THE

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INAUGURAL DISSERTATION

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| FOR THE

DEGREE OF Pu.D. IN THE UNIVERSITY OF LEIPZIG,

BY
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C. HERBERT. HURST,
pees IN THE VicrorIA UNIVERSITY,
Assistant LECTURER IN ZOOLOGY IN THE OWENS COLLEGE,

MANCHESTER. )
WETH ONE] PLAT Re

: MANCHESTER :
€ GuARDIAN ” PRINTING WORKS, BLACKFRIARS STREET.

1890.

PUPAL STAGE OF CULEX,

PUPAL STAGE OF CULEX,

INAUGURAL DISSERTATION

FOR THE

DEGREE OF Pa.D. IN THE UNIVERSITY OF LEIPZIG,

BY
C. HERBERT HURST,

LECTURER IN THE VICTORIA UNIVERSITY,
ASSISTANT LECTURER IN ZOOLOGY IN THE OWENS COLLEGE,

MANCHESTER.

/ i, MOOD
\ OCT 15 1890
CS

WITH ONE PLATE. NY

MANCHESTER :
‘GUARDIAN ” PRINTING WORKS, BLACKFRIARS STREET.

1890.

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THE PUPAL STAGE OF CULEX.

AN INAUGURAL DISSERTATION FOR THE DEGREE OF PH.D. IN
THE UNIVERSITY OF LEIPZIG.

By C. Hersert Hurst, Lecturer in the Victoria University, Assistant
Lecturer in Zoology in the Owens College, Manchester.

[WiTH ONE PLATE. |

SHALLOW pools in most parts of Europe, and especially the
smaller pools in woods, swarm in early spring with larve of Culex,
hatched from eggs laid in floating masses by the impregnated
females which have lived through the winter. These larve have
been described by Swammerdam (1) and others, and most recently
by Raschke (2).

After a few weeks the pupa escapes from the larval cuticle, and
four days later the perfect insect flies free.

Though the pupal stage is the one of which I propose to give a
fuller account than has yet appeared, it is necessary to the proper
understanding of it that some account of the preceding and following
states should also be given, and especially of the mode of life in each
state.

The larva is an exceedingly active creature, swimming by a
wriggling movement of the body, this being aided by a median
fin-like series of setze beneath the last segment. The head is provided
with jaws and sete, by means of which the solid food is collected
and masticated. A pair of unjointed antennz of considerable length
arise from the sides of the head, and behind the base of each is a
compound eye and behind this an ocellus.

The head is moveably attached by a neck to the broad, rounded
thorax. The abdomen is long and slender, and composed,

4 C. HERBERT HURST.

apparently, of nine segments. The ninth segment bears four gill-
plates surrounding the anus, and on its ventral surface, the median
series of long setze which serves as a propeller.

Respiration, according to Raschke, is performed in a threefold
manner; by the gills just mentioned, by the rectum, and directly,
the air being taken into the tracheze by a conspicuous siphon pro-
jecting upwards from the eighth abdominal segment. The trachex
of the abdomen serve not only as organs of respiration, but also by
virtue of their great size as a float, keeping the larva when at rest
at the surface of the water, with the hinder end uppermost and the
end of the siphon touching the surface.

The alimentary canal is practically straight. The cesophagus is
narrow. The stomach is wide, and extends from the anterior part
of the thorax to the sixth abdominal segment. Its walls are very
thick, and the epithelial cells very large, and in the thorax it has
eight diverticula or pouches. From its hinder end the small intestine
runs backwards to open into the wide rectum at the anterior
end of the eighth abdominal segment, and this leads direct to the
anus at the end of the body.

Five Malpighian ceca lie in the hinder segments of the abdomen,
and open into the anterior end of the small intestine.

A pair of sac-like salivary glands lie at the sides of the stomach in
the anterior part of the thorax, and their ducts, according to Raschke,
unite, and have their opening “ oben am Beginn des Cisophagus.”

So far I have followed Raschke’s account, except in ascribing a
hydrostatic function to the “colossal” tracheal system, which is
apparently much larger than would be necessary for respiratory
purposes alone.

To Dr. Raschke’s account I would add that, in addition to the
head appendages, there appear during the larval period not less than
eight other pairs of appendages beneath the larval cuticle. Of these
six pairs are thoracic and two abdominal. The thoracic appendages
are three pairs dorsal, the future pupal siphons, the wings and the
halteres ; and three pairs ventral, the future legs. The two abdo-
minal pairs belong to the two last segments. Those of the eighth
abdominal segment lie in the larval siphon, and form the fins of the
pupa ; the hindmost pair form the outer gonapophyses of the adult,
which are accessory organs of copulation.

THE PUPAL STAGE OF CULEX. 5

All these eight pairs alike arise as foldings of the epidermis
(“hypodermis”) outwards. All alike are completely hidden by the
larval cuticle.

The antennz, moreover, are much larger in an advanced larva
than they appear to be. Their growth forwards being prevented by
the unyielding cuticle, they grow backwards, and their basal portion
is folded, and even “telescoped.”

Towards the end of larval life the animal becomes sluggish ;
profound changes in its mouth-parts deprive it of the power of
eating, and it floats with its siphon-stigma at the surface. Shortly
the cuticle bursts in the thoracic region, along the mid-dorsal line ;
the pupal “horns” or siphons are protruded, the abdominal tracheze
appear to collapse, and the animal floats with the anterior end
upwards, the new siphons coming to the surface. The old larval
siphon, or rather its soft parts, are withdrawn from the cuticle and
invaginated into the eighth segment of the abdomen ,; the intima of the
abdominal and thoracic tracheal trunks breaks up into pieces,
which in the abdomen correspond to body-segments. The body of
the escaping pupa is gradually withdrawn from the larval cuticle, and
the eighteen fragments of the old tracheal intima are drawn out of
the body by nine pairs of stigmata, and cast off with the exuvie.
These nine pairs of stigmata are situated, one in the hinder part of
the thorax, one in each of the first seven segments of the abdomen,
and the ninth pair are united to form a single aperture, the old
respiratory opening at the end of the larval siphon.

With the larval exuviz are also cast off the cuticular portions
of the jaws and antenne, and all the hairs and spines with which
the larval cuticle was beset.

