Miss C. A. Smith.
The Development of Anopheles punctipennis Say.
Psyche 21:1-19, 1914.

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. PSYCHE
VOL. XXI. FEBRUARY, 1914. NO. 1.
THE DEVELOPMENT OF ANOPHELES PUNCTIPENNIS SAY
Anopheles punctipennis Say, is a.strictly American form, of the so-called malaria mosquito. A. punctipennis and A. maculi- pennis, as well as A. crucians, have been constantly associated as malaria carriers, especially since Dr. Dupree's discovery of the parasites of malaria in the salivary glands of all three. But in the case of A. punctipennis there is growing up a reasonable doubt as tA whether in the north it is really a malaria carrier, or at least whether the malaria carried by it is not a different form from that conveyed by A. maculipennis. In 1903 Dr. J. B. Smith of New Jersey stated that in that state only A. maculipennis had been actually demonstrated to be a malaria carrier; and so far as known this has not been proved to be otherwise. In 1903, also, Hirshberg of Johns Hopkins published the account of his note- worthy inoculations of fifty-eight females of A. punctipennis. He allowed them to bite patients afflicted with estivo-autumnal mala- ria, without finding the parasites in the walls of the stomach, or intestine, in the body cavity, or in the salivary glands. In the opinion of the experimenter himself, however, the fact that out of forty-eight similar inoculations of A. maculipennis only eight were infected detracts from the certainty of the results with A. punti- pennis.
Breeding Places.
Larvae, pupae, and eggs have been taken from seven different pools near Ithaca, at Forest Home, from October to the middle of August. The people living close by do not have malaria, and on the evidence of reliable citizens, have not had it 1 This study was carried on in the entomological laboratories of Cornell University under the kindly supervision of Dr. J. G. Needham, to whom I am greatly indebted for constant advice and help.
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9 Psyche [February
for at least fifteen years.
Whereas, down on the flats at the head
of Cayuga Lake, and where A. maculipennis is common, there is more or less malaria all the time. Forest Home does not pro- duce A. maculipennis at all, while A. punctipennis is there in comparatively great numbers. This is not unlike the situation at Baltimore where in 1902 Hirshberg and Dohme reported that "A. punctipennis breeds in the higher sections, while A. maculi- pennis is to be found in lower localities." Local Occurrence.
On October 91 a great many {dl-grown larvae, also many pupae, were found in pool 1, which is a drinking place for cattle, fed by a spring, and never dry. It was covered with Lemna, although not densely. A few masses of Spirogyra and Cladophora were there. Larvae of may-flies, dragon-flies and damsel-flies, various beetles, mostly Dytiscidse and Hyrophilidse, great quantitites of small crustacea and spring-tails, a great many chironomids of various species, and with some oligochsete worms, a few hydrachnids, and hydras-all these and some other forms, including Culex, were present in the pool. Water-striders and whirligig beetles were very few. The Lemna certainly furnished some food to the larvae of A. punctipennis, for they were often seen to be brushing the leaves, after being brought into the laboratory. The larvse were found in plentiful numbers, but always in separate groups, as if each had developed from a different laying of eggs. Pool 2 was of an entirely different character, and about a quarter of a mile away. It was very small, not more than four feet in width at the widest place, and about a foot deep. The bottom was covered with dead leaves, and the water was clear, and there were no visible algse or other plants in it. It was deeply shaded, and contained just one brood of larvae, of less than fifty specimens, all of about the same size, and nearly ready to change to the pupa. They were almost black with a white mid-dorsal line. They were thf largest larvae that we have found. None found in the spring were so well developed as these October forms, from which came very large adults.
All the adults developing in autumn are exceedingly hardy. One
female emerged from the pupa on November 3, and was left with no food or water. On December 2 it was still alive and eagerly drank water and fed on moistened dates. It lived for three weeks more with access to this food, never having tasted blood, having



