Annals of Tropical Medicine & Parasitology

ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19

Food of Glossina Palpalis in the Cape Lighthouse Peninsula,

Warrington Yorke & B. Blacklock

To cite this article: Warrington Yorke & B. Blacklock (1915) Food of Glossina Palpalis in the Cape Lighthouse Peninsula, Sierra Leone, Annals of Tropical Medicine & Parasitology, 9:3, 363-380, DOI: 10.1080/00034983.1915.11687686 To link to this article: http://dx.doi.org/10.1080/00034983.1915.11687686

Published online: 24 Mar 2016.

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Download by: [Australian Catholic University] Date: 26 August 2017, At: 11:20 FOOD OF GLOSSINA PALPALIS IN THE CAPE LIGHTHOUSE PENINSULA, SIERRA LEONE

[Being the Second Report of the Thirty-second Expedition of the Liverpool School of Tropical Medicine, 1914-1915.]

BY WARRINGTON YORKE

AND B. BLACKLOCK

(Received for publication 28 April, 1915)

Although there is a certain amount of evidence to show that Glossina palpalis can take up water and vegetable juices, practically all observations indicate that for its continued existence vertebrate blood is necessary. In view of the small amount of time at our disposal, and of the comparative scarcity of Glossina palpalis, we were able to examine only 200 in respect of the nature of the blood found in their intestinal tract. The tsetse were dissected immediately they were brought into the laboratory. As at the same time the flies were examined for the presence of trypanosomes, they were dissected in the manner described by Lloyd (1912). Briefly it consists in splitting the dorsum of the thorax longitudinally with a cataract knife, and then drawing out the salivary glands by gentle traction on the head. Usually the glands are removed intact attached to the head, the oesophagus breaking off at the point where

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 it enters the pharynx; occasionally they break across when only partially withdrawn, but in this case are easily caught up with a fine pair of forceps. The proventriculus is then sought at the ventral sudace of the thorax, and is drawn out with the sucking stomach and most of the intestine. The posterior portion of the hind-gut and rectum are removed by cutting off the last segment of the abdomen. The gut contents and those of the salivary glands and of the proboscis were examined between a slide and coverslip in a fresh unstained condition. Recognisable red blood cells were seen in 16 of the 200 flies thus examined. It is necessary to point out here that only those flies in which definite red blood corpuscles were seen are recorded as containing blood. Practically every had in its mid-gut a certain quantity of pigmented material, the colour of which varied from bright red to brown or black. Such pigmented material does not, however, necessarily imply the presence of red blood corpuscles ; on microscopical examination it was usually found to consist of granular debris and globules appearing red or brown in colour, and crystals which in many cases were probably haemin or haemoglobin crystals. Although it is highly probable that this pigmented material was derived from blood, still one could not be certain, and, therefore, in the following table only those flies in which definite red blood corpuscles were encountered are considered as containing blood. TABLE I.-Result of examination of freshly-caught wild Glossina palpalis for recognisable blood corpuscles

Number in which Number in which Number recognisable recognisable examined mammalian red nucleated red corpuscles were seen corpuscles were seen

