes
caught. If an Anopheline should by chance be
caught, note the striking difference in the genétal
appearance, the attitude, and the spots on the
wings.

3. Observe in stuffy, furnished rooms, offices,
etc., the presence of mosquitoes feeding actively

67

during the day. Capture some of these. They
will probably belong to the genus Stegomyia.
Note their extreme alertness. Observe that they
are black with white bands. Note the habit of
waving the hind legs, and that the tarsi of these
are kept curved. Ascertain whether the males
feed upon blood.

4. Examine stables, huts, outhouses in the
early morning.

LITERATURE

The Cambridge Natural History. ‘\nsects, Part II.’ A most useful
book for an introductory knowledge of a variety of winged life in
the tropics and elsewhere.

68

Chapter VIIT
THE OVUM

Ova are minute bodies one mm. or less in
length. When first laid they are white in colour,
but become rapidly brown or black.. They occur
on the surface of water, and if submerged do not

Fig. 16. Eggs of Stegomyia

hatch out. Mosquito eggs may be laid by the
edge of water, or on floating objects, or upon the
water. In the last case, they have some device to

RR
WKN
RY

HN

Fig. 17. Egg Raft and Eggs of Culex

ensure that they shall float, and not sink and be
destroyed. In the case of Anophelines and some
species of Stegomyia (Fig. 16), each ovum lies

69

separately upon the water, and has air cells which
keep it afloat. In the case of Culex and Taenior-
hynchus, hundreds of eggs are cemented together to
form rafts, each egg lying perpendicularly, with
its larger end pointing downwards. In Culex, the

Fig. 18. Egg Raft and Eggs of Taeniorhynchus

egg-rafts are broad and roughly oval in shape
(Fig. 17). In Taeniorhynchus, the egg-raft is
extraordinarily elongated, resembling, in shape,
a racing skiff (Fig. 18).

THe EXAMINATION OF Ova

Culex.—Examine the surface of some semi-
putrid water for egg-rafts of Culex. Egg-rafts
can almost always be found on the surface of
water containing macerating leaves, fruit, etc.
They are bodies of a blackish-brown colour, and
are readily wafted about by the wind.

1. Note that the raft is boat-shaped, measur-
ing one-fifth to one-third inch in length, and
consists of two hundred to four hundred eggs.

2. Note that the separate ova are smooth
elongated bodies, about 0'7 to o'8 mm. in length.
Note that there are no floats or other markings
as in the case of Anopheline ova.

3. Note that one end of the egg is thicker
and blunter than the other, and that to the thicker

70

end is attached a clear transparent globular body
(the micropilar apparatus). Note that this body
is readily detached, often leaving a spike-like
process projecting from the thicker end of the
ovum.

4. Make as many observations as possible
upon the egg-rafts, e.g., time necessary for hatching
of larvae, amount of desiccation they will with-
stand.

The egg stage in S. fasciata lasts twelve to twenty-four hours ;
in C. jamaicensis, twelve hours; in C. sollicitans, twelve hours
(Taytor). The eggs of Culicidae have but little resistance to
desiccation, but those of S, fasciata will hatch after being kept
‘dry’ for three months,

Anophelinae—The ova of Anophelinae are
difficult to detect in nature, but may be seen by the
aid of a lens on the margins of small pools, where
larvae abound. They are about 0°7 to 1:0 mm. long.

Examination of Anopheline Ova :—

1. Confine some female Anopheles as de-
scribed on p. 120. Endeavour to choose those in
which the ovaries are nearly mature (p.g7). Fifty
to one hundred and fifty eggs are laid. Remove the
piece of paper upon which the ova have been
deposited and place this upon a slide. Examine
with a low power in strong daylight, and the
mirror turned off.

2. Observe the remarkable resemblance of the
ova to little boats, and the presence of the two
beautiful oval air cells placed upon either side,
acting as floats. (These are absent only in one
species as yet described, viz., M. turkhudi). Observe
also the presence of a white frill or a mere ribbed
rim around what would be the gunwale of the

boat (Fig. 54).

71

3. Observe that one end of the ovum is
always stouter than the other. The stout end
contains the head of the embryo, and is the end
from which the young larva escapes. Note also
that when’ Anopheline eggs are seen at the side of
vessels drawn up by capillarity the thick end is
at the bottom. Examine the surface of the water
remaining in the hollow stopper or receptacle,
and observe that the ova of Anophelines are laid
singly without any cement substance, and float
singly or touching one another on the water.

<c
«

Fig. 19. Patterns formed by Eggs of Anophelines

omnnnnen

4. Observe star-shaped patterns formed by
some species, or the arrangement in parallel
groups assumed by the ova of others (Fig. 19).
Note that this arrangement is dependent on
physical causes (shape of the egg, etc.), and not
on the fact that the eggs are laid in such
positions. This is readily done by stirring up a
number of Anopheline ova on water, and noting
how they tend to form groups in triangles and
star shapes (Vide p. 221).

5. Ascertain that Anopheline ova, when first
laid, are white, but rapidly darken and become
black. Observe that Anopheline ova are very often
laid in heaped-up masses, which eventually
become dispersed by waves, etc. Observe that
the eggs then form patterns.

72

6. Place some half-dried mud in a flat dish,
and put this inside a piece of mosquito netting
in which some Anophelines with ripe ovaries
are placed. Observe that ova are laid upon the
mud.

7. Preserve the mud for forty-eight hours,
preventing it from becoming completely dry.

At the end of forty-eight hours’ or more,
remove a few ova to a dry slide, and place under
alow power. Allow a drop of water to flow on
to the ova. Observe the escape, within a minute
or so, of the young larvae, and the fact that a cap-
like piece of the egg-shell is pushed off.

8. Observe that Anophelines kept ina dry test
tube will occasionally lay their eggs on the side
of the tube.

g. Note the time when the eggs were laid
and the time at which the larvae emerge. This
depends greatly on the temperature. It may take
two to three days. Ce. argyvotarsis, one-and-a-
half days (Taytor).

10. Remove Anopheline ova on paper and
allow them to dry, and note that after two or
three days (t., 86°-96° F.) at the most, they will
not hatch when carefully placed on water.

Stegomyia.—Confine some gravid females of
Stegomyia mosquitoes.

1. Note that in S. fasciata the eggs are
laid singly, and much resemble, at first sight, the
ova of Anophelines. Note that in others the eggs
are laid in rafts (S. notoscripta).

2. Note that they are irregularly oval,
thicker at one end than the other, and havea
corrugated surface in which are entangled numer-
ous minute air bubbles.

ve)

3. Examine the surface of water left exposed
for several days in a tumbler, etc. Note, if
Stegomyia mosquitoes have ovi-posited, the prers-
ence of eggs occurring singly or in parallel groups.
Note that the ova are larger than those of Ano-
phelines, and that they hatch into Culex-like larvae
(see Stegomyia larvae, p. 85). .

Taeniorhynchus.—Examine natural waters, es-
pecially small pools with a dense growth of alga,
swamp pools, irrigated land, etc., for the egg-
rafts of Taeniorhynchus.

1. Observe the extreme length and narrow-
ness of the rafts. Note also how small a portion
of the raft is submerged.

2. Observe that the ova are arranged as in
Culex rafts with the thicker end downwards, and
that they are smooth and have a micropilar
apparatus.

3. Endeavour to obtain the ova of known
species of Taentorhynchus, by confining gravid
females. Note the shape of the rafts.

Mansonia.—Observe that the eggs have a
curious snout-like projection, and that they are
laid singly.

Psovophova.—The eggs are large, two mm. long.
They occur in patterns like those of Anophelines.
The eggs are covered with minute prickly scales.

Tayzor, in Havana, has made many observations on eggs,
He gives the following :—C. pipiens, raft 200-400 eggs; egg
og byor6mm. C. nigritulus, raft 200-300 eggs; egg o 6 by
org mm. U, lowii. raft 50-75 eggs. S, fasciata, Eggs laid
singly, about fifty in number,

Chapter IX

THE LARVA AND NYMPH

Toe Larva

The larvae of mosquitoes, more especially of
Culex, are well-known objects. They can be seen
by holding up to the light almost any specimen
of water that has been left undisturbed for some
days, but especially water which contains mace-

rating leaves.

Dire, Odrehra, Chiro nome’s Ephemera.

Fig. 20. Larvae that may be mistaken for Mosquito Larvae

43

Larvae which may be mistaken for those of
mosquitoes are :
1. Chivonomidae.—The larva of Chironomus
is a red worm-like creature (blood worms). On
the prothorax it has a pair of feet armed with
hooks (Tig. 20).

Larva % Unephelet and Culer.

Fig, 21. Larvae of an Anopheline (left) and Culex (right)

2. Ephemeridae (May-flies)—The larvae of
certain small Ephemridae may, at first glance, be
mistaken for mosquito larvae. There is no real
resemblance, and the triradiate tail of the ephemera
larva and the tracheal gills distinguish it (Fig. 20).

3. Dixitdae—The larva of Dixa rather closely
resembles the larva of Anophelines, though not other
mosquito larvae (Fig. 20). Ventrally it has pseudo
pods on the fourth and fifth segments.

EXAMINATION OF THE LARVA

Culex (Fig. 21)—Obtain some Culex larvae
from any source and place in a glass vessel.

76

1. Observe the hanging attitude of the larva.
Note the angle it makes with the surface of the
water, and how this varies in different species.
Note, if the larva of C. concolor is being examined,
that the position is nearly horizontal.

2. Observe the large head, the prominent
eyes and projecting antennae.

Of?
'

wh

SK

x
~

err ere

absiloer

" 6
5

Fig. 22. Respiratory Syphons of Larvae.
(1) Stegomyia ; (2) Culex ; (3) Culex with Large Syphon Tube ;
(4) Taeniorhynchus ; (5) Canniba/ Larva (? C. concosor) ;
(6) Enormous Syphon Tube (one-quarter scale of others,
genus undetermined) ; (7) Spine on Tube seen on
the flat ; (8) Spine seen sideways

3. Note the long respiratory syphon arising
from the eighth abdominal segment.

Place a half-grown larva under a coverglass
and examine under one-third inch objective.

1. Make an accurate drawing of the
antennae,

id

2. Carefully observe the length and thick-
ness of the respiratory syphon.
3. Note the absence of palmate hairs.
4. Determine whether the larvae examined
are Culex, Stegomyia, Taeniorhynchus, etc.
The characteristics of the larvae of Culex
appear to be :—
(i) Antennae not markedly curved, or sickle-
shaped (Fig. 23).
(ii) The respiratory syphon long, but not
extremely slender as in Taeniorhynchus
(Fig. 22).
Determine the relative length and breadth
of the various larvae :—
In Culex, the length is to the breadth as 4 : 1.
In Taentovhynchus _,, we i Q-12 = 1,
In Stegomyia a a ae 1,
In any particular species there will be deviations
from these, but broadly speaking, these differences
hold good.

Tue LARVAE oF ANOPHELINES

To collect the larvae-——Necessary apparatus :—

1. An ordinary spoon, dessert or table-spoon
size is most convenient, but a tea-spoon does well.

2. A white enamel tin or large cup, or an
ordinary bath tin.

3. Bottles, specimen tubes, paper, pencils,
etc.

Procedure.

1. By inspection—Inspect closely the surface
of any small puddles that have been in existence
some time. Examineespeciallysmall rock puddles,

78

small shallow pools in ‘nallahs’ and river-beds,
in the dry season.

Examine especially the edges where larvae
are fond of resting, with the head facing the open
water and the tail touching the bank. Note also
how larvae tend to cling to foating twigs, etc.
If no larvae are seen, stir up vigorously the
bottom of the pool with the spoon. This will
dislodge larvae from the edges, etc.

Examine the surface of the pool again and
observe the larvae now plainly visible against the
muddy water. Wait a few minutes for the
appearance of the larger larvae, which remain
below longer than the younger forms. Examine
carefully for nymphae, which easily escape de-
tection.

Dip out the larvae and nymphae with the
spoon as they appear. The thinner the edge of
the spoon the less disturbance is caused, and the
more readily are larvae removed.

Place the larvae as caught in bottles, tubes,
etc. If desirous of labelling these, write in pencil
on the paper, and put into the bottle.

2. By Dipping—Choose any water with
grassy or weedy edges, eg., the edges of rivers,
streams, ditches, lake margins, swamps, etc.

With the least possible disturbance, dip out
water from the most sheltered positions, and as
close to the vegetation as possible, bringing up
water and weeds in the can. Allow the specimen
of water to remain a few seconds, and remove
any larvae or nymphae as they appear on the top
with the spoon.

79

EXAMINATION OF LARVAE

Anopheline larvae cannot be mistaken for any
other mosquito larvae (Fig. 21).

tr. When undisturbed they lie flat along
the top of the water, and on every segment
certain hairs (palmate hairs) actually indent the
surface film. Observe that when viewed in certain
lights from one side these identationscan be plainly
seen. The appearance may even be as though the
dorsum of the larvae projected from the water.
This, however, is not the case.

This appearance is diagnostic of the Anopheline
larva. One species (M. turkhudi) does not, how-
ever, rest in this position, but after rising to the
surface in a horizontal position slowly sinks until
the tail only touches the surface.

Anopheline larvae, which are about to turn
into nymphae, also sometimes tend to sink, so
that the head is directed obliquely downwards
(often seen in M. vossz‘).

One species of Culex, at least (C. concolor),
adopts a nearly horizontal attitude. The line of
indentations of the surface film mentioned above
is not, however, seen.

2. When disturbed, Anopheline larvae dart
into the water, or what is véry characteristic, if
not greatly disturbed, they pass by a series of
wriggling jerks along the surface of the water.

When moving up towards the surface, an
Anopheline moves in a much more irregular and
jerky manner than a Culex larva. This is well
demonstrated when living larvae in a cell are
projected upon the screen of the lantern. ‘The
Culex larvae are seen rising by a series of regular

80

lateral sweeps, whereas Anopheline larvae exhibit
only irregular jerks.

When kept in a vessel, Anopheline larvae
tend to collect round the edges with their heads
pointing towards the centre. In this position
the body of the larvae is often bent as it lies along
the curved surface of the capillarity film at the
edge. Culex larvae and the larvae of M. turkhudi
frequently do not go to the side, but remain in
the more central portions of the fluid.

4. Anopheline larvae, when full grown, possess
very small heads in proportion to the size of the
larvae (about eight mm. in length). In most of the
Culicidae the head is very large, with very a
minent and large antennae.

5. Anopheline larvae have no ‘spiracle tube,’
the tracheae open into a pit on the dorsum of the
eighth abdominal segment. In Stegomyza, se
spiracle tube is short and thick (Fig. 22), 1
Taentorhynchus (Fig. 22) very long and dienien
but in Anopheline larvae alone is this structure
absent (Fig. 21).

Procure a considerable number of Anopheline
larvae, and ascertain the following points :—

1. The Moulting of Anophelines—Note that as
Anopheline larvae grow in size they cast their skins.
Remove a cast skin by floating it upon a slide.
Note the perfect nature of the ‘skin,’ and how all
the chitinous structures are represented, even air
tubes. Dry the specimen upon the slide and
mount in a drop of balsam. Observe how beauti-
fully certain hairs resembling fan-palm leaves are
shewn (palmate hairs). (Vide p. 235).

2. The Method of Feeding of Anophelines.—
Observe with a lens the action of the feeding

81

brushes and the currents they produce on the sur-
face of the water. Note the rotation of the head
so that, whilst feeding, the ventral surface of th
head is uppermost.

3. The Food of Larvae.—Tear a larva to pieces
with a needle and remove a small portion of the
dark central mass of food material filling the
straight alimentary canal. Place in a drop of
clean water and crush under a coverglass. Note
what organisms form the chief bulk of the food.
Note the presence of sand grains—unicellular
plants and animals—short lengths of alga, diatoms,
etc. Also bacilli.

Determine the common foods of several species
of Anophelines.

4. Desiccation of Larvae —Cr1ii and Casa-
GRANDI have found that Anopheline larvae can only
resist desiccation at 20° C. for two days, at 35° C.
“ for one day, and 40° C. for two minutes only.
Larvae of Anophelines stranded on moist mud will
live as long as four days, but in the tropics as
soon as the mud loses its glistening surface they
die.

Culicine Larvae.—The larvae of the Culicidae,
with the exception of those of Anophelines and
possibly some other genera, are superficially much
alike. The conspicuous hairs and spines, and
even the complicated terminal segment, are very
similar in the different genera. There are, how-
ever, marked differences in some features on closer
examination. These differences are mainly to be
found in the syphon tube, the antennae, and
mental plate, but to a less extent in other
structures.

G

82

Note differences in naked eye appearance ;
note the long worm-like Stegomyia larva and its
wriggling mode of progression ; note the trans-

arent and spiny appearance of some larvae
foray Taentorhynchus); note that some larvae
adopt a nearly horizontal attitude (C. concolor and
others), others a vertical attitude (Stegomyza), whilst
the majority form a small angle with the vertical.
Examine larvae under a low objective. Note the
head with eyes, large feeding brushes, antennae and
various hairs; note the large bunches of hairs
arising from the thorax and abdominal segments ;
note the last segment bearing four large clear
papillae and two systems of hairs; note the penul-
timate segment which carries the syphon tube and
some curious claw-like spines.

Note especially the following :—

(1) The syphon tube.
(ii) The antennae. |
(ii1) The mouth parts.
(iv) The anal papillae.
(v) The hairs of the thorax and abdomen.

The Syphon Tube.—This is formed of a single
cylindrical piece of chitin, and contains the origin
of the two main tracheae of the body. Note the
small flap-like pieces of chitin forming a closing
apparatus at the extreme tip. Measure (by eye-
piece micrometer) the length and greatest breadth
of the syphon tube ; note that in different species,
and especially in different genera, the syphon tube
varies greatly in its measurement. By dividing
the length by the breadth a figure may be obtained
which is useful, and may be termed the syphonic
index number; note that in Stegomyia ‘this
number is about two. In Culex,fourto seven. In

83

Taeniorhynchus, as much as twelve in some cases.
Draw accurately by measurement (eye-piece micro-
meter) a number of syphon tubes of different
Culex larvae. Note that marked variations in
different species exist (Fig. 22).

Note two rows of spines on the posterior
aspect of the syphon tube, starting from the base
and extending a variable (in different species)
distance up the syphon tube; note that they
differ in number and length, etc., in different
species. The spines appear serrated or compound,
according to the angle they are viewed from, and
differences may be supposed to exist which depend
upon this fact. In some species (certain carnivorous
or cannibal larvae) a large fan of hairs projects
posteriorly in the medium line from the syphon
tube. In certain species the syphon tube is of
enormous size, and may.attain to one-third the
length of the larva.

The Antennae.—Note in the case of most
typical Culex larvae that the antennae are large
conspicuous objects; note a basal, medial, and
terminal portion, and a large bunch of feathered
hairs arising at the junction of the two first-
named portions ; note also large single and stout
hairs from the more terminal portion ; note spines
on the body of antenna.

Examine the Antennae of various Lavvae.—Note
in some cases that the antennae are more rudi-
mentary (Stegomyia, Anopheles). In the case of
Stegomyia (as far as described) they are small and
spineless, and possess only a small hair arising
from a papilla, which may be single or in three
or four branches. Make drawings (using eye-

84

piece micrometer), and note gréat variation in
different genera and species.

The Mouth Parts——Note the characters of the
claw-like mandibles, and especially the exact
character of the triangular mental plate, which
forms a conspicuous dark triangular body on the
under surface of the head (Fig. 23).

Note that in diflerent species the plate varies
in appearance, especially in the size and number
of notches in its margin. In some species the
plate is like a shark’s tooth, in others it 1s comb-
lke.

The Anal Papillae—Note the tracheae ramify- *

ing in these, the papillae being possibly gill-like
in function. In Megarhinus, Toxorhynchites,
Mucidus, Psovophova, Lutzia, C. concolor, and C.
tigripes they are quite rudimentary.

The Large Body Hairs.—These are long in
some larvae, much shorter in others; their
arrangement is very similar in the different larvae.

5. Cannabalism of Larvae.—Add some large
Culex larvae toa small bottle containing some
small larvae or Anopheles larvae. The Anopheles
larvae or small Culex larvae will be devoured by
the large forms. Mucidus sp., C. concolor, and
Psorophora sp., are especially cannabalistic.

6. Observe the occurrence in nature of the
two forms, Culex and Anopheles, also what Culex
larvae are found living together.

7. The Enemies of Larvae—Add small fish,
waterbeetles, and their larvae (Dytiscidae, Hydro-
philidae), Libellula larvae, Corysca, Nepa, tad-
poles, and other water animals, respectively, to a
series of wide-mouthed bottles containing equal
numbers of larvae. Note the rate at which they

85

are devoured, if at all. The carnivorous forms
Nepa, Corysca, Libellula rapidly devour larvae.
Hydrophilidae beetles, tadpoles, etc., do not destroy
larvae. Observe that some species of fish are
much more active devourers of larvae than others.
Note that weeds often protect larvae from being
consumed by small fish.

8. Make experiments with different chemical
and other bodies, and note the absence or presence
of culicidal power.

(a) Note that chemical bodies in solution kill
only with difficulty, as a rule, e.g., corrosive sub-
limate. Ammonia, however (1 in 4,000 of water)
will kill mature larvae according to WADDELL.

(b) Note that oils rapidly kill larvae by blocking
the air tubes. Treat larvae by pouring a little
olive oil upon the water. Stain with osmic acid
and note globules of oil within the air tubes.

Add some paraffin oil to a small Anopheline
pool, observe the presence next morning of dead
female mosquitoes that have come to lay their
eggs. Observe the effect of paraffin on different
kinds of natural water, and the great efficacy in
some cases and futility in others.

10. Observe that pools covered with Lemna
are very frequently, if not always, free from
larvae. The action of the Lemna is said to be
mechanical.

EXAMINATION OF OTHER LARVAE

Dixa—In its movement along the surface
of the water the larva of Dixa resembles Anopheline
larvae, and this larva also rests horizontally just
beneath the surface film.

86

In Dixa there is no globular thorax, and the
whole larva is longer and thinner than an
Anopheline larva (8 mm.) Moreover, Dixa larva
only indents the surface film at the head and tail,
there being no palmate hairs on any of the
segments. Dixa larvae move very rapidly, and
have a habit of climbing above the surface of the
water and resting ina loop with the head and tail
downwards. When placed in a specimen tube it
climbs up the side and becomes lodged in crevices
in the cork.

It is found frequently in running water
(Fig. 20).

Mochlonyx Larva.—Note absence of palmate
hairs on dorsum and presence of respiratorysyphon,
but less developed than in a Culicine. They are
extremely voracious. They lie deep in the water.

Corvethva Larva.—These are the so-called
‘phantom’ larvae. They are extremely trans-
parent, and lie horizontally rather deep in the
water. The head is smaller than in Anophelines.
There is no syphon communicating with the
external air. They are extremely voracious. Add
some Corethra larvae to a tumbler containing Culex
larvae.

Stegomyia.—The larva of Stegomyia is rather
longer than that of Culex. When disturbed it ex-
hibits a rather lashing movement like that of cer-
tain small aquatic worms. When at rest at the
surface, the attitude of the body is almost vertical
The larva, however, spends a good deal of its time
browsing at the bottom of the water, and then
lies for the most part horizontally.

The head is small in proportion to the rest
of the body, and the thorax is less conspicuously
marked off from the abdomen than in Culex.

87

The antennae resemble those of Anophelines
larvae, more, than those of Culex. The large
branched hair of Culex is represented by a
short inconspicuous simple hair (or as many as
three) projecting from the side of the antennae

(Fig. 23)

JF, Stigomyia
4 Tos =
4 C.Concolor(t)

2 hs &, Stigomice,

Fig. 23. Antennae and Mental Plates of Larvae

The syphon tube is characteristic, being very
short and stout (Fig. 22), only twice as long as
broad, whereas in Culex the syphon tube is four
or more times as long as broad (Fig. 22).

1. Examine domestic utensils, disused water
pots, and tins containing water. Examine the
water which collects in the axils of the banana
leaves, collections of water 1n tree-stumps in the
jungle. If larvae are present—

(i) Note the very short and broad spiracle
tube and its dark colour. Compare with that of
Culex and Taeniorhynchus.

88

(ii) The larva is longer and more worm-like
than most mosquito larvae. ‘The ‘wriggling’
motion is also very markedly shewn owing to the
length of the body.

Taeniorhynchus.—In natural waters, especially
shallow trickling water forming pools, with a
dense growth of spirogyra, etc., swamp water and
river margins, the larvae of Taentorhynchus will be
readily found.

1. Note that the larva les often embedded
in the masses of green spirogyra or other thread-
like algae.

2. Note the great transparency of the larva
and the frequency with which brilhantly green
specimens are found.

N

Culex Fatigans. Taeniorhynchus
Fig. 23a

Under a low objective note the following,
which appear to be characteristic of this genus :—

1. The enormous horn-like and curved
antennae (Fig. 23a).

89

2. The extreme length and slenderness of
the syphon tube (Fig. 22).

Psovrophora——The larvae are large, half-an-
inch in length. They are extremely cannabalistic.

THe NyMpH

The nymphae of mosquitoes are extremely
characteristic bodies. Wherever a number of
fully-developed larvae are found there will gene-
rally also be seen numbers of bulbous comma-
shaped creatures, having a large globular body
(head and thorax) and a small tail, kept more or
less tucked in beneath. When disturbed they
dart downwards with great speed, but very soon
reappear at the surface.

Onopheres Culex

Fig. 24. Nymphae of an Anopheline and Culex

Nymphs are not so easily seen in pools as
larvae.

The differences in the nymphae of different
genera of the Culicidae is not nearly so great as in
the case of the larvae.

gO

By keeping under observation a number of
nymphae, some will be seen to become less
inclined for active movement, and the abdominal
segments (tail) may be extended horizontally.
Soon after these changes the adult insect emerges
through a crack in the chitin of the back of the
thorax. The process as seen in Anopheles is very
fully described by Nuttavy and SHIPLEY.’

EXAMINATION OF NYMPHAE

t. Note the effect of tapping the glass vessel
and the rapidity with which the nymphs regain
the surface.

2. Observe that when first they appear the
nymphs are light in colour, but darken very con-
siderably later.

3. Note that just before the hatching of
mosquitoes the nymph lies with the tail extended,
and that silvery marks may be seen, due to air
lying under the chitin.

4. Observe the emergence of the imago.

Examine the nymphs of Anophelines, Culex,
Taeniorhynchus, etc., and observe that to the naked
eye they are very similar.

t. Note that the nymphae of Anophelines lie
less vertically in the water than those of Culex.

2. Observe that the nymphs of Anophelines are
more elongated antero-posteriorly and compressed
laterally than those of Culex and Taentorhynchus.

3. Observe the very large nymphs of some
common species of Taeniorhynchus and the great
length of air tubes which are directed straight
forwards in a very characteristic manner.

1. Fournal Hygiene, vol. 1, part IL.

gI

Place nymphae in drops of water on a slide
and examine the air syphons. Note—

1. In Anophelines the syphons have a square
truncated end, and are proportionally much shorter
than in Culex, and project from about the middle
of the thorax (Figs. 24 and 25).

3. Jatnuarrhyncus 4. Qnatheles

6 Jaenss y rhgn evs

nopheles Colex Covethra,
Fig. 25. Nymphal Syphon Tubes
2. In Culex the syphons are long and narrow,

and have a slit-like opening, and project from the
posterior portion of the thorax (Figs. 24 and 25).

g2

3. In Stegomyia the syphons are broadly tri-
angular, and are characteristic. Note the marked
contrast in appearance to those of Culex (Fig. 25a).

Fig. 25a. Nymphal Syphon Tube of Stegomyia

Examine the nymphs of Corethva and Moch-
lonyx when they are encountered.

(a) Note in Covethra the pointed syphons and
the straight tail (Figs. 25 and 26).

(b) Note in Mochlonyx the Culex-like nymph
and the thin roundedand pointed syphons (Fig. 25).

Fig. 26. Nymphs of Chivonomus and Corethra

w3

Examine the bottoms of pools of polluted
water, and note in the mud the brilliant red
nymphs and larvae of Chivonomus (Fig. 26).

1. Observe that the Chivonomus nymph has
a large globular body (head and thorax) and bears
a general resemblance to mosquito nymphs.

2. Note, however, the presence of the con-
spicuous white feathery gills which form tufts at
the side of the head.

3. Note that the Chivonomus nymph and
larvae do not rise to the surface to breathe as do
those of mosquitoes.

4. Note the curious rhythmic bending move-
ment of the larvae and nymph of Chivonomus
which, when they are present in numbers, give the
mud at the bottom of the pool a curious appear-
ance.

LITERATURE

Miall. Aquatic Insects.

94

Chapter X

TO CAPTURE, PRESERVE ALIVE, AND
EXPERIMENTALLY FEED MOSQUITOES

To CaprurrE ANOPHELINES

Necessary Apparatus.—One or two small
collecting tubes (I*ig. 27), a clean and perfectly
dry bottle (whiskey bottle), some cotton wool.

To Detect ANOPHELINES

Choose a suitable native village, z.e., in Africa,
any bush village; or in India, any village near a
nallah or other source of Anopheline larvae.

Determine whether Anophelines exist in any of
the following situations :—

1. In the dark corners of sheds, cow-houses,
or other out-houses.

2. Under the eaves (in darkish parts) of the
huts.

3. Inthe huts themselves, hanging to straws,
stalactites of soot, etc., etc.

4. Any other likely situations, e.g.,collections
of dry grass; in the undergrowth in the bush
(capture in this situation is difficult).

Pyoceduve.—If, on inspection, none of the
insects can be detected by careful scrutiny (the

95

most concentrated attention is, as a rule, needed),
the thatch should be carefully disturbed with the
hand or a short stick.

Observe carefully any insects which fly out,
and note where they settle. Choose especially
portions of the thatch which are not too dark to
prevent one seeing clearly, but are not too much
exposed to light.

Train, if possible, one or more intelligent
natives to detect the insects and to collect them,
as shortly described. It is a good thing, if even
only a very few Anophelines have been found by a
personal inspection, to offer a small reward to any
persons in the village who will undertake to
collect them. One or two tubes should be left
for this purpose.

To Detect CuLEx

Examine the walls of houses, out-houses, and
native huts. Especially examine clothes hung up
in native huts. Many specimens of Culex, resting
in their characteristic hunchback attitude, will
probably be detected. Especially on dark clothing,
old blankets, inside leather boots or boxes.

Mosquitoes seem especially fond of the smell (?)
of leather.

To Detect TAENIORHYNCHUS

These are best caught by sitting, with a light,
near a marsh or grassy land. A wall, or tent, or
cloth hung up should be at hand, and kept slightly
illuminated with a lamp. They may be captured
as they settle upon the sheet or upon oneself.

96

To Capture MosQuiToEs

1. Placea collecting tube very slowly over
the mosquito.

2. Insert a fnger underneath, and so rapidly
block the tube; or a piece of cardboard or wool
may be carefully sipped underneath.

Breeding oul”.

Cott eeling :

Fig. 27. Method of Collecting and Breeding-out Mosquitoes

3. Place a plug of cotton wool in the mouth
of the tube.

4. Transfer to the large bottle by placing
the tube over the mouth of the bottle and with-
drawing carefully the cotton wool. Keep the
bottle closed with a plug of cotton wool.

5. Capture as many specimens as required,
and transfer them as caught to the bottle.

97

A wide-mouth bottle, over which a piece: of
stout paper has been tied, with a small trap-door
cut slightly larger than the tube, may be used.
This has the advantage that mosquitoes do not so
easily fly out into the tube during the act of
transference. It has the disadvantage that the
paper tears, and the mosquitoes are more likely
to escape through accidental circumstances.

To BreEp Out MosguiToEs

(Fig. 27)

Collect a number of full-grown larvae and
nymphae of both Anophelines and Culex.

I. Separate the nymphae from the larvae
and place them ina jar or wide-mouthed bottle
half-full of water, leaving room for the insects
when hatched. Cover the jar with a piece of
thick cardboard or a lid, the central portion of
which is replaced by mosquito netting.

2. Place the larvae where they will receive
plenty of light, but will not be subject to great
heat.

. Remove the nymphae as they are seen at
the end of each day.

To Krzep MosguiToEs ALIVE

The length of time mosquitoes remain alive
in captivity depends almost entirely upon the
suitability of the conditions under which they are
kept.

Except for special purposes, mosquitoes (espe-
cially Anophelines) should not be kept in open
spaces, 7.e., frames covered with mosquito netting.

Procure several ‘chutney jars’ with hollow

H

98

glass stoppers. These can be obtained generally
from the native bazaar in India for a few annas
(Fig. 28). This form of jar is very convenient,

but any other jar will serve. ue
Cut a piece of thick cardboard so that it will,

when forced down into the jar, remain supported
on the shoulders of the jar.

lea ul

Fig. 28. Method of keeping Mosquitoes alive

\

li

Fill the stopper nearly to the brim with water.
Cut a thin slice of cork and place it on the surface
of the water. Upon the cork place a piece of clean
white paper. The paper should not quite occupy
the whole of the space in the mouth of the stopper.

1. Invert the chutney jar (prepared as above)
over the top of a jar in which some mosquitoes
have hatched. Remove the cardboard and gently
tap the glass. The mosquitoes will fly upwards
into the chutney jar. Place the chutney jar con-
taining mosquitoes upon its stopper prepared as
above.

o9

Place the whole, after labelling, in a dark cup-
board or other convenient place (incubator).

At the end of the first day or so, the males
will be found dead upon the piece of paper, and
can be removed. On the second night after
hatching, most of the insects will feed, and the jar
is ready for use.

2. Place the inverted (chutney) jar, prepared
with cardboard as above, over a bottle in which
Anophelines, caught in a village or elsewhere, have
been placed. Remove the cotton plug and shake
the bottle gently to drive the insects out. Replace
jar upon the prepared stopper. Place in a dark
spot. Next morning remove the stopper and re-
move any dead mosquitoes and ova by taking out
the piece of paper.

On the second night after the mosquitoes
have been collected, the bottle is ready for feeding
experiments. On the third day, generally, the
mosquitoes have no longer any blood remaining
in the mid-gut, and are ready for dissection.

The glands of any mosquitoes that may die
before this may of course be dissected, if desired,
on the chance of finding sporozoits.

In the use of village-caught Anophelines, it must
be borne in mind that any subject upon which
they are fed is liable to a fresh infection. In the
case of natives (who sleep without hesitation in
any village), the employment of village-caught
mosquitoes cannot, however, be very prejudicial.

The advantages of the above way of keeping
mosquitoes are :—

t. The mosquitoes will keep alive longer
than in any other way known to us.

2. The immense convenience in feeding.

TOO

3. Any mosquitoes that may have died in
the night can be recovered, and are not dried up.

4. It is an extremely convenient way of
obtaining and examining the ova.

5. Mosquitoes which have become feeble are
given the best possible chance of living, and will
be found resting all day on the piece of paper.

If boxes and net-covered frames be used, an
enormous mortality usually results. The dead
bodies dry up and get lost in the folds of netting,
or, unless special precautions are taken, are eaten
up by ants.

If chutney jars with hollow stoppers cannot
be procured :—

Procure any form of wide-mouthed jar or
bottle, such as a prune jar, preserved fruit bottle,
etc. Insert a piece of stout cardboard as before.

1. Prepare the metal top of a screw-top
bottle or some other suitable small receptacle
with water, cork,and paper asabove. Place upon
a square piece of very stout cardboard or wood.
Invert the jar over this (Fig. 28).

2. Prepare a saucer by adding a few tea-
spoonfuls of water and placing on this cork and
paper. Invert the jar over the saucer. This is
rather more convenient than the last mentioned
method, as mosquitoes are less liable to escape in
the process of lifting the bottle.

To FEED MosguiToEs

Select a bottle in which the mosquitoes (twenty
to thirty, or at least a dozen, in each bottle) are
seady for feeding, 7.e., the second evening after
hatching or collecting. Lift the bottle from the

IOI

stopper, first disturbing any mosquitoes which
may be resting on the stopper, and place it mouth
downwards on the table.

Slip underneath the mouth of the bottle a
small piece of mosquito netting of rather a fine
mesh. Tie this around the neck of the bottle
with twine. The bottle is then ready for
feeding.

Fig. 28a. Method of Feeding Mosquitoes j'

At, or shortly after dark, take as many
bottles as may be desired to the ward or dormi-
tory. Slightly damp the forearm (or the calf) of
the patient, and, turning the bottle right side up,
let the patient’s arm rest upon the mouth of the
bottle (Fig. 28a).

In from a few minutes to half-an-hour or
more the bottle will be noticed to have splashes
of blood upon the bottom and sides (in the case
of Anophelines only). If possible wait till all the
Anophelines have fed.

Remove the bottle, invert upon a table, untie
the twine, and remove the netting. Replace the
bottle upon the prepared stopper.

102

Repeat the process every night, allowing the
mosquitoes to feed by preference on the same case
throughout. Where it is uncertain which of several
cases may or may not have mature sexual forms
in the blood, a bottle may be fed on alternate
nights on the different cases.

Add clean water and a fresh piece of paper
each time the bottle is used.

To PREPARE FED MosQuiToEs FOR DISSECTION

After having fed the mosquitoes in a bottle
for a certain number of days, place it apart from
others, and allow it to remain undisturbed
(merely changing the water, etc.) for several
days.

Ascertain each day whether the mosquitoes
have completely got rid of the blood in the mid-
gut. When they are quite free from any dark
colouration of the ventral aspect of the abdomen
they are ready for dissection.

N.B.—If chloroform, and especially if tobacco smoke is used

to kill the mosquito, it is essential to well wash the jars before
again keeping mosquitoes in them.

To Freep MosguIToEs oN Birbs, ETC.

1. Prepare a framework of wood and book-
binder’s cardboard. Cover two sides with card-
board. Cover one end with netting drawn tight,
and to the other attach a ‘sleeve’ of netting.
Catch or breed out a number of Culex (e.g., Culex
fatigans), and place in the frame. Keep the frame
ina dark place, and. place a saucer of water
10, 1.

103

Before placing the bird in the cage, a small
bag of netting should be tied around its head, as
it then remains perfectly quiet, and further, the
legs may be fastened. Small birds, such as spar-
rows, should be carefully treated, as, otherwise,
they are very liable to succumb. Pigeons should
be treated in the same way, if necessary.

2. Mosquitoes may be fed singly on pigeons
and other large birds by placing the end of the
test tube, in which the mosquito is confined,
against an area of skin denuded of feathers.

FEEDING EXPERIMENTS ON BIRDS

1. Feed anumber of Culex, e.g., C. fatigans,
on sparrows (in which have been detected proteo-
soma in the blood), by placing these for a time in
the mosquito cage.

After feeding one or two days, place those
mosquitoes, which obviously have fed and are
gorged with blood, in a prepared chutney jar, and
keep until ready for dissection.

Note (i) the zygotes of proteosoma which
generally occur in large numbers in the stomach
wall, and in which very coarse and dark pigment
is seen.

(ii) Feed some Anophelines on proteosoma
sparrows, and note that no zygotes are formed.

(iii) Feed some Taentorhynchus on proteosoma
sparrows, and note the negative result.

(iv) Feed some Culex upon pigeons con-
taining halteridium, and note negative result.

Sparrows containing halteridium so frequently
(in India) contain proteosoma that, even if the
latter is not observed under the microscope, it is
difficult to be sure of their absence.

104

Chapter XI

DISSECTION AND EXAMINATION OF
MOSQUITOES FOR THE MALARIAL
PARASITE

DissECTION OF MOSQUITOES

Necessary Apparatus.

1. Slides and coverglasses.

2. Two needles, preferably the straight
surgical needles described for making blood films,
as they have a cutting edge.

3. Some salt solution, 0o°5 grammes per cent.

4. It is convenient to have a board, twelve
by three inches, covered half with white and half
with black paper.

Some mosquitoes are caught by slipping
over the top of the jar used for feeding another
empty jar of the same size, and they may be kept
alive in a dark cupboard for two or three days,
until their stomachs are quite free from blood
(seen by the complete disappearance of black from
the ventral portion of the abdomen).

A few specimens are killed by chloroform or
oe smokey or by ‘concussion’ simply, in a test
tube.

1. Observe (if a gravid female) two whitish
areas on either side of the hinder portion of the
abdomen (ripening ovaries). If the blood in the
stomach be not digested, a dark mass will be seen

105

in front of these, and possibly the extreme
anterior portion of the abdomen will appear trans-
parent (air containing oesophageal diverticulum).

(Fig. 29.)

To Dissect Our tue Mrp-Gut (Stomacn)

1. Pull off, with forceps, the legs and wings
(and remove most of the scales by a few strokes of
a small camel hair brush).

2. Placea drop of salt solution on a slide,
and place the slide on a light background.

Ovary

Fig. 29

Turn the mosquito upon its back and witha
needle, held in the left hand, transfix the thorax.

Carry the mosquito transfixed on the needle
to the shde, and lower the tip of the abdomen
into the drop of salt solution.

Keeping the transfixing needle in position,
make with the other needle a nick upon either
side between the sixth and seventh abdominal
segments, which point corresponds to the division
between the mid and hind-guts. After thus
loosening the last few segments, place the point
of the needle upon them, slowly dragging them
away from the rest of the abdomen.

: 106

3. After separating the segments a very
short distance, remove the preparation to a dark
background. Again draw apart and note the
white viscera stretching between them. Make

pana ~¥ Jha
membranous
‘ oesophagus,
Anlerior P {
orCion —
Wud got
At, conlaining
diverliculume
Toslitor of oesophagus
povlion 4
Trick -que

; Tylorus and
ongin of fbules

tralpryhéan
fubules ! 4 T qut

Ovary

Fig. 30. Dissection of the Viscera of a Mosquito

steady traction until a central, rather transparent
body is alone left between the two portions of
abdomen.

Cut across the anterior attachments of the
mid-gut.

107

4. Draw the body of the mosquito away
from the separated segments; the mid-gut and
sundry other viscera will be left attached to the
latter floating in the salt solution.

_ Observe that when the tension is relieved,
the structure last to leave the abdomen of the
mosquito assumes a saccular appearance. This is
the mid-gut.

THe VISCERA
(Fig. 30)

1. Unless the mosquito is newly hatched,
note two opaque white oval bodies (the ovaries)
attached to the separated segments. If the
ovaries are near maturity, masses of white ova
are seen.

2. The Mid-gut—This extends from the
level of the first pair of legs to the posterior
border of the sixth abdominal segment.

(i) An anterior narrow portion resembling
an oesophagus.

(ii) A posterior dilated portion at the level
of the sixth (and fifth) abdominal segments in
which, if the last meal of blood is not quite
digested, a black mass will be seen. If any blood
remains in this portion, 7.e., ‘the stomach,’ dis-
card the specimen for one kept longer without
food.

(i11) At the commencement of the mid-gut
a ring-like, thickened portion (the proventriculus).
It acts as a valve between the oesophagus and
mid-gut (Fig. 30).

3. Passing between the mid-gut and the
separated segments, note five brilliantly white
threads—the malpighian tubules (Fig 30).

108

4. Between the malpighian tubules the
transparent intestine which may exhibit active
peristalsis (Fig. 30).

5. Attached to the proventriculus an exceed-
ingly delicate membrane, the dilated oesophagus
and three diverticula of the same, which usually
contain gas bubbles. ScHaupinn has shewn by
adding Baryta water to these bubbles that they
are really carbonic acid gas. Further, also, the
bacteria in these diverticula produce enzymes which
are the cause of the ‘irritation’ of the bite, as may
be shewn by rubbing a diverticulum into a scratch
on the skin. The salivary secretion as has been
generally supposed has not this property.

The ventral diverticulum extends as far back
as the fifth abdominal segment.

To PREPARE THE Mip-GuT For EXAMINATION

1. Cut (by pressing with the needle) across
the intestine and malpighian tubes just below
the termination of the saccular mid-gut. This
will separate the mid-gut from the rest of the
viscera.

Remove everything from the slide but the
mid-gut. Remove excess of fluid, and see that
no ova or extraneous matters are left upon the
slide. Add asmall drop of clean salt solution,
and place a thin coverglass upon the preparation.
The mid-gut will flatten out considerably. Re-
move with filter paper applied to the edge of the
coverglass any excess of Auid. Examine under

one-third inch objective and afterwards under
one-twelfth.

109

If the mid-gut has been removed in ‘toto, and
the preparation not too much compressed, the
following appearances are seen :—

1. The narrow anterior portion of the mid-
gut, with the calyx-like proventriculus at its free
end.

2. If a portion of the extremely thin mem-
brane of the true oesophagus or its diverticula be
included in the preparation, it will probably be
seen to exhibit peculiar markings, due probably to
muscular fibres in the membrane, but resembling
rather closely sporozoits. It is essential that this
structure should be recognized when seen, and
that the resemblance of its markings to sporozoits
should not lead the beginner astray (Fig. 32).

Irosele fibres

Swollen.
Epi thelwm cells

Large 34 90le aes capillaries
Fig. 31. Microscopic appearance of Mid-gut, shewing

Cell Structure and Zygotes

. The expanded posterior portion of the
mid-gut. This body forms the main mass of the

IIO

preparation, and is all important in relation to
malarial studies.

The following appearances are seen in a good
preparation :—

1. Well-defined tubes with spiral lining (air
tubes or tracheae). Note that these branch and
ramify upon the surface of the mid-gut and mal-
pighian tubes (Fig. 31).

2. Large muscular fibres, together with
elastic fibres, forming a check pattern. Note that
they are circular and longitudinal (external).
Note that at the edge of the viscus they are seen
in optical section (Fig. 31).

3. Large cells with large nuclei and granular
protoplasm (epithelium of mid-gut). Note some
in situ forming a single layer of polygonal epi-
thelium, and others detached and in process of
being carried along by fluid streaming from interior
of mid-gut. Note that in some places these cells
are undergoing vacuolization with dancing of the
protoplasm granules (Fig. 31).

4. Note any contents of the stomach—

(1) Remains of blood.
(11) Crystals of various kinds.
(111) Gregarines, flagellates, bacteria, etc.

5. Note that in focussing downwards one
passes through a double thickness of wall. Note:
that the air tubes are focussed on the upper and
lower surfaces of the preparation, and the epithe-
hum and crystals in the middle.

6. Trace several of the finer air tubes to
their apparent termination, and note that when
they lose their spiral lining they are continued as
very fine transparent tubules (air capillaries).

IIl

Note that at the point of breaking up, one can
generally make out large stellate cells (tracheal
cells). (Fig. 31.)

Observe in some preparations, large oval
cells of brownish colour lying upon the outer
surface of the stomach. Note that they are rather
opaque, and contain a certain amount of diffuse
yellowish pigment. They areso called pericardial
cells (see Fig. 32).

8. Observe, in most preparations, one or
more large clear cells with a small nucleus and
filled with oil globules (cells of the fat body)
(Fig 32.) These lie upon the stomach and, in
common with the last named cells, are accidental
in this situation.

Tue EXAMINATION OF THE Mip-GuT FOR THE
ZYGOTE OR Oocyst STAGE OF THE
MALARIAL PARASITE

(The examination of the stomach blood for flagellating and
the motile ov vermicule forms is deferved to a later Chapter).

Obtain a number of Anophelines (not M. rvossiz)
from some native quarter (see p. 92), or better,
those specially fed. Keep these alive for two
or three days until no blood remains in the
mid-gut (for methods of keeping alive, see p. 95).

Prepare the mid-gut as described above.
considerable number may prove negative, but a
variable percentage will be positive. Examine
with one-twelfth inch.

Carefully note the presence of small collections
‘of pigment of the nature of malarial pigment.
By careful focussing, the younger forms may be

II2

seen as clear oval or round bodies, 6-7 w, in which
the distinct and clearly defined pigment occurs.
The more advanced forms can scarcely be missed.
It is necessary to bear in mind the normal structures
and the fact that, until the parasite reaches a
considerable size and has a very sharply defined
cyst wall, pigment, of the characters belonging to
the species of parasite concerned, is present.

Fig. 32. (Left to vight) Pericardial Cell, Fat Body Cell,
Swollen Epithelium Cell, Thin Membrane shewing
Spovozoit-like appeavance

1. Zygotes of crescent tertian shew, when
young, a clump of pigment resembling black
pepper grains (Fig. 33).

2. Zygotes of simple tertian shew yellowish
or golden pigment in wisps (Fig. 33).

3. Zygotes of quartan shew rather coarse
pigment in a clump (Fig. 33).

The older zygotes (40-60 «) are indistinguish-
able as regards the species of parasite concerned.
They may shew :—

1. A very clear and distinct cyst wall (ad-
ventitious).

2. The formation of sporoblasts.

PLATE II
Fig. 1—Young zygotes of the benign tertian parasite.
Fig. 2—Young zygotes of the malignant tertian parasite.

Fig. 3.—Mature zygote, outside epithelium of mid-gut (Section,
haematein).

Fig. 4——Transverse section of salivary gland shewing sporozoits
in situ (haematein),

Fig. 5——Sporozoits from the salivary gland (Romanowsky
stain).

Plate Ll.

a

i)

FGy Ne FabiGs, <2

FIG. 3. FIG 4.

FIG,5

113

3. In still more developed forms, the sporo-
blasts are seen to be surrounded by a radiating
arrangement of young sporozoits or blasts (Figs. 9
and 31).

4. Fully developed forms are large cysts
packed with many hundreds of fine sickle-shaped
bodies, and, if they are ruptured, these latter escape
into the surrounding fluid, and are readily dis-
tinguished as sporozoits (Fig. 37).

Fig. 33. (Left to Right) Zygotes of Malignant Tertian,
Simple Tertian, Quartan, and Proteosoma

To Maxe PERMANENT PREPARATIONS OF
ZYGOTES

Method 1.—In case of a specimen shewing
zygotes, place a large drop of two per cent. formalin
on one side of the coverglass, and draw this
through by filter paper placed on the other side.
Repeat several times. Remove excess of the
formalin with filter paper. Ring edges of cover-
glass with black varnish or Canada balsam.

The zygotes will retain their appearance as
seen in the fresh specimen.

Method 2—Run formalin through as in
Method 1. When an excess of fluid is present,
slide off the coverglass. The flattened mid-gut
will probably remain attached to the coverglass.

I

114

Wash in water, and stain lightly with methy-
lene blue. Wash in water, and allow to dry.
Warm gently to ensure complete dryness, and
place the coverglass, mid-gut downwards, upon a
drop of balsam upon a slide.

The muscular fibres and other structures of
the mid-gut will be well exhibited. The zygotes.
will be stained rather a dark blue. If not too
darkly stained, the pigment of the zygotes will
have the appearance it had in the fresh specimen.

3. Mount directly in glycerine.

. For more minute histological examina-
tion, imbed the stomachs in paraffin or the
whole mosquito (vide p. 123).

To DissEct OuT THE SALIVARY GLANDS

This is quite a simple proceeding if it be re-
membered where they lie. They are intra-thoracic
structures, and they commence at the hinder por-
tion of the neck and end opposite the first pair of
legs. They he ventrally, in fact, roughly speaking,
they he just above the origin of the first pair of
legs (Fig. 34).

The simplest and most rapid method, and the
one that hardly ever fails, is the following :—

1. Place the mosquito in a drop of salt solu-
tion on its right side, with the head pointing to-
wards you, as you dissect.

2. Place the needle of the left hand on the
thorax to steady it, and place the needle of the
right hand on the back of the head and make
steady gentle traction.

3. If done carefully, it will be seen that the
head has pulled out a httle mass of white tissue

II5

from the thorax (the dissection is best done on a
dull-black surface).

4. Examine the piece of tissue under a half-
inch lens. (The diaphragm should be as nearly
closed as possible). The glands will be seen
hanging on to the neck as finger-like, transparent,
glistening bodies. Muscle has a greyish look,
and even the fat body is not so refractile as the
glands.

Sheng poston. ca Salivery Giends

Fig. 34. Shewing position of Salivary Glands

Now place one needle on the head, and
with the other make a transverse cut between the
head and the attached portion of the glands.

6. Examine again the now separated
glands. Generally all six are with certainty got
in this way.

7. Ifthe glands are not found on the neck,
proceed with the dissection by Method 2.

8. When dissected out in this way, they
are generally quite free from surrounding tissues,

116

but if found necessary they can be teased out further
and placed in fresh drops of salt solution.

g. At allstages of the dissection make sure
that the glands are really present and that they
have not floated to the side or stuck to the
needles.

to. By this certain and rapid method, as
many as one hundred glands may be dissected
out, put under a coverglass, and examined micro-
scopically in a day’s work.

Duel of Mer side.

eB Clivel sland.

_ Inet, =
and

Fig. 35. The Salivary Glands of one side

Method 2.—Consists in isolating, by a series
of cuts, the anterior ventral portion of the thorax
in which the glands lie.

1. Make a cut obliquely in an antero-
posterior direction, so as to sever the main mass
of thoracic muscle.

2. Makeacut at right angles to this, passing
just behind the attachment of the first pair of
legs.

3. Cut through the neck.

4. The glands lie in the portion thus iso-
lated. Considerable teasing out is still required
to isolate them from the surrounding tissues.

117

Examine each portion of tissue separated out, and
remove to a fresh clear drop of salt solution.

Remember that in examining under the
microscope the apparent right hand is really the
left, and vice versd.

This method, which is longer than Method 1,
requires more dissection and teasing out in order
to isolate the glands cleanly, and, as we have
said, may still be followed, even if No. 1 has
failed ; but our experience has been that Method 1
is learned at once without any difficulty.

Seevelion.
dislind mg cell

hucleus of-
Sa livery cell

Grenulay profaples rm
cell

Fig. 36. Microscopic Structure of Salivavy Acinus and that of
a Newly-Hatched Mosquito (vight)

Ascertain that the glands of either side
consist of three acini, the ducts of which join
almost immediately after leaving the acinus to
form a single long duct.

1. Observe that of the three glands of each
side (Figs. 35 and 38) :— .

(2) Two are highly refractile, and the cells
in these are very distinct and clearly defined
(lateral glands).

(ii) One is much less refractile, and the
component cells are much less easily defined
(central gland).

2. Observe that each acinus has a duct

118

running through its whole length, and that the
secretory cells form a single row around this.

3. Observe that each secretory cell has a
large mass of clear secretion within it, forming
the chief bulk of the cell, and that the nucleus is
flattened and pushed to the periphery (Fig. 36).
Pressure tends to force the secretion out of the cell
in viscid looking droplets. The secretion of the
lateral glands is far more refractile than that of
the central (Fig. 38).

Proloblasmm
of Salivary cell

Seerelian *

bring mol

Revelions
forced vl

by pressuve.

Lporegaulé

Fig. 37. Spovozoits in the Salivary Gland
(Fresh preparation)

4. Ascertain that the duct formed by the
junction of the three intra-acinar ducts joins,
eventually, the similar duct from the other side, to
form a common salivary duct which passes into
the salivary receptacle. The duct is thick walled,
and is lined with a spiral thread resembling a
tracheal tube.

119

EXAMINATION OF THE SPOROZOIT FoRM OF THE
MALARIAL PARASITE

Obtain a number of Anophelines (not M. rossi?)
from a native quarter (five per cent. to twenty per
cent. or more have sporozoits in the glands), or
Anophelines fed for twelve days or more at a tem-
perature of 80°F. Prepare specimens of the glands,
as described above.. Having placed one or more
lobes under a low power, press with the point of
a needle on the coverglass, so that the gland is
ruptured, and the secretion poured out as droplets
into the surrounding fluid.

Examine with one-sixth inch. If sporozoits
are present they are generally very numerous, and
large numbers of fine, very distinct curved ‘rods,
will be easily seen with this power, lying through-
out the fluid around the gland and packed in large
numbers in the substance of the gland. Finally,
examine with one-twelfth inch (Fig. 37).

The sporozoits have a mean length of 14,

and vary between Io » and 20 w; and are I-2, in
width.

’ EXAMINATION: OF MoTION OF SPOROZOITS

Dissect out the glands and, when isolated
cleanly, transfer to a drop of human serum,
previously got ready by allowing blood to clot in
a small tube. Three kinds of motion may be
observed :—

1. Formation of curves.
2. Formation of ring-formed contractions.
3. Locomotion. Forward motion.

Penetration of Red Cell by Sporozotts—This

has not been seen in case of sporozoits of the

I20

salivary glands, but has been observed twice by
ScHAUDINN in the case of sporozoits from a
ruptured cyst in the stomach.

To PREPARE PERMANENT PREPARATIONS OF
SPOROZOITS

Pressing firmly upon the coverglass, draw it
along the slide, so that a film is made on cover-
glass and slide.

Dry by rapidly waving the slide and the
coverglass in the air. Fix both in alcohol, and
stain with Romanowsky. Wash, dry, and examine
without coverglass with an oil immersion.

Dy Dorel Gland

Rescpletes Cole se. Sporsoile mm glx.

Fig. 38. Sections of Salivary Glands, shewing differences between
the Central and Lateral Glands and between those of
Anophelines and Culex, also Sporozoits in the
Glands of an Anopheline

,

I2I

The sporozoits appear as fusiform bodies with
a central red mass of chromatin.. They are about
14min length, with one end often more pointed
than the other.

Wash off the oil with xylol, dry and label.

THE REPRODUCTIVE SYSTEM

To Examine the Spermatheca (Fig. 39).—By
pressing with the edge of the triangular needle
cut off the extreme tip of the abdomen—the last
or eighth segment only. Place this ina very small
drop of salt solution, and tease the fragment
carefully apart. A small black granule (In Culex,
three) will be seen. Isolate this as much as
possible from other tissue and cover with a cover-
glass.