The pupa which thus escapes differs from the larva very widely.
It is a little under 1 cm. in length when fully extended. It consists
of a bulky, laterally compressed mass made up of head and thorax
with their appendages, and of a slender flexible abdomen, which
when at rest is curved under the thorax. In a specimen measuring
9 mm., which is nearly the maximum size, the thorax measures
2°5 mm. and the abdomen 6°5 mm., but the thorax appears to be
much longer on account of the wings which extend downwards and
backwards from its sides.

The head lies below the thorax, and so adds nothing to the length

6 Cc. HERBERT HURST.

of the animal. It is broad from side to side, short from back to
front, while ventrally it is drawn out into a long process which
extends backwards under the thorax as far as the anterior part of
the abdomen, where it curves upwards. This process is made up of
what are usually spoken of as the “ mouth-parts,” including labrum,
epipharynx, one pair of mandibles, two pairs of maxillx, and the
hypopharynx. The second pair of maxille are fused together to
form the labium.

In describing an animal which is coiled up so that head and tail
almost meet, the terms ‘‘ dorsal,” “ventral,” ‘“ anterior,” and “ pos-
terior” are liable to be misleading. To avoid this as far as possible
I shall apply the terms to those. parts to which they would be
respectively applicable in the fully-developed insect in the act of
sucking blood, 7.¢., I shall regard the general direction of the mouth-
parts as downward, their distal ends as ventral, and I shall speak of
the labrum as being in front of the mouth.

From the sides of the epicranial region the antennz run Gu inraede
to the sides of the thorax, and then downwards, one beneath the
anterior margin of each wing. The head and all its appendages are
immoveable during pupal life.

The thorax is rounded, but somewhat compressed from side to
side. From the sides of its summit arise the respiratory siphons,
a pair of conspicuous organs whose position has led to the name
“horns” being applied to them. The wings are nearly flat oblong
plates, arising behind the bases of the siphons and extending down-
wards and backwards. Immediately behind them are the halteres,
a pair of triangular plates enclosing the halteres of the future gnat.
I have endeavoured to show the forms of these parts in Fig. 1.

The legs are almost completely hidden by the wings, but the
femur, tibia, and first joint of the tarsus of the first leg, and the
tibia and first joint of the tarsus of the second are visible (Figs. 1
and 2).

The abdomen is flattened dorso-ventrally, and when at rest is
curved under the thorax. It is jointed and flexible, and forms with
the pair of large flat fins borne by its eighth segment the only
locomotor organ of the pupa, the wings and legs lying immoveable
and even adhering to one another, though they are easily separated
in specimens which have been kept in alcohol,

THE PUPAL STAGE OF CULEX. if

The pupa does not eat. It breathes air through the apertures at
the ends of its siphons. It floats, thorax uppermost, by virtue of a
large air cavity lying under the hinder part of the thorax and the
anterior part of the abdomen. This cavity is bounded in front by
the legs, at the sides by the wings, and below. by the mouth-parts.
It extends up at each side of the first segment of the abdomen,
where it is covered by the halteres, and into this part of the cavity
at each side opens a large stigma, held open by the fairly well-
developed cuticular lining (“intima”), and guarded near its entrance
by numerous spines. These two stigmata belong to the first
abdominal segment, and put the air-cavity just described into direct
communication with the tracheal system. As already mentioned, I
regard this cavity and these stigmata as being mainly, if not
exclusively, hydrostatic in function, serving not only to make the
pupa float when at rest, but to make it float in a definite position,
with the thorax uppermost and the apertures of the siphons at the
surface of the water. |

The pupa is sensitive to light, and immediately darts backwards
when a shadow falls upon it suddenly. The movements, however,
though very rapid, are devoid of anything like steering. The larva
had to steer in its search for food, but the pupa has simply to get
out of the way of danger, and the direction of its flight is of little
importance, though, since the movement is always backward with
reference to the pupa, it is chiefly downward with reference to the
outer world.

A sudden very loud noise, or a very gentle tap upon the vessel
containing the pups, causes those at the surface to dart downwards,
but as slight sounds of various kinds produce no effect upon them,
I conclude that the tremor of the surface of the water, and not the
sound itself, was recognised by them. The setz on the first segment
of the abdomen are probably the organs by which this movement
is felt.

As to the anatomy of the pupa, it is only necessary now to state
that at the beginning of pupal life the internal arrangements are
those of the larva; at the end of that period they are those of the
imago.

At the beginning of the fifth day of pupal life, the cuticle splits
along the mid-dorsal line of the thorax ; the thorax of the imago

8 C. HERBERT HURST.

protrudes, and the head, then the abdomen, and lastly the wings,
legs, and proboscis are drawn out of the pupal cuticle, which is left
floating in the water while the imago flies away.

With the cuticle are cast off nine pairs of fragments of tracheal
intima, two pairs being drawn out through the thoracic stigmata,
the others through the stigmata of the first seven segments of the
abdomen,

These fragments differ as follows. The first pair are well developed,
and have the spiral thickenings very well marked. ‘They are con-
tinuous with the lining of the respiratory siphons, and formed
during pupal life the connection between these organs and the
tracheal system generally.

The first abdominal pair are not so well developed, but the spiral
thickening is recognisable in them, and the terminal portion of each
is better developed than the rest, and is beset internally with
numerous small spines. It was through these that the tracheal
system communicated during pupal life with the air-cavity beneath
the thorax. The remaining fragments, 7.¢., the hinder thoracic pair
and all the abdominal pairs except the first, are very thin and
delicate, and were functionless during pupal life.

The cuticular lining of the anterior and posterior portions of the
alimentary canal and the cuticle of the invaginated larval siphon are
also shed, together with all sete: and the whole of the pupal siphons
and fins. These three last alone involve the loss of portions of
tissues other than cuticle.

The imago, with its long, slender body, wings, legs, and proboscis,
hardly needs to be described. Like pupa and larva it breathes air,
but now by more numerous stigmata, and unlike them it flies in the
air. The larva fed upon solids ; the pupa did not eat at all. The
imago feeds upon fluids, and the female, at least, upon the hot blood
of man and other mammals. The male is short-lived, and his food
is said to consist of the sweet juices of flowers. To find the female
and to impregnate her are the real objects of his short life. His
antenn are provided with long hairs, which A. M. Mayer (8) has
shown to be sensitive to a particular sound when the head is turned
towards the source from which it proceeds, and he has further
shown that sound to correspond to the note emitted by the vocal
organs which Landois (4) has described on the sides of the thorax

THE PUPAL STAGE OF CULEX. 9

of the female, beneath the halteres. The male, therefore, would
appear to be endowed with a special and very largely developed pair
of organs for detecting the whereabouts of the female.