Males ...... 113 8 0

Females ...... 87 6 1

Total ...... 100 14 1

From the limited number of observations made, it appears that eight per cent. of freshly caught Glossina palpalis in this Peninsula contain recognisable red blood corpuscles-mammalian blood in Downloaded by [Australian Catholic University] at 11:20 26 August 2017 seven per cent., and nucleated red cells of an undetermined nature in one per cent. The chief source of mammalian blood on the Peninsula is probably man and his domestic stock-goats, sheep, and dogs. In addition there are a few antelope-bushbuck and duiker were seen-and a considerable number of monkeys (Cercopithecus sp.) squirrels and bats, rats and mice. It is interesting to note that the mammalian red cells seen in the fourteen flies observed to contain these, were of the large type and readily distinguishable from the small variety found in sheep and goats. The source of nucleated blood corpuscles 1s apparently much larger. Birds and lizards are numerous, and in the mangrove swamps there are vast numbers of mud-fish, crabs, shell-fish, snails, wading birds and sea birds. No crocodiles occur. In view of the fact that the reservoir of nucleated blood corpuscles is probably much greater than that of non-nucleated red cells, it is interesting to find mammalian blood in fourteen Glossina palpalis and nucleated red cells in only two, an observation which appears to indicate that Glossina palpalis either prefers mammalian blood or finds it more easy to obtain. The flies were captured at various parts of the Peninsula, but owing to its small size none of these localities was more than a quarter of a mile from habitation-either the village of Aberdeen or the settlements at the isolation camp and the Cape Lighthouse. In order to appreciate the real meaning of the results obtained by such an examination of the gut of freshly caught tsetse in respect of blood corpuscles, it is of importance to have at our disposal some data regarding the length of time red blood cells can be recognised in the intestine of a fly after feeding. To obtain this information freshly caught tsetse were fed on a rat or fowl and dissected after various intervals, and the gut contents examined for the presence qf red blood cells. The results are given in the Tables II and III. In the case of mammalian blood it is seen that in over go per cent. of the flies red cells can be recognised 24 hours after a feed, but in only 40 per cent. of those examined after 48 hours, whilst in no instance were definite red cells noted after 72 hours. The fowl red blood cells were found to be recognisable for longer periods ; after 24 hours 100 per cent. of the flies showed nucleated red blood cells, whilst after 48 hours red blood cells were seen in 6o per cent., and

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 after 72 hours in 40 per cent. The temperature was that of the laboratory, 8o0 -86° F. Possibly to a certain extent this difference is to be explained by the fact that the nucleated red blood cells of the fowl are more characteristic, and hence more easily recognised than are the non-nucleated red cells of mammals. Of course, these figures are approximate, as only 64 tsetse were employed in the experiment: they are, however, sufficiently accurate for practical purposes, although some observers have recorded, in the case of isolated flies, that blood could be found after much longer intervals. Thus in the case of Glossina morsitans, Lloyd (1913) observed fowl red cells in a clot in the sucking stomach several weeks after the fly had last fed on a fowl, monkeys having been used as blood donors in the interval. Nevertheless, these figures show

TABU H.-Experiment to aacertain the length of time rat red blood corpuadea can be recognised in G. palpalis

No. of Result of Date hours examination. Percentage of flies No. Date fed examined after Sex Recognisable in which feed blood cells blood was found present or absent

I 17.12.14 18.1Z.14 :1.4 ~ +++ z 17.12.14 18.1:1..14 Z4 ~ +++ 3 17-12.14 18.1:1..14 :1.4 ~ +++ 4 '7·1:1..14 18.1:1..14 24 ~ +++ 5 '7·1:1..14 18.1:1..14 Z4 ~ +++ 6 17.1:1..14 18.1Z.14 :1.4 ~ +++ 92 7 17-1:1..14 18.12.14 :1.4 ~ +++ 8 '7·1:1..14 18.1:1..14 :1.4 ~ ++ 9 z8.1.15 29· 1•15 Z4 ~ - IO 28.1.15 :1.9· 1•15 24 ~ +++ II 2.2.15 3·:!..15 :1.4 ~ +++ 1:1. 2.:1..15 3-:1.·'5 24 ~ +++

13 :!.8.1.15 30-1.15 48 ~ - 14 :!.8.1.15 30.1.15 48 ~ - 15 28.1.15 30.1.15 48 ~ +++ 16 28.1.15 30.1.15 l 48 ~ - 17 28.1.15 30.1.15 48 ~ +++ 40 18 :1..:1..15 4-2.15 48 ~ - 19 :1..:1..15 4.2.15 48 ~ - :1.0 :1..:1..15 4.2.15 48 ~ + 21 2.2.15 4.2.15 48 ~ - j 2:1. :1..:1..15 4.2.15 48 ~ +++

23 28.1.15 31.1- 15 72 ~ - :1.4 28.!.15 31.1.15 72 ~ - 25 28.1.15 3'·'·'5 72 ~ - 26 2.2.15 5·2.15 72 ~ - 27 2.2.15 5·2.15 72 ~ - 28 2.:!..15 5·Z.I5 72 ~ - 0 Downloaded by [Australian Catholic University] at 11:20 26 August 2017 :1.9 2.2.15 5-Z-15 72 ~ - 30 :1..2.15 5·:!..15 7Z ~ - 31 2.:1..15 5·2·15 7:!. ~ - 32 2.2.15 5·2.15 7& ~ - 33 :1..2.15 5·2.15 72 ~ - I 34 21.1.15 25·1·15 ¢ ~ - J