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19141 Smith- Tk e Development of Anopheles punctipennis Say 3 spent its whole life in the cage.
In the spring and summer they
die within three days, under the same treatment of water and food.
A larva of A. punctipennis was frozen solid on December 3, was thawed out on December 4, and in an hour was apparently normal. The full-grown larvae found in the autumn differed more from one another than those found in the spring and summer, when they are mostly green or brown. In autumn there were many more of the striped and speckled forms. They all developed into Ano- pheles punctipennis, with some differences in size. Pool 2 did not yield larvae of A. punctipennis in the spring, having dried up completely, and pool 1 was much later in devel- oping them than 3 which was near by-within a hundred feet. Pool 3 on May 24 yielded larvae of all sizes. Most of them,
however, were ready to pupate.
They represented the first laying
of the spring.
The water of this pool was several feet deep at one end, and clear and cool.
No larvae have been found at this spot,
in the shade of an overhanging bank where Spirogyra grows deep, giving its clear green hue from below the surface. A few feet away, in shallow water with Spirogyra protruding from the surface, were plenty of larvae. With the Spirogyra,knd also in fruiting condition, was plenty of Zygnema. In and above these mats of fruiting algae, many of them feeding down among the fila- ments, were larvae of A. punctipennis, nostly full grown. And with them in great numbers were several species of Chironomid larvae, weaving their houses of the delicate threads, and feeding there as well. This pool was visited at intervals, until on June 10 there were scarcely any larvas, but a few pupae and plenty of empty pupa cases. On June 17, however, great numbers-of very small ones from 1 to 2 mm. and less were to be found again in pools 3 and 1. In 1 they were found only among the Lemna, and in 3 only in mats of Spirogyra and Zygnema. Meanwhile, multitudes of toad tadpoles had developed in pool 3, and along with their growth went the disappearance of the mats of algae from the sur- face, and also of Anopheles punctipennis from that part of the pool. They were found a month later in almost the same place in great numbers among fruiting Chara, which had spread from the deeper part of the pool. There was protection and food among its filaments.




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4 Psyche [February
Pool 4 was a temporary pool beside Fall Creek, about six feet in length and three feet or less in width. It was sheltered by an
overhanging stump, but was in the bright sunlight nearly all day. Pools 3 and 1 were sunny also. The only alga in it was Mougeotia which was in vigorous fruiting condition. Embedded within it were many masses of Chironomid eggs, as well as many full-sized Chironomid larvae within their filmy homes. Many very small larvae of A. punctipennis were at the surface of the water among the delicate filaments. A great many minnows were darting about below the algse, also small Coleoptera. There were no water- striders or whirligig beetles, or other surface feeders. After two weeks the Mougeotia had sunken to the bottom and the larvae of A. punctipennis had disappeared, excepting a few large ones. Pool 5 was near 4 but in the deep shade, the larvae being con- fined to a mat of Spirogyra.
Here the water flowed slowly. Pool
6 was near, but in sunlight all day, and the larvae were concen- trated among the filaments of a mass of Mougeotia, and were to be found nowhere else in the pool. The water flowed quite rapidly by, but the Mougeotia was anchored.
All the larvae found in the Mougeotia were exceptionally trans- parent, even in the older stages. Just after molting, they were almost as clear as glass, and were at all times the best ones to study. On the other hand, larvae found in pool 7, a muddy dark pool in a very shady place, were always very dark and opaque. Here the larvae were found among the large floating leaves of a Polygonum, and nowhere else in the pool. Feeding Habits. A number of larvae of all ages were put in a watch crystal with small masses of Zygnema and Mougeotia, all in fruiting codition. Cladophora was added also. A good deal of surface material was devoured by all the larvae. The younger ones were more particular in the matter of particles, rejecting a good many. They moved around among the filaments, head bent downward, finding plenty to their liking on the Cladophora, as well as on the others. They merely brushed off the particles from the larger filaments. A larva of only 1.5 mm. was seen to swallow a filament of Zygnema, and to make an effort to swallow large ones of Spirogyra, of which it would merely chew the broken ends. Many small larvae were seen eating the delicate Mougeotia. When the food is merely surface particles the head is turned a