The female imago, after impregnation, has to find a suitable place
to lay her eggs, i.e, the surface of a stagnant pool; and having
found it, has to lay the eggs at a suitable season, and this in the
case of females escaping from the pupa late in the summer involves
the necessity of living through the winter. Hence the mouth
appendages are specially adapted for piercing the skin and extracting
the blood of mammals, and this is stored in her capacious stomach.
The structure of the mouth-parts in the two sexes has been
described by Dimmock (5); but as he appears uncertain as to the
injection of “saliva” into the wound, I shall add that a special
apparatus developes during pupal life by which the saliva is dis-
charged near the tip of the hypopharynx (“lingua ”).

Having now given a brief outline of the life-history of the
gnat, I will proceed to describe the pupa and the changes which it
undergoes in more detail. As, however, I have directed my attention
almost exclusively to the more important organs of the body, and
not to hairs and the like, I shall not make any distinction of species.
What I have to record is probably applicable to all species alike.

DESCRIPTION OF THE PUPA OF CULEX.

THE EXTERNAL CHARACTERS.

The head is broad from side to side ; the epicranium has a well-
marked median groove ; the clypeus, broad above, is gradually
narrowed below, and continued without any distinct line of demar-
cation into the labrum. At the sides are a pair of compound eyes,
to be regarded rather as the rudiments of the eyes of the future
gnat than as the visual organs of the pupa itself. Their form and
size in the earliest stage are shown in Fig. 1. During pupal life they
increase in size till they almost encircle the head. Corneal facets
are never formed in the pupal cuticle, but beneath it the convex
facets of the imaginal cornea are formed during pupal life.

10 C. HERBERT HURST.

Behind the compound eye, on each side of the head, is an ocellus
with fully-developed lens, ete. In the youngest pup it is separated
by a small interval from the compound eye (see Fig. 1); but the
growth of the latter obliterates this interval, and the ocellus is in
the older pupzx not readily distinguishable except in sections. The
statement, found in systematic works, that the Tipulariz are devoid
of ocelli is, however, not strictly true; in Culex, at least, they are
well developed, though, as they abut upon the compound eye, they
are in the imago so inconspicuous that they may easily be over-
looked.

In the mouth-parts, the labrum, epipharynx, mandible, maxille with
their palps, labium and hypo- and epipharynx are present, though
the two last can only be seen on dissection.

Of their mode of origin in the larva I as yet know nothing, At
the time of escape of the pupa from the larval cuticle they are
of the full size, which is considerably greater than in the adult.
The form of most of these parts is shown in Figs. 1 and 2. That I
may not have to refer to these parts again, I will at once say that
the chief changes which occur in them during pupal life are :—
(1) The development of a cuticle within the pupal cuticle, and this,
in the case of the labium (fused second maxille), is covered with
scales closely resembling those found in Lepidoptera; (2) a con-
siderable shrinking ; (3) in the male only, atrophy of the mandibles,
which in a young pupa are as large as in the female, but in the
adult are not recognisable.

The antennw, which were folded and telescoped at their bases in
the larva, are in the pupa laid upon the sides of the thorax, as seen
in Fig. 2. Their hinder (distal) extremities are hidden by the wings.
The swollen basal joint of the antenna of the imago is hardly
recognisable on the surface, although it is already a conspicuous
object in sections of the youngest pupz, and even in the larval state.
I shall describe it with the other sense organs. The shaft of the
antenna is segmented, but the external segmentation loses its cor-
respondence with the segmentation of the developing antenna
within it early in pupal life.

The thoraa is large and rounded, but somewhat compressed from
side to side. Mid-dorsally the cuticle of the prothorax is marked by
fine transverse corrugations, and this is the part which ruptures to

THE PUPAL STAGE OF CULEX. 1

allow the imago to escape. A pair of branched sete arise from the
dorsal region of the hinder part of the thorax.

The respiratory siphons (AT, Fig. 1) are nearly cylindrical, nar-
rowed at their bases and curved forwards to be attached by
flexible membranes to slight prominences on the sides of the pro-
thorax. Above they are obliquely truncate and open, and the
margin is slightly notched on the inner side. The outer surface is
marked so as to resemble imbricated scales, each with a minute spine
at its apex. The cavity of the siphon communicates directly with
that of a tracheal trunk at its base. Palmen (6) says that after a
“close investigation” he has found that there is no opening. The
tone of assurance in which he contradicts all previous observers led
me to put the question to the test. I removed the side wall of the
thorax, with some of the underlying muscles and trachex, from a
specimen preserved in alcohol. I drew out the alcohol from the
cavity of the siphon by means of blotting-paper, and then touched
the tip with a minute drop of glycerin. I watched the effect under
the microscope, and saw the glycerin force its way into the siphon,
driving the air before it into the trachex. Palmén, moreover, says
the organs are gills! Each is a thick chitinous tube, the cavity
guarded by numerous hooked spines, the walls consisting of hardly
anything but the chitinous cuticle, the epidermis (“ hypodermis ”)
between its two layers being barely recognisable on account of its
thinness. The ‘tracheal gills” on which Palmén lays much stress
have absolutely no existence.

The wings of the pupa, that is the organs within which the wings
of the imago are developing, are a pair of oblong plates about 23 mm.
in length. They are closely applied to the sides of the hinder part
of the thorax, and directed downwards and backwards. ‘They are
immoveable.

The halteres are a pair of elongated triangular plates lying along
the dorsal and hinder border of the wings.

All these three pairs of dorsal appendages arise within the larva
in the same way, and their bases or points of attachment all lie in
the same horizontal plane. Each is at first (in the larva) a fold of
the epidermis; each acquires a cuticular covering (like all other
parts of the body), and the first pair become rolled up to form
tubes, the respiratory siphons, while the other two remain flat plates.

12 C. HERBERT HURST.

The three pairs of ventral appendages of the thorax, or legs, are
long cylindrical bodies folded upon themselves, and lying beneath
the thorax and between the wings. The same segmentation into
femur, tibia, etc., is recognisable as in the adult gnat, but the
segments are more nearly equal in the pupa, and the joints of the
developing and shrinking legs of the future imago soon lose their
correspondence with those of the pupal cuticle enclosing them.
They arise in the larva, like other appendages, as folds of epidermis
enclosing mesoblastic tissues.

