The Function of the Anal Gills of the Mosquito Larva

THE FUNCTION OF THE ANAL GILLS OF

THE MOSQUITO LARVA

BY V. B. WIGGLESWORTH, M.A, M.D.

(From the Department of Entomology, London School of Hygiene and Tropical Medicine.)

(Received 15th April, 1932.)

(With Four Text-figures.)

IN a recent paper (Wigglesworth, 1932 a) the theory was put forward that the chief function of the so-called rectal glands of terrestrial insects is the reabsorption of water from the excreta in the rectum, and it was suggested that many of the sup-posedly respiratory structures in aquatic insects, such as the anal gills of the mos-quito larva (which may perhaps be looked upon as homologous with rectal glands that have prolapsed through the anus), might also be concerned in absorbing water, not from the excreta, but from the surrounding medium. It has been shown in another place (Wigglesworth, 1932^) that the properties of the anal gills are such that they must almost certainly absorb water, but in the present paper this hypo-thesis will be proved experimentally.

Full-grown larvae of the yellow-fever mosquito (Aedes (Stegomyid) argenteus Poir) have been used for all the experiments. The internal structure of this larva is readily seen in living specimens, but this is made easier if the larvae are kept for a week in clear water without food, so as to reduce the quantity of fat globules in the fat-body.

GENERAL STRUCTURE OF THE LARVA.

Function of the Anal Gills of the Mosquito Larva iy

protrude from the anus. There are five Malpighian tubes, which discharge into the "pyloric chamber." After a short loop running forwards on the surface of the mid-gut, they turn back and end around the rectum.

pv

Fig. i. A, anatomy of larva (semidiagrammatic): ac, anal canal; ag, anal gills; c, caeca; int, intestine (hind-gut); mg, mid-gut; mt, Malpighian tubes; pc, pyloric chamber (hind-gut); pv, proventriculus; r, rectum; rt, respiratory siphon; tr, one of the main tracheal trunks. The figures i, ii, etc., indicate the respective abdominal segments. B and C show cros9 sections of the hind-gut, B through the intestine (int), C through the rectum (r).

Fig. 2. A, larva with ligature between fourth and fifth abdominal segments. B, the same after im-mersion for 6 hours in iM glycerol. C, larva with ligatures round neck and between sixth and seventh abdominal segments. D, the same after immersion in tap water for 2 hours. (From camera lucida drawings.)

PERMEABILITY OF THE CUTICLE TO WATER.

If the larva is immersed in hypertonic (2M) dextrose or glycerol, it quickly shrinks, showing that the cuticle is permeable to water.

But if this experiment is repeated after a ligature of fine hair has been tied round the middle of the body (at, say, the fourth abdominal segment), although the part

i 8

of the body behind the ligature shrinks rapidly as before, being appreciably shrunken in 15 min., the part in front of the ligature shrinks very slowly and is scarcely altered in 6 hours (Fig. 2A, B).

These experiments show that the hinder end of the larva, i.e. presumably the anal gills, is far more permeable to water than the other parts of the body.

UPTAKE OF WATER THROUGH THE GILLS.

If the larva is ligatured between the fifth and sixth abdominal segments, it is clear from Fig. 1 that no fluid can be discharged by the Malpighian tubes into the hind-gut; and if it is ligatured also round the neck, it will be unable to swallow water. Fig. 2 C and D show the changes occurring in a larva treated in this way and immersed in fresh water. The Malpighian tubes become enormously distended above the obstruction, and the hindmost part of the body gradually swells; some-times the swelling is so intense that the anal canal may prolapse, or spontaneous rupture of the gut may occur at the anus. The segments of the body between the ligatures show no change, or swell comparatively slightly. Table I shows the course of the swelling expressed by linear measurements between arbitrary points in the larva.

Table I. Successive measurements of larva after ligature between sixth and seventh

abdominal segments at 11-25. A between two arbitrary points in front of ligature: B,from ligature to tip of gills. Units are divisions of micrometer eyepiece.

A B 11.25 ajn. 7i 86 11.30 a.m. 72 87 Time

11.45 a-In

-71 89 12.15 p.m. 71 90 1.15 p.m. 71 91 2.30 p.m. 71 92

Function of the Anal Gills of the Mosquito Larva

EXCRETION OF WATER BY THE MALPIGHIAN TUBES.

If the hind-gut of a larva is watched under the microscope, it can be seen that fluid accumulates in the "pyloric chamber" (see Fig. 1) until a small drop has collected, which is then carried down to the rectum by a wave of peristalsis. This process is repeated every 2 or 3 min., and sometimes a fragment of the contents from the mid-gut is also carried down to the rectum. The fluid is evidently secreted by the Malpighian tubes, for the contents of the mid-gut are almost solid.

