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Physiology of excretion in the larva of Corcyra cephalonica Stainton (Lepidoptera, Pyralidae)
J, Ins. P&y&l.,
1962, Vol. 8, pp. 223 to 232. Pergamon Press Ltd.
Printed in Great Britain
PHYSIOLOGY OF EXCRETION IN THE LARVA CORCYRA ~~~HA~~~~~A STAINTON (LEPIDOPTERA, PYRALIDAE~
OF
P. N. SRIVASTAVA Department
of Zoology, University of Lucknow, Lucknow, India
(Received 8 May
1961; revised 1 h’owmbeu
1961)
Abstract-In the larva of Corcyra ce~ha~o~a Stainton the six ~alpi~hi~ tubules are cryptonephric. Each tubule is physiologic~ly and histologically divided into the proximal, the middle, and the distal portions. The haemolymph diffuses in through the middle portion without any discrimination between metabolically useful and excretory substances. The proximal portion retains the excretory products in the lumen but allows water and other useful substances to pass out to the body fluid. The distal portion absorbs moisture from the excreta which is used to flush out the solid matter from the lumen of the tubule. The cycle is maintained and the nitrogenous wastes are eliminated efficiently in this manner.
INTRODUCTION A CO~~PREHENSIVE account of the physiology of excretion in ~~o~n~us has been given by WIGELESWORTH(193 l), and in the larva of Tenebrio m&or by PATTON and CRAIG(1939). Recently RAMSEY(1953, 1954, 1955, 1956, 1958) has described the excretory mechanism in the stick insect Dixippus morosus; he maintains that the whole process of excretion involves the contributory processes of diffusion, ;rbsorption, and secretion, of which the reabsorption takes place mainly in the rectal glands; the other two processes taking place in the iVIalpighian tubules. It has been shown in Sitophilus gram&us by GUPTA and SINHA (1960) and in Pwiplnneta americana by SRIVASTAVA and GUPTA (1961) that the main organic constituent of urine, namely uric acid, is not excreted through the ~alpighian tubules but through the anterior portion of the hind-gut. These facts tend to prove that the process of excretion does not necessarily involve a uniform mechanism throughout insects. In view of these facts, and also because of existing controversy regarding excretion in insects, detailed investigations on the excretory system and excretion of ~~~lllot~~p~ africana, Au~~~opho~~ foveicollis, Fo~~~u~a sp., and the larva of C?orcyrn cephalonica were undertaken by the author to elucidate a mechanism common to insects belonging to different groups. In this paper, however, the excretion in the larva of Corcyra cephalonica has been dealt with in detail, while only the results on the other species are included in the discussion.
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Distribution of uric acid in various tissues gave a clue regarding the organs involved in the excretion of the nitrogenous wastes; chemical analysis of the food and excreta made it possible to assess the composition of the urine; injection of neutral red and methylene blue dyes into the body cavity revealed the course taken by the substances during excretion; and a study of the glycogen in the various regions of the Malpighian tubules helped to show if metabolically useful substances make their way into the Malpighian tubules. MATERIALS
AND METHODS
Larvae of the rice moth Corcyra cephalonica were reared in the laboratory on raw milled rice, and the last instar larvae were used for the experiments. For histological studies 5 p thick serial sections were cut from tissues fixed in alcoholic Bouin’s and Carnoy’s fluids. Iron haematoxylin, Delafield’s haematoxylin and eosin, and Mallory’s triple stains were used.
WIGGLESVVORTH’S (1931), HOLLANDE’S(in COWDRY,1952), and DE GALAKTHA’S (in LILLIE, 1954) histochemical methods were employed for the detection of uric acid and urates in the sections. Benedict’s test (BALDWIN and BELL, 1955), however, proved quite sensitive and handy in the biochemical detection of uric acid and urates in various tissues and excreta. Positive reactions with all these tests have been adopted as the criterion for the presence of uric acid in the materials examined. Semi-micro methods (VOGEL, 1954) were used for the analysis of the inorganic constituents of the food and the excreta, whereas methods given in HAWK et al. (1947), BALDWIN and BELL (1955), and also those used by WICGLE~WORTH (1931) were employed to detect the organic constituents. Moisture in the excreta was determined as described by FISHER et aZ. (1945). The distribution of the giycogen in the different portions of the Malpighian tubules was demonstrated with the help Solutions (0.001 per cent) of of Best’s carmine, iodine, and PAS reactions. neutral red and methylene blue were injected into the body cavity of the larva by means of an ’ Agla’ micrometer syringe in order to see the course of fluids taken during excretion. OBSERVATIONS
Anatomy and histology of Malpighian tubule The larvae possess six cryptonephric Malpighian tubules which are arranged in two groups. The tubules do not directly open into the gut, but the three on either side originate separately from a common, thin-walled tri-radiate sac-like urinary bladder which communicates with the alimentary canal by a short duct situated near the junction of the mid- and bind-gut (Fig. 8). All the six tubules are similar to one another and no evidence could be found that they functioned differently. Each tubule extends forwards along the mid-gut almost up to its middle and then turns backwards closely following the same course right up to the colon. It then enlarges and forms a short bulb-like ampulla and again becomes narrow. ‘Ihe narrow portion enters beneath the fascia surrounding the rectum and follows a zigza.g course over the rectal wall. The length of each Malpighian
PHYSIOLOGY
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CEP~ALONICA
22.5
tubule from its origin to the ampulla in freshly dissected specimens is, on an average, 11 mm. On the basis of location and structure a tubule can be divided in three parts: (i) the proximal, (ii) the middle, and (iii) the distal.
4
--I
O-05
mm
A
0
0*05
mm
FIG. 1. A transverse section through the proximal portion of the ~Ialpighian tubule of Corcyru larva. br. bor. = brush border; nu. =nucleus. FIG. 2. A transverse section through the middle portion of the Malpighian tubule of Corcyra larva. hon. bor. = honeycomb border; nu. =nucIeus.