Observe, under a low power, a brown chitinous
ball. Press firmly on the coverglass so as to rup-
ture it. Examine under one-twelfth inch.

Observe the masses of fine hair-like actively
motile bodies, if (as is probably the case) the
mosquito has been fertilized. Isolate some of
these ; they possess the characters of spermatozoa
(Fig. 39). |

Examine the spermatheca of a mosquito
newly hatched ; it does not contain spermatozoa.

To EXAMINE THE OVARIES

Examine the ovaries of a number of mos-
quitoes caught in the room, etc.

Observe that when the ovaries nearly reach
maturity they are readily detected as white areas

122

on either side of the posterior part of the abdo-
men, and that when fully developed they occupy
the whole of the lateral and dorsal portions of
this.

Drag off the last few segments of the ab-
domen in a drop of salt solution, and allow the
ovaries to float out in this. Observe that they are
pyriform bodies, the apex being above (Fig. 39).

lev minal tu
| folfteviar lube

Sper ma theca

Fig. 39. Spermatheca, with Spermatozoa
Structure of Ovaries

Ascertain that each ovary consists of a large
number of follicular tubes, commencing as fine
threads and ending in the oviduct. Observe
especially the follicular tube forming the apex of
the ovary, as here, this is most readily made out.

Ascertain that each follicular tube contains
several egg follicles, the lowest of which is the
most advanced in development.

123

Note that these latter show various stages of

_ development in the different mosquitoes. Note

that each consists of an outer layer of small
cubical cells (follicular epithelium) and an inner
mass composed of from four to eight large cells.
Note the first appearance of the yelk as small oil
globules in the lowest cell (the ovum), and how
this increases until it nearly fiils the follicle by
encroaching on the other cells (nurse cells).

Observe in. the mature ovum that the outer
covering is formed of the original layer of outer
cells (follicular epithelium).

Ae
9? ®>
aor as) Gf
&)\)
i O
Oclo-Sporegoa Yelk of ovum Aeflaced Sporeom in ov ebeut hy

by Plispe razon. Salivary Clawds

Vegettal Godes im hind gut”

Fig. 40. Pyotozoa other than the Malarial Parasite
found in Anophelines

The following organisms may be found in
mosquitoes apart from the various stages of the
malarial parasite :—

1. Encysted Trematodes, mostly found in the
tissues, near the neck; also free in the stomach.

124

2. Nematodes in the thorax or abdominal
cavity.

3. Sporozoa. (a) Sausage-shaped bodies in
masses, sometimes in close connexion with the
salivary glands (Fig. 40).

(b) Octosporozoa, consisting of eight small
sausage-shaped bodies in small cysts, enormous
numbers of which replace the yelk of the ovum
(Fig. 40). —_

(c) Flagellate bodies in the rectum and hind-
gut, frequently in enormous numbers, and shewing,
when stained, large numbers of developmental
forms (Vide under T. noctuae).

(d) Gregarines. Occasionally on the outside
of the stomach or encysted in the malpighian tubes.
In the larvae, the worm-like gregarine will be
found actively motile in the malpighian tubules.

4. The developing embryosof filariae. These
are seen as Sausage-shaped bodies with a terminal
spike in the dissection of the salivary glands. In
sections of mosquitoes they are seen in the muscles,
especially in those of the thorax.

5. The developed embryos of ftlariae in the
labium or about the base of the neck.

6. In the oesophageal diverticula masses of
micro-organisms and sporozoa (Nosema)? will be
found.

To Cur Srections or MosouitoEs

t. Killsome Anophelines by tobacco smoke or
chloroform, or allow them to fall directly into
absolute alcohol, by pouring a few drops into the
tube containing them. Avoid, if possible, mosqui-
toes containing blood, as the blood becomes very
hard.

125

2. Allow to remain in alcohol fifteen minutes
to harden somewhat.

3. Remove one by one. Cut off with a fine
scissors the legs and wings. Make a minute
incision into both the thorax and abdomen, 0.£.,
holding the mosquito carefully between the finger
and thumb, slice off with a sharp razor a
portion of the dorsum of the thorax, and a
minute portion of one side of the abdomen.
This allows more perfect penetration of fluids.

4. Replace in absolute alcohol’ (some authors
recommend boiling in alcohol, as the air in the
tracheae is expelled, and the alcohol then pene-
trates completely), allow to remain one to two
hours. This, which is to ensure complete dehydra-
tion, is the most important of operations, and upon
it depends the success of the embedding. Make
two or three changes, using alcohol to which CuSo,
has been added.

5. Remove from alcohol and place for ten
minutes or soinxylol. When the thorax becomes
transparent, the mosquito should be removed, as
too long a time in xylol produces much hardening.

6. Place for ten minutes in paraffin (vide p. 46)
kept melted in a watch glass on the heated metal
slab.

. Smear a clean watch glass with a little
glycerine, and fill with paraffin heated somewhat
over melting point; transfer the specimen to this
with a warm forceps to avoid cooling the paraffin.

8. Arrange the specimen as required. As
soon as the surface of the paraffin has set, plunge
the whole massinto water, as paraffin rapidly
cooled is more homogenous.

1. For finer work pass through thirty, fifty, and seventy-five per cent. to
absolute. See, however, examination of separate organs.

126

If the watch glass has been smeared with
glycerine it will be easy to remove the block.

g. Cut out the specimen, arrange as desired
for cutting transversely, vertically, or horizontally.
Take care that the top and bottom. edges of the
block are parallel. Smear these with a little
melted soft paraffin (made by melting a little hard
paraffin with vaseline). This gives an unbroken
ribbon of sections.

To Mount Sections oF MosQuiToEs

Unless the sections are thick it is necessary to
flatten them.

Heat some water in a dish a little below the
melting point of the paraffin. Test the. right
temperature by dropping a section on the surface.
If it melts, the water is too hot. If it does not
flatten out, the water is not hot enough.

Drop upon the surface a ribbon of five or six
sections; it will become perfectly flat if the
temperature is right.

tr. Smear some slides with solution of
pyroxylon in oil of cloves (Appendix), taking care
to avoid an excess. Lower the slide rapidly into
the water, and with the aid of a needle draw it
out again with the sections lying flat upon it.

2. Press lightly, but firmly, between smooth
blotting paper. Protect from dust, and allow to
dry for twenty-four hours, or dry more rapidly
over the flame, but avoid melting the paraffin.

When dry, hold the slide a few seconds over
the flame, this drives off the oil of cloves and
melts the paraffin.

Pour over the slide first xylol, then alcohol,
and finally place in water,

127

Mosquito tissues are so delicate that in
mounting it is difficult to avoid the separation of
portions or the whole of the section. This is
especially so in the case of the chitin, which
frequently breaks away.

OsBREGGIA’S method is recommended as giving
excellent results :—

1. Acslide is smeared with a thin film of a
mixture, consisting of two parts of commercial
liquid glucose and one part of a solution of
dextrin (dextrin, 16 0z.; water, 174 0z.; thymol,
15 grains).

2. Place the sections directly on this layer
on the slides.

3. Heat at 40°C. for some hours until the
glucose becomes hard.

. Dissolve off the paraffin in xylol and pass
through absolute alcohol. If the alcohol is not
quite absolute, the glucose will dissolve and the
film come off.

5. Dry.

6. Pour on thin film of photoxylin, and
allow to dry until edges of film become slightly
crumpled.

. Put slide in water. Sections come away
in the film of photoxylin.

8. Stain in situ, dehydrate and clear in
carbol xylol (xylol, 3 parts; absolute phenol,
1 part). Mount in balsam.

To Stain.—The best stain for general use in
sections is haematein (vide p. 50).

THe EXAMINATION OF SEPARATE ORGANS

Remove the mid-gut or the ovaries or other
organs desired, and place in a saturated solution

128

of corrosive sublimate (saturated whilst hot and
allowed to cool).

As this is a watery solution, practically no
shrinking occurs. It, unfortunately, cannot be
used for the whole mosquito, as watery solutions
will not penetrate the tissues.

Allow to remain in corrosive solution ten
minutes. Place in large volume of water for
twelve hours, and in weak watery iodine solution
(sherry colour) for one hour. Pass through 30 per
cent., 50 per cent., 75 per cent., to absolute
alcohol. Allow to remain in absolute alcohol
fifteen minutes. Transfer to xylol, three minutes ;
paraffin two minutes. Cut in ribbons and treat
as already described.

To Cut SECTIONS OF THE SALIVARY GLANDS

Embed only the head and front half of the
thorax. Cut sections as near as possible hori-
zontally, this will give sections in the length of
the glands, and both sides at the same time.

Stain with various stains : HEIDENHAIN’S hae-
matoxylin, gold chloride, etc. Much work remains
to be done upon the structure and nature of the
glands, and the method by which the sporozoits
reach the salivary cells.

LITERATURE :
Grassi. The Siudies of a Zoolgist (in German or Italian).

Chapter XII
INTERNAL ANATOMY OF MOSQUITOES

Tue ALIMENTARY CANAL

The alimentary canal is specialized on account
of the blood-sucking habits of the mosquito. It
differs from many insects in not possessing any
caecal diverticula of the mid-gut. It also differs
in the possession of five malpighian tubules, these
being in insects usually even in number (Fig. 30).

The parts of the alimentary canals are as
follows :—

The mouth
The pharynx with pumping organ ( The fore-
The oesophagus gut.

The oesophageal diverticula
The homologue of the ee re
The stomach (so-called)

The pylorus

The pyloric dilatation

The small intestine The hind-
The colon gut.
The rectum with rectal papillae

The mouth, pharynx, and oesophagus are
ectodermal in origin, and both the mouth and
pharynx are lined with chitin. The hind-gut is
also ectodermal in origin; it does not possess,
however, any portion lined with chitin. The
- kK

gut.

130

mid-gut is the true digestive portion of the
tract.

The Pharynx.—The pharynx, which is lined
throughout its extent with chitin, passes upwards
and backwards through the ganglionic ring
formed by the supra and _ infra-oesophageal
ganglia and their commissures. At first it is
narrow, but posteriorly becomes a large chamber
(the pumping organ). (Fig. 43a).

The pumping organ occupies with its muscles
a large portion of the head behind the level of the
cerebral ganglia. In the state of rest its lumen is-
triradiate in transverse section. The walls are
formed of three large and thick chitinous plates,one
placed on either side and one superiorly. Into each
of these plates powerful muscles are inserted. Those
of the superior plate consist of two muscular masses,
taking their origin from the occiput. Those of
the lateral plates consist on each side of a single
large muscular mass arising from the lateral
portions of the head. The plates are connected
by thin non-chitinous membrane, and their edges
are rolled so that they form a spring capable of
returning to their original position so soon as the
separating force of the muscles ceases.

Posteriorly, where the pharynx becomes very
narrow, a sharp bend occurs and a valvular action
is produced. The whole forms a very powerful
suctorial apparatus.

The Oesophagus.—Immediately beyond the
pumping organ the chitinous layer ceases, and
the rest of the fore-gut is formed of excessively
thin membrane. At the junction of the two
portions a sharp bend occurs, and the floor pro-
jects so as to form a valvular flap.

131

The thin-walled oesophagus is a large dilated
sac, whose-walls are supported by surrounding
structures. Into the posterior wall of the dilated
and thin-walled oesophagus projects the papilla-
like anterior portion of the mid-gut.

The Diverticula of the Oesophagus.—From the
oesophagus two or three diverticula, similar in
nature to the oesophagus, extend backwards. Of
these, one is of great size, and usually contains
gas. This most usually extends into the abdo-
men, and is a prominent object in dissections and
sections. In the newly-hatched mosquito it is
small, but rapidly becomes large enough to extend
into the abdomen (Fig. 30).

The Homologue of the Proventiculus.—There is
no true proventriculus as in many insects. There
is, however, an interesting fold of the fore-gut
into the mid-gut which represents this organ.
The muscular bundles are here increased, and the
whole forms a valvular muscular organ (Fig. 30).

The Mechanism of Feeding— The powerful
pumping action which must result from a draw-
ing asunder of the three large chitinous plates of
the pumping organ is very evident. These plates,
also, when drawn apart must, by reason of their
spring-like shape, revert to their original posi-
tions close together, without any muscular aid.
Posteriorly the valve-like arrangement mentioned
before prevents regurgitation. Further, when the
blood reaches the junction of the oesophagus and
mid-gut the invaginated portion is withdrawn,
and is distended by the entering blood into a dis-
tinct ‘crop.’ The valvular function is suspended
and the blood flows onward.

The Mid-gut.—The mid-gut extends from th

132

proventriculus to the origin of the malpighian
tubes. It consists of two portions which merge
into one another—an anterior narrow portion,
and a large dilated posterior portion, which
becomes greatly distended after feeding. Unhke
most insects there are no caecal appendages in
the mosquito. Posteriorly there is a marked con-
striction, with strong muscular bundles, which
forms a very marked pylorus (Fig. 30).

The anterior narrow portion of the mid-gut
lies in the thorax, and does not become distended
with blood. The posterior portion when fully
dilated fills the greater portion of the abdomen,
theviscera being pushed into the last few segments.

The Hind-gut.—The hind-gut is short and
passes in one or two bends from the pylorus to
the anus. Immediately beyond the pylorus there
is a considerable dilatation which is poorly sup-
phed with muscular fibres: into this open the
five malpighian tubules. For a short distance
beyond this the lumen is narrow (small intestine),
but becomes gradually larger (colon). At the
termination of the colon there is a slight constric-
tion, after which the canal dilates again to form
the rectum (Fig. 30).

Into the rectum project six solid growths,
the so-called rectal glands, which are, however,
papillae. Posteriorly the rectum ends in the anus
close above the gynaephoric canal.

The appendages of the alimentary canal
are :—

The Salivary Glands.—The salivary glands
consist of six tubular acini lying three upon either
side. Those of one side le generally one above
the other in the long axis of the body, their

ts)

anterior ends lying close against the prosternum,
where the ducts coming from each acinus unite to
form a single duct. The upper and middle acini
generally le with their distal ends close to the
proventriculus. The lower acinus passes towards
the thoracic ganglion. Occasionally, an acinus
becomes bifid at a short distance from its termina-
tion. A common abnormality also, is a small
accessory acinus near the proximal end of an
acinus. A duct can be seen traversing almost the
entire length of each acinus. Shortly after leaving
the acinus, the three unite to form a single duct.
The duct of each side passes up into the neck, and
lies close to the nerve cords passing between the
thoracic and thecerebral ganglia. Beneath, and in
contact with the lower surface of the suboesopha-
geal ganglion, the ducts of each side unite to form
a common salivary duct which passes forwards
and enters the chitinous first portion of the
alimentary canal close to the base of the proboscis
(Fig. 34). .

The Malpighian Tubules.—These are five in
number and open into the first portion of the hind-
gut immediately beyond the pylorus. Their blind
ends are held in position in the neighbourhood of
the rectum by tracheal branches. They pass
forwards in loops above their origin, so that, in
transverse section, as many as ten may be seen
cut across.

THe MuscuLtar SYSTEM

The chief muscular masses in the mosquito
are contained in the thorax. They are chiefly
muscles moving the wings and legs.

Wing Muscles.—There are two large muscular

134

masses on either side of the thorax, passing from
the dorsal to the ventral body wall. Between
these bundles there is a space, in the lower portion
of which lies the alimentary canal, main air tubes,
and other structures. The upper portion of the
space is occupied by a second series of large
muscular bundles, passing from the front to the
back of the thorax. Neither of these large masses
of muscle are inserted directly into the wings, the
up and down movement of the wings being
caused by alterations in the shape of the thorax,
consequent on the contractions of the vertical and
horizontal fibres, respectively.

There are, however, a few fibres arising from
the lateral portions of the thorax, and inserted
about the base of the wings.

Leg Muscles.—These occupy but little space
in the thorax. They rise, to a large extent, from
the internal processes of the exoskeleton (apodemes),
and are inserted into neighbouring portions of
the limbs. They arise, also, from one segment of
a limb and are inserted into another.

The Muscles of the Body Segments.—These
arise from one segment and are inserted into the
next. They are arranged dorsally and ventrally
in lateral groups throughout the abdomen.

A small muscle is also situated on each side,
passing vertically from the tergum to the sternum.
These on contracting flatten the abdomen.

Muscles in Association with the Alimentary
Canal.—Several important muscular masses are
connected with the largechitinous pumping organ.
A pair of muscles arises from the occipital region
of the exoskeleton, and is inserted into the upper

133

plate of the organ. A large muscle arises on each
side, and is inserted into each of the lateral plates.

In the thorax, a small muscular band rises
from the neighbourhood of the first pair of legs,
and passes upwards close to and outside the sali-
vary glands of each side. The contraction of this
band must exert pressure upon the salivary glands.

Anteriorly and posteriorly small muscular
bundles pass from the dilated portion of the mid-
gut to the abdominal wall.

THe TRACHEAL SYSTEM

Respiration is entirely carried on by tracheae.
These. take their origin from external openings—
the spiracles—and eventually terminate in minute
capillaries in the actual tissues of the insect. In
the Culicidae there is no development of large or
multiple air sacs in connexion with the tracheal
system, as in many insects.

The spiracles are placed both in the thorax
and in the abdomen. ‘The thoracic spiracles are
two in number, situated in the meso-thoracic and
meta-thoracic segment, respectively. Of these the
anterior one is the largest in the body. ‘The
second thoracic spiracle is also much larger than
the abdominal spiracles. The abdominal spiracles
are situated in the pleural membrane, one in each
segment (Fig. 42).

The Tvacheae.—Very large tracheae pass
inwards from the anterior thoracic spiracles.

1. Alarge branch passes forwards towards the
neck and gives off a branch which passes down on
either side of the middle line to the two anterior
coxae and the salivary glands. The main branch

136

continues on through the neck, and supplies the
head with numerous large branches.

2. A large branch passes upwards and back-
wards along the edge of the meso-scutum, and
gives off branches which supply the wing muscles.
A smaller branch also passes forwards and supplies
the muscles of the thorax.

3. The largest trachea in the body (main
trachea) passes downwards, backwards, and in-
wards, so as to le on either side of the anterior
portion of the alimentary canal. Numerous
branches are given off from this trunk to the
thoracic muscles, the alimentary canal, and legs.
Posteriorly the trunk is continuous with a
trachea passing forwards from the second thoracic
spiracle, thus forming on either side a large
tracheal loop.

Large tracheae also pass inwards from the
posterior thoracic spiracles.

1. Branches pass forwards and join in a
loop with the main trachea, also backwards to
join the abdominal system.

2. Branches pass downwards to the meta-
thorax and posterior pair of legs.

3. Branches pass inwards to the muscles and
mid-gut.

From each abdominal spiracle a short thick
trunk passes inwards, which gives rise to the
following branches :—

A dorsal branch ramifying beneath the
tergum and joining the branch of the opposite
side.

A sternal branch supplying the sternal plate
ae muscles, also joining the branch of the other
side.

ey

Loop branches passing to the trunks, anterior
and posterior.

Branches passing inwards and supplying
visccra. Branches from the first, second, third,
and fourth abdominal tracheae supply mainly the
mid-gut, those from the fourth and fifth the
ovaries, those from the sixth and seventh the
genital organs.

The Vascular System.—As in most insects
where the respiratory system ramifies throughout
the whole body, the vascular system is not well
developed. A dorsal vessel or heart. and an
anterior prolongation of this (aorta) are the only
closed blood-vessels. Apart from the dorsal vessel
the blood circulates in large blood spaces, which
lie between the lobes of the fat-body and among
the muscles and viscera.

The dorsal vessel passes close beneath the
tergal plates throughout the abdomen. It is very
thin walled, and is not provided with valves. The
upper portion is attached to the dorsum at
intervals by suspensory fibres (muscular), so that
a festooned appearance is given in longitudinal
section. There is, however, no true division into
compartments. Laterally large cells (pericardial
cells) are arranged throughout its entire extent,
and fibres of a muscular nature (alary muscle)
pass from the body wall and end in branches in
close connexion with the dorsal vessel.

At the first abdominal segment the dorsal
vessel dips down beneath the mesophragma,
lying as it does so in direct contact with the
cuticule. In the thorax it again arches upwards,
and lies between the lower portions of the
antero-posterior wing muscles close above the
anterior portion of the mid-gut.

138

In the anterior third of the thorax it divides
into two smaller portions which pass outwards,
and coming in contact with the salivary ducts
enter the neck.

Blood spaces without definite walls occur
throughout the body. The thorax especially con-
tains large spaces among the muscles, and the
complex fat-body which hes between and sup-
ports the organ is everywhere bathed. with blood
fluid.

Tue Nervous SYSTEM

The ganglionic system in the Culicidae is con-
siderably developed. The head ganglia are large
and complex. The thoracic ganglia are large
and compressed so as to form a large ganglionic
mass. The gangha of this system are as fol-
lows :—

(a) Lying around the pharynx is a gang-
lionic ring composed of large supra and infra
oesophageal ganghawith theircommissures. From
these, large nerves go to the eyes, antennae, and
mouth parts.

(b) In the thorax, lying below the oesopha-
gael diverticulum and close to the sterna, is a
large compound ganglion showing evidence of its
origin from the conjoined ganglia. Between this
and the head ganglia are two long slender nerve
cords, which pass in the neck in close relation
with the salivary ducts. From the thoracic gang-
lion large nerves pass to the limbs, and posteriorly
nerve cords connect it with the first abdominal
ganglion.

(c) The abdominal ganglia lie with their
connecting commissures close upon the abdominal

139

sterna. The last ganglion lies just below the
junction of the oviducts to form the common ovi-
duct. A large nerve passes from it among the
viscera of the last few segments.

The Visceral System.—Small ganglia con-
nected with the main ganglionic system occur in
connexion with the viscera. The most important
of these are two small groups of large nerve cells
lying in front of and above the thoracic ganglion,
with the middle portion of which they are con-
nected by nerves. They he laterally beneath the
oesophageal diverticulum and anterior portion of
the mid-gut, and are not far removed from the

salivary glands. Another small ganglion occurs
above and in front of the proventriculus (Fig. 34).

Tue REPRODUCTIVE SYSTEM

The organs of the reproductive system are :—
1. Ovaries.
2. Oviducts and common oviduct.
3. Mucus gland and duct.
4. Spermathecae and ducts.

The ovaries occupy a variable position depend-
ent upon the state of their development. In the
newly-hatched mosquito they are small bodies
lying in the fourth and fifth abdominal segments
close by the posterior portion of the mid-gut, and
attached to the body wall by numerous tracheae.
As they enlarge they push the mid-gut, hind-gut,
and malpighian tubes towards the ventrum, so
that eventually the ovaries occupy nearly the
whole of the posterior portion of the abdomen.
Each ovary consists of very many follicular tubes,
each containing egg follicles in different stages of

140

development. In the mature ovary the lower
follicles have in every tube become the large
completely-formed egg (Fig. 39).

The oviducts are muscular tubes passing from
the ovaries. They join beneath the rectum to
form the common oviduct, which is still more
abundantly supplied with muscle fibres, and which
eventually opens beneath the anus.

The spermatheca is a chitinous sac, which in
the impregnated female is filled with a mass of
spermatozoa. Its duct is long and twisted and
opens into the common oviduct near its termina-
tion. (In Culex there are three spermathecae.)

The mucus gland, globular or ovoid in shape,
opens by a short duct into the same region.

The Fat-body.—The adipose tissue is disposed
in two ways.

tr. As a general hning to the body wall,
being nearly everywhere present directly beneath
the cuticle, aud

2. As lobular masses lying in among the
organs and muscles. Thus a large pad lies over
the compound thoracic ganglion, and sends pro-
cesses which lie in among the salivary glands and
other viscera. Other smaller masses lie in the
head and abdomen.

HisToLoGy

Methods.—The examination of the fresh tissues
frequently reveals structures not easily seen in fixed
preparations. The tissues are best dissected out
in normal saline of low tonicity, O°3 OF O'4 per
cent., as insect juices have a lower isotonic point
than those of mammals. Better preparations of

141

both tissues and included parasites are usually to
be obtained by the use of fixed tissues. Several
tissues (including the salivary glands and mid-
gut) may, when dissected out, be spread by means
of the edge of a slide or cover-glass, and rapidly
dried. These, fixed and stained, give beautiful
preparations of sporozoits, as well as certain
parasites in the mid-gut, hind-gut, etc.

For fixing mosquitoes as a whole, watery
solutions are not generally so good as alcohol, on
account of the difficulty of penetration from the
nature of the exoskeleton and the large amount of
air contained in insect tissues; very good results
are obtained by fixing and hardening in abso-
lute alcohol, and proceeding at once to embed in
paraffin. It is best, so soon as considerable
hardening has taken place, to make a minute
incision into both the thorax and abdomen. For
fixing portions or isolated organs of mosquitoes
saturated solution of perchloride has advantages
over alcohol and fixes the cells of the mid-gut
extremely well. It does not penetrate, however,
well into undissected mosquitoes. Picric acid
gives good results with isolated organs. The
changes in the mid-gut cells during digestion are
well shown.

Both Culicines and Anophelines, but especially
the latter, cut readily in paraffin or celloidin. For
staining smear preparations and sections hae-
matein gives very good results; sporocysts and
sporozoits, as well as the normal tissues, are well
stained.

The stellate cells in connexion with the
tracheal endings upon the mid-gut, etc., are fre-
quently well shown by gold chloride (Fig. 32).

142

Hempennain’s haematoxylin gives good results
with the salivary glands, and also the muscle
fibres in connexion with the alimentary canal.

Tue HistoLtoGy oF THE ALIMENTARY CANAL AND
APPENDAGES

The epithelial lining differs considerably in
the mid-gut from either the fore-gut or hind-gut.
In the mid-gut the possession of a marked striated
border by the epithelial cells is characteristic.
The muscular fibres of the alimentary canal are
striated throughout.

The Fove-gut.—The anterior portion of the
fore-gut is lined by chitin and does not differ from
the cuticle in structure. It consists of a single
layer of cubical cellsof small size. The oesophageal
dilatation and its diverticula resemble one another
in structure. Inthe adult mosquito they consist of
an extremely delicate membrane formed of a single
layer of flattened cells, with externally some
scattered muscular fibres. In fresh preparations
peculiar wrinklings of this membrane are seen,
which may appear like bundles of sporozoits. A
similar appearance is seen in the dilated portion
of the hind-gut just beyond the pylorus.

In the pupa, the oesophageal diverticulum is
seen passing backwards as a narrow tubular organ
lying beneath the mid-gut. It isin this stage
lined with well-marked cubical epithelium. Ina
freshly-hatched mosquito this organ is frequently
undistended, and shows anarrow lumen surrounded
by a single layer of large cells. These cells re-
tain very little trace of protoplasm, which, how-
ever, may still be present in fine strands, and

143

around the nucleus, which is pushed to the outer
portion of the cell.

In the majority of mosquitoes the walls of
the oesophageal diverticulum are crowded with
micro-organisms and bodies which appear to be
protozoal in nature.

The Mid-gut.—There is but little structural
difference between the narrow anterior portion of
the mid-gut, which lies in the thorax, and the
posterior dilated portion, which lies in the abdo-
men. In many insects there are caecal tubes or
pouches opening into the anterior portion of the
mid-gut. These are, however, quite absent in the
adult mosquito. The main thickness of the wall
consists of epithelium; external to this is a thin
coat of muscle fibres (Fig. 32).

The epithelium consists of a single layer of
large cells, which are columnar in the undistended
organ, but become flat and pavement-like when
the organ is full of blood. They have a finely-
reticulated protoplasm, which stains more deeply
towards thefree border. Stained with HEIDENHAIN’S
haematoxylin, alcohol-hardened specimens are
seen to contain numerous stained granules,
collected especially in the outer portion of the cell.
These are especially abundant in the anterior
portion of the mid-gut. They have also, very
frequently, a number of small clear vacuoles (drop-
lets), which become more frequent and of larger
size towards the free border of the cell. The most
marked feature of the cell is the clear striated
border which is present in all the cells of the mid-
gut, but absent in all other portions of the alli-
mentary canal. The striated border is_ best
marked in the undistended organ, and becomes

144

almost invisible in the fully distended state, when
the cells are much flattened.

The nucleus of these cells is large and centrally
situated. The chromatin is arranged in small
stellate masses arranged circumferentially and
centrally, and connected with one another by
fine threads of chromatin. ‘There isa body which
stains less deeply, generally to be made out
(karyosome) in the centre of the nucleus.

Occasionally young cells are to be seen near
the basement membrane.

The muscular coat 1s very thin. It consists
of an open mesh-work of long muscular fibres
running longitudinally and circularly. In the
large posterior portion of the mid-gut, these fibres
form a very regular series of large square or
rhomboidal meshes. In the narrow anterior
portion they are more closely approximated, so
that the muscular layer here is more evident in
sections.

The individual muscle fibres are very long,
fusiform, striated fibres. On the outer surface of
the mid-gut le numerous large branched cells in
which the small tracheae end, and from which
bundles of minute structureless air tubes pass into
the wall of the mid-gut. These cells are frequently
well shown in gold chloride specimens. Similar
cells occur throughout the viscera in connexion
with the tracheal endings (See‘ Tracheal Endings’).

The Homologue of the Proventriculus.—Mention
has been made of a fold occurring at the anterior
extremity of the mid-gut. This consists of an
invagination of a portion of the fore-gut into the
mid-gut. The mid-gut is also folded in with the
portion of fore-gut, so that in this region there is

145

a double thickness of mid-gut wall as well as the
fore-gut. There is an increase in the muscular
fibres of the mid-gut at this point, especially the
circular fibres, so that a very distinct mass is
formed homologous to the proventriculus of many
insects. There is no chitinous development, how-
ever, and the structure would appear to act only
as a muscular sphincter (Fig. 30).

The Hind-gut.—The nature of the epithelium
and arrangement of the muscular fibres differs
somewhat in different portions of the hind-gut.
Structurally the small and large intestine are
similar, whilst the dilatation beyond the pylorus,
and especially the rectum, differs from these.

The dilatation which occurs at the origin of
the malpighian tubules is thin-walled and poorly
supplied with muscle fibres. The cells lining it
are small and flattened.

The intestine is lined with a single layer of
large cubical cells ; external to these is a muscular
coat. The cells of the intestine have large nuclei
which have a similar, though more open, arrange-
ment of the chromatin than the nuclei of the mid-
gut. The protoplasm is finely reticular, and stains
less deeply than the cells of the mid-gut. Stained
with HempEnuAin’s haematoxylin, no granules are
present as in the cells of the mid-gut. They have
no striated border.

In the rectum the cells become small and
flattened. There are here, however, bodies usually
termed rectal glands. These are papillae covered
with a single layer of much hypertrophied cells
resembling those lining the small intestine and
colon.

The muscular system of the hind-gut is very

i

146

similar to that of the mid-gut, consisting of very
large fusiform, straited cells arranged circularly
and longitudinally. The circular fibres in the
small intestine lie outside the longitudinal, and
pass spirally around the mid-gut. Towards the
termination of the intestine longitudinal fibres
also lie outside the circular. In the rectum and
extending throughout the hind-gut and mid-gut
in the Culicidae, there are, in a large proportion
of specimens, swarms of a flagellate organism
a ee | =

The Salivary Glands.—The salivary acini le
in a cleft in the fat-body, which latter comes in
close contact with the glands. Each gland acinus
consists of a single layer of large cells, limited
externally by a delicate sheath (basement mem-
brane) and internally by the intra-glandular duct
wall.

In Anophelines the intra-glandular duct
becomes larger as it approaches the termination
of the acinus, and forms a large cavity.

In Culicines the duct remains of the same dia-
meter throughout the acinus, and terminates
abruptly near the end of the acinus without any
dilatation.

In both Culicines and Anophelines there are
two types of gland acinus. These are recognizable
both in the fresh gland and in fixed specimens.

From their appearance in the latter they may be

termed (1) The granular type.

(2) The clear or colloid-like type.

The Granulay Type—The greater portion of
the acinus consists of cells whose nucleus and
protoplasm has been pushed to the outer portion

147

of the cell by a large mass of secretion, which
occupies almost the whole of the cell. In the
fresh gland this secretion appears as a clear,
refractile substance, and can, by pressure, be made
to exude from the cell in refractile globules. In
specimens hardened in alcohol, this clear secretion
appears as a granular mass, occupying the greater
portion of the cell. It stains faintly with haema-
tein, and shows under high powers (one-sixteenth
oil immersion) a coarse reticulum and _ isolated
globules, an appearance probably due to the pre-
cipitation or coagulation of the secretion by the
alcohol. Considerable variations exist, however,
in the appearance of this granular secretion, both
in the different mosquitoes and in different parts
of thesame gland. In Anophelines the greater por-
tion of the gland contains cells densely crowded
with granular material. Very frequently, how-
ever, the terminal portion contains cells in which
only a few large globular masses exist (Fig. 38).

The protoplasm of the cell occupies, in the
fully-matured gland, only the extreme periphery,
and the nucleus, which is much degenerated, is
pushed to the outer portion of the cell, and usually
lies in the angular interval left at the base of two
or more contiguous cells. In the granular type
of gland this disappearance of the protoplasm and
nucleus from view is more pronounced than in the
clear type of gland.

The Clear or Colloid-like Type.-—Of the last-
mentioned type there are two acini upon either
side; of the present type there is but a single
acinus upon either side, which usually lies between
the two acini of granular type (Fig. 38).

In the fresh gland the cell outlines are not so

148

distinct as in the granular type, and the secretion,
when extended by pressure, is much less refractive.
In alcohol-hardened specimens, the acinar cells
contain a large mass of clear, homogenous -secre-
tion which, as in the last- mentioned type, fills
almost the entire cell, and pushes the protoplasm
and nucleus to the periphery.

In the clear type, however, the protoplasm is
always in greater amount than is the case with
the granular type, and the nucleus never becomes
so greatly degenerated. The clear, homogeneous
secretion stains readily with haematein, and may
even stain quite deeply. With HerpENHAIN’s
haematoxylin it frequently becomes almost black.
It resembles very much in appearance colloid
substance as it is seen in the mammalian thyroid.

In Anophelines this substance also distends the
central duct space within the acinus. In this
situation an appearance is sometimes produced
which resembles faintly-stained sporozoits, but
which is a normal condition.

The Maturation of the Glands.—In freshly-
hatched mosquitoes both types of acinus consist
of large glandular cells arranged round the lumen.
These contain a large, centrally situated nucleus,
and have protoplasm containing a large number
of coarse granules, staining with haematein. In
the portion of the cell nearest the lumen a vacuole
of varying size is situated. This is the commence-
ment of the large mass of secretion which, in the
mature gland, occupies the entire cell. In the
granular type of acinus the vacuole contains
granules ; in the clear type it resembles the colloid-
like secretion (Fig. 36).

Further Variations in the Cells of the Salivary

149

Acini.—In the granular type of gland the greater
portion of the acinus is composed of cells of the
character described above. A portion, however,
usually exists which differs considerably in struc-
ture. This portion adjoins the duct, and may, in
Anophelines, reach as much as one-quarter of the
entire gland in length. In this portion of the
gland the cells are much smaller than those con-
taining the granular secretion, so that the diameter
of the acinus is much less here, and a sudden in-
crease takes place when the portion containing
the granular secretion is reached. The cells
lying towards the duct differ from those lying
towards the acinar end of this portion. There
is, however, no line of demarcation between them,
the one gradually becoming changed into the
other. In the centre of each cell is a clear body,
pushing the nucleus and protoplasm to the outer
portion of the cell. Towards the duct end in the
centre of this clear substance is a darker portion
continuous with the duct lumen. As the cells
come to lie nearer the distal portion, this central
‘dark lumen becomes obliterated. This structure,
though present in Anophelines, may be absent in
Culex. In certain Culex another variation in the
gland cells frequently occurs. The portion of the
gland lying close to the duct, instead of being
less in diameter is greater. The cells composing
this portion are columnar in shape, with centrally
situated nuclei and no contained secretion.

In certain specimens it is not uncommon to
find cells occupying a peripheral position, and not
approaching the lumen, which contain a substance
resembling the colloid-like secretion of the clear
type of gland.

I50

Changes after Feeding.—Very little change
occurs in the glands after feeding. They are for
the most part still quite full of secretion. Probably
a very small amount only of secretion is used
with each puncture.

The Ducts.——The intra-acinar ducts vary in
Culicines and Anophelines. In Culicines they remain
narrow and tubular throughout the entire length
of the gland. In Anophelines they become large
spaces in both types of acini, but especially in the
clear type. The duct is lined throughout by a
clear homogeneous skeletal material, which is
continuous with a similar substance dividing the
cells of the gland from one another. Into the duct
the secretion-filled cell opens by means of a small
opening.

The duct, after leaving the acinus, consists of
a thick-walled tube, with a central spiral thread
resembling the spirals in the trachea. The wall
is homogeneous, but contains many nuclei.

The Malpighian Tubules—The malpighian
tubules are tubular bodies with caecal ends,
which open into the hind-gut. The cells are
extremely large, being, next to the pericardial
cells, the largest in the body. Each cell contains
a large nucleus, and contains numerous large
granules, which stain feebly with haematein, but
powerfully with HerrpEnHarIn’s haematoxylin.
Numerous fatty granules are also present. Each
cell is wrapped round a central lumen, the cells
being arranged alternately, so that a zig-zag
appearance is given in section. The inner portion
of each cell is markedly striated, the lumen being
thus bounded by a striated area. In relation
with these tubules, a large number of tracheae and
tracheal end-cells exist.

151

In certain conditions the malpighian tubule
cells may be found quite free from granules,
though otherwise unchanged. This change occurs
in mosquitoes with large numbers of a flagellate
organism (previously noted) in the rectum and
hind-gut.

The Muscular System-—The muscular fibres
of the mosquito are without exception striated.
Those of the wings differ in structure very much
from those of the limbs and body segments. The
muscle fibres of the alimentary canal are large
fusiform cells, with a single large nucleus with some
surrounding protoplasm. «The muscle fibres in
connexion with the heart are much branched.

Many of the fibres contain a very marked
sarcolemma and space between this latter and the
fibre. This space is usually seen occupied by
extremely delicate branching threads, which stain
feebly with haematein.

In the pupae there exist some large cells of
peculiar nature in association with the sheaths of
the muscle fibres.

The structure of insect muscle is described
in many works on histology, and does not need
repetition here.

The Tracheal System.—The larger tracheal ves-
sels consist of a single layer of flattened cells with
an inner chitinous layer. In smaller tubes the cells
embrace the entire vessel, the nucleus frequently
being bent around the lumen. ‘The cells of the
tracheal vessels contain numerous small clear
vacuoles (chitin formation). The chitinous hning
possesses a thickening in the form of a spiral thread,
which may become unwound and lie stretched as
a wavy thread in fresh preparations.

152

The smaller tubes contain the spiral thread
until they become from 2 to 5 1n diameter.
They then divide to form bundles of excessively
minute air capillaries, which enter among the
tissue cells. The division into capillaries takes
place in the substance of large branched cells
situated at the termination of the tracheal vessels.
The cells often appear cribriform in section from
the number of air capillaries. These cribriform
cells in connexion with the tracheal endings are
well seen in the mid-gut and malpighian tubules.
They are, however, seen best of all in the unde-
veloped ovary of the newly-hatched mosquito,
which is extremely rich in bundles of capillary
air tubes.

The Vascular System.—The dorsal vessel is a
delicate walled tube composed of longitudinal
and oblique fibres with a nucleated inner layer.
The fibres may be traced directly from the termi- —
nations of the branched alary muscle fibres. ‘The
alary fibres break up into fibres which pass in
close connexion with the large pericardial cells, and
eventually form (1) fibres passing into the dorsal
vessel as longitudinal fibres, (2) fibres joining in
an anastomosis in connexion with the floor of
the dorsal vessel.

The pericardial cells are extremely large cells
lying on either side of the dorsal vessel through-
out its whole extent. They are by far the largest
cells in the mosquito, varying from 30 w to 50 win
longitudinal diameter. They are elongate or pear-
shape in form, and contain several nuclei. The
nuclei usually show signs of degeneration. The
peripheral portion of the cell stains more deeply
than the central portion, which contains the

12

nuclei and small stained granules. There are a
considerable number of masses of a light yellowish
pigment resembling that found in the large vis-
ceral ganglia cells. The fibres from the branches
of the alary muscles pass over and around the
pericardial cells to reach the dorsal vessel. From
their structure and situation the pericardial cells
appear to be of the nature of ganglion cells
(Fig. 32).

The Fat-body.—The fat-body, both where it
occurs as a portion of the body wall and where it
les as free lobulated masses, consists of cells con-
taining numerous oil globules. The cells are of
considerable size, and their borders may be fre-
quently traced as polygonal areas. The nuclei
are oval in shape with a central mass of chro-
matin and chromatin threads. Besides oil globules
the cells contain granules staining with haematein,
and minute droplets of a highly refractile, dark
substance, which gives the appearance of pigment.
These droplets are larger in amount in old mos-
quitoes than in those freshly hatched (Fig. 32).

The Nervous System.—The gangha of the
ganglionic system consist of an outer portion of
nerve cells and an inner portion of non-medullated
nerve fibres. Considerable complexity exists in
the larger ganglia, especially the head ganglia.

The ganglia of the visceral system differ
greatly from those of the ganglionic system. The
ganglion cells are few in number and of large
size. They possess clear reticular protoplasm, a
little denser around the periphery than in the
centre. Around the inner margin of the denser per1-
pheral portion small stained points are arranged.
In the centre a variable number of granules of
yellowish pigment exist.

154

The Reproductive System.—Each ovary con-
sists of a large number of follicular tubes whose
lower ends open into the ovarian tube, and
whose upper ends terminate in a delicate sup-
porting filament (terminal filament). The apex
of the ovary is formed of a single follicular tube,
whose filament is attached to the fat-body of the
fourth segment.

Around the whole ovary there is a delicate
nucleated sheath.

Each follicular tube contains one or more
egg-follicles in different stages of development.
In the freshly-hatched mosquito each follicular
tube contains an undeveloped egg-follicle. As
this develops, a second and a third undeveloped
follicle appear above it, which again undergo
development into mature eggs. The follicle at
first consists of two to four large cells, with large
nuclei surrounded by a single layer of smaller
epithelial cells (Fig. 39).

The central cells then increase in size and
number, so that many very large cells are con-
tained in the now enlarged follicle. The sur-
rounding epithelial cells also become larger, and
rapidly increase in number so as to form a layer
of regular cubical cells surrounding the follicle.
The central cell nearest the ovarian tube is the
ovum, the rest are nurse cells, and eventually dis-
appear. Both the ovum and the nurse cells in-
crease greatly in size. The nurse cells have clear
protoplasm and extremely large nuclei, which
exhibit karyokinetic figures. The ovum contains
very numerous yolk granules, which occupy the
whole of its substance, except a thin coating
of granular protoplasm. Still later this thin

ES

external layer can only with difficulty be made
out (Fig. 39).

The nucleus of the ovum undergoes very pro-
nounced changes. It appears as an irregular
mass, staining uniformly with nuclear stains.
This mass becomes more and more distorted and
broken up, and eventually disappears.. It may
frequently, however, be seen as irregular masses
of staining material even in the mature egg. A
portion of the nucleus is seen very early to be
separated off from the rest, often surrounded by
the latter. This portion (female pronucleus) is
small and difficult to detect in sections in the
more mature ovum. As the ovum increases still
more rapidly in bulk, the nurse cells become
crowded into the distal portion of the follicle and
eventually disappear, so that, in the mature egg,
no trace of them is to be seen. The epithelial
layer surrounding the follicle becomes much
flattened, and forms eventually a covering to the
egg (chorion). The outer portion of this covering
(exochorion) is transparent, and marked with
oblique parallel markings. Over the proximal
end, 7.e., the end lying towards the ovarian tube,
the chorion forms a globular mass ornamented
with rows of pits. This is the micropylar appara-
tus through which the spermatozoa penetrate the
ovum.

Frequently in Anophelines a large portion or
the whole of the adult ovum consists of a mass of
sporozoa. These consist of numerous small cysts,
each containing eight round or crescent-shaped
bodies,each with a central chromatin spot (Fig. 40).

The ovarian tube arises in the centre of the
ovary, and receives on all sides the follicular

150

tubes. It is lined with a single layer of small
cubical epithelium. After passing out of the
ovary, a considerable number of striated muscular
fibres are arranged in a loose network around it,
and pass from it to surrounding structures. There
are also muscular fibres in the ovary itself in con-
nexion with the ovarian tube and egg-follicles.
The spermatheca consists of a chitinous sac,
with large cells lying externally. These resemble
the cells of the cuticle, and contain droplets.
They do not cover the whole of the surface of the
spermatheca. The contents of the spermatheca
in the fertilized insect consist of a mass of sper-
matozoa, which, in the fresh state, may be seen
revolving with great rapidity within the sac. The
spermatozoa have a narrow, slightly-curved head
and a long tail. The duct of the spermatheca is
narrow and thick-walled, and contains muscular
fibres. Certain large cells lie in connexion with
the duct externally. The mucus gland contains
cells filled with secretion. There are small nuclei
in connexion with the intra-acinar duct (Fig. 39).

LF

Chapter XIII

TO COLLECT AND PRESERVE
MOSQUITOES

How to CoLiect MosgulToEs

Mosquitoes may be collected in two way :—
1. By capturing the adult flies.
2. By breeding out from larvae and
nymphae.

rt. Search in the daytime in houses, huts,
and out-houses, at the base of large trees, amidst
brushwood, and other dark or shaded places.
Anophelines, however, are rarely caught except in
huts and out-houses. They are especially fond of
cow-sheds and the darker portions of the eaves of
huts.

Some species of mosquitoes may be caught by
sitting with a light near a white wall or suspended
sheet, or inside a tent, near a jungle or marsh.
Culex and Taentorhynchus may be found sitting
on the surface just beyond the brightly illuminated
area. Anophelines are rarely caught in this way,
but, one species at least (My. barbivostris), appears
to be attracted by light, and was caught by us on
an illuminated sheet at night, near swampy land.

In searching for adult Anophelines, as many
places as possible should be examined, as the
distribution of some species is very local.

158

If the captured insects appear to have fully
matured ovaries, some of these should be placed
in bottles, as previously described (p. 97), and
allowed to lay their eggs.

If care is taken to place only one species in a
bottle, the characters of the ovum may be noted,
in addition to the adult insect.

Some of the ova should be placed in fresh
water, and an attempt made to determine the
characters of the larva (p. 73), when it has hatched
out and is sufficiently grown.

2. Breeding out.—Full-grown larvae, and
especially nymphae, are collected. These are
collected from every possible source. Scarcely
any water will be found free from some form of
mosquito larvae. Even strongly brackish waters,
containing over one per cent. of salt, often contain
large numbers.

Examine water from the following sources :—

(1) Domestic utensils, cisterns, tins, pots,
calabashes, in which there has been water for
three or four days. The larvae of Stegomyia,
Culex, etc., and only rarely Anophelines, will be
found.

(ii) Cess pits, pools full of decaying leaves,
etc., sewage ditches. Note larvae of certain species
of Culex, etc.

(1) Observe presence of the larvae of
Stegomyia and Culex in the water which collects
in the axils of banana leaves and other plants.
Also, occasionally, Anophelines in large collections
of water of this kind,

(iv) Puddles of all kinds, with and without
algae, ponds, tanks, swamps, rice fields, ditches,
canals, rivers, streams, lake margins, and wells,

132

-and observe that in all, Anopheline, as well as
Culicine larvae, nay abound.

Note that in waters covered with certain
species of Lemna, Anophelines are rarely found.

(v) Place any larvae (nearly adult specimens
if possible,) and nymphae in collecting tubes with
‘a note as to where they have been obtained.
If it is necessary to cork the tubes, some air
space should be left and the corks loosened as
often as possible. Larvae, as a rule, survive
carriage in small collecting tubes better than they
do in bottles or larger vessels. They may be
carried long distances, e.g., in a train or on horse-
back, provided that occasionally the tube is un-
corked and they areallowed breathing time. Larvae
survive rough treatment better than pupae, and
when apparently dead, may often be revived by
floating out on the surface of the water. Examine
the larvae (Chap. IX), and roughly divide them
into as many groups as possible, observing the
main characters of each.

(vi) Place each variety in small bottles, over
the neck of which a piece of mosquito netting
must be tied as soon as the larvae have turned
into nymphae.

When the adult insect has hatched out, note
its attitude and any other special features.

To Kitt MosguIToEs

1. A mosquito that has just hatched out from
the nympha should not be killed for some hours
until its exoskeleton has hardened. If it is killed
immediately, the wings on drying will shrivel, and
possibly the whole insect become distorted.

160

2. Allow the insect or insects to escape into
a clean, dry, glass vessel.

Pour some chloroform on a pellet of wool
and place under the jar, but take care that the
mosquitoes do not get wetted by the chloroform.
Leave them exposed to the vapour of chloroform
some little time after apparent death. Tobacco
smoke may also be used for killing.

After killing, turn the mosquitoes out upon
a sheet of clean paper.

To Mount MosgQuIToEs

Necessary apparatus—
Fine silver pins. No. 20.
Thin cardboard or thick paper.
Large entomological or ordinary pins.
Specimen tubes with corks.

1. Prepare a disc by cutting with scissors
a circular piece of Bristol board (or very thick
paper). The diameter should be slightly less than
that of the specimen tube.

2. Push a fine ‘silver’ pin two-thirds of its
length through the centre of this.

3. Place the mosquito upon its back ona
clean sheet of paper. (In this and other manipula-
tions use a pin for moving or steadying the
mosquito.)

4. Take the head of the fine pin in the
finger and thumb, or hold it near the head end
with a pair of forceps. Endeavour to place the
point of the pin exactly in the centre of the origin
of the legs, which all arise very close together
from the under surface of the thorax. Bear in
mind, that the more the insect is touched the more

I61

scales are rubbed off, and that a crookedly
‘mounted specimen is better than a ‘rubbed’ one.

5. Push the pin steadily through the thorax,
so that it emerges as near the centre of the dorsum
of the thorax as possible. [Practise mounting by
forcing the fine pin through without aid from the
other hand. ]

6. Having transfixed the mosquito, force the
point of the pin one millimetre beyond the back,
by pressing it against the smooth surface of a cork
or tissue paper. The pin should not be pushed
through too far, as it prevents the lens of the
microscope being brought near enough for

examination.

Fig. 41. Authors’ Method of Preserving Mosquitoes

7. Placing the disk against a cork, pass
carefully through the edge a large entomological
pin. This is passed in the reverse direction to
the fine pin. Force three-quarters of the length
M

162

of the large pin through the cardboard disk, and
then firmly press the point into the cork of a
specimen tube, so that when the tube is corked
the mosquito is inside (Fig. 41).

In damp climates, it may be necessary to
carefully dry the tube and insect in a dessicator
(over sulphuric acid or hme), or by placing in the
sun or warm place to prevent mould. This, how-
ever, is but seldom required. Mites are rarely
seen in insects preserved in tubes as described.

Write any information, e.g., locality, date or
reference number upon the outer surface of the cork
and on the edge of the cardboard disc. For trans-
mission, all that is necessary is to pack the tubes
in wool in a box and send by post. Packed in
this way they are far more secure than when
mounted in the ordinary way in an entomological
box. Mosquitoes for the British Museum should
be addressed :—

The Director
The British Museum
(Natural History)
Cromwell Road, London, S.W.

Endeavour always to send both male and
female, at least two of each, and, what is of the
greatest possible importance for the advance of
our knowledge of mosquito classification, the
careful description of ova and larvae.

Note.—lIf from any cause it is impossible to pin and
mount mosquitoes in this way they may instead be simply
placed between layers of tissue paper in a pill box, etc. This
is'far better than placing them in any fluid such as spirit, by
which treatment they are rendered useless for identification.

163

To Mount Portions or MosqQuiTogEs
PERMANENTLY

1.—Wings.—Clip off one or both wings as
near as possible to the thorax, so as to avoid
cutting the base of the wing itself.

2. Allow the severed wing to fall in the
centre of a clean glass slide. See that the dorsal
surface of the wing is upwards.

Place one or two very minute drops of
thick Canada balsam at some distance from the
wing, but within the area of a coverglass. This
is merely to hold the coverglass firmly in place ;
the balsam must not be allowed to touch the
wing.

4. Press a coverglass firmly down on the
Canada balsam. If it is desired, the coverglass
may be ringed with paraffin or some material
which will not run by capillarity beneath the
coverglass.

Legs.—Mount the legs of one side in order in
a similar way.

Mount the palps and proboscis.

Mount (a) The male ungues.

(b) Scales from head, scutellum, etc.
(c) Wing denuded of scales.

In Canada balsam, by placing a drop of bal-

sam on these and mounting in the ordinary way.

Chapter XIV

ANOPHELINAE. EXTERNAL ANATOMY
OF THE IMAGO

Tue Heap

The -head is composed mainly of the two
large compound eyes. These meet below and
approach one another very closely above.

Parts of the head. The following are the
usual names for the different regions of the head
(Fig. 42).

. The nape: the extreme back of the head.

The occiput: the portion behind the eyes.

The vertex : the space between the eyes.

The frons: the space in front of the eyes.

The gena: the side of the head below
the eyes.

The frons is triangular in shape, with one
angle directed downwards. From the upper two
angles arise the antennae, and from the lower
projects the clypeus, lying over the base of the
proboscis.

The Clypeus.—Projects over the base of the
proboscis as a prolongation of the frons. The
character of the clypeus is of specific importance.
It is

1. Hairy in Culex.

2. Scaly in Stegomyza.

3. Nude in Joblotia.

ees Os. ISS

165

The Antennae.—Consists of fourteen to six-
teen segments, of which the basal one is large and
globular. The plumose antennae of the male
readily distinguishes it from the female.

Beotosers

hos

@nknna — Proburets

. Clypers

rons eee eee
Oces ol”
Tape

Seytvne
@

reso hora &

Scott lum
Biz seufellum

Fig. 42 External Analomy of Female Anopheles

The Proboscis.—The proboscis consists of the
very highly specialized mouth parts, ensheathed

166

in the lower lip or labium. The proboscis con-
sists of (Fig. 43) :—

1. The labium, forming the sheath.

2. The labrum and epipharynx, }

or upper lip | forming
3. The hypopharynx, or tongue > the
4. Two mandibles | stylets.
5. Two maxillae J
6. Two maxillary palps.

Fig. 43. The Proboscis (Labium and Stylets), after NUTTALL
and SurpLey. fight hand, cross section of Proboscis
The Palpt ave not shown

The Labium.—The labium forms the thick
and scaly proboscis as usually seen. On its dorsal
surface it 1s hollowed out, and in this hollow run,
as in a sheath, the piercing mouth parts or stylets
(Fig. 43). The labium itself does not penetrate
the skin, but becomes sharply bent during the act
of biting, just as when a cane walking stick is
pushed against the ground. This may easily be
seen if a mosquito is watched during the process
of biting.

167

The Labellae—Attached to the end of the
labium by a hinge joint on either side are two
leaf-like processes, the labellae (Fig. 43). It is
through the angle made by the two labellae, that
the’ stylets pass, asa billiard cue, between the
first and second fingers (NUTTALL and SHIPLEY).

The labium proper stops short at the point of
junction of the labellae, but is continued on its
upper surface as a blunt point covered with fine
hairs (Dutton). We may liken it to a pen con-
tinued on beyond the penholder, the junction of
pen and penholder being the point at which the
labellae are hinged on.

Dutton’s Membrane.—The area between the
end of the labium proper and the extreme tip is
covered by an extremely thin membrane (Durron).
In the act of biting, when the labellae are sepa-
rated, this membrane is somewhat stretched, and
applied to the skin.

Tue Escape or THE FILARIAL EMBRYO

It has been shewn by Low and Jamgs that
the filarial embryo occurred in the proboscis,
according to Low among the stylets. According
to Dutton, the embryo lies really in the tissue of
the fleshy labium, moreover with its head at the
level of the membrane described above, and
that it is by the rupture of this excessively thin
membrane that the embryo escapes. Grassi and
Noe think that the embryo escapes through the
middle of the bent-up labium through a rupture
at this point, but Durron’s explanation seems
more likely.

The epipharynx is the central tube through
which the blood is sucked. Its point slopes off

168

somewhat like the tip of a hypodermic needle.
In cross section it has the shape of an ©, the com-
pletion of the tube being formed by the apposi-
tion below of the hypopharynx. The labrum is
blended with the epipharynx, but does not extend
to the tip.

The hypopharynx is a thin, flat two-edged
lamella closely applied to the under surface of the
epipharynx. It is pierced by the salivary duct
down which the salivary secretion and sporozoits
pass. The opening of the duct is continued as a
groove reaching almost to the tip of the hypo-
pharynx.

Ot saphapeat = Labrom [fe Sharyrn,

fs ae 2
: MLL “pp Manyen

Labium

Fig. 43a. Showing relation of Pharyngeal and Salivary Pumps
to the Probosctis

The mandibles are very fine chitinous rods in
cross section, crescentic in shape. At the tip of
the mandibles are about thirty serrations, though
in certain species of Culex these appear to be
absent.

The mandibles are closely applied to the sides
of the epipharynx.

The maxillae are stouter than the mandibles,
and fit around the outer side of these and the

169

hypopharynx. They have about twelve serrations
at the extremity, coarser than those of the mandi-
bles. In some culices, papillae replace the serra-
tlOrs.