The abdomen is dorso-ventrally compressed and exceedingly flexible
dorso-ventrally, though not from side to side. It is the only part
of the pupa in which the segmentation of the body is readily recog-
nisable, and as I shall very frequently have to refer to the various
segments by number, J shall use the terms “first segment,” etc., to signify

«“ first segment of the abdomen,” etc.

Nine segments are readily recognised in the abdomen, and the
last one, though it is probably composed of no less than three
condensed and highly modified segments, I shall call simply “ninth
segment.”

Each abdominal segment has a chitinous tergum and sternum, and
setee are distributed sparingly over them, being almost confined to
the hinder parts of the terga. The terga and sterna of successive
segments are united by soft arthrodial membranes.

Of the setw, only one pair need special mention. These are
placed on the hinder part of the first segment, the base of each
being a triangular plate attached by one angle to a soft membrane, and
the distal side of the plate is divided into a number of bars which,
by repeated division or branching, give rise to about one hundred
sete all lying in one plane parallel to the median plane of the body.
Each seta bears a few fine hairs. When at rest, the pupa floats with
the tips of these setze, and the tips of the respiratory siphons, at the
surface of the water, and these set probably assist in maintaining the
equilibrium of the animal in this position, as well as serving as sensory
organs by means of which any disturbance of the surface is felt.

The eighth segment bears a pair of large fins, thin oval plates about
1:2 mm. in length, attached by the narrow end beneath the tergum
behind. Each is stiffened by a midrib which projects beyond the
hinder border of the fin as a spine. (Fig. 2.

THE PUPAL STAGE OF CULEX. 13

Beneath the fins and behind the eighth segment is the “ninth
segment” with its appendages. Though this region is probably made
up of more than one segment, its composite nature is not easy to
recognise, as the plates supposed in other insects to represent the
terga and sterna of tenth and eleventh segments |[see, for instance,
Huxley (7) and Miall and Denny (8)] are not developed in the
young pupa, nor, indeed, is there in any stage any such development
of the pupal cuticle, though plates developed within as parts of the
imaginal cuticle may perhaps represent some of these parts.

The appendages of the “ninth segment” of the pupa are a pair of
blunt processes arising below and in front of the anus, and directed
backwards below the fins. They are much larger in the male than
in the female. A pair of appendages are already recognisable in
this region in sections of the larva, and I think even two pairs, but
this portion of the larva is particularly difficult to cut, and I am
not yet certain as to the hinder of the two pairs. Of the existence
of one pair I have no doubt.

THE DIGESTIVE SYSTEM.

The alimentary canal of the pupa runs almost direct from end to
end of the body, the only convolution occurring in the region of the
intestine.

In the youngest pupa the condition is practically that of the larva.
(See Raschke, op. cit.) The narrow cesophagus projects slightly into
the stomach. The stomach extends from the anterior part of the
thorax to the end of the fifth segment (abdominal): it is very wide,
and the eight diverticula found in the thoracic region of the larva
are still present. The walls are very thick, and the cells of its
epithelium large.

The stomach opens behind into the intestine, which is slightly’
coiled and opens into the wide rectum, which ends at the hinder
end of the abdomen. The epithelium of the rectum consists of very
large cells, and is thrown into longitudinal folds.

The salivary glands are unbranched sac-like glands in the anterior
part of the thorax at the sides of the alimentary canal. Their ducts
unite beneath the sub-cesophageal ganglia, and from this point the
single median duct runs forwards to open in the floor of the mouth.

14 C. HERBERT HURST.

The five Malpighian ceca open into the anterior end of the
intestine. They are nearly cylindrical bodies of an intense white
colour ; their closed ends lie in the seventh or eighth segment, and
measure about 0°13 mm. in diameter. They run forwards, almost
straight, to near the anterior end of the fourth segment, and then
backwards to their point of opening into the intestine immediately
behind the constriction dividing the latter from the stomach.
The diameter of each near its opening is about 0°03 mm. Each
cecum is made up of two rows of cells alternating more or less
regularly on the two sides, the narrow lumen taking a zigzag course
between them. The individual cells are very large, the long diameter
of each being the diameter of the organ itself. The nucleus is large
and transparent, but the rest of the cell contains a large quantity of
a granular deposit which gives the organs their intense white colour.
During pupal life I have noted no important changes in these organs.

Such is the structure of the alimentary canal and its appendages
at the commencement of pupal life—a structure adapted to the life
of the larva, but not to that of the imago, and the changes which it
undergoes during the pupal period are so great that at the end of
that period no part of the whole canal corresponds in structure to
the above description.

The most striking change is the reduction in thickness of the
epithelium which occurs throughout, but which is perhaps best
shown in the stomach. Four stages of the change are shown in
Figs, 3, 4, 5, and 6, which are drawn from the epithelium of the
hinder part of the stomach. The beginning of the change has
already occurred before the pupa leaves the larval exuvie, but the
first stage here shown (Fig. 3) is from a young pupa. At the base
of each of the large epithelial cells may be seen one or two nuclei ;
a little later the protoplasm of the cell divides into a small portion

around the new nuclei, and a much larger portion, which rapidly
undergoes degeneration and, separating from the basal layer (the
new epithelium), is apparently digested. The outer surface of the
stomach is covered by an exceedingly thin layer in which I could
not make out any structure, but which is presumably muscular, and
is at first folded longitudinally (Figs. 4 and 5), but afterwards
becomes even, the new cells at the same time becoming flattened
(Fig. 6).

THE PUPAL STAGE OF CULEX. £5

A similar change occurs in the intestine. The epithelium divides
into a thin outer and a thick inner layer. The latter becomes
loosened, breaks up, and appears to be digested.

In the rectum more complex changes occur, though here also the
superficial portion of the epithelium is thrown off, but it breaks up
later and more slowly than elsewhere ; in fact, the disintegration
and digestion appears to commence in the anterior part of the
stomach, and progress gradually backwards. Before the epithelium
shows any sign of disintegration in this region, the rectum becomes
differentiated into two parts: an anterior very wide part, the “ rectal
pouch,” and a narrower hinder portion, to which alone I shall apply
the term “rectum” from this stage onwards. The wall of the
rectal pouch rises up into four very large and prominent papille,
the “rectal glands”: one ventral at the anterior end of the pouch,
just below the opening of the intestine into it; one dorsal and
posterior, and two lateral, intermediate in position between the other
two. Nerves and trachee push their way into the axis of each
papilla. The epithelium of the papilla undergoes division into two
layers as elsewhere, but the distal layer, which is ultimately shed, is
very thin, and the basal or permanent epithelium consists of very
large columnar cells, while the opposite is the case everywhere else,
and especially in the rectum, where the permanent epithelium is so
thin that I had difficulty in detecting it, and Chun (Q) states that it
is absent in Musca and other insects.