that as a general rule mammalian (rat) red blood cells are no longer recognisable after a period of 72 hours, whilst fowl red cells are observed in only 40 per cent. of cases after a similar period. This information is of importance in connection with that obtained by Downloaded by [Australian Catholic University] at 11:20 26 August 2017 examining the blood found in freshly caught tsetse-fly. When the statement is made that only seven per cent. of such flies contain mammalian blood, it must be borne in mind that if the flies had all fed on a mammal 48 hours previously, we would expect to fi.nd red cells in only 40 per cent., whilst if they had fed 72 hours previously very few would contain recognisable red cells.

TABLE III.-Experiment to ascertain the length of time fowl red blood corpuscles can be recognised in G. palpalis

! No. of Result of Date i hours examination. Percentage of flies No. Date fed examined after Sex Recognisable in which feed blood cells blood was found I present or absent I I I "2.8.1.15 2.9·•·•5 2.4

The question whether tsetse-fly take up other food than blood is one which is difficult to decide. Stuhlmann (1907) and Degen (1909) came to the conclusion that they did not, but Maugham (191 1) states that he has seen tsetse-flies sucking vegetable juices on two occasions. In 1905 he observed a Glossina morsitans alight on a stem of young marsh grass (Phragmites communis) and deliberately insert its proboscis and unmistakably suck for a period of about three and a half minutes. At this stage Mangham caught the fly, and found on examination that it was partly full of the moisture from the plant. Again in 1908, in an absolutely gameless and practically waterless country, he observed Glossina morsitans feeding on a piece of sugar-cane at a point where the pith was exposed. He attempted to catch this fly, but unfortunately failed to do so. Taute (1912) investigated the point experimentally in the following manner. Two hundred tsetse-flies which had been fed daily on blood for a period of two months were starved for fi.ve days, and subsequently were given an opportunity of feeding on small pieces of mango fruit. Taute records that in three cases Glossina morsitans buried its proboscis completely in the fruit and remained in this position for several minutes, but defi.nite sucking did not take place. One of the flies was killed and dissected immediately after this operation. Neither in the lumen of the proboscis nor in the oesophagus or remainder of the alimentary canal could the smallest trace of mango juice be recognised. The work of Carpenter (1912-13), however, affords strong support to the view that Glossina palpalis does take up other food than blood. Carpenter examined the intestinal contents of a large number of freshly caught G. palpalis, and found in a proportion of them small fragments of tissue of an obviously vegetable origin, e.g., pieces of vegetable parenchyma, starch grains, pieces of alga, and a minute fungus. Apart from the solitary instance recorded by Mangham, there appears to be no direct evidence that tsetse-fly will imbibe anything other than vertebrate blood, and, moreover, the evidence offered by

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 him is by no means convincing, as he omits to state the manner in which he examined the fly or how he detected that it was partly fi.lled with moisture from the plant. Stuhlmann affirms that Glossina lives exclusively on living blood, and that they refuse to take up shed blood, water or syrups; and Degen attempted to feed them on fruit, saccharine fluids, meat, etc., without success. Rodhain, Pons, Vandenbranden and Bequaert (1912) observe that it is generally admitted that tsetses cannot engorge themselves with blood unless they obtain it directly from the capillaries in which the fluid is maintained under a definite pressure. Leaving pressure out of the question for the moment-Rodhain and his collaborators have since shown that this is not an essential factor-all experimental evidence shows that Glossina are unable to imbibe shed blood. In the experiments described below we have demonstrated that they will not absorb certain other fluids offered to them in open vessels. Fourteen flies which had been starved for 48 hours were placed in each of two cages. In one of these was a petri dish filled with the following solution :- Sodium chloride... o·g g. Sugar 2·og. Neutral red o·1 g. Water 100 c.c and in the other a petri dish containing the following :- Methylene blue . . . o·o5 g. Sodium carbonate o·o25 g. Wa~ 100~~ Small twigs and leaves were floated on the surface of the solutions. The flies were dissected as they died; all those that were still alive at the end of 48 hours were killed and examined. There was no indication that any of the flies had imbibed fluid. These results are conclusive because, as will be seen later, solutions. of neutral red and of methylene blue in the concentrations used, if taken up by Glossina, stain the tissues densely. Such work as we have done, therefore, confi.rms the generally accepted view that Glossina is unable to imbibe exposed fluids directly. The work of Rodhain and his collaborators showed, however, that tsetses can suck up citrated blood through a membrane consisting of the freshly removed skin of a mouse. We made use of this observation to prove that not only will tsetses take up citrated blood Downloaded by [Australian Catholic University] at 11:20 26 August 2017 through such a membrane, but that they will also imbibe various other fluids. The apparatus used by us was a slight modification of that figured and described by Rodhain. It consisted of a short glass cylinder about three-quarters of an inch in diameter, the lower end of which was closed by a cork through which the shorter limb of an U-shaped piece of glass tubing passed ; the upper end of the cylinder was covered by the membrane. The pressure of the fluid in the cylinder was indicated by its level in the longer limb of the glass tube. 37° In our first series of experiments flies were fed on defibrinated goat-blood through a membrane of fresh rat's skin. It was observed that (within the limits of the experiment) the pressure made no difference to the manner in which the flies engorged themselves. They distended themselves without the least difficulty at pressures ranging between + or - 6o mm. of blood. It was further noted that full distension of the flies occurred at least as rapidly and regularly as when the flies were allowed to feed on live rats. On the