The Maxillary Palps.—These lie upon either
side and somewhat dorsally to the proboscis. In
the act of biting they take no part, but are then
separated from and he at right angles to the pro-
boscis. Differences in the palpi are of both
specific and generic importance.

The expanded ends of the palpi in the male
Anophelines are even more conspicuous than the
plumose antennae. .

The Pyvothovax.—The main portion of the
thorax is mesothoracic; anteriorly, however, there
is a collar-like piece of chitin, the prothorax. To
this are attached two moveable bodies, the pata-
gla.

The prothorax is of importance in classifica-
tion, ¢e.g., in the new genus of the Anophelinae
Stethomyia the prothoracic lobes are mammillated.

The Mesothorax (Fig. 42).—The scutum of the
mesothorax forms the large globular mass of the
thorax. Behind the scutum, and just behind the
origin of the wings, is a transverse bar of chitin,
the scutellum. Behind the scutellum is a convex
triangular area extending as far as the first
abdominal segment, the post-scutellum (Fig. 42).

The scutellum and post-scutellum are of
importance in classification. Thus the scutellum,
with its ‘posterior border bristles,’ is often of
specific value, whilst the post-scullem may be—

1. Bare. Culex and Anophelinae.
2. With hairs. Wyeomyza.
3. With scales and hairs. Joblotia.

170

THe WING

The wings shew :—

1. An anterior straight, thick, and strong
border or costa. .

2. A posterior curved and thin border,
carrying a fringe.

Two small folds at the base of the wing

(squama and alula).

4. Nervures, or veins.

The costa in Anophelines is generally covered in
part with white, and in part with black, scales
(spotted).

Anlénov forked.
cell

Bace

5 bongftdinal
anf lorgifodinal
a? langiledmol
3? long ibidinal
~ olenov
forked cell
Fig. 44. Wing of Anophelinae :—
Upper = Supernumary cross-vein
Lower = Posterior cvoss-vein ’
Anterior forked cell = First forked ‘cell
First posterior forked cell = Second posterior cell
Second posterioy forked cell = Anal cell

The fringe in Anophelines has most frequently
light and darker portions, the number and position
of which are of specific importance (Fig. 51).

171

The Nervures.—-The nervuration of the wing
is of considerable importance. Several nomen-
clatures are in use. That used in the accompanying
diagram is, however, the simplest (Fig. 44).

In classification, the relative position of the
apices of the two forked cells are frequently used.
Also the relative positions of the point where the
auxiliary vein cuts the costal vein, and the point
where the fifth vein cuts the posterior margin.
As a rule, the position of this first point is much
nearer the base than that of the second point, but
in a few instances, e.g., My. sinensis, they almost
coincide.

Also the positions of the upper, middle, and
cross veins. It will be found, however, that even
in the same species there is no constancy in these
latter, and they can hardly be given as of specific
importance as has been done. Dénitrz has made
the same criticism, and indeed, finds that the
position in each wing of the same mosquito may
be different.

Tue Lecs

These consist of the following segments :-—

1. Coxa and trochanter. Small pieces at
the origin of the legs (Fig. 42).

2. Femur.

3. “Tibia.

4. Tarsus, consisting of five segments, the
last of which carries the claw or ungues.

Fig. 45. Fove Ungues of M. funesta ( & ) the larger Uniservate.

Fore Ungues of M. rossii ( 8 ) the larger Biserrate.
(After THEOBALD)

172

The Ungues.—The ungues vary in the male
and female and in the different legs. They may
be simple, uniserrated or biserrated (rarely tri-
serrated). (Fig. 45.) They are of specific value
(THEOBALD).

Tue ABDOMEN

The abdomen consists of nine segments. To
the ninth segment are attached the genitalia.

The genitalia are variously shaped lobed
appendages. In the male they are provided at
their free end with claspers. The claspers in the
male are of specific value (Fig. 46).

GQ. funesus a pseudo preles

Irate Gendalre
Fig. 46. Male Genitalia

Technique ;—

1. The constituent parts of the proboscis may be readily
separated by dropping the living mosquito into alcohol
(Curisty).

2. The study of the chitinous parts is much facilitated
by soaking in oil of cloves.

173

Chapter AV

. CLASSIFICATION AND IDENTIFICATION
OF THE CULICIDAE |

SCALES

‘THEOBALD has attached to scale structure the
greatest importance from the point of view of
generic’ and specific classification. Hence it is
necessary to consider somewhat in detail these
structures. THEOBALD gives the following :—

_ Head Scales.—Three forms of scale occur
(Fig. 47). |
1. Narrow curved scales.
2. Upright forked scales.
3. Flat scales overlying one another
hike the tiles of a roof.

No. 1, 2, and 3 scales found, ¢.g., Culex.

No. 2 and 3 scales only, found, e.g., Stegomyza.

No. 3 scales only, found, e.g., "Megarhinus.

Toxorhynchites.

Thoracic Scales.—TuHEoBALD describes five
forms (Fig. 47).

1. Narrow Hatr-like Curved Scales.—They
often form a dense feltwork over the mesothorax.

2. Narrow Curved Scales.-—They may occur
all over the mesothorax and scutellum, or at the
sides of the scutum and in front of the scutellum.

3. Spindle-ShapedScales.—These lie scattered
about, and never form a complete covering.

174

4. Flat Scales like those on the head. They
cover the scutellum in Stegomyia, whereas in Culex
the scutellar scales are of the narrow curved type.

5. Long Twisted Scales.—Characteristic of
Mucidus, a genus of mouldy-looking mosquitoes.

Abdominal Scales.—The scales covering the
abdomen in all Culicidae, except the Anophelinae,
are overlapping flat scales. In the Anophelinae
they are not found, except to some extent in
Myzorhynchus and Nyssorhynchus. In the genus
Aldrichia, however, of Anophelina, the abdomen is
covered with flat scales as in Culex.

cloccey

Sh orace Seales

Head Scales

Ta noplles

Myzorkynchus Gelolifedeplinn
Ing - ven Seales

Af Healt

Fig. 47. Varieties of Scales (after THEOBALD).
Panoplites = Mansonia

Wing Scales.—Scales clothe the veins, except
the cross veins. Flat scales are arranged in a double
row along each vein.

Many, or in some species all, of the veins
have also lateral scales,

7

The lateral scales are very variable in shape,
€.8.,

1. In Mansonia they are broad asymmetrical
flat scales.

2. In Aedomyza the scales are similar.

3. In Mucidus the wing scales are quite
characteristic, being pyriform or inflated and half-
dark, half- white.

4. In Megarhinus the scales may be azure
green or blue.

The Wing Fringes consist ol—

1. Long narrow-pointed scales attached to
the edge of the wing by a narrow stalk.

2. Smaller scales similar in shape.

3. Border scales. Small flat scales.

Leg Scales.—The legs are covered with flat
scales in nearly all Culices.

t. In Sabethes the scales are hair-like and
occur in tufts.

2. In Mucidus, Psovophora, the scales are
elongated and project from the legs.

The sub-family Anophelina contains, as we
shall see, twelve genera, the Culicinae twenty-five,
and the Aedeomyina seventeen. When we consider,
further, the large number of species in some of these
genera, e.g., Culex, it is impossible to attempt here
to describe each mosquito, however briefly. Con-
sidering the great importance, however, of the
Anophelinae, we shall attempt to give the charac-
teristic specific points for each of the species, as an
aid to a detailed examination by means of
THEOBALD’S monograph. With regard to the
other sub-families we shall attempt only to give
characteristics of each genus.

The Culicidae are divided into the following

176

sub-families, based mainly upon the length of the
palpi in male and female :— _
1. Palpi long in both sexes, as long as the
proboscis in the female Anophelina
2. Palpi long in both sexes, shorter than the
proboscis in the female Megarhinina
3. Palpi short in female, long in male—
Culicina
Palpi short in female, long in male. Post-
scutellum with hairs (chaetae) and scales, Joblotina —
[= Trichoprosopina
5. Seven (not six) longitudinal veins with

scales Heptaphlebomyina
6. Palpi very short in female and male—

Aedeomyina

7. Proboscis short, not formed for pierc-

ing Covethrina

Sus-Famity ANOPHELINA (vide next Chapter)

Sus-Famity MEGARHININA

Genus 1. Megarhinus.—First sub-marginal
cell much smaller than second posterior cell. Palpi

Ww an i

wp
—

Fig. 48. Head and Scutellay Scalesof Aedes (left)
Megarhinus (right)

ai

five-jointed in ¢ (M. purpureus only four). Readily
recognized: (i) by their large size, often called
‘elephant mosquitoes’; (ii) by their brilliant
metallic colours ; (iii) by a caudal tuft of hairs on
each side of the abdomen; (iv) by the long and
curved proboscis; (v) head is elsthed with flat
scales only (Fig. 48).

They may be found resting on the trunk of
trees in the forest, also in houses in the bush.
Species, about six.

Genus 2. Toxorhynchites—Palpimuch shorter
than proboscis in 2, three-jointed. Supernumerary
cross-vein nearer the apex of the wing than the
mid cross-vein. Species, four. .

SusB-Famity Cuticina

First sub-marginal cell equal to or longer
than the second posterior cell.

Genus 1. Janthinosoma.—Hind legs densely
scaled, giving a characteristic appearance. Species,
five.

Genus 2. Psovophova.—Characterized by (1)
great length of é palpi, five-jointed; (11) densely
long scaled legs ; (iii) posterior cross-vein a little
nearer the base than the mid; (iv) proboscis
curved in?. Species, four.

Genus 3. Mucidus.—Easily recognized by
their curious mouldy appearance. Posterior cross-
vein nearer apex of wing than mid. Wing scales
large, pyriform, parti-coloured. Head and thoracic
scales long and twisted, expanded at the apex.
Legs densely scaled with projecting scales. Species,
five.

N

178

Genus 4. Desvoidea= Armigeres.—Head, flat
scales, a few upright-forked. Differs from Stego-
myia, (i) is longer, with unbanded tarsi and
abdomen. 3 palpi, untufted. 9 palpi, very pointed
and provided with bristles only. Species, two.

Genus 5. Stegomyia. — Head completely
clothed with broad flat scales (Fig. 49) and a
few upright forked. Palpi four-jointed in the °,
five-jointed in the 3. Scutellar scales flat, mostly
black and white mosquitoes with banded legs and
abdomen. Species, twenty.

narrow-corved
Sidley

fiat scales.

upright forbed- a

Ste omy7a.
ae Cae

Fig. 49. Head scales of Stegomyia (left) and Culex (right)

S. fasciata—Transmits yellow fever.
1. Tarsi basally banded white.
2. Proboscis unbanded.
3. Thorax. A pure white broad curved
band on each side, and two median
. pale parallel lines.
4. Ungues of 2 toothed.

Genus 6. Theobaldia——Palpi in ¢ clubbed as
in thelAnophelinae. Palpiin 2 five-jointed, apical
joint mammilliform, wings in both sexes densely

179

scaled, collected into spots, thus forming a spotted
wing group of mosquitoes. Species, five.
Genus 7. Lutzia.—Resembles Theobaldia.
Palpi in ? three-jointed, apical joint not
mammilliform.
Palpi in ¢ not clubbed, three-jointed.
Wings spotted by ecules similar to those
of Taeniorhynchus. Species, one.
Genus 8. Culex.—Head scales: narrow curved
and upright forked; laterally flat scales (Fig 49).
Palpi in @, three-jointed. Third palpal joint
usually as long or longer than the other two.
Wing: first sub-marginal cell longer and
narrower than the second posterior. Posterior
cross-vein nearer the base than the mid wing-
veins. Scales small, lateral ones linear.
Scutellum: narrow curved or spindle scales
(Fig. 49).
C. mimeticus has spotted wings. Species,
very numerous.
Genus 9. Gilesia.—Related to Culex and
Stegomyia.
(i) Scutellum with small flat scales, some
spindle scales.
(11) Head: broad, flat, spindle scales.
(iii) Basal joint of antennae, hairy and scaly.
(iv) Claws short and thick with a blunt

tooth:
(v) Wing scales like those of Taeniorhynchus.

Species, one.

Genus 10. Lastoconops.—
(i) Head scales as in Culex. Basal joint of

antennae, a few scales.

180

(ii) Abdomen: large projecting flat scales with
deeply dentate apices, giving these mosquitoes a
ragged appearance. Species, one.

Genus 11. Melanoconion.— Distinguished from
Culex by the dense broad scales on the costa and
apex, and by the black spine-like scales along the
upper border. Small dark mosquitoes. Species,
SIX.

Genus 12. Gvrabhamia.—Allied to Culex and
Taeniorhynchus. Palpiin 2 four-jointed. Apical
joint minute. Penultimate longand thick. Wing
scales not so long or dense as in Taeniorhynchus.
Scales mottled. Wings short and stumpy. Legs
mottled and spotted. Species, ten.

Genus 13. Acartomyia.—Allied to Culex and
Grabhamia, Distinguished from Grabhamia by
having flat irregularly disposed scales all over the
head, from Culex in the 8 palpi. Two terminal
segments and the apex of the antipenultimate
swollen. Terminalsegment club-shaped. Ragged
appearance of head, well marked. Species, one.

Genus 14. Taeniorhynchus.—Palpi five-jointed
in é, the fifth segment minute. Characterized
by the wing scales. They are thick elongated
scales ending with a broad sloping convexity or
blunt point ; median linear scales often absent,
proboscis usually banded. Species, about sixteen.

Genus 15. Mansonia = Panoplites.— Palpi
four-jointed in ¢, more than one-third the
length of the proboscis. Characterized by wings
densely scaled along the veins with broad asym-
metrical flat scales. No median scales. The genus
resembles Aedeomyia, but the palpi in the ¢ are
long in members of this genus, short in the
Aedomyina. Species, eight.

181

Genus 16. Finlaya.—Three ventral abdominal
scale tufts. Scutellum, four median bristles. Wing
scales, large and broad, pyriform. Species, two.

Genus 17. Howarvdia.—Resembles Aedes, but
scutellum has only four bristles. Palpi, four
segments, apical, one minute, not mammilliform.
Species, two.

Genus 18. Skusea.—-Head, flat scales only.
Anterior and posterior forked cells densely scaled.
Palpi in@?, three segments. Scutellum, six bristles
and narrow curved scales. Species, three.

Genus 19. Katageiomyia.—Differs from Steg-
omyia in having (1) narrow-curved scales on back
of head ; (2) narrow-curved scales on lateral lobes
of scutellum. Species, one.

Genus 20. Macleaya.—Differs from Stegomyia
in having (1) narrow-curved scales on the centre
of the head, and (2) on the lateral lobes of the
scutellum. Species, one.

Genus 21. Hodgesita.—Resembles Stegomyza.
Differs in having the lateral vein scales long and
almost overlapping those of neighbouring veins.
Apices of scales have marked lateral spines. Palpi
very short (one-jointed ?), possibly an Aedeomyine.
Species, one.

Genus 22. Scutomyra.—Diflers from Stegomyia
in having narrow-curved scales on head. From
Macleaya, in having scutellum entirely clothed
with flat scales. From Leicesteria, in having all
scutellar scales flat.

Genus 23. Danielsia—Distinguished from
Macleaya and Scutomyia by the narrow-curved
scutellar scales, and from Katageiomyza by the long
male palpi.

Genus 24. LEvetmapodites—Head, flat and

182

upright forked scales. Scutellum with flat scales
on mid lobe. Palpi in 2 four-jointed, in ¢ five-
jointed, long, thin, and hairless. Last two hind
tarsi in 3 densely scaled, forming a paddle in one
species. Species two.

Genus 25. Hulecoetomyia.—Head a. marked
median area of narrow-curved scales extending
backwards. Scutellum with a rosette of flat and
somewhat spindle-shaped scales on mid-lobe.
The scutellar scales are vounded apically not
pointed. Distinguished from Stegomyia. by head
and scutellar scales. Species, one. Malay.

Genus 26. Lercesteria.—Unpublished.

SusB-FaMILy JOBLOTINA

Genus 1. Joblotia——Metanotum (= Post-
scutellum) with a tuft of chaetae and with flat
scales. Clypeus and base of antennae bristly.
Second long vein carried nearly to the base of the
wing. Second posterior fork cell (anal cell) very
large. Mid cross-vein nearer the apex than the
anterior (supernumerary). Wings densely scaled ;
scales shorter than in Taeniorhynchus. Species,
one.

Sus-Famity HEPTAPHLEBOMYINA

Genus 1. Heptaphlebomyia.—Like Culex, but
has a distinct scaled seventh vein. Species, one.

Susp-Famity AEDEOMYINA

Genus 1. Deinocerites = [Brachiomyia].—
Characterized by the 2 antennae. Much longer
than the proboscis. Second segment as long as the

183

three terminal segments. Antennae scaled. Antennae
in é pilose and longer than the whole body.
Species, two.

Genus 2. Aedes.—Head, narrow curved scales
form a broad median line only. Other scales flat.
Scutellum, narrow curved scales, six bristles. Palpi
in@, four segments, apical segment minute, mam-
milliform. Traces ofa fifthsegment. Species, two
(ig. .48).

Genus 3. Aedimorphus.—Head, mostly flat
scales, narrow curved behind. Scutellum, flat
scales, eight (?) bristles. Has no flat thoracic scales
as Uranotaenia; probably a Culicine. Species, one.

Genus 4. Verrallina.—Head as in Skusea.
Palpi, two segments only (trace of a third), apical
segment large. Scutellum, four bristles and narrow
curved scales. Species, three.

Genus 5. Ficalbia.—Intermediate between
last two and next genus. Head scales, no narrow
curved, almost entirely flat. Scutellum, flat scales
as in Uvanotaenia, but thoracic scales narrow curved.
Palpi, two segments. Species, two.

Genus 6. Uvanotaenta.—Head, flat scales,
upright forked may or may not be present.
Scutellum flat scales. Thorax, narrow curved and
flat scales. Wings, small forked cells. Metallic
scales at the base of the wings. Related to Aedes,
but more brilliant (metallic) and stouter mos-
quitoes. Species, fourteen.

Genus 7. Mimomyia.—Resembles Urano-
taenia. Has no flat scutellar or thoracic scales.
Forked cells larger than Uvanotaenia. No metallic
scales at the base of the wings. Species, two.

Genus 8. Aedeomyia.—Alled to Aedes. Dis-
tinguished by (1) head scales upright, fan-shaped ;

184
clypeus scaly; (ii) thorax, broad, flat spindle
scales ; (iii) scutellum, broad flat scales ; (iv) legs,
densely scaled; (v) wings, densely scaled as in
Mansonia, also with long lateral scales. Species,
three.

Genus g. Haemagogus.—Related to Aedes,
but palpi ‘five segments. Head covered with flat
scales. Brilliant metallic (blue) mosquitoes.
Species, two.

Genus 10. Wyeomyia.—Chaetae on the post-
scutellum. Head, flat scales. Thorax, spindle
and flat scales. Scutellum, flat scales. Palpi
short. Proboscis not as long as whole body. Species,
two.

Genus 11. Phoniomyia.—Resembles W yeomyia,
but distinguished by (i) wing scales broad, lateral
scales as in Taentorhynchus ; (11) proboscis longer
than the whole body. Species, two.

Genus 12. Dendvomyia.—Resembles W yeomyia,
distinguished by (1) scutellar scales small, flat,
rounded apically ; (11) wings more densely scaled
than in Phoniomyia, scales Taeniorhynchus-like ;
(111) proboscis moderately long. Species, five.

Genus 13. Runchomy1a.—Allied to Dendromyia.
Characterized by (i) frons projecting as a blunt
spine; (ii) proboscis as long as the body in @ ;
(111) ventral apical tuft of bristles; (iv) wings
covered with rather broad scales. Species, one.

Genus 14. Sabethes——Distinguished from
Wyeomyia by the asymmetrical wing scales. One-
or more legs with dense paddle-like structures in
both sexes. Mid cross-vein nearer the apex than
the anterior. Posterior nearer the apex than the
mid inthe é. Third long vein carried through
into the basalcell. Brilliant metallic mosquitoes.
Species, four.

185

Genus 15. Sabethoides.—Closely resembles
Sabethes. Distinguished (i) by much smaller
palpi; (11) unpaddled legs. Species, one.

Genus 16. Goeldia.—Post-scutellum with
chaetae and scales. Wing scales asin Runchomyia,
dense, elongated. Wing venation as in Culex,
Proboscis short and thick; not as long as body.
Palpi in ¢ one-third length of the proboscis. In
@ quite short. Species, one.

Genus 17. Limatus.—Characterized by the
proboscis bent in the middle; densely scaled at
the bend. Species, one.

Sus-FAMILY CORETHRINA

Genus 1. Covethva.—-First tarsal segment
longer than the second tarsal.

Genus 2. Mochlonyx.—First tarsal segment
shorter than the first tarsal.

LITERATURE

A Monograth of the Culicidae. Vols. LIU. FI. V. Turo-
carp. . Brit, Mus. Nat. Hist. The.data. in this chapter have
been taken almost entirely from THEoBALD’s work.

186

Chapter XVI

THE CLASSIFICATION AND IDENTIFI-
CATION OF THE ANOPHELINAE

It is by no means an easy matter to fix
definitely the species of an Anopheline, and yet the
identification of species is essential in connexion
with malarial studies. It does not suffice merely
to ascertain that Anophelines are present in any
given locality, but it must be clearly. made out
what the species are. It is, indeed, only by accu-
rately observing the relation of Anophelines to
malaria that we can hope for the explanation of
many of the difficulties surrounding the anomalous
distribution of endemic malaria. Ina _ later
chapter the extreme importance of the species of
Anophelines in this connexion will be evident. In
the description of the habits of Anophelines, their
breeding places, occurrence apart from man, etc.,
it is no longer sufficient to ascribe these to the
whole genus, but they must be ascribed to the
actual species involved. The study of the
Anophelines from the point of view of classification
and identification is, therefore, of importance.

CLASSIFICATION OF ANOPHELINAE

THEOBALD has sub-divided the old genus
Anopheles into twelve new genera. These genera

187

comprise over eighty different species. The assign-
ing of an Anopheles (old sense) to its proper
genus, simplifies, therefore, very much the ultimate
determination of the particular species. THEO-
BALD’S Classification is the following :—

Thorax and ab-|Prothoracic lobes/Wing scales

domen with] simple; no flat} lanceolate Anopheles
hair-like head scales
curved scales Wings _ scales

mostly long
and narrow- Myzomyia

Wing _ scales

partly large

and inflated
Cyclolepidopteron

Prothoracic lobes;Wing _ scales

mammillated: | lanceolate Stethomyia
median flat
scales

Thorax with |Wing scales small, lanceolate or
narrow curved] narrowed Pyvetophorus
scales ; abdo-
men hairy

Thorax with hair-like curved scales; some
narrow curved ones in front; abdomen with
apical lateral scale tufts and scaly venter,
no ventral tuft Arvibalzagia

Thorax with hair-like curved scales ; abdominal
scales on venter only with a distinct ventral
apical tuft, no lateral tufts -Myzorhynchus

188

Thorax and ab-
domen with
true scales

Abdominal scales with dorsal

patches of small or narrow flat

scales ; thoracic narrow curved

or spindle-shaped Nyssorhynchus
Abdomen nearly completely

scaled with irregular scales

and with lateral tufts Cellia
Abdomen completely scaled with
large flat scales, as in Culex Aldrichia

These features are shewn in the accompanying
diagram (Fig. 50). The thoracic scales in Cyclolept-
dopteron are not sufficiently hair-like (THEOBALD).

Place the mounted or unmounted mosquito

under a low

power of the microscope, and deter-

mine carefully the characters of the hairs or scales
on the head, thorax and abdomen. By this means
the Anopheline is assigned to its proper genus.

Anopheles

Myromy ca

ed

C
4

a

1

Fig. 50 Thoracic, Scutellar, Abdominal, and Wing Scales of
the Anophelinae (after THEOBALD)

189

Cy. ladspedepleron Slathomy ca Pi retophorus

es

piyenndi peed My ssorhynchus Callte

Mig. 50 (contd.)—Thoracic, Scutellay, Abdominal, and Wing
Scales cf the Anophelinae (after THEoBaLp)

1gO

Tue DIFFERENTIATION OF SPECIES

Many features are of value in determining the
species.

1. The Wings :—

(a) ‘They may shew areas of dark scales on
the costa, auxiliary, and first longitudinal veins,
producing the main spots of the wing.

(b) Small areas of scales on the second to sixth
long veins; less dark and distinct than the large
costal spots.

(c) Pale areas on the wing fringe.

(a) The markings on the wing are fairly constant in each
species, but variations occur, so that the spot may be longer
or shorter, giving the wing a darker or lighter aspect in the
same species. Thus in N. stephensi the following variations
in the second costal spot may be encountered (Fig. 51).
Especially does. this variation occur in the wing of males.
The costal spots may also be confluent. They may depart
from their typical shape, as is frequently seen in the T spot
of M. rossi.

(b) The smaller spots on the wing field along the course
of the veins are also useful for determining species. Thus
M. leucophyrus has six spots on the sixth long vein, while
MM. elegans has only four. The. extent to which the third
longitudinal vein is scaled is also of specific importance
(Fig. 52).

(c) The wing fringe has at the points, where the long
veins cut the margin, a variable number of light areas. Thus
A. punctipennis has only one’ pale area, while A. pseudo-
punctipennis has many. Another example of this means of
distinguishing species is given in the figure (Fig. 51).

2. Leg Markings :—

(a) Uniformly coloured as in the second
division of Myzomyia.

(6) Speckled or banded, chiefly in the genera
Nyssorhynchus and Cellia. The banding of the
legs is of great importance in distinguishing the

IgI

species (Fig. 53); thus (1) banded tarsi, eg., N.
maculatus ; (2) tarsi pure white, e.g., N. fuliginosus,
N. jamesii.

Fig. 51) [—Wing fringesfof (1)JM. rhodesiensis, (2) M. funestus,
(3) AL. listont, (4) M. culicifacies

Variations in Wing Spots of M. vossti, N. stephensi,
and P. costalis

Variations of Cross-veins of M. rossti

3. Palpi:—Similarly the bands or collection
of white scales on the palpi is a convenient means
of separating the members of a particular genus
(Figs. 52, 53). It should not be forgotten that in
these two characteristics there is a certain amount

192

of variation, possibly seasonal, and a slight differ-
ence in the bands on the palpi and legs is. not
sufficient in itself to constitute a difference in
species.

Other characteristics that are useful for the
determination of species are the, male genitalia,
and the character of the ungues in the male,
whether having one or more teeth. The position
of the cross-veins has also been used, but this is
so variable in the same species that it has httle
value.

Characters of the Larvae and Ova.—tn the
Anophelina, as in the rest of the Culiczdae, this is
a most important means of differentiation. Mos-
quitoes that otherwise are almost indistinguish-
able are readily separated by their larvae being
different.

One precaution must be taken. It must be
quite certain that it is the larva of the mosquito
in question that is being examined. The easiest
way to make sure of this is to carefully examine
the larva first, and then to hatch out the mosquito
and then examine it. The examination of the
larvae is considered later. :

Genus 1. Anopheles—Wings unspotted or
slightly spotted. Mostly belong to temperate
climes or hill districts.

A. bifurcatus and A. maculipennis transmit
malaria. A. punctipennis has experimentally given
negative results.

Costa Uniform, Wings spotted.

Le. Gia maculipennis. -—-Wing with four spots ;
apex of first tarsal joint spotted. ‘Europe.
2. A. crucians.—White spots on brown

193

veins. Three black spots on sixth vein. Costa

uniformly dark; tarsi unbanded. North America.
A. eisenit.—Resembles A. maculipennis.

Apices of hind tarsi, yellowish. Gautemala.

Costa Spotted

4. A. punctipennis—Two yellow spots, one
at the apex ; the second on the apical third. One
fringe spot. North America.

5. A. pseudo-punctipennis——Wings as in pre-
vious species ; but wing fringe with several yellow
spots. North America.

6. A. punctipennis (Var. A).—Three costal
spots.

7. A. franciscanus.—Costa, a pure yellow
apical spot ; a second spot about middle of costa.

8. A. gigas——Costa, two large black spots.
Length, five to six mm. A large hill species. India.

g. A. lindesayit—Costa black, apical white
spot. Femora have acharacteristic broad median
white band. A hill species. India.

Wings Unspotted

to. A. bifurcatus——Thorax. Golden hairs
arranged so as to leave two broad bare lines on
the front. Abdominal haivs golden. Europe.

A. walkeri is regarded by ‘THEOBALD as
identical with this.

11. A.algeriensis—Lateral vein scales longer
and finer than in A. bifurcatus. Anterior and
posterior cross-veins in same line in both sexes.
In A. bifurcatus the posterior is internal in ?, the
anterior in ¢.

12. A. nigripes—A black mosquito. No
bands on tarsus. Europe. America.

fo)

194

13. A. immaculatus——Ash-grey in colour.
Slight apical bandings to tarsi. Palpi and pro-
boscis lighter at apex. Ennur, Madras.

14. A. aitkeniti—Uniformly dark. No mark-
ings on palpi or legs. Bombay Presidency.

15. A. stigmaticus.—Light brown; tarsi un-
banded. Australia.

16. A. annulipalpis.—Tarsi banded. Last
tarsus pure white. S. America.

Genus Myzomyia.—To this genus belong those
species which are associated in the tropics with
the most severe endemic malaria, e.g., M. funesta
in Africa and M. listont and M. cultcifacies in
India. The group includes, however, several
species, one at least of which has, as far as our
knowledge extends, no power of transmitting
malaria in nature, viz., M. ross71.

The malaria transmitters form a natural
group: they are small, dark mosquitoes, with
unbanded legs, and they breed in fresh natural
waters, e.g., streams, river beds, etc.; whereas
we also have in the group domestic mosquitoes,
i.e., those that breed in foul pools about houses.
M. vossti is the type of this class.

Whether in this genus any others than the
three mentioned above convey malaria there are
at present no facts to shew, and the larval characters
of only the Indian species are at present known.

‘The type species is M. funesta, which is a
typical spring and fresh-water breeder. It is
noteworthy that M. funesta is associated with a
higher malarial endemicity than P. costalis,

which is a typical domestic mosquito breeding in
foul pools.

195

Group I

Small dark mosquitoes breeding in natural
waters.

1. M. funesta.—Costa: six white spots.
Basal spots with pale interruption. Wing fringe:
pale spots at ends of all the veins, except sixth.
Palpi: three bands, the basal one further from
the middle one than the apical. A variable
species : third long vein may be dark. Resembles
listont and rhodestensis (Fig. 51).

2. M., listont—Third long vein light. Wing
fringe, four or more hight spots (ig. 51). Palpi,
two broad apical bands further apart than in
funesta, one narrow basal. Basal portion of
costa uniformly black (characteristic). Attitude,
Anopheline-like. Associated with high endemic
index in the Duars, Bengal. Larval characters:
antennae with simple hair. Clypeal hairs simple.
Palmate hairs on thorax and on all abdominal
segments.

M. aconitta. —aconita = unspeckled, be-
cause at the commencement of the third long vein
the usual dark spot is absent. Palpi, four bands.
Costa, four spots, ight interruption in basal spot.
Fringe, several pale areas. Anterior forked cell
much longer and narrower than posterior. Differs
from listont in palpi. Sumatra, Java.

4. M. culicifactes——Third longitudinal vein
dark. Wing fringe, three spots at most. Palpi,
three equal bands, two at the joints, one at the
apex. Attitude, Culex-like. Associated with high
endemic index of malaria in the Punjab and
Madras. Larval characters as in M. listont. Ova:
floats do not touch margin of upper surface

(Type 1) (Fig. 54).

196

M. leptomeves—Base of first long vein
white. Anterior forked cell much longer and
narrower than posterior. Costa, two spots, thus
differing from Hebes. Fringe, pale areas at all
the veins.

DOLL UTR, PLLA |.

: RRC HK 1.
XK *-

LILI ELLA ULE 2.

y TL VE 3.
CAMILLE. OU ELLL Yer 7

NU MELE LLM CLIT a

Fig. 52
PalpijofiM. funesta (1), M. listoni (2), M. culicifacies (3),
M. vhodesiensis (4), M. hispaniola (5), M. turkhudi (6),
Third Long Veins of M. funesta (7), M. listoni (8),
| M. culictfacies (9)

6. M. hebes.—Hebes=inconspicuous, a small
species resembling vhodestensis. Wing costa, four
spots; wing fringe, seven ight areas. Vein six,
one long spot. Palpi, first and second segments
covered with white scales. End of third segment
is dark, fourth segment quite white. Distinguished
from M. vhodesiensis by palpi and wing fringe. East
Africa.

Group I

Larger species than the above. Generally
lighter. Wings not so covered with dark scales.

Habits: Domestic species breeding in foul
puddles, etc., near houses

197

Larval characters (M. vossiz): Antenna with-
out branched lateral hairs. Clypeal hairs simple.
Palmate hairs second to seventh segments, and
often rudimentary hairs on thorax and first and
second abdominal segments. Ova (type 2), 7e.,
floats, touching margin of anterior surface.

. M. albivostvis—Characterized by the
banded proboscis; pale scaled to about half its
length. N. deceptor has also a banded proboscis.
Malaysia. Length, two to five mm.

8. M. longipalpis—Palpi long, thin; three
narrow white rings; wing costa, black, four
almost equal yellow spots; wings mostly brown
scaled; hind legs only, banded; narrow apical
and basal yellow bands. British Central Africa.
Length, three mm.

9g. M. ludlowit—Palpi, apex broad white
band; a second small one close to it; a third
basal band. Wing costa, four large spots, one
or two small basal. Legs, femora, tibiae and
metatarsi, especially in hind legs, spotted with
yellow. Tarsi, broad apical and basal pale
banding, especially in hind legs. Philippine
Isles. Length, four to five mm.

10. M. rvossii—Probably =A. vagus (DéniTz).
Sumatra. Palpi, somewhat like those of M.
ludlowii, but easily distinguished; the apical
white band is broader; the second band is much
nearer the base than in M. ludlowi1, so that the
black area between is longer. Wings, four
spots, and some basal spots. The second large
spot has the characteristic T. shape, but is very
variable. Tarsi, slight pale apical and basal
bands to some of tarsi. India, Malaysia.

>

198

tr. M. litziit.—Characterized by the linear
ornamentation on the thorax, and marked bands
(five in number) on the fore and mid metatarsi.
Wings, three distinct pale spots; two smaller ones,
3 to 3.5 mm. Rio de Janeiro.

12. M. elegans.—Possibly = M. leucosphyva
(Donttz). Leukos = white, sphyron = ankle-
joint. Palpi, four white bands. Wing costa,
four large black-scaled areas, three small. Wing
fringe, six pale interruptions. Legs speckled with
white scales. Femora and tibiae speckled in hind
legs. Characterized by a large tibio-metatarsal
band on the hind legs. Resembles N. stephens: ;
differs in the palpi; has four, not three, spots on
the sixth longitudinal vein. Differs from N.
leucosphyrus in having four, not six, spots on the
sixth vein. Possibly belongs to Nyssovrhynchus
genus. India.

13. M. tesselatum.—Costa, four large, four
small spots. Fore tarsi apically and_ basally
banded. Mid and hind tarsi apically only.
Thorax, two dark spots in front and a dark area
near the scutellum. Malay.

14. M. punctulata—Costa, four large spots
and numerous dark gnd white spots. Malay;
closely resembles the former.

15. M. leucosphyra—Related to the two
former. Distinguished by prominent. tibio-

metatarsal band and by the prominent median
dark spot on costa. Sumatra.

16. M. impuncta.—Costa, four small dark
spots. Fringe spotted. Sixth vein, three spots.
Relationship doubtful; not fully described.

Egypt.

199
Group III

Medium size dark mosquitoes. Apex of palpi
black.

17. M. turkhudi.—Palpi, apices black, the
band not so broad as in Hispaniola; third long vein
mostly dark, but varies; pale interruption in
basal costal spot. India. Larvae resemble
Culex. Ova, very peculiar, type 3 (vide p. 222).

18. M. hispaniola (TuHEo.) Spain. Third
longitudinal vein, mostly pale yellow, except at
the base and apex. Wing fringe with spots, except
where lower branch of fifth and sixth join the
costa. Basal portion of costa uniformly black.

19. M. rhodesiensis (THEO.) Rhodesia.—
Third longitudinal vein dark. Palps with only
two conspicuous bands. The palpi are much
longer and thinner than in M. funesta. The
veins are all dusky scaled. Base of the costa
black. In M. funesta there is a white inter-
ruption. Wings, costa three small white spots
and a yellow apical spot. Fringe unspotted,
except an apical spot (Fig. 51).

Genus Cycloleppteron.— Wings with numerous
large inflated scales; collected in patches or
irregularly disposed.

Larval Characters (THEOBALD).—Antenna
without lateral branched hair. Clypeal hairs
simple. Palmate hairs, six pairs. Lanceolate.

tr. C. grabhamit.—Palpi unbanded. Jamaica.

2. C. mediopunctatus.—Palpi banded, black
and gold. Brazil.

Genus Stethomyia(ornQos = breast).—Head with
a median patch of flat scales. Palpi very thin.

1. S. nimba.—Wings unspotted. Thorax
brilliant silvery median band. 3S. America.

200

2. S. fragilis—Wings unspotted. Thorax
greenish-brown. Malay.

Genus Pyretophorus (rvperopopos = fever pro-
ducing).

1. P. superpictus.— Larva has branched
frontal hairs. Wing costa, four distinct spots
and additional basal spots. Legs dark brown
with apical white tarsal bands. Palpi, apical
white band and two narrower bands, the second
slightly nearer the first than the second. Eur ope
and Mashonaland.

2. P. costalis—Wing costa, four large and
two small spots. On the first long vein there are
two broken spots, under the two middle large
spots, giving a pattern only found in P. marshalliz
besides. This arrangement is, however, variable.
Femora and tibiae mottled with yellow. Tarsal
banding involves to some extent both sides of the
joints. Palpi, three narrow bands at the joints.
West Africa, Uganda.

P. costalis v. melas. Pale costal spots are
absent, but the arrangement on the first long vein
is the same. P. costalis conveys malaria.

P. cineveus—Wings, three white spots
on the black costa; wing fringe brown, with
yellowish patches. Palpi, four white rings; legs
very thin, jet black; apex of femora and tibiae
pure white spot; apices of fore and hind meta-
tarsi, minute apical bands; length five mm.
South Africa. B.C.A.

. PP. marshallit. — Wing markings very
similar to P. costalis, distinguished by the palpi;
two broad apical bands; one small basal one ;
apex white. Mashonaland.

5. P. jeypovensis.—Costa black, two large

201

white spots on the apical half, and two small
ones at the base. Fringe spotted. Palpi black,
with three white bands, the broadest apical.
Apex white. Madras.

6. P. chaudoyet.—Wing, six black costal

spots ; legs unbanded, a pale knee and tibial spot
on the hind legs. Palpi, apex black and with
three narrow white bands. Algeria.
7. P. palestinensis—Wings, five large black
costal spots and five yellowish ones of unequal
length. Legs brown, a pale spot at junction of
tibiae and femora, and tibiae and metatarsi.
Palpi, three pale bands, the apex white. Differs
from P. chaudoyei in the form of the large costal
spot in the apical half of the sixth long vein being
dark, and in presence of a deep brown medium
thoracic line. Resembles closely P. superpictus,
but the legs are unbanded ; spotted wing fringe,
and uniserrated large fore ungues in the ‘male.

8. P. minimus.—Wings, three nearly equal
creamy spots and an apical spot. Fringe, spotted
except at the sixth vein, thus distinguished from
P. superpictus. Legs, no trace of banding or pale
knee spots. Mid ungues straight; fore ungues
curved. Hong Kong.

P. atvatipes—Clypeus, trilobed, costa
uniformly black, six spots on the veins. Australha.

10. P. merus.—Resembles P. cinereus, but
distinguished by the spotted and banded femora
and tibiae, also by its broader fringe spots.

11. P. pitchfordi—Three main costal spots,
two basal interruptions. Sixth vein two spots.
Thorax broad white median band. Zululand.

Genus Arribalzagia.—Related to Myzorhyn-
chus, but has no distinct lateral scale tufts.

202

1. A. Maculipes—Hind and mid legs much
banded and speckled. Almost certainly transmits
malaria (Lutz).

Genus Myzorhynchus.—mifw to suck, pryxos, pro-
boscis.

Palpi densely scaled in the 2, also the pro-
boscis. These are ‘wild’ mosquitoes found in
situations remote from the dwellings of man.
They breed in swamps and large bodies of water,
especially those containing weeds. They do not
usually frequent houses. M. sinensis is, however,
attracted by light. They feed readily on human
blood when occasion offers.

(A) Palpi unbanded.—Last hind tarsus brown:

1. M. barbivostris, one fringe spot. India,

Malaysia.

tA. M. pseudo-barbivostris. — Distinguished
from former by its speckled femora and tibiae.
Philippine isles.

2. M. bancrofti, several fringe spots. Aus-

traha.

3. M. umbrosus, no fringe spot, only one

costal spot. Malaysia.
Last hind tarsus white:
4. M. albotaeniatus, other hind tarsi much
banded. Malay.
Last two hind tarsi white:
. M. coustant. Madagascar.
(B) Palpi banded.—Last hind tarsus brown :
6. M. sinensis, wing fringe, one pale spot.
China.

Palpi banded, last hind tarsus brown, wing
fringe unspotted. Apex of palpi white:

7. M. vanus, costa two yellow spots, wings

distinctly spotted. India, Malay,
Philippines, etc.

203

8. M. pseudopictus, wings without prominent

spots. Europe.

g. M. minutus, wings, two white costal spots.

Punjab.
Apex of palpi black :
10. M. nigerrimus. India.
(C) Palpi banded, last hind tarsi white :
11. M. mauritianus, two hind tarsi white.
12. M. paludis, three hind tarsi white. Africa.
13. M. ziemanni.Two and two-thirds hind
tarsi white. Africa.

Genus Nyssorhynchus.—woow, to puncture,
‘bite,’ pvyxos, proboscis.

Mosquitoes mostly with legs spotted and
banded, or one or more tarsal segments pure
white. They are both domestic and wild mos-
quitoes. They breed chiefly in pools with algae,
and in lakes. N. Stephenst will, however, breed
in pots and tins.

A. Legs unspotted. Larva with outer pair of
clypeal hairs markedly branched. Leaflets of pal-
mate hairs with long filament.

1. N. fuliginosus. — Probably = leucopus,
Dénitz. Costa, four large and one or more small
pale spots. Femora, pale band near the apex.
Hind tarsi, three and one-fifth pure white. Palpi,
two narrow white bands, apex white (Fig. 53).
India.

2. N. karwavi—Legs not speckled, one and
one-fourth hind tarsal joints white. In fore and
mid legs, tarsal joints, except fourth and fifth, have
apical white band. In hind legs, tibia, first and
second tarsus, have apical bands, third and fourth
have both apical and basal bands ; the fifth is
white. Palps, four white bands, two terminal

204

broad and equal, two basal narrow, apex white.
India.

B. Legs markedly spotted. Larva with simple
or slightly branched frontal hairs. Filaments of
palmate leaflets very short.

6 CCD .

6 CC ETT RE Ty,

\. LLL TOT
es
. Wu ia BD
ee eee ae . MU fi

4 ou a NLL ary

+ OTE
Fe es NS MN BY aa 3

f WWE MU [omspeckled L172 s
CTT Tombeked 77 UTM __ Ye)

IRB LALLA LLL CL

Fig. 53
Hind Tarsi and Palpi of N. kavwari (6), N. maculata (1),
N, theobaldi (2), N. fuliginosus (3), N. fuliginosus (nag-

purensis) (4), N. maculipalpis (5), N. jamesii (7),
N. pretoviensis (8), N. willmori (9)

\

3. N. stephensi.—Syn = A. metaboles, THEO-
BALD. Tarsus without any segment of hind leg
white. Legs brown, speckled with white ; joints of
fore-and hind tarsi with apical spots. Wing costa,
four broad prominent black spots and two smaller
basal ones. The third largest spot has three
typical spots beneath it on the first long vein.
Fringe dark, with pale areas. Palpi, two broad
apical white bands, one narfow basal; white
scales between the last two bands. India.

205

N. maculatus——Resembles N. stephenst,
but is easily distinguished by tarsi. Wings, costa
four large and two small basal spots. Under the
third largest spot are three black spots on the frst
long vein. Legs, with femora, tibiae, and meta-
tarsi with broken creamy bands and spots. Fore
and mid tarsi with narrow yellow bands. Hind
tarsi with broad white ones. Last segment pure
white. Palpi, four bands, two unequal white
apical bands, then a small white one, and a
second towards the base.

5. N. theobaldi.—Wings, jet black with the
costa interrupted by five white spots and an apical
spot. Legs, brindled with white scales and a
large sub-apical white patch on the femora. Two
-‘and a quarter hind tarsi pure white, then a black
band, then a small white one. Palpi, three white
bands, apex white, two apical bands equal, a
third narrow.

A Nagpur variety which THEOBALD considers
may be a distinct species has two-and-a-half
hind tarsi white and the tips of the palpi black.
. India.

6. N. maculipalpis = A. jamesti in Reports
to Royal Society, STEPHENS and CHRISTOPHERS.
Wing, costa black with five white spots. Legs,
black spotted with white, last three hind tarsi
pure white, and apex of next. Palpi, two broad
white bands, one apical,a third narrow one towards
the base. The rest of the palpi spotted with white.
Length, 5°55 mm. India, Africa.

N. maculipalpis, v. Indiensis—Hind legs not
quite so banded as in the type. Some variation
in wing markings.

7. N. jamesit.—Costa; four large and two

206

small dark spots. Legs brown, fore femora and
tibiae more or less spotted. Hind legs, femora
and tibiae with an apical white spot, last three
tarsi white, and apex of next (Fig. 53). The first
tarsal segment of the fore leg has an indistinct
median band. Palpi black, with white rings and
white apical joint, closely related to A. fuliginosus,
but easily distinguished. Length, 3 to 3°5 mm.
Cp. A. maculipalpis, length, 5°5 mm.

8. N. pretoriensis—Clypeal hairs of larva
simple. Palps not mottled, otherwise like N.
maculipalpis. The two white apical bands are
further apart. Second hind tarsus has a small
black patch near its base. Metatarsus, mottled
with white and black, and has a broad white
apical band lke the first tarsal. The last two
hind tarsi only white.

g. N. deceptor (Dé6niTz). Sumatra. Deceptor
because very hke M. punctulata or leucosphyra.
Terminal half of proboscis white. Terminal half
of palpi white, with a narrow black ring at the
commencement of the third and fourth joint. The
ring around the lght end of the second joint,
possessed by M. punctulatus, is wanting. Legs
similarly marked as in leucophyrus, excepting the
hind legs, which have only a small light spot at
the end of the tibia, and not a broad white band.

10. N. willmori (JAMES). Punjab, Kashmir.
Wings, four large and three small black areas.
Palpi, three white bands, the two terminal ones
are equal and broad, the third narrow and basal.
Legs, dark brown, thickly speckled with white
spots. The last tarsal segment of hind leg pure
white.

t1. N.annulipes. Femora and tibiae banded.

204

Tarsi have apical and basal bands. Palpi: apices
of last three segments banded. First and second
segments have white scales. Australia.

12. N.mastert. Distinguished from former
by the proboscis having pale apex in 2. It isalso
smaller. Australia.

13. N. philippinensis. Pale spot at apex of
tibiae. Three anda fifth hind tarsi white. Palpi
golden brown, three bands.

14. N. nivipes. Wing resembles that of N.
stephensi, but legs not speckled. Resembles N.
maculatus but has three and a fifth hind tarsi
white. Malay.

Genus Cellia— Wings densely scaled. Palpi
of @ densely scaled. Easily recognized by the
dense coating of irregular scales.

Larvae (C. pulcheryima).— Antennal hair
simple. Clypeal hairs, outer pair branched. Ova,
type 2.

Last hind tarsi white:

1. C. pulcherrima, 33 white. Punjab.

4. C. bigotiz, 3. Chili.

3. C. pharoensis, 1[$]. Egypt, Gambia.

4. C. argyrotarsis, 4. Palpi, three bands,

deep black basal band to last tarsus.
Acts as a host for F. nocturna
(VINCENT). West Indies.
5. C. albipes, 3. Palpi, two bands. West
Indies, Brazil.
Last hind tarsi yellow:
6. C. kochit, 3. Malay.
Last hind tarsus black:
C, squamosa. Africa.

Genus Aldrichia—Wings much as in Myzo-

myta, for which genus it was originally mistaken.

208

1. Al. ervor.—Resembles M. rvossii. Easily
distinguished by the abdominal scales. India.

Genus Lophoscelomyia.—Resembles Nyssorhyn-
chus, but differs in having (1) long curved hair-like
scales on thorax instead of narrow curved and
spindle scales ; (2) dense apical tufts on the hind
femora in both sexes.

tr. L. astatica—Very small. Wings, five

black spots and four yellow costal spots. Malay.

Genus Christya.—Thorax, hair-like scales and
narrow curved laterally. Wings, dense lanceolate
scales. Abdomen with characteristic dense long
lateral apical tufts of hair-like scales.

1. Ch. itmplexa.—Fore femora with white
spots and a prominent pale band. Hind tarsi
black, apex of leg white. Uganda.

Addenda

A. wellcomei :—Palpi, basal third black, apical two-thirds
ochreous. Apical band almost white, and a second white band.
Wings resemble those of A. gigas. Costa, black with two
yellow spots on apical half. Fringe, yellow spots at junction
of all the veins. Sudan.

M. Nili. Resembles M. funesta. It is darker. Palpi
have only one small apical band. Palpi and proboscis much
shorter than the body. Sudan.

209

Chapter XVII

THE HABITS OF ANOPHELINES

GEOGRAPHICAL DISTRIBUTION

This is as yet far too imperfectly known for
a close consideration of the subject to be of much
value. We may consider, however, that the
Anophelinae of some portions of Africa and some
portions of India are known with a sufficient
degree of exactitude to make a comparison of
interest. The following is a complete list of the
known Anophelinae :—

Europe
A. maculipennis Mym. hispaniola (and
A. bifurcatus Teneriffe)
A. nigripes ; Myzo. pseudopictus
Pyr. superpictus
Palestine
Pyr. palestinensis A. maculipennis
M. pseudopictus P. superpictus
North America

A. maculipennis A. bifurcatus

(? European species) A. punctipennis
A. nigripes A. crucians
A. pseudo-punctipennis A. franciscanus

South America and West Indies

C. argyrotarsis Cy. grabhami
C. bigotii Cyclo. medio-punctatus
Mym. lutzii Steth. nimba
Arri. maculipes C. albipes
A. eiseni A. annulipalpis

P

Mym. funesta

Myzo. paludis

Mym. rhodesiensis

C. squamosas

Pyret. superpictus (West

Coast and Mashonaland)

Pyret. costalis

C. pharoensis (Gambia, Egypt)

Mym. longipalpis

Pyret. marshallii (Mashonaland)

P. pitchfordi (Zululand)
Ch. implexa (Uganda)
M. coustani (Madagascar)

A. welcomei (Sudan and Aden)

Myzo. nigerrimus
Myzo. barbirostris
Nysso. maculipalpis
Nysso. jamesii
Nysso. stephensi
Nysso. theobaldi
Nysso. maculatus
Nysso. willmori
Nysso. karwari
Nysso. fuliginosus
Mym. rossii (everywhere)
Mym. listoni

Mym. leptomeres

Mym. aconita
Mym. ludlowii
Mym. albirostris
Mym. punctulata
Mym. kochii
Mym. rossii
Mym. listoni
Mym. vara

Mym. minuta
Myzo. barbirostris
Myzo. sinensis (China)
Nysso. maculatus
M. tesselata

S. fragilis

Africa

Pyret. cinereus (S. Africa)

Mym. mauritianas

Mym. hebes (E. A. and
S. W. A.)

Pyret. merus

Nys. maculipalpis

Nys. pretoriensis

Myzo. barbirostris

A. algeriensis

Mym. impunctas (Egypt)

Pyret. chaudoyei (Sahara)

M. zeimanni (Cameroons)

M. rossii (Egypt ?)

M. indiensis

Mym. culicifacies

Mym. turkhudi

Mym. elegans

Myzo. minutus

Myzo. varus

Ce. pulcherrima (Punjab)
A. lindesayii (hill species)
A. immaculatus (very rare)
A. aitkenii (Goa)

A. gigas (hill species)
Ald. error

P. jeyporensis

Malaysia

L. asiatica
P. minimus (China)
Nysso. deceptor
Mym. umbrosa
Mym. albotaeniata
Myzo. plumiger or [64]
(Dénitz)

Mym. tenebrosa (Dénitz)
Mym. leucosphyra

i (Dénitz)
Nyss. leucopus (Dénitz)
Pyret. gracilis (Dénitz)
Mym. vaga (Dénitz)
N. nivipes

211

Australia
Myzorhynchus bancroftii N. masteri
A. stigmaticus P. atratipes

N. annulipes
Philippine Isles

M. pseudo-barbirostris N. philippinensis

Seasonal Prevalence.—Few observations have
been made on this point. It would appear that
there is one simple explanation which, at least
in part, will account for the prevalence of a
particular species at a particular time, and its
appearance or disappearance at others. We have
already shewn how selective the Anophelinae are
in their choice of breeding- grounds, consequently,
if at any time, eg., the dry season, a suitable
breeding-ground does not exist,a particular species
or genus of the Anophelinae may be absent.

Thus we found in Nagpur (India, C.P.), during
the dry season, in those places where shallow
puddles had dried up, Mym. rossii was rare, but it
abounded wherever puddles still remained. Where
weedy lakes existed, Nyss. fuliginosus was common,
elsewhere rare. Now these conditions are directly
dependent on the rainy season, and where vast
areas of weedy swamp are formed during the rains,
then M. nigerrimus prevailed, to disappear when
the swamps dried up. In temperate climes, the
temperature is, no doubt, an important factor, the
onset of the cold weather causing a general hiber-
nation. We may quote the following observa-
tions :—

Nyssorhynchus pretoviensis.—This species first
appeared February 10, and gradually became
more prevalent, superseding the other common

212

species (Pyvet. cineveus) in April (THEOBALD,
Monograph of the Culicidae, p. 99).

Pyvetopherus chaudoyei.— In the winter up to
June one only sees C. pipiens. These then dis-
appear and Pyret. chaudoyei appears’ (THEOBALD,

» FO),
a ‘tne incidence of Pyret. chaudoyei is said to be
accompanied by the recrudescence of severe
malaria, but it ought also to be definitely shewn
that sporozoits are present in this species, and
presumably that the sporozoit rate increases with
the outbreak of malaria.

Tue HIBERNATION OF ANOPHELINES

1. Hibernation of the Adult Insects. ANNETT
and Dutton describe the finding of A. macult-
pennis during the winter in England in cellars,
lumber-rooms, and other cold places, but not in
stables where the temperature is higher.

They observed the following points :—

(i) The attitude is peculiar, the insect lying
quite flat upon the surface with its legs spread
out. In this position the under surface of the
thorax touches, or nearly touches, the wall.

(ii) Only females are found, and these are
always fertilized, and have the spermatheca filled
with spermatoza.

(ii1) The insects are difficult to arouse, and
very sluggish in any movements they make. .

(iv) They do not feed unless the temperature
is raised. If kept at a low temperature (provided
the air is moist) they remain for weeks without
feeding.

(v) Ifroused by raising the temperature they

213

feed readily, and the ovaries rapidly develop.
Eggs are laid, and, in most cases, the female dies
after their deposition.

The adults of A. bifurcatus do not hibernate,
or only rarely.

2. Hibernation of the Lavva.—The larvae of
certain Anophelines, e.g., A. bifurcatus, appear able
to resist low temperatures, and are found even
when parts of the water are frozen over. Under
these circumstances they grow extremely slowly,
if at all.

So also in the tropics, different species tide
over the ‘cold weather’ in different ways. Thus
James found that M. culicifacies hibernated by
means of larvae only, little or no growth occurring
in these (t. 55° F. about); whereas Ce. pulcherrima
and N. fuliginosus laid eggs which developed into
pupae and imagines.

- 3. Hibernation of Eggs.—There is a certain
‘amount of evidence to shew that eggs can survive
for some months in moist earth, exposed to frost,
etc. For young larvae have been found in fresh
pools in the winter, under conditions that made
it unlikely that the eggs had been deposited there
on the appearance Of water. The resistance of
eggs to drying under a tropical sun is, however,
practically nil.

Move oF DISPERSAL OF ANOPHELINAE

There is no evidence existing at present to
show that mosquitoes habitually disperse any con-
siderable distance from their breeding-grounds. In
fact, the evidence is completely against such a dis-
persal, and, broadly speaking, the Anophelinae

214

remain where they were developed, and in the
native huts where they find abundant food.

That various accidental modes of distribution
occur is equally certain, e.g. :—

1. On trains, boats, and even ocean-going
steamers, they may be carried long distances, ¢.g.,
from West Africa and South America to England,
but it remains to be shewn that Anophelinae, thus
introduced, ever effect a permanent habitation,
even when the removal by this means is from one
portion of the tropics to another.

2. Locally, streams and canals may carry
larvae and ova long distances, perhaps miles.

3. Winds.—The maximum distance that the
Anophelinae can be carried in this way is quite un-
certain. Nearly all of the excessive distances that
have been given as: possible flights refer to Culex.
It appears certain, moreover, that the Anophelinae
dishke wind and seek shelter from it.