The ~r~xirn~~ portion. This comprises an inverted U-shaped loop having an ascending and a descending limb situated in the region of mid-gut. It has a milkvwhite appearance and measures 8.0~ 0.09 mm. Both the limbs are similar In histology, being made up of five to six cells whose boundaries are not clearly demarcated (Fig. 1). The free end of the cells is provided with a brush border, similar to that of the lower segment of ~~o~~~~~ (~~GGLESWORTH, 1931). The lumen is quite large and remains filled with solid excretory matter, mainly uric acid. The middle portion. The distal continuation of the descending limb in the region of the ileum and colon forms the middle portion. It comprises a narrow tubular part and the ampulla, both of which are translucent. The tubular part measures 2.5 x 0.05 mm, whereas the ampulla is about 1-3 mm in diameter. The histology is different from the proximal portion inasmuch as the free end of the cells has a honeycomb border (Fig. 2). No vaIvular arrangement could be detected at either end of the ampulla. The dista~p~~tio~. The convoluted part of the Malpighian tubule following the ampulla and situated beneath the fascia constitutes the distal portion, Proximally it communicates with the ampulla of the middle portion, whereas distally it ends blindly. Histologically it is similar to the middle portion. ~istyi~ti~~
of uric acid in various ttsfues
Malpighian
tzdwles. Histochemical tests for uric acid revealed a complete picture of its distribution in the various regions of the Malpighian tubules. A heavy deposit is present in the lumen of the proximal portion of the tubule (Fig. 3),
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but it is completely absent from its cells. Spheres of uric acid with radial striations as shown in Rhodnius (WIGGLESWORTH,1931) could clearly be seen in sections of this portion of the tubule prepared by WIGGLESWORTH’S method (Fig. 5). On the other hand, uric acid in crystallized form could be detected only in the cells (Fig. 4) and never in the lumen of the middle portion of the tubule, whereas no trace of it could be found either in the cells or in the lumen of the ampulla and the distal portion of the tubule. Fat body. Sometimes the fat body gave a faint reaction for uric acid with Benedict’s reagent, but all other tests were negative. Under the circumstances it may not be inappropriate to presume that the faint reaction is due to the small amount of uric acid produced during metabolism of the fat cells themselves. Uric acid does not accumulate in the fat body of Corcyra larva, unlike cockroaches (CulkoT, 1895) and certain other insects (WIGGLESWORTH,1953; ROEDER,1953). Wall of the mid- and hind-guts.
Both of these gave negative results.
Contents of the mid- and hind-guts. A negative reaction was obtained with the contents of the mid-gut, whereas those of the hind-gut gave a positive reaction. Excreta.
A strong positive reaction for uric acid was obtained with the excreta.
Chemical composition of food and excreta Inorganic constituents. The inorganic constituents of food (raw milled rice) and excreta of Corcyra larvae fed on rice are given in Table 1. TABLE 1
Constituents
Food (raw milled rice)
Carbonate Chloride Nitrate Phosphate Sulphate Calcium Iron Magnesium Potassium Sodium Ammonia -I- = Positive;
Excreta
+ + (Weak) + + + ? - = Negative;
? = Doubtful.
Organic c~tit~nts. The data given by SUBRAHMANYAN et al. (1954) indicate that the main constituents of raw milled rice are 79.6 per cent carbohydrate, 12.6 per cent moisture, and 6.6 per cent protein.
6 FIG. 3. A photograph of a transverse section through the proximal portion of the Malpighian tubule of a Corcyra larva. Uric acid (ur.) is seen only in the lumen of the proximal portion. FIc. 4. A photograph of a transverse section through the middle portion of the Malpighian tubule of a Corcyra larva, showing uric acid (ur.) inside the cells. FIG. 5. A photograph of a transverse section of the proximal portion of the Malpighian tubule of a Corcyra larva, showing uric acid spheres (ur. ac. sph.) with radial striations. FIG. 6. A photograph of a transverse section through the middle portion of the Malpighian tubule of a Corcyra larva, showing glycogen in the lumen (gly. 1.) as well as in the cells (gly. c.), and indicating its (glycogen) entry from the blood. FI(;. 7. A photograph of a transverse section through the proximal portion of the Malpighian tubule of a Corcyra larva, showing glycogen in the cells (gly. c.) which is diffusing out.
{~ : ~
L~I~ ~ ~ t~roxim~pc~t~n
~-~
N,t Toqenous w~,~tes
l
~ " Mwto~caHy useful substances and C o n c e n f raF'~d ,o~,d "~tmosr d r y U w l n ~
•
A
r
\
.~ ,~C_ U r i n a r y b l a d d e r
\ Hoernollfmph( e x e r t p~O~lin ond other substances of Ior~r molecules) is dlf fusing |n
//J b~ °'~°° I ~Hor~'yc~ l
,
M~ddl~ pore,on
B
~u|la
\
J ?)'.'::
I Moisture ~rom the ¢xcreto ~s absorbed h~re and passed i n t o ~ middle portion)
FiG. 8. Diagrammatic representation of the process of excretion in a Corc3'ra larva. Arrows indicate the course taken by substances during excretion. Figs. A and B show the details of the histology of the proximal and middle portions respectively.
PHYSIOLOGY
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Analysis of the excreta shows that protein, glycogen, dextrin, reducing sugars, cellulose, acetone, aceto-acetic acid, lactic acid, creatin and creatinine, xanthine, cystine, and guanine are absent. Its main constituents are a large quantity of uric acid, traces of urea, a certain amount of starch, and about 9-6 per cent water. The chemical composition of food and excreta described above provides evidence that all the protein and most of the carbohydrate and water get utilized and the nitrogenous waste is excreted out mainly as uric acid. Carbonates, chlorides, calcium, magnesium, iron, a certain amount of starch, and some water together with large quantities of uric acid form the excreta of Corcyra larva. Excretion 5fdyes
The course taken by the injected dyes (neutral red and methylene blue) and their excretion through the Malpighian tubules is described in Table 2. TABLE
2
Interval
Observation
5 min
Middle portion of the tubule fills with the dye, but the ascending and major portion of the descending limb, the distal portion, and the ampulla do not take up the dye.
10 min
The dye has almost filled the lumen of the entire proximal portion of the tubule; there is no indication of the dye in the distal portion or the gut. The dye appears to have ascended from the middle portion and not passed in directly from the body cavity, as the cells of this portion remain transparent.
30 nun
The lumen of the proximal portion becomes turgid due to the inflow of the dye, while the middle portion becomes colourless, showing that almost all the dye from the body fluid has been removed.
4 hr
About one-third of the lower portion of the descending limb is colourless and empty while the remainder is still full of coloured dye solution.
24 hr
No trace of the coloured solution remains in any part of the tubules, but fine granules of the injected dye are still present in the lumen of the ascending limb and the urinary bladder. Some granules are seen in the lumen of the hind-gut.