Besides this shedding of epithelium, changes of form occur in
various parts of the alimentary canal.

The anterior part of the cesophagus expands, especially in the
female, and acquires a thick chitinous lining. In cross section it
becomes triangular, and the sides and roof, all of which are convex
inwards, are supplied with muscles arising from the walls of the
head, which by their contraction increase the size of this cavity, and
serve to produce the sucking action by which the imago draws the
blood of its victims up through its proboscis. This apparatus is not
well-developed in the male.

The posterior part of the wsophagus gives off ventrally a large
diverticulum (“crop”), which runs backwards under the stomach as
far as the hinder end of the thorax, its walls developing numerous
small sacculations along its two sides. Sometimes a forwardly-

16 C. HERBERT HURST.

directed diverticulum of this crop is found arising from its ventral
wall. The crop appears at a later stage than that shown in Fig. 7.

The cavity of the stomach becomes wider, while the part behind it
becomes narrower, with the exception of the rectal pouch.

The salivary glands, which at the beginning of pupal life were
a pair of hollow unbranched club-shaped organs lying at the sides of
the alimentary canal in the anterior part of the thorax, become during
pupal life divided into about four branches, and the cavity almost
disappears, and acquires a pretty thick chitinous lining. The ducts
run downwards to the neck, which they traverse at the sides of the
nerve cords. Just below the hinder border of the sub-cesophageal
ganglia they unite to form a median duct, which runs forwards to
open into a pit at the base of the hypopharynx. This pit becomes
deeper during pupal life, and acquires a very thick chitinous lining.
From it a deep groove, also very strongly chitinised, runs downwards
along the middle line of the anterior surface of the hypopharynx to
its extremity. This is true of both male and female ; but the hypo-
pharynx of the male is inseparable from the labium.

THE CIRCULATORY SYSTEM.

The heart lies in the abdomen in a median space between the
extensor muscles and close beneath the dorsal wall of the body. It
arises at the anterior end of the eighth segment, and ends suddenly
at the anterior end of the first segment, giving off the aorta from the
ventral border of its anterior end. From its sides ‘alee cordis” run
outwards beneath the extensor muscles and between the main
tracheal trunks and the stomach, to be attached to the ‘‘ peritoneal ”
covering of the tracheal trunks, or to the outer layers of the wall of
the stomach. Each “ala” consists of a dorsal and a ventral lamina,
the two running together some distance from the heart. The space
between them has been called “ pericardium”: it contains the
‘‘ pericardial cells,” and communicates freely with the body cavity by
the spaces between the alw. The ventral lamina of each is con-
tinuous with the corresponding lamina of the other side of the
body, and all the ventral laminze together thus form an imperfect
‘pericardial septum” (Graber). The dorsal laminz are attached to
the sides of the heart. near the dorsal surface, their fibres taking a

THE PUPAL STAGE OF CULEX. 1%

longitudinal direction on the heart, and forming its outermost layer.
The heart is further bound by fibrous strands to the dorsal body
wall.

Graber (10) appears to believe that the septum is invariably
attached to the outer wall of the abdomen, dividing the cavity of the
abdomen into two cavities, a small dorsal “ pericardium ” containing
only the heart and pericardial cells, and a large ventral cavity con-
taining all the other organs of the abdomen. His figure of Acridium
is reproduced in the most popular text-book (Claus), and his view
that this arrangement is universal, and that the “septum ” serves as
a pump driving blood from the large abdominal cavity to the pericar-
dium, is reproduced in other text-books, in such form as to lead to the
belief that the arrangement is the same in all insects. Whatever may
be the case in other insects, this view is certainly not applicable to
Culex. Here the “septum” does not extend to the body wall, and
if a “ pericardium,” in Graber’s sense of the term, exist at all, the
extensor muscles and the main tracheal trunks lie in it, and the
septum cannot, judging from its anatomical relations, have the
function ascribed to it.

The heart itself is a more or less cylindrical tube, about 0:06 mm.
in diameter. Its hinder end at the anterior limit of the eighth
segment is open, but I am unable to give an account of any valvular
apparatus which may be present here. There is no sharp division
into chambers either by constrictions or by valves. In the first
segment a pair of valved ostia opens backwards ; in segments III to
VII paired ostia are present, their margins being turned in and
directed forwards to form the valves. I have not detected any
aperture or valve in the second segment. The ostia are small paired
slits in the sides of the heart, and between the two laminz of the
ale, putting the cavity of the heart in communication with the
“ pericardial” cavity. The infolded margins of the slits serve as
valves in two ways ; firstly, they prevent the blood from flowing out
through the ostia ; and, secondly, they prevent the blood within the
heart from flowing backwards during systole.

Of the histology of the heart I would speak with the greatest
caution. Graber (op. cit.) has made the subject his own, and has
applied very special methods to the investigation. My object has
been rather to record the anatomical structure and the development

18 C. HERBERT HURST.

of the pupa, and I simply note histological results incidentally,
referring those who wish to learn the histological structure of insect
hearts to the classical work just mentioned.

Without the application of special methods, I have recognised
three layers in the wall of the heart.

The inmost layer, or endocardium, is an exceedingly thin layer
of flat cells. Their nuclei are conspicuous objects, occurring with
striking regularity in pairs, four pairs to each segment of the
abdomen, and a similar but smaller nucleus is to be seen in each
flap of each valve, from which I conclude that this endocardium
extends also to the valves. Whether the other layers also extend
into the valves or not, I cannot say with certainty.

The middle layer consists of encircling fibres, slightly oblique in
direction, and probably muscular.

The outer layer is also fibrous, its fibres being on the whole longi-
tudinal in direction, but they curve outwards to be continuous with
the fibres of the dorsal laminz of the alee.

