i6

THE FUNCTION OF THE ANAL GILLS OFTHE MOSQUITO LARVA

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

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

(Received 15th April, 1932.)

(With Four Text-figures.)

IN a recent paper (Wigglesworth, 1932 a) the theory was put forward that the chieffunction of the so-called rectal glands of terrestrial insects is the reabsorption ofwater 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 glandsthat 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 inanother place (Wigglesworth, 1932^) that the properties of the anal gills are suchthat 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) argenteusPoir) have been used for all the experiments. The internal structure of this larva isreadily seen in living specimens, but this is made easier if the larvae are kept for aweek in clear water without food, so as to reduce the quantity of fat globules in thefat-body.

GENERAL STRUCTURE OF THE LARVA.

The anatomy of the larva is shown in Fig. 1 A. The anal gills arise from the thinmembrane around the anus, and the blood in their lumen communicates freely withthe general body cavity; the detailed structure of the gills has already been de-scribed (Wigglesworth, 1932 b). The alimentary canal consists of a fore-gut endingat the proventriculus, a straight mid-gut with six conspicuous caeca at its com-mencement, and a hind-gut. The hind-gut consists of a "pyloric chamber," a'' small intestine'' which runs a slightly sinuous course, a distensible '' rectum,'' andan "anal canal." Fig. iB and C represent transverse sections of the "intestine"and "rectum" respectively. The former is thin-walled, the latter is bounded byrelatively large epithelial cells; both, of course, are lined with chitin. The pro-ventriculus secretes a cylindrical chitinous sheath, the " peritrophic membrane " (seeWigglesworth, 1930 a), which extends unbroken throughout the gut, and may even

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 themid-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., indicatethe respective abdominal segments. B and C show cros9 sections of the hind-gut, B through theintestine (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 seventhabdominal segments. D, the same after immersion in tap water for 2 hours. (From camera lucidadrawings.)

PERMEABILITY OF THE CUTICLE TO WATER.

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

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

JBB-Zi 2

i 8 V. B. WlGGLESWORTH

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

These experiments show that the hinder end of the larva, i.e. presumably theanal 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 isclear from Fig. 1 that no fluid can be discharged by the Malpighian tubes into thehind-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 andimmersed in fresh water. The Malpighian tubes become enormously distendedabove 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 spontaneousrupture of the gut may occur at the anus. The segments of the body between theligatures show no change, or swell comparatively slightly. Table I shows the courseof the swelling expressed by linear measurements between arbitrary points in thelarva.

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

AB

11.25 ajn.

7i86

11.30 a.m.

7287

Time

11.45 a-In-

7189

12.15 p.m.

7190

1.15 p.m.

7191

2.30 p.m.

7192

This experiment shows that water is absorbed by the hind end of the larva. Theanus is kept closed, and it has just been shown that absorption through the generalsurface of the body does not occur, so that the absorption must be through the gills.

Additional evidence of this is afforded by the behaviour of the fluid in the trache-oles of the gills. It has been shown (Wigglesworth, 19306) that the limit to whichair extends in these tracheoles is probably determined by the osmotic pressure ofthe fluid around their endings; and if the larva is asphyxiated by keeping it underwater, the increased osmotic pressure of the blood, due to muscular contractions,extracts fluid from the tracheal endings and air extends downwards towards the cells.But Fig. 3 shows that when the larva is ligatured near the hind end of the body, theair actually retreats in the tracheoles, indicating a fall in osmotic pressure in the gills.In this particular experiment the fluid continued to rise in the tracheoles; doubtlessbecause the larva concerned was very placid. But in some cases there may be suchactive twitching of the muscles in the hinder segments of the body that the rise inosmotic pressure which this causes exceeds the fall due to the entry of water, and airextends down the tracheoles again.

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 thatfluid accumulates in the "pyloric chamber" (see Fig. 1) until a small drop hascollected, which is then carried down to the rectum by a wave of peristalsis. Thisprocess is repeated every 2 or 3 min., and sometimes a fragment of the contentsfrom the mid-gut is also carried down to the rectum. The fluid is evidently secretedby 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 bodybehind the ligature. Fluid is evacuated from the rectum from time to time; eitherin small quantities every few minutes or in a larger quantity at intervals of15-20 min. In either case it seems as though the quantity of fluid passing downthe intestine is less than that evacuated by the anus; and this suggests that fluid isbeing reabsorbed in the rectum. In a later paper (Wigglesworth, 1932c) conclusiveevidence 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 thisfluid is difficult to estimate, but it certainly does not exceed the cubic capacity of

3-2

2 0 V. B. WlGGLESWORTH

two anal gills per hour. None the less, this fluid is ample to keep the Malpighiantubes well flushed with water, and they never contain any solid uric acid, even whenthe larva is on a pure protein diet. This state of affairs is in marked contrast with theadult mosquito (in which the larval tubes persist) where the supply of water is solimited that, except just after a meal, the Malpighian tubes always contain muchsolid 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. in5 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 purewater the gills of many of them blacken and slough away. In a few days they havehealed 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 tubesand the elimination of fluid from the anus observed. Immediately after ligaturingthey usually discharge some of the solid material from the mid-gut, and sometimesa little fluid may be passed during the next 20 min. or so, but thereafter, althoughthey have been watched for nearly 2 hours, they pass very little fluid and in somecases none at all. It is noteworthy that even when no fluid is being discharged fromthe anus, occasional drops pass down the hind-gut. This affords additional evidencefor the reabsorption of water in the rectum.