Appearance of the dye solution in the middle portion of the tubule almost immediately after injection into the body cavity presumably indicates that the dye enters the fumen of the middle portion of the tubule at the same concentration as that in the haemolymph. The course taken by the dyes during excretion shows that each portion of the tubuleis functionally different from the other and allows only one-way permeability of the fluids. No substance from the body fluid enters the proximal portion. The
228
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solid excretory matter is retained in its lumen, but water is allowed to return to the body fluid. On the other hand, dye in solution enters the middle portion through its cells but nothing can pass out into the body fluid from this region. The distal portion does not seem to have any connexion with the body fluid as the dye could never be seen in this portion of the tubule. The experiments, therefore, reveal that also on a physiological basis the Malpighian tubule of Corcyra larva is divided into the same three portions, viz. the proximal, the middle, and the distal. Distribution of glycogen in the Malpighian tubules Little is known about glycogen in the Malpighian tubules of insects, and this seems to be the first record. As with uric acid, a differential distribution of glycogen in the Malpighian tubules of Corcyra larva could be seen. The results are described below. (1) Glycogen could not be detected distal portion of the tubule.
either
(2) A heavy deposit (Fig. 6) of glycogen portion, while a fair amount of this substance within the cells of this region.
in the cells or in the lumen
of the
is present in the lumen of the middle could be detected in a state of diffusion
(3) The amount of glycogen both in the lumen as well as in the cells of the proximal portion (Fig. 7) gradually decreases in the descending limb, becoming less and less in the ascending limb till it is altogether absent by the time it covers about half or two-thirds of the ascending limb. No glycogen could be detected in the urinary bladder. DISCUSSION
There is ample evidence for the fact that in Corcyra larvae the nitrogenous wastes are excreted mainly through the Malpighian tubules and do not accumulate in the fat body. A distinct morphological and physiological differentiation between the different portions of each tubule is clearly indicated. The experiments with the injected dyes suggest that the middle and proximal portions of the tubules perform different functions during the process of excretion. Practically the whole plasma (except the protein and certain other larger molecules) containing both metabolically useful and excretory substances presumably enters the lumen of the tubule. No selection seems to be exercised and the process is more or less a passive diffusion of the haemolymph, similar to that described by RAMSAY(1958) in Dixippus morosus and PATTONand CRAIG(1939) in the larva of Tenebrio molitor. The fluid gradually ascends into the proximal portion in which a selective reabsorption appears to take place. Almost all the water and metabolically useful substances are permitted to pass out to be restored to the haemolymph, while the nitrogenous wastes and other excretory matter, like the solid dyes, are retained in the lumen. As the time advances more and more water is absorbed and the urine becomes highly concentrated till it reaches the ascending limb. The urine from all the three tubules of one side collects into the corresponding urinary bladder and is ultimately voided into the lumen of the gut (see Fig. 8).
PHYSIOLOGY
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The distal portion does not seem to take any active part in the excretion of nitrogenous wastes since at no time could the dyes or uric acid be found in this region. This is in agreement with METALNIKOV(1908), who found that in GalZeria larvae the tubes covered over by the fascia could never take up the dye. This portion of the tubule appears to be mainly osmoregulatory, reabsorbing water from the excreta in the rectum. Its honeycomb border, similar to that of the middle portion of the tubule, leads me to believe that the water absorbed from the excreta is not allowed to pass out and return to the body fluid but is collected in the ampulla from which it is forced into the middle portion of the tubule. This water may thus help flush the spheres of uric acid from the tubules into the gut. This finding is in conformity with that of POLL (1938, in WIGGLES~~O~TH,1953) who stated that the water absorbed by the perirectal tubes is used to flush out the solid matter from the lumen of the tubules of caterpillars. The diffusion and reabsorption cycle is shown diagrammatically in Fig. 8. The suggestion based on the injection experiments that there is a division of labour in the tubules of Corcyra larva finds strong support from the study of the distribution of glycogen and uric acid. The absence of both glycogen and uric acid from the distal portion of the tubule and the ampulla clearly indicates that no substance from the body cavity enters this portion. At the same time, the presence of large quantities of glycogen in the cells as well as in the lumen of the middle portion of the tubuies strengthens the contention that not only the nitrogenous wastes and water but in fact almost the whole plasma minus the protein enters through this region. The gradual decrease in the quantity of glycogen from the lumen of the descending limb of the proximal portion and its absence from the ascending limb coupled with the fact that uric acid was never found in the cells of both the limbs, although it is present in large quantity into the lumen, is evidence that selective reabsorption of only the metabolically useful substances along with water takes place through this portion of the Malpighian tubule. The postulate presented here that the different segments of a Malpighian tubule perform separate functions is in conformity with the findings of WIGGLESWORTH(193 1) on Rho&us. The proximal and middle portions of the tubules in Corcyra larvae are not only similar in structure to the corresponding regions, i.e. the Iower and upper segments of the tubule of Rho~~i~s, but also exhibit the same functions. However, the tubules of ~~a~nius are not cryptonephric, hence none of its parts corresponds to the distal portion of the tubule of Corcyra larva. But the present view differs from that of WIGGLESWORTH (1931) in that there is no selective filtration of the body fluid through the Malpighian tubules in Corcyra larva, as it has been established that practically the whole plasma (except proteins and larger molecules), and not only the uric acid in the form of sodium and potassium urates as reported in Rho&&s, enters the middle portion of the Ma~pighian tubule of Corcyra larvae. The author’s views are further supported by the findings of RAMSAY(1958}, who has shown that in ~~~~pus morosus metabolically useful substances enter into the Malpighian tubules by passive diffusion and are reabsorbed in the rectum.
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P. N. SRIVASTAVA
The observation of glycogen in the Malpighian tubules of Corcyra larva finds support in the experimental findings of PATTONand CRAIG(1939) on T. molitor, as they have definitely shown that the inward movement through the tubes is not confined to water and nitrogenous wastes but includes electrolytes and a number of other compounds like amino acids. Absence of any selective absorption of water, salts, and amino acids, as demonstrated by their ingenious experiments, should in my opinion be equally applicable to simpler carbohydrates. The digested and absorbed carbohydrates along with the other constituents (except proteins) of the body fluid should be penetrating the Malpighian tubules of Co~cy~~ larvae in a similar manner, as the amino acids, glycine and glutamic acids, urea, etc., readily enter the tubes of T. molitor when added to the perfusion medium. As in Corcyra larvae, uric acid has been reported to be the main constituent of the nitrogenous waste in Galleria larvae, in Heterogenea, in Tineola biselliella, in ~n~~e~eff, and in silkworms by ~~ALNI~OV (1908), SAMSON (1908), HOLLANDE and CARDEBARD (1926), LEIFERT(1935), GRASSEand LESPERON(1936), and KUWANA (1937) respectively. Ammonia is absent from the excreta of Corcyra larvae as in that of Rhodnius. Absence of sulphur from the food of Corcyra larvae as in that of Rhodnius explains the absence of ammonia from their excreta. The contention that ammonia is present only in excreta of those insects which take food rich in sulphur is further supported by the observations of PRADHAN(1949) on ~nthy~~s fasciatus. The presence of a certain amount of undigested
carbohydrate (starch) in the excreta of Corcyra larvae is interesting and calls for comment. The food of Corcyra larvae, with which it always remains surrounded, is available in plenty. But since it contains only 12.9 per cent of moisture out of which some has to be excreted, as is clear from the analysis of the excreta, there appears to be a reasonable possibility that the larva is induced to ingest large quantities in order to obtain the requisite amount of moisture. It is suggested that the carbohydrate ingested in excess of requirements remains undigested and is disposed of in the excreta.