If the larva is ligatured between the fourth and fifth segments of the abdomen, i.e. in front of the forward loop of the Malpighian tubes, this continuous excretion

Fit?. 3 • Movements of fluid in tracheoles of gill in larva ligatured between sixth and seventh ab-dominal segments and immersed in tap water. A, at commencement of experiment (io.oajn.); B, 10.05 a.m.; C, 10.10 a.m.; D, 10.15 ajn.; E, 10.30 ajn.; F, 12.0 noon.

of fluid still occurs; and under these conditions there is no swelling of the body behind the ligature. Fluid is evacuated from the rectum from time to time; either in small quantities every few minutes or in a larger quantity at intervals of 15-20 min. In either case it seems as though the quantity of fluid passing down the intestine is less than that evacuated by the anus; and this suggests that fluid is being reabsorbed in the rectum. In a later paper (Wigglesworth, 1932c) conclusive evidence will be given that this is actually the case.

Since there is no noticeable change in the volume of the larva behind the ligature, the fluid eliminated must represent fluid absorbed by the gills. The volume of this fluid is difficult to estimate, but it certainly does not exceed the cubic capacity of

two anal gills per hour. None the less, this fluid is ample to keep the Malpighian tubes well flushed with water, and they never contain any solid uric acid, even when the larva is on a pure protein diet. This state of affairs is in marked contrast with the adult mosquito (in which the larval tubes persist) where the supply of water is so limited that, except just after a meal, the Malpighian tubes always contain much solid uric acid (Wigglesworth, 1932 a).

PROPERTIES OF LARVAE WITHOUT GILLS.

Larvae can be readily deprived of gills by immersing them for 2 or 3 min. in 5 per cent. NaCl or in ./V/50 NaOH. As already described (Wigglesworth, 1932 b), these reagents destroy the cells lining the gills, and on restoring the larvae to pure water the gills of many of them blacken and slough away. In a few days they have healed completely, leaving only four little scars. These gill-less larvae can live and be-come adult, but they seem to grow more slowly than normal larvae in the same culture.

Larvae devoid of gills have been ligatured just in front of the Malpighian tubes and the elimination of fluid from the anus observed. Immediately after ligaturing they usually discharge some of the solid material from the mid-gut, and sometimes a little fluid may be passed during the next 20 min. or so, but thereafter, although they have been watched for nearly 2 hours, they pass very little fluid and in some cases none at all. It is noteworthy that even when no fluid is being discharged from the anus, occasional drops pass down the hind-gut. This affords additional evidence for the reabsorption of water in the rectum.

If gill-less larvae are ligatured at the sixth abdominal segment, there is not the excessive distension of the Malpighian tubes above the obstruction which occurs in the normal larva, and the swelling of the body behind the ligature is almost negligible even after 2 hours (see Table II, and contrast with Table I).

Table I I . Successive measurements of gill-less larva after ligature between sixth and

seventh abdominal segments at 2-15. A, from ligature to tip of siphon; B, from ligature to tip of anal segment. Units are divisions of micrometer eyepiece.

A

2.15 p.m.

72 SO

2-45 p.m.

72 5°

Time

3.15 pjn.

72 SO

3-45

P-m-72 5°

4.15 p.m.

73 Si

These experiments show that only a negligible quantity of fluid is taken in through the skin apart from the anal gills.

INGESTION OF FLUID BY THE MOUTH.

Function of the Anal Gills of the Mosquito Larva 21

brushes and mouth-parts, and collect in the buccal cavity. When a considerable bolus has accumulated, the mouth-parts are drawn in, and the bolus is swallowed without any noticeable amount of fluid; although, of course, the particles must be moist with water.

The particles of dye ingested in this way are confined within the peritrophic membrane as a solid black column. But the dye quickly appears in solution outside the peritrophic membrane, both in the caeca and in the uniform part of the gut behind; and this blue solution is subject to the peristaltic waves which pass over the mid-gut. These waves usually run from behind forwards, and will therefore tend to carry the fluid to the caeca (see Fig. 1). Trypan blue is not absorbed by the mos-quito larva and the contents of the caeca become gradually darker, and in 2 or 3 hours they consist of blue-black masses of solid dye. There is no solid dye elsewhere in the gut outside the peritrophic membrane.

Similar results may be obtained with indigo carmine, which is not absorbed from the intestine, and ammonia carmine, which is absorbed very slowly.

It is evident from these observations that there must be a continuous absorption of fluid in the caeca. We have seen that no appreciable amount of water is swallowed by the mouth; therefore the fluid that is being absorbed must have been secreted by the intestine into the lumen and carried forwards to the caeca.

Now, as shown by Frederici (1922) and Samtleben (1929), the mid-gut behind the caeca consists of two distinct segments with clearly defined histologicaL differ-ences. These correspond with the regions observed by Van Gehuchten (1890) in his classical work on Ptychoptera, the anterior half being regarded as "absorbing cells" and the posterior half as "secreting cells."