T.uu: IV.-Giving the results of feeding G. palpalis on defibrinated blood and various dilutions of this with normal salt solution, through a membrane consisting of rat skin I No. Sex Membrane i Nature of fluid Result Remarks -- I I c! Rat skin ...... j Undiluted goat blood Complete distension Immediately 2 c! , ...... I , , , 3 !j! , ...... ! , , 4 !j! , ...... , , " 5 c! , ...... " ," 6 !j! ... " " , 7 c! ," ... :::t so% goat'blood ... ," 8 " c! ...... 1 , 9 !j! ," ...... ,, ," ," 10 c! , ... :::I 25% goat,;lood ... , II !j! , ... " 12. c! , ...... ," ," '3 c! , ...... 10% goat" blood ... , 14 !j! , ...... Partial distension ... After "15 minutes 15 !j! , ...... " 16 c! , ...... 10%. rat blood" .. . Complete" distension Immediately" 17 c! ...... , , , 18 c! " ...... , , , 19 c! ", ...... 5 % goat blood ... No visible distension After 15 minutes goats' cells seen in anterior gut 20 !j! , ...... , , Mter 15 minutes no goats' cells seen in anterior gut :n !j! , ...... 5 % rat blood ...... , Mter 15 minutes rats' cells seen in anterior gut 22. !j! , ...... , Partial distension ... Mter 15 minutes 23 c! , ...... , No visible distension After 15 ~nutes. red cells seen In antenor gut 24 c! , ...... Marked distension ... After 15 minutes

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 "

other hand the flies attacked the membrane with less alacrity than they do the skin of the living . Nevertheless, many flies settled on and pierced the membrane at once, and, as a rule, a little patience was sufficient to induce a large proportion of the flies to feed. An experiment was conducted to determine the effect of diluting the defibrinated blood with salt solution in various degrees; the results are given in Table IV. It was found that Glossina palpalis 371 engorged itself with the following dilutions, viz., so per cent. blood, 2S per cent. blood, and 10 per cent. blood, but the dilution consisting of S per cent .. blood and 9S per cent. salt solution was not taken up by the tsetses with the same rapidity and regularity as the other solutions. Of the six flies which were offered the last dilution of blood, only two partially distended themselves; in the other four no distension was observed, but in three of these red cells were seen on dissection. Physiological salt solution alone was then tried, but although the flies attacked the membrane with the same eagerness as before, no visible distension was noted, except in one instance where the fly appeared to become partially engorged. Tsetses frequently inserted their proboscis and appeared to endeavour to feed; in some instances the membrane was pierced probably at least 100 times with apparently no result. We shall return later to the question whether these flies had actually taken up any salt solution, and simply note here that no distension comparable to that observed when defibrinated blood is offered took place. We next turned our attention to the question of what element of the blood proves so attractive to the fly that it engorges itself with this fluid to such a marked degree. Fresh defibrinated goat-blood was centrifugalised, and the red cells separated from the plasma; the red cells were then washed free from plasma with normal sodium chloride solution and a so per cent. suspension of them made in salt solution-a concentration which corresponds approximately to that of red cells in normal goat-blood. It was found that Glossina palpalis engorged itself readily with the red cell suspension. By the addition of normal salt solution, suspensions containing 2 S per cent., 1 o per cent. and S per cent. of red blood cells were made. The Downloaded by [Australian Catholic University] at 11:20 26 August 2017 results of offering these to the flies are given in Table V. The tsetse engorged themselves completely with all the higher concentrations, but in the case of the S per cent. suspension of red blood cells only one of three flies became completely distended, whilst in the other two no distension was observed, although red cells were found in the gut on dissection. Attempts were made to feed a number of Glossina palpalis on fresh defibrinated plasma. The plasma was obtained by shaking up goat's blood in a bottle with beads. Mter separation of the fibrin 372 by straining through gauze, the red cells were thrown down by centrifugalisation and the plasma siphoned off. Plasma thus prepared is always of a slightly reddish tint, owing to a certain amount of damage to red cells during the process of defi.brination, but the amount of haemoglobin dissolved is so small as to be