There are six Malphighian tubules, three on each side of the gut, The tubules are cryptonephric and are physiologically and histologically divided into the distal, the middle, and the proximal portions. The distal and the middle portions have a honeycomb border, the proximal a brush border. Experimental evidence shows that each portion of the tubule has one-way permeability. The haemolymph ~excluding proteins and certain other larger molecules) is constantly diffusing in the middle portion of the Malpighian tubules, the excretory products are retained in the lumen of the proximal portion, while water and metabolically useful substances pass out into the body fluid. The distal portion absorbs water from the excreta. This water does not pass back to the body fluid but helps to flush out the solid matter from the lumen of the tubule. Further evidence for this diffusion and reabsorption cycle has been provided as a result of the study of the distribution of uric acid and glycogen in various portions of the Malpighian tubules. Besides
PHYSIOLOGYOF EXCRETIONIN THE LARVAOF CORCYRA
CEPHALONICA
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231
uric acid, carbonates, chlorides, calcium, iron, magnesium, and a certain amount of undigested starch constitute the excreta of Corcyra cephalonicalarvae. Acknowledgements-The work was done under the guidance of Dr. P. D. GUPTA to whom I am greatly indebted. I take this opportunity to express my gratitude to Professor M. B. LAL for kindly providing me with facilities for research work, and, finally, I wish to thank the Government of India for the award of the Research Training Scholarship. REFERENCES BALDWIN E. andBELL D. J. (1955) Cole’s Practical Phy~oZa~~c~~Chemistry. Heffer, Cambridge.
COWDRY E. V. (1952) ~aborato~ Technique in ~ioZogy and Medicine. Williams & Wilkins, Baltimore. C&NOT L. (1895) Etudes physiologiques sur les Orthopteres. Arch. Biol., Paris 14,293-341. FKSHERH. J., WARRENL. E., SALE J. W., Ross W. H., RINDOLLARW. F., and MARIANL. 0. (1945) Oficial and Tentative Methods of Analysis of the Association of Official Agricultural Chemists. Benjamin Franklin Station, Washington. GRASSE P. P. and LESPERON L. (1936) Mue et excretion chez le ver a soie. C.R. Sot. Biol., Paris 122,1013-1015. GUPTA P. D. and SINHA R. N. (1960) Excretion and its products in some stored-graininfesting beetles. Ann. ent. Sot. Amer. 53, 632-638. HAWK P. B., OSER B. L., and SUMMERSONW. H. (1947) Practical Physiological Chemistry. Blakiston, Philadelphia. HOLLANDEA. C. and CARDEBARD N. (1926) Notes chimiques et physiologiques se rapportant aux excrements de la teigne du crin (Tineola ~se~liella Hummel ; Syn. cr~ne~laTreitscheDuponchol). Bull. Sot. Chim. bioE., Paris 8, 631-635. K~JWANA2. (1937) Uric acid excretion in silkworm. yap. J. Zool. 7, 305-309. LEIFERT H. (1935) Untersuchungen iiber den Exkretstofbvechsel bei eiren Raupen und puppen von Antheraea pernyi. Zool. Jb. (Physiol.) 55, 13 l-l 90. LILLIER. D. (1954)HistopathoZogic TechnicandPracticalHistochemistry. Blakiston, NewYork. METALNIKOVS. (1908) Recherches experimentales sur les chenilles de Galleria mellonella. Arch. Zool. exp. g&z. 8, 489-588. PATTONR. L. and CRAIG R. (1939) The rates of excretion of certain substances by the larvae of Tenebrio molitor L. J. exp. ZooE. 81, 437-457. PRADHANK. S. (1949) On the physiology of digestion in the larva of woolly bear, Anthremds fasciatus Herbst. J. zool. Sot. India 1, 107-119. RAMSAYJ. A. (1953) Active transport of potassium by the Malpighian tubules of insects. J. exp. Biol. 30, 358-369. RAMSAYJ. A. (1954) Active transport of water by the Malpighi~ tubules of the stick insect, Dixippus mo~osus (Orthoptera, Phasmidae). J. exp. Biol. 31, 104-l 13. RAMSAYJ. A. (1955) The excretory system of the stick insect, Dixippzrs morosus (Orthoptera, Phasmidae). r. exp. Biol. 32, 183-216. RAMSAY J. A. (1956) Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae) : Calcium, magnesium, chloride, phosphate and hydrogen ions. J. exp. Biol. 33, 697-891. RAMSAYJ. A. (1958) Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae): Amino acids, sugars and urea. r. exp. Biol. 35, 871-891. ROEDER K. D. (1953) Insect Physiology. Wiley, New York. SAMPSONK. (1908) Uber des Verhalten der Vasa Malpighii und die exkretorische Funcktion der Fettzellen wahrend der Metamorphose von Heterogenia limacodes Hiifn. Zool. Jb. (Anat.) 26, 403-422. SRIVA~TAVA P. N. and GUPTA P. D. (1961) Excretion of uric acid in P~~~aneta americana L. 3’. Ins. Phys~oZ. 6, 163-167.
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SUBRAHMANY~ V., BAINSG. S., SWAMINATHAN J., and BHATIAD. S. (1954) Investigations on grain substitute-V. The nutritive value of synthetic rice. SUE. cent. food tech. res. Inst. Mysore 4, 55-57. VOCEL A. I. (1954) A Textbook of Macro and Seµ Qualitative Inorganic Analysis. Longmans, London. WIGGLESWORTH V. B. (1931) The physiology of excretion in the blood-sucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). J. exp. BioE. 8, 41 l-451. WIGGLESWORTH V. B. (1953) The Principles ofInsect Physiology. Methuen, London.
