The granular appearance in the stained cell is caused by an accumulation of secretion by which the nucleus is displaced to the periphery of the cell. This secretion in the fresh gland exudes from the cell in clear, refractile globules. The appearance is then an artificial one. In the hardened gland the majority of the cells have this fine granular appearance, but in some of the cells of the terminal portion of the acinus a few globules only of secretory matter may be evident. These granular cells do not stain deeply with hematoxylin, and the nuclei, pushed to the periphery, show apparently signs of degeneration. The portion of the acinus lying near the duct may, however, show considerable variation from the type in the case of Anopheles. The cells are smaller; the nucleus and protoplasm are displaced to the periphery by a clear body in the center of which is a dark area continuous with the cluct lumen. This is not always present in Culex. In the latter, however, in the same portion of the gland, that is, the portion nearest the duct, the epithelium may be columnar, with centrally placed nuclei and no secretory substance. Sporozoites occur in all three acini in Anopheles, but, according to some observers, most abundantly in the central lobe. The secretion of the cells of the acini passes into the lumen by means of minute openings into the cell.
A band of muscle arises close to the origin of the first pair of legs and passes outside the salivary gland on either side. It is probable that the contraction of these bands would exert pressure on the glands and so promote the excretion of saliva.
Before considering this question, on which Schaudinn has made some interesting experiments, it will be necessary to return to the esophageal diverticula. Besides the gas bubbles which they contain, there are also present a trace of fluid and also bacteria, or "molds." They are always present in greater or less quantity. Before sucking they are few, but while digestion is going on in the stomach they increase immensely, forming a thick layer over the wall in all stages of proliferation. Even in the newly hatched mosquito they can be found, and, in fact, in the egg, in the larva? (in the gut), and in the nymphae. Where scanty, as in the newly hatched mosquito, they can be developed in large quantities by feeding the mosquito on sugar solutions, and Schaudinn was led to believe that they were the agents which produced the carbonic acid with which the diverticula are filled. When a mosquito is thus fed on sugar solutions, the gas development sometimes reaches such a pitch that the gut is enormously distended with gas and death of the mosquito may ensue. Such mosquitos are often seen in the tropics; they look as if they were suffering from some emphysematous affection. Schaudinn next observed what took place when an insect punctures. This may readily be studied under the microscope by confining the mosquito under a watch glass or other suitable transparent covering. Observed under the microscope, the respiratory movements of the mosquito are seen to be going on regularly and moderately. Suddenly a violent contraction of the whole abdomen sets in, which it is interesting to observe is not seen if the mosquito is feeding on fluids of any kind. What, then, is the meaning of this enforced contraction? Schaudinn supposes that it is induced by the presence of carbonic acid gas, which is present on the superficial layers of the skin, and that it leads, by its presence in the tissues when inspired through the stigmata of the body, to violent muscular contraction. This idea he sought to confirm experimentally by confining a mosquito in a hollow slide and then producing an atmosphere of carbonic acid gas by the addition of acid to some particles of chalk placed in the hollow slide. This violent contraction he was actually able to bring about occasionally in this way. What, now, is happening in the pharynx and proboscis? This can be readily studied at the same time by carefully confining the proboscis under a cover glass in a drop of fluid, such as glycerin or salt solution. Observing under the microscope, it is then seen that during this violent contraction of the abdomen the gas bubbles of the diverticula are extruded from the tip of the proboscis, and, together with them, large quantities of the " molds" that the diverticula also contain. Probably at the same time the secretion from the salivary gland also reaches the site of puncture, when the mosquito is piercing the skin, the .flow being promoted by the pressure of the salivary muscle on the glands. These substances have then been ejected; and, if the process is further watched, it is seen that they float away from the tip of the proboscis; but (and this is a very important fact), both gas bubbles and bacteria may be sucked back again almost completely. What is the function of these secretions ? It has always been taken for granted that the irritation of the mosquito puncture is due to