4. Trees, Plantations, ‘ Bush’ Jungle—These
elements undoubtedly hinder the flight of Ano-
phelines, and, on the contrary, open spaces promote
their diffusion. It is necessary to bear this fact in
mind, where a belt of jungle screens off a source
of Anophelines (larvae), which may find an oppor-
tunity of becoming infected later.

*‘DoMESTIC’ AND ‘ WILD’ SPECIES OF ANOPHELES

Anophelines are mostly found in association
with native dwellings where there is abundance of
food (blood). Anophelines are also generally abun-
dant where cattle are kept.

Certain species are distinctly ‘domestic’ in
their habits, e.g., Mym. rvossii, Pyr. costalis, Nyss.

\

215

stephensi, and others. They are found resting in
the daytime in the thatch of huts, and they
breed close at hand in the nearest puddle. They
may, however, fly up to half-a-mile if there are
no breeding places closer.

Other species are not peculiar to houses, but
are also found breeding in streams and pools in
the jungle far from habitations. Such species are
Nyss. maculatus, Nyss. theobaldt.

The mosquitoes of the genus Myzorhynchus,
on the contrary, are ‘wild’ :\nophelines. They are
only occasionally found in houses. They breed
in extensive bodies of water, swamps, rivers,
jungle pools, etc. It is Anophelines of this type
which chiefly frequent one’s tent when this is
pitched in remote and especially in swampy jungle.
The more common species of these wild Anophelines
are M. barbirvostris, M. sinensis, M. paludis.

NaTuRE OF Foop

The normal food of the female (domestic)
Anophelines is blood. In nature they appear to
feed every night, the stomach never becoming
empty. In Anophel/nes caught under natural con-
ditions, the stomach contents generally shew
blood in two or three stages of digestion.

Female Anophelines readily drink water, espec-
ially if they have been kept for some time in a
dry bottle. It seems doubtful whether vegetable
juices form an important article of food as appears
to be the case with some of the Culicidae. Male
Anophelines can be seen feeding upon banana and
other fruit juices, but are, notwithstanding, found
dead about the second or third day of captivity.

216

Under some conditions the females do not
feed upon blood, e.g., A. maculipennis in England
(THEOBALD). .

Bancrort states that Nyss. annulipes will
live for a month on dates, but only for three days
on bananas.

TIME OF FEEDING

The usual time for feeding of Anophelines is
after dark, more especially in the early night and
before dawn. Occasionally some Anophelines may
be found biting in broad daylight, and ANNETT
and DutTon state that Anophelines feed readily in
certain parts of Nigeria by day. Possibly certain
species feed more readily by day than others.

We have ourselves seen on rare occasions
M. rossii attempting to feed in the daytime, and
Gray, B.C.A., says ‘ that Ce. albipes when disturbed
will bite at any time of the day or night.’

On the whole, however, the Anophelinae are
strictly nocturnal in their habits. Nor do they
hover around lamps as has been supposed. Of
A. bifurcatus, BLANCHARD states that it bites
fiercely at dusk, but at night practically not at
all. At dawn, however, it begins again, and it
bites at all times in shady places, outhouses, etc.

DISTANCE OF FLIGHT

The maximum distance that Anophelines can
fly requires further study. In questions of flight,
the species of mosquito should always be noted.
Observations upon the flight of mosquitoes have,
so far, been vague and uncritical. With regard
to Anophelines on ships, it must be borne in mind
that they have not necessarily come from the land

B19

on the night upon which they appear, but may
have come on board when the ship was in port or
even have been bred on board. In certain villages
in India studied by us, Mym. culicifacies, Nyss.

stephenst,and Nyss. fuliginosus were always present
in abundance, if there were extensive breeding-

grounds within quarter-of-a-mile. Where villages
were distant half-a-mile from extensive breeding-
grounds, they contained few or no Anophelines. The
only exceptions to this rule were when breeding-
places had only recently dried up. In the case of
the above species they undoubtedly fly fairly
readily quarter-of-a-mile, but half-a-mile appears
to be beyond the normal distance of flight..

RELATION TO CoLour, ODOUR oF OBJECTS
Etre.

Anyone who, in the tropics, has left his ward-
robe open at sunrise and then closed it, and again
examined it some time later, will have often
observed the well-known fact that, on his white
clothes, few or no mosquitoes are resting, but that
on his blue serge clothes there may be dozens.
He will have noted, too, that outside his mosquito
net it is on the shady side that the mosquitoes
remain longest, until from here also they fly away
as the fierce sun rises.

Hewill have noted, too, that Anophelines aswell
as Culicines have a predilection for certain smells.
Old boots and blacking attract them strongly,
and the leather of a saddle room 1s their favourite
haunt. Anophelines, too, much prefer the odoriferous
skin of the native to that of the European, as
experiments made by us in Sierra Leone clearly
shewed.

218

NuTTaLE and SuipLrey have made some
laboratory experiments on the influence of colour,
and find that navy blue is the colour most pre-
ferred by A. maculipennis, and yellow the one most
shunned.

As, however, the Anophelinae at least are
nocturnal in their habits, and prefer biting un-
clothed portions of the body, the colour of one’s
clothing will not be much protection. If white
or yellow socks can prevent the persistent attacks
of Stegomyza, it would indeed bea practical boon.
To various trees and plants has been ascribed a
repellent effect upon mosquitoes. None of these
statements has, so far, borne a critical examination.

LENGTH OF LIFE oF MosQuITOES

The length of life of mosquitoes, under suit-
able conditions, is probably considerable ; several
weeks to months. In captivity they may, if suit-
ably housed and constantly fed, be kept alive for
days, weeks, and even months. A mosquito kept
some time in captivity becomes infirm, and readily
falls into the water whilst laying its eggs. It also
finds difficulty in hanging on to smooth glass,
and even though a rough surface is supplied the
insect is constantly found on the bottom of the
cage resting in a horizontal position. After laying
eggs, such infirm mosquitoes generally die the
same night. In nature, Anophelines certainly remain
alive in huts for one or two months and possibly
longer. After the drying up of all breeding-
places, the winged Anophelines do not muchdiminish
in number for several weeks. If the drying up
continues, the numbers gradually diminish, but
specimens may be caught up to two months or
more afterwards.

219

‘ AESTIVATION’ OF MosQuiToES

In very hot and dry countries, the Anophelines
which remain through the dry season appear to
exhibit some peculiarities in their habits :—

1. Unlike hibernating mosquitoes, they feed
regularly and are found full of blood.

2. The ovaries are in the majority large and
the ova fully developed.

3. They do not lay their eggs even when
‘test pools’ are made near the houses in which
they abound.

The Significance of Breeding-Places in the Dry
Season.—It is usual in hot and dry climates to find
in the dry season at most a few breeding-places.
It is a mistake to conclude that these represent the
distribution of Anophelines in the area in question.
Under such conditions careful search will demon-
strate that Anophelines are present in small numbers
in nearly every hut. After the first downpour of
‘the rains,’ young larvae will be found,in from
three to four days, in puddles throughout the
district, shewing that numbers of Anophelines have
survived the period of drought which may have
been two months or more. One cannot, therefore,
by measures directed solely against a few pools
that remain in the dry season, hope to effectually
get rid of the Anophelines in a district.

Tue Mate ANOPHELINE

Little is known as to the habits of the male
Anophelines. When they are found in numbers it is
probably a sign that breeding is going on at the
time. At times the number of males caught in
native huts exceeds that of the females. Copula-

220

tion is said only to occur after the first meal of
blood. Annett and Dutton describe many hun-
dreds of male Anophelines dancing together in
midge-like fashion in the villages at dusk.

FECUNDATION

Fecundation takes place it is thought gener-
ally on the wing. It is also effected in captivity
when the Anophelinae are confined in test tubes.
In the case of Stegomyia, Low describes fertiliza-
tion as taking place soon after the flies emerge
from the pupae.

Proportion of Males to Females of Reeaved Insects.
One is often struck by the large proportion of
males among. mosquitoes artificially raised, a
troublesome fact when feeding experiments are
being conducted. According to BERKELEY, if the
larvae are kept supped with abundant food the
proportion of males is much reduced.

221

Chapter XVIII

ANOPHELINAE—THE OVUM

Tue Ovum

Anophelines in captivity generally lay their eggs
on some floating object, but also upon the surface
of the water. When laid ona solid object, and
even when laid on the water, the eggs are deposited
in a piled up mass. Later, the ova, if on water,
often form very regular and beautiful patterns.
Brick-red masses of eggs are sometimes laid.
These do not develop further.

Observe (i) the arrangement in equilateral
triangles and star patterns (Fig. 19).

(11) The arrangement in rows of eggs lying
side by side.

Both patterns are dependent upon the shape
of the individual ovum; ova belonging to type 1
forming stars, and ova belonging to type 2, rows.

The number of ova varies, but is usually about
one hundred. The size of the ovum varies with
different species from about 0°6 to 1:0 mm.

Duration of Egg Stage-——TVemperature is no
doubt an important factor. Thus the egg stage
in A. macultpennis lasts from two to four days,
whereas in Ce argyvotarsis it is one-and-a-half
days in Havana. In M. vossi it is about forty-
eight hours.

Anopheline ova (with one exception as yet des-
cribed) are boat shaped, with an approximately

222

flat upper surface and a deeply convex lower sur-
face. One end which contains the head of the
embryo is blunter and broader than the other.
During the act of hatching this end is forced open
by the escaping larvae.

1. TheUpper Surface—Observe that the upper
surface is generally granular or tuberculated in
appearance. At either extremity it 1s continuous
with the pointed ends of the ovum, and in this
position there are usually several small polygonal
areas. The width of the upper surface and the
extent to which it is encroached upon by the floats
varies in different species.

2. The Lower Surface.—The lower surface is
generally smooth and dark grey. In damaged ova
a silvery membrane will be seen partly detached,
shewing a deep shiny-black surface beneath. The
silvery membrane is the outer covering of the egg,
and formed by the layer of follicular epithelium
(Fig. 39). In some species the lower surface is
marked with silvery lines forming a reticular
pattern.

The Floats—Occupying about the middle
third of the side of the ovum is a remarkable
structure—the float. This consists of a very
delicate membrane continuous with the chitinous
cuticle covering the whole ovum and containing
air cells.

The floats are generally oval in shape and
shew regular transverse corrugations. ‘The shape
and position of the floats vary considerably in
the different species.

The Frill—Around the margin of the
upper surface (forming the gunwale of the boat)
there is in some species a gleaming white frill-like

ao

structure. This is striated in appearance, but
portions of it may (in some species) be free
from striations. In other species the appearance
is rather that of a white striated rim. In all
species of Anopheles ova yet described, a striated
frill or rim is present. The width and extent of
the frill vary in different species.
Type 1—Ova have the upper surface very
narrow, with the lateral floats not touching the
margin (Fig. 54: 1).
he species with ova of this type are-—
M. barbivostris M. culictfacies
M. sinensis M, listont
A. bifurcatus

Type 2—Ova having a more or less broad
upper surface, with the lateral floats touching the
margin (Fig. 54: 2, 3, 4, and 6).

Species having ova of this type are—

M. vosst1 N. fuliginosus
Ce. pulchervima N. stephensi
A. maculipennts A, algeriensis

Type 3.—Ova with no floats, and with upper
surface rudimentary (Fig. 54: 5).
One species only as yet described has ova of
this type, viz. :—
M. turkhudi

Species having ova of the first type have in
all cases been species breeding in either open
natural waters or running streams.

Species with ova of the second type are in
general found breeding in pools.

The only ova as yet systematically described
are those of the Indian Anophelines. Furtherobserva-
tions will probably add further types to the above.

<nm
SU Pe .
>»,
>

aR

Fig. 54. Ova of Anophelinae

6. N. maculipalpis

3. M. vossit

2. C. pulcherrima
5. M. turkhudi

8

33
= §
28
ae
Ses
et)
Hot

255

Within each type great variation usually
exists in the different species. The following are
the most notable variations found :—

1. The Frill.—The width. The continuity
of the frill around the whole of the margin of .
the upper surface or its replacement in the middle
third by the floats. The extent of striation of the
frill. ‘The presence of a striated rim only.

2. The Floats.—The position, placed forwards
and encroaching on the upper surface, or laterally
situated. The shape, oval, globular, or scallop-
shell.

3. The Lower Surface.—Whether ornamented
or not with silvery reticulated pattern.

The following is a brief résumé of the
characters of the ova of Anopheles, as far as these
have been described :—

Type t. M. sinensis, sub-sp. nigervimus

Ovum.—Upper surface very narrow. Floats
do not touch margin of upper surface. Lower
surface of ovum ornamented with polygonal
markings.

M. barbtvostris

Ovum.—Upper surface very narrow. Floats
do not touch margin of upper surface. Lower
surface of ovum ornamented with polygonal
markings.

M. culictfacies

Ovum.—Upper surface very narrow. Floats
do not touch margin of upper surface. Lower
surface not ornamented. A short but distinct
fringe is continued around margin of upper
surface::

Q

226

M. listont

Ovum.—Upper surface very narrow. Floats
do not touch margin of upper surface. Lower
surface not ornamented. A small inne passes
around margin of upper-surface. *

Type 2. M. vossii ©

Ovum.—Upper surface prgad. Fringe very
well developed and striated throughout whole
length. Floats scallop-shell shape and touch
margin of anterior surface. Lower surface not
ornamented.

Ce. pulcherrima

Ovum.—Upper surface broad. Floats touch
margin of upper surface. Fringe well developed
around margin.of upper surface. Striations are
not present in that portion of the fringe lying
over the floats. Lower surface not ornamented.

N. fuliginosus

Ovum.-—Upper surface moderately broad.
Floats touch margin of upper surface. Floats
long and-narrow. Fringe around upper surface
only indicated by white border. Lower surface
not ornamented.

N. maculipalpis

Ovum,.—-The uppér surface is rather narrow.
The floats are rather short and oval, and. are
placed far forwards as in the ovum of N. stephensz,
though less markedly so. The fringe is fairly
developed, but is not continued over the floats.

may

N. stephensi

Ovum.—Upper surface broad, except in central
portion where encroached upon by floats. _Floats
placed on margin of upper surface so that they
touch, or nearly touch, one another in middle line.
Floats short and almost globular. Fringe not
well developed. Lower surface not ornamented.

N. theobaldi

Ovum.---As the females of this species have
only been very occasionally caught by us in
houses, we have not been able to describe the
ovum as deposited by the insect. Fully developed
ova removed from a bred specimen showed, how-
ever, that the ovum resembled that of N. maculi-
palpis. ‘The floats were rather short and situated
far forwards as in N. stephenst. The fringe is
fairly developed, but does not pass over the floats.

Type 3. M., turkhudt

M. turkhudi is a very aberrant type; so far as
the ovum and larva are concerned. Both the
ovum and larva approach to the characters of the
Culex ovum and larva. The eggs were laid upon
a floating object. When placed upon water they
sank. They were laid in the heaped-up manner
sometimes adopted by Anophelines, especially M.
vossit and N.maculipalpis. The chief characters
of the ovum are :—

1. No separation of an upper surface as in
all other Anopheline ova. At the thicker end of
the ovum there is an oval area about a quarter
the length of the whole egg. This is glistening

228

white and striated, and probably represents the
upper surface of other Anopheline ova.

2. There areno floats or any markings
representing them.

3. There is a pale area at the thicker end of
the egg with a scalloped edge.

4. The ovum is otherwise without markings.

It is obvious that the characters of the ovum
are of considerable importance in the classification
of Anophelines, and every care should be taken to
describe these in as great detail as possible.

In making drawings of the ova of Anophelines,
it is convenient to use an eyepiece micrometer.

To Mount Ova

No thoroughly satisfactory method is known
to us, but although imperfect, any of the following
methods will give specimens in which some, at
least, of the ova preserve most of their charac-
teristics.

1. Place the eggs on a slide which has been
made slightly sticky with balsam, and then mount
them in a drop of balsam and place a cover-glass
over them.

2. Mount in two per cent. formalin solution
and ring the coverglass with balsam or shellac.

3. Mount in glycerine and ring the specimen.

4. Mount in a drop of cedar-wood oil.

229

Chapter XIX

ANOPHELINAE—THE LARVA AND NYMPH

THe Larva

The larva of Anophelines when first hatched out
are minute characteristic creatures, with very black
heads and transparent bodies. They move witha
very active wriggling movement. They can, even
at this stage, be distinguished from the larvae of
Culex, especially with the aid of a lens, as they take
up a horizontal position. At first the heads of all
species are dark and very conspicuous. After a
certain number of days, however, the head becomes
lighter in colour, and characteristic markings can
be made out on the dorsum. When first hatched
the head is very large in proportion to the thorax,
later, it is smaller, and finally, it is the thorax
which is the larger.

During the first few days the palmate hairs
are simple lanceolate structures, and cannot be
used as specific characters.

Duration of Larval Stage.—This is determined by at least
two factors. (1) Food.—Thus larvae kept in tap-water in the
laboratory grow very slowly, if at all; (2) temperature—Thus
the larval stage of A. maculipennis varies from sixteen to twenty-
two days at air temperatures of 68°-78° F., while in the tropics
the time is much shorter, e.g., twelve days for Ce. Argyrotarsis
in Havana, and eleven days for M. vossti, where the tempera-
ture of the water varied from 96°-102° F.

230

Examination of the Full-Grown Larva.—Cover
the larvae in a drop of water, with a coverglass,
and examine with one-fourth or one-sixth objec-
tive; the following points can be readily made out.
Observe that old larvae are often almost totally
enveloped in vorticellae or other infusoria.

1. The Head.—The head is globular in shape,
and is for the most part enclosed ina hard and
continuous chitinous case. Anteriorly, there are the
rather complicated mouth parts. Posteriorly,
there is an opening into which the neck is inserted,
around this isa pigmented border resembling a
collar. There is a gap in this dark border in the
middle line posteriorly, and here two diverging
bands of chitin form a ‘V’ on the back of the head.
Grouped around this ‘V’ mark are more or less
continuous patches of pigment, which shew
differences in their arrangement, to some extent,
specific.

2. The Antennae.—Arise from two prominent
lateral protrusions, they are freely movable at
their articulation. Each antenna is a rod-shaped
unjointed body. At its terminatian are two leaf-
shaped bodies, and a branched hair arises between
the leaflets. The antenna is covered with small
spines, which are particularly developed in pairs
along the inner border. In most species of. Ano-.
phelines a hair can be made out arising from a
papilla situated at the junction of the proximal and
middle third of the antennae.

This hair is of specific importance.

(i) Inthe majority of Anophelines it is simple
and unbranched.

(ii) In A. lindesayii, M. nigerrimus, M. bar-
bivostvis it is branched, and in the last two very
large and conspicuous. (Fig. 55).

Pea

~ 3:. The Eyes. The eyes are situated later-
ally, and can be seen both from the dorsal and
ventral surface. Their size and appearance vary
with the age of the larva. In the full-grown
larva a-crescentic compound eye is seen on either
side, and behind this a single pigment mass
(simple eye). The compound eye is absent in the
first stages, and becomes more prominent as the
larva approaches maturity.

4. The Mouth Parts.

I 2 3 4
Fig. 55. Lateral Hairs of Antennae
1. M. rossit. 2. N. stephenst. 3. A. lindesayti

4. M. nigerrimus

(a) Two very conspicuous bodies resembling
somewhat shaving brushes are protruded or with-
drawn under the overhanging clypeus. These
are the feeding-brushes, and are employed in col-
lecting the minute food particles on which the
larva feeds.

(b) On either side of the mouth is a broad
blade-like structure carrying several leaflets and
some hairs (maxillary palps).

232

(c) Below the feeding brushes, and not so
easily visible, are two stout bodies with comb-
like projections (mandibles).

(d) In the middle inferior line lies a conical
toothed structure. The under lip of Meinert
(Fig. 23).

(e) In the fully-grown larva a snout-like
process covered with short hairs-projects forwards
in the middle line between the brushes.

The front portion of the head projects between
the antennae as a semi-circular smooth area. In
front of this is a protrusion overhanging the base
of the brushes (the clypeus).

5. The Clypeal Haivs.—These are four or six
in number. Two spring from the extreme front
of the clypeus near the middle line ; two from the
outer corner of the .clypeus immediately over
the feeding-brushes, and two usually very small
and not always present behind the origin of the
others.

The clypeal hairs are best seen when the
feeding brushes are retracted. They must not be
confounded with certain other hairs on the larval
head. These are :—

(i) Six large branched hairs arising from
the prominence lying between the bases of the
antennae.

(ii) Four similar branched hairs, but smaller,
situated further back (NUTTALL and SHIPLEY).

Nore.—Ep. and Ev. Sercent describe variations in these
hairs in A. algeviensis. In eighteen out of forty-six examined
both were simple. In three out of forty-six both were slightly
branched. In twenty-five out of forty-six the central hair had
two or three small terminal branches.

233

The hairs exhibit great variation in different
species, but are quite constant in the one species.
A minute description of these hairs is of great
importance in describing the specific characters of
the larva.

Clypeal Hairs of Larvae :—

(i) The four anterior hairs may be quite
simple and unbranched. M. rossii, N. stephensi,
M. culictfacies, M. listoni, M. turkhudi, A. bifurcatus.

(ii) All four anterior hairs may shew small
lateral branches. .P. jeyporensis.

In A. maculipennis all four hairs are branched,
the outer pair form distinct tufts.

(iii) The outer pair may be markedly
branched, e.g., Ce. pulcherrima and M. pseudopictus.

(iv) The outer pair may be alee into
a close tuft (cockade), e.g., M.barbivostris, A..puncti-
pennis.

—— |

\1I \t \ WI

Fig. BS Clypeat Hairs of Taveae

1. M. vossii, N. stephensi, M. culictfacies, M. listont
23 IN, maculipalpis. 3. P. jeypovensis. 4. Ce. pulcherrima
5. M. sinensis, M. barbirostris

The two hairs situated behind these may,
instead of being very short and inconspicuous, be
long and prominent, e.g., M. turkhudt.

234

6. The Thovax.—The thorax, in the adult
larva is large and globular. - In the young larva:
it is not,so. broad as the head, but becomes, pro+.
portionately larger as the larva, advances, in age.
Numerous hairs arise from the front and sides. of
the thorax. None of these vary perceptibly: in
different species. A number of large hairs arising
from papillae on the lower surface are capable of
being used almost as a means of propression when
the larva is in very shallow water.

(a) Observe on the dorsum of the thorax, a
short but extremely stout and strong hair, unlike
the others, projecting outwards and forwards.

(b) A flap-like body may, with : careful
focussing, be seen lying at the base of the most
anterior hairs on either side.

(c) Insome species of Anophelines a single pair
of palmate hairs, similar to those on the abdo-
minal segments, are found upon the thorax. In
others they are rudimentary or absent. The
presence of well-developed palmate hairs on the
thorax is of specific importance.

(i). It is well developed and_functionally
active in M. culictfacies, M. listont, P, jeyporensis.

(Gi) + It is rudimentary, or absent i in all other
larvae as yet described. :

7. The Abdomen.—The first seven 1 segments are
very similar in shape. The eighth carries the
opening of the air-tube, and the ninth some
curious papillae and large hairs.

Each of the first two segments carries on each
side a pair of long feathered hairs. The third
carries a single similar hair. On the other
segments there are much smaller and unfeathered
hairs. On all the segments there are groups of

230

small hairs which do not appear to vary’ and
which are not of sufficient importance to describe
here. None of the above structures appear, to
vary in different species.

SKELETON LARVAE

Examination of Palmate Hativs-—The most
important appendages of the abdominal segments
are certain small fan or palm-leaf-shaped' hairs
attached by a short stalk to the outer’ dorsal
portions of certain of the segments’ (Fig. 57).
The number of segments bearing well-developed
palmate hairs varies in different species.

Place the larva under a coverglass in a drop
of water or use a permanently mounted larva.

Soph so

Fig. 574. Palmate Hairs of Larvae:
1. M.vossii. 2. M. nigervimus. 3. M. listoni
_ 4. N. maculatus

tr. Determine the number of segments which
carry distinct and large palmate hairs and those
carrying ill-developed ones.

2, Carefully cut out witha néadle two or
three central abdominal segments. Crush these.

236

The following arrangement of these hairs is
found in Indian species of larvae, the only larvae
as yet systematically described.

1. Fully developed hairs on all segments
(one to seven) and on the thorax.

P. jeyporensis
M. listont
M. culicifactes

2. Fully developed hairs. on the second
to seventh, or third to seventh segments. Rudi-
mentary hairs on the second or even first abdominal
segments and on the thorax.

N. stephenst
N. maculatus
N. theobaldi
. Palmate hairs confined to the third, fourth,
fifth, sixth, and seventh segments.
M. sinensis
M. barbivostris
A. maculipennis (NuTTALL and SHIPLEY)
Palmate hairs confined to the fourth,
fifth, and sixth segments.
M. turkhudi

The Leaflets —In the well-grown larvae each
palmate hair consists, as a rule, of nineteen or |
twenty leaflets arising close together from a short
stalk, ‘and forming a semi-circular fan. When
collapsed, as is the case when the larvae is beneath
the surface, these hairs are inconspicuous. When,
however, the larvae takes up its characteristic
attitude at the surface of the water, these spread
out fan-like, and are very striking objects under
the microscope. In the freshly hatched larva,
the separate leaflets appear to be folded together,
so that the hair has the appearance of a single

237

lanceolate structure. About the third day, the
hairs are seen with seven to eight uniformly lanceo-
late leaves. Very soon after this, they take on
the characters séen in the hairs of the mature
larva.

In the mature larva the leaflets shew much
variation in, the different species. In most species,
the leaflets terminate rather suddenly in a number
of jagged points-or notches, whilst the central
portion continues as a more or less fine filament.

ran

Fig. 578. Leaflets of Palmate Hairs

1. M. sinensis, M. barbivostris. 2. A. lindesayii
3. N. theobaldi, N. stephensi. 4. M. listoni, M. culictfacies
5. M. vossiit. 6. M. turkhudi

The character of the notching and the relative
length of the filament to the leaflet are of specific
importance. The following types of leaflets are
known :—

1. The leaflets are unbrokenly lanceolate in
shape, with saw-like notches along the edge of the
outer half. There is no distinct terminal filament.

M. sinensis
M. barbivostris

238

2. The filament is long and filamentous, as
M.vossti se, ead
M. culicifacies
M. listont
N. fuliginosus
Further differences are seen in the case of most
of the above species. In M. vossiz the filament is
as long as the leaflet, and there is scarcely any

notching where the two join. In N. theobaldz,
the notching is well marked (Fig. 578).

3. The filament is very short, a mere spike-
like process.
N. stephensi

N. maculata

N. theobaldi
N. maculipalpis

The Stigmatic Syphon.—The eighth segment
bears the stigmatic opening. This is a large
quadrilateral space, with hard comb-like chitinous
processes on either side. These have the teeth
projecting backwards, and are capable of being
approximated so as to obliterate the cavity. Into
the anterior portion of the space, under cover of a
lip-like process, the two main air tubes open.

‘The ninth segment is cylindrical in shape, and
is chiefly notable from the fact that it carries four
large transparent papillae well supphed with air
tubes and certain long curved hairs. Of the hairs
one series projects downwards so as to resemble a
rudder. ‘The others project posteriorly.’ «Phere
does not appear to be much variation in the
different species.

230

EXAMINATION OF THE LARVA

1. Some features,e.g.,feeding, are conveniently
studied by placing the larvae in a drop of water
in a watch glass. .

2. For examining under a high power, the
activity of the larva must be restrained by a cover-
glass. .

3. Permanent preparations’ may be made at
once by placing in strong formalin, then alcohol,
then oil of cloves, then balsam (vide p. 250).

4. Beautiful preparations of the palmate
hairs, etc., are got by mounting the larval skeleton
thrown off at the time of pupation.

PupaTION

Just before this process the larva becomes
quieter. The attitude also frequently alters,
becoming a hanging one, somewhat like that of
a Culex larva.

In this condition larvae are very readily
killed by agitating the water (and it is difficult
to carry larvae in this stage without killing them).

The change into the nymph is very sudden.
A few rapid motions and the larval skin is cast
off, leaving the characteristic nymph.

THe NyMPHA

This stage in the tropics usually lasts about
forty-eight hours. When first the larval coat is
cast the nymph is light in colour, and may be
readily overlooked. Later, the nymph becomes
darker, and towards the end and immediately
prior to the emergence of the imago, silvery patches

240

due to collections of air are-seen beneath the
cuticle.

Pupae taken out of the water and kept on
moist blotting-paper will still develop into winged
insects (NUTTALL and SHIPLEY).

Egg to Imago.—The developmental cycle for A. maculi-
pennis is about thirty days at a temperature of 20°-25°C.: In
the tropics it is much less. Thus the minimum time for Ce.
argyvotarsis, M. rossii, M. culicifacies is fourteen days.

241

Chapter XX

THE BREEDING-PLACES OF
ANOPHELINES

Larvae should be sought for in the most
diverse situations and, after being examined and
described, be allowed to hatch out. New species
of Anophelines are often obtained in this way. It
is the case in India, and almost certainly will
be found to be so in other countries, that certain
kinds of breeding-places are preferred by certain
species.* A collection of larvae made from shallow
puddles will be found to yield quite a different
set of species to one made from a streamlet or
pool full of vegetation, even though close to the
puddles (Fig. 58).

The following tabular statement gives the
more common situations of Anopheline breeding-
places and the species, as far as known, found in
each. It is obvious that a great deal of work
yet remains to be done.

1. Foul puddles near habitations M. rossil
2. Clean puddles without much

alga and often turbid with

suspended matter :—
(M. rossii

(a) Pits and puddles near houses iP costalis

* Thus M. lutzi is said to breed only in the water’collected
in the leaves of the parasitic Bromeliaceae, and N. annulipes is
said to breed in the sea.

R

(b) Roadside puddles ip
(c) Cattle footmarks N
(d) Shallow, muddy sheets of water M.
(e) Pools in sandy river beds i
(f) Large pools in quarries, etc. \M.
3. Puddles and pools with much
alga. Common in stream
beds, water trickling over
rocks N
N
4. Earthenware vessels, empty paraf-
fin tins, boats, water barrels N
P
5. Wells, springs N
P,
A
6. Swamps.
(a) Deep water with much aquatic
' vegetation M
M
M
(b) Rice-fields, wet cultivation of
all kinds M
P
N
N
A
7. Running water
(a) Swiftly flowing streams M.
(6) Sluggish irrigation channels,
ditches, muddy trickles, edges
of rivers M.
M.
N.
(c) With much weed and alga M.
M.
N.
N.
N.
(d) Stony and shallow M.
N.
M.

. maculatus
. costalis

. stephensi

rossii

culicifacies
turkhudi

. fuliginosus
. maculatus

. stephensi
. costalis

. stephensi

costalis

. lindesayii

. sinensis
. barbirostris
. paludis

. TOSSII

. jeyporensis

. maculatus

. maculipalpis
. lindesayii

listoni

funesta
culicifacies
maculipalpis
sinensis
barbirostris
fuliginosus
maculatus
theobaldi
culicifacies
theobaldi
turkhudi

N. fuliginosus

A. lindesayii
N. maculatus

243
Lakes with weedy margins
9. Hill species
A gigas

8.

ay AYN HIAAN MTT SL . aot lp PTT MN
NTN —_e AWS

nt Wetihy
we ey,
see APOE EELEAO

vue

swiliitdins Sliding ily,
Up

7) M lity, wt Wy, is

aan CL ey

ANAL

ow " = sit

Ra
»
~

S
=

wis ili ith IN
1A ll
“at it is Nile
fila "

es on a Tea Plantation, to re

M. listoni and N. maculatus

N. maculatus

places of M. vossti, M. listoni, and
M. rossit. e

different breeding-

io)

. 58. Portion of Coolie Lin

¢

Fig

244

Chapter XXI

THE IDENTIFICATION OF ANOPHELINE
LARVAE

1. Naked Eye Chavacters—Some larvae may
be identified by the naked eye. The distinction,
however, between most species is insufficient to
allow of separation by this means.

2. Observe that the colour of larvae is not
dependent on species but on the nature of the food,
amount of light they have been exposed to in
nature, the colour of the water, and other general
conditions.

The most distinctive of Anopheles larvae
are those of M. sinensis and M. barbivostris. These
are very large larvae, most frequently black, or
black speckled with white, but also brown or
vivid green in colour. One of their characteristics
is a peculiar ‘stick-like’ appearance, and the
assumption of a bent or contorted attitude.

The larvae of M. turkhudi can be detected
by their attitude, which is almost Culex-like.
Larvae about to change into nymphae, however,
also frequently adopt this position.

Naked eye examination always requires
verification by the microscope.

(A) Larvae may be bred from ova deposited
by females of a known species. To successfully
accomplish this requires a good deal of care.

245

1. Remove the paper upon which the ova
have been laid (p. 96), and place in a small
bottle containing some filtered fresh water from a
pool or rain puddle.

2. Place in a good light, but take care that
the sun, by the focussing action of the glass, does
not heat the water, otherwise the larvae will be
killed.

3. When the larvae are hatched, transfer
them (after a day or two) to a larger vessel of
fresh water containing some weed. When the
fresh natural appearance of the water disappears,
more fresh water from a pool should be added.

4. By keeping larvae in a not too porous
earthenware vessel, they may be placed with
impunity all day in the direct sun. It is necessary,
however, to watch carefully, to guard against
desiccation and consequent death of the larvae.

Larvae kept in flat, partially glazed earthen-
ware vessels, with a certain amount of mud, and
placed in the sun, develop more quickly than those
kept in bottles.

It is of course necessary to make certain that
foreign ova or young larvae are not introduced
with the fresh water.

Some larvae are exceedingly difficult to rear
artificially, notably those of M. barbivostvis and
M. sinensis. They remain for long periods with-
outperceptibly increasing in size, and frequently
die.”

(B) An alternative and less tedious way is
to examine nearly adult larvae found in nature,
and to observe, after accurately noting the larval
characters, what species of Anopheles eventually
hatches out.

246

By examining the larva on a slide without a
coverglass,the main characters may be noted with-
out in any way damaging the larva, which later
becomes a nymph and eventually animago. As
a rule, however, many specimens of the same
species are found together. By a preliminary
examination, larvae shewing the same characters
may besorted out, and some specimens afterwards -
mounted and subjected to a more detailed exami-
nation, whilst the rest are allowed to hatch out
in due course.

The characteristics of the larvae which are
of specific importance are, as we have seen—
1. The antennae.
2. The clypeal hairs.
3. The leaflets of the palmate hairs.
4. The.segments carrying palmate hairs.
By means of these characters most species of
Anopheline larvae can be identified. So far as

Indian Anophelines are concerned, the following
characters hold good :—

Type 1.—Larvae with the external pair of
clypeal hairs converted into a cockade-like tuft
(Fig. 56).

Species having larvae of this type are—

M. barbivostris
M. sinensis, sub-sp. nigerrimus
Larvae of this type also have a large branched
hair upon the antenna, and the leaflets of the
palmate hairs differ markedly from all other
larvae (Fig. 578). ’
Type 2.—Larvae with the external frontal

247

hairs branched but not developed into tufts
(Fig. 56). N. fuliginosus
Ce. pulchervima

Type 3.—Larvae with the external pair of
frontal hairs simple and unbranched, and with
palmate hairs on every abdominal segment and
on the thorax (Fig. 56).

M. culictfacies
M. listoni

Type 4.—Larvae with the external pair of
frontal hairs simple and unbranched, but with no
developed palmate hairs on thorax or first abdo-
minal segment (Fig. 56).

M. rossit
N. stephenst

Type 5.—Larvae with two large additional
hairs placed behind those already mentioned. Also
with first three abdominal segments free from

palmate hairs. IM. fuvbheads

Larva of M. barbivostvis—-Antenna with large
branched hair. External pair of frontal hairs
developed into cockades. Palmate hairs on second
toseventhabdominal segments. Leaflets of palmate
hairs lanceolate in shape and deeply serrated in
outer half. Head of larva without pigmented
markings.

Larva of M. sinensis—Antenna with large
branched hair. External pair of frontal hairs
developed into cockades. Palmate hairs (?).
Leaflets of palmate hairs lanceolate with serrations
in outer half. Head of larva without pigmented
markings.

248

The habits of both these species, M. barbivostvis
and M. sinensis, sub-sp. nigervimus,are very similar.

The larvae are found in water with .much
aquatic vegetation—rivers, lakes, ponds, and
swamps. They are only caught singly, but are
generally widespread in their occurrence where
large bodies of water are present.

Larva of N. fuliginosus.—Antenna without
large branched hair. External pair of frontal
hairs branched (branches usually six in number).
Palmate hairs on second to seventh abdominal
segments. Leaflets of palmate hairs with very
marked ‘shoulder’ at origin of terminal filament.
Terminal filament from one-half to two-thirds the
length of basal portion. Head of larva with dis-
tinctive markings.

Larva of M. culictfacies—Antenna without
large branched hair. Frontal hairs all unbranched.
Palmate hairs on first to seventh abdominal seg-
ments, and a pair of fairly developed ones upon |
the thorax. Palmate hairs with terminal filament
nearly as long as basal portion. Head with
markings.

Larva of M. listont—Antenna without large
branched hair. Frontal hairs simple. Palmate
hairs on all segments, and very well-developed
pair on thorax. The palmate hairs in this species
are very large. The terminal filament is nearly
as long as basal portion.

Larva of Ce. pulcherrima.—Antenna without
large branched hair. Outer pair of frontal hairs
branched (six branches). Palmate hairs on second
to seventh abdominal segments. Filament of pal-
mate hair nearly as long as basal portion. Head
markings present. .

249

Nature of breeding-place unknown.

Larva of M. vossti.—Antenna without large
branched hair. Frontal hairs unbranched. Pal-
mate hairs second to seventh abdominal segments.
Terminal filament of palmate hair very long;
often longer than basal portion. The ‘shoulder’
at the origin of the filament is very slightly
marked. There are markings upon the head
(Fig. 574).

Breeds nearly always in small pools near
houses. These pools are frequently foul and
nearly always muddy. The female lays her eggs
very readily in captivity.

Larva of N. maculipalpis.—Antenna without
large branched lateral hair. Frontal hairs are
peculiar and show a condition intermediate
between the branched hairs of M. barbivostris,
N. fuliginosus, and the unbranched hairs of M.
vossit and other species (Fig. 56). Palmate hairs
on second to seventh segments. Leaflets of pal-
mate hairs have very short filaments. ‘The notch-
ing at the termination of the leaflet is not so
marked as in N. theobaldz.

Larva of N. theobaldi (GiLes).—Antenna
without large branched lateral hair. Frontal
hairs unbranched. Palmate hairs on second to
seventh segments. Leaflets of palmate hairs
have very short filaments. There are marked
notches at the ending of the leaflet in the filament
(lig. 578). j

The larvae of this species frequent especially
sluggish streams with much growth of alga. They
were found by us in Nagpur in association with
N. fuliginosus, M. barbivostris, and M. listonz.

250

Larva of M. turkhudi—rThe larva is Culex-
like in some of its characters, though undoubtedly
much more nearly related to the Anopheline type.

The full-grown larva is distinguished by the
adoption of the slightly hanging attitude. The
chief characters of the larva are :—

1. Two large additional frontal hairs are
developed, which reach as far forward as the
longest of the hairs described in other larvae.

2. The shape of the head differs from that
of the ordinary Anopheline larva.

3. The palmate hairs are only represented
on two or three abdominal segments, namely,
the fourth, fifth, and sixth. They are absent on
the first three abdominal segments.

4. The palmate hairs are small and poorly
developed. The leaflets are irregular and the
terminal filament blunt.

This species must be looked upon as a form
which in its egg and larval stages has lost many
of the characteristics of Anopheline eggs and larvae,
and has approached in these stages the characters
of the eggs and larvae of Culex.

251

CLASSIFICATION OF INDIAN ANOPHELINES ACCORD-

ING TO LarvAL CHARACTERISTICS *

Pa

Antennae without branched lateral hair

3
Z ee M. sinensis Leaflets of palmate hairs
5. a2 lanceolate and serrated
as 3 M. barbirostris Ditto
23 ~
cae = : at :
25 A. lindesayii Leaflets of palmate hairs
as .
E 2's ; shewing regular and
4 e deep notching (Fig.578)
wn
M. rossii Palmate hairs well deve-
F loped on third to
e | § seventh segments
2) 8 ee :
& | se |) M. culicifacies Palmate hairs on all seg-
5 wie ments and on thorax
co | & M. listoni Palmate hairs very large
uv =
a on all segments and on
E thorax
2
i= w
2/4
e|é N. stephensi
| Sie N. maculatus
er N. theobaldi
E
a
i
eo ||) ey A 5
a a5 N. maculipalpis
So | zm
BE! P. jeyporensis Palmate hairs very large
I 8 bp on all segments and
m | 8 thorax
23 N. fuliginosus Filaments of leaflets
23 long
ao Ce. pulcherrimus
)
S238 M. turkhudi Palmate hairs on fourth,
$23. fifth,andsixthsegments
088 only ; filaments short,
ae leaflets rudimentary

* The Larvae of the remaining In:lian species are, so far, undescribed.

252

To Mount. LarvarE

1. Place a drop of formalin in a hollow
ground slide. The drop must be just sufficient to
fill the cell when the coverglass i is in position.

By means of a pipette or spoon take up a
larva and, removing the excess of water, allow |
the larva to float off into the drop of formalin.

Place the coverglass in position, avoiding air
bubbles, and ring with Canada balsam, etc.

It is important that no air bubbles are
included, as a white deposit forms around them.

If too much formalin has been added, the
excess must be carefully removed before ringing.
If hollow ground slides are not available, a ring
of balsam may be made on the slide and allowed
to become somewhat hard. Fill the cavity with
formalin, place the larva therein, and cover care-
fully with a coverglass. Avoid excess of fluid! or
air bubbles. It is best to allow the Canada
balsam to be just soft enough to stick to the
coverglass.

Larvae mounted in this way retain their
characters very well, and the clypeal and palmate
hairs can be examined with ease.

2. 0 Mount in Balsam.—If placed in alcohol,
oil of cloves or xylol, and balsam in the ordinary
way, the shrinkage of the soft pars and even
of the hairs is very great.

On no account touch the larvae with forceps,
and only occasionally, and with the utmost care,
with a needle point.

Place a number of larvae in a covered watch-
glass containing formalin. Leave for twenty-four
hours at least.

253

Lift each larva carefully by means of a strip
of cigarette paper. Drain off the excess of forma-
lin, and place with the greatest care in absolute
alcohol. Allow the specimen to remain for at
least ten minutes in alcohol.

Remove with cigarette paper to a watch
glass containing oil of cloves. With cigarette
paper transfer to a slide. Remove excess of oil
of cloves, mount in a large drop of balsam, taking
care that the dorsum of the larva is upwards.

If great care is taken not to detach the hairs
by handling, the larval characters are beautifully
displayed in this way.

254

Chapter XXII

THE RELATION OF SPECIES OF ANO-
PHELINAE TO MALARIAL ENDEMICITY

Species undoubtedly play an important part
in the development of blood parasites in the
mosquito. _ .

Proteosoma, for instance, develops in certain
species of Culex, e.g., C. nemovusus was used by
Kocu in Europe. It does not, however, develo
in certain species of Taentorhynchus (S. P. Temes),

The malaria parasite does not develop in
species of Culex, Taeniorhynchus, Stegomyia, or
other blood-sucking flies, e.g., Phlebotomus, Simu-
lium, etc. In the case of Culex fatigans placed
under absolutely identical conditions with Ano-
pheles, no sign of zygote formation occurs on the
second or third day.

Similarly with regard to filaria, it is only in
certain species of Culicidae that certain species of
filaria will develop, thus Ce. argyrotarsis is an
efficient host for F. nocturna, but inefficient for
PF, demarquatt.

The malarial endemicity or endemic index may
be defined as the percentage of infected children -
(under ten years of age) in any district, and repre-
sents the liability of immigrants to contract
malaria.

. A. Rossii
5a N

256

It is well known in a general way that in
one country malaria is more intense than in
another, but here we have a means of exactly
measuring this difference, and, moreover, in the
different parts of any particular district. We
may illustrate this by the differences we found in
Bengal in an extent of country where, as far as
we could judge, the climatic conditions were
practically identical, yet we find in the environs of
Calcutta the endemic index is 0, while in the
Duars (at the foot of the Himalayas) it is as high
as. seventy-two (Fig. 59). We found, however,
that there was one important matter in which
the Duars differed from Calcutta, and that was
in its Anopheline fauna. Whereas in Calcutta
M. rossii was the predominant species, in the
Duars M. listont was the commonest Anopheline.

Again, in the Jeypore district (Madras), we had
a district of uniformly high endemic index, fifty
to one hundred, and here we found an Anopheline,
P. jeyporensis, which we had not encountered
elsewhere, so that the view seemed tenable that
the high endemicity of these districts was
dependent on their special Anopheline fauna. To
test to what extent species was concerned in
determining endemicity, we then made use of
another more exact method, viz., determining by
dissection whether any difference occurred amongst ©
the different species in the percentage of infected
specimens: we were able to carry this out in the
case of M. vossii and M. culicifacies. We caught
these species in the same huts in the same villages
at the same time, and determined by actual dis-
section the percentage of glands infected with
sporozoits. The results were most striking, and

.

Tay

“

fully confirmed our previous idea, based on more

general
species.

IL

Mian Mir (Punsas)

considerations of the importance of
They were as follows :—

Number

Number

dissected with sporozoits Forseatage
M. culicifacies 259 12 4°6
M. rossii 496 fo) fo)

II]. Ennur (Mapras)

Number Number p ;

dissected with sporozoits SRSER BES
M. culicifacies 69 6 8°6
M. rossii 364 fe) °

Undoubtedly then, under natural conditions,
the species is here a very important factor.

Again, under artificial conditions (feeding
experiments), we found that there was a difference
in the number of zygotes found in the stomach as
the result of feeding.

The species which appeared to be most active

WETE :—

M. culicifacies
N. stephenst
N. theobaldi

258

Those in which zygote formation seemed
less abundant were :—
M. vosstt
M. turkhudi
M. barbirostris

It should be noted, however, that in these
experiments M. rossit became infected, while in
nature it has never been found infected by us.

There are, moreover, many considerations
which lead to the conclusion that in nature all
species of Anopheline are not equally concerned in
the transmission of malaria.

We may have countless numbers of M. vossi1,
as in Calcutta (environs), and get a malarial index
of o, and this appears to hold good in Madras,
Bombay, and, as far as our observations go,
universally. On the other hand, where we find
M. listont, M. culictfacies, P. jeyporensis, in India,
we have a high endemic index.

The group of mosquitoes, those associated
with intense malaria, are small dark mosquitoes
with unbanded legs (Myzomyia, group 1).

M. FUNESTA AND P. COSTALIS IN AFRICA

The former mosquito is, hke M. l:stonz,
which it closely resembles, a breeder in clean
waters, streams, springs, etc., while P. costalzs is
found breeding in shallow pools about houses and
frequents towns (in Africa), which M. funesta
does not.

M. funesta was found by us to be infected

in the Lagos hinterland to the extent of twenty-
five to filty per cent.

259

P. costalis, in Lagos itself, contained only
three per cent. of sporozoits.

It is important then to determine precisely
the species in a district and to determine the
percentage of infection sporozoits.

A. maculipennis and A. punctipennis in America.
Both these species were fed on the same case of-
malignant tertian malaria by HirsHBERG, and kept
at the same temperature—30° C.

Number fed Number infected
A. maculipennis 48 8
A. punctipennis 58 fe)

Similar results (unpublished) have been ob-
tained in Japan.

Anophelinae that ave known to transmit malaria.—

Although we have over eighty species, it has been deter-
mined, only in a very few cases, which of these actually do
transmit malaria in nature. Thus we know that the following
species do :—

Europe.—A. maculipennis (mainly); <A. bifurcatus (less
concerned).
P. superpictus and M. pseudopictus (in some parts
especially).
Africa.—M, funesta, P. costalis, A, maculipennis (Algeria).
N. America.—A. maculipennis.
W. Indies.—Ce. albipes (according to Paysos).
India.—M. listoni, M. culicifacies, N. maculatus (?).

LITERATURE

Stephens and Christophers. Malarial Reports to the Royal
Society. Series VI and VII. Harrison and Sons, London.

260

Chapter XXIII
TO MAKE A MALARIAL SURVEY

ENDEMIC MALARIA

The clue to the epidemiology of malaria in
the tropics is to be found in the infection of the
native population of a country. The malaria of
Europeans is merely the result of their exposure
to infection from this source. Investigation into
the natural history of malaria, therefore, resolves
itself largely into the study of native or endemic
malaria. It has always been recognized that in
a particular country certain districts are more
malarial than others. It was not, however, till
Kocu used the percentage of infected children as
the test of the malarial intensity of a place that
accurate measurement of this became possible.

To INVESTIGATE THE ENDEMIC MALARIA OF aA
DISTRICT

(A) The Breeding-Places of Anophelines—

1. Examine all collections of water within
half-a-mile. Stir up the mud of small puddles,
and use a dipper where the water is weedy or
difficult of access. Examine wells, ‘chatties,’
streams, and swamps, as well as pools of every
description. Take specimens of larvae from each,
placing in specimen tubes and labelling.

261

2. Determine the species of the larvae
collected.
3. Make a map of the neighbourhood,
noting—
8) All breeding grounds.
b) What species are found breeding in
those examined (T°ig. 60).

se e a wn ps oat
Hill ss anew = = so Hl"
o si Ly) SNH VW SS SS Fy a
reatea ayye rE STIS aan == Zope onmnnes®

uy
\

\

Z

mys ANS

5 anys
ZINN LASS
ge dtay see

WA spleeris 50:

parasites70.

ing grounds of \
A. Jeyporensi}

RAC
Re
FM) ii5
4 eS Ml) ii

oD

w SS ‘6
Ro ntti
oS” Mies
STS Ny

~
a \

te PRS RTLLE EN

jon wn >

ww
AW uw aap
BRAND Wt av
= 0 go HULL.

wi

Fig. 60. Map shewing how to make a Malarial Survey

(B) The Presence of Winged Anophelines—

1. Search in outhouses, under eaves, etc., as
described in Chap. XIII, for Anophelines. Deter-
mine the species, note relative numbers of each
species on map. The relation'of Anophelines to
native dwellings will probably be evident.

262

2. In the dry season the search for Anophelines
may be negative, and there may be no breeding-
places. Make in the most sheltered places small
cement pools, and keep these filled with water.
After a certain number of days they may contain
young Anopheline larvae if the adults are present
in the houses. (It is necessary to be sure one’s
water supply does not contain young larvae or
eggs). The absence of the larvae in the pools
does not necessarily mean, however, that adult -
Anophelines are not present in the houses (see choice
of breeding-grounds by different species of Ano-
phelines).

_ Note the result in the case of each test pool.

3. Inthe conditions just described observe
the pools made by the first shower of rain of the
on-coming ‘rains.’ Note after three days have
passed the presence of larvae in many of these.
Note the presence of these on the map. The
distribution of Anophelines at the end of the pry
season will usually be found to correspond to that
of native huts.

THe PREVALENCE OF MALARIA

If we proceed to-ascertain to what extent
malaria prevails in a district we may attempt to
do so in several ways. _

1. We may consult hospital statistics and
returns of death from malaria. This method is
open to such grave error that it is extremely
doubtful whether it is worth the labour bestowed
upon it.

2. We may determine to what extent en-
largement of the spleen occurs. This method has
been largely used.

263

PRECAUTIONS NECESSARY IN APPLYING THE
SPLEEN TEST

1. The Age of the Individuals Examined.—
The enlargement of the spleen due to ordinary
malarial infection tends to disappear once the
individual has ceased to suffer from malarial
infection. In very malarious countries, where
each individual, after childhood, has become
highly immune, the adult population usually shew
no splenic enlargement (Tropical Africa).

In less malarious regions the adults have not
become highly immunized, and a certain number
of them will be found with enlarged spleens and
malarial infection. The use then of the percent-
age of adults with enlarged spleens is not a
reliable method of determining the’ real intensity
of malaria.

In children, the spleen enlargement appears
to require a certain time to become apparent, and
it takes a certain time to disappear, as the
malarial infection disappears with ensuing im-
munity. :

In the examination of children for splenic
enlargement and the presence of parasites in their
blood, we found :—

(i) In the early ages, one to two years, the
number infected is usually in excess of those
shewing splenic enlargement.

(ii) Above two years, the spleen rate is
usually somewhat in excess of the parasite rate.

(iii) Above ten years, the spleen rate is
usually considerably in excess of the parasite rate.

In the use of a spleen census one should then
avoid a mixed adult and child count, and children

264.

between two years and ten years of age should be
chosen.

2. The District in Question.—It seems clear
that the comparison of the malaria of widely
different regions by means of the percentage of
enlarged spleens in the children is not possible.
We have, however, found that in Bengal, the
parasite rate and the spleen rate in children varied
proportionally, the spleen rate was, however,
nearly always about double that of the parasite
rate.

3. Time of Year. Seasonal Variations.—We
may determine by actual blood examination how
many individuals have parasites in the peripheral
circulation. By the use of the parasite rate in
children up to ten years of age we get a definite
and true index of endemicity which may be used
in the comparison of one locality with another.

4. To the last method we would add, asa
complimentary one, the determination of the per-
centage of infected Anopheles as giving the actual
risk of infection in a district.

THE DETERMINATION OF THE ENDEMIC INDEX OF
A PLACE

1. Place a number of cleaned slides in a
slide box. Take a straight surgical needle, paper
and pencil.

2. Choose any village or quarter of a town.
Get the assistance of a native with local influence,
the native magistrate in an Indian bustee, the
chief in an African village. Instruct him to
muster the children of the village. The free dis-
play of ‘ pice,’ half-pence, etc., will greatly aid one,

265

and by palpating a few spleens previously to
taking blood specimens the children will come
readily. It is well first to take the blood of one
or two adults or big boys so as to allay fears. In
all cases it will be found best to take for granted
the willingness of the child, and if the operation
is quickly and quietly performed there is little.
objection, especially when each receives payment.

3. Make dry blood films by the method des-
cribed in the early part of the book.

4. At the same time a spleen census may
with advantage be made.

On examining the films determine :—

(i) Number shewing parasites or pigmented
leucocytes in the blood.

(11) The species of each parasite present and
the percentage value for each if the numbers are
large enough.

To DETERMINE THE INFECTION IN THE
ANOPHELES

(THE SporozoiT Rate)

1. Collect as large a number of Anophelines as
convenient from the village in and around which
the previous observations have been made.

2. Dissect as many specimens as possible,
noting in each case the species dissected, and
noting in which species, if any, sporozoits are
found.

In many cases the sporozoit rate is extra-
ordinarily low, eg., two per cent., although
Anophelines are ‘abundant and the malarial index

266

is not low. In others, especially in African bush
stations, the percentage may reach fifty per
cent.

3. Leave specimens not dissected for several
days and examine the mid-gut for zygotes.

-MALARIAL INFECTION OF EUROPEANS

Although malaria is an infectious disease,
and can arise only from an original human
source, yet in the tropics we can no longer con-
sider the origin of infection as occasional and due
to the presence of other cases of ‘fever.’ In the
tropics, and especially in Africa, we are dealing
with a disease which is a normal condition of
childhood, and which, with the coincident infec-
tion of Anophelines, is the usual accompaniment of
every native hut.

European malaria in the tropics is, indeed,
chiefly dependent on two factors—

1. The degree of exposure to native malaria,
z.e., the proximity to native dwellings.

2. The endemic index of the native dwel-
lings in question.

To INVESTIGATE EuRopEAN MALARIA

1. Examine the blood of as many Europeans
as possible. Enquire carefully whether the person
is taking quinine at the time, also take the
temperature.

(1) ‘The number shewing parasites or cres-
cents.

(ii) The presence of pigmented leucocytes.

(111) The presence of an increase of the large
mononuclear leucocytes.

‘207

In every case make a differential count of the
leucocytes and keep the record.

Observe especially, any community of Euro-
peans shewing a larger percentage than usual of
malarial infection. Note the conditions under
which these are living, and note also the probable
greater prevalence of blackwater fever in these
communities, e.g., Roman Catholic Fathers, West
African miners, railway communities, Europeans
in poor circumstances living in the slums of native
towns, etc., Syrian hawkers, etc. Note those
communities habitually taking quinine.

Infected ra.
Anopheles / ‘

Jw ©
oe
YS
ceo” a ‘
Anophetes €o 9 {(¢
Infected Anopheles. deme Ce
aor ~ Ny
me
{0 ow
oe

Nanee’

scale

Large Raitway camp
European quarters =D
Native quarters -

Fig. 61. Shews how Europeans ave infected with Malaria
from the native (children)

2. Note the usual relation between the degree
of ill-health and the proximity of native huts.
Make a map shewing European dwellings and
shewing huts and hovels in relation with these
(Fig. 61).

Make a thorough investigation of the
conditions in these huts.

268

(i) The percentage of infected children in
each group.

(11) ‘The degree of infection of the adults.

(iii) Roughly estimate the number of Ano-
pheles present, whetherswarming, abundant, scanty,
or impossible to detect by search. In the latter
case make several ‘ test pools.’

(iv) Determine the species present and the
relative numbers of each.

(v) Determine the sporozoit rate for each
species.

(vi) Carefully mapall breeding-places, noting
what larvae are found.

4. Captureas many Anophelines as possible in
the European houses, especially in the morning,
and by looking within the nets. Determine the
species, sporozoit rate, and from where probably
derived. Examinethe ovaries andspermatheca, and
note. whether freshly hatched or impregnated
females are chiefly found. Note the presence or
absefice of males.