1962, Vol. 8, pp. 223 to 232. Pergamon Press Ltd.
Printed in Great Britain
PHYSIOLOGY OF EXCRETION IN THE LARVA CORCYRA ~~~HA~~~~~A STAINTON (LEPIDOPTERA, PYRALIDAE~
OF
P. N. SRIVASTAVA Department
of Zoology, University of Lucknow, Lucknow, India
(Received 8 May
1961; revised 1 h’owmbeu
1961)
Abstract-In the larva of Corcyra ce~ha~o~a Stainton the six ~alpi~hi~ tubules are cryptonephric. Each tubule is physiologic~ly and histologically divided into the proximal, the middle, and the distal portions. The haemolymph diffuses in through the middle portion without any discrimination between metabolically useful and excretory substances. The proximal portion retains the excretory products in the lumen but allows water and other useful substances to pass out to the body fluid. The distal portion absorbs moisture from the excreta which is used to flush out the solid matter from the lumen of the tubule. The cycle is maintained and the nitrogenous wastes are eliminated efficiently in this manner.
INTRODUCTION A CO~~PREHENSIVE account of the physiology of excretion in ~~o~n~us has been given by WIGELESWORTH(193 l), and in the larva of Tenebrio m&or by PATTON and CRAIG(1939). Recently RAMSEY(1953, 1954, 1955, 1956, 1958) has described the excretory mechanism in the stick insect Dixippus morosus; he maintains that the whole process of excretion involves the contributory processes of diffusion, ;rbsorption, and secretion, of which the reabsorption takes place mainly in the rectal glands; the other two processes taking place in the iVIalpighian tubules. It has been shown in Sitophilus gram&us by GUPTA and SINHA (1960) and in Pwiplnneta americana by SRIVASTAVA and GUPTA (1961) that the main organic constituent of urine, namely uric acid, is not excreted through the ~alpighian tubules but through the anterior portion of the hind-gut. These facts tend to prove that the process of excretion does not necessarily involve a uniform mechanism throughout insects. In view of these facts, and also because of existing controversy regarding excretion in insects, detailed investigations on the excretory system and excretion of ~~~lllot~~p~ africana, Au~~~opho~~ foveicollis, Fo~~~u~a sp., and the larva of C?orcyrn cephalonica were undertaken by the author to elucidate a mechanism common to insects belonging to different groups. In this paper, however, the excretion in the larva of Corcyra cephalonica has been dealt with in detail, while only the results on the other species are included in the discussion.
223
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Distribution of uric acid in various tissues gave a clue regarding the organs involved in the excretion of the nitrogenous wastes; chemical analysis of the food and excreta made it possible to assess the composition of the urine; injection of neutral red and methylene blue dyes into the body cavity revealed the course taken by the substances during excretion; and a study of the glycogen in the various regions of the Malpighian tubules helped to show if metabolically useful substances make their way into the Malpighian tubules. MATERIALS
AND METHODS
Larvae of the rice moth Corcyra cephalonica were reared in the laboratory on raw milled rice, and the last instar larvae were used for the experiments. For histological studies 5 p thick serial sections were cut from tissues fixed in alcoholic Bouin’s and Carnoy’s fluids. Iron haematoxylin, Delafield’s haematoxylin and eosin, and Mallory’s triple stains were used.
WIGGLESVVORTH’S (1931), HOLLANDE’S(in COWDRY,1952), and DE GALAKTHA’S (in LILLIE, 1954) histochemical methods were employed for the detection of uric acid and urates in the sections. Benedict’s test (BALDWIN and BELL, 1955), however, proved quite sensitive and handy in the biochemical detection of uric acid and urates in various tissues and excreta. Positive reactions with all these tests have been adopted as the criterion for the presence of uric acid in the materials examined. Semi-micro methods (VOGEL, 1954) were used for the analysis of the inorganic constituents of the food and the excreta, whereas methods given in HAWK et al. (1947), BALDWIN and BELL (1955), and also those used by WICGLE~WORTH (1931) were employed to detect the organic constituents. Moisture in the excreta was determined as described by FISHER et aZ. (1945). The distribution of the giycogen in the different portions of the Malpighian tubules was demonstrated with the help Solutions (0.001 per cent) of of Best’s carmine, iodine, and PAS reactions. neutral red and methylene blue were injected into the body cavity of the larva by means of an ’ Agla’ micrometer syringe in order to see the course of fluids taken during excretion. OBSERVATIONS
Anatomy and histology of Malpighian tubule The larvae possess six cryptonephric Malpighian tubules which are arranged in two groups. The tubules do not directly open into the gut, but the three on either side originate separately from a common, thin-walled tri-radiate sac-like urinary bladder which communicates with the alimentary canal by a short duct situated near the junction of the mid- and bind-gut (Fig. 8). All the six tubules are similar to one another and no evidence could be found that they functioned differently. Each tubule extends forwards along the mid-gut almost up to its middle and then turns backwards closely following the same course right up to the colon. It then enlarges and forms a short bulb-like ampulla and again becomes narrow. ‘Ihe narrow portion enters beneath the fascia surrounding the rectum and follows a zigza.g course over the rectal wall. The length of each Malpighian
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tubule from its origin to the ampulla in freshly dissected specimens is, on an average, 11 mm. On the basis of location and structure a tubule can be divided in three parts: (i) the proximal, (ii) the middle, and (iii) the distal.
4
--I
O-05
mm
A
0
0*05
mm
FIG. 1. A transverse section through the proximal portion of the ~Ialpighian tubule of Corcyru larva. br. bor. = brush border; nu. =nucleus. FIG. 2. A transverse section through the middle portion of the Malpighian tubule of Corcyra larva. hon. bor. = honeycomb border; nu. =nucIeus.