If gill-less larvae are ligatured at the sixth abdominal segment, there is not theexcessive distension of the Malpighian tubes above the obstruction which occursin the normal larva, and the swelling of the body behind the ligature is almostnegligible 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 andseventh abdominal segments at 2-15. A, from ligature to tip of siphon; B, fromligature to tip of anal segment. Units are divisions of micrometer eyepiece.

A

2.15 p.m.

72SO

2-45 p.m.

725°

Time

3.15 pjn.

72SO

3-45 P-m-

725°

4.15 p.m.

73Si

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

INGESTION OF FLUID BY THE MOUTH.

It is improbable that the larva normally swallows fluid, though it may sometimesbe seen to do so while compressed beneath a coverslip. The normal method of feed-ing can be readily observed if larvae are immersed in a suspension of trypan blue inwater. The solid particles of dye are swept into the mouth by means of the feeding

Function of the Anal Gills of the Mosquito Larva 21

brushes and mouth-parts, and collect in the buccal cavity. When a considerablebolus has accumulated, the mouth-parts are drawn in, and the bolus is swallowedwithout any noticeable amount of fluid; although, of course, the particles must bemoist with water.

The particles of dye ingested in this way are confined within the peritrophicmembrane as a solid black column. But the dye quickly appears in solution outsidethe peritrophic membrane, both in the caeca and in the uniform part of the gutbehind; and this blue solution is subject to the peristaltic waves which pass over themid-gut. These waves usually run from behind forwards, and will therefore tendto 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 3hours they consist of blue-black masses of solid dye. There is no solid dye elsewherein the gut outside the peritrophic membrane.

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

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

Now, as shown by Frederici (1922) and Samtleben (1929), the mid-gut behindthe caeca consists of two distinct segments with clearly defined histologicaL differ-ences. These correspond with the regions observed by Van Gehuchten (1890) in hisclassical 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 thefluid to be seen in the mid-gut of the larva has not been ingested by the mouth butsecreted by the cells; and that the circulation of this fluid is from the hind part ofthe mid-gut forwards to the caeca. (There is at present no evidence as to which partof the mid-gut produces the digestive enzymes, though there is no doubt that muchof the actual digestion takes place in the caeca, as may be judged by feeding thelarvae 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 similarforward circulation of fluid in the gut of the Chironomus larva.

RESPIRATORY FUNCTION OF THE ANAL GILLS.

As their name implies, the anal gills are usually regarded as respiratory organs," tracheal gills," which take up oxygen from the water into the tracheal system; buthow important this respiratory function may be is a debated question (see Wiggles-worth, 1931 a). Mosquito larvae breathe primarily at the water surface throughspiracles opening at the eighth abdominal segment; yet many species can remainsubmerged for long periods. For instance, da Costa Lima (1914) and Macfie (1917)found that Stegomyia fasciata (= Aedes argenteus) could live indefinitely andapparently normally beneath the surface if the water were sufficiently aerated.

22 V. B. WlGGLESWORTH

This larva, as we have seen, has very well-developed anal gills, and this has ledda Costa Lima and others to state generally that larvae with long gills are better ableto withstand submersion. But this is not entirely true; for Macfie (1917) found thatCulex thalassius, a larva with extremely small gills, can survive indefinitely underwater, like Aedes argenteus, whereas Culex fatigans, which has well-developed gills,could not survive a day under the same conditions. Hence Macfie suggests that theoxygen absorption by submerged larvae is "mainly through the general cutaneoussurface and only secondarily through the papillae." Koch (1920), working withCulex pipiens, found that cutaneous respiration was of small importance in thisspecies, 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 thetracheal system is entirely closed. On these grounds Martini (1923) maintains thatspecies adapted to live under water can be divided into "skin breathers " and "gillbreathers."

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

The question has been reinvestigated by using the spontaneous aggregation ofthe flagellate Polytoma as an index of oxygen tension (and so of the site of oxygenuptake) 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 ofPolytoma 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 tailend is considerably thinner than the head and thorax, it cannot be held still by gentlecompression beneath a coverslip. This difficulty has been got over by placing thelarva between two wisps of cotton-wool under the coverslip. It is then kept perfectlystill, while the movements of the flagellates are not affected.