In investigating the malaria of any such
settlement, native and European, continue the
observations if possible throughout the year.
Make observations on—

1. Seasonal variations in the endemic index
(percentage of infected children).

2. Seasonal variations in the number of
cases among Europeans.

3. Prevalence of any particular species of
Anophelines at any time of the year.

4. Distance of flight of Anophelines from
breeding- grounds, etc.

5. Sporozoit rate of Anophelines at different
times of the year.

269

6. Examine especially the conditions where
Anophelines, breeding-places, native huts, oppor-
tunity for constant importation of malaria and
numerous susceptible children exist, and yet there
is a complete absence of endemic malaria. In
Africa it will probably be impossible to find such
places, but they occur in India.

Enpemic AREAS OF A CoUNTRY

The map (p. 253) shews how the endemicity
of large areas of a country is a very variable one.
When opportunity offers, the endemic index should
be determined for each locahty, and, as far as
possible, all the other facts detailed above. But
the simple taking of the blood of a number of
children (under ten) in any village gives at once
valuable information as to malaria of the district,
information which often is quite unsuspected.
Thus, as is shewn in the map, the endemic index
of Calcutta is 0, that is to say, in the immediate
environs (not in the town itself) where practically
the condition is one of a number of isolated
villages, there is no malaria among the native
children. At Jalpaiguri the figure is low, twelve
per cent., but on reaching the foot of the Hima-
layas,we find the extremely high figure seventy-two
per cent. In this case we were able among other
differences to find a different species of Anopheles,
which, as we have seen, is undoubtedly an import-
ant factor.

In other cases, however, all the conditions
may be apparently identical, but within a distance
of even ten miles we may get a change from an
endemic index of o (Madras) to ninety (Ennur).

270

These differences hold good in other countries,
e.g.,in Italy. Here the mortality from malaria
in the north is comparatively trifling, while in
the south and the islands it is severe.

Here the difference may be due to differences
in climate, but this explanation does not suffice
in the examples in India we have mentioned.

Again, we have great irregularities in the
distribution of the species of parasite. The quar-
tan, for instance, in the Duars (Bengal) is
exceedingly common amongst the native children,
but in. Lahore it is rare.

Similar differences have been noted in Algeria,
where over large areas the quartan parasite is
extremely rare, yet in a few localities it occurs in
seventy per cent. of cases (BILLET).

So in India, as a whole, we have certain
small areas where malaria is intense, e.g., the
Duars, Jeypore (Madras), and Kanara (Bombay)
(Curisty), where we also find blackwater fever; yet
in others, as in the Central Provinces, where
apparently all the conditions are favourable, we
have only a moderate intensity.

We require, then, to examine carefully the
endemic indices over large areas in order to get
an accurate idea of the variations in endemic
malaria. The instances we have given will shew
how erroneous it is to say broadly, ‘such and
such a country is highly malarial,’ for while this
may be true of one district it might be quite untrue
of another.

_ Further, after having established these broad
data, it will be necessary to make a close survey
of each individual district in order to endeavour
to explain the factors at work.

271

Chapter XXIV

CLINICAL STUDY OF MALARIA

ENUMERATION oF RED CELLS

In blood counting, much practice only can
give accurate results; inaccurate results are mis-
leading and useless. For comparative purposes,
counts should be made always at the same time,
if possible, to obviate the effect of food, etc.

For diluting the blood, og per cent. salt
solution may be used, or preferably, Torson’ s fluid,
which has the following formula :—

H,O » rmoce,

Glycerin ce @one

Sodium sulphate 8 grammes

Sodium chloride 1 gramme

Methyl. violet (or other stain sufficient to

colour the nuclei of the leucocytes)

The diluting fluid must always be poured into a
watch glass, and it should not be sucked up out of
the stock solution.

No pressure must be used to make the blood
drop exude from the finger.

Blood is then sucked up to the mark 1 on the
pipette, and the end of the pipette carefully wiped
before plunging it into the Totson’s fluid. The
Torson issucked up to the mark 101 exactly. The
pipette is then rotated between finger and thumb,

27%

so as to ensure thorough mixing. After rejecting
the first few drops, which consist of diluting fluid
simply, a small drop of the mixture is blown on
to the counting chamber, and the coverglass
applied as rapidly as possible. No fluid should
escape into the moat or under the coverglass.
When NewrtTon’s rings are seen between the cover-
glass and side of the chamber, the former is in its
right, closely-applied position.

In counting the corpuscles in each square,
include, of those that touch or overlap the sides,
only those on the left hand and top side or right
hand and bottom side. Count at least 1,000 cells,
the error is then only about two per cent.

The number of corpuscles per mm.

_ No. of corpuscles counted x dilution (100) x 4,000
Number of squares counted.

The normal average values are for man 5,000,000,

for woman 4,500,000.

TotaL Leucocyte Count

The leucocytes may also be counted at the
same time as the red cells, 7.e., from the pipette
used for the red. This method has the advantage
that all errors effect both counts equally, and the
true ratio of red to white may still be got. Now
for counting the leucocytes much larger fields are
necessary than in the case of the red, as for every
five hundred red cells there is only one white, so
that counting chambers especially ruled are often
sold for this purpose, but their use is unnecessary
and may be obviated by the following method.

We require simply to determine the area of the whole

microscope field with a given eye-piece, objective and given
length of tube.

273

1. One division of a Thoma Zeiss square = ‘o5 milli-
metre.

z. So that if the diameter happened to cover eight divi-
sions the diameter would be 4 millimetres, or the radius ‘2
millimetres.

3. The avea of the field will therefore be wr?, and the

I

corresponding volume of blood mr? x is == depth between

coverglass and chamber, i.e., rr? 5 = ere a millimetres.
4. So that to get the ane se of leucocytes in the cubic

millimetre we must multiply by 79°51, but it would be much

more convenient to multiply by 100. In that case where

wy x 2 == +, y = ‘1785 millimetres, or diameter of field is
357 millimetres. We require, therefore, to arrange our micro-
scope so that the diameter is of this value, and this is readily
done.

Two observations only are necessary.

1. Draw the tube of the microscope out so
that diameter of field covers exactly eight divisions
of the Thoma Zeiss chamber ; note length of tube.
Let this=2, .

2. Draw tube out so that diameter covers
exactly seven divisions. Note length of tube=y.

3. Therefore an increase in tube length of y — x
reduces diameter from eight divisions (‘4 millimetre)
to seven divisions (‘35 millimetre); difference = °05
millimetre.

4. Required to calculate what increase in
length will cause reduction from “4 to °357
(diffetence = bales

This calculation is a once for all, by the
observer for his microscope, and the tube is drawn
out the required amount, using, of course, the
same eye-piece and objective.

T

274

To find total number of leucocytes per mm.*-—
Total number counted x 100 x dilution (100)
Number of microscope fields counted

Count one hundred if possible.

The leucocytes may also be counted in the
special pipette for white cells, but here again the
method of counting by using the whole micro-
scope field should be used. If the ‘ white’ counter
is used, the diluting fluid should be acetic acid
o°3 percent. Sufficient gentian violet or methyl.
violet is added to this to colour the nuclei.

To CrLean PIPETTES

For any accuracy of observation the pipettes
should be scrupulously clean. There should not
be the slightest tendency for the glass ball to stick
to the sides. After acount has been made, the
rubber tube is removed and the contents ejected
by blowing from the pointed end.

1. Suck up dilute acetic acid-so that all
traces of stain are removed.

2. Suck up several lots of clean water to
remove the acid.

3. Then absolute alcohol two to three times
to remove the water.

4. Then ether two to three times to remove
the alcohol. .

5. Finally, blow hot air through with a
syringe, the glass barrel of which may be heated
in a flame (or simply suck air through).

These procedures take a very short time, and
it is a satisfaction to know that the pipette has
been put away perfectly clean and ready for the
next observation.

os)

THe EstTIMATION OF THE HAEMOGLOBIN

GoweEr’s haemoglobinometer is the simplest
and best. In sucking up the blood take care not
to hold the tube too vertical, as the blood readily
flows out from the rather large calibre of the tube.
Order from a good maker, as several inferior
instruments are on the market. The round form
of tube is more easy to manipulate than the flat.

The standard of comparison in this apparatus
is pikro-carmine gelatine, the colour of which
corresponds to a one per cent. watery solution
of normal blood.

All blood counting apparatus, etc., can be
got from T. Hawsxiey, 357 Oxford Street,
London, W.

Dare’s haemoglobinometer is accurate. It
possesses the advantage of dispensing with a
pipette. It costs £4.

To Count PLATELETS

Diluting fluid: glycerine saturated with
dahlia, and two per cent. saline solution, take
equal parts of these.

Or better, a freshly-made five per cent. solution in water
of crystallized sodium metaphosphate, Dilute the exuding
drop of blood with five to ten times its bulk of fluid. The
exact amount need not be known. Count the ratio of platelets
to red cells in an ordinary coverglass preparation.

The ratio of platelets to red cells is 1:8 about.
The absolute value per mm.} 635,000 about.

Differential Counting of Leucocytes (vide
page 41).

276

Tue LreucocyTes IN MALARIA

We shall consider (1) the total leucocytes,
(2) the percentage value of each kind.
The Total Leucocytes.—We may take 10,000
as the normal value per mm.’, and as 5,000,000
is the normal value for red cells, the proportion
of white to red is
WC _ 10,000 _ I
RC 5,000,000 500
Now, in malaria, we may find two conditions,
either that the total number of leucocytes is con-
siderably below the normal value, 10,000, -7.e.,
there is leucopenia or hypoleucocytosis, or that
the total number is much above 10,000, 7.e., leu-
cocytosis. If there is leucopenia, say, for instance,
the total number is 5,000 instead of 10,000, then
WC_ 5,000 _ iI WC
RC ~ 5,000,000 1,000’ RC
_is smaller than normal.
If, on the contrary, the total leucocytes are
20,000 instead of 10,000, 7.e., leucocytosis, then
es t.e., the fraction wt is
RC 5,000,000 250° ~~ ”’ RC
greater than normal.

z.e., the fraction

Itis this ratio a that it 1s important to

determine, for unless the red cells are counted as
well as the white, little value attaches to the
leucocytic value.
Turning now to malaria, we find that we get
changes of the following kinds :—
(1) Ira.m., rigor. Red cells=2,900,000. WC_ 1
White cells 10,000 RC ~ 290
z.e., leucocytosis.

277

(2) 11.30 a.m., rigor completed ROT
1.e., leucopenia.
aie WC _ I
(3) 2 p.m., temperature 38°2 RC 568

1.e., increased leucopenia.
The leucocytosis was, in this case, quite tran-
sient, followed by a marked leucopenia.
During the course of an.attack, we may have
changes of this kind :—
1. Some days before the attack and before
parasites appear in the blood, instead of
Ji WC_ rss ;
RG = 500 RO reco he leucopenia.
2. During the shivering attack and height
of the pyrexia, the condition changes to one of
leucocytosis, so that
WC_ it I
RC 300” 200’
This leucocytosis may not last long, but
is followed again by a marked leucopenia which is
at its maximum before the sae of the next attack.

I
or even —
O

WC I
RC instead of ae may be cee

BILuet (Fig. 62), who has traced out hourly
the relation of the leucocyte curve to the tempera-
ture curve, has shewn that in regular curves of
the tertian or quartan type, the leucocytic curve
follows closely the variations in the temperature.
Thus, before the febrile attack in a quartan, there

may be a leucopenia represented by Wes ax

at the time of the attack, however, there is a
WC_ it

leucocytosis of reer

278
This gradually disappears, passing through

I :
the normal value —, and again reaching a
00

marked leucopenia before the next attack. The
variations are of the same kind in irregular
temperatures, the leucocytosis corresponding to the
rise of temperature, and the leucopenia to the
apyretic intervals.

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279

The Percentage Value of the Leucocytes.—If
we now make a differential count in a stained
specimen we shall be able to ascertain what
change, if any, there is in the relative percentage
of a different kinds (for normal values vide

. 42).

7 1. The main characteristic change is that
there is an increase in the percentage of large
mononuclears, so that at times they may even
outnumber the polynuclear.

2. The change is especially well-marked in
the periods of apyrexia (7.e., when there is a leuco-
penia). When there is a leucocytosis the increase
in the mononuclears may not be apparent.

As examples of this leucocytic change, we
may give the following :—

(1) Small mononuclear 18°I per cent.
Large mononuclear and .
transitional 314 "
Polynuclear 50°2 -
Eosinophil O"4 %
A fatal case of malignant tertian (BASTIANELLI).
(ii) Small mononuclear IQ'I per cent.
Large mononuclear and
transitional rae a
Polynuclear 39°0 ‘5
Eosinophil 06
A fatal case of comatose malignant tertian (Bas-
TIANELLI).
* Panse.—Malignant tertian fever, t. 37°2° C
(111) Small mononuclear 18° per cent.
_ Large mononuclear and
transitional 26°4 .
Polynuclear 55°3 .

* Zeitschrift fiir Hygiene, 1903 p. 592.

280

t.97°6° F. Malignant tertian :

(iv) Small mononuclear 14°8 per cent.
Large mononuclear and
transitional ASG!
Polynuclear 38°5 ro

The figures are by no means always as high
as this, but, as we have already said (p. 41),
we consider a value above fifteen per cent. as
diagnostic of malaria. The higher values are
appreciated at once by an inspection of the
slide where the large mononuclears seem to occur
in every field, and may be pigmented. For the
low values a careful count is required.

An increase in the large mononuclears has
been found in one case of human trypanosomiasis.
This has not, so far, been confirmed, but if it is, it
can but slightly effect the value of the counts in
malaria as a diagnostic means, for the clinical fea-
tures of trypanosomiasis, so far as known, are
extremely characteristic, and the chance of an
European being infected with the disease does not
appear to be great, the two known cases having
occurred in tropical Africa. Further, together
with the increase of the mononuclears in malaria
there are, if thorough search is made, also pig-
mented leucocytes to be found. The relative
count of malaria is of great assistance in at least
two conditions, (1) in those cases where quinine
has been taken, (2) where consequently the diag-
nosis is uncertain and the question of typhoid
fever arises. As we shall now see, the relative
count in typhoid is quite different ‘from that of
malaria.

281

Typuoip FEVER

During the first week (of uncomplicated
cases) the leucocytes are normal.

During the second week there is a leucopenia,
e.g., 2,000, and the leucopenia is in proportion to
the severity of the disease.

During the third and fourth weeks the
leucopenia is still more marked, though also a
leucocytosis may be found without any apparent
cause.

Relative Leucocyte Valwes—During the third,
fourth, and fifth weeks the mononuclears, large
and small, may reach the values of forty to sixty
per cent., and among these the proportion of
small mononuclears is very striking.

PNEUMONIA

There is very early a leucocytosis, e.g., 25,000,
four hours after the initial chill. The maximum
occurs, as a rule, just before the crisis. The

‘number may fall from a high value to normal in
twenty-four hours. Leucocytosis is said to bear
a relation to the amount of exudation (Z.e., lobes
involved).

Relative count—

Large and small mono-

nuclear 2 to.4 per cent.
Polynuclear gotog5 ,,
Eosinophil rare.

THe WipaL ReEAcTION IN TYPHOID

While we consider, it is not going too far to
say that typhoid and malaria can be readily dis-
tinguished by the leucocytic count, yet seeing that

282

in the Wrpat reaction we have an easy means of
diagnosing typhoid, the application of this test is
of the greatest service in those cases where the
diagnosis of malaria or typhoid remains doubtful.
We shall describe briefly how the test is carried out.
There is no necessity for specially constructed bulbs
or graduated pipettes, as often thought.

1. Draw out a piece of glass tubing, so as
to make a pipette, having a fine end about the
diameter of a hypodermic needle.

2. Collect enough blood to fill the pipette
to the height of about half-an-inch. The blood
will readily flow in if the pipette is held sloping
downwards. Seal off the fine end in a flame.
Centrifugalize, if convenient, but abundance of
serum can be got without by allowing to clot.

3. To Dilute the Sevrum.—Draw out a piece of
glass tubing into a long fine filament; take a
piece about six inches long; make an ink mark
about half-an-inch from the end of the tube;
insert this marked end into the tube containing
serum (and clot) and allow serum to flow up to
the ink mark; then let a distinct bubble of air
follow (the size of this bubble does not signify) ;
next allow broth to flow up to ink mark; repeat
this procedure until nine drops of broth are in the
tube; these are now each separated by an air
bubble, and also by a bubble from the serum.
The dilution is now one in ten.

Blow out all the drops on to a slide or watch
glass, and mix by sucking up and blowing out a
few times.

4. Take a drop of the diluted serum in a
fresh piece of tubing, make a mark as before, and
then allow broth to flow up; this gives a dilution

283

of one in twenty, a second drop one in thirty, third
drop one in forty, and finally a drop of typhoid
emulsion ; this gives a dilution of serum of one
in fifty, containing typhord bacilli. The whole
aoe of dilution takes less than five minutes.

A bacillus should be
used “that is known to be active. Take a fresh
over-night agar culture and make a fairly thick
emulsion in broth or salt solution.

6. Dilution and Time Reaction.—A dilution
of one in fifty with a time limit of half-an-hour
may be used. With a less diluted serum the time
limit must be less.

7. Whatever time hmit and diesen be used,
it is very necessary to perform controls from time
to time with a variety of other cases to make sure
that the agglutination, if produced, is not produced
by normal sera.

THe Isoronic Point or JONICITY OF THE
BLoop

If a drop of blood is allowed to drop into a
one per cent. solution of salt in a small test tube
and stirred up, the uniformly turbid solution will
eventually become clear when the corpuscles have
settled at the bottom and the supernatant fluid
will be unchanged; if, on the contrary, we add
another drop of blood to a little water in a test
tube the whole drop is immediately laked, and we
have resulting a solution of haemoglobin. The
former solution of salt is called hypertonic, the
latter solution of water hypotonic. Now, if we
start with such a hypertonic solution, one per cent.
salt, and proceed gradually to dilute it, we shall

284

eventually reach a strength where the hypotonic,
i.e., haemolysing effect begins to appear. The
strength of salt solution just above this where
no change occurs is the isotonic point for the
particular blood in question. This point then
gives us information as to the resistance to a
haemolytic action of the corpuscles. The blood
in various diseases is found to vary in regard to
the strength of salt required to prevent haemo-
lysis. So that if a normal blood is unchanged by
a o'5 per cent. salt solution, whereas an abnormal
requires o'6 per cent. to protect it, the latter
blood is described as having a less resistance than
the former, but it has a higher isotonic point.

The determination of the isotonic point then
gives us a more definite notion of the state of the
blood in disease than does a mere determination
of the haemoglobin. The isotonic point of human
blood is about 0°41 per cent. salt solution.

To DETERMINE THE Isotonic PoINT

1. Measure out one c.c. of each salt solution
of descending strengths, 0°43 per cent., 041 per
cent., 0°39 per cent., etc., into four small test tubes
and one c.c. of water into a fifth tube.

2. Add to each the amount of blood con-
tained in two divisions of the stem of a THoma-
ZEISS pipette (the whole stem contains ten divi-
sions).

Allow to stand for some time. Some of
the solutions will have haemoglobin in solution.

The amount of haemoglobin in each tube
can be estimated by adding the amount of a normal
blood in two divisions to onec.c. of water. Call

285

this = too percent. Dilute this solution, so that
a nuinber of tubes equal to ninety, eighty, seventy,
etc., per cent. are’ got. Compare the tubes con-
taining the salt solutions directly with these.

In malaria, the resistance of the blood is
markedly lowered, thus, whereas in a control
normal blood a o41 per cent. salt solution gave
no haemolysis ; in the case of two malaria patients,
the haemolysis was equal to twenty-five per cent.
and forty per cent., respectively.

In blackwater fever, on the contrary, a raised
resistance of the blood may be found.

CLINICAL StuDy oF MALARIA

The Uvine.-—While not proposing here to con-
sider the general reactions of the urine in malaria,
for which we must refer the reader to any standard
text-book, yet we think it useful to consider some
points which are of more particular interest. It
is especially in blackwater that we still require
complete analyses of the urine, and more especially
in those who are constantly subject to malarial
attacks and are at the same time taking quinine.
It is possible that such analyses might give us
indications which would enable us to avert the
danger of an attack of blackwater fever and _ to
determine when quinine should not be given. We
have not considered here the method of examining
the urine by ‘cryoscopy,’ as it is not at present a

ractical clinical method, but its possibilities
should not be forgotten.

Albuminuria.—The occurrence of albuminuria
in malaria varies according to the particular
country ; thus in Rome it is uncommon, in Senegal,

286

on the contrary, exceedingly common. This is an
illustration of the often neglected fact that tropical
malaria differs in many ways from malaria of
temperate climes. ;

Filter the urine if morphological constituents
are present, as is the case in blackwater fever,
through two thicknesses of filter paper, or add some
calcined magnesia, then filter. Place some urine
in a urine glass and, with a pipette reaching to the.
bottom, allow half as much nitric acid to slowly
trickle in (Stmon). A white cloud at the junction
layer indicates serum albumin (globulin or pep-
tones). Urea nitrate crystals will often separate
out at this junction layer.

Serum Globulin.—Make the urine alkaline with
ammonia ; filter off any precipitated phosphates ;
to the urine add an equal volume of saturated
solution of ammonia sulphate. A precipitate
indicates globulins; or the formation of the pre-
cipitate may be seen at the junction layer. ‘Test
filtrate for albumin by adding excess of acetic
acid and boiling.

Albumoses.—Acidify the urine with acetic acid;
add an equal volume of a saturated solution of
salt ; boil; if a precipitate occurs (albumen) filter
hot. Albumoses separate out-on cooling; or to
the hot filtrate add caustic soda solution, then
dilute copper solution gradually; a red colour
signifies albumoses.

Notge.—Urines rich in urobilin (e.g., malaria
and blackwater fever) will give this biuret reaction.

In presence of urobilin: to ten c.c. of urine
add eight grammes of powdered ammonium sul-
phate until dissolved ;. boil for a few seconds ; the
albumoses are precipitated on the sides of the

287

test tube; pour off the urine, and wash the pre-
cipitate with alcohol, then chloroform; dissolve
in water and apply the biuret test. Test the
alcoholic extract for urobilin.

Nucleo-Albumens.—Filter the urine carefully ;
boil to remove albumen; then add gradually
excess of strong acetic acid. A turbidity indicates
nucleo-albumens.

Broop (HAEMOGLOBIN, ETC.)

1. Examine Shectroscopically (Fig. 63).-—If
the bands of methaemoglobin or oxyhaemoglobin
are seen, confirm by adding ammonium sulphide
when the bands of reduced haemoglobin are got.

2. Heller's Test—Make the urine strongly
alkaline with caustic soda; boil; the precipi-
tate in the presence of haemoglobin is bright
red ; confirm by dissolving the filtered precipitate
in acetic acid, a red solution is formed (spectro-
scopically this gives the characteristic bands of
haemachromogen).

3. Guaiacum Test—Equal parts of tincture
of guaiacum and oi] of turpentine (which has been
exposed to the air) are taken; add slowly to the
urine. A blue ring is formed at the junction
layer.

METHAEMOGLOBIN

The urine in blackwater fever when examined
early, most frequently contains blood pigment in
this form, later oxyhaemoglobin. ‘This, according
to Hoppe-SEYLER, also holds good for every urine
with haemoglobin in solution.

Fig. 63. Spectra of (1) Oxyhaemoglobin ; (2) Haemoglobin ;
(3) Alkaline Methaemoglobin; (4) Alethaemoglobin in
Neutral or Acid Solution ; (5) Alkaline Haematin ;

(6) Reduced Haematin Haemachromogen ;

(7) Uvobilin ; (8) Zinc-Urobilin

289

The Characters of Methaemoglobin are :—

In acid solution the oxyhaemoglobin bands
are weak or invisible. There is a band between C
and D, nearer the former. The band of acid
haematin is similar in position. It is, however,
close to C.

In alkaline solution the acid band disappears,
and a faint band on the red side of D takes its
place (compare with alkaline haematin).

Reduced by ammonium sulphide, the bands
of reduced haemoglobin are got. It differs from
oxyhaemoglobin in its chemical reactions by the
fact that it is precipitated by basic or neutral lead
acetate solution, whereas oxyhaemoglobin is not.

Detection :—

1. In presence of oxyhaemoglobin. Ppt.
with basic lead acetate; filter, decompose the
precipitate with carbonate of soda solution ; ex-
amine for the bands of alk-methaemoglobin.

' 2. In presence of urobilin. Proceed in the
same way.

In presence of bile pigment. Precipitate
these by making the solution alkaline with
amimonia after adding CaCl..

4. In neutral solutions its spectrum is iden-
tical with that of haematin in natural solutions
(NEUBAUER and VoGEL). Reduced by (NH,),S,
methaemoglobin is changed to reduced haemo-
globin and haematin to reduced haematin, the
bands of which are easily recognized.

URoBILIN

Frequently occurs in the urine in jaundice
instead of bile pigment.
U

290

According to Hayem, it is associated with
methaemoglobinaemia. Its occurrence in black-
water fever is very common, occasionally before
the attacks, but more constantly after the oxy-
haemoglobin has disappeared or together with it.

Characteristics :—

1. In acid urine a band near F occurs, be-
tween 88 and ror.

2. In alkaline urine a band between 81
and 95.

3. Make the urine strongly alkaline with
ammonia filter, add ZnCl, solution, but not suffi-
cient to form a permanent precipitate.

A green fluorescence occurs, and the much
clearer band nearer ‘b’ than the acid band.

Detection :—

1. Hoxyhaemoglobin is present. Precipitate
the urobilin with basic lead acetate, then acidify
the precipitate, when the urobilin goes into
solution.

2. If methaemoglobin is present. Neutva-
lize the urine with carbonate of soda; precipitate
the methaemoglobin with neutral lead acetate.
Filter ; test the filtrate for urobilin.

Bite PIGMENTS

Where urobilin is present, as in blackwater,
the colour of the foam on shaking the urine, the
staining of the filter paper, etc., cannot be
regarded as satisfactory texts.

Detection :—

1. Gmelin-Rosenbach Test.—Filter the urine
through filter paper (Swedish). Dry ; apply a
drop of nitric acid (fuming) to this, a play of
colours is got.

291

2. Huppert’s Test.—Precipitate the urine
with BaCl,. Filter; wash the residue off the
filter (perforated) with acidulated H,SO, alcohol.
Boil. A bright green colour indicates bilirubin.

3. Smith's Test.—To ten c.c. of the urine add
two c.c. of dilute tincture of iodine (tincture of
iodine 1, alcohol 10). A green ring forms at the
junction zone.

BILIRUBIN AND HAEMATOIDIN (IN URINARY
SEDIMENT)

1. Bilirubin crystals form yellowish-brown
rhomboidal plates or needles.

Easily soluble in CHCl, Gives GME in’s
reaction, green, under the microscope.

2. Haematoidin, dark-red in colour or
' greenish if impure, with nitric acid they give a
transient blue.

According to Hoppre-SEyLer, however, they
are identical.

HAEMATOPORPHYRIN

Occurs 1n urine as alkaline haematoporphyrin
(Fig. 64). In urate sediments a similar form occurs.
It is soluble in chloroform, giving bands similar
to those of oxyhaemoglobin, but acid converts
this into acid haematoporphyrin bands. Solu-
tions have a brilliant red fluorescence. It is
‘found in the urine in toxic conditions, such as
chronic sulphonal poisoning. It is precipitated
‘by lead acetate, while oxyhaemoglobin is not.

SUGAR

Before testing for sugar, boil to remove all
proteids. ;

292

Reduction of copper solution is effected by
bile pigments. Reduction occurs also in patients
taking salicylic acid, sulphonal, and quinine
(Simon), so that it may be necessary to use—

1. Fermentation Test or

_ 2. Phenyl-Hydvazine Test—Take a_ pinch
of=pure:phenyl-hydrazine, ten drops glacial acetic
acid, one c.c. of a saturated solution of common

Fig. 64. Spectva of (1) Haematoporphyrin (in Acid Solution);

zy

(2) Haematoporphyrin in Alkaline Solution ; (3) in Neutral
Solution ; (4) Haematoporphyrin (urvate-sediments)

203

salt; add three cc. of urine; boil for three
minutes; cool; crystals separate out in a few
minutes up to one hour. This is an exceedingly
delicate test.

Tue DETECTION OF QUININE IN THE URINE *

The detection of quinine in the urine is of
importance in connexion with the property that
this drug has of inducing attacks of haemoglo-
binuria (blackwater fever) in patients resident in
regions where malaria is especially virulent, and
where generally the parasite form is the malignant
tertian associated with an extremely high endemic
index of native (children) malaria.

Two hundred c.c. of urine are acidified with
some drops of sulphuric acid. A spoonful of solid
picric acid is then added. The solution is allowed
to stand for an hour and then filtered. The solu-
tion should be quite clear and should give with
a saturated solution of picric acid no turbidity. If
there is difficulty in getting a clear filtrate adda
trace of egg albumen and filter again. The half-dry
residue is then digested in an Erlenmeyer flask
with fifty c.c. of 30 per cent. soda solution for
half-an-hour on the water bath. Now add
sixty c.c. chloroform; shake for two hours in a
shaking apparatus. The solution of chloroform
is now removed by means of a separating funnel
and. collected in a weighed flask. The flask
should have a long neck to prevent spurting.
Evaporate in a water bath and dry at 120°C. The
residue is quinine. The experimental error is
only one to two per cent.

* Kleine. Zeitschrift fur Hygiene. Bd. xxxviii, H. 3, s. 460.

294

DETERMINATION OF THE PERIODICITY OF
PaRASITE DEVELOPMENT

The inspection of a temperature chart is not in
itself sufficient to determine the cycle of develop-
ment of a parasite. Thus, as is well known, a
quotidian temperature chart may be produced by
a double tertian (simple) or by a triple quartan
infection. If then, in the case of the double
tertian, we made microscopical examinations at
definite intervals for forty-eight hours, we should
find in the blood at any particular time parasites
in two phases of development corresponding to
each cycle. The accompanying chart shews how,
in the case of what proved to be the malignant
tertian parasite, we were able to establish the
cycle of development. We proceeded to make blood
examinations at frequent intervals (four hours).
We found that at any particular time parasites
of various sizes might be found, but by counting
several hundred parasites in each film and esti-
mating their size with a micrometer we found
that at any particular time there was a prepon-
derance of parasites of one size. Thus, at ten
p.m. on the 2nd, there are numerous small forms,
z.e., about one-seventh to one-eighth of a red cell
in diameter, and it is not till ten p.m. (about) on
the ath that the same condition of blood is found
again, accordingly the parasite had a develop-
mental cycle of forty-eight hours (approximately),
And, further, we determined the periods taken to
develop from small forms to largest forms in the
peripheral blood (about eighteen hours) and the
disappearance of these and the reappearance of
numerous youngest parasites (about thirty hours).

295

. MIP-MIAM]PMIAM .| A.M.[ P AMP ™.1AM,
Time AeWZawle ee 60/2] 10/2 [6 [tol2 16 lolz 16 [0 216 tol2 [60/2 (6 flol2 [6
By Fladralsikes |-d exrelss| OF alta dla frekal |
S 2: OS E bG }
| =I L) (5)
wv
106
105
5 SH : ; { 5
104 rhl I 5
SCs
Lit is]
Hatt f
103] ttt
ALS qY t S
Ne ; a iy is
° ti HAE g HY
102 ;
i \
BEECH Eth
i BLN
WW ml
101 s f iS NE
is ‘a
_ \ s \
iS \ nt S
wi 5 ut
“al \ { 9 \
a 1 5 t SP
St 1 y
9f] po. c 1-H \
R) tT
Nor! Temp iz eo \
oF body. HEH | |
98 “Coy | i
WW \
MI Vr
| iN
97 Wy
Lay of Dis\— 2 3% ae 5 GA Za Bt
Date |_3/8/39 4/8/99 Baas _[ 6/éss | 7/8/99 _| 8/8/99 3/8/99

Fig. 65. Illustrating method of determining the developmental Cycle
of a Parasite. (The figures within the circles vepresent the
size of the Pavasites—thus 7 signifies the Parasite ts
one-seventh the diameter of a red cell)

296

“So that by determining these three periods we
were able to conclude that the parasite. was the
malignant tertian.

In order then to determine the cycle of a
parasite it is necessary :—

1. To estimate the size and percentage of
parasites of each size at any particular time, ¢.g.,
starting with the onset of the attack.

2. To follow each group to its period of
maximum development in the circulation.

3. To estimate the time between this period
and the next appearance of young forms.

4. Toestimate the time between the appear-
ance of an outburst of young forms (No. 1) and a
second similar outburst (No. 4).

The interval between one and four should be
equal to the sum of the intervals of periods two
and three. It is more accurate to use a micrometer
scale for measuring, but the estimation can be made
with considerable accuracy without.

If we are dealing with three generations of
parasites as in a triple quartan the principle is.
precisely the same, though it may require careful
observation to separate the different groups,
though in this particular case the process is facili-
tated by the presence of segmenting and _pre-
segmenting bodies which are easily counted.
In order then to establish a parasite cycle, repeated
observations at definite intervals are necessary,
and also the temperature should be-. carefully
recorded every four hours or two hours as con-
siderable variations may otherwise escape obser-
vation.

297

SIMPLE TERTIAN

Examined during the commencement of
apyrexia, young parasites are found one-fifth to
one-ninth the size of the red cell. The corpuscles
may be slightly larger than normal. On the
following day the parasites occupy one-half to
two-thirds of the corpuscle, much pigmented, but
not so actively motile as the smaller ones. The
red cells are much enlarged at the end of the
second day; presegmenting forms are found.
Division into four to six or more parts may take
place six hours before an attack, but true fission
forms are only found two to three hours before an
attack. These forms have sixteen to twenty
spores; so that, here again, the multiplication of a
group of parasites may be shown, microscopically,
to coincide with a febrile attack. But, as opposed
to quartan, the actual number of fission forms in
the peripheral circulation is small; they are far
more numerous in the spleen.

DouBLeE TERTIAN

The interval between the development of the
two groups of parasites is about twenty-four
hours. So that if a blood examination be made
just before an attack, fission forms will be found,
and parasites about half-grown. Here again, by
following out the development periodically, these
latter forms will be found to sporulate on the next
day.

QUARTAN PARASITE

If the blood be examined as soon as apyrexia
sets in, the corpuscles will contain young parasites

298.

one-fifth to one-sixth as large as the red cells;
growth is progressive during the apyrexia, and
six to ten hours before the next attack ‘ preseg-
menting’ forms will be found. The parasite now
fills the cell which is not enlarged. The pigment
is arranged in radiating bands. The next stage
is the concentration of the pigment in a central
mass, and it is seen in fresh or stained specimens
that the cytoplasm is divided into eight to ten
segments or oval bodies (daisy form). This seg-
mentation is nearly coincident with the next
attack, but parasites in complete fission may be
found five to six hours earlier. As the tempera-
ture rises these sporulating forms disappear and,
again, young forms are found in the red cells. The
quartan then goes through all its stages in the
peripheral circulation.

DouBLE QUARTAN

Two groups of parasites going through the
above regular cycle, with about a day’s interval,
may be followed in the blood.

TRIPLE QUARTAN

There are three distinct groups sporulating
on successive days.

MALIGNANT TERTIAN (TROPICAL)

During the pyrexia, small forms, one-eighth
[one-fifth] of the red cell, may be found. They
persist during the pyrexia, i.e., for the greater part
foaday. The parasites at this stage may be ex-
traordinarily few.

a9.

During the apyrexia, forms one-fourth to one-
third of a red cell are found, and the parasites are
found in greatest numbers. Fission and preseg-
menting forms are extremely rare in the tropics.
During the next attack the young forms are again
found, and so the time of the cycle, as we have
shown above, may be deduced, and may be con-
trolled by observation of intermediate stages.

QUOTIDIAN

Parasites have been described which com-
plete their development in twenty-four hours
(about). Thus, at the pyrexia young forms occur.
During the apyretic interval large forms and pre-
segmenting forms, and, again, at the next attack
young forms, thus developing in twenty-four hours.
As we have stated above, to establish accurately this
cycle three periods would have to be traced :—

No. 1. (? Twelve hours) from young forms

to largest forms.

No. 2. (? Twelve hours) from largest forins

to young forms.

No. 3. Twenty-four hours from young forms

to young forms.

While some consider that the quotidian tem-
perature is due to the fact that the malgnant
tertian has a very variable period of development,
viz., twenty-four to forty-eight hours, and, in fact,
all intermediate times, others consider that with
one generation of parasites there is a second
accumulation of young forms in sufficient quantity
to produce a quotidian attack.

In quotidian fever, due to the malignant
tertian parasite, the characteristic febrile attack,

300

with its preliminary pseudo-crisis, is lost. The
attack instead of lasting about a day lasts a few
hours only, as in the simple tertian, and instead
of a pseudo-crisis there is a. true crisis, but the
young parasites, as in the malignant tertian attack,
are. still coming into the circulation, and there
follows a rise which replaces the apyretic day in
the ordinary malignant tertian.

According to Maurer, in the case of the
quotidian chart produced by one generation of
malignant tertian parasites we have the febrile
attack produced by the division of the majority
of the segmenting forms, and then a fall to normal
occurs; when, however, there is a_ sufficient
accumulation of young forms arising from the
same generation there is again a rise giving the
quotidian chart. As we have seen, the young
forms of the malignant tertian parasite persist
during the pyrexia. If, however, by any means
they are destroyed or cease appearing temporarily
during the day of pyrexia, we should get a fall to
normal, and then as soon as this inhibiting cause
was removed, again a rise, giving a quotidian chart
produced by the malignant tertian parasite.

IRREGULAR TEMPERATURES

Besides the typical malignant tertian tem-
perature chart and the quotidian chart, various
irregular temperatures may occur, due solely to
the malignant tertian parasite. Such charts are
not at all uncommon in first attacks in the tropics,
and may be followed by charts with regular
curves.

The malignant tertian parasite has a develop-
mentalcycle of about forty-eight hours, and itseems

301

more likely that these irregular charts are produced
by an irregular irruption of young forms into,the
circulation than that the parasite has a variable
time of development. If we suppose that young
fission forms exist in the internal organs, but do
not commence their growth in the circulating red
cells, but come into the circulation irregularly,
then we should have still a constant time of de-
velopment, but an inconstant time at which the
development started. If, however, a quotidian
parasite exists, there should be no difficulty, as
we have stated above, in determining the fact by
a series of measurements at fixed intervals.

ACTION OF QUININE

The data of different investigators into the
absorption and elimination of quinine exhibit
considerable differences dependent upon the dif-
ferent conditions of experiment and the mode of
estimation employed. The following statements
must therefore be received with caution :—

1. According to KERNER the elimination of—
Quinine hydrochlorate begins in fifteen minutes
and ends in forty-eight hours.
Sulphate (neutral) begins in thirty minutes and
ends in forty-eight hours.
Sulphate (basic) begins in forty-five minutes and
ends in sixty hours.
2. Mode of Administration—
Per os, quinine appears in the urine in thirty to
fifty minutes.
Per rectum, quinine appears in the urine in eighteen
to twenty minutes.

302

Subcutaneously, quinine appears in the urine in
twelve to twenty minutes.

3. Duration of Elimination—

According to Garorato it lasts one-and-a-half
to seven and three-quarter hours.
According to Diett it lasts forty-eight hours.
According to Byasson it lasts seventy-two hours.
According to Personne it lasts eight days.

4. The Acme of Elimination—

According to THau and Kerner after the first six
hours.

According to GAROFALO after the first one-and-a-
half to four hours.

According to KLEINE after the first three to
six hours.

5. Hypodermic Injection—GarRoFaLo states
that the elimination is rapid, and that larger doses
can be accumulated in the blood in a shorter time
by this method than by doses given by the mouth,
while KLz1ne states that the absorption by this
‘method is slow. KLeEINeE’s figures will be given
below.

6. Amount of Quinine Eliminated—

WELITSCHOWSKI 100 per cent. about.
KERNER 95
BYASSON” - 75 oh. od
KLEINE 9-27 ,, (vide later)
PERSONNE ..- TEs aes
MERKEL 13 "
Marianl, during first day 18°7 per cent.
5 second ,, 6°3 a5
~~ ‘itd, 1°3 s
4 rourkh ,,, O'7 is

Total 27 per cent.

393

7. Kverne’s data as to the amounteliminated
in twenty-four hours :—
Per os Administration—
(i) 25°34 per cent.
(i) I9y7I
(ili) 27729 ,,
Ue) Oe 5,
This low value, No. 4, is explained’ by the fact
that the quinine was given on a full stomach,
whereas in the three other results the quinine had
been given to the patient fasting.
Per Clysma— (i) 17°66 per cent.
M1) PFI5 oy
(iii) 17°84 i.

Subcutaneously—(i) 11°37 ”

Gi) 970

Now, although proportionately a smaller
amount is excreted in this way (and this is pos-
sibly in conformity with the clinical experience
that ringing in the ears and other unpleasant
symptoms of quinine are generally absent after
subcutaneous injection), yet it is probable that the
excretion 1s a more prolonged one than by the
other methods, for deposits of quinine can still be
found at the site of injection some weeks later,
and so the undoubted efficacy of this mode of
treament may really be due to its prolonged action
(end elimination).

Mariani's results also shew that after an
injection of quinine into the muscles of a rabbit,
about twenty-four hours later, half the amount
could still be extracted from the muscles. KLEINE
and Marrani's results shew that a full stomach
inhibits markedly the absorption of quinine, so
also any catarrhal state is prejudicial.

304

8. Action of Quinine on Pavasites.—Quinine

although it does not prevent fission yet destroys
the young ring forms.

As is well pointed out by Marcuiarava and
Bicnam1, the ensuing attack may still lack nothing
in severity, although parasites are exceedingly
scanty.

Although this may be considered as the
typical action of quinine, yet there are cases, as
anybody who has observed really severe cases of
tropical fever, e.g., in West Africa, well knows, in
which quinine has not always this inhibitory
effect.

In such cases the number of parasites may
be exceedingly small or even absent, and yet the
severity of the symptoms persist. To those cases
where with severe symptoms and yet an absence
of parasites and to those cases where other
factors promote the rapid disappearance of para-
sites we shall refer in the succeeding section.

1. On Young Parasites (malignant tertain).—
When quinine is given at the time of their
first appearance in the circulation, the parasites
continue in the circulation for a variable period
of time depending upon the amount. of quinine
and probably on other unknown factors. Al-
though. parasites are still found yet their growth
is arrested, and the outburst is not followed
by large forms, presegmenting, and eventually
fission forms. It must be noted that quinine
may have no such inhibitory effect at all.

2. On Large Parasites.—The parasites still
go on developing as far as presegmenting and
segmenting forms, but generally there is no
subsequent production of young forms.

fe

3. On Presegmenting and Segmenting Forms.
These are, as we have said, rarely found in
the circulation, but if quinine is given at the
time that corresponds to this stage, the sub-
sequent effect is that very few young rings
appear:at the next attack.

QUININE HAEMOGLOBINURIA

Between this phenomenon and_ blackwater
fever there is, in. our opinion, practically no
difference. It is apparently true that cases of
blackwater fever do rarely occur in which no
quinine has been previously administered, and
in which we have the exciting cause of ‘chill,’
other drugs, ‘exertion,’ etc., but it does not
effect the position that quinine, not necessarily
in large doses, is the common cause of this
phenomenon. ‘We cannot here enter into the
evidence for this fact, but must refer to the
literature of the subject.

Blackwater fever is then a quinine intoxi-
cation, but it is something more. It occurs
only in those who have previously suffered from
malaria, and, in fact, there is considerable
evidence to shew that it occurs frequently in
divect association with a malarial infection. It
has often been denied that blackwater fever is
malarial at all, on account of the scarcity or
fréquent absence of parasites, but, as we shall
shew on page 308, this depends upon when the
examination is made. Regarding the haemo-
globinuria attack :—

1. The haemoglobinuria follows the admini-
stration of quinine after a certain variable interval,

Vv

306

two to three frequently, five to six or possibly
twenty-four hours.

2. The amount of quinine does not deter-
‘mine whether the haemoglobinuria is slight or
severe.

3. After haemoglobinuria has been produced
by quinine, a second administration does not
necessarily produce a second attack of haemo-
globinuria.

These facts clearly shew that it is not the
quinine, pey se, but a condition of blood in the
particular malarial patient which is the determin-
ing factor whether quinine will produce an
attack.

This is further borne out by the well. known
fact that the aborigines rarely, if ever, suffer from _
haemoglobinuria, but it is in Europeans subjected” .
to unnatural climatic conditions and subjected to
virulent malaria that the disease is most frequently
found.

We would only add, finally, that it is quite
illogical to abstain from quinine in malaria, on
the contrary, its adequate administration’ would
prevent the occurrence of these attacks.

As we have already said, an accurate study
of the urine in these cases and in allied cases of
malaria where quinine produces urobilinuria is
necessary.

Especially important is the study of the
urine and the blood in the prehaemoglobinuric
-state. It would, of course, involve an accurate
study of all possible subjects of the disease, and _
more especially those who had already had an
attack,

307

Post-MorTEM CHANGES IN MararIa
(MARCHIAFAVA AND BiGNAMr)

Brain :—
1. Punctiform haemorrhages of the men-
inges.

2. Punctiform haemorrhages of the white
substance of the brain.

3. The brain capillaries may contain nearly
every red cell infected. Sporulating forms are
especially common.

: sxenwean fe Sy) Zo [-
oe re Pe a
ie RR ZA pe) S
fellow pment Longe cls rated ih pollen

em Leven celle SSG olhek, Biante

Fig. 66, Shewing deposition of Pigment in Liver (left),
Spleen (vight), and Sporulating Parasites in
Brain Capillary (bottom)

4. The capillary endotheliuin may show fatty
degeneration, together with pigmentation, and
sometimes parasites.

5. Similar appearances are also found in the
vessel of the pia mater.

Lungs :—

1. Large pigmented mononuclears in the
capillaries, but especially in the veins; in the
lungs especially phagocytosis is proceeding.

308

2. There is a terminal infection with. the
diplococcus pneumoniae.

Spleen.—The trabeculae of the pulp are dis-
tended by infected red cells, and pigmented large
mononuclears are abundant. The malpighian
follicles, on the contrary, are non-pigmented.

Liver.--Endothelium of capillaries is swollen
and pigmented. Pigment is also found in KupFER’s
cells. The liver cells contain only haemosiderin,
not melanin. Pigmentation is most intense around
the central veins.

Kidneys.—Pigmentation is much less marked.
Changes may occur in the epithelium of the tubules,
independent of the presence of parasites.

Bone Marrow.—Parasites and melanin, free,
and in large mononuclear, leucocytes and macro-
phages are found. Crescents may be found here
when absent or scanty elsewhere, as in the spleen
and brain ; it 1s consequently supposed that they
principally develop here.

In cases of malaria of long standing the yellow
marrow becomes red.

Stomach and Intestines—In malaria with
choleraic or haemorrhagic symptoms, parasites
may abound in the capillaries of the villi.

Curonic MALARIA

Spleen.—As is well known, the spleen may
in these cases fill the whole abdomen. Dilata-
tion of the various lacunae occurs with a
thickening of the splenic reticulum. The pigment
tends to become deposited eventually in the
connective tissue surrounding the follicles. The
splenic septa become thickened.

a9

Liver—The pigment is found mainly in
the periphery of the lobules, and pigment in
the form of blocks in the perivascular con-
nective tissue.

The capillaries are much dilated, and the
epithelium contains blocks of pigment. Atrophy
of the liver cells and their nuclei occurs.

Bone Marvow.—The marrow of the long
bones is usually red, due to a large develop-
ment of haematoblastic tissue. Normoblasts are
common.

Pigment disappears rapidly from the bone
marrow.

LITERATURE

Marchiafava and Bignami. Twentieth Century Practice
of Medicine. Malaria. Vol. XIX. S. Low, Marston and Co.
This comprehensive and learned treatise is incomparably the
best in the English language, dealing with all aspects of
malaria and also blackwater fever.

310

Chapter XXV
BLACKWATER FEVER

Diagnosis.—Attention should be paid to the
following points :—

(1) Haemoglobinuria.—The colour of the urine
may vary from a very light red to a dark porter
colour.

(2) Jaundice.—Varying from a pale lemon
yellow to a deep bronze.

(3) Constitutional disturbance.— Slight, or
extremely severe, with a high temperature, vomit-
ing of green bile, sudden anaemia, pain over
kidneys and gall bladder, collapse.

We believe that careful observation of these
points will reveal the existence of blackwater
fever in many places where it is not supposed to
exist.

EXAMINATION OF THE BLoop IN BLACKWATER
FEVER

1. Note the difficulty in obtaining a full-
sized drop of blood.

2. Observe the ‘thin’ nature of the blood
drop, its ‘oily’ nature, and the difficulty with
which it adheres to the slide. These properties
are best seen in severe cases.

3. Collect a specimen in a fine pipette and

Sit

allow the serum to separate. Observe whether
the serum is yellow (cholaemia) or reddish
(haemoglobinaemia), using the spectroscope if
necessary.

. To some of patient’s serum add normal
blood. Observe whether there is any haemolysis
(using a haemocytometer if necessary).

5. Determine tonicity of patient’s blood.
Rate of coagulation approximately by placing
several drops on a glass slide.

6. Count the red and white cells. The red
cells are, as a rule, quite normal in shape.

7. Determine the amount of haemoglobin.

8. Make films every two hours if possible
(as early as possible), noting accurately the time
and temperature at which the films are made.

g. Examine films for parasites; if these are
absent, search carefully several large films for
pigmented leucocytes, as these, as also in ordinary
malaria, may require long search.

10. Make careful differential counts of the
leucocytes, especially when the temperature is
falling, as it is then that the mononuclear increase
is most marked. When the temperature is raised
(e.g., 103° to 105°) the polynuclears may reach
ninety per cent.

11. Observe presence of normoblasts, megalo-
blasts, various abnormal staining reactions, e.g.,
polychromatophilia of the red cell, especially dur-
ing recovery.

12, Make careful blood counts immediately
before and after administering quinine when
no haemoglobinuria results. According to PANSsE*
there may result a blood destruction due to the

*Panse. Zeitschrift fur Hygiene, 1903, s. 1.

312

quinine, which does not shew itself as haemoglo-
binuria.

' Microscopical investigations in this disease »
are frequently negative as regards malaria para-
sites, but it is all important when the examina-
tion is made, as the following analysis of over one
hundred cases microscopically examined shows :—

Parasites are present the day before the attack
in ninety-five per cent. of cases.

Parasites are present the day of the attack.in
seventy per cent. of cases.

Parasites are present the day after the attack
in twenty per cent. of cases.

In a series of cases examined by ourselves in
British Central Africa we found malaria parasites
only in 12°5 per centi, but, as we have already
shown, we have two further tests for a malarial
infection :—

(1) The increase in: the percentage of large

mononuclear leucocytes.

(2) The presence of pigmented large mono-

nuclear leucocytes.
By using these tests we were able to prove that
93°7 per cent., not 12°5 per cent., of our cases were
due toa malarial infection.

Further, in the only case of blackwater fever
seen by us before the onset of haemoglobinuria,
parasites weve present in abundance, afterwards
they rapidly disappeared.

For the details of the proof we must refer to
the original papers, where also the causative action
of quinine is discussed. That quinine is the factor
which, in the large majority of cases, determines:

the onset of haemoglobinuria appears to us equally
certain.

foe

EXAMINATION OF THE URINE IN BLACKWATER
FEVER

‘1. Before the attack (if possible) examine
for albumen, urobilin, reducing bodies, etc.

2. Examine so-called ‘high-coloured’ urines.
As a rule these do not shew bile pigment,

3. Examine urine during an attack for
methaemoglobin (or haematin), oxyhaemoglobin,
urobilin, bile pigment (unusual), bilirubin crystals,
haemoglobin casts, granular or hyaline casts,
blood cells (rare).

4. Centrifugalize the urine. Examine the
clear layer (as in 3), and make films of the
sediment.

The sediment may contain hyaline and
granular casts stained with haemoglobin. The
mass of the sediment, however, consists of masses
of haemoglobin of a yellowish-red colour.

UROBILINURIA

As we have indicated elsewhere, the occur-
rence of urobilin may be an important indication
in cases where a_ susceptibility to quinine
haemoglobinuria exists: thus in Murrt’s case, a
girl had haemoglobinuria eight times between
August 3, 1894, and April 6, 1895, following
upon the administration eight times of small
doses of quinine. From 1895 to 1897, the girl
remained well. On March 27, 1897, she was
given o'5 grammes of quinine, to see whether
her disposition to quinine poisoning still re-
mained. The result was fever, vomiting of
bile, etc., albuminuria, peptonuria, and uro-
bilinuria (not haemoglobinuria).

314

A. PLeHN, in a recent paper, points out a
peculiar property of the urine sometimes ob-
served in blackwater cases. On boiling the
urine and allowing to stand for some time, a
bright purple colour appears. .

We have observed that blackwater urines
made alkaline with potash, and then boiled
produce a purple colour, giving the bands of
haemochromogen (reduced haematin),, shewing
that the urine itself contained reducing bodies.

’ Whether PLeun’s purple colour is the same
we cannot say.

Post-MorTEM EXAMINATION

1. Make smear preparations of spleen,
kidney, liver, bone marrow, brain, etc. Examine
for parasites and pigmented leucocytes. Parasites
are generally absent, but pigmented leucocytes
may occur in large numbers in the spleen. Fine

igment is also found in the liver.in endothelial
capillary cells (Fig. 66).

2. Cut sections, especially of brain tissue, as

parasites may be found in the capillaries and

nowhere else.

Spleen —Malarial pigment (melanin) occurs in large mono-
nuclear cells and in giant cells (macrophages). Melanin may
also occur in the stroma or even beneath the capsule.

Liver.—Melanin occurs in endothelial cells, and especially
in macrophages. Yellow pigment (haemosiderin) occurs in
the liver cells, also, to a certain extent, in the same situations
as thelanin. Apply the iron reaction ‘(vide Appendix) to the
sections, Haemosiderin gives the blue colour, melanin does not.

Kidney.—Necrosis and desquamation of the epithelium of
the convoluted tubes. The straight tubules are blocked with
masses of granular matter, staining dark red with eosin. Inter-
stitial nephritis usually not present.

Bone marrow.—Evidence of malarial infection (pigment).
Proliferation of normoblasts,

St)
LITERATURE.

Kocu. Zeitschrift fir. Hygiene (Bd. XXX, 1899, p. 295).

STEPHENS and CurisToPHErs. fRepoyts to the Malarial
Committee of the Royal Society. Harrison & Son, London.

Panse. Zeitschrift fir Hygiene (1903,'s. 1).

Twentieth Century Dictionary of Medicine. Malaria.
The fullest and best account in the English language.

Chapter XAVE

THE HAEMOCYTOZOA—Continued

The haemocytozoa, or endoglobular haema-
tazoa, are divided by LaverRaAN into three genera :

1. Genus Haemamoeba.
2. Genus Pivoplasma.
3. Genus Haemogregarina.

The genus haemamoeba includes the malaria
parasites with which we have already fully dealt.
We now proceed to the other members of the genus,
and then to the other two genera, which go to
make up the endoglobular haematazoa.

GENUS HAEMAMOEBA

HAEMAMOEBAE IN BIRDS

1. H. velicta (Proteosoma gvrassii).—Dis-
covered by Grassi in the blood of birds in Italy.
In certain regions sparrows and goldfinches are
commonly infected. Sparrows are frequently in-
fected in India. In Africa numerous small birds
were examined by us, but proteosoma was never
found (only halteridium). Transmission from one
bird to another by inoculation is readily effected.
Canaries are extremely susceptible. Pigeons,
among other birds, are immune. Birds that have
recovered from an infection have acquired a well-
marked immunity against a subsequent inoculation

S17

The parasite is closely allied to the malaria para-
site, and is especially suitable for the study of the
exogenous mosquito cycle.

Endogenous Cycle (Fig. 67)—The parasite in
its earliest stage is unpigmented. Coincident with
growth a grain or two of pigment appears, and
the characteristic property of the parasite shows
itself, viz., the displacement of the nucleus of the
red cell, so that the nucleus may take up a position
at right angles and away from the normal one.
All stages of development up to segmenting forms
are found in the blood at the same time, so that
no cycle of development can here be followed ;
nor is there any intermission in the clinical
symptoms (temperature, etc.) of infected birds.

Fig. 67. (Upper line) Proteosoma shewing medium-size Parasite
and Segmenting Form. (Lower line) Halteridium young
form, Female and Male Gametes, and Vermicule

Exogenous Cycle.—Besides the asexual, sexual
forms occur in the blood. They are spherical
‘hyaline bodies of two varieties, characterized in
stained specimens by the same general differences

318

which distinguish the male and female gametes
of the malaria parasite. ow

(i) The male cell possesses a mass of compact
chromatin and faintly staining protoplasm.

(ii) The female cell possesses but little chro-
matin, but stains deep blue(ROMANOWSKY). -

Flagellation.— (i) ‘This can be observed in’
a simple wet film preparation. Make stained
specimens according to the method given on p. 31, °
Or

(ii) Use artificial serum (bird’s serum, one
part ; salt solution, o°6 per cent., nine parts), and
to this add a trace of bird’s blood. Make a series
_of hanging dropsin moist chambers. Dry, fix, and
stain, from time to time, according to stage of ©
development, observed microscopically.

Further stages of development (vermiculi)
have not been observed on the slide.