TABu V.-Giving the results of feeding G. palpalis on suspensions of red blood cells of various concentration through a membrane of rat skin

No. Sex I Membrane Nature of suspension Result Remarks I

I cJ Rat skin 000 ... so% goat erythrocytes Complete distension Immediately 2. ~ , ...... , , , 3 cJ , ...... , , , 4 cJ , ...... , , , 5 cJ , 000 ... , , 6 ~ ...... " , , 7 cJ ," ...... 2.5% goat "erythrocytes , , 8 cJ ...... , , , 9 cJ ," ...... 10% goat erythrocytes Partial distension ... After 15 minutes IO ~ , ...... II cJ ...... " Complete" distension Immediately" 12. cJ " 000 ... 5 % goat erythrocytes" , After 15 minutes 13 ~ ," 000 ... No visible distension After 15 minutes goats' " erythrocytes seen in gu 14 , ...... cJ " " "

TABLE VI.-Giving the results of feeding G. palpalis on defibrinated plasma through a membrane of rat skin

No. Sex Membrane Nature of fluid Result Remarks ! --

I cJ Rat skin 000 ... 70 % plasma and 30% No visible distension After 15 minutes normal saline 2. ~ ...... Slight distension ... " ...... " No visible distension " 3 cJ " ...... " " 4 cJ " " " Downloaded by [Australian Catholic University] at 11:20 26 August 2017 5 ~ ...... " 6 cJ " 000 ... ", Almost complete" ," " distension 7 cJ , 000 ... No visible distension 8 000 ... " Complete distension " ~ " " "

practically negligible. Two of eight flies, all of which had made repeated efforts to feed, succeeded in completely engorging themselves. In only one of the remaining six was slight distension noticeable. The results are given in Table VI. 373 From these experiments it is apparent that the most attractive element in the blood is the red corpuscle. In order to carry the matter further, the washed red cells of the goat were laked by the addition of three parts of distilled water to two parts of red cells. This solution was offered to three Glossina palpalis, all of which quickly and completely engorged themselves, thus proving that the integrity of the red corpuscles is not an essential factor. An attempt was then made to separate the red cell stromata from those constituents of the corpuscle which are soluble in water. For this purpose sufficient sodium chloride was added to the laked red cell solution to render it isotonic, and the stromata were subsequently precipitated by prolonged centrifugalisation. Unfortunately, we did not succeed in obtaining complete separation of the stromata, as we had no high power centrifuge at our disposal, and owing to the minute size of the goat's erythrocytes precipitation of the stromata is a matter of no small difficulty. Nevertheless, although complete separation was not obtained, a considerable fraction of the total amount of stromata was precipitated and a solution of goat's haemoglobin, fairly free from solid matter, was obtained. The resulting solution contained approximately as much haemoglobin as that found in a 40 per cent. suspension of goat's red cells. Four flies to which this solution was offered rapidly and completely distended themselves. The solution was then diluted with an equal volume of physiological saline. It was found that the six flies to which this solution was offered did not feed with the same avidity as did those which were offered the more concentrated solution; one became completely distended, three others partially, whilst in two no distension occurred. Having thus determined that Glossina palpalis feeds readily on