Between the lamin of the al cordis, that is in the pericardial
cavity, are large ovoid masses of brown cells, the “pericardial cells.”
Of these masses there are two pairs near the anterior, and two near
the posterior end of each segment of the abdomen ; but the number
increases towards the end of the pupal stage, and still further in the
imago, by the division of some of them into two or more masses,
The protoplasm of these cells is extraordinarily spongy, and contains
numerous granules, which stain deeply with borax carmine. The
nuclei vary in number from three or four to ten in each mass,
though the boundaries of so many cells cannot be made out. The
cells appear to be undergoing division very slowly. The excretory
function of these cells has recently been shown by Kowalevsky (12).

The aorta runs from the ventral border of the anterior end of the
heart forwards above the stomach and cesophagus to the head, where
it ends, the end being open. In transverse sections of the thorax,
the aorta is seen as a laterally compressed tube. I have not seen

any branches given off from it.

THE RESPIRATORY SYSTEM.

Culex, as already mentioned, breathes air in all three states—
larva, pupa, and imago—and also breathes it directly, but the air is

THE PUPAL STAGE OF CULEX. 19

taken in at different apertures in the three states. The larva, accord-
ing to Raschke (op. cit.), breathes also by gills and by the rectum.

I have already described the respiratory siphons of the pupa, and
given evidence to show that they really do lead directly into the
trachez, in spite of Palmén’s contention to the contrary.

From the base of each siphon, trachez run to various parts of the
body and head. Amongst these may be mentioned specially one
transverse trunk running across the thorax between the alimentary
canal and the nerve-chain, and putting the two siphons in direct
communication with each other ; and a pair of longitudinal trunks
running backwards to the hinder end of the body, and giving off
branches to the various organs, and also a trunk to each of the
stigmata. As already mentioned, these stigmata are present in the
hinder region of the thorax, and in each of the first seven segments
of the abdomen ; but the stigmata, except the first abdominal pair,
are closed, and the pupal intima of the trachez connecting them with
the main trunks is thin and collapsed. The widely open stigmata of
the first segment, with their spines and their probable function, |
have already commented upon ; but while insisting on the importance
of the hydrostatic function of the tracheal system in both larva and
pupa, I would again say that I do not consider this a sufficient
ground for the view that the hydrostatic function is the primitive
one. In Culex larva and pupa, it is important only inasmuch as it
subserves respiration by bringing the animal to the surface and
maintaining it there in the only position in which air can be breathed
directly.

The cuticular lining (“intima”) of the chief trunks and their
branches is well developed even at the commencement of pupal life,
and has the usual spiral thickening. The trunks connecting the
stigmata with the main trunks are the only ones that undergo any
marked change during the pupal condition. These widen around
their separated and collapsed intima, and a new and strongly
thickened intima is formed. In the main trunks no new intima is
formed, and when the imago escapes from the pupal cuticle no
portion of the intima is shed from any part. of the system which has
been functional during pupal life, excepting the portions connecting
the siphons and the first abdominal stigmata with the main trunks.
These fragments are, in the case of the siphons, well developed, and

20 C. HERBERT HURST.

have a fully-developed spiral thickening. The portions connected
with the first abdominal stigmata, though better developed than the
portions connected with the other stigmata of the abdomen, have
the spiral thickening only slightly developed. The terminal portion
is beset with very numerous small spines.

The fate of the invaginated portion of the larval siphon is
interesting. The whole of the tissues composing it break up and
undergo complete absorption, so that no trace of it is discoverable in
the advanced pupa.

Before dismissing the respiratory system, I will again state that
the pupa breathes air only, and breathes it through the open
stigmatic horns or siphons alone. The tracheal gills of Palmén have

no existence.

THE MUSCULAR SYSTEM.

Concerning this I have nothing new to communicate. The muscles
of the pupa are those of the imago. All the chief ones are present
in the young pupa, but they increase greatly in size, and this is
especially true of the thoracic muscles.

THE NERVOUS SYSTEM.

The nervous system is particularly interesting. Within the short
space of four days, certain ganglia increase enormously in size by
the addition of cells apparently derived directly from the epidermis ;
and other ganglia, already well developed and functional, shift bodily
from their original positions, and in some cases fuse with ganglia
originally remote from them.

Raschke (op. cit.) says that in the larva each of the first eight
segments (of the abdomen) has a pair of ganglia, and this statement is
certainly true of all the larve I have examined, and yet a pupa which
I killed when only half escaped from the larval cuticle had already
four pairs in the thorax, and none in the first segment of the abdomen.
During pupal life these four ganglionic masses fuse into one compact
mass, though its composite nature is always recognisable in sections.

The ganglia of the eighth segment (of the abdomen) at the
beginning of pupal life oceupy their typical position in the anterior
part of the segment, and are connected with the ganglia of the
seventh segment by connectives (or ‘“‘commissures”) nearly equal in

THE PUPAL STAGE OF CULEX. 21

length to the seventh segment. During the first two days of pupal
life these connectives vanish completely, and the ganglia migrate to
the anterior part of the seventh segment to fuse with the ganglia
of that segment. As with other composite ganglionic masses, the
composite nature of the ganglionic mass so formed is easily recognised
in sections, especially horizontal sections, even in the imago. In the
female the process goes a stage further. A pupa almost ready to
burst and give exit to the imago has still the arrangement just
described, but the imago, killed immediately after its escape, is
found to have no ganglia in the seventh or eighth segment, but in
the sixth segment are two masses; the first, the pair properly
belonging to the segment, lying at its anterior end; the other, the
‘double ganglionic mass formed by the fusion of the seventh and
eighth ganglia, lying at the hinder end of the segment.

In the male imago, however, the arrangement is the same as in
the advanced pupa.

A detailed description of the ganglia of the head and the changes
they undergo during pupal life would take me too far. The most
striking change is the very great increase in size which these ganglia
undergo, and the most interesting point is the way in which this
increase is brought about. The epidermal (‘“ hypodermal”) cells,
especially those near the borders of the eyes, proliferate freely, and
the cells budded off from their inner surfaces migrate inwards and
form the new cells of the ganglia. By this process the ganglia, which
at the beginning of pupal life were comparatively inconspicuous,
grow till they almost fill the head, and there are places in the
advanced pupa where the ganglia and the epidermis appear to be
continuous.

The sense-organs of the pupa itself are not of special interest,
that is, the organs which serve during pupal life as sense-organs.
The set have already been mentioned. The ocelli are those of the
larva, but they persist to the adult condition, the chief change
which they undergo being the development of an exceedingly dense
pigment. I have already referred to the common but erroneous
statement that the imago is devoid of ocelli.