A typical result is shown in Fig. 4. The flagellates quickly congregate at the baseof the gills, especially on the inner surface, round the anus. At first sight this lookedlike a chemotactic response to the excreta. But that is certainly not the case, for ifany faeces that are passed are caused to fall to one side, the flagellates immediatelyforsake them and cluster round the base of the gills again. In a few minutes theyleave the anus and form a small sphere with the anus as centre. This sphere enlargesuntil it reaches the coverslip and slide; thenceforward it enlarges in two dimensionsand 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 givenstage the band stands rather further out from the gills than from the general bodysurface. There is often a heavy aggregation around the head, but probably this isdue largely to the movements of the mouth-parts, for it does not give rise to a rapidlyretreating band.

These results are similar to those obtained by Thorpe (1930) on the parasiticlarva, Cryptochaetum iceryae, and they show that although the uptake of oxygen

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. Thefailure 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, thediffusion of oxygen along their length will occur chiefly in the blood; and as there isno 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, anyactive consumption of oxygen within them, it follows that the tension of oxygen inthe terminal region of the gills will be practically the same as that of the surroundingwater, 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 thebody wall of aquatic insects can be of comparatively little use in respiration unlessthey are richly supplied with tracheae or unless there is an active circulation of bloodwithin them.

The elimination of carbon dioxide has been investigated by keeping the larvaebeneath 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 withparasites, the evolution of carbon dioxide occurs more or less equally over the bodysurface; and there is no indication of a greater elimination at the gills. This differencebetween oxygen uptake and carbon dioxide discharge is doubtless due to the fargreater rapidity with which carbon dioxide diffuses through chitin (for discussionsee Wigglesworth, 1931 a).

1 The tracheal supply of the anal gills is very sparse as compared with that of undoubted respira-tory organs such as the tracheal gills of Ephemerid larvae or the skin of parasitic Hymenopterous larvae.

24 V. B. WlGGLESWORTH

DISCUSSION.

Many years ago Overton (1902) put forward the hypothesis that in fresh-wateranimals, water is constantly absorbed through the skin by osmosis and constantlyeliminated from the body by the kidneys, which thereby maintain the osmoticpressure 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 subjectof Overton's experiments), it would seem that the skin itself is responsible forregulating the water content of the body, while the excretory organs eliminate anamount 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 concentrationsdepends entirely upon the output (of water) and not upon the intake." This view isadopted 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 theterminal bladder of the water vascular system of Cercariae and the contractilevacuole 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 thestudy of the physiology of excretion in certain insects (Wigglesworth, 19316) showsthat this is in fact the case. This same property might be expected to persist in theaquatic forms (which are certainly derived from terrestrial ancestors), and if so, itis 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 casewith the mosquito larva. For if the excretory organs are precluded from getting ridof 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 areligatured, the uptake of water is soon arrested. From this difference it appears asthough 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.

The skin of the frog behaves, in fact, as though it harboured certain forces ofan unknown nature which resist the endosmosis of water, whereas the rate of exos-mosis is directly proportional to the osmotic pressure difference (Adolph, 1930).Whether a similar difference in permeability in the two directions occurs in the gillsof the mosquito larva, or whether the gill membrane does work by resisting theinflow of water, cannot be decided from the facts available; but one thing is certain,the inner and outer surfaces of the cells have such divergent properties (Wiggles-worth, 19326)—the cytoplasm of the cells being dispersed when hypertonic solu-tions of certain salts are applied externally—that very different results wouldassuredly be obtained between endomosis and exosmosis. It is conceivable thatcertain of the cases of "unidirectional permeability" that have been described inother animals may be explicable in a similar fashion; indeed, Bauer (1925) has

Function of the Anal Gills of. the Mosqtdto Larva 25

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

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

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

Anal gills are exceptionally well developed in the larva of Aedes argenteus. Inmany allied larvae they are much smaller and their respiratory function is likely tobe still less important. Thus Fox (1920) observed that in the larva of Chironomus nomore oxygen is absorbed by the anal gills than elsewhere on the body surface, andother 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 totreat the anal "gills" primarily as water-absorbing organs, and to regard theirrespiratory function as secondary or merely incidental.

But it is very difficult to judge how important this water-absorbing function maybe. It is not essential, for the larva can mature without the "gills." Why then dothey exist ? Perhaps their presence is to be associated with the method of feeding onsolid particles filtered from the water; perhaps it is advantageous that water shouldbe absorbed parenterally, and thus be made available for the elimination of wasteproducts, without the dilution of the digestive juices which copious water drinkingwould entail.

SUMMARY.

The anal gills of the mosquito larva {Aedes argenteus) are the only region of thebody 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 absorbedby the gills and later excreted by the Malpighian tubes. The absorption of waterappears to be effected mainly by osmosis.

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

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

Some of the water excreted by the Malpighian tubes is reabsorbed in the rectum.

26 V. B. WlGGLESWORTH

As judged by the spontaneous aggregation of the flagellate Polytonia, oxygen isabsorbed by submerged larvae all over the body surface, but most actively at thebase 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 areonly incidentally concerned in respiration.

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