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19141 Smith-The Development of Anopheles punctipennis Say 5 half circle with mouth parts upward, next to the surface film; but when feeding on a filament, usually the mouth parts are under- neath and the head is bent downward. When feeding in this position, the antennae and maxillary palpi are spread wide apart, bristles and hairs all extended. The large lateral bristles, outside the antennae are held out like a fan, and the six branched hairs on the top of the head are raised, making a complete hedge above the rotating brushes.
If particles of food are scarce in the surface film, the young larvae bend and run their brushes over their own bodies as far as possible, the limit of their reach being apparent by the Vorticellae and diatoms clinging to the anterior parts of some unlucky individuals. Naturally these are not so active in their movements as the others. In eating the filaments they swing the brushes furiously until a filament is brought within reach. Or they dive in among the filaments, head down and brushes rotating. Sometimes they merely crush out the contents of the cells, at least in the case of large zygospores of Spirogyra, leaving the empty filaments. In that case they grasp the filament anywhere and run it through the mandibles, swallowing only the green parts. The delicate filaments of Zygnema and Mougeotia are swallowed entirely, also slender filaments of Spirogyra. They show considerable prefer- ence for the more delicate filaments. Usually they draw the fila- ment into the mouth with the rotating brushes, bite it in two, and then rapidly draw in one broken end. They rarely leave a fila- ment partly consumed, and often go back to the other broken end. . The food of the larva of A. punctipennis, is not necessarily entirely of an herbivorous nature. Once I saw two young water fleas (Simocephalus) almost dragged into the abyss. They strug- gled valiantly and escaped by setting up counter currents with their feet. The three currents could be seen close to one another. One went too near and lost an antenna which was broken and swept in by the brushes. It drew back but was temporarily in- capacitated and unable at once to leave the vicinity. Finally, it escaped. A little Chydorus all but lost its life, being drawn in by the brushes, but escaped.
Fatalities.
Dr. J. B. Smith in his report on the Mosquitoes of New Jersey (1904) gave a detailed account of the enemies of mos- quitoes. He remarked that the larva actually faces greater dan-



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6 Psyche [February
gers than does the adult. He placed as enemies, first weather conditions; then diseases,
of which we know little. We have
seen several conditions which relate to this subject, however. Four young larvae that had passed their second molting were every one of them afflicted with a protruding intestine, which ex- tended out from the body for fully half the length of the larva. This condition lasted for more than a day after they were taken from the pool. A scarcity of food,-for they were kept in clear water for twenty-four hours-evidently cured them. They were normal on the second day after.
Spirogyra furnishes food for the growing larva, but it also shel- ters enemies. It is the favorite habitat of several species of chi- ronomids, and in just so far is it a check to mosquito development. The chironomid larvae build their tubes of the algae filaments, and seemingly of other things too.
In six different instances we have had evidence that some chi- ronomid larvae destroy the larva of Anopheles punctipennis, and feed upon its tissues, as well as using portions of its body to fill in the crevices of their houses.
In all the cases, the Anopheles.
was in the quiescent state just preceding or following the molting process. In one case the chironomid built its tube close up beside the body of the dead A. punctipennis, and gradually transferred the tissues into the walls of its home. In another case a chirono- mid was seen to swallow a part of the dark mass of the dead mos- quito larva, and the digestion in the chironomid was watched through the transparent organs. A healthy larva of* A. puncti- pennis was supposedly alone in a dish of water with algae for food. It was left over night. In the morning its head was torn from its body and was floating at a distance. The only living animal in the dish was a chironomid larva.
Development.
Careful observations on the swarming of Anoph- eles punctipennis have been reported by Knab (1907). The males were seen to swarm a little before 5 o'clock of a sunny after- noon in October. They came from different directions to form the swarm which contained less than a hundred mosquitoes. They circled about above a projecting mass of foliage. Mating of a number of individuals occurred, and by 5.30 o'clock the swarm began to diminsh.
In the opinion of Kulagin (1907), based upon at least six years