The compound eyes belong properly to the imago, not to the pupa,
though they are probably sensitive to light in the pupal condition.
At first they are small (see Fig. 1) and devoid of corneal facets, but

22 C. HERBERT HURST.

they grow till they occupy the greater part of the surface of the
head.

So much has of late been written upon the eyes of insects, that
one should hesitate to add to that literature without having made
very special study of the organs in question. Still, one of the most
remarkable papers of the day (Patten, 11) has attracted so much
attention, and is so strongly opposed to the views of previous
observers, that the little I have already observed may be of interest.

Each eye is made up of a very large number of ‘“ elements.”
Growth of the eye consists in the addition of new “elements” at
its edge. Each new element is formed directly from the previously
unmodified epidermis at the margin of thé eye, and each arises
independently of the rest of the eye, as a separate invagination of
the epidermis. The cells, four in number, around the margin of
each invagination, persist as the “nuclei of Semper,” ‘corneal hypo-
dermis,” ‘corneal epidermis,” ‘‘cellules cristallines,” ‘“cellules de
Semper,” ‘refractive globules” or “spherules.” The invaginated
portion gives rise to all the other parts lying outside the limiting
membrane, with the possible exception of the pigment cells. The
elements are at first devoid of pigment.

The details of the development I have not yet worked out, and I
think it best to reserve further description for a future paper.

Other sense-organs developed during pupal life are antenne.
Antenne are, it is true, present already in the larva, but they have
no resemblance to those of the imago, and they are functionless
during pupal life.

The epidermis round the base of each antenna of the larva grows
rapidly, and as it is prevented, by the rigid and unyielding cuticle
of the shaft of the antenna, from growing forwards, it grows back-
wards, and becomes “telescoped” and much folded, and sections
through the larva show that the differentiation of the epidermis of
the different parts has already begun.

When the pupa escapes from the larval cuticle, much of the
folding is undone, but a portion of the telescoping persists at the
base of the organ, and this part gives rise to the large hemispherical
basal joint of the antenna of the imago.

This remarkable organ was described in the imago thirty-five
years ago by Johnston (13), but very imperfectly. Externally it is

THE PUPAL STAGE OF CULEX. 23

not conspicuous in the pupa, though it is just recognisable. During
pupal life its parts undergo considerable change, and these will be
best understood if I describe the adult structure first.

In the imago the antennz differ markedly in the two sexes. In
the female the shaft is longer than in the male, and the hairs with
which it is beset are less numerous and very much smaller. In both
sexes the basal joint is enlarged, and forms a nearly hemispherical
cup, with small cavity and very thick walls, covered and lined
with chitin. The shaft of the antenna arises from the centre of
the cup, and the chitinous floor of the cup is strengthened by a
series of radial thickenings. In the female the edge of the cup is
turned in, so that the apérture of the cup is narrower than the cavity
immediately below. The structure in the male is really an exaggera-
tion of this ; the edge is folded in so completely that it unites with
the floor, and the walls of the cavity of the cup of the female thus
come to be represented by a concave double disc, the two lamin of
which are closely united, and the space between them, the equivalent
of the cavity of the cup in the female, is here obsolete. The attach-
ment of the shaft to the floor of the cup appears to be rigid, and the
organ would appear to be adapted for the perception of sound-waves
coming in the direction of the axis of the shaft alone.

A section taken along the axis of the organ shows the following
structures: A layer of flattened epidermal cells, next to the cuticle of
the outer wall; then a layer of cells I shall call “ganglionic,” thickest
at the base of the cup, and continuous with the antennary lobe of
the “brain.” Between this layer and the inner wall of the cup
is a double (perhaps treble) layer of long narrow .rod-like cells, at
right angles to the surface, that is, radiating from the centre of the
cup.

These structures form a thick ring round the cup, perforated at
the base by the nerve supplying the shaft of the antenna.

The basal joint is supplied by an enormously large nerve arising
from the ventral portion of the supracesophageal ganglion at the side
of the esophagus. This nerve is broader than the abdominal double
nerve cord, and is independent of the nerve supplying the shaft of
the antenna, which lies ventral to it. The nerve, after entering the
organ, divides, one layer penetrating the “ganglionic” layer; another
runs between the ganglionic layer and the layer of rods, and a third

24 C. HERBERT HURST.

on the inner surface of this layer, supplying certain small rounded
cells lying between this layer and the base of the shaft.

All the cellular layers of this organ are epidermal in origin, but
the layer which I have called ganglionic stands, during the later
part of pupal life, in direct continuity with the superficial layer of
cells of the “brain,” and this layer in turn is continuous with the
deep layer of the epidermis of the head immediately behind the
base of the antenna. Whether the continuity of the ganglionic
layer of the organ with the brain is due to identity of origin, both
being budded off from overlying epidermal cells, or to migration of
cells from the brain into this organ, is difficult to determine, but
the cells of the cerebral lobes (‘‘ hemispheres ”) are larger than those
of the cup-like organ, while the latter resemble the cells of the
inner optic lobes very closely in size and in mode of staining.

This organ is already a conspicuous object in sections of the larva,
more conspicuous indeed than the “brain,” but the differentiation
of the layers is only completed late in pupal life.

THE REPRODUCTIVE SYSTEM.

1. The male generative organs of the adult consist of testes,
vasa deferentia, “‘ prostatic glands,” copulatory organ with a common
pouch at its base, and two pairs of gonapophyses. Of these last
the outer ones are a large pair of forceps for holding the female.
Both pairs are probably segmental appendages, and I have already
spoken of their origin in the larva.

The testes are a pair of cylindrical bodies already present in the
larva at the sides of the intestine in the sixth segment. They are
chambered, and the spermatic elements in the hinder chambers are
more advanced than those in the anterior chambers. The length of
each testis is that of the segment in which they lie.

The vas deferens of each side is a direct continuation of the wall
of the testis, and is a very narrow tube running backwards, quite
distinct from its fellow of the opposite side, but the two are closely
bound together in their hinder parts, and they open behind into the
common pouch.

The prostatic glands are a pair of elongated glandular tubes,
apparently simple, but seen in sections to be double, though the
cavities communicate behind before opening into the common pouch.

THE PUPAL STAGE OF CULEX. 25

The common pouch is a dilatation of the ejaculatory duct at the base
of the copulatory organ, and the latter is perhaps derived from a
pair of abdominal appendages.