Development of Vermiculii—(i) Determine
what species of Culex is the suitable one for the
process of development. C. nemorosus was used
by Koc, in Italy. C. fatigans is also a carrier.

(ii) Collect the Culex that have fed on
sparrows, etc., roosting at night in trees. The
Culex can be caught in large numbers in shaded
drains, under bridges, in outhouses, etc., and ex-
cellent material is in this way easily got. Identify
the species of Culex that is infected.

(iii) For the method of feeding mosquitoes
on birds’ blood, vide p. 102.

Twelve to fifteen hours.—Vermiculi in all
stages of development are found in the stomach ;
a conical projection arises from the fertilized
gamete. This gradually elongates, forming a long,.
curved, oval body, the complete vermiculus. The

es coe)

protoplasm is vacuolated, and a nucleus (chro-
matin) is readily shown by staining (RoMANOWSKY).

The proteosoma vermiculi are larger and more
slender than those of halteridium.

Development of Zygotes (one to two days).—
The vermiculi have disappeared, butin the stomach
wall are now found transparent, spherical, Pig:
mented bodies.

Three to four days.—The zygotes have in-
creased in size, and sporoblasts appear in their
interior. In the larger forms, signs of further
division are seen (striation), formation of sporo-
zoits.

Development of Spovozoits (nine to ten days).—
By this time the sporozoits have reached the
salivary glands. Somewhat earlier they can still
be found amidst the thoracic muscle. Earlier
still, they can be pressed dut of the ripe oocysts
in the stomach wall. The sporozoits occupy
chiefly the middle lobe of the gland (Kocn).

Black Spores are found in the larger zygotes.
They also occur free.in the thoracic region (or,
possibly, in the gland substance). They are
brownish-black, curved, sausage-shaped bodies,
suggesting a mycelial nature. It is believed by
Grassi that they are degenerated sporozoits, as
they are found within the large sporoblast cysts.
We have, however, found them in or about the
salivary elands i in ‘Myzomyia VOSSI1,

2. H. danilewskyi (Halteridium). — = Oratits
almost exclusively, in the blood .of ‘passerine’
birds. Pigeons are very commonly infected, also
sparrows, finches, ‘ paddy’ birds, parrots, ete,

The parasite is characterized by its peculiar
curved halter shape, embracing the oval nucleus

320

of the red cell without any displacement of the
latter (Fig. 67). Young forms are occasionally
seen, but whether these are young sexual or asexual
forms is not determined. Segmenting forms and
those corresponding to an asexual cycle, as in
proteosoma, are unknown.

Two varieties of parasites, the male and
female gametes, are easily distinguished.

(i) Note that the male gamete has a clear
hyaline appearance. On staining (RomaNnowsky)
a central mass of chromatin is distinguished,
while the protoplasm is a faint blue. Five or
more oval pigment grains are placed generally
at either extremity.

(11) In fresh specimens the female gamete is
finely granular, and the pigment is frequently
scattered throughout. On staining, a small
amount of chromatin is shewn, while the proto-
plasm takes on a deep blue colour.

Flagellation.—Select an infected bird that
shews numerous gametes in each field. Proceed
in the same way as in proteosoma. The gametes
first become spherical and then escape from the
red cell. The pigment of the male gamete dis-
plays violent movement, and in a few minutes
four to eight flagella are extended. The motion
of these is at first so rapid that they cannot be
distinguished, but the corpuscles in the neigh-
bourhood are seen moving. In .a few minutes one
or more breaks off, and if, fortunately, a female
gamete is in the same field, the loose flagellum
(mikrogamete) can be seen entering the female.
The pigment of the latter shews active movements
at this stage.

Vermicul1—The formation can readily be

peu

observed on the slide. A conical projection
forms at one point of the fertilized gamete (copula).
This elongates slowly and gets curved, forming
an egg-shaped or spindle-shaped mass. The
conical portion eventually separates, leaving
behind the remains of the cell with the pigment.
The vermiculus is thus at first unpigmented, but
later again it is pigmented (Kocu). In the fresh
specimen the protoplasm appears vacuolated, and
has a nucleus which is readily stained by Roman-
OWSKY stain.

Note that the vermiculus (or ookinet) shews
forward, rotatory, and peristaltic motions. The
further development of the vermiculi is completely
unknown.

Post-mortem. — Pigment is found in the
kidney, intestine, bone marrow, liver, and especially
thé spleen. The brain, on the contrary, is almost
entirely free from it.

It is probable that the halteridia of all birds
are not of the same species. Inoculation from
one bird to another is extremely difficult, if not
impossible. This may be due to the fact that the
parasites in the blood are in all sexual forms.
In monkeys we appear to have a parallel condition,
viz., gametes only in the blood, the asexual forms
being unknown.

HaAEMAMOEBAE IN MONKEYS

H. kochi—These haemamoebae occur in
monkeys. The forms usually met with are
sexual forms. Asexual forms resembling young
malaria parasites are very rare. Flagellation
can be seen in fresh specimens. ‘The parasites

Ww

998

in the fresh film are spherical pale bodies
containing brownish-yellow pigment. On stain-
ing, two types can be distinguished. The male
(mikrogametocyte), pale homogeneous blue with
much chromatin; the female, deep blue, granular,
with little chromatin.

No temperature changes occur in the in-
fected animals. The infection is not transmissible
by inoculation (cp. halteridium).

Post-movtem.—The spleen is pigmented, the
capsule thickened. Pigment also occurs in the
marrow.”*

e

HAEMAMOEBAE IN BATS

Dionist has described in bats parasites
which have a general resemblance to malarial
parasites, but almost certainly have no real
relation thereto. He distinguishes the following

forms :—

Fig. 68. (1) H. muvinus (above) medium size ; (below) free form
(2) H. melanipherus (above) medium size ; (below) large Ovoid
form. (3) H. vesperuginis (above) irregular form ;

(below) Ring form. (After Dionts1) ©

4. H. melanipherus (polychromophilus mel-
anipherus).—This occurs in the blood of Mint-
opterus schreibersii, and is so called on account of

*Kosser. Zeitschrift fur Hygiene. Bd. XXXII.

323

its staining reactions with Romanowsky, and
because it is pigmented. It somewhat resembles
the quartan parasite (lig. 68).

H.. muvinus(polychromophilus murinus).—
Found in the blood of Vespertilio muvinus. It
is also pigmented. It shews like the former a
variety of ‘polychrome’ effects with Roman-
owsky. In this, as in the former, Dionis1
figures a great variety of forms (Fig. 68).

6. H. vesperuginis (achromaticus vesper-
uginis).—In the blood of Vesperugo noctula. ‘The
young forms resemble those of the malignant
tertian parasite (ig. 68). It occurs in large
numbers in the blood, but forms no pigment
during its development. It produces considerable
anaemia and degenerative changes in the red
cell.

How bats are infected is quite unknown.
The parasite ‘find’ differs according to
whether the animal is hibernating or not.

HAEMAMOEBAE IN CATTLE

7. [H. bovis].—Parasites in the blood of
cattle, described by Korie in South Africa.
They have a general resemblance to malaria
parasites, but are quite distinct from Piroplasma
bovis. They produce remittent fever and severe
anaemia, but not haemoglobinuria. Ko. also
describes pigment in red cells (independently
of parasites), but what this means is not clear.*

“Korte. Zeitschrift fur Hygiene. 18098.

24,
HaEMAMOEBAE IN TORTOISES

8. H. metchnikow?.—Found in the blood of
Tvionyx indica, or Chitya indica, a large fresh-
water tortoise, in many Indian rivers. All adult
specimens of this tortoise from the Jumna were
infected.

The parasite resembles H. danilewskyi (hal-
teridium), in that two forms are easily dis-
tinguished in the blood—(1) a hyaline form with
large pigment grains, staining very slightly with
methylene blue; (2) a granular form, with fine
pigment, staining deeply with methylene blue.
These forms cozrespond to the male and female
gametes respectively. In one of Simonp’s figures
it is interesting to observe a male and female
gamete in the same red cell, which, so far as we
know, has never been observed in the case of
H. danilewskyi. But besides these pigmented
forms there are also found unpigmented forms,

Fig. 69. H. metchnikowi, Gametes and Vermicule

which have the typical gregarine look, that is to
say, curved, worm-like bodies. The exact relation-
ship of the haemogregarine to the haemaebae forms
is not understood. Simonp,' however, points out
that halteridium has a vermicule stage, and there
is the possibility of the relationship being similar
in this case (Fig. 69).

325
Genus HAEMOGREGARINA*

The haemogregarines are unpigmented uni-
cellular organisms, which, at one stage of their
development, have a worm-like form. They occur
as endoglobular parasites, and also as free forms
in the plasma. ‘The vermicule stage may be both
endoglobular and free. .The sexual and asexual
cycles occur, as far as is known, in the same host.
They occur in fish, amphibians, and reptiles, but
not in mammals, and, unlike the gregarines, not
in invertebrates. They are, so far as is known,
non-pathogenic, and they cannot be transmitted
by inoculation from one animal to another. The
cycle of development, as far as it is known, will be
described under the various species.

Fig. 70. H. vanavum (or Drepanidium ranarum) young
form, Gametes free in the Plasma, and Fission forms
in Spleen. (Partly after Mincutn)

1. H. vanarum (= Lankesterella ranarum).
Found in the blood of Rana esculenta (edible
frog). This species includes, according to
LAVERAN, two species, H. princeps and H. montilis,
described by Lappe. Here, as in other species of

Nore,—Ample material for the study of these parasites is
readily obtainable in the tropics, Frogs, toads, lizards, snakes,
tortoises, etc., are commonly infected, Examine especially the
liver and internal organs for developmental forms. Examine

carefully for ticks, lice, etc., and possible cycles in these.
Preserve all ecto parasites in spirit for subsequent identification.

326

haemogregarines, the sexual and asexual cycles
occur in the same animal. The cycle of develop-
ment is as follows :—

(i) Sexuals Form or Schizonts.—These are
endoglobular, four to eight «in length. Increase
in size takes place, and eventually they become
spherical and divide into a number of segments
(schizonts). According to some observers seg-
menting forms are only found in the spleen.

(ii) Sexual Forms.—Free in the plasma,
twelve to fifteen long. These are male and
female, and are characterized by the same general
differences as other gametes ; the male mikrogame-
tocyte is slender and finely granular; the female
makrogametocyte is fat and coarsely granular.

(111) A mikrogamete in the form of a small
mass of chromatin separates off and fertilizes
the (now) makrogamete.

(iv) Azygote results, which is at first motile.
This becomes encysted as the

(v) Oocyst, which is found in the epithelial
cells of the intestine. This passes out eventually
in the faeces of the frog. Sporoblasts are formed
as in the malarial cycle, and from these result

(vi) Spovozoits——These would gain access to
a fresh frog which had swallowed an oocyst.
Hintze has shewn that frogs confined in pools are
especially liable to infection.

2. H, splendens (= Dactylosoma splendens).
—Found in the blood of R. esculenta.

_ The following forms are figured by Lasse
(Hig. 91) i—

(1) Amoeboid forms.

(11) Forms resembling in shape a finger-
glove.

327

(iii) Segmenting forms as in Haemamoeba
velicta (Proteosoma).

The protoplasm contains no pigment but
refractile granules.

This differs from the typical development of
haemogregarines, and it is probable that its posi-
tion requires revision. According to Hinrzz, it is
a variety of H. ranarum,

Fig. 71. H. splendens—Adult form with
Refractile Granules

3. H. magna—Described by Grassi and
Fevetti in R, esculenta. M1INncuIn thinks it may
be the makrogamete of H. ranarum or H. monilis,

In frogs not uncommonly curious rod-shaped bodies are
found lying in a vacuole in the red cell. When these occur
further search will show cysts filled with these rod-like bodies,
Originally described as protozoan parasites Cytamoeba, they are
considered by LAvERAN to be bacterial in nature.

4. H, viedyi—Occurs in a Californian
Salamander, Batracheseps attenuatus.

5. H. stepanow1,—It is found in the tortoise,
Cistudo europaea. ‘This may be taken as the type
haemogregarine. It presents the following forms
(Fig. 72) —

(i) Reniform parasites, ten to fourteen «
long. Curved and thickened at each end,

328

granular, non-pigmented. Intermediate forms
occur between this and the next developmental
stage.

(ii) Vermicule forms, also endoglobular,
but after examining a fresh specimen of blood
for some time, free forms are seen thirty to
forty « long and three to four » broad. These
are actively motile, and constrictions can .be
seen travelling down their length during the
motion. Young forms and reproductive forms
are not seen in the circulation. These are
found in the liver. The reproductive forms are
at first endoglobular, but later free. They occur
as

(iii) Ovoid forms, ten to sixteen » long
by four to six » broad, shewing as many as
six nuclei (chromatin masses). The protoplasm
finally segments and there is formed

(iv) An actively amoeboid young form.

Fig. 72. H, stepanowi, Endoglobular and free Vermicules ;
H. lacertarum, shewing disintegration of Nucleus of
Red Cell ; H. lacazei, H. lacertarum, Cyst
with Makromerozotts

The spores that are found in the kidneys
of tortoises belong, according to LAvERAN, not
to the haemogregarine at all, but are those of
a Myxosporidium (M. danilewskyi).

329

6. H. lacertarum (=Karyolysus lacertarum)
(Fig. 72)—Found in the blood of Lacerta
agilis, LL. muralis, and L. ocellata.

The parasite has a more compact form
than some of the other haemogregarines. They
exert a marked. action on the red cells, which
become much enlarged and anaemic, and, as
the name of the species implies, a disintegrating
action on the nucleus is one of its effects. The
nucleus is either pressed to the side or broken
up into fragments.

8
S/.
Jp

Fig. 73. (1) H. mesnili, shewing characteristic looped Vermicule ;
(2) H. lavevani, shewing characteristic hooked Vermicule and
two bright Granules (after Simonp) ; 3 H. bigemina
in Blood of Blennies (after MincHIN)

The parasite in its endoglobular stage
becomes encysted, and is then called a cytocyst.
Further, it appears as if at this stage sexual
differences appeared, for some of these cysts-
divide up into about a dozen macromerozoits
(Fig. 72), while others divide up into twice
as many or more micromerozoits. Corresponding
to these we have free forms in the liver, twelve
by three w» and eight by two » respectively.
According to ScHAUDINN it is transmitted by
an Acarine.

7. H. lacagei (= Haemocytozoon clavatum).

+330

In the blood of lizards. The vermicules have a
peculiar shape (Fig. 72). Here also cyst forma-
tion has been described in the spleen by LABBE.

8. H. mesnili—tIn the blood of a tortoise,
Emys tectum (Fig. 73).

Amoeboid forms, reniform, and vermicule
forms occur. Besides these, free merozoits, but
their origin is obscure. The form of the
vermicule is characteristic at one stage of its
development.

H. laverani—tIn the blood of Indian
tortoises, Cryptopus granosus. Similar forms occur
to those of the last species. The vermicule is
characterized by a blunt hook-like appendage,
and the presence of two bright granules.

The parasite is endoglobular in all its stages.

10. H. bigemina.—Discovered by Laveran
in the blood of blennies. A vermicule form occurs
free in the plasma.

The endoglobular parasite divides by simple
binary fission. In fishes we also have H. delagei
in two species of ray and H. simondi in the sole.

11. H.mauritanica.—In Testudo mauritanica.
Resembles H. stepanowd (Fig. 72). Two forms
occur: (i) very granular, smaller forms, with two
large refractile granules at each end ; (ii) larger,
uniformly pale forms. In stained specimens the
smaller forms appear oval or reniform, with a
nucleus transversely placed. The nucleus of the
red cell is displaced. The larger forms have at
one of the poles a pale blue mass, staining with
difficulty. Division forms are found in the liver.

12. H. tunistensis—In Bufo mauritanicus.
(i) Vermicule form. The two limbs of the ver-
micule are equal; the vermicule is encysted ;

Jo

at one end of the cyst is a deep staining mass.
According to BitLer there is a second cyst
included in the first. This second cyst shows
stippling with Romanowsky, but the red cell is
not hypertrophied. (ii) Elongated halter-forin,
18 « by 4-5 .. Parasites especially abundant in
the liver.

13. A. vipervini—Or Karyolysus viperini. The
parasite bores into the nucleus, and, eventually,
encysts there.

lig. 73a. H. vipevini. (1) young parasite; (2) the parasite
is encysted in the nucleus, the cyst and the red cell show stippling
(Romanowsky). H. bagensis. (1) vermicule; (2) encysted stage.

14. H. bagensis.—In a tortoise (Emys leprosa)
in Tunis. (i) Young stage does not exceed three-
quarters the length of red cell. (ii) Vermicule
stage encysted. The cyst does not stain. Nucleus
variable in position, always central according to
Bituet. (ili) Large oval forms. (iv) Division
forms in liver.

15. H. sergentium—tIn a lizard, Gongylus
ocellatus. Has a destructive action on the nuclei
of the red cells. They flatten and elongate, and

332

also fragment. Parasite is reniform, 15-18 » by
5-6. The protoplasm has numerous granules,

16. H. curvivostris.-—In Lacerta ocellata var.
pater. Differs from the previous one. (1) The
vermicule form destroys the nucleus; (2) The
encysted form becomes embedded in the nucleus.

“. A curvirostlrrs.

Fig. 73Be shewing disintegrating action on nucleus.

Genus PIROPLASMA

SPECIES HOST
P, bovis Cattle (Texas fever organism)
P, canis Dogs (Italy, Senegal)

P. ovis Sheep (Italy, Roumania)
P. equi Horse (S. Africa)
P. hominis Man (producing ‘ spotted fever ’)

1. P. bovis—‘ The parasite of Texas fever of
cattle clearly does not belong to the proper group
of malaria parasites, although it occurs in.the red
cell. It forms no pigment, and differs in its
development from the malaria parasite’ (Kocu).

333

Technique—Examine the blood of young calves, especially
those snowing some emaciation, The blood is most readily
got from the ear. Wash the ear witha wet cloth, dry, and rub
until the veins become prominent, Wash stained films moment-
arily in acetic acid, 1 in 400H,O, otherwise the deep blue of
the red cells somewhat obscures the parasites. Examine very
carefully, as parasites may be scanty in chronic cases,

The parasites are two to four » in length, one
to two » in width. Various forms occur in the
circulation—(i) a spherical or ovoid form, (ii) a
piriform parasite in pairs or fours. These are
characteristic, and give the name. Intermediate
stages between (i) and (ii) occur. The spherical
forms show a chromatic particle, and closely
resemble ‘young rings.’ The chromatic body
(nucleus) divides into two portions, one going to
each end; the parasite elongates, and by this means
the piriform body is got. The piriform parasites
are two to three » long and about one » in
diameter.

(iii) Bacillary forms showing, however, a red
chromatic spot and blue protoplasm (Fig. 75).

(iv) Large double forms having a curious
resistent appearance (Vide Plate) probably gamete
forms of P. bovis.

The number of parasites in the peripheral
circulation is proportionate to the severity of the
disease—one to two per cent. of corpuscles are in-
fected, at the end of the disease five to ten per
cent. (or even twenty-five to thirty per cent.) The
number in the blood is not so great as the number
in the spleen (ten per cent.), liver (thirty per cent.),
and especially kidneys (eighty per cent.)

' Free parasites are found in the blood in the

334

later stages of the disease, but especially in the
kidneys.

Post-movtem.—Haemorrhagic oedema about
the stomach, kidneys, and retroperitoneal tissue.
Intense hyperaemia of the spleen and kidneys, the
latter are nearly black. Haemorrhagic erosions.
Ulcers in various portions of the alimentary canal.
Ecchymoses in pelvis of kidney.

OO

Fig. 74. Piroplasma canis (left), typical Piviform
Parasites ; (vight) Amoeboid forms

Transmission by Ticks——According to Motas
(Bucharest), Pivoplasma ovis can be transmitted by
transferring adult ticks from an infected to a non-
infected animal. He did not succeed in trans-
mitting the disease by larvae or nymphae de-
veloped from ticks taken from infected animals.
This is in direct opposition to the classical re-
searches of SmitH and KiILBorNnE on Pivoplasma
bovis, corroborated by Kocu. SmitH and KILBorNE
hatched young ticks from the eggs of ticks that
dropped off infected cattle. It is these young ticks
that communicate the disease.

2. Pivoplasma canis.—The parasite is mor-
phologically identical with Pivoplasma bovis. It
is a strictly specific parasite, and has not been
transferred to any other animal than the dog.
Native dogs in the tropics may harbour the para-
site without shewing any symptoms.

Fig.
Fig.
Fig.
Fig.
Fig.
Fig.

PLATE III

Pivoplasma bovis

(As seen in calves in Madras)
1-3.—Amoeboid forms.
4.—Dividing forms.
5-8.—Typical piriform parasites,
g.—Free forms.
10.—Gamete form (?)

t1.—Bacillary form, fresh film.

Fig. 12-15,—Bacillary forms (Romanowsky).

Plate HT.

335

Technique.—Examine a number of pariah dogs, especially
puppies and sickly dogs. With a scissors trim the hair off the
tip of the ear and then snip the skin,

The disease exists in France, Italy, India, Africa,
etc. In the chronic forms the parasite is rare in the
circulation, but in the acute form with high fever,
icterus, and haemoglobinuria, the parasite (typical
pirosoma form) is found with great ease in the
blood. Four to six parasites often occur in each
cell. Kidney blood post-mortem is extremely rich
in parasites. Young'dogs, two to twelve weeks old,
are the most easily infected, by intravenous in-
jection. The tick, Demacentor veticulatus, is sup-
posed to convey infection in Europe, and Haema-
physalis laechi in South Africa. Both these ticks
appear to pass their larval stages on other hosts
than the dog.

3. Ptvoplasma ovis.—The disease in Hungary
is known as carceag. Bases considers that the
organism forms a connecting link between the
bacteria and protozoa. Sheep that have recovered
have a marked immunity.*

4. P: [Rochi].—‘ African Coast Fever,’ de-
scribed by Kocu, is an exceedingly virulent form
of piroplasma infection in Rhodesia and South
Africa, eighty to ninety per cent. of infected
cattle die. A pecuharity of the disease is that the
anaemia is slight and, correspondingly, haemo-
globinuria rare. The parasite is smaller than
that of the piroplasma of Texas fever. The
parasites are disc-shaped or leaf-shaped, and as
the disease progresses may be found in almost
every cell. Pear-shaped organisms are rare. It is
suspected that Ehipicephalus decolovatus, or the

*Motas. Soc. d: Biol, 1903, y- 1,523.

336

blue tick, transmits the disease, for this tick
transmits also Texas fever in South Africa.

It is in this form of bovine piroplasma espe-
cially that atypical forms have been. described by
THEILER, and subsequently more fully by
LAVERAN.

(1) Forms resembling straight or curved
bacilli, one to three « long. They are thicker at
one end, which contains a chromatin particle.
One to four may occur in the same cell.

(i1) Forms resembling cocci, singly or in
pairs. Two to four may occur in the same cell.

OY OW

Fig. 75. Atypical forms of Pivoplasma
(After LavERAN)

It is important to note that together with the
atypical forms typical {forms are always found,
though these latter may be rare. ©

These atypical forms occur in the severe cases.
Similar cocci-like forms, described by SmitH and
KILBORNE in Texas fever, occurred in the slight
cases. The post-mortem lesions, characteristic of
this form of pirosomal disease, are local infarcts in
various organs.

5. Pivroplasma hominis.—This species of piro-
plasma is responsible for the disease known as
‘spotted fever,’ occurring in Montana and Idaho,
U.S.A., and possibly in Egypt. As the name
implies, there accompanies the fever an eruption
of spots. The mortality in the United States is
as high as seventy to eighty per cent. It is much

337

less in the cases described in Alexandria, Egypt.
It is possible that they are not the same diseases,
and the subject requires elucidation. In the Mon-
tana disease, piriform, ring-shaped, and cocci-like
forms occur.

6. Pivoplasma equi (LAVERAN).—Found in
horses in South Africa.

Ticks

Life History.—The female, after satiating her-
self with blood, falls to the ground and, in a few
days or weeks, lays eggs.

Eggs.—The eggs are laid in masses of several
thousands (Ixodidae), of some hundreds (lvgasidae).
The process lasts about a week. They are small,
oval, opaque bodies. They may take weeks or
months to hatch out. From the eggs is developed—

The Larva.—These are hexapod. They cling
to blades of grass, etc., and may do so for several
months before attacking a host. The larval stage
lasts six to ten days. The moult then takes place,
and there emerges from the skin—

The Nymph.—These are octopod. They re-
semble adult females. They have respiratory
stigmata, but no sexual organs. The nymphalstage
lasts seven to ten days. The nymph moults, and
there emerges—

The Adult-—These again attach themselves
to the host, and ina few days copulation takes
place. The female gradually distends and remains.
attached for about nine to eleven days. The female
then drops off. What the male does is uncertain.
The female’s whole cycle on the host is thus
twenty-two to thirty-one days. The entive life
cycle takes, probably, about two to three months.

x

338

The dimensions of the various stages of
I. vicinus are :-—

Eggs ... 04 by 03 millimetres.
Larva .. 06 by 04 millimetres.
Nymph... 1-3 by 06-2 millimetres.
Adult... .... 2°5 by 1°5 (male) millimetres.

Adult... ... ro-11 by 6-7 (female full

grown) millimetres.

Ticks have a predilection for certain hosts,

but the same tick may be found on different

animals, and, further, the larval and adult stages
may be passed on different animals.

ExTrERNAL ANATOMY

Technique.—Collect ticks from the ears of dogs, bellies of
cattle, sheep, etc. Preserve in spirit. Make out in fresh speci-
mens the following points, dissecting, if necessary :—

1. The Rostyum, capitulum or head, is the
small anterior projecting’ portion, it is joined on
by a short neck to the scutum; on its ventral sur-
face is seen—

2. The Labium or hypostome, a bi-laterally
symmetrical structure, furnished with a number of
teeth directed backwards. The number of rows
of teeth and their disposition are very important
in classification.

The Mandibles lie dorsally to the labium.
The terminal portion (digit) terminates in two
or three processes, apophyses, which bear hooked
teeth directed backwards. They are important
in classification.

4. The Mandibular Sheath lies above the
mandibles. The anterior extremity is notched
corresponding to the two halves of the sheath.

339
(2), (3), and (4) form the piercing organ or

haustellum.

. The Palpi are four-jointed and form a
kind of sheath for the haustellum. The shape
of the palpi, their spines and processes are of
the greatest importance in classification.

Fig. 76. Eggs, Larva, and Adult Tick. (After MaasEn)

6. The Scutum is a dorsal structure, situated
behind the base of of the rostrum. It is a
hard leathery plate. In the male it practically
covers the whole of the dorsum. In the female
it is confined to a roughly triangular anterior
portion of the dorsum. The males are thus
readily distinguished from the females. It is
absent in the :Argasidae.

7. The Povose areas are dorsal structures
forming two oval depressions one on each side of
the middle line at the base of the rostrum. They
are most conspicuous in the female, but exist in
both sexes.

' 8. The Eyes, not always present, are small,
almost globular structures, situated laterally at
the margin of the scutum in the Ixodidae, or as
punctiform structures on the supracoxal fold of
the first leg in the Argasidae.

. The Stigmata.—Situated ventre' and
laterally behind the level of the fourth les a the

340

pee

hh esis

Digit

“ne”

phasis '

( feth)

Pocbeaal
.. / Fal pus projection
Ornitho doves taemophysalrs
— Falbres,

Rosbum- 3 - For0se o7€e42}

Am bl omma

Rast Sarr of legs —
ie,

b: Ventral Shields

Lao des #hipiceb bolas

’

Fig. 76a. Illustrating external anatomy of ticks.
(Adapted from NEUMANN)

341

Ixodidae open into a stigmal plate or peritreme.
In the Argasidae they lie between the third and
fourth legs. The shape of the plates are important
in classification.

10. The Anus isa little way in front of the
posterior ventral margin. It has a valve.

11. The Anal plates or clype7, four in number,
on either side of the anus, in the male. They are
used for classifying. Not always present.

12. The genital orifice isin the middle line,
a little way behind the rostrum.

13. The Legs are six in the larva, eight
in the nymphs and adult. The claws have
ventrally a well-marked pad or pulvillum. The
coxa (with which the trochanter articulates) may
have spines or teeth or larger ‘shields.’ These are
used in classification.

INTERNAL ANATOMY AND DISSECTION OF TicKs

1. Take a large gravid female which has
been kept a few days until it shews signs of
shrinkage.

2. Taking care not to compress the tick, snip
tangentially round the body.

3. Place the tick in normal saline, and now
separate the dorsal from the ventral surface, and
pull it gently over the head with a forceps.

. The various viscera may now be fairly
easily displayed. Observe—

(a) The very complicated diverticula contain-
ing blood (d). ,

(b) The alimentary canal similar in appear-
ance passing direct from the head to the anus (a, c).

6) The rectum. A terminal portion white in
appearance (rect).

val glands *

i ie
Vike ;
Nive No if
OME AN Ds
SG
a ae

“yh
AEG W,
Ly yyy

ayy
a
Ht ve

Ovum

Fig. 77. Ilustvating internal anatomyfof ticks. B the viscera;
C. a longitudinal section; D. transverse section of diverticulum ;
E. nearly mature ova.

343

(d) The salivary glands. A number of small
tubules near the rostrum on either side.

(e) The ovary. A brownish elongated organ
(0). Examine with a lens and note the large ova.

(f) The malpighian tubules ? (t).

5. Lo cut sections. Snip off portions.of the
chitin before placing in alcohol. Do not over-
harden. Use asharp razor. Fix the sections to
the slide by the hot water method. Stain with
haematein. Observe the following tissues :—

(a) An external chitinous layer and a single
row of columnar cells forming the cuticle.

(b) The alimentary canal and its appendages
cut in various planes.

In the diverticula, distended with blood, the
epithelium is flattened; in the empty diverticula
the epithelium is columnar, and the cells often
have processes projecting into the lumen. Note
the peculiar crystalline contents of the diverticula
(altered blood).

(c) Large cells with large nuclei (ova). These
are arranged around branches of the follicular tubes
as in the mosquito.

(d) Undeveloped follicular tubes containing
large cells.

(ce) Tissue, rich in nuclei, especially in the
posterior portion of the body (= Fat body of
insects).

(f) A large nerve ganglion anteriorly.

(¢) Muscle fibres passing chiefly ventrad and
dorsad, tracheae supplying the viscera.

CLASSIFICATION OF TICKS

Ticks are divided into two families—
1. Argasidae, scutum absent.
2. Ixodidae, scutum present.

344

The Ixodidae are divided into two sub-families—
1. ERhipicephalinae.—Palpi not longer than
broad, rostrum short. Anterior portion
of body emarginate to receive the

rostrum.

2. Ixodinae——Palpi longer than broad, ros-
trum long. Anterior portion of body
straight or emarginate.

The Rhipicephalinae are the most important
from our standpoint, as to the genus Rhipicephalus
belong most of the ticks that are known to trans-
mit parasites. The various genera are Rhipice-
phalus, [Boophilus], Haemaphysalis, and Derma-

centor.

Fig. 78. Tick under surface, shewing Anatomy and parts
used for classification. (After SaLMon and STILES)

Genus RHIPICEPHALUS

_ Eyes present. Base of rostrum hexagonal
(dorsally), forming on each side a projecting
angle. Palpi short and broad. Stigmata, comma-
shaped ; clypei, two pairs in the male. Coxae i,
two large teeth. The genus includes nearly thirty
species. Some of these, including the carriers of
piroplasma, we may classify in the following
way :—

345

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SYIBWIY Ul el, sTVJL Ul Winynsg SaTqrpuryy wniqe’T tinynsg jo smolln y saisadg

SN TIVHdHOIdINY SONAL) JO Lavd dO NOILVOMISSVTZ)

346

Genus ‘BooPuiLus

Not admitted by NEUMANN as a genus.

Genus HAEMAPHYSALIS

Eyes wanting. Base of rostrum rectangular,
twice as long as broad, palpi conical. Second
segment of palpi has a_ well-marked. lateral
conical projection. Stigmata comma-shaped or
circular. Anal shields absent in male. Coxa i
not bifid. Coxa iv in male, a_ well-marked
spur. There are about twenty-six species.

H. leachi (South Africa)—The dog is the
usual host. Possibly transmits Pivoplasma canis,
occurs also on cattle.

Labium.—F our rows of teeth in 9, five rows
1 #.

Palpi—Dorsal surface as broad as long.
Second palpal segment has a sharp lateral spine.

Coxa iv.—Has a tuberosity.

GENUS DERMACENTOR

Eyes present. Base of rostrum broader than
long. Palpi short and thick. Stigmata, comma-
shaped. Anal shields absent in male. Coxa i
bidentate in ¢ and ?. Scutum ornamented.
About twenty-four species.

D. Electus is the American dog tick.

The subfamily Jxodinae consists of five
genera, Ixodes, Haemalastor, Aponomma, Ambly-
omma and Hyalomma,

347
Genus IxoDEs

Eyes absent. Palpi long. Tarsi without
terminal spurs. Anal groove surrounds anus
anteriorly and opens posteriorly. Scutum in
male does not cover the body laterally and
posteriorly. Stigmata oval in ¢, circular in &.
Male ventrally covered with six shields; two
lateral, embracing the origins of the legs and
the stigmata; one median, between the genital
opening and the anus; two on each side of
the anus (perianal); and one triangular posterior
shield, carrying the anal orifice at its anterior
corner. Female has dorsally three longitudinal
grooves on the abdomen, ventrally two bell-
shaped grooves, the first has its apex at the
‘vulva, the second at the anus. There are a
large number of species.

ts
mon on sheep, goats, cattle: Europe.

2. I. hexagonus.—The European dog tick.

Genus HAEMALASTOR

Eyes wanting. Rostrum long. Palpi piri-
form ¢, claviform 2. Anal grooves as in Ixodes.
Stigmata circular in both sexes. Legs very long.
Dorsal: and ventral chitinous thickenings in the
male; fine grooves in the female. There are
seven species.

GENusS APONOMMA

Eyes wanting. Anal groove surrounds anus
posteriorly, and opens anteriorly. Anal plates
absent. Base of rostrum pentagonal. Scutum

348

covers the dorsum entirely ; usually marked with
green spots. Stigmata, comma-shaped. Female,
scutum shorter than broad, three green spots.
The species are parasitic on reptiles.

GENUS AMBLYOMMA

Eyes present, conspicuous. Anal groove as in
Aponomma, anal plates absent. Rostrum long.
Scutum often has coloured designs. Stigmata
usually triangular, nearly always eleven posterior
marginal festoons in the male. There are over
eighty species.

A. variegatum.—Is frequent on cattle in
Rhodesia.

Genus HyatomMa

Eyes present, conspicuous. Rostrum long.
Anal groove opens anteriorly. Body elongate
oval. Colour deep-brown. Male, two pairs of,
ventral shields, two perianal, large, triangular,
and two small external. Scutum covering nearly
the whole of the dorsum. Crenellated or festooned
posteriorly. Male, stigmata comma-shaped, with
a long tail; female, stigmata with a short tail.
Three species only.

H, aegyptium.—Attacks cattle especially, also
dogsandcats. Occurs in Egypt, North and South
Africa.

Argasidae.—Scutum absent, rostrum inferior
(except in larva). Stigmata between third and
fourth legs. Pulvillum of tarsi wanting in adult.
Palpi, free, short, filiform, four segments. Tegu-
ment leathery, without dorsal or ventral shields.
Sexual dimorphism not marked.

Genus ARGAS
Eyes absent. Rostrum, which is concealed

349

by the cephalo-thorax, is situated at least its own
length behind the anterior margin. No pro-
jecting hood. Body oval or orbicular. The
species are nocturnal in their habits, infest birds
mainly. ‘There are eleven species. A species of
Argas is the transmitter of ‘spirillar fever’ of
poultry.

A. reflexus infests pigeons, European.

A. persicus =Garib-Guez of Persia. The bite
is said to produce severe local and constitutional
effects.

A. tholozant = Kéné of Persians, similar
effects ascribed to it.

GENUS ORNITHODORUS

Eyes present or absent. Rostrum hidden
under a projecting beak (hood), close to the
anterior margin of the body, so that the tips of
the palpi are visible from above. Lateral borders
of body generally straight, sometimes concave,
tegument mammillated.

O. moubata = ‘ Garrapata,’ tick of TETE on
the Zambesi. The bite is said to occasion severe
local and general disturbances. It is the cause of
‘tick fever’ in Uganda (Cristy).

LITERATURE

1. Cattle Ticks of the United States. Salmon and Stiles,
Washington, 1902. An excellent compendium on ticks, many
illustrations. Price, a few shillings.

2. L.G. Neumann. Révision de la famille des Ixodidés,
Mémoires. Soc. Zool. France. Complete monographs.

. Texas Fever. Theobald Smith and F. L. Kilborne,
1893. Washington. Bulletin No. 1.
4. Wasielewski. Sporozoenkunde.
5. The Spovozoa. Minchin in Lankester’s Zoology.

350

Chapter XXVITI
THE TRYPANOSOMIDAE

‘TRYPANOSOMATA AND TRYPANOPLASMATA |

The Trypanosomidae comprise two genera—
(1) Trypanosoma, (2) Trypanoplasma. The genus
Trypanosoma is characterized by the possession
of a longitudinal undulating membrane, the
thickened border of which takes’ its origin pos-
terlorly from a blepharoplast, and terminates an-
teriorly in a free flagellum. Division takes place
longitudinally. The genus Trypanoplasma has
two flagella, one anterior the other posterior. Both
arise from one blepharoplast ; the anterior forms
the thickened border of the undulating membrane;
the posterior flagellum curves around the posterior
end of the parasite, and then is prolonged into a
flagellum about equal in length to the anterior
one.

The Trypanosomidae occur in fish, amphibia,
reptiles, birds, and mammals. Most of these are
very incompletely known, and it is only some
species in mammals that have been at all closely
studied.

Trypanosomata of fish—These are common in
tropical fish, but have not yet been accurately
described. Those recorded below occur in fish of
temperate climes. Among marine fish they occur
chiefly in the cartilaginous fish. The sole is the
only osseous marine fish so far found infected.

554

Here, as in the case of other trypanosomes, mor-
phological appearances often make it a difficult
matter to distinguish species. The fact, however,
that the trypanosome of one species of fish cannot
be transmitted to another species of fish is in
favour of the specific nature of the trypanosomes
of ‘each species of fish.

Mode of infection.—This, probably, is effected
by ecto-parasites of the fish, especially leeches.
Experimentally, it has been shown that leeches
can transmit the infection. Inoculation of fish
is most successful when made intra-peritoneally.

1. T. vemaki.—In the pike (Esox luctus). Never
very numerous. ‘Two varieties or species occur :
(i) T. vemaki, var. parva, 28-30 u long, flagellum.
included, by 1°4 # broad. The blepharoplast. is
rather small. (11) T. vemaki, var. magna, 4°5 » long,
by 2°5« broad. .These two varieties resemble one
another very closely, except in size.

2. T. danilewskyi.—In the carp (Cyprinus
carpio). 35-45long, by 3“ broad. Blepharoblast
large. The protoplasm in stained specimens has
many chromatic granules.

. T. tincae.—In the tench (Tinca tinca).
Generally scanty in the blood. Motility very
active, and curls on itself. 35 « long, by 2°5-3 »
broad. Posterior end is blunt.

T. abvamis.—In the bream (Abramis
bvama). Recorded by Laveran and MEsni, but
undescribed.

T. granulosum.—In the eel (Anguilla vul-
gavis). They vary in size from 44-80 » in length,
and 2°5-3in width. Very active in their move-
ments... Undulating membrane broad. Posterior
end sharply pointed. Stained specimens show

be

large chromatic granules. They live in blood kept
at a temperature of 10-19° c. for a week.

6. T. soleae—-In the sole (Solea vulgaris).
40 & sas Flagellum very short, 8 » (Fig. 82).

7. T. scyllit.—In dog-fish (Scyllium canicula,
etc). The trypanosome is curved on itself. 70-75»
long, by 5-6 #« broad. Posterior end conical.
Undulating membrane much folded.

8. T. vajae.—In rays (Raja punctata, etc.)
Like the last it is curved on itself. 75-80 « long,
by 6 broad. Posterior end much drawn out in
some forms, blunter in others.

Fig. 79. T. votatovium, T. cobitis, T. cavassii, Tp. danilewshki.
Left to right. (After LAVERAN and MESNIL,
MirraPHANow and DaANILEWSK1)

T. cavassit (MITRAPHANOW).—Besides the
forms with undulating membrane and flagellum,
disk-like forms are described. In the isle of fish
(Carassius vulgaris) ; in the tench (Tinca vulgaris).
Also described in the blood of the stickleback,

pike, etc. (Fig. 79).

PLATE IV
SENEGAMBIAN TRYPANOSOMES

Birps
‘ig. 1—T. johnstoni, n. sp. From the blood of Estrelda
estvelda. xX 2,000.

‘ig. 2—Trypanosoma, sp. incert. x 2,000.

Frocs
‘ig. 3.—T. votatorium. x 2,000.
‘ig. 4.—T. mega. n. sp. X 2,000,

‘ig. 5.—T. Rkaryozeukton. x 2,000. Showing chromatic granules
extending ina chain from the centrosome to the

nucleus.

a

AI FLV 1d

i

10. T. cobit’s (MirrapHANow).—In the blood
of the mud-fish (Cobitis fossilis). Thirty to forty
long, one to two u broad. Flagellum ten to
fifteen. Itis long andthin. Forms without an
undulating membrane are described, and also
without a flagellum (Fig. 79).

TRYPANOSOMATA OF BATRACHIANS

1. T. votatorium.—Occurs in various species
of frogs: Rana esculenta, R. temporaria, Hyla
arborea, etc. It is characterised by extreme varia-
tion both in dimensions and in appearance. Thus
we have (i) forms with striation (Fig. 79); (ii)
forms without striation; (iii) spherical forms in
which the undulating membrane is retracted.

The position of the blepharoplast or centro-
some is also variable. Generally, it les close to
the nucleus, and, consequently, the membrane
does not extend further back than this point; at
other times it hes near to posterior end, with a
consequent more extensive development of the
membrane.

Dimensions: 40-60 « long, by 5-40 # broad.

It is doubtful whether the trypanosome
occurring in these frogs belongs to one or to
several species.

2. T. mega.—-In a Gambian frog. (Vide
Plate IV, Fig. 4).

3. T. karyozeukton.—In a Gambian frog. (Vide
Plate IV, Fig. 5).

Laveran and Mesniv think that these may
only be varieties of T. votatovium.

4. T. tnopinatum.—Occurs in Rana esculenta
in Algeria. 25-30 # long (including flagellum),

Y

354

34 broad. Resembles 7. lewisi and T. vemakz.
Bituet thinks it may be a developmental form of
a haemogregarine occurring with it in the blood.

5. T.nelspruitense—In frogs in the Trans-
vaal, characterized by its long flagellum, 20-35 u,
giving a total length of 40-70 yp.

TRYPANOSOMATA OF BIRDS

Though first described in 1888 by DaniLew-
SKy, our more precise knowledge is due to
LAVERAN.

1. T. avium.—oOccurs in the owl (Syrnium
aluco). 33-45 long (flagellum included). The
undulating membrane is well-developed, and has
several folds. The posterior extremity is pointed.

2. T. johnstoni.—In the blood of Estvelda
estvelda in Gambia. It resembles a spirochaete
in appearance. There is no free flagellum. (Vide
Plate II, Fig. 1). 36-38 » long, by 1°4-1°6 broad.

3. T. paddae.—In the blood of Padda ory-
givova. 30-40 « long, by 5-7 broad. Posterior
end very pointed. Undulating membrane narrow
and folded, but difficult to stain. Division takes
place longitudinally. Pathogenic (?).

Other trypanosomes have been described by
various observers in many different birds. The
trypanosomes are, generally, scarce. DANILEWSKY
states that while rare in the blood, trypanosomes
may be abundant in the bone-marrow.

4. T.noctuae.—In the blood of an owl (Athene
noctua) occur halteridium-like bodies. They are
male and female (and hermaphrodite), and have
the appearances in fresh and stained preparations
which characterize other gametes. According to
the remarkable work of ScHauninn, these so-called

bee

halteridia are simply stages in the life history of
a trypanosome. The ‘halteridia’ undergo their
development in Culex pipiens. (Vide note, p. 368).
The following stages occur :—

(1) The blood is sucked into the stomach, the gametes
become spherical, and the mikrogametes (flagella), (which
themselves are built on the same fundamental plan as trypano-
somes) fertilise the makrogamete (female).

(2) The result, as in the case of the malaria parasite, is a
zygote or copula which in eight to thirty-six hours becomes a
vermicule or ookinet.

(3) These ookinets are of three kinds—hermaphrodite,
male, and female.

A. Development of the ookinet into an indifferent try panosome.—

The ookinet now develops into a typical trypanosome with
nucleus, blepharoplast, undulating membrane, and flagellum.

[The locomotor apparatus of a trypanosome arises, accord-
ing to ScHAuDINN, from the mitotic processes which take place
in the nucleus of the ookinet. Three nuclear spindles are
formed by successive divisions. The third of these becomes the
locomotor apparatus. The excentric spindle fibres become the
thickened edge of the undulating membrane, the central fibres
form the free flagellum, while the contractile mantle fibres,
eight in number, which direct the movements of the nuclear
chromosomes on the spindle, become ‘ myonemes,’ four of which
run on either side in the (Ektoplasmic) undulating membrane].

We thus get an indifferent trypanosome.

B. Development of the indifferent trypanosome in the stomach of
Culex pipiens.
(1) Multiplication by longitudinal division takes place ; a
fresh locomotor apparatus is developed, the old one remaining
unchanged. The resulting trypanosomes are small in size.

(2) The trypanosome has a resting or ‘gregarine’ stage,
It bores its way into and even through the epithelial wall of
the stomach, contracts, and the flagellum becomes a short rod-
like process of attachment. It may divide in this stage, forming
extensive clusters. This stage 1s adopted when the blood
supply is scanty in the stomach. If blood is imbibed the
parasites again become actively motile.

356

C. Development of the indifferent trypanosomes in the blood.’

t. Some of the larger forms sub-divide in the blood.

2. The smaller forms apply themselves closely to the sur-
face of the red cells and gradually penetrate, they lose their
flagellum and now resemble young halteridia.

3. Pigment appears in twenty-four hours.

The parasites, however, again leave the red cell, chiefly
at night time, and again become trypanosomes though now
increased in sizes These changes also occur especially in the
internal organs, bone marrow, spleen, kidney, and liver. This
process is repeated several times till after six days the fully-
developed trypanosome and halterida stages are reached.

5. ‘The fully-developed trypanosome divides rapidly till
a largenumber of young flagellates are formed, which go through
the same process until a massive infection of the red cells is
produced. The developmental cycle in the blood thus takes six
days. Whether in other species a formation of spores as in
malaria, and as originally described by DaniLtewsxy, takes
place remains to be seen.

A. The development of the ookinet (9 ) into the female trypanosome.

1. By a similar process the female trypanosome results.
It is larger and stains more deeply than the indifferent form.
The membrane is less well-developed, the blepharoplast is
smaller, and the flagellum is shorter. It moves more slowly
and soon takes on the gregarine form in the stomach,

B. The development in the mosquito.

1. They are the most resistent forms and can be found in
the stomach two to three weeks after feeding, when all the
other forms have died.

2. In this fasting condition they have penetrated the
epithelium, and resemble malarial zygotes. It is these forms
also by which the infection is transmitted to the egg as they
remain alive in the ovary, and these forms can also, by partheno-
genesis, give rise to all three forms, either in the stomach or in
its halteridium form in the blood.

C. Development of Q trypanosome in the blood.

1. They enter the blood cells and grow slowly.
2. They leave the red cell not as a trypanosome but in a
worm-like gregarine form.
They eventually form the characteristic makrogamete.
At the end of the acute infection they are the only forms

gor

remaining in the blood, but they may again, by parthenogenesis,
produce all the other forms and so a relapse of the disease (Cf.,
relapses in malaria by parthenogenesis of the makrogamete),

Blephoraples a

Try pa \nosome,

2,

2,3.6 - Festing Stages (Aaltendio)
5.7 * Trypanosome Stages.

Fig. 79a. Upper figure: shewing the three kinds of ookinet and

the three trypanosomes developed from them in the mosquito’s

' stomach. Lower figure: sherwing the development of the indifferent
trypanosomes into ‘ halteridia’ in the blood (aftey SCHAUDINN).

A: Development of the male trypanosome from the ookinet.

The male ookinet is hyaline without reserve stuffs, coarsely
vacuolar and refractile. It is smaller than the two other
ookinets, but has a relatively larger nucleus rich in chromatin,
By similar nuclear changes trypanosomes are developed.

358

The male trypanosome is much smaller than the other two
forms, but has a more fully-developed locomotor apparatus.
The blepharoplast is relatively much larger, and the flagellum
is longer, they are consequently very actively motile.

B. Development in the mosquito.

They do not develop further. They only become functional
when they leave the blood and fertilize the female in the
mosquito’s stomach.

C. Development in the blood.

As the males rapidly die out in the stomach it is not often
that they reach the blood. If they do they probably die quickly.
The mikrogametocytes of the blood are developed from the in-
different trypanosomes.

The fertilization of the makrogamete (female trypanosome)
by the mikrogamete (male trypanosome) takes place when blood
is sucked into the mosquito’s stomach, and thus the cycle is
completed.

The civculation of the trypanosomes 1n the mosquito.

1. The gametes in the blood of the owl are sucked in.
The formation of the ookinet, and development into trypano-
somes takes place in the mid-gut in about twenty-four hours,

2. If no more blood is given the parasites (female) are
now found in the resting (gregarine) stage attached to the mid-
gut, and they may occur in such quantity as to kill the
mosquito,

3. If asecond meal of blood is given development into
the motile stage occurs, followed by the resting stage. The
parasites are now collected in enormous masses (thousands)
around the proventriculus and commencement of the mid-gut.

If a third meal of blood is given these masses are
washed onwards by the entering blood, eventually reach the
ileum and the great upward curvature of the colon,

5. Here they penetrate the epithelium, reach the blood
stream, passing some backwards to the ovaries, some forwards
until they reach the blood spaces surrounding the pharyngeal
pump. They accumulate here in such masses that they even-
tually pass through the epithelium of the pharynx and so reach
the lumen. 2

6. They are then washed out when the contents of the
diverticula, carbonic acid gas, bacteria, etc., are ejected prior
to the sucking act.

7. The parasites that reach the ovaries develop there, and

so can be transmitted through,the larval stages to the newly-
hatched mosquito.

302
TRYPANOSOMATA OF MAMMALS

Examine the blood or the oedema fluid, if present, of
camels, cattle, horses, dogs, etc, especially those showing
emaciation, oedema of any part, watery discharge from the eye,
skin eruptions, etc, Apparently healthy animals may not in-
frequently harbour trypanosomes, ¢.g., calves in India (Madras),
the big game in Africa, etc. Compare carefully the trypano-
somes found.

Doin,

1 £OY 4

Fig. 80. T. lewisit—(1) Dividing form with two blepharoplasts ;
(2) Adult form; (3) Young forms resulting from
division (fresh preparation)

Trypanosomes are bodies easily detected in
fresh blood with a one-sixth or one-seventh lens.
They are actively motile, and may be seen dis-
placing the red cells by their motions. As they
come to rest the undulating membrane and flagel-
lum are visible. They are bodies about twenty
w long. In stained specimens (RoMANOWSKY) an
oval nucleus lies about the middle of its length,
and near the blunt posterior end a small stained
particle is clearly seen, the centrosome, or rather
blepharoplast. From this, the flagellum starts,
‘and can be seen as a distinct wavy thick red
line extending the whole length of the organism
and continued beyond as the long (anterior)
free flagellum. The portion (unstained) between
this external wavy margin and the blue stained
body of the organism is the undulating membrane

(Fig. 80).

360

1. T. lewisi.—Is found in a small percentage
of ordinary sewer rats in England. It .is non-
pathogenic. It is common in rats of tropical
countries. White rats are easily infected, but
never spontaneously show infection. Possibly
there are various species of this trypanosome. It
is Slenderer than 7. brucei. Its undulating mem-
brane is less wide. It is stated that infectton can
be transmitted by a rat flea. .

2. TT. brucet.—This is the highly pathogenic
trypanosome of Ngana, or tsetse fly disease.
Lengthf27 «by 2u. TT. brucei is fatal to nearly
all, if{not all, mammals. In the horse there occurs
watery discharge from the eyes and nose; puffy
swelling under the belly; sheath of penis, etc.,
marked anaemia, wasting.

Fig. 81. T. gambiense, T. lewisi, T. brucei, T. equiperdum,
and dividing form of T. brucei, shewing two nuclei,
two blepharoplasts

The disease was shown by Bruce to be con-
veyed from infected animals to healthy ones by
means of Glossina morsitans. The fly after biting
remains infective from twelve to forty-eight hours.

361

3. T. evanst—The trypanosome of Surra. A
common disease in many parts of India, e.g.,
Bombay, at certain seasons especially, though
probably always in a latent condition. It is
possible that its increase at a particular time is
associated with the prevalence of a biting fly.
Surra is characterized by a similar train of symp-
toms to those of Ngana. Rocers states that the
disease in India is conveyed by Tabanidae (horse
flies). This statement has not yet been confirmed.

Whether the ‘surra’ of camels in India is
produced by the same trypanosome there is no
evidence to shew.

LaverAN and MeEsnit, who have recently
been able to make a comparison of T. brucez and
T. evansi, state that T. brucei is shorter and more
compact than T. evansi. The movements of
T. brucei are also less extensive. The posterior
end of T. brucet is also blunter than that of
T. evansi. The free portion of the flagellum is
shorter in T. brucei than T. evans, and the pro-
toplasm of T. brucei has more numerous and
larger granules than that of T. evans. The
nuclei and the blepharoplasts are morphologically
indistinguishable. Further, the mean length of
T. brucei is less than that of T. evansi, and the
width of T. brucei is greater. The distinction
between Surra and Ngana is, however, best proved
by the fact that an animal immunized against
Ngana is yet susceptible to inoculation with
Surra.’

T. equinum (Mal de Caderas).—In Central
and South America. A disease affecting horses.

The symptoms—remittent fever, oedema,
wasting—resemble those of Ngana and Surra.

362

Most characteristic is the paralysis of the hind
legs, from which the disease takes its name.

It runs a chronic course, two to six months.
In donkeys six to twelve months. There is occa-
sionally haemoglobinuria.

Mice, rats, rabbits, dogs, guinea pigs, etc.,
are susceptible. Incubation period, five to eight
days. Horned cattle are the most refractory.

It is thought that the infection is transmitted
by a biting fly (Stomoxys calcitrans). There appears
to be, however, some connection between Mal de
caderas and a disease affecting a rodent (Hydro-
chaerus capybara), allied to the guinea-pig. When
an epidemic occurs among these, then Mal de
caderas breaks out among the horses (LavERAN),
The parasite morphologically resembles T. brucez.
In the latter the centrosome is, however, larger.
In T. equinum it is so small that its existence has
been denied. Moreover, an animal immunized
against T. equinum is still susceptible to T. brucet.

5. T.equiperdum (Fig.81).—This trypanosome
is the cause of the disease among horses in Algeria
and India, known as Dourine. In asses the
symptoms are slight. In horses, and especially
stallions, the symptoms are much more marked.
It is conveyed, as far as is known, under natural
conditions by ‘coitus’ only, and not by means of
flies.

In eleven to twenty days after coitus,
oedematous swellings of the genitalia appear.

In forty to fifty days characteristic ‘ plaques’
on the skin. These are very occasionally absent,
as in asses, but when present are pathognomonic.
These ‘plaques’ last only one to eight days.
Around these there is oedema. The animals

PLATE V

T. gambiense

Fig. 1.—T. gambiense in Gambian native. x 2,000.
Fig. 2—T. gambiense in tame rat, ‘long form.’ x 2,000.

Fig. 3—T. gambiense in tame rat, showing longitudinal
division. x 2,000.

Fig. 4.—T. gambiense in tame rat, from a specimen taken one
week before death, ‘stumpy form,’ showing chromatin
granules. xX 2,000.

Fig. 5.—T. gambiense in tame rat, from a specimen taken one
week before death, ‘ round form,’ showing granules.
X 2,000,

T. dimorphum

Fig. 6.—T. dimorphum. The small ‘tadpole-shaped’ parasite
in the early stage of the disease. x 2,000.

Fig. 7.—T. dimorphum, ‘stumpy form,’ in tame rat. x 2,000.

Fig. 8—T. dimorphum, showing longitudinal division of
‘tadpole-shaped’ parasite. x 2,000.

Fig. 9.—T. dimorphum, ‘long form,’ showing longitudinal
division. x 2,000.

Fig.10.—T. dimorphum, ‘long form.’

614

A AaLV 1d

363

become anaemic, complete paraplegia sets in, and
death in two to ten months.

Trypanosomes are most easily found in the
‘plaques,’ with difficulty in the blood.

Post-Mortem.—There is inflammation of the
urogenital mucosa, and in two cases areas of
softening have been found in the spinal cord.
Ruminants are refractory (to T. brucei they are
very susceptible). Dogs which have been immu-
nized against T. equiperdum yet succumb to
T. brucei, so that Dourine and Ngana are distinct.

6. T. gambiense (Dutton).—This, the first
human trypanosome to be described, was discovered
by Durron in the blood of a European in the Gam-
bia. The clinical symptoms of the case were :—
1) Irregular relapsing fever.
2) Oedema, especially about the eyes.
(3) Congestion of the skin.
(4) Erythematous patches, associated with

thickening of the skin.
(5) Increased pulse and respirations. Loss
of flesh.