The ~r~xirn~~ portion. This comprises an inverted U-shaped loop having an ascending and a descending limb situated in the region of mid-gut. It has a milkvwhite appearance and measures 8.0~ 0.09 mm. Both the limbs are similar In histology, being made up of five to six cells whose boundaries are not clearly demarcated (Fig. 1). The free end of the cells is provided with a brush border, similar to that of the lower segment of ~~o~~~~~ (~~GGLESWORTH, 1931). The lumen is quite large and remains filled with solid excretory matter, mainly uric acid. The middle portion. The distal continuation of the descending limb in the region of the ileum and colon forms the middle portion. It comprises a narrow tubular part and the ampulla, both of which are translucent. The tubular part measures 2.5 x 0.05 mm, whereas the ampulla is about 1-3 mm in diameter. The histology is different from the proximal portion inasmuch as the free end of the cells has a honeycomb border (Fig. 2). No vaIvular arrangement could be detected at either end of the ampulla. The dista~p~~tio~. The convoluted part of the Malpighian tubule following the ampulla and situated beneath the fascia constitutes the distal portion, Proximally it communicates with the ampulla of the middle portion, whereas distally it ends blindly. Histologically it is similar to the middle portion. ~istyi~ti~~
of uric acid in various ttsfues
Malpighian
tzdwles. Histochemical tests for uric acid revealed a complete picture of its distribution in the various regions of the Malpighian tubules. A heavy deposit is present in the lumen of the proximal portion of the tubule (Fig. 3),
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but it is completely absent from its cells. Spheres of uric acid with radial striations as shown in Rhodnius (WIGGLESWORTH,1931) could clearly be seen in sections of this portion of the tubule prepared by WIGGLESWORTH’S method (Fig. 5). On the other hand, uric acid in crystallized form could be detected only in the cells (Fig. 4) and never in the lumen of the middle portion of the tubule, whereas no trace of it could be found either in the cells or in the lumen of the ampulla and the distal portion of the tubule. Fat body. Sometimes the fat body gave a faint reaction for uric acid with Benedict’s reagent, but all other tests were negative. Under the circumstances it may not be inappropriate to presume that the faint reaction is due to the small amount of uric acid produced during metabolism of the fat cells themselves. Uric acid does not accumulate in the fat body of Corcyra larva, unlike cockroaches (CulkoT, 1895) and certain other insects (WIGGLESWORTH,1953; ROEDER,1953). Wall of the mid- and hind-guts.
Both of these gave negative results.
Contents of the mid- and hind-guts. A negative reaction was obtained with the contents of the mid-gut, whereas those of the hind-gut gave a positive reaction. Excreta.
A strong positive reaction for uric acid was obtained with the excreta.
Chemical composition of food and excreta Inorganic constituents. The inorganic constituents of food (raw milled rice) and excreta of Corcyra larvae fed on rice are given in Table 1. TABLE 1
Constituents
Food (raw milled rice)
Carbonate Chloride Nitrate Phosphate Sulphate Calcium Iron Magnesium Potassium Sodium Ammonia -I- = Positive;
Excreta
+ + (Weak) + + + ? - = Negative;
? = Doubtful.
Organic c~tit~nts. The data given by SUBRAHMANYAN et al. (1954) indicate that the main constituents of raw milled rice are 79.6 per cent carbohydrate, 12.6 per cent moisture, and 6.6 per cent protein.
6 FIG. 3. A photograph of a transverse section through the proximal portion of the Malpighian tubule of a Corcyra larva. Uric acid (ur.) is seen only in the lumen of the proximal portion. FIc. 4. A photograph of a transverse section through the middle portion of the Malpighian tubule of a Corcyra larva, showing uric acid (ur.) inside the cells. FIG. 5. A photograph of a transverse section of the proximal portion of the Malpighian tubule of a Corcyra larva, showing uric acid spheres (ur. ac. sph.) with radial striations. FIG. 6. A photograph of a transverse section through the middle portion of the Malpighian tubule of a Corcyra larva, showing glycogen in the lumen (gly. 1.) as well as in the cells (gly. c.), and indicating its (glycogen) entry from the blood. FI(;. 7. A photograph of a transverse section through the proximal portion of the Malpighian tubule of a Corcyra larva, showing glycogen in the cells (gly. c.) which is diffusing out.
{~ : ~
L~I~ ~ ~ t~roxim~pc~t~n
~-~
N,t Toqenous w~,~tes
l
~ " Mwto~caHy useful substances and C o n c e n f raF'~d ,o~,d "~tmosr d r y U w l n ~
•
A
r
\
.~ ,~C_ U r i n a r y b l a d d e r
\ Hoernollfmph( e x e r t p~O~lin ond other substances of Ior~r molecules) is dlf fusing |n
//J b~ °'~°° I ~Hor~'yc~ l
,
M~ddl~ pore,on
B
~u|la
\
J ?)'.'::
I Moisture ~rom the ¢xcreto ~s absorbed h~re and passed i n t o ~ middle portion)
FiG. 8. Diagrammatic representation of the process of excretion in a Corc3'ra larva. Arrows indicate the course taken by substances during excretion. Figs. A and B show the details of the histology of the proximal and middle portions respectively.
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Analysis of the excreta shows that protein, glycogen, dextrin, reducing sugars, cellulose, acetone, aceto-acetic acid, lactic acid, creatin and creatinine, xanthine, cystine, and guanine are absent. Its main constituents are a large quantity of uric acid, traces of urea, a certain amount of starch, and about 9-6 per cent water. The chemical composition of food and excreta described above provides evidence that all the protein and most of the carbohydrate and water get utilized and the nitrogenous waste is excreted out mainly as uric acid. Carbonates, chlorides, calcium, magnesium, iron, a certain amount of starch, and some water together with large quantities of uric acid form the excreta of Corcyra larva. Excretion 5fdyes
The course taken by the injected dyes (neutral red and methylene blue) and their excretion through the Malpighian tubules is described in Table 2. TABLE
2
Interval
Observation
5 min
Middle portion of the tubule fills with the dye, but the ascending and major portion of the descending limb, the distal portion, and the ampulla do not take up the dye.
10 min
The dye has almost filled the lumen of the entire proximal portion of the tubule; there is no indication of the dye in the distal portion or the gut. The dye appears to have ascended from the middle portion and not passed in directly from the body cavity, as the cells of this portion remain transparent.
30 nun
The lumen of the proximal portion becomes turgid due to the inflow of the dye, while the middle portion becomes colourless, showing that almost all the dye from the body fluid has been removed.
4 hr
About one-third of the lower portion of the descending limb is colourless and empty while the remainder is still full of coloured dye solution.
24 hr
No trace of the coloured solution remains in any part of the tubules, but fine granules of the injected dye are still present in the lumen of the ascending limb and the urinary bladder. Some granules are seen in the lumen of the hind-gut.