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19141 Smith-The Development of Anopheles punctipennis Say 7 of observation of Anopheles in Russia, mating occurs in the autumn except in a few isolated cases, where it occurs after hibernation. The hibernating females deposit the eggs during the whole of the next spring and summer, and the September and October larvae result from the isolated cases. Accordingly, he is of the opinion that there is but one generation in a season, the newly developed forms not depositing eggs until after hibernation. There has been insufficient study of this subject.
The eggs are laid singly and a number of times by each indi- vidual during a single breeding season. Dr. Dupr6e found that specimens of A. punctipennis, which were kept in the laboratory and supplied with blood, would lay at six or seven different periods, with a total number of more than 2,000 eggs. As many as nine layings were noted in one case. The eggs, from 100 to 300 at a be, are deposited separately, or sometimes in clusters of just a few. They are apt to float below the surface, although some of them are on the surface. Stirring the water will bring a good many eggs to the surface, from which they soon disappear again. They seem not to possess so perfect a means of keeping afloat as is recorded of the eggs of Anopheles maculipennis, which has the light clasping membrane about the whole rim of the egg. In A. punctipennis this clasping membrane is restricted to the sides. It is very delicate, apparently, for in several instances it was badly broken, or had perhaps been eaten away. A young crustacean was seen to devour the clasping membrane from one side of an egg.
The egg (fig. 1) is .55 mm. in length, mottled in appearance, and dark brown in color with blotches of silvery white. At the end are scattered light and dark spots, the former of which have been called "knobs" and which are arranged in a more or less regular pattern. Sometimes the eggs lose nearly all their dark color, and float. They float with the concave surface underneath. The larva breaks through the convex surface at the larger end, leaving a little fragment of the shell cut out on three sides, and bending over like a little canopy at one end of a little boat. First Stage of the Larva (fig. 2). At first the young larva spends periods of time quietly.
Suddenly it will start a strong current of water by means of the rotary brushes, which are large in propor- tion to the remainder of the body. These are fully developed



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8 Psyche [February
from the first.
With these it sweeps in any small particles in the surface film.
It takes but a short time to exhaust the immedi- ate supply, and so the larva moves from place to place. Fre-
quently it bends and brushes over its whole body as far as it can reach, removing anything like vorticellae or diatoms. Sometimes these develop too rapidly and it is not at all uncommon to find larvae less than three days old, thickly fringed with these sessile forms. Of course, such as attach themselves to the head and anterior part of the thorax remain there, being out of range of the jaws.
If food particles are scarce, the larva will brush over any algae or other plants that may be at the surface. One very small larva was seen to remain down below the surface film for about half a minute in search of food. One of these very young ones was seen to eat a short filament of fine Spirogyra, when the larva was less than two days old. It refused a row of diatoms. It started back from an on-coming rotifer immediately in front of it. The same
rotifer ran into and over the long anterior bristles from the thorax and the young larva gave no response. But when it happened again from the front, the larva darted away. The most con- spicuous movement of an Anopheles larva of any age is its turning of the head through an angle of 180 degrees, when feeding. The young larva does this with energy whenever feeding on surface particles.
It turns the head always in the same direction-counter- clock-wise, as was determined by the periodical disappearance of a Vorticella which was attached to the side of the head. This always moved downward and out of sight before appearing at the other side.
The young larva measures from .7 mm. to .8 mm. in length, not including the hairs at either end of the body. The second and third segments of the thorax have not yet united and the head is very prominent. The dorsal surface is dark brown in color, except where there are conspicuous yellow spots, which are caused by the cenocytes within. On the segments of the thorax and on five of the segments of the abdomen these yellow spots are very noticeable, especially on the third segment of the abdomen. The
head is colored an even gray, with a dark spot in the center, and one small spot on each side. The single eyes and cccollar" are reddish in color and remain so during the first day. The branched