Taking all these observations into account, it seems probable that most of the fluid to be seen in the mid-gut of the larva has not been ingested by the mouth but secreted by the cells; and that the circulation of this fluid is from the hind part of the mid-gut forwards to the caeca. (There is at present no evidence as to which part of the mid-gut produces the digestive enzymes, though there is no doubt that much of the actual digestion takes place in the caeca, as may be judged by feeding the larvae on blood and noting the gradual darkening of the haemoglobin in the caeca.) It may be recalled that Miall and Hammond (1900) suggested a somewhat similar forward circulation of fluid in the gut of the Chironomus larva.

RESPIRATORY FUNCTION OF THE ANAL GILLS.

This larva, as we have seen, has very well-developed anal gills, and this has led da Costa Lima and others to state generally that larvae with long gills are better able to withstand submersion. But this is not entirely true; for Macfie (1917) found that Culex thalassius, a larva with extremely small gills, can survive indefinitely under water, like Aedes argenteus, whereas Culex fatigans, which has well-developed gills, could not survive a day under the same conditions. Hence Macfie suggests that the oxygen absorption by submerged larvae is "mainly through the general cutaneous surface and only secondarily through the papillae." Koch (1920), working with Culex pipiens, found that cutaneous respiration was of small importance in this species, but that larvae deprive* of their gills became still less resistant to submersion.

Again, the papillae are quite small in the allied larva Mochlonyx, in which cutane-ous respiration is relatively more important than in most mosquito larvae (Koch, 1918), and they are quite small in Corethra and many Chironomids, in which the tracheal system is entirely closed. On these grounds Martini (1923) maintains that species adapted to live under water can be divided into "skin breathers " and "gill breathers."

These facts do not support the idea that the anal papillae are primarily respiratory organs; but if their respiratory function is important it should be manifest in Aedes argenteus, where the papillae are conspicuous structures.

The question has been reinvestigated by using the spontaneous aggregation of the flagellate Polytoma as an index of oxygen tension (and so of the site of oxygen uptake) as originally done by Fox (1920), using Bodo, and more recently by Thorpe (1930, 1932). Dr W. H. Thorpe very kindly provided me with a suitable culture of Polytoma uvella, and also gave me the benefit of his experience with the method. The chief difficulty in using this method on a mosquito larva is that, since the tail end is considerably thinner than the head and thorax, it cannot be held still by gentle compression beneath a coverslip. This difficulty has been got over by placing the larva between two wisps of cotton-wool under the coverslip. It is then kept perfectly still, while the movements of the flagellates are not affected.

A typical result is shown in Fig. 4. The flagellates quickly congregate at the base of the gills, especially on the inner surface, round the anus. At first sight this looked like a chemotactic response to the excreta. But that is certainly not the case, for if any faeces that are passed are caused to fall to one side, the flagellates immediately forsake them and cluster round the base of the gills again. In a few minutes they leave the anus and form a small sphere with the anus as centre. This sphere enlarges until it reaches the coverslip and slide; thenceforward it enlarges in two dimensions and the flagellates form a band which moves gradually away from the gills. Mean-while a band forms all round the body and moves away from it; but at any given stage the band stands rather further out from the gills than from the general body surface. There is often a heavy aggregation around the head, but probably this is due largely to the movements of the mouth-parts, for it does not give rise to a rapidly retreating band.

Function of the Anal Gills of the Mosquito Larva 23

occurs all over the body surface it is somewhat more active at the anal gills. The failure of the flagellates to congregate all over the surface of the gills is easily in-telligible from the fact that as the gills are poorly supplied with tracheae1, the diffusion of oxygen along their length will occur chiefly in the blood; and as there is no active circulation of blood in the gills (as may be seen from the sluggish move-ments of the occasional amoebocytes which occur in them), nor, presumably, any active consumption of oxygen within them, it follows that the tension of oxygen in the terminal region of the gills will be practically the same as that of the surrounding water, i.e. above the tension at which the flagellates collect.

Fig. 4. Aggregation oiPolytoma uvella around larva immersed in culture of this flagellate at 2.30 p.m. A, 2.40 p.m.; B, 3.10 p.m.; C, 3.40 p.m.; D, 4.0 p.m.

From the same argument it follows that these finger-like outgrowths from the body wall of aquatic insects can be of comparatively little use in respiration unless they are richly supplied with tracheae or unless there is an active circulation of blood within them.

The elimination of carbon dioxide has been investigated by keeping the larvae beneath a coverslip in suitable indicators (phenol red, bromothymol blue, indo-phenol blue). Baryta cannot be used because it destroys the gill epithelium.