Appearance of the dye solution in the middle portion of the tubule almost immediately after injection into the body cavity presumably indicates that the dye enters the fumen of the middle portion of the tubule at the same concentration as that in the haemolymph. The course taken by the dyes during excretion shows that each portion of the tubuleis functionally different from the other and allows only one-way permeability of the fluids. No substance from the body fluid enters the proximal portion. The
228
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solid excretory matter is retained in its lumen, but water is allowed to return to the body fluid. On the other hand, dye in solution enters the middle portion through its cells but nothing can pass out into the body fluid from this region. The distal portion does not seem to have any connexion with the body fluid as the dye could never be seen in this portion of the tubule. The experiments, therefore, reveal that also on a physiological basis the Malpighian tubule of Corcyra larva is divided into the same three portions, viz. the proximal, the middle, and the distal. Distribution of glycogen in the Malpighian tubules Little is known about glycogen in the Malpighian tubules of insects, and this seems to be the first record. As with uric acid, a differential distribution of glycogen in the Malpighian tubules of Corcyra larva could be seen. The results are described below. (1) Glycogen could not be detected distal portion of the tubule.
either
(2) A heavy deposit (Fig. 6) of glycogen portion, while a fair amount of this substance within the cells of this region.
in the cells or in the lumen
of the
is present in the lumen of the middle could be detected in a state of diffusion
(3) The amount of glycogen both in the lumen as well as in the cells of the proximal portion (Fig. 7) gradually decreases in the descending limb, becoming less and less in the ascending limb till it is altogether absent by the time it covers about half or two-thirds of the ascending limb. No glycogen could be detected in the urinary bladder. DISCUSSION
There is ample evidence for the fact that in Corcyra larvae the nitrogenous wastes are excreted mainly through the Malpighian tubules and do not accumulate in the fat body. A distinct morphological and physiological differentiation between the different portions of each tubule is clearly indicated. The experiments with the injected dyes suggest that the middle and proximal portions of the tubules perform different functions during the process of excretion. Practically the whole plasma (except the protein and certain other larger molecules) containing both metabolically useful and excretory substances presumably enters the lumen of the tubule. No selection seems to be exercised and the process is more or less a passive diffusion of the haemolymph, similar to that described by RAMSAY(1958) in Dixippus morosus and PATTONand CRAIG(1939) in the larva of Tenebrio molitor. The fluid gradually ascends into the proximal portion in which a selective reabsorption appears to take place. Almost all the water and metabolically useful substances are permitted to pass out to be restored to the haemolymph, while the nitrogenous wastes and other excretory matter, like the solid dyes, are retained in the lumen. As the time advances more and more water is absorbed and the urine becomes highly concentrated till it reaches the ascending limb. The urine from all the three tubules of one side collects into the corresponding urinary bladder and is ultimately voided into the lumen of the gut (see Fig. 8).
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The distal portion does not seem to take any active part in the excretion of nitrogenous wastes since at no time could the dyes or uric acid be found in this region. This is in agreement with METALNIKOV(1908), who found that in GalZeria larvae the tubes covered over by the fascia could never take up the dye. This portion of the tubule appears to be mainly osmoregulatory, reabsorbing water from the excreta in the rectum. Its honeycomb border, similar to that of the middle portion of the tubule, leads me to believe that the water absorbed from the excreta is not allowed to pass out and return to the body fluid but is collected in the ampulla from which it is forced into the middle portion of the tubule. This water may thus help flush the spheres of uric acid from the tubules into the gut. This finding is in conformity with that of POLL (1938, in WIGGLES~~O~TH,1953) who stated that the water absorbed by the perirectal tubes is used to flush out the solid matter from the lumen of the tubules of caterpillars. The diffusion and reabsorption cycle is shown diagrammatically in Fig. 8. The suggestion based on the injection experiments that there is a division of labour in the tubules of Corcyra larva finds strong support from the study of the distribution of glycogen and uric acid. The absence of both glycogen and uric acid from the distal portion of the tubule and the ampulla clearly indicates that no substance from the body cavity enters this portion. At the same time, the presence of large quantities of glycogen in the cells as well as in the lumen of the middle portion of the tubuies strengthens the contention that not only the nitrogenous wastes and water but in fact almost the whole plasma minus the protein enters through this region. The gradual decrease in the quantity of glycogen from the lumen of the descending limb of the proximal portion and its absence from the ascending limb coupled with the fact that uric acid was never found in the cells of both the limbs, although it is present in large quantity into the lumen, is evidence that selective reabsorption of only the metabolically useful substances along with water takes place through this portion of the Malpighian tubule. The postulate presented here that the different segments of a Malpighian tubule perform separate functions is in conformity with the findings of WIGGLESWORTH(193 1) on Rho&us. The proximal and middle portions of the tubules in Corcyra larvae are not only similar in structure to the corresponding regions, i.e. the Iower and upper segments of the tubule of Rho~~i~s, but also exhibit the same functions. However, the tubules of ~~a~nius are not cryptonephric, hence none of its parts corresponds to the distal portion of the tubule of Corcyra larva. But the present view differs from that of WIGGLESWORTH (1931) in that there is no selective filtration of the body fluid through the Malpighian tubules in Corcyra larva, as it has been established that practically the whole plasma (except proteins and larger molecules), and not only the uric acid in the form of sodium and potassium urates as reported in Rho&&s, enters the middle portion of the Ma~pighian tubule of Corcyra larvae. The author’s views are further supported by the findings of RAMSAY(1958}, who has shown that in ~~~~pus morosus metabolically useful substances enter into the Malpighian tubules by passive diffusion and are reabsorbed in the rectum.
230
P. N. SRIVASTAVA
The observation of glycogen in the Malpighian tubules of Corcyra larva finds support in the experimental findings of PATTONand CRAIG(1939) on T. molitor, as they have definitely shown that the inward movement through the tubes is not confined to water and nitrogenous wastes but includes electrolytes and a number of other compounds like amino acids. Absence of any selective absorption of water, salts, and amino acids, as demonstrated by their ingenious experiments, should in my opinion be equally applicable to simpler carbohydrates. The digested and absorbed carbohydrates along with the other constituents (except proteins) of the body fluid should be penetrating the Malpighian tubules of Co~cy~~ larvae in a similar manner, as the amino acids, glycine and glutamic acids, urea, etc., readily enter the tubes of T. molitor when added to the perfusion medium. As in Corcyra larvae, uric acid has been reported to be the main constituent of the nitrogenous waste in Galleria larvae, in Heterogenea, in Tineola biselliella, in ~n~~e~eff, and in silkworms by ~~ALNI~OV (1908), SAMSON (1908), HOLLANDE and CARDEBARD (1926), LEIFERT(1935), GRASSEand LESPERON(1936), and KUWANA (1937) respectively. Ammonia is absent from the excreta of Corcyra larvae as in that of Rhodnius. Absence of sulphur from the food of Corcyra larvae as in that of Rhodnius explains the absence of ammonia from their excreta. The contention that ammonia is present only in excreta of those insects which take food rich in sulphur is further supported by the observations of PRADHAN(1949) on ~nthy~~s fasciatus. The presence of a certain amount of undigested
carbohydrate (starch) in the excreta of Corcyra larvae is interesting and calls for comment. The food of Corcyra larvae, with which it always remains surrounded, is available in plenty. But since it contains only 12.9 per cent of moisture out of which some has to be excreted, as is clear from the analysis of the excreta, there appears to be a reasonable possibility that the larva is induced to ingest large quantities in order to obtain the requisite amount of moisture. It is suggested that the carbohydrate ingested in excess of requirements remains undigested and is disposed of in the excreta.