a drop of "poisonous" saliva injected into the wound, and that probably an additional function of the saliva was. to prevent the coagulation of the blood in the same way as it is generally accepted that the secretions from the pharynx of a leech act on the blood during the process of sucking, but Schaudinn's observations lend no support to this view; for if the saliva has this function, then presumably, by isolating the salivary glands and rubbing them into the skin in a superficial wound, the irritative effect should easily be got. The glands, as we shall see under the section Technic (p. 217), are easily isolated, and they may be rubbed up in a little salt solution and then rubbed into the wound. The result is entirely negative: no trace of the irritative effect and reddening is obtained. Experiments have been made also by Schaudinn and others to test what the action of the salivary secretion is on the blood. The results are somewhat contradictory, some observing no trace of a hemolytic effect, whereas Schaudinn obtained some evidence of this. As to any anticlotting power, the experiments are also not conclusive. The esophageal diverticula, as we have seen, contain carbonic acid gas, and it is to this constituent that Schaudinn is inclined to attribute the fact that the blood does not clot at the site of puncture; experimental proof of this requires still to be obtained. To what constituent must we attribute the irritation, if it is not due to the saliva? Schaudinn was able to show that it is due to the bacteria contained in the diverticula. He isolated the diverticula, pressed out the carbonic acid gas, and then transferred them on the point of a needle to a minute scratch made in the skin, rubbing them well in. At once the characteristic irritative effect of a mosquito bite was experienced, accompanied by redness. The effect was indeed more marked than in the case of the mosquito, and lasted longer, and in one experiment, where he used the contents of a diverticulum filled with the bacteria, a thick and painful swelling an inch in diameter was produced, which lasted a week. Schaudinn then attributes the irritation of the mosquito's puncture to the enzymes of these commensual "molds," which, he says, are always present in the mosquito in all stages of its life history-egg, larva, nympha, imago. This most interesting discovery will, of course, require much further work devoted to it; at present, however, he considers that these " fungi " belong to the Mycetae, and possibly are nearly allied to the Entomophthorece. It is interesting to note that at the end of suction a few only of these commensual molds are found in the diverticula, together with traces of blood. The blood, however, furnishes sufficient sugar for the molds to thrive upon and multiply in adequate supply for the next puncture. The molds that have been ejected may be, as we have seen, sucked up again and find their way into the stomach, and, in fact, here during digestion they produce mycelial forms and very minute fruit organs, which also can be found in the egg. What, finally, are the processes involved in the act of suction? As soon as the stilets have pierced the skin and blood bathes the end of the proboscis, a powerful abdominal contraction occurs, which has the effect, as we have seen, of expelling the carbonic acid bubbles from the diverticula, and, together with this, the bacterial enzyme and the salivary secretion. The bubbles are then covered with a layer of blood, in which are mixed up the enzyme and the salivary juice. The enzyme produces irritation at the site of puncture, resulting in increased pressure and flow of blood, while the carbonic acid prevents coagulation of the blood. As soon, now, as the abdominal contraction is ended, a state of negative pressure exists in the cavity of the proboscis and the bubbles, with their covering of blood, molds, and salivary secretion, are sucked up by the capillarity of the tube of the epipharynx. The action of the pump then comes into play, and the contents are passed on into the stomach, aided also by the peristalsis of the esophagus. This peristaltic and pumping action takes place in the interval between two abdominal contractions, and is repeated four or five times, so that the spacious esophagus, the crop, and the diverticula become completely filled. Then ensues another abdominal contraction: the pump returns to its normal position, the valve between the esophagus and pharynx closes, and the blood is forced on into the midgut by the pressure of the abdominal contraction. During this passage, as we have seen, the crop is expanded and filled with blood, and the esophageal valve is opened, so that the flow into the midgut is unimpeded. Often this process proceeds so rapidly and effectively that it is a common sight, in feeding experiments with the Anophelince, to see the blood ejected from the anus of the mosquito in considerable splashes on the sides of the vessel in which they are kept.