As in Fox's experiments with aquatic larvae and Thorpe's experiments with parasites, the evolution of carbon dioxide occurs more or less equally over the body surface; and there is no indication of a greater elimination at the gills. This difference between oxygen uptake and carbon dioxide discharge is doubtless due to the far greater rapidity with which carbon dioxide diffuses through chitin (for discussion see Wigglesworth, 1931 a).

1

DISCUSSION.

Many years ago Overton (1902) put forward the hypothesis that in fresh-water animals, water is constantly absorbed through the skin by osmosis and constantly eliminated from the body by the kidneys, which thereby maintain the osmotic pressure of the blood. In recent years much doubt has been cast upon this generali-sation ; and in a number of animals, notably the frog (which was the chief subject of Overton's experiments), it would seem that the skin itself is responsible for regulating the water content of the body, while the excretory organs eliminate an amount of fluid which bears no relation to the needs of the organism as a whole (Przylecki, 1922; Adolph, 1930). Indeed, Adolph (1927) goes so far as to state that "no case has yet been found in which the maintenance of unequal concentrations depends entirely upon the output (of water) and not upon the intake." This view is adopted also by Schlieper (1930), although he admits that the observations of West-blad (1922), on the contractile vesicle of Turbellarians, and of Herfs (1922), on the terminal bladder of the water vascular system of Cercariae and the contractile vacuole of various Protozoa, fit in well with Overton's hypothesis.

The regulation of the water content of the blood is regarded (Przylecki, 1922) as a function which has been acquired by the kidney very late in evolution, namely, in the terrestrial vertebrates. But it would not be surprising to find the same pro-perty independently evolved in the other great terrestrial group, the insects; and the study of the physiology of excretion in certain insects (Wigglesworth, 19316) shows that this is in fact the case. This same property might be expected to persist in the aquatic forms (which are certainly derived from terrestrial ancestors), and if so, it is in aquatic insects that Overton's conception is most likely to be realised.

From the results recorded in this paper it appears that this is in fact the case with the mosquito larva. For if the excretory organs are precluded from getting rid of the water taken in by the anal gills, the body continues to swell and may ulti-mately burst. This is not so in the frog (Przylecki, 1922), in which, if the ureters are ligatured, the uptake of water is soon arrested. From this difference it appears as though the uptake of water in the mosquito larva were simply due to osmosis, whereas in the frog the uptake is controlled according to the needs of the animal.

Function of the Anal Gills of. the Mosqtdto Larva 25

published observations which suggest that this may be the case with the skin of the frog.

The intake of water is admittedly slow, but this does not necessarily signify of course that work is being done; it may be that the intact membrane is only slightly permeable.

The importance of the anal gills in the life of the larva is difficult to assess. Their respiratory function certainly does not seem very important; for they contribute only a small part of the general cutaneous respiration, and, under normal circum-stances, cutaneous respiration is probably of small account as compared with respiration through the spiracles. Were the larva more dependent on the anal gills for respiration, they might be expected to be better adapted to the purpose, either by the richness of their tracheal supply, or by the efficiency of the circulation of the blood within them.

Anal gills are exceptionally well developed in the larva of Aedes argenteus. In many allied larvae they are much smaller and their respiratory function is likely to be still less important. Thus Fox (1920) observed that in the larva of Chironomus no more oxygen is absorbed by the anal gills than elsewhere on the body surface, and other observations pointing to the same conclusion have already been discussed

(p. 22).

On the other hand, the uptake of water by the gills is very active, whereas else-where in the body it is almost negligible. It is therefore probably more correct to treat the anal "gills" primarily as water-absorbing organs, and to regard their respiratory function as secondary or merely incidental.

But it is very difficult to judge how important this water-absorbing function may be. It is not essential, for the larva can mature without the "gills." Why then do they exist ? Perhaps their presence is to be associated with the method of feeding on solid particles filtered from the water; perhaps it is advantageous that water should be absorbed parenterally, and thus be made available for the elimination of waste products, without the dilution of the digestive juices which copious water drinking would entail.

SUMMARY.

The anal gills of the mosquito larva {Aedes argenteus) are the only region of the body that is freely permeable to water. In hypertonic solutions of sugar or glycerol, water is extracted from the gills and the larva shrinks. In pure water this is absorbed by the gills and later excreted by the Malpighian tubes. The absorption of water appears to be effected mainly by osmosis.

Larvae can mature without the gills, but they seem to grow more slowly, and show almost no parenteral absorption of water.

Normally the larva swallows very little fluid. The fluid in the gut is probably secreted in the posterior part of the mid-gut and reabsorbed in the anterior part and in the caeca.

As judged by the spontaneous aggregation of the flagellate Polytonia, oxygen is absorbed by submerged larvae all over the body surface, but most actively at the base of the gills. Carbon dioxide is given off equally all over the body surface.

It is concluded that the anal gills are primarily water-absorbing organs, and are only incidentally concerned in respiration.

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