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 fresh haemoglobin solution, further experiments were performed with solutions made from the crystallised haemoglobin of commerce (Griibler's dried haemoglobin). Solutions made from this prepara­ tion are of a brownish-red colour, and give rather indistinct absorption bands of oxyhaemoglobin. The strength of the solution used was between 1 and 2 per cent. Four of the fi.ve flies to which this solution was offered became partly distended, whilst in the case of the other, although no distension was visible, the solution was seen in the gut on dissection. None were found to engorge 374

themselves completely in a manner ~omparable to that seen with haemoglobin solution made from fresh blood. Details are given in Table VII. Owing to the lack of suitable apparatus, we were unable to determine the result of offering to Glossina palpalis the washed stromata derived from red blood cells.

TABU VII.-Giving the results of feeding G. palpalis on haemoglobin solutions of various concentration through a rat skin membrane -- No. Sex Membrane I Nature of fluid Result Remarks

I !j1 Rat skin ...... 40 °/0 • solution of goat Complete distension Immediately haemoglobin in 0·9 o/o NaCl 2 <1 ...... , , After S minutes 3 J " ...... , , Immediately 4 !j1 " ...... , , , s !j1 ," ...... 20%• solution of goat Partial distension ... After IS minutes haemoglobin in 0·9% NaCl 6 !j1 , ...... , Slight distension ... , 7 ~ , ...... , Almost complete distension " 8 J ...... , No visible distension 9 !j1 ," ...... Slight distension ... " 10 ,J ...... " No visible distension " 11 !j1 " ...... 1-2.% solution" of dry Partial distension ... ," " crystalline haemoglobin 12. J ...... , , 13 <1 " ...... ," 14 <1 ," ...... , No visible" distension After IS" minutes Hb. solution seen in anterior gut IS & , ...... , Slight distension ... After 1 5 minutes

• By this is meant that the solutions contained respectively as much haemoglobin as do 40 and 2.0 per cent. suspensions of goat red blood cells.

Summarising the results of these experiments, we find that

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 Glossina palpalis feeds with avidity, through rat's skin, on fresh defibrinated blood and also on suspensions (so to 5 per cent.) of washed red blood cells in normal saline, and on solutions containing as much dissolved haemoglobin as is present in 40 and 20 per cent. suspensions of red blood cells. Defibrinated plasma does not appear to have the same attraction for them, nor does sodium chloride solution alone, but the latter, containing a small proportion of the dried haemoglobin of commerce in solution is to a certain extent 375 taken up. Although these experiments are not quite so conclusive­ especially as regards separation of the constituents of the red blood cells which are soluble and insoluble in water-as could be desired, nevertheless, we consider they suggest strongly that the element in the blood which is attractive to Glossina palpalis is that fraction of the erythrocyte which is soluble in water, most probably haemoglobin. Having completed these observations on blood, we decided to make use of the same technique with a view to determining whether Glossina palpalis will take up other solutions through fresh rat-skin. Reference has already been made to the fact that when sodium chloride solution was offered, although the flies repeatedly pierced the membrane, they did not appear to take up any of the fluid. At all events, no distension was noticed as a rule, although in one instance partial engorgement did apparently occur; it was by no means easy to state definitely whether or not any of the solution had been imbibed, as small quantities of a clear fluid cannot be recognised in the gut of the fly. In order to determine the point, the solution must be coloured with some dye that can be readily recognised. For this purpose methylene blue, neutral red and fuchsin were used. It was found that when physiological saline, containing these dyes in solution, was offered to Glossina palpalis small quantities were taken up by the flies in a proportion of the instances. Dissection showed that the intestinal tract was deeply stained, red or blue according to the dye used. In some cases the stain had spread to the salivary glands and to the fat bodies. In fact, so deeply were certain flies coloured that even before dissection the dye could be recognised through the integument of the abdomen. After obtaining this information various other solutions were offered to Glossina palpalis, details of which are given in Table VIII. It is