The trypanosomes are generally scanty in the

blood, and it may be necessary to centrifugalize

and examine the leucocytic layer.

N.B.—Europeans in an area where trypanosomiasis (or
sleeping sickness) is endemic in the native population, may be
infected with trypanosomes and show none of these signs, at
least at first, except, perhaps, a daily rise of temperature in
the evening. Examine repeatedly and carefully Europeans with
an iregular intermittent temperature on which quinine has no
effect,

TRYPANOSOMIASIS (SLEEPING SICKN ESS)

In 1902 CaSTELLANI found a trypanosome in
the cerebro-spinal fluid of a case of sleeping

364

sickness. Bruce furnished the proof of the causal
connection of this trypanosome with the disease.
Bruce further showed that the disease was trans-
mitted by a particular ‘tsetse’ fly, Glossina palpalis.
It has since been shown that the trypanosome in
this disease is identical, morphologically and in
its pathogenic properties, with T. gambiense. In
fatal cases a streptococcus can often be isolated
from the organs, but death may occur with all the
typical symptoms and yet the organs be completely
sterile; so that the streptococcus occurs only as a
terminal infection.

1. Blood examination.—Parasites may be
absent from the peripheral blood for a month or
more at a time, and even if abundant, seventy to
a cover-slip, may again completely disappear.
The number of parasites bears no relation to the
severity of the symptoms.

2. Cerebro-spinal fluid Obtained by lumbar
puncture. Seldom more than one to five trypano-
somes occur in the centrifugalized sediment.

Cervical lymphatic glands——Greic and
Graystate that trypanosomescan be most certainly
found by puncturing the cervical glands.

4. Post-mortem.—Parasites are often found
in the pericardial, pleural, and peritoneal fluids
even without centrifugalizing.

T. gambiense is from 18-25 » in length, by
2-28 broad. It occurs in two main forms—(1) a
‘long form’ with a pointed posterior end, and (2)
a short stumpy form with many chromatic eranules
(Vide Plate V).

Pathogenic action.—It is pathogenic for many
mammals, é.g., rats, guinea-pigs, rabbits, monkeys
(except Cynocephalus), etc. On the whole, the

365

disease is a;chronic one in animals, and recovery
may take place. White rats are easily infected,
best by intraperitoneal inoculation;. while guinea-
pigs are most convenient for experimental work.
About half the inoculations into animals fail, and
post-mortem material never gives a positive result.

Morbid anatomy.—The changes in the central nervous
system are those of a chronic meningo-encephalitis and
myelitis (Morr). The pia-arachnoid is sometimes opaque, and
the vessels are considerably congested. Purulent meningitis is
the most frequent complication. The brain in these cases is
covered with lymph, and the vessels of the pia-arachnoid much
injected,

Microscopically.—The pia-arachnoid shows a mononuclear
infiltration most marked over the cerebellum and medulla.
The blood in the vessels also shows the mononuclear character,

T. theilevi.—This is found in the blood of
cattle in South Africa, subject to a_ disease
known as ‘gal ziekte,’ 7.e., gall sickness. Length,
thirty to sixty-five #; width, two to four w.

THEILER states that a biting fly, Hippobosca
rufipes, transmits the disease.

T tvransvaliense—Found in the blood of
oxen, eighteen to fifty # long by four to six »
broad. ‘The blepharoplast of this trypanosome
almost touches the nucleus. The undulating
membrane is consequently little developed.

THEILER considers this to be only a variety of
T. theileri. T. theileri is infective for cattle only.

8. T. dimorphum.—In the horse in Gambia.
The disease is characterized by progressive weak-
ness and emaciation. ‘There are no oedematous
swellings as in Ngana. Various animals are
‘ susceptible. It occurs in three forms (PI. V, Figs.
6-10).

366

Various other trypanosomes have been des-—
cribed in cattle, horses, and camels in Uganda,
Algeria, Soudan, Somaliland, Togoland, German
East Africa, etc. Their relationships are at present
doubtful.

Trypanosomes have also been recorded from time to time
in many other mammals, e.g., rabbit, hamster, dormouse, bat,
squirrel, lemming, souslik, guinea-pig, Gambian mouse, field
mouse, and mole. In the Gambian mouse the trypanosome is
said to possess no undulating membrane. The trypanosomes
in these various animals have a general resemblance to T. lewis1,
but most are insufficiently described.

DIMENSIONS OF TRYPANOSOMATA FouND IN

MAMMALS

1. T.lewisit - - 24725 by I-4

2. T. brucei - 25-30 m by 1°5-2°5 mu
3. T. equiperdum 18-26 wu by 2-2°5 u
4. T.evansi - - 20-30 by 1-24

5. I. equinum 20-25" by 2-3

6. T. gambiense 18-25 w by 2-2°8 w
7. T. theileri 30-65 wu by 2-4 pw

8. T. transvaliense 18-50 « by 4-6 u

Considerable variation exists between the
data of observers, and though these figures can
be considered as ‘approximately correct, they do
not suffice for distinguishing the various species.

Whether it will be possible to distinguish
nearly allied species morphologically, e.g., T. brucei
and T. evansi, remains to be seen. Differences in
the position of the blepharoplast and differences in
staining properties hardly suffice in similar species
that resemble one another closely, and at present
the only certain method is their pathogenic pro-
perties.

Inoculation—The most certain and rapid
method is intraperitoneal, e.g., in the case of

367

T. lewisii, but subcutaneous is almost equally
certain, and in T. brucei scratch inoculations
nearly always succeed.

Blood Examination.—If present in fair quan-
tity there is no difficulty in detecting them fresh
with a low power. If very scanty, it may be
necessary to centrifugalize the blood. The most
delicate test of a successful infection which may
have resulted, even though no parasites be found,
is a subinoculation into a highly susceptible
animal.

Fig. 82. T. soleae, T. avium (after DANILEWSKY)
Tp. borvelt (after LAVERAN)

Where parasites cannot be found by an
ordinary examination in the blood, they may,
however, be readily discovered in the oedematous
swellings so often found in trypanosomiasis.
Thus it is often extremely difficult, if not im-
possible, to detect trypanosomes in the blood of ,
a rabbit infected with T. Brucet, yet they are
easily found in the oedematous fluid about the
ears, muzzle, etc.

Cultivation of trypanosomes——Novy and McNeav have
succeeded in growing trypanosomes in vitvo. Of those yet tried
T. lewisi is the most readily cultivated. The medium consists
of a mixture of nutrient agar and defibrinated blood. The pro-
portion of blood to agar is 2:1, though T. lewis: will also grow

368

when the proportion is 1:2, or event:10. T. brucet is much
more difficult to grow, the best proportions of blood to agar
are 2:1 or 3:1.

The culture medium is best poured into flasks so as to get
a large but shallow layer of condensation water. After inocu-
lation the cultures are kept at a temperature of 25°C. Assoon
as a good growth is obtained subcultures should be made.
Bacterial contamination must be scrupulously avoided.

In cultures of T. lewist forms 1-2 w (excluding flagellum)
in length are found, and filtrates that have passed through ‘a
Berkefeld filter are infective. The virulence of cultures may be
completely lost, though the trypanosomes are still active and
growing in subcultures,

GENUS TRYPANOPLASMA

1. Tvypanoplasma borreli.— Twenty u long,
three to four # broad. Each flagellum, fifteen »
long. It is curved in shape. ‘he undulating
inembrane on the convexity. The anterior end
is more pointed than the posterior. Found in the
blood of the red eye (Leuciscus erythrophthalmus).
Also a similar, if not identical, species in min-
nows (Phoxinus laevis). ‘They may cause anaemia,
wasting, and death of the fish (Fig. 82).

2. T.cyprvini.—In the carp (Cyprinus carpio).
Ten to twenty to thirty » long. The flagella are.
ofunequal length. The anterior flagellum is twice
as long as the posterior. Pathogenic (?).

Addendum.

T. noctuae (Vide p. 345).—Drs. Ed. and Et. Sergent have
confirmed Schaudinn’s results. Culex pipiens were fed on an
owl containing ‘ halteridia.’. A month later they were allowed
to bite on four occasions a non-infected owl. Two days after
the last meal, the owl showed ‘ halteridia’ in its blood.

LITERATURE

Trypanosomes et Trypanosomiasis. Laveran and Mesnil.
1904.

369

Chapter XXVIII

“Tue Leisuman-Donovan Bopiss
Leishmania donovant

1. 'The parasites known by this name were
discovered in the spleen in the disease so common
in India, known as chronic ‘ malarial’ cachexia,
Dam-dam fever, Kala-azar, tropicalspleno-megaly,
etc. They have since been found by splenic
puncture in a few cases of chronic fever from
Africa. .

2. The same parasites also have been found
in the granulation tissue of Tropical ulcer, Delhi
boil, Aleppo button, Scinde sore, Oriental sore,
etc.

CLINICAL CHARACTERS OF THE FEVER

1. Great enlargement of the spleen, which
frequently reaches the umbilicus and even the
pubis. This is the most distinctive character of
the disease.

2. Emaciation.—Usually present in advanced
cases, and in fatal cases it 1s extreme.

3. Irregular pyrexia. — Uninfluenced by
quinine, the accompanying chart indicates its
character in a well-marked case.

4.—Abdominal symptoms.—Dysenteric ulcera-
tion with blood and mucus in thestools inadvanced
cases. Death from peritonitis following perfora-
tion is not uncommon.

5. Ulcevations.—Cancrum oris, noma vulvae,
or other phagedaenic processes are common.
Small ulcers occur about the knees and elbows, or

Z

i

larger ulcers on the leg. The occurrence of these
ulcers should arouse suspicion of a systemic infec-
tion with.the parasite, for in Madras all cases
affected with noma or cancrum oris yielded
parasites on splenic puncture.

6. Skin lesions.—Epecially in advanced.cases,
papular eruptions occur about the thighs and
scrotum.

7, Haemorvhages, epistaxis, petechiae, purpura,
etc,

8. O0edema of the feet.-—Occasionally but not
constantly present.

g. Pigmentation of the skin—Not usually in
excess of the normal.

Technique.—(1) For puncturing the spleen use a hypo-
dermic needle. Boil it previously in normal saline, or in normal
saline containing o°1 per cent. ammonium oxalate. Puncture
between the ribs if the splenic enlargement is not great, other-
wise where it is most prominent. Make a number of dry and
wet films. (2) To examine the granulation tissue of ‘ Tropical
ulcer’ snip off with a curved svissors pieces of tissue from
papules or ulcers. Crush a fragment on a slide by means of
another slide and make thin smears. Imbed other pieces for
section cutting.

Examine films made by splenic puncture and
in stained specimens (RoMANowsky) ; observe the
following characters of the parasite (Pl. VI) :—

1. ‘The presence of small round or oval bodies
containing two chromatin masses—a large and a
small. These are so distinctive that they cannot
be mistaken, and could not possibly be confused
with platelets (Figs. 1-6).

2. Observe that some of these bodies are free
but that the majority occur in leucocytes, and in
fragments of the cytoplasm of splenic cells (matrix
of Ross, zooglea of Manson), which have a close
resemblance to unaltered red cells (Figs. 12-14).

Fig.

Fig
Fig
Fig
Fig
Fig

Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.

Fig.
Fig.

Fig.

Fig.

PLATE VI
THE LEISHMAN-DONOVAN BODIES

1.—A rare form of the parasite without vacuole or
secondary chromatin body.

. 2.—Small form of parasite.

. 3.—Parasite with ‘ tail’ joining the chromatin masses.
. 4.—Pear-shaped form.

. 5.—A large form with very large chromatin mass.

. 6.—Tail-like structures distinct from that of Fig. 3.
7-11.—Dividing forms.

12._-Parasites in apparently altered blood cells.

13.—Parasite in matrix, the remains of protoplasm of a
leucocyte.

14.—Parasites in bodies after treatment with hypotonic
ammonium oxalate solution.

15.—Parasites and pigment in apparently an altered red
cell.

16.—Parasites in a polynuclear leucocyte.
17.—Parasites in a large mononuclear leucocyte.

18.—Endothelial cell containing parasites, from the femoral
vein.

19.—A macrophage containing parasites.
20.—Endothelial cell from testis with parasites.

21.—Swollen endothelial cell from granulation tissue with
parasites.

22.—Necrotic macrophage from spleen with parasites.

23.—A similar cell reduced to a mere pellicle with
parasites.

24.—Section of liver shewing macrophages in the capillaries
with parasites.

25.—Young granulation tissue from an ulcer shewing two
parasites.

371

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‘Ajesouousds teotdo1, jo aseo pooueapy

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Sass aur

oFOl

oGOT

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oLOT

31VOS LIAHNAYHVG

af”

3. Further observe that polynuclear Ieuco-
cytes contain only one or two of these bodies
(Fig. 16);'large mononuclear leucocytes one to
six (Fig. 17); cells of an endothelial type one to
twelve (I'ig. 18); large cells with a hyaline or
finely granular or vacuolated cytoplasm (macro-
phages) up to several hundreds (Fig. 19).

4. The parasites are approximately circular
or oval, 2°5-3°5 » in size, clearly outlined, and
appear to possess a distinct cuticle, as they retain
their shape and are rarely seen distorted in films.

5. The two chromatin masses are character-
istic, the large one staining lightly and the small
one intensely with Romanowsxky. The masses are
usually situate opposite each other in the short
axis of the parasite. The larger chromatin mass
always forms part of the periphery of the parasite.

6. Most of the parasites contain one or two
vacuoles which may displace the cytoplasm of the
parasites to the periphery.

7. Developmental forms. Division com-
mences at the thick end of the parasite, and the
large chromatin masses may be widely separated
before the small chromatin mass has begun to
divide. As many as three to six bodies are formed
in this way, the large nuclei being arranged peri-
pherally, and the smaller centrally (Figs. 7-11).

OccURRENCE IN PERIPHERAL BLoop

In two cases only, approaching a_ fatal
termination, have we found parasites in the peri-
pheral biood. The parasites were of the typical
structure, but were all included in leucocytes.
During a count of five hundred leucocytes we
counted altogether thirty-seven parasites.

373
LeucocyTic CHANGES

Leucopenia is the most marked change. So
much is this so, that it is necessary to take several
large films in order to make accurate leucocytic
counts. Two thousand leucocytes per mm? is a
common value and still smaller numbers are not
uncommon. The relative leucocytic values, how-
ever, do not vary much from the normal.

Case I | Case II} Case HI} Case IV
Large mononuclear =) Ey 86 10°6 16°0
Small mononuclear -| 24’0 20°4 292 70
Transitional -| 18 o'6 — —
Intermediate - - 6 o'o — —
Myelocytes -| 12 o'2 o'6 ; —
Polynuclear -| 60°0 69°8 56°0 73°0
Eosinophil — -| ro O'4 36 4/0

POST-MORTEM CHANGES

N

Spleen.—The appearance of the spleen and the
liver are almost pathognomonic. The spleen re-
tains its shape when removed from the body as if
hardened in situ. It is firm but friable, not tough
like a fibroid spleen.

Livey.—Firm but friable, retaining its shape
like the spleen on removal. On cutting into it

374

an arborescent appearance is noticed, due to the
deposit of a white tissue (macrophages containing
parasites) in the centre of the lobules.

Large intestine.—Extensive multiple ulceration
is almost constantly present. Fungating granu-
lation tissue occurs in association with the ulcers.
Purulent peritonitis, broncho-pneumonia, septic
infarcts,are commonly met with. The other organs
show no particular change to the naked eye.

MICROSCOPICAL CHANGES

1. Make thin smears of spleen pulp, liver,
bone marrow, lung, kidney, testis, lymphatic gland,
suprarenal. Stain with Romanowsky. Parasites
occur In immense numbers in the spleeu, liver, and
bone marrow. ‘To a less extent in the lungs and
testis. ‘They are present also in the suprarenals
and lymphatic glands.

2. Make thin smears from granulation tissue
of ulcers of the skin and intestine. Parasites are
present in both situations; in the skin they are
scanty, in the intestine they may be very numerous.

3. Place small pieces of these tissues on cover
glasses. Harden in alcohol or corrosive sublimate.
Embed in paraffin. Cut sections. Stain by the
modified Romanowsky method (p. 51), or with
haematein. The study of sections is essential for
a clear understanding of the relation of the
parasite to the tissues. Observe the following
conditions :—

Livery.—In the lumen of the capillaries of the lobule, often
applied closely to the capillary wall, occur numerous large
cells crowded with parasites. These cells are sometimes
retracted and globular, but more usually they are characteristic-
ally extended, and suggest the idea that they are actually

B75

moving inside the capillaries. These cells are of doubtful
nature but resemble the macrophages seen in the organs in
malaria, In some cases these cells contain melanin. The
parasites in these cells have the characteristic structure. They
appear to lie in vacuoles, but these are undoubtedly the body
of the parasites (Fig. 24).

Spleen—The parasites occur in similar cells. They are
very conspicuous in sections, Large mononuclear cells con-
taining parasites are more abundant than in the liver. Neither
do the red cells contain parasites, nor do free forms occur.

In contrast to what is seen in blood films made
by spleen or liver puncture where most of the para-
sites are either free or contained in a matrix, in
sections no such relation exists; the parasites lie
in cells. These cells are of various types.

(a) But slightly modified endothelial cells. These have
an oval nucleus and extensive protoplasm showing vacuolization
(Fig, 20). The protoplasm may show buds or protrusions.
These cells contain six to twelve parasites, Identical cells are
seen in the capillaries of the testis and of granulation tissue.

(b) Large round cells with a large nucleus. The protoplasm
has a ground glass appearance and is vacuolated. In the testis
and in granulation tissue these cells are attached at one point
to the capillary wall, the rest of the cell projecting freely.
They also occur in the blood taken post-mortem from the large
veins. They contain twenty or more parasites (Fig. 21).

(c) Very large cells with one or two vesicular nuclei, They
occur in the liver and spleen in immense numbers. ‘They occur
either extended along the capillary wall or in a retracted form,
In the spleen their processes extend among the smaller cells of
the pulp. They contain numerous parasites,

.. (d) Large cells staining more intensely than the last and
sometimes showing signs of necrosis. -The nucleus is pushed to
the side The centre of the cell is occupied by a large vacuo-
lated space, around which are arranged numerous parasites.
The cells, in fact, contain so many parasites that they appear
to be on the point of rupture, and such cells are rarely seen
whole in films unless fixed extremely carefully with osmic acid
vapour. They contain as many as two hundred and fifty bodies
(Figs. 22, 23).

376

Bone marrow.—In films the parasites occur in macrophages
in immense quantity. Tosome extent also in large mononuclear
cells, and a few in polynuclear cells, and in myelocytes. _

Large intestine-—Parasites occur in large numbers in the
granulations, and in the mucous membrane in the early stages
of infiltration. They occur in similar cells to those found in
other situations,

Granulation tissue.—Sections of papules or ulcers of the
skin show a few parasites in what are apparently endothelial
cells of the fine capillaries. In larger capillaries cells may con-
tain three or four parasites, while in small vessels large cells
similar to those in the liver and spleen are found crowded with
parasites. These cells are attached at one point to the capillary
wall,

Lymphatic glands——In those draining the area of a skin
lesion parasites are found, They occur in large cells in the
lymph sinuses and in cells of the reticulum.

We have thus in an infection caused by these
parasites two processes—(1) ‘Tropical ulcer,’ a
local invasion of the nature of a granuloma; (2)a
systemic infection of the nature of a septicaemia,
involving chiefly the visceral endothelia. The
endothelial cells increase in size, and become so
distended with parasites as eventually to undergo
necrosis. Such cells would appear eventually to
rupture, and the parasites set free to be again taken
up by other cells.

Development of the pavasites——Rocers states that he has
observed further development of these bodies in vitro To
splenic blood from a case of Kala-azar he added some citrate

of soda to prevent coagulation, and then incubated the solution
at about 20°C. He then observed :—

(1) Multiplication of the parasites so that they became far
more numerous,

(2) In a few days flagellates made their appearance, These

closely resembled trypanosomes except that they had no undu-
lating membrane,

One of us, S.R.C., confirmed the observation
that flagellates are formed, but these flagellates
are certainly not trypanosomes.

ave

Chapter XXIX
SPIRILLAR. FEVER

It seems highly ‘probable that the ‘spiro-
chaetes’ found in the blood are protozoa and not
bacteria. Firstly, they will not grow on any
medium ; secondly, the character of the tempera-
ture chart is unlike that associated with any
bacterium ; thirdly, the spirillar disease of fowls
is conveyed by ticks; and fourthly, ScHAauDINN’s
remarkable work on Spivochaete ziemanni, if we
can extend it to others, seems to complete the
proof of their protozoan nature.

1. Sptrochaete obermeievi.mIn man occurs as
a fine spiral thread-like body ten to forty m in
length, by, at most, one # broad. The number of
spirals is on an average about ten. Examined
with high powers they appear to be uniform in
structure, or at most present minute unstained
spots. ‘They have been said to possess flagella but
the observation has not been confirmed. ‘The
formation of tangles or rosettes is an uncommon
phenomenon. The presence of spirochaetes in the
blood can be detected with comparatively low
powers by the disturbance among the red cells.
The blood should be examined during the pyretic
attack; they disappear entirely during the apyrexia.
Stain with RoMANowsky. Outside the body the
spirilla, at a temperature of 20°C., will survive as
long as afortnight. The only susceptible animals
are monkeys.

378

2. Spivochaete ansevina.—Highly pathogenic
to geese, death occurring in about a week. The
spirochaete closely resembles the former morpho-
logically. The blood shows immense quantities
of the spirochaetes, and tangles are common.
They appear with the pyretic attack and disappear
as the temperature falls. Besides geese, ducks are .
susceptible to inoculation, and, to some extent,
hens.

Shrrockvele (gallinarum)
Fig. 84

3. Spivochaete (gallinavum).—Affects fowls in
Rio de Janeiro. The spirochaetes are found during
the pyrexia. Tanglesare numerous. Death takes
place in four to five days. The spleen and liver
are much enlarged. The disease is conveyed by
ticks of the genus Argas. When subcutaneous.
inoculations are made spirochaetes do not appear ~
in the blood until the second day. They then
increase until the fifth or sixth day, when they
suddenly disappear.

4. Spivochaete theileyi.—In cattle in Transvaal
and Cameroons. The spirochaetes are actively
motile, twenty to thirty «long. Small forms only
eight “alsooccur. Insome of the infected animals
bacilliform Pivoplasma were present. In others,

|

Pivoplasma, T. theilevi, and spirochaetes. The
spirochaetes are probably the cause of death..

Tal,

Try pancsomes,

Ookinet Tangle

Resling Form

(s=1)

Smell a wochoele.

7

Asy lomera Ped

Dividing ¥ gf tante Forms

é Spirocheele

Fig, 85. Shewing acveropment of the onkinet uf Sp. zicmanni into
trypanosomes and then spirochaetes, in the mosquito (after
SCHAUDINN)

5. Sptrochaete ziemannit.—In the blood of
Athene noctua besides the halteridia described in
the previous chapter, ZEIMANN has described
peculiar large fusiform parasites. Male and female
forms occur and flagellation has been observed.

380

ScHAUDINN has traced their further develop:nent in
Culex pipiens. The general development is similar
to that of T. noctuae, for these also have a trypano-
some stage.

a ay / Rest Sage ~ ;

Ty paretems, gusphong wor miblast eae rig

Fig, 86. Development in the blood of Sp. ziemanni and change
to vesting forms (after SCHAUDINN)

Development in mosquito.

1. Ookinets of three kinds are developed in the stomach.

2. From these are developed trypanosomes which, in this
case, are: minute, and the males so much so that they are very
difficult to see except when agglomerated in rosettes,

3. The backward and forward movement of spirochaetes
results from the fact that two of these trypanosomes, after
division, remain attached.

4. Multiplication goes on in the stomach, and the result-
ing forms are so extraordinarily minute that they are invisible
except in agglomerated masses.

5. These trypanosomes now penetrate the epithelium of
the malpighian tubes, multiply here, and come to rest. They
eventually pass out with the malpighian secretion, reach the
great curvature of the colon, and then follow the same course
as I’. noctuae, ‘The mosquitoes can infect a fresh owl after their
third meal of blood. ,

381

Development in the blood.

1. A large development of the indifferent trypanosome
takes place, causing an acute infection,

2, The sexual forms appear. These parasites infect
leucocytes or rather young Hgb-free erythroblasts, so that
development goes on mainly in the internal organs, ‘The sexual
forms are distinguished from the indifferent forms by their much
greater size They are much larger than the cells they attack,
and, in fact, take up the cell into their protoplasm ‘They are
found in the blood as in T. noctuae, both in the resting stage
and in the motile trypanosome stage When the blood enters
the mosquito’s stomach flagellation takes place, the makro-
gamete is fertilized, and the ookinet results.

Trypanosomes and spirochaetes have been
found together with piroplasmata in cases of red
water in cattle, and it is suggested that possibly
piroplasmata develop in ticks by means of a
trypanosome stage.

6. Tick Fevery.—Ross and Mine have found
spirilla (scanty) in all cases examined by them

(vide p. 349).

382

Chapter XXX
FILARIA

The Filavitdae form one of the families into
which the Nematodes are divided. Other families
in this sub-order are the Ascaridae, Strongylidae,
Anguillulidae, etc. The Filariidae are divided into
several genera, only one of which concerns us
immediately, viz., the Filavia, and first, we shall
consider those species of filaria which have their
embryos in human blood. They are the following :

1. fF. bancrofti, syn, F. nocturna (Manson).
. diurna.

. perstans.

. o22ardt.

demarquat.

loa.

. megalhaesi (adults).

. gigas (Prout).

CONT OUrifp WwW bd
ry ay PS Py Ny hy

The following are the characters of the em-
bryos of each species :— :

F. bancroftiz.—Occurs at night in the peri-
pheral blood, found in the internal organs by day,
especially in the vessels of the lungs. Ce. argyvo-
tarsis and Ce. albipes are efficient hosts.

1. Length about three hundred « fresh (one
hundred and eighty «, stained specimens) by seven
to eleven » wide.

383

2. Enclosed in a sheath considerably longer
than body of embryo.

- 3. If placedrapidly beneath microscope shews
at first active progressive movement (ANNETT and
Dutton), later the anterior tip of the sheath ap-
pears to become attached to the glass, and move-
ments of the embryo, though active, are not pro-
gressive.

4. The embryo shews an anterior abruptly
rounded off end and a posterior, tapering for two-
fifths the length. There is a six-tipped prepuce
and a short very fine fang.

5. Thestained specimen shews(i)an irregular
transverse break about twenty-one per cent. of the
length.

(ii) A V-shaped spot or transverse irregular
break at a distance of about thirty per cent. of the
length from anterior end. Nearly always present.

(iii) An area of varying length with cells
loosely arranged, sixty-three per cent. length.
This is constant and represents the central ag-
gregation of fresh specimens.

(iv) An irregular, sometimes oval spot, often
present, eighty-five per cent. length.

(v) Asmall central bright spot occasionally,
ninety-one per cent. length.

F. diurna.—No ditferences are distinguish-
able between the embryos of fF. diuyna and
F. noctuyna either in the fresh or stained specimen
(AnneTT and Dutron). Dutron and ANNnrETT
have found the embryos taken from the adult
female F. loa to be practically identical with those
of F. diurna, and describe a case in which infec-
tion with F. loa was associated with embryos
present in the blood during the day and not to
the same extent at night.

384

Embryos of F. loa, taken from the female, are
described by AnNETT and DutTTon.

1. Length; 208 #.

2. Possess a sheath.

3. Spots as follows :—

(1) An oval or diamond-shaped spot, twenty-
four per cent. length from anterior end.

(ii) An indistinct lateral area containing |
scattered nuclei, thirty-seven per cent. length.

(iii) A longer portion of worm which stains
badly, sometimes divided into anterior and
posterior portions.

(iv) A small lateral bay, eighty-six per cent.
length.

I’. perstans—Embryos present in peripheral
blood day and night.

1. Length, two hundred « by four » to
five « breadth in fresh, and about ninety » in
- stained preparations.

2. Do not possess a sheath.

3. Movements extremely active, and pro-
gressive movement continues for many hours.
They possess the power of considerable elonga-
tion and shortening.

4. No hooked prepuce, a fang is generally
observed protruded and retracted. The. body
tapers gradually for two-thirds of length, and is
abruptly truncated at the tail and slightly
bulbous.

5. In stained specimens the following spots
are made out :—

(i) A narrow irregular transverse band at
distance of 26:4 per cent. length, nearly always
present.

385

_ (ii) A wider irregular transverse spot, at
thirty-six per cent. length. Occasionally.

(iii) The largest of the spots an irregular
transverse area, sixty-three per cent. length. Not
always present.

(iv) A very inconstant central bright speck
at eighty-three per cent. length.

i

iti

Fig. 87. F. bancrofti, Embryo shewing sheath; F. perstans,
Embryo (sheathless); F. bancrofti (Embryo), prepuce.and
fang (above); F. perstans (Embryo), fang (below)

6. They are readily distinguished from the
embryos of F. oggardi by their blunt tails.

7. ‘The observations of Firxet and Curisty
point to the fact that there is more than one
species of F. perstans.

F. ozzardt :—

1. Length, one hundred and seventy-three
to two hundred and forty » by four to five #.

AI

386

2. ‘They are sharp-tailed and sheathless.

3. They have no periodicity,

F. demarquatt :—

1. Length, two hundred and five # by
five m.

2. Tail sharp and sheathless.

3. Cephalic armature, ill-developed prepuce
and spine.

. A V spot exists fifty-two « from the
head (seen in wet films).

5. There is no periodicity.

6. Ce. argyvotarsis and Ce. albipes are in-
efficient hosts.

F. loa—Two cases of infection with the adult
F. loa have been described in which F. diurna
occurred in the blood. It is possible then that
F. diurna is the embryonic form of F. loa (vide
F. diurna antea). On the other hand, F. diurna
embryos are indistinguishable from those of F.
bancrofti, the adult forms of which are well
known.

F. megalhaesi—Adults only known.

PF. gigas :—

1. Blunt tailed.

2. Has no sheath.

THe CHARACTERS OF THE GENUS FILARIA

They are long slender worms of almost
uniform breadth throughout their length. The
anterior extremity is rounded, and the mouth
often has no lips. The males are distinctly
smaller than the females. They have an incurved
or spiral tail, the latter sometimes having lateral
membranous outgrowths. They usually have

387

four pre-anal and a variable number of post-anal
papillae and spicules, which vary in size and
appearance. In the females the vulva opens in the
neighbourhood of the mouth. The host in which
the filaria reaches full maturity, giving rise to
embryos, is the definitive host, the other host is the
intermediary or secondary host. - Thus F. bancrofti
has for its definitive host, man, for its inter-
mediary host, certain species of Culicidae.

F. vecondita.—Definitive host, dogs. Inter-
mediary host, Ct. canis (dog-flea).

ADULT FILARIAE

1. F. bancvoft?.—The adult male and females
are found together, sometimes in the lymphatics
or in cyst-like dilatations of these. The embryos
gain access to the circulation by the thoracic duct.

2. F. diuvna.—Adult form doubtful. Accord-
ing to AnNETT and Dutton it is F. loa.

3. F. perstans—The adults were found by
DaniELs at the root of the mesentery, behind the
abdominal aorta, and beneath the pericardium.

F. ozzavdi—Adults found by DanieEts
in the sub-peritoneal tissue.

5. F. demarquait.— Adults doubtful. <A
female form has been described differing slightly
from that of F. ozzardz.

6. F.loa.—Adults found in the subcutaneous
areolar tissue, also in the eyelids, and beneath the
conjunctiva.

F. megalhaesit—Adults only known, found
in the left ventricle of the heart by Ficuerra DE
SABOIA.

8. F. gigas Adults unknown.

388

The following are the characters of the re-
spective species :-—

F. bancrofti—Males and females found to-
gether in lymphatics.

g 1. Length, eighty-five to one hundred and
fifty mm.

2. Distinct neck; one-third width of body.

3. Body plain, tapering somewhat abruptly
to neck, and tapering towards tail.

4. Cuticle with striations.

5. Tail ends bluntly, and has a small de-
pression, surrounded by two small lips.

6. Mouth simple, minute, terminal.

7. Ova twenty-five to thirty-eight »« by
fifteen u.

8. Anus ventral opening on summit of a
bilobed papilla.

3 1. .Length, eighty mm.

2. Body cylindrical, tapering to tail. No
neck.

3. Mouth circular, simple, terminal.

4. Cloaca ventral, four pairs pre-anal, four
post-anal, papillae (Manson doubts the presence
of these). Two unequal spicules.

5. Genital tube simple. Ocsophagus thick-
walled. 4

6. ‘Tail vine-tendril like, with one or two
spirals.

F. perstans :

9 1. Length, seventy to eighty mm.

2. Neck longer than F. bancroftt.

3. Body without markings.

4. Tail incurvated. Tip of tail mitred. This
is characteristic of this species.

5- Mouth minute, simple.

389

6. Embryos in utero, blunt tailed, not
sheathed.

g i. Length, forty-five mm.

2. Head end as in female.

3. Two caudal ends much coiled.

4. One spicule and two papillae.

5. Low describes four pairs of pre-anal and
one pair of post-anal, minute papillae.

F. ozzavdi.—Adult.

t. Dimensions much the same as those of
F. bancrofti.

2. Distinguished by the bulbous tail; in F.
bancroftt 1t is not bulbous but circular.

F. loa.—Adult forms travel about in con-
nective tissue.

? 1. Length, thirty to forty mm. (average).
Varies from sixteen to seventy mm. Breadth,
0°57 mm.

2. Noneck. Head cone shaped.

3. Body cylindrical, tapers sharply towards
head and tail.

4. Cuticle with bosses, except over the head.

5. Tail terminates in a short incurved por-
tion, and has two small tubercles at its extremity.

6. Mouth simple.

7. Ova, containing embryos, thirty-five « by
twenty-five u.

8. Anal orifice on low, broad papillae, 0-3
mm. from the tip.

é 1. Length, twenty-five to thirty mm.

2. Uniform thickness except at head and tail.
Cuticle with bosses, but not so numerous
as in the female.
4. Tailnot spirally twisted, merely incurved.
It possesses well-marked lateral alae.

399

5. Three well-marked pre-anal papillae and
two unequal post-anal papillae. ‘Two slender
unequal spicules.

F. megalhaesi—Adult males and females in
left ventricle.

9 1. Length, one hundred and fifty-five mm.
by o-7 mm.

2. Club-shaped oral end.

3. Swollen oesophagus well marked.

4. Mouth simple.

5. Cuticle, fine striations.

é 1. Length, eighty-three mm. by o'4 mmm.

2. Four pairs pre-anal,four post-anal papillae,

and two spicules.

To Examine Bioop For FiLariA EMBRyos

The technique varies somewhat according
to what end the observer has in view.

1. To facilitate detection, it is well, as
Manson advises, to make thick films of blood.
Dry. Then wash out the haemoglobin with water °
or one-third per cent. acetic acid, and stain with
haematin, or gentian violet, or fuchsin.

For the latter stains, a few drops of a satu-
rated alcoholic solution of the dye are added to
half a watch-glassful of water.

Search the slides stained (or fresh) with a
half-inch lens.

2. (For studying the minute structure of
the embryos, the above method is not advisable).
Make a film in the ordinary way. Fix in alcohol
and stain with haematein. The ‘spots’ and
granules of the embryo are most beautifully
shewn.

391

FILARIA IN MAMMALS °

F. immitis—Adults found in right ventricle
of dog, fox, and wolf. Embryos in blood.
Embryos develop in the malpighian tubes of
Anophelines. Afterwards they enter the general
body cavity and pass towards the labium.

Dogs in the tropics commonly harbour this
filaria. The filariae are most numerous at
_ night.

F. vecondita.—A single female adult has been
found in kidney of dog.

Embryos in Blood.-—Embryos develop in Ct.
canis (dog-flea), and P. irvvitans (man and
dog); also in a dog tick. They are found in the
intestine and body cavity. ‘The filaria has, how-
ever, not been transmitted from infected fleas to
healthy dogs.

PF. equina.—Serous cavities, intestines, and
liver of horses, donkeys, and mules.

Embryos in Blood.—-Appear like F. bancroftt,
but smaller.

F. haemorrhagica——Male and female live
together in tissues of horse and donkey. They
form hemispherical tumours the size of a nut be-
neath the skin. These burst and discharge blood.
Fresh tumours appear in from one to two days.

F. iyvitans—Found in ‘summer sores’ of
horses and donkeys.

' F. evansi—Lung and mesentery of camel.
Embryos in blood.

F. lacrymalis and F. palpebralis.—About the
eyes of horses and cattle.

F. oslevi—The adults cause broncho-pneu-
monia in dogs.

392

AVIAN FILARIAE

Filarial embryos are very common in the
blood of birds, and the adult forms are found
in the most diverse positions, notably in the sub-
cutaneous tissues. In some form the embryos
appear to be confined to the lymph.

In the description of Avian filaviae the
following should be noted :—

1. The species of bird concerned.

2. The site of the adult filariae.

3. The description of the adult filariae,
female and male; the use of Cozss’s formula
gives uniformity to descriptions. The measure-
ments are taken with the animal in profile from
the anterior end.

(i) To the base of the oesophagus.

(ii) To the nerve ring.

(11) To the cardiac constriction.

(iv) To the vulva in the female, or to the

middle in male.

(v) To the anus, noting when this is ter-

minal.

At each of these points transverse measure-
ments are taken and noted below the above, so :—

Longitudinal.
Transverse.

The unit of measurement is one-hundreth
part of the length of the worm. .
This formula should be used with caution,
since it rests on the assumption that the pro-
portions of the various parts of the body are
constant in different individuals (SuipLey)

393

Drawings should be made of the head and
tail, and the mouth, anus, and vaginal orifice
carefully described.

The description of the embryo. Where
found, blood or lymph. Presence of a sheath.
Length and breadth of embryo and sheath. The
exact description of spots and the distance of
these from the anterior extremity. The following
spots and markings may be seen :—

(i) A transverse slit, about twenty-five per
cent. length. Sometimes not seen.

(ii) A clear, sometimes lateral, sometimes
transverse spot, about thirty to forty per cent.
length.

(ii1) A long space in which the nuclei are
loosely arranged, often ending anteriorly and pos-
teriorly in clear space. About sixty-five per cent.
length.

(iv) Asmall spot, about seventy-six per cent.
length.

(v) Very small lateral spot or slit, ninety per
cent. length.

DEVELOPMENT OF FILARIA IN THE MosQuitTo

Experiments made so far have been chiefly
with F. nocturna (F. bancroftt).

Both Anopheline and Culicine mosquitoes may
act as the hosts of F. bancrofti. Certain species
of both genera, however, do not act as hosts.
The following have been shewn to act as hosts :—

C. pipiens P. costalis
C. ciliaris Mym. rossit
C. fatigans Mya. sinensis

394

The following have been shewn by BANCROFT
not to allow full development to take place. In
some species partial development occurs, the
larva, however, eventually disappearing :—

Oo

notoscriptus (SKUSE)
C. annulirostris
C. hispidosus

C. vigilax
é
C.

”

9

. nigrothorax (Macouart)
procax (SKUSE)
[A.] musivus ,,

Grass1 and Noe’s experiments shew that
F.immitis is capable of developing in A. claviger.
As regards the re-infection of a healthy dog, the
experiments are somewhat inconclusive, for, in
the dog used, a single immature worm only was
found, about. sixteen days after the period of
‘biting.’ They state, however, that of a batch
of Anophelines dissected before the ‘ biting,’ many of
the labia contained filaria, whereas, of a batch
dissected after the ‘ biting,’ none contained filaria,
the conclusion being that the filaria had escaped
through the labia into the blood of the dog
during the ‘biting.’

Seven stages of development of the embryo
are usually described. The following is a resumé
of the changes undergone in Culex. pipiens :—

Fiyst Stage-—One hour after removal of
blood by mosquito, the sheath is cast and the
embryos exhibit ‘active locomotive movements.
In twelve to eighteen hours many have bored
through the stomach wall, and have reached the

BUS

. muscles. Some die within the stomach. In the
muscles the cuticular striation disappears, move-
ment ceases, and the body becomes thicker.

Second Stage.—For two to three days the
embryo becomes much thicker, and the mouth
begins to be faintly indicated.

Third Stage-—An anus appears in front of
the. tail, and a mouth is very distinct with four
fleshy lips. Cells are seen in the body, and
these form an alimentary and tegumentary layer.
The embryo is now about 073 mm. long.

Fourth Stage.-—Rapid growth takes place,
and the tail becomes relatively smaller.

Fifth Stage.—Lengthening takes place. The
whole worm becomes fibrous and transparent in
appearance. It hascast the cuticle. Some large
cells at the end of the tail form papillae which
-are characteristic of this stage of the larva.
The parasite is now about 1°5 mm. (one-six-
teenth inch). Time, about seventh day.

Sixth and Seventh Stages.--Movements become
more active and, when the filariae have reached
their highest stage of development in the thoracic
muscles, they leave that tissue and travel forward
in the direction of the head of the mosquito
(Low and James). They reach the loose tissue
about the salivary glands and pass into the
“neck. Some are found in the abdomen. Num-
bers of the filaria larvae euter the lower part of
the head, lying beneath the large head ganglia.
Eventually one or more worms pass into the
substance of the labium, where they are readily
found by dissection. The larva at this stage
measures about one-sixteenth inch in length.

396

Tue TRANSMISSION TO Man

According to Dutton, who has very minutely
described the structure of the proboscis, the
worms can only leave the labium at one point,
i.e., by perforating an extremely delicate mem-
brane, which closes in the extreme end of the
labium (see p. 167). If they escape elsewhere,
they must penetrate the dense and hard chitinous
envelope of the labium—a very improbable
occurrence.

DEVELOPMENT OF FILARIA IN THE LOUSE

In the lymph, e.g., of the subcutaneous tissue, of the swift
(Cypselus affinis) occur the embryos of F. cypseli. Dutton has
traced the development of these, up to an almost mature stage,
in a louse, Leiothina sp., which infests these birds. The mode
of escape of the filariae from the louse and infection of a fresh
bird is uncertain.

FILARIASIS AND EOSINOPHILIA

Wurtz and Clerc found in a case of F. loa (no embryos
present in the blood) a marked eosinophilia. A similar increase
has been found by us in a case of ‘ tropical swellings’ of doubt-
ful nature, but resembling somewhat Calabar swellings. The
leucocytic counts were :—

F. loa Bae ie
Large mononuclear - 5 I
Small mononuclear - 13 23
Polynuclear - 29 26
Eosinophil - 53 50

LITERATURE -

Railliet, A. Traité de zoologie médicale et agricole. Paris,
1895. 2nd edition.

Annctt, Dutton, and Elliott. Liverpool School of Tropical
Medicine. Memoir IV, Part II. Filariasis.

APPENDIX

BLOOD-SUCKING FLIES

_ The Diptera or flies are two-winged insects (the posterior
pair of wings are transformed into halteves), and are so dis-
tinguished for example from the Hemiptera or bugs which
generally have four wings. In the Diptera the metamorphosis
is complete, eggs, larva, pupa, insect; in the Hemiptera it
isnot so. The following have blood-sucking habits :—

The Nematocera (vnua, thread; xépas, antenna),

1.—Blepharoceridae.

Wings irridescent, ample, bare, with creases, no ‘discal’
cell on wing (the discal cell lies between the second posterior
cell and the second basal cell). Posterior tibiae with stout
spines, anterior tibiae unarmed, ‘The fourth vein is the one
immediately preceding the large posterior fork, the incomplete
vein not being counted. They resemble midges. The larvae
have suckers, and are found attached to stones in the water.

Genus Curupira (? blood-sucking).

No incomplete vein A long vein between the first and
fourth. Eyes contiguous. C. torrentium, Brazil.
Genus Snowia (? blood-sucking).

Eyes separated by a broad frons. Palpi four-jointed, well
developed.
2.—Culicidae. Mosquitoes or gnats.

3.—Chivonomidae. (Midges).

Head small, often retracted under thorax, which has no
transverse suture, Simple eyes (ocelli) absent or rudimentary.
Antennae up to fifteen segments, densely pectinate in 3, often
simple in 9 andsmaller Legs longand slender Tibiae and
tarsi nearly cylindrical, Costal vein ends at apex of wing.

il

[Genus Chivonomus] not blood-sucking. Larvae are ‘ blood
worms.’ ‘Vers de Vase.’

»
‘ Mp
a
"HT ReonoTnnnaRA ARENT

Wing of Chivanemns plumesus.

Fig. 88. The avvow indicates the point at which the costal vein
ends.

Genus Cevatopogon.

Very minute midges. Wings generally spotted. Head
depressed in front, produced into a short rostrum. Antennae
thirteen segments, the first eight bead-like, the rest elliptical.
Sub-costal vein ends beyond half the length of the wing.
Second long vein ends near the tip, third long vein at the tip.
Femora armed beneath with spines. Larvae mostly non-
aquatic. C. varius. A pest in Scotland.

Genus Tersesthes. New Mexico.

4.—Psychodidae (Moth flies).

Very small. Antennae very hairy. Wings very hairy
(Vide Fig. 12). Larvae of some genera amphibious. The
larvae and pupae resemble those of Ceratopogon. The eggs are
laid in a cluster on the water. .

Genus Phlebotomus. Europe and tropics.

¥

Wing of Phlebotomus sf
Fig. 89

5.—Simulidae (Sandflies, Buffalo-gnats).
Small hump-backed flies. Antennae destitute of hairs.
Wings relatively large. Proboscis short, thick, consisting of

ill

epipharynx and hypopharynx. Antenna eleven segments.
Palpi four segments.

Simple eyes (ocelli) absent; thus distinguished from
Bibionidae. Eyes in ¢ joined together (holoptic). The facets
on the upper part of the eye are the larger.

Egg.—Deposited in a compact layer on stones and grass.
Egg measures 0'40-0'18 mm.

Lavrva—Twelve segments. On under side of anterior
portion is a subconical retractile process crowned with bristles.
Anal extremity bristly, with three short retractile tentacles.

Pupa—Has a respiratory tuft on each side of thorax.
Pupa has spines by which it anchors itself to the cocoon.
These can be found under small stones. Pupal stage about
five days. :

Genus Simulium.

Body small, hump-backed, with a hairy felt-work (to-
mentum). Head small. Palpi four segments, the fourth com-
posed of numerous annuli; largerin @ thanin ¢ Antennae
eleven segments, narrowing to the tip, a little longer than the
head. Wings large. First, second, and third veins dark,
remainder pale. g generally black, @ cinereous. Iyes con-
tiguous in ¢ (holoptic) remote in @ (dichoptic).

Wing of Hodrus sp

Fig. 90. Wing of Lepidoselaga (Hadrus), a Tabanid. a.c.v. =

anterioy cross vein; .c.v. = posterior cross vein

The Brachyceva (8paxus short, xépag antenna) include the
following :—

1.—Tabanidae (Horse-flies or gad (=sting) flies).

Large flies. Antenna three-jointed, not terminating ina
style or arista (the arista (when bristle-like) or style (when
thick) being an appendage of the terminal portion (flagellum)
of the antenna). Third segment of- antenna annulated.

iv

Labium enclosing four stylets in ¢, six in 9. The terminal
joint of the palpi is inflated, and the palpi hang down in front
of the proboscis. Eyes in g holoptic (contiguous), occupying
most of head area. In @ dichoptic (separate). The male fly
does not bite. :

Egg.—Spindle-shaped. They are laid in spherical or flat
groups on the stems of grass, etc.

Larva.—Are aquatic or live in damp earth. They are
carnivorous. ‘They are about an inch long.

Pupa—Aquatic or terrestrial. Over an inch long.

The Tabanidae are divided into two divisions, comprising
more than thirty genera and over thirteen hundred species. It
is only possible to mention here the commonest genera.

1. Hind tibiae with spurs at the tip; ocelli in most cases

present. Pangoninae.
2. Hind tibiae without spurs at the tip; no ocelli (simple
eyes). - - Tabaninae.

t Labium
Anskeathing Stylels
= Poboscrs

Heéd of Ta banus 5 (aftér Dele fond)
Fig. gt

1. Pangoninae
Genus Pangonia.

Face and front in 9 without tubercles or callosities.
Proboscis often long, thin, horizontal. In some, three to
four times length of body, piercing, even when the fly is on
the wing. Third segment of antenna, eight rings. Species
about two hundred and fifty.

Genus Chrysops.

Front with a tubercle or callosity. Three ocelli. Second
segment of antenna almost as long or as long as first. Eyes
golden green. Flightsilent. Wings widely separated ; spotted.
Hind tibiae spurred. Species about a hundred and fifty.

Ch. caecutiens attacks the eyes especially.

Genus Silvius,

_ Second antennal segment very much shorter than first.
Wings without any spots. Third antennal segment five rings
as in Chrysops. Species about twenty-six.

Sub costal Contol

Wing of Haemolopota plavralis.
Fig. 92

2. Tabaninae

The two most numerous genera are Tabanus and
Haematapota.

(a) Front much longer than broad. Frontal tubercle
when present not transverse.

Genus Tabanus.

Proboscis short and thick ; vertical in the female, oblique
in the male. Antennae scarcely longer than head. Third
segment five rings. First ring is characteristically notched in
shape of a crescent with a basal process (Vide Fig. gt). Eyes bare.
Large flies with humming flight. Species about a thousand.

(b) Front as broad as it is long or broader. Frontal
tubercle transverse, about four times as broad as long.

Genus Haematapota.

Terminal segment of antennae not crescentic. Third segment
has four rings. Wings adjacent like the side of aroof. They
have transparent markings. No ocelli. Flight silent. About
fifty species. ;

BI

vl

H. pluvialis. Common in woody lanes in England in the’
summer.

The third group (Cyclorrhapha Schizophora) include the
muscinae, sarcophagidae, and oestridae. ‘The last two groups
are not blood-suckers, but are included here for ‘their
pathological interest ; also only some of the first group are
blood-sucking.

Labaum
a ne i =

Bread of “Slomox -p

Fig. 93
1.—Muscinae== Muscidae (restricted).

Antennae dependent in front of head. They have three
segments. The third segment is flattened and pod-like in
shape, with an arista plumose generally to the tip. Hind body
devoid of stiff bristles.

(a) Genus Musca.
House flies (not blood-suckers).
(b) Genus Calliphora.

Blow flies or blue bottles (not blood-sucking).
(c) Genus Lucilia.

Green bottles (not blood-sucking),.

L. macellavia. The larva of this fly is the American ‘ screw
worm, infesting the nasal fossae and frontal sinuses of man.
(d) Genus Auchmeromyia.

A. luteola, The larva is the blood-sucking floor maggot
of the Congo, etc.

va

3!

vil

BLoop-Suck1nG GENERA

Genus Haematobia.

(a) Palpi shorter than proboscis, partly ensheathing it.
(6) Labellae fleshy, easily visible. (c) Arista plumose dorsally ;
three to four hairs ventrally. (d) Third and fourth long veins
reach the apex of wing. Small mottled flies.

— ee
KEES yt Post Cell

Wing of “Stomoxys.sp-

Fig. 94. Shewing the first posterior cell open at the margin of
the wing.
Genus Lyperosia.
(a) Palpi long and flattened, ensheathing the proboscis.
(b) Arista plumose dorsally. (c) Wing asin Stomoxys. Differs
thus from Glossina. These flies are common on camels. L.
ivvitans is the ‘horn-fly’ termed H. serrata in U.S.A.

Genus Beccarimyia.
(a) Palpi shorter than proboscis. (b) The first post-cell of

the wing is closed before the margin.

Fig. 95. Stomoxys, shewing vesting position
of Wings, x 2. (After AusTEN)
Genus Stomoxys.

(a) Palpi very small, bearing some hairs; not projecting
beyond the epistome. (b) Proboscis is bent at its base like
an elbow joint. (c) Arista plumose dorsally, distally forms a
fine hair. (d) Third and fourth long veins reach the apex.
The fourth is bent beyond the posterior cross vein. Wings
diverge widely. S. calcitrans, the‘ stable’ fly, is common about
farm-yards.

vill

GENUS GLOSSINA

TSEISE FLIES*

Abdomen generally, but not always, has pale
but well-marked dark-brown bands interrupted in
the middle.

t. Dull-coloured, brownish flies, seven to
twelve mm. long (excluding proboscis and
wings).

2. Wings in resting position, closed flat, one
over the other, scissors-like, projecting beyond
the abdomen.

3. Proboscis ensheathed in palpi, projecting
horizontally in front.

4. Base of proboscis suddenly expanded into
a large onion-shaped bulb.

5. Arista feathered on upper side only.

6. Male genitalia (hypopygium) highly cha-
racteristic, oval and tumid, with a vulviform
median groove (anus) running from anterior
margin to beyond the middle. Sex easily distin-
guished by this mark.

7. Wings absolutely characteristic, especially
in the course of the fourth longitudinal vein
(vide Fig. 96, iv). “The anterior transverse vein is
very oblique. The bend in the course of the
fourth vein, before it meets the anterior transverse
vein, is absolutely diagnostic.

* The data of this section are compiled from 4 Monograph of the Tsetse Flies,
by E. E. Austen, and from an article in the British Medical Journal, September 17,
1904, by E. E. Austen.

1X

_ Larva and Pupa.—According to Bruce, tsetse
flies (or at least one species) do not lay eggs, but
extrude a yellow-coloured larva. After a few
hours this changes into a pupa. The pupa is
six mm. long and three mm. broad. It consists
of twelve segments. The twelfth segment is pro-
duced into two large lips, enclosing a pit, the site
of the respiratory stigmata in the larva. At the
anterior end is a longitudinal groove, through
which the fly eventually emerges.

Costa

Fig. 96. Wing Venation of Glossina, and antenna with
Feathered Avista. (After AUSTEN)

CLASSIFICATION OF SPECIES

(1) Hind tarsi entirely dark.

(a) Abdominal segments: sharply defined pale
hind borders. Second segment: a conspicuous
square or oblong pale area in the centre.

1. Gl. tachinoides.—The smallest tsetse fly
(8mm,). ¢é smaller. In the ? the tarsi basally
somewhat pale.

(6) Abdominal segments: hind borders, if
lighter, extremely narrow.. Second segment: pale
area triangular. Larger species than (a).

2. Gl. palpalis—Darkest of all species of
Glossina. Third joint of antenna dusky-brown
to cinereous black.

x

3. Gl. pallicera—Third joint of antenna
orange-buff. Front in both sexes narrower than in
Gl. palpalis. In ¢ thearista is stouter and longer
than that in Gl. palpalis.

(2) Hind tarsi not entirely dark.

Small species, length is rarely ten-and-a-half
mm. (A) Last two joints of front and middle tarsi
have sharply defined dark-brown tips.

Fig. 97. Glossina, shewing scissors position of Wings
when at rest, x 2. | (After AuSTEN)

4. Gl. morsitans.—

1. Smaller than G. longipalpis.

2. Head narrower.

3. Front paler and wider.

4. Eyes in éand 2 distinctly converging
towards vertex.

5. Abdominal bands less deep, pale
hind margins of segments there-
fore deeper.

6. Hypopygium in ¢ larger, paler, some-_
what more oval in outline, and
clothed with fewer hairs.

7- Tip of é abdomen less hairy laterally.

xi

8. Bristles on sixth segment in ¢ stouter
and more conspicuous than in longi-
palpis.

5. Gl. longipalpis. —

(B) Last two joints of front and middle tarsi
entirely pale.

_ 6. Gl. pallidipes.—

Large species. Length, at least ten-and-a-half
mm. (in this respect they contrast markedly with
the other small species).

7. Gl. longipennis.—

1. Thorax with four sharply defined
dark-brown oval spots.

2. Ocellar spot, dark-brown, very con-
spicuous compared with the body.

3. Proboscis shorter than in G. fusca,
and relatively shorter, compared with
the body, than in any other species.
In both sexes the front is broader
than in Gl. fusca.

8. Gl. fusca.—Thorax without spots.
2.—[Sarcophagidae].

Not blood-sucking. Arista feathery at the base, bare
at the tip. Large flies, about 14 millimetres long.

Genus Sarcophaga. Elongated thorax, three black bands,
abdomen spotted. Third segment of antenna three times the
second segment.

S. carnaria, S. magnifica, and S. vuficornis (India), give rise
to terrible forms of myiasis in man and animals.
3.—[Oestridae] (Bot. (=Larva) Flies).

Not blood-sucking. Large flies. Proboscis rudimentary.
Antenna very short. Arista segmented. Flight humming.

(a) Genus Gastrophilus, e.g., G. equi. The white eggs can be
easily seen on the horse’s hair. The larvae are swallowed
and they attach themselves to the mucosa of the stomach.

(b) Genus Hypoderma, e.g., H. lineata. Larvae produce ox
warbles (=tumours) in the ox.

(c) Genus Oestrus, e.g., O. Ovis. Larvae in the respiratory
passages of the sheep.

Xu

(d) Genus Cephalomyia, e.g., C. maculata. In the camel.

(e) Genus Cephenomyia, eg., C. vufibarbis. In red deer.
Scotland, .

(f) Genus Dermatobia, ¢.g., D. cyaniventris. Larva is the
‘ver macaque’ (America), producing myiasis in man and
cattle.

(g) Genus Ochromyia, ¢.g., O. anthvopophaga. Larva is the
‘ver de Cayor’ (Senegal), producing myiasis in man.