The hinder parts of the vasa deferentia appear to be developed as
a forward outgrowth of the ventral wall of the common pouch, and
the prostatic glands are lateral outgrowths of the same. The
hinder part of each vas deferens is in some Culicide expanded to
form a vesicula seminalis of considerable size, but this is not the
case in Culex.

ul. The female generative organs are a pair of ovaries, oviducts
uniting behind to form a median oviduct, a median copulatory
pouch and three spermathecx opening into the last.

The ovaries correspond in size and position with the testes.

The median oviduct is formed by invagination in the region which
I take to be the ninth sternum, while the anus opens at the posterior
end of what I take to be the eleventh segment, so that there is no
common cloaca. This invagination is already far advanced at the
beginning of pupal life (Fig. 7), and during this period it grows
forwards, keeping pace with the forward shifting of the last pair of
ganglia, and at all stages lying just behind it, till the final ecdysis,
when the rapid shifting of the ganglia leaves it behind. Its anterior
end is, in the adult, near the anterior end of the seventh segment.

In the youngest pupe three flattened invaginations, the future
spermathec, lie upon the dorsal wall of this median oviduct.
During the pupal period the anterior end of each becomes spherical
and acquires a strong chitinous lining, The anterior ends of these
organs remain stationary in the eighth segment throughout.

The bursa copulatrix is a dorsal outgrowth of the invagination
which gives rise to the median oviduct, and is a small pouch lying
just behind and above the genital aperture.

I am painfully conscious of the fact that the foregoing account of
this interesting pupa is far from complete, but the pressure of other
work prevents my adding anything considerable to it at present.
As soon as I have time to do so, I intend to work out the details of
the development of the eye, but fear it will not be possible before
next summer.

26 C. HERBERT HURST.

In conclusion, I would express my best thanks, firstly, to the Council
of the Owens College, through whose generosity I have been able to
leave my work in Manchester; and, secondly, to my honoured
teacher, Herr Geheimrath Professor Dr. Leuckart, to whom I am
indebted for many kind hints, especially as to the literature of the
subject, and also for the loan of very numerous books and papers.

I subjoin a list of the books and papers to which I have referred

in the foregoing dissertation :—

1. SWAMMERDAM.—“ Bibel der Natur.”

2, RASCHKE.—‘ Die larve von Culex nemorosus.” Berlin, 1887.

3. A. M. Mayver.— Researches in Acoustics,” ‘“‘ American Journal
of Science,” 1874.

4. Lanpois.—“ Die Ton- und Stimm-Apparate der Insecten,”
“ Zeitsch. f. wiss. Zool.,” xvil., 1867.

5. Dimmock.—“ Mouth-parts of some Diptera.” Boston, 1881.

6. Paumin.—“ Zur Morphologie des Tracheensystems.” Helsing-
fors, 1877.

7. Huxtey.—“ Anatomy of Invertebrated Animals.”

8. MraLt and Denny.—“ The Life-history and Structure of the
Cockroach.” London, 1886.

9. Cuun.—-“Bau, etc., der Rectal-driisen bei den Insekten.”
Frankfurt a/M., 1875.

10. GRrABER.—‘“ Ueber den propulsatorischen Apparat der Insekten,”
“ Arch. f. Mikr, Anat.,” ix., 1873.

11. Parren.—“‘ The Eyes of nee: and Arthropods,” - « Mitth,
aus d. zool. Stat. zu Neapel,” 1886.

12. KowALevsky.—‘ Ein Beitrag zur Kenntnis der Excretions-
Organe,” “ Biolog. Central-blatt,” ix., 1889.

13. Jounston.—“ Auditory Apparatus of the Culex Mosquito,”
“Journ. Microscopical Science” (old series), vol. iii., 1855.

I was born in Littleborough, in Lancashire, September 6th, 1855.
After elementary education in private schools, I spent three and a half
years at the Manchester Grammar School ; three years as apprentice
to a firm of manufacturing pharmaceutical chemists; one year as
assistant to an analytical chemist ; one year as student of chemistry
at the Royal School of Science, South Kensington; one year as
science-teacher in a boarding school; three semesters as student of
biology and geology at the Royal School of Science, South Ken-
sington, under Professors Huxley, Thistleton-Dyer, McNab, and
Judd ; and one year at Owens College as student of zoology and
embryology, under Professor Milnes Marshall. I then entered the
University of Leipzig (1882), and while there was appointed
Assistant Lecturer and Demonstrator in Zoology in the Owens
College, Manchester, which post I still hold, along with the position

of Lecturer of the Victoria University.

The Council of the College generously gave me leave of absence
for the whole of the summer session of the present year to enable
me to return to Leipzig, where I have made nearly the whole of the

observations recorded in the accompanying dissertation.

CHARLES HERBERT HURST.
October 20th, 1889.

DESCRIPTION OF PLATE,

Illustrating Mr. Hurst’s Paper on the Pupa of Culex.

Fig. 1. Side view of the male pupa (x 10).

Fig. 2. Ventral view of the female pupa partially extended (x 10).

Fig. 3 to 6. Successive stages in the metamorphosis of the epithelium
of the hinder part of the stomach (Xx 225).

Fig. 7, Sagittal section of a very young female pupa (x 50).

Ant. Antenna. do. Aorta. Aft. Respiratory siphon. B. Buccal
chamber. CG. Cerebral ganglion. D. Gastric pouch. F. Fin. Fe 1.
Femur of the first leg. G. Ganglia. Gn. Outgrowth of “ninth
segment,” within which the gonapophyses develop. Hr. Halter.
H. Head. Ht. Heart. Jn. Intestine. Lb. Labium. Lobr., Zr. Labrum.
M. Malpighian tube. M.Ap. Its opening into the intestine. MS.
Mesosternum. Mi. Metasternum. Mz. Maxilla (first). Map. Its
palp. NC. Nerve commissures and ventral cord. Oc. Ocellus.
Od. Median oviduct. Op. Compound eye. J. Prosternum. Jf.
Rectum. S. Aperture of salivary duct. SD. Salivary duct. SG.
Subcesophageal ganglion. $i. Larval respiratory siphon introverted
into eighth segment. Sp. Spermatheca. Sf. Stomach. Ta 1, Ta 2.
Proximal joints of tarsi. 771, 7i 2, 713. Tibie. Tr. Trachea.
W. Wing.

I., IL, II1., etc. First to eighth segments of abdomen.

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