There are six Malphighian tubules, three on each side of the gut, The tubules are cryptonephric and are physiologically and histologically divided into the distal, the middle, and the proximal portions. The distal and the middle portions have a honeycomb border, the proximal a brush border. Experimental evidence shows that each portion of the tubule has one-way permeability. The haemolymph ~excluding proteins and certain other larger molecules) is constantly diffusing in the middle portion of the Malpighian tubules, the excretory products are retained in the lumen of the proximal portion, while water and metabolically useful substances pass out into the body fluid. The distal portion absorbs water from the excreta. This water does not pass back to the body fluid but helps to flush out the solid matter from the lumen of the tubule. Further evidence for this diffusion and reabsorption cycle has been provided as a result of the study of the distribution of uric acid and glycogen in various portions of the Malpighian tubules. Besides
PHYSIOLOGYOF EXCRETIONIN THE LARVAOF CORCYRA
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uric acid, carbonates, chlorides, calcium, iron, magnesium, and a certain amount of undigested starch constitute the excreta of Corcyra cephalonicalarvae. Acknowledgements-The work was done under the guidance of Dr. P. D. GUPTA to whom I am greatly indebted. I take this opportunity to express my gratitude to Professor M. B. LAL for kindly providing me with facilities for research work, and, finally, I wish to thank the Government of India for the award of the Research Training Scholarship. REFERENCES BALDWIN E. andBELL D. J. (1955) Cole’s Practical Phy~oZa~~c~~Chemistry. Heffer, Cambridge.
COWDRY E. V. (1952) ~aborato~ Technique in ~ioZogy and Medicine. Williams & Wilkins, Baltimore. C&NOT L. (1895) Etudes physiologiques sur les Orthopteres. Arch. Biol., Paris 14,293-341. FKSHERH. J., WARRENL. E., SALE J. W., Ross W. H., RINDOLLARW. F., and MARIANL. 0. (1945) Oficial and Tentative Methods of Analysis of the Association of Official Agricultural Chemists. Benjamin Franklin Station, Washington. GRASSE P. P. and LESPERON L. (1936) Mue et excretion chez le ver a soie. C.R. Sot. Biol., Paris 122,1013-1015. GUPTA P. D. and SINHA R. N. (1960) Excretion and its products in some stored-graininfesting beetles. Ann. ent. Sot. Amer. 53, 632-638. HAWK P. B., OSER B. L., and SUMMERSONW. H. (1947) Practical Physiological Chemistry. Blakiston, Philadelphia. HOLLANDEA. C. and CARDEBARD N. (1926) Notes chimiques et physiologiques se rapportant aux excrements de la teigne du crin (Tineola ~se~liella Hummel ; Syn. cr~ne~laTreitscheDuponchol). Bull. Sot. Chim. bioE., Paris 8, 631-635. K~JWANA2. (1937) Uric acid excretion in silkworm. yap. J. Zool. 7, 305-309. LEIFERT H. (1935) Untersuchungen iiber den Exkretstofbvechsel bei eiren Raupen und puppen von Antheraea pernyi. Zool. Jb. (Physiol.) 55, 13 l-l 90. LILLIER. D. (1954)HistopathoZogic TechnicandPracticalHistochemistry. Blakiston, NewYork. METALNIKOVS. (1908) Recherches experimentales sur les chenilles de Galleria mellonella. Arch. Zool. exp. g&z. 8, 489-588. PATTONR. L. and CRAIG R. (1939) The rates of excretion of certain substances by the larvae of Tenebrio molitor L. J. exp. ZooE. 81, 437-457. PRADHANK. S. (1949) On the physiology of digestion in the larva of woolly bear, Anthremds fasciatus Herbst. J. zool. Sot. India 1, 107-119. RAMSAYJ. A. (1953) Active transport of potassium by the Malpighian tubules of insects. J. exp. Biol. 30, 358-369. RAMSAYJ. A. (1954) Active transport of water by the Malpighi~ tubules of the stick insect, Dixippus mo~osus (Orthoptera, Phasmidae). J. exp. Biol. 31, 104-l 13. RAMSAYJ. A. (1955) The excretory system of the stick insect, Dixippzrs morosus (Orthoptera, Phasmidae). r. exp. Biol. 32, 183-216. RAMSAY J. A. (1956) Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae) : Calcium, magnesium, chloride, phosphate and hydrogen ions. J. exp. Biol. 33, 697-891. RAMSAYJ. A. (1958) Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae): Amino acids, sugars and urea. r. exp. Biol. 35, 871-891. ROEDER K. D. (1953) Insect Physiology. Wiley, New York. SAMPSONK. (1908) Uber des Verhalten der Vasa Malpighii und die exkretorische Funcktion der Fettzellen wahrend der Metamorphose von Heterogenia limacodes Hiifn. Zool. Jb. (Anat.) 26, 403-422. SRIVA~TAVA P. N. and GUPTA P. D. (1961) Excretion of uric acid in P~~~aneta americana L. 3’. Ins. Phys~oZ. 6, 163-167.
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SUBRAHMANY~ V., BAINSG. S., SWAMINATHAN J., and BHATIAD. S. (1954) Investigations on grain substitute-V. The nutritive value of synthetic rice. SUE. cent. food tech. res. Inst. Mysore 4, 55-57. VOCEL A. I. (1954) A Textbook of Macro and Seµ Qualitative Inorganic Analysis. Longmans, London. WIGGLESWORTH V. B. (1931) The physiology of excretion in the blood-sucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). J. exp. BioE. 8, 41 l-451. WIGGLESWORTH V. B. (1953) The Principles ofInsect Physiology. Methuen, London.