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 interesting to note that three flies became completely engorged with o·9 per cent. sodium chloride solution to which had been added about 5 per cent. of cane-sugar and a little neutral red. Another fly became partially distended on a 2 5 per cent. solution of glycerine in water coloured with methylene blue. These observations demonstrate that under certain conditions Glossina palpalis will imbibe and even completely distend itself with fluids other than blood. Although in all the experiments just described the membrane used was the freshly removed skin of a rat or rabbit, nevertheless we found that this tsetse will feed through other membranes, e.g., sheep's bladder, peritoneal tissue and membrane composed of a thin sheet of rubber. As regards the last, one fly out of five completely distended itself with defibrinated blood through this membrane. Although the flies had not the slightest

TABLE VIII.-Giving the result of feeding G. palpalis on various fluids through a membrane of skin -----··--- --·--- No. Sex Membrane Nature of fluid Result Remarks

Fresh rabbit skin ... Methylene blue No visible distension, Anterior and mid-gut blue. solution• but abdomen of Posterior gut green. bluish tinge Salivary glands, mal- pighian tubules and fat bodies blue 1. ~ No visible distension ~ Same rabbit" skin 1.4 " No staining" seen on dis- hours old " " section 4 ~ " " 5

Downloaded by [Australian Catholic University] at 11:20 26 August 2017 glycerine 18 ~ Partial distension ... Anterior and mid-gut and " " also salivary glands and fat bodies stained 19 ~ Anterior gut blue I " " " • The methylene blue solution used was the weak solution employed in Romanowsky's stain; its formula is given on page 369. difficulty in piercing the rubber, they did not seem able in the majority of cases to imbibe blood through it; possibly owing to the extremely elastic nature of the rubber the lumen of the proboscis was occluded, thus preventing the passage of fluid through it. 377 The practical point that we have to decide is, does Glossina in nature obtain food other than blood ? Although this question is not answered by the experiments described above, yet the results obtained are very suggestive. They show that under favourable conditions Glossina will take up solutions of vegetable origin, such as sugar and water. Apparently one essential is that the fluid should be presented enclosed by a membrane. The reason for this

TABLE IX.-Giving the results of feeding G. palpalis on blood through membranes other than freshly-removed skin

No. Sex Membrane Nature of fluid Result Remarks -- I

I <:! Rabbit skin removed Defibrinated goat blood Complete distension Immediately 2.4 hours previously and partially decomposed 2. ~ , 3 ~ " Partial distension" ... After "1 5 minutes 4 <:! Ox bladder" ...... 1 " Complete distension Immediately 5 ~ " 6 <:! " " Partial distension" ... After "15 minutes 7 <:! " " Slight distension ... 8 <:! Peritoneal" " Partial distension ... " membrane, ox " " 9 ~ Slight distension ... 10 <:! " " " II ~ Thin rubber" sheeting " " " 12. <:! " Complete" distension " 13 ~ " " No VIsible distension " 14 J " " " 15 <:! " " " " 16 ~ " ," ," " " I "

is not that the flies require the fluid to be at a positive pressure before they can imbibe; it is possibly a mechanical difficulty, the fly being unable to take up fluid unless its proboscis is buried in the membrane or tissue. The experiments show, moreover, that even when the Downloaded by [Australian Catholic University] at 11:20 26 August 2017 proboscis is inserted into a membrane, it does not necessarily follow that the fluid enclosed will be imbibed to any considerable extent. The results obtained prove most definitely that Glossina palpalis exhibits a preference for certain fluids, especially blood, red cells and haemoglobin. The physical character of the fluid does not, within limits, appear to matter. Defibrinated blood, a 50 per cent. suspension of red cells in saline, and a solution obtained by laking two parts of washed red cells with three of distilled water, are taken up with equal readiness, whilst plasma and normal salt solution are only imbibed in small quantities as a rule. Turning to the question whether Glossina takes up vegetable juices in nature, we may state at once that we have not had the opportunity of deciding the point absolutely. Glossina of both sexes were frequently seen to plunge their proboscis into bananas, oranges and mangoes which had had their skin removed, and also into the skin itself. The insertion of the proboscis was repeated again and again, and the organ often remained buried in the fruit for a minute or more; the whole procedure certainly gave one the impression that the flies were endeavouring to obtain food from the fruit. Furthermore, we noticed that Glossina palpalis will pierce leaves placed on the surface of a fluid with the object apparently of imbibing the fluid below. Unfortunately, we had not time to extend our observations on this subject, but in view of the results already detailed, and of the observations of Carpenter who found vegetable tissue in a proportion of wild G. palpalis dissected by him, we consider that it is highly probable that tsetse do take up food of vegetable nature. The frequency with which a hungry fly will insert its proboscis, or at least attempt to do so, into practically any object presented-they can even be seen trying to pierce the glass vessel in which they are kept when no skin is in immediate contact with the glass-suggests that, in the absence of blood, they are on the look out for other food, for there is every reason to believe that Glossina recognises when it is in the presence of the skin of a living animal. Assuming Glossina palpalis cannot reproduce itself or live for any long period in the absence of blood, it is possible that food of a vegetable nature may suffice to enable it to exist in a locality which for a certain period in the year is denuded of vertebrates. Downloaded by [Australian Catholic University] at 11:20 26 August 2017