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19141 Smith-The Development of Anopheles punctipennis Say 9 hairs from the sides of the head, outside the antennae, are rela- tively longer than in any succeeding stage. The six bristles across the dorsal surface of the head (a) are unbranched, as are the two . hairs which lie between the brushes and the central terminal hairs, which remain unbranched throughout the larval life. The most conspi~uous structures of the head of the young larva are the long simple hairs on the top, and the long lateral hairs at the side of the head, which with hairs of the antennae form a barricade. Four simple hairs and the absence of a rudder-like tuft on the ninth segment (a) of the abdomen, are characteristic of the first stage.
When about two days old the larva will have changed con- siderably in appearance. It will measure by this time about 1.5 mm. in length. The thorax will have become distinctly formed and the long hairs will appear somewhat shorter. The head will have quite a different shape from that of the larva of one day. It will have become very dark, especially the "collar" which will be almost black. The head is much narrower and deeper. The integument has a snug, tight-fitting look (fig. 3). This figure shows a larva in the act of molting for the first time. In this case it died in the process, the integument of the thorax having failed to split apart.
For some time previous to each of the molting processes in the larval life, the head shows this peculiar narrowing, with the in- creased width and deepening color of the "collar. " 'The larva measures just the same after the molting process as before it. In this case it was 1.5 mm.
Second Stage 01 the Larva.
The changes at the time of the first
molting are conspicuous. Although the thorax is distinct and large, the head is even larger (fig. 4). The four dorsal hairs of
the posterior end have become eight (a), and the ventral tuft (b) has appeared, consisting of two rows of long branched hairs with a fan-like arrangement. On the head the simple hair just inside of each rotary brush (d) has become much branched, and is used for combing out the brush. The six simple hairs lying back of the brushes, on the top of the head, have become very much branched.
The eyes are still little developed.
The yellow spots on the dorsal surface have increased in number and density, being most conspicuous on the third, fifth, and eighth segments of the abdomen, and on the thorax.



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10 Psyche [February
Within the thorax a clear specimen shows a number of parts: the anterior lobes of the heart with a large valve, the contractions of which synchronize with the throbbing between the tracheae of the abdomen; the beginning of the arch of the tracheae, surround- ing the food tube, and from which branches are later given to the organs of the thorax; the three pairs of imaginal buds. Within a few hours after the first molt, one larva was eating vigorously of fine filaments of algae, and preferably of surface particles. It swallowed the filaments from the end, just as does the full-grown larva.
By the time the larva is six days old, it begins to show signs of the approaching second molt, which occurs from the seventh to ninth day. As before, the head becomes narrow and the "collar" dark and broad. One measured 3.2. mm. at this time, making a growth of .7 mm. in about two days.
Third Stage of the Larva (fig. 5).
The most important changes
which are evident after the second molt are: the rapid increase in the size of the thorax accompanied by the enlarged internal organs; the appearance of the ommatidia of the adult eye, around the larval eye; the disappearance of two pairs of the long hairs and the general shortening of all the thoracic hairs; the darkening of the integument of the head, which shows an unexpected pattern in the maculation. Eight specimens examined at this age showed almost exactly the same arrangement of pigment on the head. It may not be constant, but it is certainly more so than that of any other stage.
Respiratory Siphons (figs. 17,18, 19).
At this time it is possible
to see, along with the rapid growth of the wing buds, the forma- tion of the pupal respiratory siphons in the prothorax. From the time of the first molt, a pair of straight tubes in each side of the thorax has been evident. Now they have become more conspic- uous. They seem to change their position, sometimes being close up to the anterior wall of the thorax, and sometimes they lie with the end quite away from the wall. Developing near by, at an angle with the first tube, and penetrating also through the wall, is another structure, tube-like at first, but after a time changed to form a part of the now plainly visible respiratory siphon. The siphons, also, are frequently drawn away from the integument, at other times being pushed up close to it.



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19141 Smith-The Development of Anopheles punctipennis Say 11 Fourth Stage of the Larva.
By the twelfth or thirteenth day
the larva has increased in length to 4-5 mm., and it then shows the signs of an approaching molt, the darkening of the head and collar and the widening of the latter. Molting is getting to be a critical process and a great many lose their lives in the act. They are conspicuously large and cannot protect themselves meanwhile. Sometimes the integument of the head does not separate off easily. One was seen in which the head had not molted for more than