Myiasis is common in Africa and in the tropics, but the
larvae. have been identified in but few instances as yet.

The fourth group, the Pupipara (to which Glossina also
belongs, from the point of view of its life history), comprises :
1.—Hippoboscidae (spider flies).

They run rapidly over the body, hiding in hair or feathers.
Head circular. No distinct neck. Clypeus distinct, separated
from the head by a curved suture. Antennae lie in cavities in
its anterior angle. Antennae: one segment with or without a
style (arista). Palpi absent. Abdomen leathery, capable of
much distension in 2. Tarsi: fifth segment longest, with

two or three claws. Empodia (between the claws) distinct.
Wings large, or mere strips, or absent.

sf

~ Sd 1
Fig. 98. Hippobosca Rufipes, left x 2—vight, natural size
(After THEILER)

(a) Genus Hippobosca.

Wings large, obtuse. No ocelli; arista nude; legs long
and extended. Claws bidentate.
A. equini. Runs rapidly over the body; is the [New]
forest fly of England.
A. camelina. Attacks camels in Egypt.
_H. vufipes transmits Trypanosoma theileri (?).

Xlll

(6) Genus Melophagus.

Wings extremely minute. Eyes small. No arista on
antennae. Claws bidentate.

M. ovi is the sheep ‘tick.’ Four millimetres long.

(c) Genus Ornithomyia.
Wings large. Tour millimetres long.
O. aviculavia. Occurs on birds.

(d) Genus Lipoptena, e.g., L. cervi on the red deer.
(e) Genus Stenopteryx, e.g., S. hirundinis of the swallow.

Wing of Hifbobo sea ruft pes.

Fig. 99
2.—Nycteribiidae.
Found on bats. They have no wings.

FLEASt

Fleas, or Siphonaptera, are considered to be aberrant forms
of flies, and hence follow naturally after the division Pupipara,
of the flies.

Lire History

Eggs: About a dozen are laid, in floors, in cracks, etc. ;
sometimes in the hair or fur of animals. They are 0°7 by 04
millimetres (P. ivritans). The eggs hatch in about a week or
more.

Fig. 100. Larva of a Flea x 20 (after RatLuiet)

+ Rabinowitsch has obtained positive results in the transmission of the rat
trypanosomes by fleas. In this case, and in the case of Glossina there is no evidence
to show that the trypanosome undergoes any developmental change ; nor, even in
the case of Glossina, has it actually been shewn that trypanosomes occur in or on
the proboscis during the biting of an uninfected animal.

X1V

Larvae: Are worm-like, whitish, consisting of fourteen
segments. They are about one-and-a-half by one-tenth
millimetres in size. They feed on organic refusé (?) and on
blood. In about eleven days they are full grown.

Nymphs.—Have legs but are stationary. After the lapse
of eleven days the flea emerges (the evolution thus taking
about a month).

ANATOMY

1. The head is small, not distinctly separated from the
body.

os The antennae are placed in fossae behind the eyes.
They consist of two basal segments, and a third of diverse
form irregularly segmented. The maxillary palpi must not
be mistaken for them.

3. The mouth consists of (a) hypopharynx (the central
stylet) serrated above, tubular below; (6) two serrated man-
dibles hollowed on their inner surfaces and forming with (a) a
gutter, along which the blood flows ; (c) a labium single fora
short distance, then bifurcating and forming two labial palps,
which form a sheath for the piercing organs (a) and (6);
(d) two maxillae having the form of expanded plates, each
bearing a four-jointed palpus.

4. The thoracic segments, three in number, are separate.
The metanotum has a ‘ wing-like’ flap or epiphysis especially
well developed in the Sarcopsyllidae.

5. Abdomen consisting of ten segments, overlapping ;
the dorsal and ventral portions not being united, and so
allowing of distension. The terminal segments are highly
specialized forming the genital apparatus. The sexes are
readily distinguished by this means (Vide Fig.)

CapTuRE oF FLEAS

Small animals, such as rats, are best chloroformed in a box
with a glass top. ‘The fleas are subsequently carefully brushed
out. The nests of small animals, such as mice, birds, etc., are
often a rich source of fleas. The nest should be put in a bag
for subsequent examination. For lifting the fleas use the tip
of a stiff feather dipped in spirit. Very careful manipulation
is necessary to avoid damaging bristles or spines.

IDENTIFICATION OF FLEas

The number of described species already approaches two
hundred, Consequently all that we can attempt here is to
indicate some points in the structure of fleas. The external

Epbcbh acs
ih a

Maxilla,
i Max Palpe

Lakeal pol -

mai ie
Hef

Fig. 1or.

I.—+t1-t3, thoracic segments ; A1, A3 first and second abdominal segments.
IIl.—Head of a flea shewing antenna (three segments) lying in antennal
groove.

II].—Hind segments of male flea shewing claspers. d = dorsal segment,
v = ventral segment ; 1.p, immovable process; m.p, moveable process ;
m = manubrium (diagrammatic).

IV.—Hind segment of female flea (diagvammatic).

XV1

copulatory organs of the male are of great importance in class
ification. A complete description of a flea, however, involves
a description of practically every bristle or series of bristles on
the body. The following classification is taken from Baker :—

{ ‘Thoracic segments short and narrow. Labial palpi with-
out pseudo-joints, third antennal segment without clearly
separated pseudo-joints. 2.

\

Thoracic segments not short and narrow, labial palpi
with three or more pseudo-joints. Third antennal segment
with nine or more fairly distinct pseudo-joints. Maxillary
palpi almost always shorter than anterior coxa. Epiphyses
(flaps) of meso- and metathorax extending over-only one
\abdominal segment. 3-

Maxillae without, or with, very short and broad pro-
jecting laminae. Maxillary palpi extending beyond anterior
coxae. Head produced into a sharp point in front in 9 and
é Metathoracic epiphysis extending over two to three
abdominal segments. Sarcopsyllidae.

i
{
Maxillae with a narrow long curved lamina. Maxillary.
palpi as long as anterior coxae. Head evenly round. Meta-
thoracic epiphysis extends to one abdominal segment.
Hectopsyllidae.

j Labial palpi largely developed with eleven to thirteen
pseudo-joints, abdomen of gravid 2 much swollen, ante-
pygidial bristles absent Vermipsyllidae.

3.

Labial palpi three to five pseudo-joints; antepygidial
bristles present. 4.

i Fore tibiae: posterior border has a few black teeth.
Fifth tarsal segment greatly enlarged, those on forelegs as
long as rest of tarsus; claws of all legs nearly as long as
fifth joint. Fore coxae with but few long spines. Body of
gravid female swollen. -~ Megapsyllidae.

Fore tibiae: post border hasslender spines. Fifth tarsal
segment not greatly enlarged, never as long as rest of tarsus.
Fore coxae with several or numerous rows of ‘bristles.
Gravid female not swollen so as to expose membrane
between sclerites, - Pulicidae,

XV11
Sarcopsillidae ;—

1. Genus Sarcopsylla.

Maxilla without a projecting lamina, angle of head pro-
duced, metathoracjc ‘epiphysis extending to three abdominal
segments. Fifth tarsal segment without lateral heavy spines
and legs almost spineless, e.g., S. penetvans, the Chigoe or Jigger,
etc. 4

2. Genus Xestopsylla.

Maxilla with a short projecting lamina, angle of head not
produced, metathoracic epiphysis of medium size extending to
hardly two abdominal segments. [Fifth tarsal joint with the
usual spines. Legs with spines, e g., X. gallinacea, infests hens
US.A,.

Pulicidae.

1. Maxillae long triangular, acute at apex. Abdominal
segments with no ctenidia (combs). Post tibial spines in pairs
not ina very close set row. Last tarsal segment on all the
tarsi with a marginal row of four stout spines. Eyes large.
@ one antepygidial bristle on each side.

(a) Head without ctenidia. Genus Pulex, e.g. Pulex
cheopis, associated with the transmission of plague.
It is distinct from P. pallidus.

Fig. 102. P. ivvitans, x 30. (After Raiuet)

(b) Head with ctenidia. Genus Ctenocephalus, e.g.,
Ctenocephalus canis on dogs.

XV

Fleas for identification should be sent to the Hon. N. Charles
Rothschild, Tring Park, Tring, Herts.

Fig. 103. Ctenocephalus canis, x 30. (After RarLuizr)

LITERATURE
Taschenberg. Die Flohe.

C.F. Baker. A revision of American Siphonaptera or fleas,
together with a complete list and bibliography of the group.
Proceedings of the United States National Museum, Vol. XXVIII,
pp. 365-469, with Plates X-XXVI (No. 1361).

STAINS, NUMERICAL DATA, ETC.

FrIxInG AND HARDENING SOLUTIONS

Alcohol is a fixative and dehydrating medium, and for
ordinary work is the most convenient. The tissues, in small
pieces, may be placed directly in methylated spirit (ninety per
cent. alcohol), or absolute alcohol (ninety-eight per ‘cent.
alcohol). Change the alcohol a few times. Or pass through
fifty, seventy-five, ninety-five, one hundred per cent. alcohols
leaving a few hoursineach. After hardening, if the specimens
are not to be imbedded immediately, transfer to alcohol of

XIX

about eighty per cent. for preserving. After the use of other
fixatives, specimens should be washed and transferred to eighty
per cent. of alcohol for preservation.

__ Rectified spirit of the British Pharmacopaeia, is equal to
eighty-four per cent. alcohol.

_ Methylated spirit, containing wood naptha, is equal to
ninety per cent. alcohol.

Ordinary methylated spirit contains mineral naphtha, and
should not be used.

Absolute alcohol is equal to ninety-eight per cent. alcohol.
For practical purposes the dilution of alcohols is sufficiently
- accurately made by means of the diluting formula (p. xvii).

Zenker’s Fluid :—

Potassium Bichromate 2°5 grammes
Sodium Sulphate I‘O grammes
Corrosive Sublimate 50 grammes
Water 100°0 grammes

Add glacial acetic acid to this stock solution, in the pro-
portion of five grammes to one hundred c.c., before use, Fixa-
tion is complete in one to twenty-four hours. Wash thoroughly
in alcohol to which enough iodine has been added to give a
dark-brown solution. Or, if alcohol is undesirable, use tinc-
ture of iodine, two parts, potassium iodide, one part, glycerine,
fifty parts, water, fifty parts. Ienew until no further decoloura-

‘tion takes place.

Haematin or eosin and methylene blue give good results
for malarial tissues.

Orth’s Fluid—This is Muller’s fluid, to which formalin
(i.e., formaldehyde forty per cent. solution) is added in the pro-
portion of ten c.c. to one hundred c.c. of Muller before use.

Fixation of small pieces takes place in two to three hours,
if kept warm. Wash thoroughly. Pass through alcohol.

Muller’s Fluid—Potassitim Bichromate, 24 parts.
Sodium Sulphate I part.
Water 100 parts.

Add a little camphor or naphthalin to prevent the growth
of moulds. Change the fluid after twenty-four hours, and then
every few days for the first week. Tissues are ready in a fort-

\ night or three weeks. They may be left much longer. Fix

XX

in the dark. Wash thoroughly in water till colourless.
Transfer to alcohol, seventy to eighty per cent., for preservation.

Flemming’s Solution—Chromic acid, 1 per cent., 15 vols.
Osmic acid, 2 percent., 4 vols.
Glacial acetic acid I vol.

Mix in the above proportions before use. Use. very small
pieces. Fixation is complete,in about twenty-four hours.
Blackening due to the osmic may be removed by hydrogen
peroxide. Blood films may be fixed in this solution.

Tissues thus fixed may be preserved in equal parts of
alcohol and glvcerine.

Formalin (forty per cent. solution of formaldehyde).—Use

two to five per cent. solution in water. Small pieces are fixed
in twelve to twenty-four hours. They may be left in solution
or transferred to alcohol.
' Corrosive Sublimate.—Best used as a concentrated alcoholic
solution (or aqueous may be used). Fixation takes place in a
few hours. Wash thoroughly in water and transfer to iodine .
solution (vide Zenker’s fluid) till iodine no longer decolourized.

_ The concentrated alcoholic solution is a most rapid fixing
and hardening reagent, and sections can be cut in a very short
time, if small pieces are used. :

Decalcifying Solution—Tissues require fixing before and
after these solutions—

(i) Phloroglucin, one gramme, nitric acid, ten c.c., water,
one hundred c.c. ; or

(ii) One to five per cent. solution of nitric acid in water
or alcohol. Change the fluid daily. Decalcification takes
place in two to three days. .

(iii) Picric acid, a saturated solution (= about 0°75 per
cent.) containing crystals. Decalcification may take weeks or
months. Wash in alcohol.

Eau de Javelle (dissociating and decolourizing solution), —
Add to a concentrated aqueous solution of chloride of lime a
solution of potassium oxalate as long as a precipitate is
formed. Filter and dilute if necessary. This may be used for
softening the chitinous skeleton of mosquitoes and for decolour-
izing Madura fungus, etc. ,

For Fixinc PararFin SECTIONS TO THE SLIDE

I. Celloidin, one part, oil of cloves, two parts; or
2. Thin solution of white shellac in creosote ; or

XX1

3. Thoroughly mix equal parts of white of egg and
glycerine ; filter. This is one of the simplest and best means ;
or

4. Filtered white of egg, 50 c.c.

Glycerine 50 c.c.

Sodium salicylate I gramme.
Shake well and filter (this takes about a week). The solution
keeps well for six months or more; or

5. Simply use fresh white of egg; smear thinly over; dry.

MountTING MEDIA, ETC.

1. Favvant’s Solution. —

i. Take equal parts of glycerine and a saturated solution
of arsenious acid. Add powdered gum arabic till the solution
is saturated ; or

ii. Pure gum arabic, 40 grammes.

Water 40 C.C.
Glycerine 20 C.c.
Carbolic acid I gramme.
[or Thymol o'3 grammes. |

Powder the gum and dissolve in about one hundred and fifty
c.c. of water by boiling, add the carbolic acid, dissolved in a
a little water, filter through a hot filter, changing when
clogged, evaporate until it is about eighty c.c., then add the
glycerine.

2. Acetate of Potassium.—Saturated solution. Cement
the cover-glass with gold size, dammar lac. This is used for
osmic preparations, and for glycogen stained with iodine, etc.

Glycerine Jelly —Glycerine 70 Cc,
Water 60 c.c.
Gelatine 10 grammes.
Thymol o'5 grammes.

or one gramme of phenol to each one hundred grammes of the

mixture. .

Dissolve the gelatine at forty degrees in a water bath ; add
the glycerine (warmed); powder the thymo| and mix with a
littlé water, and stir in; add the beaten-up white of an egg ;
stir continuously ; warm to eighty-five degrees ; filter through
a hot filter.

To Mount DELICATE OBJECTS

Place in ten per cent. glycerine, allow this to concentrate
in the air, and then transfer to the jelly.

C1

XXL

To mount in Jelly—Remove as much water as possible ;
place the slide, coverglass, and jelly in the incubator (if
necessary). Before cementing see that the layer of jelly is not
too thick. If too thick press some out and scrapeaway; then
cool.

Dammayr Lac.—Dissolve in equal parts of benzene and oil
of turpentine. It does not render preparations as translucent
as Canada balsam.

Fixinc BLoop

As we have already stated, for practical purposes alcohol
is absolutely satisfactory. The following solutions have been
used, and may prove useful occasionally :—

1. Osmic acid——Osmic acid, 1:0; sodium chloride, o'6 ;
distilled water, 1oo‘o.

A neat and practical method of using this is to moisten a
camel’s hair brush with the solution, then to touch the blood
drop, and to immediately spread the blood out on the slide
with the brush. Wash the brush, after use, in alcohol
(Kornilowitch).

2. Chlovofovm.—Instead of heat in staining with Ehrlich’s
triacid. Fix for five minutes in chloroform (neutral to litmus
paper). Stain for five minutes or more after fixing (Josué).

3. Strong Flemming. Especially for nuclear structures
of parasites. :

4. Heat.—Heat up from one hundred to one hundred and
ten degrees C. in a hot oven, and then, when this temperature
is reached, allow to cool again in the oven. os

5. Osmic acid, two per cent., glacial acetic acid, equal
parts. Expose to the vapour. For delicate work indispensable.

STAINING SOLUTIONS FOR BLoop, ETC.

1. Romanowsky stain (p. 10).

2. Haematin (p. 50).—If the solution has become reddish
on keeping neutralize with a little ammonia.

3. Eosin and Methylene Blue (consecutively).—(a) Stain
for one to five minutes in a one-half per cent. solution of eosin
in sixty per cent. alcohol, wash, dry with blotting paper, and
stain (b) in a one per cent. solution of methylene blue for
thirty seconds to one minute.

This is a useful and simple method for studying the acido-
phil and basophil reactions of granules. ,

XX11

_ 4. Safranin.—Make a saturated solution in two per cent.
anilin water. Heat to sixty per cent. C. Filter hot. Stain
sections over night. Differentiate very carefully with one per
oe hydrochloric alcohol. Safranin is an excellent nuclear
stain,
5. Ehrlich’s Triacid. Stain for five minutes.

6. Ehrlich’s Haematoxylin-Eosin—Haematoxylin, five

grammes ; acetic acid, twenty grammes; alcohol, one hundred
grammes ; glycerine, one hundred grammes ; water, one
hundred grammes. Allow this to ripen for a month in the
sun, then add eosin to the extent of one per cent. ; stain for
twenty-four hours. ‘The solution is best got ready-made.

SOLUBILITY oF STAINS

10 c.c. of saturated alcoholic methylene blue contains
o'068 grammes. of the stain.

to c.c. of saturated aqueous methylene blue contains
o°664 grammes of the stain.

1o.c.c. of saturated alcoholic gentian violet contains
0°442. grammes of the stain,

10 c.c, of saturated aqueous gentian violet contains 0°175
grammes of the stain.

to c.c. of saturated alcoholic fuchsin (basic) contains
0'292 grammies of the stain.

“ro c.c. of saturated aqueous fuchsin (basic) contains 0'066
grammes of the stain. (Hewlett).

Loffler’s Methylene Blue—30 c.c. saturated alcoholic
methylene blue.

100 ¢.c. of o'r per cent. caustic potash.

Ordinary Methylene Blue—A saturated alcoholic solution
is used as the stock, and a few drops added to a watch-glass-
ful of water, according to strength required ; or ten per cent.
solution is a convenient strength.

Borax Methylene Blue (Sahli’s)—Saturated aqueous solu-
tion of methylene blue, twenty-four parts ; borax five per cent.
solution, sixteen parts ; water, forty parts.

The times necessary for staining are best judged by the
appearance of the films or tissues.

TissuzE STAINS

1. Haematein (p. 50).—Stain for about five minutes,
according to the ripeness of the solution. Sections do not
readily overstain. Decolourize, if necessary, with one per cent.

XX1V

alum solution. Counterstain, if required, with a weak, watery
solution of eosin, one-half to one per cent. ; or the sections
may be stained first with a strong eosin solution, five to one
hundred per cent., for five to twenty minutes. Combination.
of eosin and methylene blue can be used in a similar way.

‘2, Alum Carmine.—Carmine, two grammes ; alum, five
grammes ; water, one hundred c.c. Boil together for one
hour. Filter. Does not overstain.

3. Ehrlich Biondi—Saturated aqueous solution of acid
fuchsin, four parts ; orange g., seven parts ; methylene green,
eight parts. Dilute fifty to one hundred times before using.
Stain for twenty-four hours. Wash in alcohol. The sections
may, before staining, be treated with acetic acid (two parts in
one thousand of water) for a few hours.

Iron Reaction (HAEMOSIDERIN) IN MALARIAL TISSUES

1. Fix in alcohol.

2. Two per cent. aqueous solution of potassium ferro-
cyanide, five to twenty minutes.

3. Acid alcohol (HCI, one part, seventy per cent. alcohol,
one hundred parts), five to ten minutes.

4. Wash in water.

5. Counterstain with alum carmine.

STAINING OF AMOEBA Colt IN TISSUES
(MaLLory aND WRIGHT)

1. Fix in alcohol.
_ 2. Saturated aqueous solutions of thionin, three to five
minutes.
_ 3. Two per cent. solution of oxalic acid, one-half to one
minute. :
4. Wash in water.
5. Dehydrate in alcohol.
6. Clean in oleum origanum cretici.
7. Wash in xyol.
8. Balsam.
Nuclei of the amoebae are brownish red, the nuclei of the
mastzellen ’ are blue.

WEIGHTS AND MEASURES, ETC.

1. Conversion from one temrerature scale to another—
C _ R _ F—32

5 4 9

XXKV

Thus to convert 100° F, to centigrade—
Too; 37 c

= 37.7

9 5
2. Formula for dilution of Solutions. ~The number of parts
required to dilute from one part of a solution of strength x per

cent. to another strength y per cent. is~ —r1,
y

Thus, to dilute a solution from 20 per cent. to 5 per cent., add

to each volume of solution 72 — 1 = 3 of the diluting fluid.
3. Approximate Values:—

1 cubic centimetre = 17 minim. I minim: = ‘059 c.c.

100 os = 3 ounces, 4 drachms, 20 minims.

1 drachm = 3°55 c.c.

1000 cubic centimetres = 1°76 pints. 1 fluid ounce =
28'4 c.c,

4 litres = 7 pints. 1 pint = 568 cc.

I gramme = 153ths grains (avoirdupois). 1 grain
(avoirdupois) = ‘0648 grammes.

1 kilogramme = 2 pounds, 3 ounces, 1198 grains (avoir-
dupois). 1 drachm = 1°77 grammes.

5 kilogrammes = 11 pounds. 1 oz. = 28°35 grammes.

I grain apothecaries’ = ‘0648 grammes.

1 drachm apothecaries’ 3 = 3°88 grammes.

I ounce apothecaries’ % = 31°I grammes.

15°4 grains apothecaries’ = I gramme.
4. 1 millimetre = ‘039 (#s) inches. I inch = 2°5
centimetres.
1 centimetre = °39 ($) inches. advo inch = ‘0253 milli-
metres.

t metre = 3°28 feet. xév inch = ¢ millimetre.
reso millimetre (u) = ‘oooo4 inches.
5M == sooo inch, iM = rovsoo inches.
5. @ = 314159.
Circumference of a circle = 2 Ty = 7d.
Area of circle = rr’.

LIST OF APPARATUS

s. d.
1. A Beck microscope, including oil immersion, ¢¢ 13 10. 0
Glass slides,f 3 x 1, ground edges, per gross o 3 6

+ Slides and coverglasses should be kept in spirit or alcohol to prevent their
becoming opaque, as only too frequently occurs in the tropics.

oS

H

II.

oo

BO) GO SS One

ry

XXV1

Cover glasses, No. 1, $-in., $-0z.

Straight surgical needles, for making blood
films and dissecting, 4 doz. (Weiss & 0s a
Oxford Street)

Stoppered jars (4), 13 x 74 centimetres

Porcelain dishes, square, flat, 1 doz.

Specimen tubes, flat- bottomed, corked, 3 in.
x 2 in., 50 -

Slide box to hold 25, sliding, cardboard

Measures, 100 c.c. and Io c.c,

Drop- bottle for xylol (a toothpick inserted
into the cork of a specimen tube serves
the purpose) -

Cedarwood oil bottle (a pin in the cork of a
specimen tube makes a convenient
dropper for the oil) —-

A ‘Primus’ paraffin burner, for boiling, etc.

t

STAINS, ETC.

Romanowsky.—Methylene blue, pure medical,
10 grammes, or in ‘ soloids ’
Eosin, B.A., 10 grammes, or in ‘ soloids’
Sodium carbonate, pure, in ‘ soloids’

Leishmann’s Stain—(a) In ‘soloids’ (Bur-
roughs, Wellcome & Co.) = ‘015 gramme.
Dissolve in 10 c.c. of methyl alcohol (or
methylated spirit).

Haematin, 5 grammes,
Alum, 1 oz.

Absolute alcohol, 1 lb.

Xylol, 1 Lb. -

Pasatin wax, melting points, 50°C. and 60°C.

1 lb. 2s. 6d. x Ib. 3s,

ADDITIONAL APPARATUS

A mechanical stage, fitting on to the stage
(not the column) of the microscope -
Browning’s pocket spectroscope, indispensable
for urine work in blackwater fever, etc.
Haemacytometer. Thoma-Zeiss.
Haemoglobinometer. Gower’s

oN

°

oo00

HHH

2

nf Of

bd

Dn WOnD

°

H

TOP EYOD

XXVil

FOR MOSQUITO COLLECTION

£ s.d

A lens (the lens of an eye-piece of the micro-
scope serves as well). 015 Oo
Silver pins, No. 20, } oz. 0 2 0
Entomological pins, 14 in. long, 1 oz. o o 8
Cardboard (fine Bristol board), 4 sheets 0 0 3
Specimen tubes, flat bottom, each about Oo Oo!

Pill boxes.

A dissecting board, 12 x 3 in., half covered
with black, half with white paper (made
by self).

Supplied by Messrs. C. Baker & Co., London.

LITERATURE
Encyklopidie dev Mikvoskopischen Technik. 2 vols. by

Ehrlich and others.
Methods and Theory of Physiological Histology. Mann.

Merck’s Reagentien Vergeichniss. A very useful com-
pendiurn, giving the composition of all the best known
stains and reagents.

XX1X

INDEX
A A. crucians
Abdomen of larvae . 234 eiseni .
Abdomen of mosquitoes . 172 franciscanus
Acartomyia 180 gigas .
Achromaticus vesperuginis 323 immaculatus
Aedeomyina : . 176 lindesayti
Aedeomyia ; : . 183 maculipennis
Aedes ; ; : . 183 nigvipes
Aedimorphus. 183 pseudo punctipennis
Aestivation of Anophelina 219 punctipennis
Africa, Anophelina of 210 punctipennis and
African coast fever . » 335 malaria
Africa, quartan parasite in 270 stigmaticus
Albuminuria in malaria . 285 walkert
Alcohol as fixative . XVIII wellcomei
Alcohol to dehydrate . 47 | Anophelinae
Aldrichia, genus . 188, 208 » aestivation of
Al. error . : . 208 » breeding-places of
Aleppo button : . 369 » Characters of
Alum carmine . XXIV » Classification of
Amblyomma. ‘ 348 », dispersal of
America, Anophelina of . 209 5, domestic
Amoeba Coli, staining of XXIV » eggs.
Ammonia as larvicide . 85 » fecundation of
Amphigony . : 57 » food of
Anal papillae . . 84 » flight of
Anophelines, attitude of . 63 3 geographical dis-
i genus . 187, 192 tribution
5 larva 75,77 Seq. » hibernation of
Ss nympha 89 » larva of :
Ms species of 192 length of life of
i the male . 219 Anoph elinae of Africa
5) unspotted 55 America
wings 63 is Australia
A. aitkentt , ; . 194 5 Europe
algeriensis . 193 5 India . |
annulipalpis 194 % Malaysia
bifurcatus . «~~. :193 re Palestine

. 192

193

» 193

193
194

. 193

192

» 193

193

- 193
» 259

194

. 193
. 208

. 176
. 219

241
62

186

213

214
68

220

215
216

. 209

212

. 229
. 218

. 210

209

1 BET
. 209
, BIO

210

. 209

XXX

Anophelinae, ova of . . 221
45 palpi of « 62
ie post-scutellum

of . . 169

Anophelinae, relation to col-

our of. . 217
s salivary glands
of . 114
3 scales of . 187
» seasonal prev-
alence of . 211

4 species, differ-
entiation of 190
4 that transmit

malaria . 259
3 wing of . 170
wild 214
Antennae, of larva . 83, 230
53 of mosquitoes. 165
Aponomma i 347
Apparatus, list of XVII
Argas. . . 348
Argasidae - 337, 343
Armigeres ‘ . 178
Arribalzagia, genus . 187, 201
<4 maculipes . 202
Artificial appearances
Auchmeromyia VI
Australia, Anophelina of . arr
Avian filariae F . 392
B
Basophil staining . 26
Bats, parasites in . . 322
Beccarimyia VII
Bile pigments in urine 290
Bilirubin in urine 291

Birds, feeding experiments
on. ‘ 103

» filaviae in . 392
» Parasites in 316
_» to feed mosquitoes

On « ; . 103

Black spores . 319
Blackwater fever and quin-
ine . 305
45 blood in. at, 310
Blackwater fever, kidneys
in . 314
c leucocytes in 312
“ Methaemo- ;
globin in 287
Pr parasites in . 312
5 pigment in . 312
3 post-mortem
changes . 314
‘5 urine in . 313,
55 urobilinuria
in. . 313
Blepharoceridae =»
Blood, anaemic ‘ . 5
» dust. . 20
» films, dry, to pre-
pare . 2 seq.
» films,to examine. 23

» films, to fix . 8 seq.
» films, to label 6 seq.
» films, to stain . 9
» films, wet, to pre- .-
pare . 5
» fixatives for . XXII

» guaiacum test for. 287
» Heller’s testfor . 287
, in blackwater fever
21, 310
» isotonic point of . 283
» normalconstituents 17
»» Spectroscopic test
for. F 287
» -Sucking flies I
» sucking maggot VI
» to examine for Fil-

aria . 390
,, to examine for Try-
panosomes . . 367

Boophilus . 346

XXXi

Borax methylene blue XXIII
Bot-flies : . XI
Brachiomyia ; 182
Brachyceva . ‘ . Ul
Breeding out of mosquitoes 158

Breeding-places in dry
season . 219
» Of Anophelinae 241
Buffalo gnats ; . tI

C

Calliphora ‘ é VI
Cannibal larvae . 84
Carmine, alum XXIV
Cecidomyidae . ‘ . 59
Cellia, genus 188, 207
» Species . . 207
5, albipes . 207
» argyvotarsis . 207
» Oigotit . . 207
» ochii . 207
» pharoensis 207
» pulcherrima . . 207
squamosa 207

Centr ifugalization ofblood
in trypanosomiasis 367

Cephalomyia . XI
Cephenomyia . XII
Ceratopogon teow
Chigger flea XVII
Chivonomidae . : |
Chivonomus : . 58
$5 larva of . 75
is nymph of . 92
Chloroform as fixative XXII
Christya . . 208
Ch. implexa 208
Chronic malaria, changes
in : 308
Chrysops . Vv
Classification of Anophe-
linae. 186
Cleaning of pipettes 274
Clypeal hairs of larvae 232

Clypeus of mosquitoes . 164
Collection of mosquitoes 157
Colour, relation to Anophe-

linae Pag)

Copula F 57

Corethva . 185

Corethra, larva of 74, 86

5 nympha of amaycr)

Covethrina ‘ . 176
Corrosive sublimate as

fixative . XX
Counting by microscope,

* fields . 272

. leucocytes ~ 272

. platelets . 275

in red cells . BYI

Crenations 15, 28

Crescent, male and female 29

Cross-veins of M. Rossii 191
Ctenocephalus . XVI
Culex . 179
» eggs ‘ . 68
» larva of 75,77
» nympha 89
» pipiens and

T. noctuae 354
post-scutellum of . 169
Culicidae, attitude of . 64
3 larvae 81
+3 palpi of . . 63

= salivary glands
of . : « LEY
6 spotted wings . 63
Culicina . 176, 177
Curupira I

Cycle of a parasite, deter-
mination of . 294

Cycle of malignant tertian
parasite ; 298

Cyclolepidopteron, genus .
187, 199
» grvabhamii 199
», mediopunctatus 199
y> Species . 199

XXX

D
Dactylosoma splendens . 326
Dammar lac XXII
Danielsia . 181

Dare’ shaemoglobinometer 275
Decalcifying solutions . XX

Deinocerites 182
Delhi boil 369
Dendromyia 184
Dermacentor . . 340

3 veticulatus . 335
Dermatobia XII

Désvoidea 178
Dilution, formula for xxv
Diplococcus Pneumoniae and

_ malaria . 308

Diptera . ; . . 58
is Cambridge Nat.

History . « OF
Dissection of salivary

glands . 114

a mosquitoes 104

Distribution of Anophelinae 209
Diverticula of oesophagus 131

ies enzymes in . 108
Pe gas in . 108
Dixa, larva of . 75, 85
Domestic Anophelinae . 214

Donovan-Leishman bodies 369
Dourine, trypanosome of 362

Drepanidium vanarum . 325
Dry season, breeding-
places in 219
Dutton’s membrane and
Filavia . . 167, 396
E
Eau de Javelle XX
Eggs, Anophelinae . 69 seq
» Classification by . 192
» Culex, . . 68
» examinationof . 69
» hibernation of 213

Eggs, Mansonia ; o 93
» LPsovophova . - 73

» rafts 2 x WF

» stage, duration of 70,221

» stegomyia 68, 72

,» Taeniorhynchus 69, 73
to imago . 240

Ehrlich Biondi stain XXIV
Embedding apparatus . 46
Endemic areas of a country 269

3 index 254

ss index, determina-
tion of . 264.

Endemic malaria, to investi-
gate . 260
Endemicity, malarial. 254

Eosin, B.A... é
55 cP eee 6 aii
Eosinophil leucocyte . 19
Eosinophilia and filariasis 396
3 », tropical

swellings 396
Ephemera, larva of . ~ 75
Epipharynx of mosquito. 166
Evetmapodites . . 181
Europe, Anophelinae of . 209
Europeans, how infected ~
with malaria 267

European malaria, to in- .
vestigate 266

F

Farrant’s solution . XX
Fat-body of mosquito 140
Fat cells . III

Fecundation of Anophelinae220

Feeding experiments on
birds

102
Female and male mos-
quitoes, proportion
between . 220
Female crescent 5 Or
Ficalbia 183

XXX11

Filaria. : . . 382
» and Dutton’s mem-
brane . 396
» Characters of genus 386
» embryo, develop-
ment of 393
5 embryo, escape of. 167
” » im mosquito 124
» €xamination of
blood for . 390
» in birds . 392
» in louse . 396
» 1n mosquitoes . 393
» nocturna, host of . 207
» bancrofti . 388, 393
» demarquait 386
» atuyna . . 383
» equina . . 391
» «6-evansi . 391
» gigas : 386
» haemorrhagica . 391
» immitis » 391
» tvvitans 3g1
5 lacrymalis . 391
» loa . . 386, 389
» ‘Itmegalhaesi .. 386, 390
» oslert : . 391
» oRzardt . 385, 389
» palpebralis . 391
» perstans 384, 388
vecondita . 387, 391
Filariae, adults of . 387
Filaviasis and
Eosinophilia 396
Filariasis and fleas . . 391
Finlaya . 181
Fish, trypanosomes | in . 350
5 parasitesin . 330

Fixative, chloroform as =e
Fixatives

- for blood . XXII

for sections XVIII

Flagella ; . 54
Flagellata of mosquitoes . 124

Flagellate bodies, tostain 31

Flagellation . 30
A of haltevidium 320
Fleas XIII
» anatomy of XIV

» and Filariasis . 391

» and trypanosome XIII
Flea, life history of . XIII
Flemming’s solution . XX

Flies, blood-sucking ey lal

Flight of Anophelinae . 216
Floats of ova . , 222
Food of Anophelinae . 215
Formalin as fixative . XX
Formula for diluting XXV
Frill of ova . 222
Irog, trypanosome in 353
G
‘ Gal-ziekte’ 365
Gamete, female , 35

5 male .
" malignant tertian 36

s of halteridium . 320
re retrogression of . 57
3 simple tertian . 35
young forms 36
Garrapaia tick 349
Gastrophilus XI
Genitalia of mosquitoes . 172
Gilesia. . 179
Glycerine jelly XXI
Glossina ’ VI
me larva of . IX
3 species of . . IX
- wing’of . . Ix
Goeldia : 185
Golgi, cycle of ; 53
Gower’s haemoglobino-
meter . 275
Grabhamia 180
Gregarines of mosquitoes. 124
Guaiacum, test for blood. 287

XXXIV

H
Haemagogus . 184
Haemalastor . 347
Haemamoeba bovis . 323
" dantlewsky . 319
ie gametes of . 320.
45 genus. . 316
‘3 Rochi . . 321

” melanipherus 322
» metchnikow: 324

. murinus 323
48 velicta 316
vesperuginis 32 3
Haemaphysalis . ‘ 346
55 leachi 335-346
Haemutapota . Vv
Haematein stain . 50, XIV
Haematobia ; . VII
Haematoidin in urine . 291
ee le a in
urine . 291
Haematoxylin eosin XXIII

Haemoglobinuria, due to
quinine . h . 305
Haemoglobin, toestimate 275

Haemogregarina bagensis 331
. bigemina 330
- curvivostvis 332
55 genus » 325
lacazet 329
5 lacertarum 329
» ° laverani:. 330
3 mauritanica 330
i mesnili 330
5 vanavum . 325
v5 medyt 327
rm serygentium 331%
% splendens . 326
‘4 stepunowt 327
- tunisiensis 330

vipevint . 331

Halteridium : . 319
a flagellation of 320

.

Haltevidium, gametes of . 320
Fr in sparrows . 103
Hatching of larvae . 244
Head of larva . . 230
» parts of mosquito . 164
Heat as fixative . 9
Hectopsyllidae . ; XVI
Heller’s test for blood . 287
Heptaphlebomyia 182
Heptaphlebomyina . 176
Hibernation of Anophelina 212
si of eggs. 213
of larvae . 213
Hind- cut of mosquito . 132
Hip pobosca . XII
, and trypanosomes . 365
» Vufipes . Xil
Histology of mosquito 140- 156
Hodgesia . . 181 |
Horse flies and Surra . 360
Howardia . 181
Hulecoetomyia 182
Human trypanosome 363
Hyalomma 348
Hypederma XI

‘Hypopharynx of mosquito 168

I
Identification of Anophelinae
186 seq.
55 Culicidae 173 seq.
5 larvae . . 244
Index, endemic . 254.
» syphonic ; 82
India, Anophelinae of # 210
», Quartan parasite in 270
Indian Anophelinae, larvae
of . 246, 251
Infection of Europeans
with malaria . 266
Intermediate leucocyte . 19

XXXV

Iron,reaction in malaria XXIV

Isotonic point of blood — 283
Ixodes . 347
Ixodidae 343
Ixodinae 344
re J
Janthinosoma 1977
Javelle, eau de ; . XX
Joblotina . . 176, 182
Joblotia, post-scutellum of 169
K
Kala-azar . 369
Karyolysus lacertarum 329
Katageiomyia . 181
Kidneys in blackwater
fever 314
L
Labellae of mosquito 167
Labium of 53 166
Lankesterella vanavum 325
Larvae, abdonien of 234
% antennae of . 230
i cannibal . 84

sc classification of 192
4 clypeal hairs of . 233

3 desiccation of . 81
‘a enemies of . . 84
a examination of . 239
3 food of . . I
55 hatching of 244
5 head of 230

35 hibernation of .
identification of 244
mental plates of 84
mounting of . 252
of Anophelinae 77, 229

9 of Chivonomus 75
». of Corethva 75, 86
* of Culex 75. 77
3 of Culicidae . 81
5s of Dixa 75, 85

Larvae, of Ephemera . 75
“e of Indian Ano-
phelinae 246, 251
“ of Mochlonyx 86
$y of Psovophora 89:
$5 of Stegomyta 86
re of Taeniorhynchus
77, 88
+5 palmate hairs of 235
xi respiratory syphons
of . » 76,82
Larval stage, duration of 22g
Larvicides ‘ , . 84
Lasioconops . 179
Leaflets of palmate hairs, 236
Leeches and trypanosomes 351
Legs, banded . 190
Legs of mosquitoes . Ras bas!
Leishman-Donovan bodies 369

Leishman’s stain g, 12
Length of life of Anophe-
linae 218

Leucocytes, counting of 272
» differential count 41

», -dropsical . 5 22
Leucocytes, eosinophil 19
» in blackwater

fever SLL
» in malaria 256
» impneumonia . 281

» insplenomegaly 373
» intermediate . 19

» in typhoid . 281

» large mononu-
clear 18, 40

a mast ; . 19

» normal values of 42
» percentage of in

malaria . . 276
» pigmented 27, 39
» polymorphonu-

clear’ . «ES

small mononu-
clear (lymphocytes) 18

XXXVI

Leucocytes, transitional . 19
Leucocytic variation in

malaria : . 40
Leucopenia in malaria . 277
Limatus . : ; . 185
Lipoptena XII
Lizards, parasites in - 329
Loffler’s methylene blue XXIII
Lophoscelomyia é . 208

ns asiatica 208
Louse, filavia in . 396
Lucilia : ; . VI
Lutzia . 179
Lymphocytes : 2 28
Lypevosia . VI

M

Macleaya . 181
Makrogamete 37, 54
Mal de caderas . 361
Malaria, albuminuria in 285

5 and = diplococcus
pneumoniae . 308

» Chronic changes
in’. : . 308

Pe endemic, to inves-
5 tigate . 260

Malaria, European, to in-
vestigate . 266

» Iron reaction in XXIV
S isotonic point of

blood in 284
» leucocytosisin . 276
» leucopenia in 275;
» parasite, life his-
1 tory , 53
» parasite, periodicity
co aero) ae ‘ . 294
» percentage of leu-
cocytesin . 279
» pigment in, post-
mortem . 307
» post-mortem
changes . 307

Malaria, prevalence of . 262
» subsidiary signs of 39

» . the urine in . 285°
Malarial endemicity - 254 —
” wn map of * 255
4 » survey
map of . 261

tissues, toexamine 43
iy » to embed 44,48
» tostain. 50

Malaysia, Anophelinae of . 210
Male Anopheline, the 219
» crescent i . 29
» and female mos-
quitoes, proportion
between 2 226
Malignant tertian stip-
pling . ‘ 13
Malignant tertian, parasite
cycle of . 298
Malpighian tubes of mos-
quito.. . 133
Mandibles of mosquito 168
Mansonia : ‘ . 180
» Sggsof . + 7
Mast leucocyte ‘ 19
Maxillary palps of mos-
quitoes . ’ . 169
Media for mounting XXI
Megaloblast . . 20
Megapsyllidae . XVI
Megarhinus . 176
Megavhinina 176
Melanin : , e 2
Melanoconion 180
Melophagus XIII
Morsiitce of Dutton 167

Mental plate of larvae 84
Methaemoglobin in

blackwater fever . 287
5 tests for . 289
Methylene blue XXIII
Microcytes . oe

Microscope, use of . . 4

XXXVI1I

Mid-gut of mosquito —._ 105
Mikrogametocyte 37> 54
Mimomyia . 183
Mochlonyx 185

5 larva 86

Mononuclear leucocytes,
large 18, 40

#5 » small . 18
Monogony : » BF
Monkeys, parasites in . 321
Mosquito, abdomen of . 172

45 antennae of . 165

apparatus for

collecting XXVIJ

breeding out

96, 97; 158
5 capture of 96 -
5 collection of 157
3 clypeus of . 164
3 dissection of 104 |
35 epipharynx of . 167 '

fat body of
filaria embryoin 124

. 140 |

me filariain.. . 393
" flagellata of = 124
7 genitalia of . 172
3 gregarines of . 124 |
a5 head parts of . 164
% hind-gut of — . 132

histology of 140, 156

hypopharynx of 168 '

” labellae of . 167.
me labium of 166 |
legs of . 171
3 male and female,
illustration . 61 |
Pe malpighian tubes.
of : . 133 |
i mandibles of . 168)
a maxillarypalps 169
os maxillae of . 168
‘4 mid-gut of . 107)
is muscular system
of . 133)

Mosquitoes, nematodes of 124
nervous system

of : 138
Pr oesophagus of . 130
55 ovaries of 121
se post-scutellum
of 169
ii pharynx of — . 130
proboscis of . 166
i proventriculus
of . 131
‘3 pumping organ
of F . 130
- reproductive
system . . 139
at salivary pump. 168
“3 scale structure. 173
- scutellum 169
s scutum . 169
s sections . 124
re spermatheca . 121
5% sporozoa in. 124
as stomach . . 105
7 to capture 65 seq.
to feed . 100

to feed on birds 102
to keepalive 97 seq.

sts to kill . 159
id to mount. 160
. trematodes in 123
‘a tracheal system
135 seq.
- vascular system 137
s viscera 106
| Mounting media XXI
ie of larvae . . 252
3 of mosquitoes 160
. of ova . 228
Muller’s fluid XIX
Musca . VI
Mucidus F . 197
Muscular system of mos-
quitoes . 133
Myelocytes in malaria 21

Myiasis

Myzomyia, genus

9

?

aca genus 187,

. 187,
species of
aconita
albivostris
culicifacies
33 Sporo-
zoits in
» atti-
tude of
elegans
funesta
yy» Sporo-
zoits in
» ungues
of
hebes
hispaniola
impuncta
leptomeres
leucosphyra
listont .
longtpalpis
ludlowt1
lutgit
nilt
punctulata
vhodesiensis
VOSSiL
5, absence of
sporozoits
in
tesselatum
turkhudi
» ova of

albotaeniatus.
bancrofti
barbivostris .
coustant
mauritianus .
minutus
nigervimus
paludis

XXXVIll

VI
194
195
195

Myzorhynchus, pseudo-

barbivostris . 202
‘3 pseudopictus . 203
er sinensis 202
53 umbrosus 202
‘a vanus 202
5 ziemanni 203
N

Nematodes in i 124
Nematocera I

Nervous system of mos-
quitoes . . 38
Ngana, trypanosome of . 360
Nycteribtidae . XII
Normoblasts 20
Nucleated red cells . x 20
Nympha of Anophelinae. 89
Nympha of Chivonomus . 92
‘5 of Corethra g2
” of Culex . 89
» » Of Taeniorhynchus go
syphon tubes gI
Nymphal stage ‘ 238
Nyssorhynchus, genus 188, 201
3 species . 203
= annulipes 206
4 deceptor 206
7 fuliginosus . 203
- jamesit . 205
‘i Rarwari . 203
a maculatus . 205
is maculipalpis, 205

» v. Indi-
ensis . 205
‘3 mastert . 207
53 metaboles . 204
3 nivipes . 207
<3 philippinensis 207
“5 pretoviensis . 206
‘i stephensi . 204
os theobaldi . 205
4 willmori , 206

XXXI1X

oO

Ochromyia XI

Oedema in trypanosomiasis

ie 367 |

Oesophagus diverticula of 131
53 of mosquito . 130
Oestrus . ‘ XI
Oil as larvicide . 85
Oocyst . ; . 56
Ookinet . 55
Ornithomyia XIII
Ornithodorus 349
Orth’s fluid XIX
Osmic acid XXII
Ova, floats of . ' . 222
» ‘frillof . 222
», Of Anophelinae . 221
» Of M. turkhudi . 229
to mount 228
Ova, types of . , 223
Ovaries of mosquito 121
Ox warbles XI

P
Palestine, Anophelinae of . 209
Palmate hairs, leaflets of. 236
» Of larvae 235

Palpi, banding of IgI

Pangonia IV

Panofplites 180

Paraffin sections, to fix XX

» Wwaxintropics . 47
Parasites, action of quinine

on . 304

3 characters of . 15

< crescent forms. 26

- crescents, fresh. 28
‘4 cycle,determina-

tion of . 294

- in bats 322

i in birds 316
+3 in blackwater

fever “a 312

| Parasites, in fish . 330
5 in lizards . 329
“5 in monkeys . 321
5 in tortoises . 324
53 large forms 26, 25
3 malaria, detec-
tion of . 24

$s 33 effect of
quinine 24

55 malignant ter-
tian. 33

% malignant ter-
tian, stippling 33

a quartan . 32, 33
' quotidian 34
i ring form. 25, 27
3 signet form 25

spherical bodies 29
Banacte tertian (malig-

nant) 32
85 (simple) 32
Pericardial cells « TEL

Periodicity of malaria

parasite . 204
Pharynx of mosquito . 130
Phlebotomus 60, II
Phloroglucin . XX
Phoniomyia . 184
Picric acid XX
Pigment in biackwater

fever . 311
3) of skin . . 15

» P.-M.inmalaria 307

Pigmented leucocytes 27, 39
Pipettes, to clean B74
Pivoplasma, genus . 332
3 bovis 332
$s canis 334
” COUE - 337
i hominis . 336
» Rocht 335
. ovis - 335
Platelets . 16, 20

Platelets, to count . = B95
Pneumonia, leucocytes in 281
Poikilocytes 21
Polar bodies 30
Polychrome staining 26
Polychvomophilus melant-
pherus. 322
‘3 MUYINUS 323
Polymorphonuclear leu-
cocyte : 18
Post-mortem changes in
blackwater fever 314
Post-mortem changes in
malaria . 307
Post-scutellum of Culex 169

of Joblotia 169
of mos-

quitoes 169

of Wyeomyia 169

”

”

”

Prevalence of malaria . 260
Proboscis of mosquito . 165
Proteosoma : . 314
‘i black spores 317
<3 cycles of . 315
insparrows . 103
vermiculi of . 316

Proventriculus of mosquito131

Psovophora 177
» eggs 73
is larvae 89
Psychodidae . I
Pulicidae . : XVII
5 and Filaria 391
Pumping organ of mos-
quito 130
Pupation . 239
Pyvetophorus, genus 187, 200
58 atratipes 201
55 chaudoyet 201
ee ‘cineveus 200
costalis 200

costalis, sporo-
zoitsin . 258

- 4, v. melas doo |

xl

Pyvetophorus, jeyporensis . 200
s marshallit . 200
sg merus . 201
4 minimus . 201
‘9 palestinensis 201
3 pitchfordt . 201
3 superpictus . 200
Quartan parasite 32, 33
4 + in Africa 270
5 am in India 270
' Quinine, absorption of 301
» action on parasites 304
, and blackwater fever ,
305
Quinine, effect on malaria
parasite . 24
» elimination of — . 301
» haemoglobinuria . 305 |
», in urine . 293
Quotidian parasite . 32, 33
R
Rat, trypanosome in . 360
Razors, sharpening of 46
Red cells, counting of . 271
55 large swollen . 22
5 nucleated 20.
5 penetration by
sporozoits 119
5 with long pro-
cesses 2 22
Reproductive system of |
mosquito, . 139
Respiratory syphons of
larvae . 76.
Rhipicephalus. . 344
Eehy phidae - 59
) Ring form of parasite 25, 27
| Romanowsky stain. 10, 126,
ee for sections 51
Ross, cycle of . . 53
Runchomyia . 184

xl

\

i)
Sabethes . . 184
Sabethoides . 185
‘Safranin . XXIII

Sahli’s methylene blue XXIII
Salivary acini . . 132
i glands, dissec-
tion of . 114
of Anophelinae 120

i » Of Culicidae 120

35 » sections of . 128

43 » Sporozoits in 118

structure | 117

pump 168

Sand-flies 59, i
Sarcophaga

Sarcopsylla a tt

Sarcopsyllidae . XVI

Scales of Anophelinae 187

» Of wings 174

» varieties of . 174

Schaudinn on Spirochaetes 379
% trypanosomes 354
Schizogony . : . 57

Schuffner’s dots 13, 33
‘Screw-worm’ larva VI
Scutellum of mosquito . 169,
Scutomyia . 181
Scutum . 169
Seasonal prevalence of
Anophelinae . . 211

Sections of mosquitoes 124 seq.

of salivary glands 128

”

o Romanowsky stain
for 51
Silvius . ‘ ‘ . Vv
Simulidae VII, 58
Skusea ; . 181
Syphonic index 3 . 82
Syphon of larvae. . 82

» tubes of nymphae QI!
Sleeping sickness and
trypanosomes 363

Slides, to clean ; 2, 23
Snowia ; ae
Sole, trypanosome it in. 352
Sparrows, halteridium . 103
55 proteosoma . 103
Species of Aldrichia . 208
+3 Anopheles . 192
sa Anophelinae, differ-
entiation of . 190
‘5 Arribalzagia. . 202
< Cellia . 207
‘a Cycloleppteron . 199
= Glossina . ,
3 Myzomyia . 195 —
55 Myzorkhynchus . 202 ~
3 Nyssorhynchus . 203
5 parasite, to de-
termine . 33
4 Pyvetophorus 200 seq.
53 Stethomyia . 199
Spectra, table of . 288

Spectroscopic test for blood 287
Spermatheca of mosquito 121

Spider flies . Xi
Spirillar fever and ticks 377
» inchickens 378
Spirochaete anserina . 378
53 (gallinarum). . 378
3 obermetert 377
35 theileri 378
“9 ziemannt 379
Spirochaetes and trypano-
somes 379
Spleen rate. . 263
Spleno-megaly tropical 369
Sporoblasts . . 50
Sporogony : : . 57
Sporozoa of mosquitoes . 124
Sporozoits ; . 56
5, -absence of, in
M. yosstti =. - 257
- false appearance
of . . 109

xhi

Sporozoitsin M. culicifacies 257

» in M. funesta,. 258
6 in P. costalis . 258
» insalivary gland 118

5 motion -of . IQ
55 penetration of
red cell by . 119
5 rate . 265
Fs to stain . 120
Sporulating forms of para- ‘
sites . ; . 34
Spotted fever . . 336
Stains XXII
Stain, Leishman’s g-12
» Romanowsky 10 seq.
» solubility of . XXIII
Staining, Amoeba coli XXIV
a basophil . . 26
a flagellate bodies 30
» .polychrome . 26
Xe spovozoits . 120
Stegomyia ‘ . 178
» eggs 68, 72
35 genus , . 65
5 larva : 86
Stenopteryx XII
Stethomyia, genus . 187,199
* fragilis . . 200
5 nimba 199
Stomach of mosquitoes 105
+ zygotes in 109
Stomoxys °. . VII
Sublimate corrosive as
fixative . XX
Sugar in urine - . 2g
Suvva and horse-flies = .._- 361
» trypanosome: . 361
T
Tabanidae 4 : » IV
Tabanus . : Vv
Taeniovhynchus . 180
» eggs = 69-73

Taeniorhynchus larva 76-87
5 aympha of go
Tersesthes . : « tl

Tertian, malignant gametes
36

= (malignant) para-

site. 32-33
3 (simple) parasite 32
Texas fever parasite . 332
Theobaldia . 178
Ticks. ; 337 seq.
» anatomy of 8

33
, and spirillar fever .. 378

» Classification of . 343
Tick fever . 349, 381
Tipulidae ‘ : « 59
Toison’s fluid . . 271
Tortoises, parasites in . 324
Toxorhynchites , . 177
Tracheal cells’. . III.

a system of mos- ;

quitoes . 135 seq,

Transitional leucocyte . 19
Trematodes in mosquitoes 123
Trichocera : js - 59
Trichoprosopina . 176
Tropical sore . 369
Tropical swellings and
eosinophilia 396

Try panoplasma ' » 350
55 borvelt . 368
95 cyprint . 368
Trypanosoma . - 350
5 and fleas XIII

Trypanosome and H: ippo-
bosca vufipes 365
+3 and leeches 351

5 and Leish-

man bodies 376

- and spiroch-
aetes 379

ss and sleeping
sickness . 363

Trypanosomeand tsetse fly 360
Trypanosomes, blood ex-

amination in 367

5) cultivation of 367

Trypanosoma abramis —. 351
avium , . 354
* brucei. . 360
- brucei and

trypanosoma evanst

compared . . 361
ii cavassit » 352
i" cobitis - 353
Pe danilewskyi . 351
Fe dimorphum 365
5 evansi . 361
5 equinum . 301
3 equiperdum . 362
5 gambiense . 363
- granulosum . 351
a inopinatum . 353
+5 johnstont . 354
n Raryozeukton 353
3 lewisti 360
9 mega " : 353
3 nelspruitense 354
oa noctuae - 354

ie noctuae and
halteridium 354
» paddae—-. 354
Ue vajae 352
“3 vemaki . 351
i votatovium . 353
+ scyllit . . 352
is soleae . . 352
, theilert . 365
$5 tincae . » 351

trvansvaliense 365
Trypanosomes, dimensions

of 366

human. 363
Trypanosomes i infrogs . 353
rs rats . 360

5 imoculation of 366
Trypanosome of Dourine, 362

xh

Trypanosome of
mal de cadevas 361

» Surra 361
Trypanosomiasis, oedema
fluidin . 367

centri-
fugalization of blood 367

Tsetse fly . VII

iy disease . 360
Types of ova . ; 5 238
Typhoid, leucocytes in . 281

» Widal reaction in 281

U

Ungues of M. funesta . 172

»» specific values of 172

» M, rossii = 72
Uranctaenia . 183
Urine, bile pigments in . 290
e bilirubin in . 291
», haematoidin in . 291

», haematoporphyrin in 291
» methaemoglobin in 287
» in blackwater fever 313

» im malaria . 285
5, quinine in . 293
» sugar in . 291
urobilin in . 289
Urobilin, test for. 290
Urobilinuria in black-
water fever . 313
Vv
Vacuoles . , 15
Vascular system of mos-
quitoes . . 137
Veins (cross) of M. rossit. | 191
Ver de Cayor . . XII
Ver macaque . XII

Vermicule of Proteosoma 318

Vermiculus 55
Vermipsyllidae XVI
Verrallina . 183
Viscera of mosquitoes 107

xliv

af ~
Weights and measures XXIV
Widal reaction intyphoid 281
Wild Anophelinae . . 214

Wings, fringe of . . 190
5 Of Anophelinae 2 170:

» scales of. é . 174
» spots on ‘ . Igo |
Wyeomyia . : . 184
» post-scutellum of 169

x
Xestopsylla . ‘ XVII

ee ,
Zenker’s fluid . ; XIX
Zygote a : :
Zygotes, malignant tertian..

7 113
55 proteosoma . 113
quartan . _.. _I13

simple tertian . 113
in stomach . 109
to mount . 113

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