CONCLUSIONS

1. About eight per cent. of the wild G. palpalis in this district contain recognisable red blood cells-seven per cent. of mammalian origin and one per cent. nucleated red cells of unknown origin. 2. Seventy-two hours after G. palpalis had completely distended itself on rat's blood recognisable red cells could no longer be found 379 in its intestine; after being fed on a fowl nucleated red blood cells could be recognised in 40 per cent. of cases at the end of a similar period. The flies were kept at a temperature of 80°-86° F. 3· Neither shed blood nor other fluid which is exposed (not covered by a membrane) can be imbibed by G. palpalis. 4· G. palpalis can take up through a membrane of fresh skin not only blood and various dilutions of it with normal saline, but also suspensions of red blood cells in normal saline, and solutions of haemoglobin (both freshly made from red blood cells, and the dried crystalline preparation of commerce) in distilled water. 5· Fluids other than blood such as solutions of sugar, sodium chloride, and glycerine, in water containing a small quantity of a dye (methylene blue, neutral red or fuchsin) are also taken up through a membrane of fresh skin by G. palpalis, but not so quickly or so readily as is blood. 6. G. palpalis exhibits a definite selective taste for the various fluids presented to it under the membrane; blood, red cells, and haemoglobin solution being much preferred. The attractive element in the blood is the fraction of the red cells soluble in water, probably haemoglobin. 7· G. palpalis which had been starved for a day or two can often be seen to insert the proboscis repeatedly into oranges, bananas or other fruits which may be offered them. 8. We are of opinion that G. palpalis in nature may under certain conditions take up fluid other than blood. Downloaded by [Australian Catholic University] at 11:20 26 August 2017 REFERENCES

C.uPJ:NTJ:R, G. D. H. (1911). Progress Report on Investigations into the Bionomics of Glossina palpalis, July 1.7, 1910, to August 5, 19II. Report of the Sleeping Sickness Commission of the Royal Society, No. XII, pp. 79-u r.

--- (1913). Second Report on the Bionomics of Glossina fuscipes (palpalis) of . Reports of the Sleeping Sickness Commission of the Royal Society, No. XIV, pp. I-37·

DEGEN (1909). Sleeping Sickness Bulletin, Vol. I, p. 471.

LLOYD, LL. (1911.). Notes on Glossina monitans in Northern Rhodesia. Bull. Ent. Res., Vol. III, pp. 95-96.

--- (1913). Glossina morsitans in the laboratory. Ann. 'Irop. Mtd. fS Parasit., Vol. VII, PP· z85-1.91..

MAUGHAM (1911). Slteping Sicknm Bulletin, Vol. III, p. 1.71.

RoDHAIN, J., PoNs, C., VANDJ:NBRANDJ:N, J., and BEQUAERT, J. (1911.). Contribution au Mecanisme de Ia Transmission des Trypanosome& par lea Glossines. Arch. f. SchiUs- u. 'Irop. Hyg., B. 16, S. 731.-739·

STUHLMANN, F. (1907). Beitrage zur Kenntnis der Tsetsefliege. Arb. a. d. Kaistrlichtn Gtsundheitsamte, B. z6, S. 301-383.

TAun, M. (1911.). Experimentelle Studien tiber die Beziehungen der Glossina morsitans zur Schlafkrankheit. Zeitschr. f. Hygim•, B. 71., S. 3 r6-po. Downloaded by [Australian Catholic University] at 11:20 26 August 2017